阅读说明：本技术 新的多肽及其医疗用途 (Novel polypeptide and medical use thereof ) 是由 M·莫格林 S·阿布迪拉西 于 2017-01-20 设计创作，主要内容包括：本发明提供了包含源自VI型胶原蛋白的氨基酸序列或者由所述氨基酸序列组成的多肽或其片段、变体、融合物或衍生物、或者所述其衍生物的片段、变体的融合物,其中所述多肽、片段、变体、融合物或衍生物能够扼杀或削弱微生物的生长。本发明的相关方面提供相应的分离的核酸分子、载体以及用来制造其的宿主细胞。另外还提供了包含本发明的多肽的药物组合物,以及将其用于治疗和/或预防微生物感染以及用于伤口护理的方法。还提供了体外杀死微生物的方法,以及与所述药物组合物结合的医疗装置。(The invention provides a fusion comprising a polypeptide derived from or consisting of the amino acid sequence of type VI collagen, or a fragment, variant, fusion or derivative thereof, or a fragment, variant of a derivative thereof, wherein the polypeptide, fragment, variant, fusion or derivative is capable of killing or attenuating the growth of a microorganism. Related aspects of the invention provide corresponding isolated nucleic acid molecules, vectors, and host cells for making the same. Also provided are pharmaceutical compositions comprising the polypeptides of the invention, and methods of using them to treat and/or prevent microbial infections and for wound care. Methods of killing microorganisms in vitro, and medical devices incorporating the pharmaceutical compositions are also provided.)

1. A fusion comprising or consisting of an amino acid sequence derived from type VI collagen, or a fragment, variant, fusion or derivative thereof, or a fragment, variant of a derivative thereof,

wherein the polypeptide, fragment, variant, fusion or derivative is capable of killing or attenuating the growth of a microorganism.

2. The polypeptide of claim 1, wherein the microorganism is selected from the group consisting of bacteria, mycoplasma, yeast, fungi, and viruses.

3. The polypeptide of any one of the preceding claims, wherein the polypeptide is capable of binding to a membrane of the microorganism.

4. The polypeptide of any one of the preceding claims, wherein the polypeptide is capable of causing rupture of a membrane of the microorganism.

5. The polypeptide of any one of the preceding claims, which is capable of promoting wound closure.

6. The polypeptide of any one of the preceding claims, wherein the polypeptide is capable of exhibiting an antimicrobial effect greater than or equal to LL-37.

7. The polypeptide of any one of the preceding claims, wherein the microorganism is a gram-positive or gram-negative bacterium.

8. The polypeptide of claim 7, wherein the microorganism is selected from the group consisting of: pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, group A streptococcus (e.g., Streptococcus pyogenes), group B streptococcus (e.g., Streptococcus agalactiae), group C streptococcus (e.g., Streptococcus dysgalactiae), group D streptococcus (e.g., enterococcus faecalis), group F streptococcus (e.g., Streptococcus angina), group G streptococcus (e.g., Streptococcus dysgalactiae), type A hemolytic streptococcus (e.g., Streptococcus viridans, Streptococcus pneumoniae), Streptococcus bovis, Streptococcus lentus, Streptococcus angina, Streptococcus sanguis, Streptococcus suis, Streptococcus mutans, Moraxella catarrhalis, non-separable Haemophilus influenzae (NTHiHih), Haemophilus influenzae type B (Hib), Neisseria nucleatum, Prevotella intermedia, Klebsiella pneumoniae, Enterobacter cloacae, enterococcus faecalis, Staphylococcus epidermidis, drug-resistant Pseudomonas aeruginosa (MRPA), Multidrug-resistant staphylococcus aureus (MRSA), multidrug-resistant escherichia coli (MREC), multidrug-resistant staphylococcus epidermidis (MRSE), and multidrug-resistant klebsiella pneumoniae (MRKP).

9. The polypeptide of any one of the preceding claims, wherein the microorganism is a bacterium that is resistant to one or more conventional antibiotic agents.

10. The polypeptide of claim 9, wherein the microorganism is selected from the group consisting of: multidrug-resistant staphylococcus aureus (MRSA), multidrug-resistant pseudomonas aeruginosa (MRSA), multidrug-resistant escherichia coli (MREC), multidrug-resistant staphylococcus epidermidis (MRSE), and multidrug-resistant klebsiella pneumoniae (MRKP).

Technical Field

The present invention relates to novel polypeptides derived from a naturally occurring extracellular matrix component protein collagen type VI, which have antimicrobial properties. The invention also relates to the use of these polypeptides in the treatment of microbial infections or in wound care. Kits, devices, and compositions that bind these polypeptides are also provided.

Background

The increase of pathogenic bacteria with resistance to conventional antibiotics has been a major threat to global health care in the last decade (1, 2). Therefore, the development of new antimicrobial agents to combat emerging infectious diseases is of great interest. To overcome the harmful effects of pathogenic bacteria, a rapid host defense mechanism is necessary. Antimicrobial peptides (AMPs) are powerful molecules of the innate immune defense system, providing a rapid and non-specific response against invading pathogens, which represents the first line of defense against invading pathogens in most multicellular organisms. AMPs exhibit a broad spectrum of activity against gram-positive bacteria, gram-negative bacteria, fungi and viruses, as well as parasites (3-8). Despite their different origins, AMPs share several physicochemical properties that are critical for their direct antimicrobial properties. These peptides are smaller molecules (10-50 amino acids) and typically carry a net positive charge in the range of +2 to +9 (4, 9-10). Another recurring feature is that they often have ≧ 30% hydrophobic residues, which enable them to adopt amphiphilic structures (4) when in contact with lipid-based environments (e.g., bacterial cell membranes). More than two thousand AMPs have been isolated from a wide range of species including plants, insects, and mammals (see antimicrobial peptide database, http:// APs. unmac. edu/AP/main. php).

It is generally accepted that AMPs have cellular membrane activity and destroy target cells by disrupting the integrity of the cellular membrane (11) or by causing intracellular changes (12). However, different modes of action have been proposed for several AMPs, but the underlying molecular mechanisms are still under investigation (13-16).

In addition to their direct antimicrobial activity, some peptides (e.g., LL-37) have immunomodulatory functions such as chemotaxis, neutralization of endotoxins (e.g., LPS), enhancement of wound healing, angiogenesis, and modulation of pro-inflammatory cytokine production (17-18). Their broad spectrum of antimicrobial activity, coupled with low bacterial resistance mechanisms, make these AMPs attractive anti-infective therapeutics (8, 19), but there remains a need for additional antimicrobial therapies and AMPs.

Extracellular matrix (ECM) proteins like collagen, fibronectin, laminin and vitronectin are attractive pathogen targets for pathogenic bacteria to adhere to, invade, and colonize the connective tissue of the host (20). Nevertheless, there is a large body of evidence that ECM proteins may have protective effects during early infection (21-24). Type VI collagen is a ubiquitous extracellular matrix component that is present in all connective tissues and is often associated with basement membrane. Type VI collagen forms a complex and extensive network of beaded microfibrils in most connective tissues. The major form of collagen type VI consists of 3 distinct polypeptide chains: α 1(VI), α 2(VI) and α 3(VI), which form triple helix monomers. Within the cell, the monomers assemble into dimers and tetramers, which are secreted into the extracellular space. There, the tetramer polymerizes end-to-end to form microfibrils, which become part of the expanded supramolecular matrix assembly. Recently, three additional chains (α 4, α 5 and α 6) have been discovered which can replace the α 3-chain in some tissues (9, 10). Structurally, each α -chain is characterized by a short extended triple-stranded helical region flanked by two large N-and C-terminal globular regions with homology to von willebrand factor type a domain (VWA) (25-27). VWA is also responsible for protein-protein interactions in the ECM (25-28). The α 1(VI) and α 2(VI) chains of type VI collagen contain one N-terminal (N1) and two C-terminal (C1 and C2) VWA domains, whereas α 3(VI) is much larger and contains some ten N-terminal (N10-N1) VWA domains and two C-terminal VWA domains. In addition, the α 3(VI) chain has three C-terminal domains (C3-C5) with homology to salivary gland protein, fibronectin type III repeats, and Kunitz family serine protease inhibitors (29). By its unique structure, type VI collagen provides strength, integrity, and structure to a wide range of tissues. It is also involved in other important biological processes such as apoptosis, autophagy, angiogenesis, fibrosis, and tissue repair (30).

It has been previously shown that peptides derived from von willebrand factor containing sequences that bind to consensus heparin (Cardin and Weintraub motifs) (31) exhibit antimicrobial activity against gram positive and gram negative bacteria (32-33).

Collagen type VI has previously been shown to bind to streptococcus pneumoniae and group a, C and D streptococci (35), killing bacteria (36). Recently, similar adhesion and bactericidal effects of type VI collagen microfibrils against moraxella catarrhalis and other gram-negative and gram-positive human pulmonary pathogens have also been reported (37). However, no structural or mechanistic explanation has been provided for this documented observation.

Thus, there remains a need to identify new antimicrobial agents, particularly agents that are capable of killing or inhibiting the growth of bacteria, such as MRSA that have developed resistance to conventional antibiotics.

Disclosure of Invention

A first aspect of the invention provides a polypeptide comprising or consisting of: an amino acid sequence derived from type VI collagen, or a fragment, variant, fusion or derivative thereof, or a fragment, variant fusion of a derivative thereof, wherein said polypeptide, fragment, variant, fusion or derivative is capable of killing or attenuating the growth of a microorganism.

It will be appreciated by those skilled in the art that the type VI collagen may be from human or non-human origin. For example, the type VI collagen may be derived (directly or indirectly) from a non-human mammal, such as a great ape (e.g., chimpanzee, bonobo, gorilla, gibbon, and chimpanzee), a monkey (e.g., macaque, baboon, and verruca), a murine (e.g., mouse, rat), or an ungulate (e.g., pig, horse, and cow).

Thus, by "type VI collagen" (also referred to as "collagen VI") we include naturally occurring human type VI collagen and homologs thereof, such as bovine type VI collagen. In a preferred embodiment, the polypeptide is derived from human type VI collagen.

By "derived from" the amino acid sequence of type VI collagen we include the amino acid sequences present in the amino acid sequence of naturally occurring type VI collagen. In particular, we include the amino acid sequence: it comprises at least 5 contiguous amino acids from the sequence of naturally occurring type VI collagen, but does not include full-length protein. For example, in one embodiment, the amino acid sequence can contain at least 5 contiguous amino acids from type VI collagen, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 contiguous amino acids from type VI collagen. Thus, the amino acid sequence derived from type VI collagen corresponds to a fragment of type VI collagen having antimicrobial activity.

By "capable of killing or attenuating the growth of a microorganism" we include polypeptides having antimicrobial activity. The antimicrobial activity may be in whole or in part, and may be dose-dependent. This can be demonstrated, for example, by radial diffusion testing.

The microorganism for which the polypeptide of the invention is effective may be selected from the group consisting of: bacteria, mycoplasma, yeast, fungi, and viruses.

In one embodiment, the polypeptide of the invention is capable of binding to a membrane of a microorganism. In another embodiment, the polypeptide can have affinity for a negatively charged surface (e.g., a bacterial membrane). The affinity can be tested, for example, by affinity for heparin, where a higher affinity for heparin indicates a higher affinity for negatively charged surfaces.

Advantageously, the affinity or binding capacity of the polypeptide is similar to or greater than that of LL-37. Thus, in one embodiment, the polypeptide is capable of exhibiting an antimicrobial effect that is greater than or equal to the antimicrobial effect of LL-37 (i.e., LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES; SEQ ID NO: 36).

In one embodiment, the polypeptide is capable of causing a structural change in the microorganism, including, for example, membrane perturbation, blebbing, or efflux of cytoplasmic components.

Thus, the polypeptide may be capable of causing membrane disruption of the microorganism. This can be quantified, for example, by studying the uptake of fluorescent molecules by the microorganisms by microscopic studies, such as electron microscopy or fluorescence microscopy.

In further embodiments, the polypeptide may be capable of promoting wound closure and/or wound healing.

By "promoting wound closure" and/or "wound healing" we include assisting the healing process of a wound by, for example, accelerating healing. For example, wound care products may be capable of enhancing epithelial regeneration and/or healing of injured epithelium and/or injured stroma. In one embodiment, the wound care product may be capable of enhancing epithelial and/or stromal cell proliferation by a non-lytic mechanism. Wound closure capacity can be quantified by, for example, a cell scraping assay.

Thus, the polypeptides may play a role in wound care by promoting wound closure/healing and/or by preventing wound infection.

Wounds to be treated by the polypeptides of the invention may be in vitro (i.e. surface wounds of the skin and underlying tissue) and/or in vivo (such as internal wounds resulting from organ transplantation or removal of tissue/parts of an organ (e.g. after colon surgery)).

It will be appreciated by those skilled in the art that the polypeptides of the invention may exert antimicrobial effects against gram positive and/or gram negative bacteria. For example, the microorganism may be a bacterium selected from the group consisting of: pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, group A streptococcus (e.g., Streptococcus pyogenes), group B streptococcus (e.g., Streptococcus agalactiae), group C streptococcus (e.g., Streptococcus dysgalactiae), group D streptococcus (e.g., enterococcus faecalis), group F streptococcus (e.g., Streptococcus angina), group G streptococcus (e.g., Streptococcus dysgalactiae), type A hemolytic streptococcus (e.g., Streptococcus viridans, Streptococcus pneumoniae), Streptococcus bovis, Streptococcus lentus, Streptococcus angina, Streptococcus sanguis, Streptococcus suis, Streptococcus mutans, Moraxella catarrhalis, non-separable Haemophilus influenzae (NTHiHih), Haemophilus influenzae type B (Hib), Neisseria nucleatum, Prevotella intermedia, Klebsiella pneumoniae, Enterobacter cloacae, enterococcus faecalis, Staphylococcus epidermidis, drug-resistant Pseudomonas aeruginosa (MRPA), Multidrug-resistant staphylococcus aureus (MRSA), multidrug-resistant escherichia coli (MREC), multidrug-resistant staphylococcus epidermidis (MRSE), and multidrug-resistant klebsiella pneumoniae (MRKP).

In one embodiment, the microorganism is a bacterium that is resistant to one or more conventional antibiotic agents.

By "conventional antibiotic agents" we include known agents capable of killing or attenuating the growth of microorganisms, such as natural and synthetic penicillins and cephalosporins, sulfa agents, erythromycin, kanamycin, tetracycline, chloramphenicol, rifampin and including gentamicin, ampicillin, benzylpenicillin, benzamincillin, benzathine, phenacillin, phenoxy-methicillin, procainazine, cloxacillin, flucloxacillin, methicillin sodium, amoxicillin, bacampicillin hydrochloride, cycloserine, mezlocillin, pivampicillin, phthalazinil hydrochloride, carpoxicillin sodium, piperacillin, ticarcillin, mecillin, pivirin (pirmiecillin), cefaclor, cefadroxil, cefotaxime, cefoxitin, cefsulodin, ceftazidime, ceftizoxime, cefuroxime, cephalexin, Cephalothin, hydroxybenzotriazole cephalosporins, cefazolin, cyclohexylamine cephalosporins, oxacepin disodium, aztreonam, chlortetracycline hydrochloride, sodium chloromycetin, demeclocycline hydrochloride, doxycycline, lysinocycline, minocycline, oxytetracycline, amikacin, neomycin B sulfate, neomycin sulfate, netilmicin, tobramycin, colistin, sodium fusidate, polymyxin B sulfate, spectinomycin, vancomycin, calcium sulfaphthalein, pyrazine sulfonic acid, sulfadiazine, sulfadimidine, sulfaguanidine, sulfaurea, capreomycin, metronidazole, tinidazole, cinoxacin, ciprofloxacin, nitrofurantoin, urotropin, streptomycin, carbenicillin, polymyxin mesylate, polymyxin B, furazolidone, nalidixic acid, trimethoprim-sulfamethoxazole, clindamycin, doxycycline hydrochloride, lysin, minocycline, doxycycline, and doxycycline, Lincomycin, cycloserine, isoniazid, ethambutol tablets, ethionamide, pyrazinamide and the like; antifungal agents, such as miconazole, ketoconazole, itraconazole, fluconazole, amphotericin, flucytosine, griseofulvin, natamycin, nystatin, and the like; and antiviral drugs such as acyclovir, AZT, didanosine (ddI), amantadine hydrochloride, isoprinosine, vidarabine, and the like.

Thus, in one embodiment, the microorganism is selected from the group consisting of: multidrug-resistant staphylococcus aureus (MRSA) (methicillin-resistant staphylococcus aureus), multidrug-resistant pseudomonas aeruginosa (MRSA), multidrug-resistant escherichia coli (MREC), multidrug-resistant staphylococcus epidermidis (MRSE), and multidrug-resistant klebsiella pneumoniae (MRKP).

Advantageously, the polypeptide according to the first aspect of the invention exhibits selective toxicity to a microbial agent. By 'selective' we mean that the polypeptide is preferably toxic to one or more microorganisms (such as bacteria, mycoplasma, yeast, fungi and/or viruses) as compared to a mammalian, e.g. human, host cell. For example, the toxicity of a polypeptide to a target microorganism is at least two times greater than the toxicity of the polypeptide to mammalian cells, more preferably at least three times, at least four times, at least five times, at least six times, at least eight times, at least ten times, at least fifteen times, or at least twenty times greater.

Suitably, the polypeptide is substantially non-toxic to cells of a mammal, such as a human.

For example, a polypeptide may not exhibit cytotoxicity to red blood cells or monocytes at concentrations that can be used to kill microorganisms such as bacteria. In one embodiment, the polypeptide does not exhibit cytotoxicity at concentrations up to 30 μ Μ, or alternatively, at concentrations up to 50 μ Μ.

For example, in this manner, when the compound is used to treat a microbial infection, the dosing regimen can be selected such that the microbial cells are destroyed in a manner that minimizes damage to healthy host tissue. Thus, the polypeptide may exhibit a "therapeutic window".

In one embodiment, the polypeptide is capable of exerting an anti-endotoxin effect.

By "anti-endotoxin effect" we include polypeptides that counteract the effects induced by endotoxin. For example, in one embodiment, the polypeptide is capable of inhibiting (at least partially inhibiting) the induction of nitrite by LPS.

In one embodiment, the polypeptide is derived from or exhibits amino acid sequence homology to a VWA domain (e.g., a spherical VWA domain). Thus, a polypeptide may comprise or consist of: an amino acid sequence corresponding to at least five (e.g. at least 10, 15, 20 or more) consecutive amino acids of a VWA domain, or an amino acid sequence having at least 70% (e.g. at least 80%, 90% or 95%) identity to such a sequence.

In further embodiments, the polypeptide may comprise or consist of the entire VWA domain.

By "VWA domain" we include the a domain of von willebrand factor and domains showing homology to the a domain of von willebrand factor, as well as regions containing the VWA-domain.

In one embodiment, the polypeptide is derived from the α 3chain of collagen type VI. Thus, the polypeptide may be derived from the α 3N or α 3C region. For example, the polypeptide may be derived from the N2, N3, or C1 domain of the alpha 3chain of collagen VI.

In an alternative embodiment, the polypeptide is derived from the α 4 chain of collagen VI.

In another alternative embodiment, the polypeptide is derived from the α 5 chain of collagen VI.

In a further alternative embodiment, the polypeptide is derived from the α 6 chain of collagen VI.

In a still further alternative embodiment, the polypeptide is derived from the α 2 chain of collagen VI, for example from the α 2N region.

It will be appreciated by those skilled in the art that the polypeptides of the invention may have cationic residues on their surface, or cationic sequence motifs therein.

Thus, in one embodiment, the polypeptide has a net positive charge. For example, the polypeptide can have a charge ranging from +2 to + 9.

In a further embodiment, the polypeptide has at least 30% hydrophobic residues.

In still further embodiments, the polypeptide can have an amphiphilic structure.

Exemplary polypeptides of the first aspect of the invention comprise or consist of: SEQ ID NO: 1 to 23 or a fragment, variant, fusion or derivative thereof, or a fusion of said fragment, variant or derivative thereof, which retains the amino acid sequence of any one of SEQ ID NOs: 1 to 23.

TABLE 1 exemplary Polypeptides of the invention

For example, a polypeptide of the invention may comprise or consist of: selected from the group consisting of SEQ ID NO: 1 to 5:

“GVR28”：GVRPDGFAHIRDFVSRIVRRLNIGPSKV[SEQ ID NO：1]

“FYL25”：FYLKTYRSQAPVLDAIRRLRLRGGS[SEQ ID NO：2]

“FFL25”：FFLKDFSTKRQIIDAINKVVYKGGR[SEQ ID NO：3]

“VTT30”：VTTEIRFADSKRKSVLLDKIKNLQVALTSK[SEQ ID NO：4]

“SFV33”：SFVARNTFKRVRNGFLMRKVAVFFSNTPTRASP[SEQ ID NO：5]

or a fragment, variant, fusion or derivative thereof, or a fusion of said fragment, variant or derivative thereof, which retains the amino acid sequence of SEQ ID NO: 1 to 5.

It will be understood by those skilled in the art that the term 'amino acid' as used herein includes the standard twenty genetically encoded amino acids and their corresponding stereoisomers in the 'D' form (relative to the natural 'L' form), omega-amino acids, other naturally occurring amino acids, unconventional amino acids (e.g., alpha-disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derivatized amino acids (see below).

When an amino acid is specifically listed, such as 'alanine' or 'Ala' or 'a', the term refers to both L-alanine and D-alanine, unless specifically indicated otherwise. Other non-conventional amino acids may also be suitable components of the polypeptides of the invention, as long as the polypeptide retains the desired functional properties. For the peptides shown, each encoded amino acid residue is indicated as appropriate by a single letter designation, corresponding to the conventional name of a conventional amino acid.

In one embodiment, the polypeptide of the invention comprises or consists of an L-amino acid.

In case the polypeptide comprises an amino acid sequence according to a reference sequence (e.g.SEQ ID NO: 1 to 23), it may comprise further amino acids at its N-terminus and/or C-terminus which are different from the amino acids of the reference sequence, e.g.the polypeptide may comprise further amino acids at its N-terminus. Similarly, where the polypeptide comprises a fragment, variant or derivative of an amino acid sequence according to the reference sequence, it may comprise additional amino acids at its N-terminus and/or C-terminus.

In one embodiment, the polypeptide comprises or consists of: a fragment of an amino acid sequence according to the reference sequence (e.g., a fragment of any one of SEQ ID NOs: 1 to 23). Thus, a polypeptide may comprise or consist of: at least 5 contiguous amino acids of a reference sequence, such as (e.g.) SEQ ID NO: at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 consecutive amino acids of any one of 1 to 23.

It will be further understood by those skilled in the art that the polypeptides of the invention may comprise or consist of: a variant of an amino acid sequence according to the reference sequence (e.g., a variant of any one of SEQ ID NOs: 1 to 23), or a fragment of said variant. Such variants may be non-naturally occurring.

By "variant" of a polypeptide we include insertions, deletions and substitutions, either conservative or non-conservative. For example, a conservative substitution refers to the substitution of an amino acid in the same general class (e.g., an acidic amino acid, a basic amino acid, a non-polar amino acid, a polar amino acid, or an aromatic amino acid) with another amino acid in the same class. Thus, the meaning of conservative amino acid substitutions and non-conservative amino acid substitutions are well known in the art. In particular, we include polypeptide variants that exhibit antimicrobial activity.

In one embodiment, a variant has an amino acid sequence that is at least 50% identical, e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical, to an amino acid sequence according to a reference sequence (e.g., SEQ ID NOS: 1 to 23) or a fragment thereof.

The percent sequence identity between two polypeptides may be determined using a suitable computer program, for example the GAP program of the Genetic Computing Group of university of wisconsin, and it will be appreciated that the percent identity is calculated in relation to polypeptides whose sequences have been optimally aligned.

Alignment may alternatively be performed using the Clustal W program (as described in Thompson et al, 1994, nucleic acids research (Nuc. acid Res.) 22: 4673-4680, which is incorporated herein by reference).

The parameters used may be as follows:

parameters for rapid pairwise alignment: k-tuple (word) size; 1, window size; 5, gap penalties; 3, the number of top diagonals; 5. the scoring method comprises the following steps: x percent.

Multiple alignment parameters: gap opening penalties; 10, gap extension penalty; 0.05.

fractional matrix: BLOSUM.

Alternatively, the BESTFIT program may be used to determine local sequence alignments.

For example, in one embodiment, the amino acids of the above reference sequence may be mutated, e.g., by modification of I, F to W, to reduce proteolytic degradation of the polypeptide (see e.g., SEQ ID NO: S)Et al, Antimicrobial Agents (Antimicrobial Agents Chemothers) 2009, 53, 593, which is incorporated herein by reference.

Variants can be made using recombinant polynucleotides using protein engineering and site-directed mutagenesis methods well known in the art (see, for example, Molecular Cloning: a Laboratory Manual, 3 rd edition, Sambrook & Russell, 2000, Cold Spring Harbor Laboratory Press, incorporated herein by reference).

In a further embodiment, the polypeptide comprises or consists of: amino acids which are species homologues of any of the above amino acid sequences (e.g. SEQ ID NOs: 1 to 23). By "species homolog" we include polypeptides corresponding to the same amino acid sequence in an equivalent protein from a non-human species, i.e. exhibiting an amino acid sequence identical to SEQ ID NO: 1 to 23 (e.g., as measured by GAP or BLAST sequence alignment). Typically, species homolog polypeptides will have the same length as the human reference sequence (i.e., SEQ ID NOs: 1 to 23).

In still further embodiments, the polypeptide comprises or consists of a fusion protein.

By "fusion" of polypeptides we include amino acid sequences corresponding to a reference sequence (e.g., any one of SEQ ID NOs: 1 to 23, or fragments or variants thereof) fused to any other polypeptide. For example, the polypeptide may be fused to a polypeptide, such as glutathione-S-transferase (GST), or protein A, to aid in purification of the polypeptide. Examples of such fusions are well known to those skilled in the art. Similarly, the polypeptide may be fused to an oligo-histidine tag (such as His6), or to an epitope recognized by an antibody (such as the well-known Myc tag epitope). In addition, fusions comprising hydrophobic oligopeptide end-tags may be used. Fusions to any variant or derivative of the polypeptide are also included within the scope of the invention. It will be appreciated that fusions (or variants or derivatives thereof) that retain a desired property, such as antimicrobial activity, are preferred.

The fusion may comprise further portions which confer a desired feature on said polypeptide of the invention; for example, the moiety can be used to detect or isolate the polypeptide, or to facilitate uptake of the polypeptide by a cell. The moiety may be, for example, a biotin group, a streptavidin group, a radioactive group, a fluorescent group, such as a small fluorophore or a Green Fluorescent Protein (GFP) fluorophore, as is well known to those skilled in the art. The group may be an immunogenic tag, such as a Myc tag, as known to those skilled in the art, or may be a lipophilic molecule or polypeptide domain capable of promoting uptake of the polypeptide by a cell, as known to those skilled in the art.

It will be appreciated by those skilled in the art that the polypeptides of the invention may comprise one or more amino acids which are modified or derivatized, for example, by pegylation, amidation, esterification, acylation, acetylation, and/or alkylation.

As known in the art, pegylated proteins may exhibit reduced renal clearance and proteolysis, reduced toxicity, reduced immunogenicity, and increased solubility [ Veronese, f.m., and j.m.harris, "advanced Drug delivery review (Adv Drug delivery Rev), 2002.54(4): p.453-6, Chapman, a.p.," advanced Drug delivery review (Adv Drug delivery Rev), 2002.54(4): p. (4): pages 531-45 ] (incorporated herein by reference). Pegylation has been used for several protein-based drugs, including the first pegylated molecular asparaginase and adenosine deaminase [ Veronese, f.m. and j.m.harris, "advanced Drug delivery review (Adv Drug delivery Rev), 2002.54(4): p. (4): 453-6, pages, Veronese, f.m., and g.pasut, "Drug discovery Today (Drug discovery), 2005.10 (21): pages 1451-8 ] (incorporated herein by reference).

In order to obtain a successfully pegylated protein with the greatest increased half-life and retained biological activity, several parameters that can influence the results are of critical importance and should be taken into account. PEG molecules may vary, and PEG variants for pegylation of proteins include PEG and monomethoxy-PEG. Furthermore, they may be linear or branched [ Wang, Y.S. et al, Adv Drug delivery review (Adv Drug Deliv Rev), 2002.54(4): p.547-70] (incorporated herein by reference). The PEG molecules used may vary in size and have attached PEG groups ranging in size between 1 and 40kDa to proteins [ Wang, y.s. et al, "advanced Drug delivery review (Adv Drug delivery Rev), 2002.54(4): p.547-70, Sato, h.," advanced Drug delivery review (Adv Drug delivery Rev), 2002.54(4): p.487-504, Bowen, s. et al, "(Exp hematology)", 1999.27(3): p.425-32, Chapman, a.p. et al, "(nature biotechnology (Nat Biotechnol)", 1999.17(8): p.780-3] (incorporated herein by reference). Furthermore, the number of PEG groups attached to a protein may vary, and examples between one and six PEG units attached to a protein have been reported [ Wang, y.s. et al, advanced Drug delivery review (Adv Drug delivery Rev), 2002.54(4): p.547-70, Bowen, s. et al, experimental hematology (Exp hemosol), 1999.27(3): p.425-32] (incorporated herein by reference). In addition, PEG for conjugation has been used, as well as linking groups, either present or absent between the various reactive groups. Thus, PEG can be linked to the N-terminal amino group, or to amino acid residues with reactive amino or hydroxyl groups (Lys, His, Ser, Thr and Tyr), either directly or by using γ -aminobutyric acid as a linking group. In addition, PEG can be coupled to a carboxyl (Asp, Glu, C-terminal) or sulfhydryl (Cys) group. Finally, the enzyme transglutaminase can be used to specifically PEGylate Gln residues, and alkylamine derivatives of PEG have been described [ Sato, H., "advanced Drug delivery review (Adv Drug Deliv Rev)," 2002.54(4) (4): p.487-504] (incorporated herein by reference).

It has been shown that increasing the degree of pegylation increases the half-life in vivo. However, it will be appreciated by those skilled in the art that the pegylation process needs to be optimized for a particular protein on an individual basis.

PEG can be coupled at a naturally occurring disulfide bond as described in WO2005/007197 (which is incorporated herein by reference). Disulfide bonds can be stabilized by the addition of chemical bridges that do not disrupt the tertiary structure of the protein. This allows the use of conjugated thiol selectivity of the two thiols containing disulfide bonds to create a bridge for site-specific attachment of PEG. Thus, the need to engineer residues into peptides for attachment to target molecules is circumvented.

Various alternative block copolymers may also be covalently conjugated as described in WO2003/059973, which is incorporated herein by reference. Therapeutic polymeric conjugates can exhibit improved thermal properties, crystallization, adhesion, swelling, coating, pH-dependent configuration, and biodistribution. Moreover, they can achieve prolonged circulation, release of biologically active substances in the proteolytic and acidic environment of the secondary lysosomes after uptake of the conjugate by the cells through pinocytosis, and more favorable physicochemical properties due to the characteristics of the macromolecules (e.g. increased drug solubility in biological fluids). The block copolymer comprising hydrophilic and hydrophobic blocks forms polymeric micelles in solution. Upon micelle dissociation, individual block copolymer molecules are safely discharged.

Chemical derivatives of one or more amino acids may also be obtained by reaction with functional side groups. Such derivatized molecules include, for example, those molecules: in which the free amino groups have been derivatized to form amine hydrochloride, p-toluenesulfonyl groups, carboxybenzoate groups, tert-butoxycarbonyl groups, chloroacetyl groups or formyl groups. The free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters and hydrazides. The free hydroxyl group can be derivatized to form an O-acyl or O-alkyl derivative. Also included are those peptides that are chemical derivatives containing naturally occurring amino acid derivatives of 20 standard amino acids. For example: 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine, and ornithine can replace lysine. Derivatives also include peptides containing one or more additions or deletions, as long as the necessary activity is retained. Other included modifications are amidation, amino-terminal acylation (e.g., acetylation or thioglycolic acid amidation), terminal carboxyamidation (e.g., with ammonia or methylamine), and similar terminal modifications.

It will be further appreciated by those skilled in the art that peptidomimetic compounds may also be useful. Thus, by "polypeptide" we include peptidomimetics having antimicrobial activity. The term "peptidomimetic" refers to a compound that mimics the configuration and desired characteristics of a particular peptide as a therapeutic agent.

For example, the polypeptide of the present invention includes not only molecules in which amino acid residues are linked by a peptide (-CO-NH-) linker, but also molecules in which peptide bonds are reversed. Such retro-trans (retro-inverso) peptidomimetics may be prepared using methods known in the art, such as those described in Meziere et al (1997) J.Immunol. 159, 3230-3237, which is incorporated herein by reference. The method involves the preparation of pseudopeptides containing alterations that involve the backbone rather than the side chain orientation. Retro-trans (retro-inverses) peptides containing an NH-CO bond rather than a CO-NH peptide bond are much more resistant to proteolysis. Alternatively, the polypeptides of the invention may be peptidomimetics, wherein one or more amino acid residues are passed through-y (CH)₂NH) -linkage rather than the conventional amide linker.

In a further alternative, peptide bonds may be omitted entirely, provided that suitable linking groups are used which leave spaces between the carbon atoms of the amino acid residues; it may be advantageous for the linker moiety to have substantially the same charge distribution and substantially the same planarity as the peptide bonds.

It will be appreciated that it may be convenient to block the polypeptide at its N-terminal or C-terminal region in order to help reduce sensitivity to exo-proteolytic digestion.

Various non-encoded or modified amino acids (such as D-amino acids and N-methyl amino acids) have also been used to modify mammalian peptides. Furthermore, the putative bioactive configuration may be stabilized by covalent modification such as cyclization, or by the incorporation of lactams or other types of bridges, see, for example, Veber et al, 1978, "proceedings of the american academy of sciences (proc. natl. acad. sci. usa) 75: 2636 and Thursell et al, 1983, communication on biochemistry and biophysics research (biochem. biophysis. Res. Comm.) 111: 166, which is incorporated herein by reference.

A common theme in many synthetic strategies is the introduction of some cyclic groups into the peptide-based backbone. The cyclic group limits the conformational space of the peptide structure, and this often results in increased specificity of the peptide for a particular biological receptor. An added advantage of this strategy is that the introduction of cyclic groups into the peptide may also result in peptides with reduced sensitivity to cellular peptidases.

Thus, exemplary polypeptides of the invention comprise a terminal cysteine amino acid. Such polypeptides may exist in heterocyclic form through the formation of disulfide bonds by thiol groups in the terminal cysteine amino acids, or in homogeneous form through the formation of amide peptide bonds between the terminal amino acids. As shown above, the cyclization of small peptides by disulfide bonds or amide bonds between N-and C-terminal cysteine regions can avoid the problems of specificity and half-life sometimes observed for linear peptides by reducing proteolysis and also by increasing structural rigidity, which can lead to more specific compounds. Polypeptides cyclized by disulfide bonds have free amino and carboxyl termini which may still be readily proteolytically degraded, whereas peptides cyclized by formation of an amide bond between the N-terminal amine and the C-terminal carboxyl group no longer contain a free amino or carboxyl terminus. Thus, the peptides of the invention may be linked via a C-N linker or via a disulfide linker.

The invention is not in any way limited by the peptide ring closure method, but includes peptides whose cyclic structure can be obtained by any suitable synthetic method. Thus, a heterolinking group can include, but is not limited to, being formed via a disulfide bridge, an alkylene bridge, or a sulfide bridge. Methods for synthesizing cyclic homo-and hetero-peptides, including disulfide bridges, sulfide bridges and alkylene bridges, are disclosed in US5,643,872, US5,643,872 being incorporated herein by reference. Other examples of ring closure methods include ring closure by click chemistry, epoxides, aldehyde-amine reactions, and the methods disclosed in US6,008,058, US6,008,058 being incorporated herein by reference.

A further approach to the synthesis of cyclic stabilized peptidomimetics is the Ring Closure Metathesis (RCM). The method involves the steps of synthesizing a peptide precursor and contacting it with an RCM catalyst to produce a conformationally constrained peptide. Suitable peptide precursors may contain two or more unsaturated C-C bonds. The method may be performed using solid phase peptide synthesis techniques. In this example, a precursor immobilized to a solid support is contacted with an RCM catalyst, and the product is then detached from the solid support, producing a conformationally constrained peptide.

Another approach disclosed by D.H. Rich in Protease Inhibitors (Protease Inhibitors), edited by Barrett and Selveson, Amur Everest publishing company (1986), which is incorporated herein by reference, is to design peptidomimetics by applying the transition state analog concept to enzyme inhibitor design. For example, it is known that secondary alcohols of staline are similar to the tetrahedral transition state of the amide bond of pepsin substrate, which is easily cleaved.

In summary, as is well known, terminal modifications can be used to reduce sensitivity to protease digestion and thus extend the half-life of the peptide in solution, particularly in biological fluids where proteases may be present. Polypeptide cyclization is also a useful modification due to the stable structure formed by cyclization and in view of the biological activity observed for cyclic peptides.

Thus, in one embodiment, the polypeptide of the first aspect of the invention is linear. However, in an alternative embodiment, the polypeptide is cyclic.

It will be appreciated by those skilled in the art that the polypeptides of the invention may be of various lengths. Polypeptides typically have a length of between 10 and 200 amino acids, such as between 10 and 150, 15 and 100, 15 and 50, 20 and 40, or 25 and 35 amino acids. For example, a polypeptide can have a length of at least 20 amino acids.

In one embodiment, the polypeptide is or comprises a recombinant polypeptide. Suitable methods for producing such recombinant polypeptides are well known in the art, e.g., expression in prokaryotic or eukaryotic host cells (see, e.g., Sambrook & Russell, 2000, Molecular Cloning, laboratory Manual, third edition, Cold Spring Harbor, new york, the relevant disclosures of which are incorporated herein by reference).

The polypeptides of the invention may also be produced using commercially available in vitro translation systems, such as rabbit reticulocyte lysate or malt lysate (available from Promega). Preferably, the translation system is rabbit reticulocyte lysate. Suitably, the translation system may be combined with a transcription system such as the TNT transcription-translation system (Promega). This system has the advantage of producing a suitable mRNA transcript from the encoding DNA polynucleotide in the same reaction as translation.

It will be appreciated by those skilled in the art that the polypeptides of the invention may alternatively be synthesised artificially, for example using well known liquid or solid phase synthesis techniques (such as t-Boc or Fmoc solid phase peptide synthesis). For example, the polypeptide may be synthesized as described in Fields, Abelson & Simon (eds.) "Solid-Phase Peptide Synthesis (1997), Academic Press (ISBN: 0-12-182190-0) (which is incorporated herein by reference).

Accordingly, the scope of the present invention includes the following related aspects.

A second aspect of the invention provides a nucleic acid molecule encoding a polypeptide according to the first aspect of the invention. In one embodiment, the nucleic acid molecule can be isolated.

A third aspect of the invention provides a vector (such as an expression vector) comprising a nucleic acid molecule according to the second aspect of the invention. In one embodiment, the vector may be adapted for replication in a eukaryotic cell, such as a mammalian cell.

A fourth aspect of the invention provides a host cell comprising a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention. In one embodiment, the host cell is recombinant. In one embodiment, the host cell is a eukaryotic cell, such as a mammalian cell.

In addition to the transformed host cells themselves, the present invention also contemplates cultures of those cells, preferably monoclonal (clonally homogeneous) cultures in nutrient media, or cultures derived from monoclonal cultures.

A fifth aspect of the invention provides a method of making a polypeptide according to the first aspect of the invention, comprising culturing a population of host cells according to the fourth aspect of the invention under conditions in which the polypeptide is expressed and isolating the polypeptide therefrom. Methods of culturing host cells and isolating recombinant proteins are well known in the art.

A sixth aspect of the invention provides a method of making a polypeptide according to the first aspect of the invention, comprising liquid or solid phase synthesis of the polypeptide.

It will be appreciated by those skilled in the art that the polypeptides of the invention may be formulated for use in clinical and/or veterinary medicine.

Accordingly, a seventh aspect of the invention provides a pharmaceutical composition comprising a polypeptide according to the first aspect of the invention, and a pharmaceutically acceptable excipient, diluent, carrier, buffer or adjuvant.

As used herein, 'pharmaceutical composition' means a therapeutically effective formulation for treating or preventing diseases and conditions associated with microorganisms and microbial infections.

Additional compounds, such as other peptides, low molecular weight immunomodulators, receptor agonists and antagonists, and antimicrobial agents may also be included in the pharmaceutical compositions. Other examples include chelating agents such as ethylenediaminetetraacetic acid (EDTA), citrate, ethylene glycol diethylenediaminetetraacetic acid (EGTA), or glutathione.

The pharmaceutical compositions may be prepared in a manner known in the art that is sufficiently storage stable and suitable for administration to humans and animals. The pharmaceutical composition may be lyophilized, for example, by freeze-drying, spray-cooling, or by using particle formation from supercritical particle formation.

By "pharmaceutically acceptable" we mean a non-toxic material that does not reduce the effectiveness of the biological activity of the active ingredient, i.e., the antimicrobial polypeptide(s). Such pharmaceutically acceptable buffers, carriers or Excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18 th edition, edited by A.R Gennaro, Mack Publishing Company (1990) and Handbook of Pharmaceutical Excipients (Handbook of Pharmaceutical Excipients), third edition, edited by a. kibbe, Pharmaceutical Press (Pharmaceutical Press) (2000), which is incorporated herein by reference).

The term "buffer" is intended to mean an aqueous solution containing a mixture of acids and bases, the purpose of which is to stabilize the pH. Examples of buffers are tris (hydroxymethyl) aminomethane, N-diglycine, trimethylglycine, 3-morpholinopropanesulfonic acid, 3- (N-morpholino) -2-hydroxypropanesulfonic acid, morpholinobutanesulfonic acid, tris (hydroxymethyl) aminomethane, hydroxyethylpiperazine ethanethiosulfonic acid, N- (2-hydroxyethyl) piperazine-N' -4-butanesulfonic acid, morpholinoethanesulfonic acid, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, N-carbamoylmethylethanesulfonic acid, N- (2-acetylamino) iminodiacetic acid, tartrate, adenosine monophosphate, aminomethylpropanediol, quinodimethallyl ester, 2- (diethanolamino) ethanesulfonic acid, 4- (cyclohexylamino) -1-butanesulfonic acid, trimethylglycine, 3-morpholinopropanesulfonic acid, 3- (N-morpholino) -2-hydroxypropanesulfonic acid, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, N-carbamoylmethylethanesulfonic acid, N- (2-acetylamino) iminodiacetic acid, tartrate, adenosine monophosphate, aminomethylpropanediol, quinaldehydo-yl acetate, 2- (diethanolamino) ethanesulfonic acid, 4- (cyclohexylamino) -1-butanesulfonic acid, and mixtures thereof, Cacodylate, N-2-cyclohexylaminoethyl sulfonic acid, 3-bis (2-hydroxyethyl) amino-2-hydroxypropanesulfonic acid, 4- (2-hydroxyethyl) -1-piperazinepropanesulfonic acid, ethanolamine, glycine, N- (2-hydroxyethyl) piperazine-N' - (2-hydroxypropanesulfonic acid), imidazole, imidazoleacetic acid, 1, 4-piperazinediethanesulfonic acid, sodium citrate buffer, sodium ethylenediaminetetraacetic acid, sodium phosphate physiological saline, dihydroxypropanesulfonic acid, trimethylolmethylaminopropanesulfonic acid, Tabes (TABS), 3-trimethylolmethylamine-2-hydroxypropanesulfonic acid, and trimethylolmethylaminoethanesulfonic acid.

The term "diluent" is intended to mean an aqueous or non-aqueous solution, the purpose of which is to dilute the peptide in the pharmaceutical formulation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol, or an oil such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil.

The term "adjuvant" is intended to mean any compound added to a formulation to increase the biological effect of the peptide. The adjuvant may be colloidal silver, or one or more of zinc, copper or silver salts with different anions, such as, but not limited to, fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate and acetate of different acyl compositions. Adjuvants may also be cationic polymers such as polyhexamethylene biguanide hydrochloride, cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly (vinylimidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

The excipient may be one or more of a carbohydrate, a polymer, a lipid, and a mineral. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrins, which are added to the composition, for example, to aid in lyophilization. Examples of polymers are starch, cellulose ethers, cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, ethylcellulose, methylcellulose, propylcellulose, alginates, carrageenan, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulfonates, polyethylene glycol/polyethylene oxide, polyethylene oxide/polypropylene oxide copolymers, polyvinyl alcohol/polyvinyl acetate with different degrees of hydrolysis, poly (lactic acid), poly (glycolic acid) or copolymers thereof with various compositions, and polyvinylpyrrolidone, all with different molecular weights, it is added to the composition, for example to control viscosity, to obtain bioadhesive, or to prevent chemical and proteolytic degradation of the active ingredients (also applicable to a-C). Examples of lipids are fatty acids, phospholipids, monoglycerides, diglycerides, triglycerides, phospholipids, sphingolipids and glycolipids (all with different acyl chain lengths and saturations), egg lecithin, soy lecithin, hydrogenated egg lecithin and hydrogenated soy lecithin, which are added to the composition for similar reasons as polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain various benefits, such as reduced accumulated liquids or advantageous pigment properties.

The pharmaceutical composition may also contain one or more mono-or disaccharides such as xylitol, sorbitol, mannitol, lactitol, xylitol, maltitol or xyloside, and/or monoglycerides such as monolaurin. The characteristics of the carrier depend on the route of administration. One route of administration is topical. For example, for topical administration, the preferred carrier is an emulsion cream containing the active peptide, although other conventional carriers may be used, such as certain petrolatum/mineral-based and vegetable-based ointments, as well as polymer gels, liquid crystal phases and microemulsions.

It will be appreciated that the pharmaceutical composition may comprise one or more polypeptides of the invention, for example one, two, three or four different peptides. Antimicrobial action can be increased by using a combination of different peptides.

As discussed above, the polypeptide can be provided as a salt, e.g., an acid adduct with an inorganic acid, such as hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, perchloric acid, thiocyanic acid, boric acid, and the like, or with an organic acid, such as formic acid, acetic acid, haloacetic acid, propionic acid, glycolic acid, citric acid, tartaric acid, succinic acid, gluconic acid, lactic acid, malonic acid, fumaric acid, anthranilic acid, benzoic acid, cinnamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, sulfanilic acid, and the like, to which inorganic salts, such as monovalent sodium, potassium, or divalent zinc, magnesium, copper, calcium, all having corresponding anions, can be added to improve the biological activity of the antimicrobial composition.

The pharmaceutical compositions of the invention may also be in the form of liposomes in which the polypeptide is associated, in addition to other pharmaceutically acceptable carriers, with amphiphilic agents such as lipids, which are present in aggregated form as micelles, insoluble monolayers and liquid crystals. Suitable lipids for liposome formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponins, bile acids, and the like. Suitable lipids also include the above lipids modified in the polar head group by poly (ethylene glycol) to prolong circulation time of the blood stream. Methods for the preparation of such liposomal formulations can be found, for example, in US4,235,871, which is incorporated herein by reference.

The pharmaceutical compositions of the present invention may also be in the form of biodegradable microspheres. Aliphatic polyesters, such as poly (lactic acid) (PLA), poly (glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly (caprolactone) (PCL) and polyanhydrides have been widely used as biodegradable polymers in the production of microspheres. The preparation of such microspheres can be found in US5,851,451 and in EP213303, which are incorporated herein by reference.

The pharmaceutical compositions of the present invention may be formulated with micellar systems formed from surfactants and block copolymers, preferably those containing poly (ethylene oxide) groups, to prolong blood circulation time.

The pharmaceutical compositions of the present invention may also be in the form of polymer gels, wherein polymers such as starch, cellulose ethers, cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, propyl cellulose, alginates, chitosan, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polyvinylimidazole, polysulfonates, polyethylene glycol/polyethylene oxide, polyethylene oxide/polypropylene oxide copolymers, polyvinyl alcohols/polyvinyl acetates of different degrees of hydrolysis, and polyvinylpyrrolidone are used to thicken peptide-containing solutions. The polymer may also comprise gelatin or collagen.

Alternatively, the polypeptides of the invention may be soluble in saline, water, polyethylene glycol, propylene glycol, ethanol or oil (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth, and/or various buffers.

The pharmaceutical composition can also include ions and defined to enhance the antimicrobial peptides effect of pH.

The above compositions of the invention may be subjected to conventional pharmaceutical procedures such as sterilization and/or may contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers and the like, for example, as disclosed elsewhere herein.

It will be appreciated by those skilled in the art that the pharmaceutical compositions of the present invention may be administered locally or systemically. Routes of administration include topical (e.g., intraocular), ocular, nasal, pulmonary, buccal, parenteral (intravenous, subcutaneous, and intramuscular), oral, vaginal, and rectal. Administration from implants is also possible. Suitable preparation forms are, for example, granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, microemulsions (defined as optically isotropic, thermodynamically stable systems consisting of water, oil and surfactant), liquid-crystalline phases (defined as systems characterized by long-range order but short-range order (examples include tablets, hexagonal and cubic phases, aqueous-phase continuous or oil-phase continuous)), or their dispersed counterparts, gels, ointments, dispersions, suspensions, creams, aerosols, drops or ampoules, and also preparations which release the active compound permanently in which excipients, diluents, adjuvants or carriers are customarily used, as described above. The pharmaceutical composition may also be provided in a bandage, plaster or in a suture or the like.

In a particular embodiment, the pharmaceutical composition is suitable for oral, parenteral or topical administration. For example, the pharmaceutical composition may be adapted for topical administration (e.g., intraocular administration in the form of a spray, liniment, paste or drops, etc.).

The pharmaceutical composition will be administered to the patient in a pharmaceutically effective dose. By "pharmaceutically effective dose" is meant a dose sufficient to produce the desired effect with respect to the condition to which it is administered. The exact dosage will depend upon the activity of the compound, the mode of administration, the nature and severity of the disease, the age and weight of the patient and may require different dosages. Administration of a dose can be carried out by a single administration in the form of a single dosage unit, or in the form of several smaller dosage units, or by multiple administrations of further subdivided doses at specific intervals.

The pharmaceutical compositions of the present invention may be administered alone, or in combination with other therapeutic agents, such as additional antibiotics, anti-inflammatory agents, immunosuppressive agents, vasoactive agents, and/or preservatives (such as antibacterial, antifungal, antiviral, and antiparasitic agents). Examples of suitable additional antibiotics include penicillins, cephalosporins, carbacephem, cephamycins, carbapenems, monoamide rings, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones. Preservatives include iodine, silver, copper, chlorhexidine, polyhexamethylene biguanide hydrochloride and other biguanides, chitosan, acetic acid, and hydrogen peroxide. Similarly, the pharmaceutical compositions may also contain anti-inflammatory agents, such as steroids and macrolactam derivatives.

Such additional therapeutic agents may be incorporated as part of the same pharmaceutical composition or may be administered separately.

It will be appreciated by those skilled in the art that the polypeptides of the invention, or pharmaceutical compositions thereof, may find application in medical devices and other articles of manufacture, the implantation or application of which to the human or animal body is accompanied by a risk of infection with microbial agents.

Accordingly, an eighth aspect of the invention provides a medical device, implant, wound care product, or material therefor, coated, impregnated, blended or otherwise associated with a pharmaceutical composition according to the seventh aspect of the invention or a polypeptide according to the first aspect of the invention.

Such medical devices, implants, wound care products, or materials therefor may be in contact with the human body or components thereof (e.g., blood).

In one embodiment, the medical device, implant, wound care article, or material therefor is used for bypass surgery, extracorporeal circulation surgery, wound care, and/or dialysis.

The composition may be coated, sprayed or otherwise applied to or blended with sutures, prostheses, implants, wound dressings, catheters, lenses, skin grafts, skin substitutes, fibrin glue or bandages and the like. In this way, the composition may impart improved antimicrobial properties or wound healing properties to the device or material.

By "implant", we include:

(a) catheters (e.g. for intravascular or urethral use).

(b) Stents (e.g., coronary stents).

(c) A shunt (e.g., a cerebrospinal shunt).

(d) Intubation or tracheotomy tubes.

(e) Intraocular devices (e.g., contact lenses, scleral buckle bands, and intraocular lenses).

(f) Prosthetic joints (i.e., the implantation of arthroplasty and other orthopedic devices).

(g) A heart valve prosthesis.

(h) A breast implant.

(i) Implantable drug delivery devices (e.g., active pumps and passive solid implants).

In one embodiment, the device or material is coated with a pharmaceutical composition of the invention (or a polypeptide component thereof). By 'coating' we mean that the pharmaceutical composition is applied to the surface of the device or material. Thus, the device or material may be coated or sprayed with a solution comprising a pharmaceutical composition of the invention (or a polypeptide thereof). Alternatively, the device or material may be immersed in a container of a solution comprising a polypeptide of the invention.

In an alternative embodiment, the device or material is impregnated with a pharmaceutical composition of the invention (or a polypeptide thereof). By 'impregnated' we mean that the pharmaceutical composition is combined or otherwise mixed with the device or material so that it is distributed throughout the mixture.

For example, the device or material may be incubated overnight at 4 ℃ in a solution comprising a polypeptide of the invention. Alternatively, the pharmaceutical composition of the invention (or a polypeptide thereof) may be immobilized onto the surface of the device or material by evaporation or by incubation at room temperature.

In a further alternative embodiment, the polypeptide of the invention is covalently attached to the device or material, for example on the outer surface of the device or material. Thus, a covalent bond is formed between an appropriate functional group on the polypeptide and a functional group on the device or material. For example, methods for covalently bonding a polypeptide to a polymeric support include covalent attachment via a diazonium salt intermediate, formation by peptide attachment, alkylation by binding phenolic, amine, and thiol groups on proteins, by using multifunctional intermediates (e.g., glutaraldehyde), and other various methods, such as the use of silylated glass or quartz, where the reaction of dialkoxysilanes and trialkoxysilanes allows derivatization of glass surfaces with many different functional groups. For details, see (1993) Butterworth-snehman press (Butterworth-Heinemann) technical Applications of Biocatalysts (technical Applications of Biocatalysts) (BIOTOL series) pp.75-118, Griffin, m., hammons, e.j. and Leach, c.k. 'immobilization of enzymes (Enzyme immobilization)', which is incorporated herein by reference. See also (1995) scientific (Science) 13: 565-.

In one embodiment, the medical device, implant, wound care product, or material comprises or consists of a polymer. Suitable polymers may be selected from the group consisting of: polyesters (e.g. polylactic acid, polyglycolic acid or polylactic acid-glycolic acid copolymers of various compositions), polyorthoesters, polyacetals, polyureas, polycarbonates, polyurethanes, polyamides) and polysaccharide materials (e.g. cross-linked alginates, hyaluronic acid, carrageenan, pectin, starch, cellulose derivatives).

Alternatively or additionally, the medical device, implant, wound care product, or material may comprise or consist of: metals (e.g. titanium, stainless steel, gold, titanium), metal oxides (silica, titania), and/or ceramics (apatite, hydroxyapatite).

Such materials may be in the form of macroscopic solids/monoliths, as chemically or physico-chemically cross-linked gels, as porous materials, or as particles.

The medical devices, implants, wound care products, and materials of the present invention can be made using methods well known in the art.

It is to be understood that the medical devices, implants, wound care products, and materials of the present invention can be used for any of the medical uses disclosed herein.

A ninth aspect of the invention provides a kit comprising a pharmaceutical composition according to the seventh aspect of the invention or a medical device, implant, wound care product, or material therefor, according to the eighth aspect of the invention.

A tenth aspect of the invention provides a polypeptide according to the first aspect of the invention, or a nucleic acid molecule according to the second aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention, for use in medicine.

An eleventh aspect of the invention provides a polypeptide according to the first aspect of the invention, or a nucleic acid molecule according to the second aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention for use in the curative and/or prophylactic treatment of a microbial infection.

The term 'prophylactic' is used to include the use of a polypeptide or agent as described herein which prevents or reduces the likelihood of a disease or condition occurring in a patient or subject.

By "microbial infection" we include infections caused by microorganisms as described above.

For example, in one embodiment the microbial infection to be treated is a bacterial infection.

The microbial infection to be treated may be an acute infection or a systemic infection.

In one embodiment, the microbial infection is resistant to one or more conventional antibiotics (as discussed above).

In one embodiment, the microbial infection to be treated is caused by a microorganism selected from the group consisting of: pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Streptococcus pyogenes.

In a further embodiment, the microbial infection is caused by a microorganism selected from the group consisting of: multidrug Resistant Staphylococcus Aureus (MRSA) (methicillin resistant staphylococcus aureus) and multidrug resistant pseudomonas aeruginosa (MRSA).

It will be appreciated by those skilled in the art that the polypeptides and formulations of the invention may be co-administered in combination with one or more known or conventional agents used to treat a particular disease or condition. By ' co-administration ' it is meant that the polypeptide of the invention is administered to a patient such that the polypeptide and co-administered compound can be found in the patient's body (e.g., in the bloodstream) at the same time, regardless of when the compounds are actually administered, including at the same time.

Thus, in one embodiment, the polypeptide, nucleic acid molecule, or pharmaceutical composition is used in combination with one or more additional antimicrobial agents (such as the conventional antibiotics described above). Alternatively or additionally, the additional antimicrobial agent may be an antimicrobial polypeptide or protein, such as LL-37 and type VI collagen, or for example selected from the group consisting of: defensins, gramicidin S, magainins, cecropin, histaminins, mycelial proteins (hypincin), cinnamycin, boolean forest 1(Burforin 1), Parasin1(Parasin 1), and protamine, as well as fragments, variants, and fusions thereof, which retain (at least in part) the antimicrobial activity of the proteins of the invention.

A twelfth aspect of the invention provides a polypeptide according to the first aspect of the invention, or a nucleic acid molecule according to the second aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention for use in wound care.

By "wound care" we include treating wounds, promoting wound closure, preventing and/or treating wound infections and/or ulcers, wherein the wound may be in vitro or in vivo. Thus, use in wound care includes polypeptides capable of aiding (e.g. accelerating) the wound healing process and/or preventing wound infection. For example, the polypeptides of the invention may be used in wound care products such as creams, gels, ointments, dressings or pastes, which are capable of enhancing epithelial regeneration and/or healing of wound epithelium and/or wound matrix. In one embodiment, the polypeptide is capable of enhancing the proliferation of epithelial and/or stromal cells by a non-lytic mechanism.

It will be appreciated that polypeptides having wound healing properties may have a primary or secondary role in the function of the wound care products of the invention.

In one embodiment of the twelfth aspect of the invention, the polypeptide, nucleic acid molecule or pharmaceutical composition is used in combination with an additional antimicrobial agent, as described above.

A thirteenth aspect of the invention provides the use of a peptide or fragment according to the first aspect of the invention, or a nucleic acid molecule according to the second aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention, in the manufacture of a medicament for the treatment of a microbial infection, as described above.

A fourteenth aspect of the invention provides the use of a peptide or fragment according to the first aspect of the invention, or a nucleic acid molecule according to the second aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention, in the manufacture of a medicament for the treatment of a wound, as described above.

A fifteenth aspect of the invention provides a method of treating an individual having a microbial infection, the method comprising the step of administering to an individual in need thereof an effective amount of a peptide or fragment according to the first aspect of the invention, or a nucleic acid molecule according to the second aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention.

A sixteenth aspect of the invention provides a method of treating a wound in an individual, the method comprising the step of administering to an individual in need thereof an effective amount of a polypeptide or fragment according to the first aspect of the invention, or a nucleic acid molecule according to the second aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention.

The term 'effective amount' is used herein to describe a concentration or amount of a polypeptide or pharmaceutical composition according to the invention that can be used to produce a beneficial change in the disease or condition being treated, depending on the disease or condition being treated, whether the change is an alleviation, a beneficial physiological outcome, a reversal or attenuation of the disease state or condition being treated, or a prevention or reduction in the likelihood of the occurrence of the condition or disease state. In the case of the combined use of the polypeptides or pharmaceutical compositions of the present invention, each polypeptide or pharmaceutical composition may be used in an effective amount, wherein an effective amount may include a synergistic amount.

It will be appreciated by those skilled in the art that the polypeptides and pharmaceutical formulations of the invention have utility in both the medical and veterinary fields. Thus, the methods of the invention can be used to treat both human and non-human animals (e.g., horses, dogs, and cats). Preferably, however, the patient is a human.

For veterinary use, the compounds of the invention are administered as suitably acceptable formulations in accordance with normal veterinary practice, and the veterinary practitioner will determine the most appropriate dosage regimen and route of administration for a particular animal.

A seventeenth aspect of the invention provides a method for killing a microorganism in vitro comprising contacting the microorganism with a polypeptide according to the first aspect of the invention or a pharmaceutical composition according to the seventh aspect of the invention. For example, the pharmaceutical composition or polypeptide may also be used in the form of a disinfecting solution or lotion to prevent microbial growth, such as on surfaces or substrates in a clinical environment (e.g., operating room) or a domestic environment (e.g., kitchen work surfaces, laundry, such as bed sheets).

In one embodiment, the antimicrobial compound may be present in the solution at a concentration of 1 to 100 μ g/ml.

In one embodiment, the solution further comprises a surface active agent or surfactant. Suitable surfactants include anionic surfactants (e.g., aliphatic sulfonates), amphoteric and/or zwitterionic surfactants (e.g., derivatives of aliphatic quaternary ammonium, phosphonium, and sulfonium compounds), and nonionic surfactants (e.g., aliphatic alcohols, acids, amides, or alkylphenols with alkylene oxide)

Suitably, the surface active agent is present at a concentration of 0.5 to 5 weight percent.

The disinfecting solution is particularly suitable for use in a hospital environment. For example, the disinfecting solution may be used to disinfect surgical instruments and surfaces of an operating room, as well as hands and gloves of operating room personnel. In addition, the disinfecting solution may be used during surgery, for example, to disinfect exposed bone. In all cases, the solution is applied to the surface to be disinfected.

The pharmaceutical composition or polypeptide may also be used to disinfect blood and blood products, and also for the diagnosis of bacterial contamination or infection.

In vitro and in vivo use, the pharmaceutical composition or polypeptide is preferably exposed to the target microorganism (or surface/area to be treated) for at least 5 minutes. For example, the exposure time may be at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 12 hours, and 24 hours.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Non-limiting examples embodying certain aspects of the present invention will now be described, with reference to the following drawings.

Drawings

Figure 1-antibacterial action of collagen type VI against different strains of gram-positive and gram-negative bacteria. (A) Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli or Pseudomonas aeruginosa (2X 10)⁶cfu/ml⁾At 37 ℃ and 5% CO₂The following incubations were performed with type VI collagen (2. mu.M) for 2 hours. Tris-HCl/glucose; buffer at pH 7.4 or bacteria incubated with LL-37 served as negative or positive controls, respectively. (B) For a clear presentation of antimicrobial activity, bacteria (2x 10)⁹cfu/ml) was treated with type VI collagen (2 μ M) at 37 ℃ for 2 hours, followed by observation by scanning electron microscopy. Compared to untreated bacteria (left panel), strong membrane disruption, blebbing and cytoplasmic component ejection were observed in the presence of type VI collagen (right panel). The scales represent 2 μm (Streptococcus pyogenes, Staphylococcus aureus) and 1 μm (Pseudomonas aeruginosa, Escherichia coli), respectively. (C) Kinetic study of bacterial membrane disruption induced by type VI collagen. Streptococcus pyogenes and Pseudomonas aeruginosa (pseudo-green) were treated with type VI collagen (2. mu.M) at 37 ℃ for 0, 30, 60 and 120 minutes and observed by scanning electron microscopy. The arrows show the film blistering. Cytoplasmic exudate is represented by a pseudo-purple color. Scale bar 1 μm. (D) For fluorescence microscopy analysis, bacteria were treated with type VI collagen as described above and tested by usingThe penetration was evaluated by an imperviousness probe FITC (lower panel). The same position was clearly visualized with optical microscopy (upper panel). LL-37 at 3. mu.M was used as a positive control for membrane disruption, while bacteria using buffer only were used as a negative control. Green color indicates bacterial lysis. Images were acquired at 1000x magnification.

FIG. 2 (A) schematic representation of the structure of the collagen type VI domain. Type VI collagen consists of 3 α -chains, α 1(VI), α 2(VI), and α 3 (VI). The N-and C-terminal globular domains of type VI collagen are numbered as previously described (51). The parentheses indicate the region where the recombinant fragment was expressed. (B) The heparin binding activity of the recombinant globular domain of type VI collagen was determined by slot blotting using biotinylated heparin. Recombinantly expressed fragments (10 μ g) of the α 1(VI), α 2(VI) or α 3(VI) chains show binding to heparin. LL-37 (5. mu.g) was used as a positive control (right panel). Unlabeled heparin (6mg/ml) inhibited the binding of biotinylated heparin to the recombinant fragment and LL-37 (left panel). (C) Binding of recombinant fragments to streptococcus pyogenes was clearly demonstrated by negative staining and transmission electron microscopy using colloidal gold labeling. Recombinant fragments at a final concentration of 2 μ M showed binding to the bacterial membrane (upper panel). The recombinant fragments did not bind to the bacterial membrane when pre-incubated with unlabeled heparin (lower panel). The scale shows 100 nm. (D) The amount of recombinant fragment bound to the bacterial surface in the absence (-) or presence (+) of heparin was calculated per μ M²Gold labeling of bacterial surfaces.

FIG. 3 is a dose-dependent killing of Streptococcus pyogenes by recombinant globular domains of collagen type VI. (A) Bacterium (2X 10)⁶cfu/ml) at the indicated concentrations, at 37 ℃ and 5% CO₂The recombinant fragments were incubated for 2 hours. (B) The recombinant globular domain of collagen type VI induces membrane disruption. Bacterium (2X 10)⁹cfu/ml) was treated with recombinant fragments and the permeabilities were visualized clearly by using scanning electron microscopy. Strong membrane rupture and leakage of cell contents were observed in the presence of these proteins and are indicated by arrows. The data shown represent at least 3 independent experiments and give an average value. Scale bar represents 5 μm.

FIG. 4 structural arrangement of VWA domains in the alpha 3-chain of human type VI collagen produced by structural stacking of VWA domain models. Under this sequence, the alpha-helix and beta-strand are indicated by rectangular boxes and black arrows, respectively. Exposed amino acids are indicated in bold letters and cationic moieties are highlighted in gray. The rectangular boxes in the sequence indicate the position of the cationic peptide as well as the control peptide (DVN 32). Sequence identifier: α 3_ N10[ SEQ ID NO: 37], α 3_ N9[ SEQ ID NO: 38], α 3_ N8[ SEQ ID NO: 39], α 3_ N7[ SEQ ID NO: 40], α 3_ N6[ SEQ ID NO: 41], α 3_ N5[ SEQ ID NO: 42], α 3_ N4[ SEQ ID NO: 43], α 3_ N3[ SEQ ID NO: 44], α 3_ N2[ SEQ ID NO: 45], α 3_ C1[ SEQ ID NO: 46],

fig. 5 (a) the surface of the VWA domain of the α 3(VI) chain appears to show electrostatic properties (black ═ positive charge; grey ═ negative charge). (B) The position of the cationic peptide and the negative control peptide (DVN32) are shown in the band plots. (C) Each peptide (GVR28[ SEQ ID NO: 1)]，FYL25[SEQ ID NO：2]，FFL25[SEQ ID NO：3]，VTT30[SEQ ID NO：4]，SFV33[SEQ ID NO：5]，DVN32[SEQ ID NO：6]) The biophysical property of (a).^aEach peptide passes through its first 3 NH₂The terminal residues are identified using the one letter code and the subsequent total number of residues that make up the peptide.^bThe ordering of the peptides is given in the single letter code.^cpI: can be used fromhttp://us.expasy.org/tools/protparam.htmlThe theoretical isoelectric point calculated by the Protparam tool obtained.

FIG. 6 antimicrobial activity of peptides derived from the α 3(VI) chain. (A) To determine antibacterial activity, the bacterial isolate shown (4X 10)⁶cfu) were seeded in agarose gels and loaded with peptide (at 100. mu.M). LL-37 and pH 7.4 buffer were used as positive and negative controls, respectively. Each clearance zone (clearance zone) corresponds to the inhibition of each peptide after 18-24 hours of incubation at 37 ℃. Representative views of the RDA gels are shown for escherichia coli (B) and staphylococcus aureus (C) using the indicated peptides.

FIG. 7 peptides derived from type VI collagen bind to the bacterial surface. (A) Binding of peptides derived from type VI collagen to pseudomonas aeruginosa, staphylococcus aureus or LPS was clearly demonstrated by negative staining and transmission electron microscopy using colloidal gold labeling. Pseudomonas aeruginosa or goldStaphylococcus aureus (2X 10)⁹cfu/ml) at 37 ℃ and 5% CO₂The cells were then incubated with LL-37, DVN32 or SFV33 conjugated with 10nm colloidal gold (see Table 2) for 2 hours. Scale 100 nm. Each peptide is shown as a black dot. (B) For LPS binding, LL-37 conjugated with 10nm colloidal gold, DVN32 and SFV33 at 37 ℃ and 5% CO₂Next, the cells were incubated with LPS (10. mu.g/ml) for 1 hour. Scale 50 nm. Each peptide is shown as a black dot. (C) CD spectra of LL-37, DVN32 and SFV33 in the presence or absence of LPS (0.2 mg/ml). The peptide concentration was 30. mu.M.

Figure 8 antimicrobial activity of peptides derived from type VI collagen in the presence of salt and plasma. In the viable bacteria count test, the antibacterial activity of peptides derived from type VI collagen against pseudomonas aeruginosa, staphylococcus aureus, escherichia coli, and streptococcus pyogenes, 2x10, was observed⁷cfu/ml bacteria in the presence of salt buffer with or without 20% human plasma (10mM Tris-HCl, 150mM NaCl and 5mM glucose; pH 7.4), at 37 ℃ and 5% CO₂The following incubations were performed with collagen VI-derived peptides (0.3, 0.6, 3, 6, 30, 60 μ M) for 2 hours. Bacteria incubated with only saline buffer with or without 20% human plasma served as negative controls. The sample with LL-37 served as a positive control. The data shown represent at least 3 independent experiments and give an average value.

FIG. 9 permeation of plasma membrane of cells by peptides derived from type VI collagen. (A) Pseudomonas aeruginosa or Staphylococcus aureus (2X 10)⁷cfu/ml) in the presence or absence of 20% human plasma salt buffer (10mM Tris-HCl, 150mM NaCl and 5mM glucose; pH 7.4) with peptides derived from type VI collagen. Propidium Iodide (PI) dye was added to the samples and incubated on ice for 30 minutes in the dark. FACS analysis of the mixture was performed using a flow cytometer. The same buffer without peptide was used as a control. As a positive control, 70% ethanol treated bacteria were used. Each experiment was performed in triplicate and the values represent the mean ± standard deviation. (B) For a clear presentation of antimicrobial activity, bacteria (2x 10)⁹cfu/ml) treatment of 2 with LL-37, DVN32 and SFV33 (30. mu.M) in the presence or absence of 20% plasma saline buffer at 37 ℃Hours, then observed by scanning electron microscopy. Intense membrane disruption, foaming and cytoplasmic component ejection were observed for LL-37 in salt and SFV33 under salt and plasma conditions. Bacteria treated with saline buffer, plasma or DVN32 showed no effect. Scale bar represents 5 μm.

Figure 10 membrane leakage levels as a function of peptide concentration. (A) Carboxyfluorescein efflux levels from liposomes composed of E.coli polar lipid extract after 45 min incubation. Each marker represents the mean leak value in 10mM Tris buffer (pH 7.4) at 37 ℃ with standard deviation for three replicates (i.e., no cumulative additions) at each peptide concentration. The curve fitting is performed by sigmoidal dose-response curve fitting, and EC₅₀The horizontal is highlighted with overlapping lines. LL-37 was used as a positive control. (B) Calculation of EC of peptides derived from type VI collagen and LL-37₅₀Value (. mu.M).

Figure 11 cytotoxicity testing of peptides derived from type VI collagen. (A) The hemolytic activity of the collagen type VI derived peptides and LL-37 was monitored by incubating 30 or 60 μ M of the peptides with human blood followed by measuring the absorbance at 540 nm. Results are expressed as% TritonX-100 induced hemolysis. (B) Serial dilutions of collagen-VI peptide and LL-37 were added to THP1 cells and cell permeation was measured by determining LDH release. All experiments were performed in triplicate.

Figure 12. anti-endotoxin activity of each peptide. The collagen VI-derived peptide or LL-37 was pretreated with LPS (10ng/ml) for 20 minutes at room temperature. The mixture was then added to RAW264.7 macrophages and incubated at 37 ℃ for 24 hours. Nitrite levels in the supernatant were determined using griiss reagent. Data are expressed as percentage of nitrite accumulation in LPS (100%) activated cells and show mean ± SEM of 3 independent experiments performed in triplicate.

Figure 13 peptide derived from collagen type VI promotes wound healing. HaCaT cells were cultured in 24-well plates and grown to confluence. The cells were kept for 24 hours in the absence of serum, then introduced into the wound by manual scraping with a sterile pipette tip, and washed twice to remove detached cells. CellsAt 37 ℃ and 5% CO₂Next, treatment with type VI collagen (10. mu.g/ml), a peptide derived from type VI collagen (10. mu.g/ml), or LL-37 (10. mu.g/ml) was carried out for up to 24 hours in the absence of serum. Cells were photographed at 0 hours of injury time and examined for cell migration within 24 hours from the addition of peptide. Controls consisted of cells treated with medium without supplements. Each image was acquired at 100x magnification. The data shown represent at least 3 independent experiments.

Figure 14 antimicrobial activity of peptides derived from type VI collagen against multi-drug resistant microorganisms. In the viable bacteria count test, the antibacterial activity of the collagen VI-derived peptide against multi-drug resistant pseudomonas aeruginosa (MRSA) and multi-drug resistant staphylococcus aureus (MRSA) was found (fig. 14A), and the antibacterial activity of the collagen VI-derived peptide against multi-drug resistant escherichia coli (MREC) and multi-drug resistant staphylococcus epidermidis (MRSE) (fig. 14B), and the antibacterial activity of the collagen VI-derived peptide against multi-drug resistant klebsiella pneumoniae (MRKP) (fig. 14C). 2x10⁷cfu/ml of bacteria were incubated with peptides derived from type VI collagen (30. mu.M) in the presence of salt buffer (10mM Tris-HCl, 150mM NaCl, 5mM glucose; pH 7.4). Bacteria incubated with salt buffer only served as controls. Samples with LL-37 served as positive controls. The data shown represent at least 3 independent experiments and give an average value.

Figure 15 type VI collagen kills multi-drug resistant pseudomonas aeruginosa (MRSA) and multi-drug resistant staphylococcus aureus (MRSA). Bacterium (2X 10)⁶cfu/ml) at 37 ℃ and 5% CO₂Next, the cells were incubated with 1. mu.M of collagen VI in a salt buffer (10mM Tris-HCl, 150mM NaCl, 5mM glucose; pH 7.4) for 2 hours. As clearly demonstrated by scanning electron microscopy, collagen VI (MRSA cVI ) -treated bacteria showed strong membrane disruption and efflux of cytoplasmic contents. Similarly, strong membrane penetration was observed in the presence of peptides derived from collagen VI (not shown). In contrast, untreated bacteria (MRPA, MRSA) showed normal structure. Scale bar represents 2 μm.

FIG. 16 type VI collagen against various gram negative and gram positive microorganismsBroad spectrum antibacterial activity of the compound. In the viable count test, 2x10⁷cfu/ml bacteria were incubated with type VI collagen (1. mu.M) in the presence of salt buffer (10mM Tris-HCl, 150mM NaCl, 5mM glucose; pH 7.4). The data shown represent at least 3 independent experiments and give an average value.

Figure 17 peptides derived from collagen VI improve survival in invasive pseudomonas aeruginosa infection models. Intraperitoneal injection of 2X10 into mice⁸cfu/ml pseudomonas aeruginosa bacteria and treated with 100 μ L SFV33, GVR28 or 100 μ L PBS (n-6/group).

Detailed Description

Examples of the invention

Example A

Introduction to the design reside in

The aim of this study was to explore whether the globular domains of collagen type VI have a role in host defense during infection and whether peptides derived from these domains have similar properties.

Materials and methods

Bacterial strains and culture conditions

Streptococcus pyogenes-producing strain AP1(40/58) of serotype M1 was from the cooperative center for review and study of the world health organization streptococcus, bragg in the czech republic of china. The Staphylococcus aureus strain 111 and the Escherichia coli strain B1351 were collected in the clinical microbiology of the university Hospital, Longde, Sweden. The pseudomonas aeruginosa strain used in this study was PAO1(ATCC, terbutonly, england) originally isolated from the wound. All bacteria were in Todd-Hewitt broth (THB)²Difco, detroit, Michigan, USA) and grown conventionally at 37 deg.C with 5% CO₂Is incubated in a humid atmosphere.

Recombinant expression and purification of N-and C-terminal VWA domains of type VI collagen alpha-chain

Type VI collagen microfibrils are extracted from bovine horn membrane by collagenase digestion as described by Abdillahi et al (36). The cDNA construct encoding the non-collagenogenic domain of collagen type VI was generated by RT-PCR on total RNA of mouse brain and cloned with NheI or XhoI at the 5 '-end and BamHI or XhoI restriction sites at the 3' -end using the following primers, see table 2 (38).

TABLE 2 primers and restriction enzymes for RT-PCR analysis of the globular region of collagen type VI.

Each amplified PCR product was inserted into a modified pCEP-Pu vector containing an N-terminal BM-40 signal peptide and a C-terminal tandem strepII-tag downstream of the restriction site (39). HEK293-EBNA cells (Invitrogen, Calsbad, Calif.) were transfected with recombinant plasmids using FuGENE6 reagent (Roche, Mannheim, Germany) according to the manufacturer's protocol. Cells were selected with puromycin (1 μ g/ml) (Sigma-Aldrich, st louis, missouri) and recombinant proteins were purified directly from Dulbecco's modified eagle medium (Invitrogen) supplemented with fetal bovine serum (Biochrom GmbH, berlin, germany). After filtration and centrifugation (1 hour, 10,000x g), the cell culture supernatant was applied to a Streptactin column (1.5ml, IBA GmbH, gottingen, germany) and eluted with 2.5mM desthiobiotin (Sigma-Aldrich), 10mM Tris-HCl, pH 8.0.

Viable count test

Bacteria at 37 ℃ and 5% CO₂Growth in THB-Medium to mid-log phase (OD)₆₂₀0.4). The bacterial solution was then washed and adjusted to 2X10 in 10mM Tris pH 7.4 containing 5mM glucose⁹cfu/ml. Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa were then incubated with 2. mu.M purified type VI collagen at 37 ℃ for 2 hours. In some experiments, streptococcus pyogenes was incubated with various concentrations (0.125, 0.25, 0.5, 1.0, and 2.0 μ M) of recombinant type VI collagen fragments for 2 hours at 37 ℃. Bacteria incubated with Tris-HCI pH 7.4 buffer or 3. mu.M LL-37(Innovagen, Longde, Sweden) were used as negative and positive, respectivelyA positive control. To quantify bactericidal activity, serial dilutions of the incubation mixture were plated onto blood agar plates, followed by incubation at 37 ℃ overnight, and the number of colony forming units (cfu) was determined. One hundred percent survival was defined as the total survival of the bacteria in the same buffer and under the same conditions in the absence of collagen type VI or recombinant protein.

Scanning electron microscopy

Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli or Pseudomonas aeruginosa (2X 10)⁹cfu/ml) purified type VI collagen at 37 ℃ and 5% CO₂Incubations were performed at 2. mu.M for 0, 30, 60 and 120 minutes. In some experiments, Streptococcus pyogenes were incubated with 2 μ M recombinant collagen type VI fragments for 2 hours at 37 ℃. LL-37 at 3. mu.M was used as a positive control, and bacteria in Tris-HCl pH 7.4 were used as a negative control. The samples were fixed with 2.5% glutaraldehyde in 0.1M sodium cacodylate (cacodylate buffer) at pH 7.4, washed with cacodylate buffer, and dehydrated serially with ascending ethanol as previously described (40). The samples were then critical point dried with carbon dioxide and absolute ethanol was used as an intermediate solvent. Tissue samples were mounted on aluminum mounts, sputtered with 20nm palladium/gold, and examined in a Philips/FEI XL 30FEG scanning electron microscope operated at 5kV acceleration voltage.

Fluorescence microscopy

The bacteria were grown to mid-log phase in THB-medium, washed and resuspended in 10mM Tris-HCl containing 5mM glucose to obtain 2X10⁷cfu/ml suspension. Mu.l of the bacterial suspension was incubated for 30 minutes at 37 ℃ with 2. mu.M purified type VI collagen or 3. mu.M LL-37, followed by addition of 200. mu.l FITC (6. mu.g/ml, Sigma-Aldrich) and incubation for 30 minutes at 37 ℃. The bacteria were washed and fixed onto poly-L-lysine (Sigma-Aldrich) coated slides by incubation at 37 ℃ for 45 minutes. The slides were washed with Tris-HCl/glucose and fixed with 4% Paraformaldehyde (PFA) by incubation at 4 ℃ for 15 minutes followed by incubation at room temperature for 45 minutes. The slides were then fixed to cover slips using Prolonggold anti-fade carrier media (Invitrogen).The bacteria were clearly visualized in a Nikon Eclipse E80i fluorescence microscope equipped with a Nikon DS-Fi1 camera, a Plan Apochromat (100x objective) and a high numerical aperture oil immersion capacitor.

Heparin binding assay

LL-37 (5. mu.g) or recombinant fragments from collagen type VI (10. mu.g) were applied to nitrocellulose membranes (Hybond-C; GE Healthcare, Uppsala, Sweden). Membranes were blocked with 2% BSA in PBS (w/v) for 2 hours at room temperature, then washed with PBST (PBS with Tween-20) and incubated overnight at 4 ℃ with 60. mu.g heparin-biotin (Sigma-Aldrich). In some experiments, unlabeled heparin (6mg/ml) was added to complete binding. After washing, the membrane was incubated with HRP streptavidin (Sigma-Aldrich) for 30 minutes at room temperature, washed, and the bands were visualized clearly by a Supersignal West Pico chemiluminescent substrate visualization system (Thermo Fischer Scientific, denmark rosskl).

Transmission electron microscopy

Binding of recombinant type VI collagen fragments to bacterial surfaces was clearly demonstrated by negative staining and transmission electron microscopy as described previously (35). Briefly, bacteria were incubated with recombinant type VI collagen fragments in the presence or absence of heparin (10. mu.g/ml) for 1 hour at 37 ℃. For clear presentation in the electron microscope, the different recombinant fragments were conjugated with 5nm colloidal gold (41). The samples were examined in a Philips/FEICM 100TWIN transmission electron microscope operated at an accelerating voltage of 60 kV. Images were recorded using a side-mounted Olympus Veleta camera and ITEM acquisition software.

Sequence and Structure analysis

The amino acid sequence of the human type VI collagen alpha-chain can be obtained from the UniProtKB database; α 1(VI) (UniProt # P12109), α 2(VI) (UniProt # P12110) and α 3(VI) (UniProt # P12111). Sequence alignment was performed using Swiss PDB Viewer DeepView version 4.1 to analyze three-dimensional structure. Only crystalline mouse α 3N5(42) was obtained using PDB code 4 IGI. The crystal structure could not be derived from the human type VI collagen VWA domain, so the graphs were generated using a model predicted from ModBase (http:// ModBase. compbio. ucsf. edu). The N1 and C2 domains of α 3(VI) have no predictive model.

Synthesis of peptides

GVR28(GVRPDGFAHIRDFVSRIVRRLNIGPSKV)[SEQ ID NO：1]、

FYL25(FYLKTYRSQAPVLDAIRRLRLRGGS)[SEQ ID NO：2]、

FFL25(FFL KDFSTKRQIIDAINKVVYKGGR)[SEQ ID NO：3]、

VTT30(VTTEIRFAD SKRKSVLLDKIKNLQVALTSK)[SEQ ID NO：4]、

SFV33(SFVARNTFKR VRNGFLMRKVAVFFSNTPTRASP) [ SEQ ID NO: 5], and

DVN32(DVNVF AIGVEDADEGALKEIASEPLNMHMFNL)[SEQ ID NO：6]

synthesized by Biopeptides (san diego, california). The purity (> 95%) and molecular weight of these peptides were confirmed by MALDI-TOF MS analysis. All peptides used were water soluble except DVN32, which was dissolved in < 0.01% DMSO.

Radial diffusion test

Radial diffusion test (RDA)²Performed substantially as described previously (43, which is incorporated herein by reference), with some minor modifications. Bacteria in 10ml full strength (3% w/v) tryptic Casein Soy Broth (TSB)²(Becton Dickinson, Franklin lake, N.J.) growth to mid-log phase (OD)₆₂₀0.4). The bacteria were then washed once with 10mM Tris-HCl (containing 5mM glucose; pH 7.4). Subsequently, 4 × 10⁶cfu/ml bacteria were added to 5ml of substrate agarose gel consisting of 0.03% (w/v) TSB, 1% (w/v) low electroosmosis type agarose and 0.02% (v/v) Tween20 (both from Sigma-Aldrich). Pouring the substrate intoIn a Petri dish. After the agarose has solidified, it is punched outTo each well, 6. mu.l of 10mM Tris-HCl buffer alone or 10mM Tris-HCl buffer containing peptide (100. mu.M) was added.Each plate was incubated at 37 ℃ for 3 hours to allow the peptide to diffuse. The substrate gel was then coated with 5ml of a cover (6% TSB and 1% agarose of the low electroosmosis type, in distilled H₂O in). Antimicrobial activity after incubation at 37 ℃ for 18 to 24 hours was seen as a clearing zone around each well.

Statistical analysis

Student T-tests were performed to determine statistical significance. Each value was expressed as mean ± standard error, significance was determined as a P value < 0.05.

Results and conclusions

Killing of gram-positive and gram-negative human pathogens by membrane permeation of type VI collagen

An integrated approach combining microbiological and biochemical tests with high resolution scanning electron microscopy was established to investigate the antimicrobial properties of collagen type VI. Viable count tests were performed by incubating the gram-positive bacteria streptococcus pyogenes and staphylococcus aureus, and the gram-negative bacteria escherichia coli and pseudomonas aeruginosa with purified type VI collagen for 2 hours at 37 ℃. Each result showed that collagen type VI did show antibacterial activity against staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa in a manner similar to that observed for streptococcus pyogenes (selected model organism) (fig. 1A). Human reference antimicrobial peptide LL-37 was used as a positive control and showed almost 100% killing of all bacterial strains. To examine whether type VI collagen disrupts the bacterial membrane, high resolution scanning electron microscopy was used to allow the bacterial structure to be clearly represented in a more three-dimensional manner during the killing process. Bacteria were incubated with buffer only or collagen type VI (fig. 1B). Each result showed strong disruption of bacterial membrane structure and extravasation of cytoplasmic components in the presence of type VI collagen, indicating disruption of bacterial membranes (fig. 1B, right panel, fig. 1C). These findings were similar to those observed after treatment with LL-37 (data not shown). In contrast, in the control sample, the bacterial cell wall structure was unaffected (fig. 1B, left panel). These observations were further confirmed by using the permeant dye FITC. Fluorescence microscopy analysis showed that uptake of FITC was only seen in samples treated with collagen VI or LL-37 (FIG. 1D), thus indicating penetration of the bacterial membrane. Similar observations were made for various other gram-positive and gram-negative human pathogens (fig. 16). Taken together, these data indicate that type VI collagen exhibits broad spectrum antimicrobial activity against gram positive and gram negative bacteria.

Spherical region of type VI collagen containing recombinant VWA domain binds to bacterial surface in a heparin-dependent manner

The affinity of a negatively charged surface on the bacterial membrane is a prerequisite for any given antimicrobial molecule to induce killing of bacteria, regardless of its mode of action. Therefore, most antimicrobial peptides and proteins are characterized by their affinity for heparin (32, 44). To determine whether type VI collagen exhibits similar properties, biotin-labeled heparin was tested in a slot blot assay for binding to immobilized type VI collagen recombinant fragments (as shown in fig. 2A). Heparin bound to different N-and C-terminal regions with different intensities (fig. 2B, left panel). Interestingly, heparin affinity with α 3C was comparable to the positive control LL-37. Binding to all fragments was blocked by unlabeled heparin (fig. 2B, right panel). To make these interactions clearly apparent, collagen type VI fragments were directly conjugated to colloidal gold and incubated with streptococcus pyogenes bacteria. Negative staining and transmission electron microscopy showed that all fragments bound to the bacterial surface in the absence of heparin (fig. 2C, upper panel). No binding was observed in the presence of heparin, instead, the gold conjugates were randomly distributed in the background (fig. 2C, lower panel). Similar results were obtained for s.aureus, e.coli and p.aeruginosa in the absence or presence of heparin (fig. 2D). The same was true for the full-length protein (data not shown).

Three spherical regions of type VI collagen alpha-chain containing recombinant VWA domain induce sterilization by membrane disruption

In order to correlate the antimicrobial activity of type VI collagen with the individual N-terminal and C-terminal globular regions, the bactericidal effect of recombinant proteins on streptococci was studied. Bacteria from streptococcus pyogenes producing strain AP1 were incubated with increasing concentrations of protein and analyzed by the viable count test. The presence of the different fragments showed dose-dependent killing of bacteria (fig. 3A). Interestingly, the N-and C-terminal regions of α 3(VI) are more potent than the corresponding domains of α 1(VI) and α 2 (VI). To analyze these findings at the ultrastructural level, bacterial samples incubated with VWA domains were examined by high resolution scanning electron microscopy. As shown in fig. 3B, the bacteria did show significant structural changes such as membrane perturbation, blebbing and efflux of cytoplasmic components. In particular, alpha 3N and alpha 3C treated streptococci showed significantly more membrane disruption. In contrast, control bacteria treated with buffer only under similar conditions were unaffected (fig. 3B, top). These results indicate that the spherical region of type VI collagen containing the VWA domain alone exhibits a pattern of killing bacteria, which is similar to whole protein.

The VWA domain of the collagen type VI alpha 3-chain contains an amphiphilic amino acid motif with putative antibacterial activity

Cationic, hydrophobic and amphiphilic properties are essential for the true core of antimicrobial peptide activity, as the combination of these properties determines the extent to which sterilization is induced (9, 45). Thus, in silico sequence analysis of the VWA domain of the α 3(VI) chain was performed in order to determine the putative antimicrobial activity for this amino acid motif. Alpha3(VI) was chosen because it was originally the most effective in sterilization as described above. First, we defined possible secondary structures by aligning the sequences of the N-terminal and C-terminal VWA domains (N10-N2 and C1, see FIG. 2A) using the Swiss-Pdb Viewer program. This analysis shows that these domains are predicted to adopt alpha-helices (FIG. 4, rectangular box) as well as beta-strands (FIG. 4, black arrows). In addition, the 3D model generated using ModBase suggested that these domains consist of a central six-stranded hydrophobic β -plate with 3 amphipathic α -helices flanking either side (data not shown). These findings are generally consistent with structural data obtained by X-ray crystallography of the mouse α 3N5 domain (42). VWA domains N1 and C2 (see fig. 2A) were not included in this study, since there is no molecular model available in any database. Next, amino acids that may be exposed to the surface are identified (fig. 4, bold letters) in order to predict the likely interaction site(s) between these domains and the bacterial membrane. By combining these results with positively charged regions in the sequence (FIG. 4, highlighted in grey), it was possible to predict putative antimicrobial regions as shown in the blue box.

Peptides derived from the VWA domain of the alpha 3-chain of collagen type VI exert bactericidal activity

Peptide sequences of putative antimicrobial regions with high overall net charge and hydrophobicity were identified, as these properties are important preconditions for AMP (46, 47). In summary, 5 peptides were selected from the N3, N2, and C1 domains (fig. 5, B and C). Surface image models were also generated in order to obtain an overview of the net charge of these domains, and the C1 and N3 domains do show a large number of cationic regions on their surface (fig. 5A). A similar pattern was observed for N2, although to a lesser extent. The N10 domain was shown to have more anionic residues on its surface (fig. 5A), and the peptide synthesized from this domain was used as a negative control (DVN 32). To verify the antibacterial activity of the selected VWA-derived peptides, all peptides were screened in the Radial Diffusion Assay (RDA) for bactericidal activity against e. All peptides exhibited varying degrees of significant bactericidal activity against all strains tested (fig. 6, a-C). Interestingly, in most cases the bacterial killing potential observed was significantly higher than that of our positive control ("classical" host defense peptide LL-37). These findings show that the VWA domain of the α 3(VI) chain contains several antimicrobial motifs.

Example B

Introduction to the design reside in

The purpose of this example was to further investigate the mode of action and immunomodulatory effects of host defense peptides derived from type VI collagen.

Materials and methods

Bacterial strains

Streptococcus pyogenes strain AP1(40/58) of serotype M1 was from the collaboration center for review and study of the world health organization streptococcus, bragg in the czech republic of china. The Staphylococcus aureus strain 111 and the Escherichia coli strain B1351 were collected in the clinical microbiology of the university Hospital, Longde, Sweden. The pseudomonas aeruginosa strain used in this study was PAO1(ATCC, terbutonly, england) originally isolated from the wound.

Growth medium

All bacteria were tested by passing them at 37 ℃ with 5% CO₂Are incubated in a humid atmosphere and grown conventionally in Todd-Hewitt solution (THB, Difco, detroit DI usa).

Extraction of type VI collagen and peptide synthesis

Type VI collagen microfibrils were extracted from bovine horn membrane by collagenase digestion as described by Spissinger et al (34), modified by Bober et al (35). Briefly, bovine horn membranes were cut into small pieces and homogenized in Tris/saline buffer containing 5mM calcium chloride and protease inhibitor. The homogeneous mixture was digested with collagenase type 1 (Wooston Biochemical Corporation, Worthington Biochemical Corporation), Rickward, N.J.. Undissolved material was pelleted by centrifugation at 48,000 Xg for 20 minutes. The supernatant was applied in 500. mu.l aliquots to 25ml Superose 6 columns equilibrated and eluted with 0.2 ml/min homogenization buffer (Amersham Biosciences, Uppsala, Sweden). Fractions of 0.5ml were collected, those containing collagen VI were pooled and stored at 4 ℃. Peptides derived from collagen type VI (see table 3) were synthesized by Biopeptides (san diego, usa). LL-37(LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) (SEQ ID NO: 36) was purchased from (Innovagen AB, Longde, Sweden). The purity (> 95%) and molecular weight of these peptides were determined by MALDI-TOFMS analysis. All peptides used were water soluble except DVN32, which was dissolved in < 0.01% DMSO (Sigma-Aldrich, st louis).

Transmission electron microscopy

The binding of each peptide to the bacterial surface and LPS was clarified by negative staining and transmission electron microscopyPresented, as previously described (35). Briefly, bacteria (2x 10)⁹cfu/ml) at 37 ℃ and 5% CO₂Next, 2. mu.M peptide conjugated with 10nm colloidal gold was incubated for 2 hours. For LPS (from E.coli 0111: B4, Sigma-Aldrich) binding, 2. mu.M peptide conjugated to 10nm colloidal gold was incubated with LPS (10. mu.g/ml) for 1 hour at room temperature. The samples were examined in a Philips/FEICM 100TWIN transmission electron microscope operated at 60kV acceleration voltage. Each image was recorded with a side-mounted Olympus Veleta camera and ITEM acquisition software.

Table 3. amino acid sequence and physicochemical properties of the peptides used in this study.

^aEach peptide passes through its first 3 NH₂The terminal residues are identified using the one letter code and the subsequent total number of residues that make up the peptide.

^bThe sequence of each peptide is given in the single letter code.

Circular dichroism

The measurement of Circular Dichroism (CD) was performed on a Jasco J-810 spectropolarimeter equipped with Jasco CDF-426S Peltier set at 25 ℃. The measurements were repeated at least twice in a 10mM quartz cuvette with stirring at a peptide concentration of 30 mM. LPS (0.2mg/ml) was added to some samples in order to investigate the effect on the secondary structure of the peptide. It was monitored in the range of 200 to 260nm (scan speed 20 nm/min). The baseline was subtracted from the average of 5 scans.

Sterilization test

Bacteria at 37 ℃ and 5% CO₂Growth in THB Medium to mid-log phase (OD)₆₂₀0.4). The bacterial solution was then washed and adjusted to 2X10 in salt buffer (10mM Tris-HCl, 150mM NaCl supplemented with 5mM glucose; pH 7.4) (both from Sigma-Aldrich)⁷cfu/ml. Peptides (0.3, 0.6, 3, 6, 30 and 60 μ M) at various concentrations were incubated with bacteria in saline buffer with or without 20% human plasma. With or without the presence ofBacteria incubated in saline buffer with plasma were used as negative controls. The sample with LL-37 served as a positive control. Samples were incubated at 37 ℃ and 5% CO₂Incubate for 2 hours. To quantify bactericidal activity, serial dilutions of the incubation mixtures were plated on THB agar plates, followed by incubation at 37 ℃ and 5% CO₂Incubate overnight and determine cfu number. The experiment was repeated three times. One hundred percent survival was defined as the total survival of the bacteria in the same buffer and under the same conditions in the absence of peptide.

Propidium iodide uptake assay

Bacterial membrane penetration was assessed using Propidium Iodide (PI) (Sigma-Aldrich) dye, as previously described (39). Briefly, bacteria were grown to mid-log phase (OD)₆₂₀Ca. 0.4), washed and adjusted to 2x10⁹cfu/ml. Bacteria (diluted 1: 100) were mixed with each peptide (final concentration 30. mu.M) in the presence of saline buffer with or with plasma and incubated at 37 ℃ with 5% CO₂Incubate for 2 hours. PI (0.5mg/ml) was added to each sample and incubated on ice for 30 minutes in the dark. The samples were analyzed on a flow cytometer (BDAccuri flow cytometer, Becton Dickinson, franklin lake). Bacteria incubated only in saline buffer with or without human plasma were used as negative controls. As a positive control, the bacteria were treated with 70% ethanol for 20 minutes at room temperature. The percentage of membrane penetration was calculated as the percentage of fluorescence intensity of the peptide-treated sample relative to the fluorescence intensity of the untreated sample.

Scanning electron microscopy

Bacterium (2X 10)⁹cfu/ml) was incubated with 30 μ M peptide at 37 ℃ for 2 hours under physiological conditions like saline buffer with or without plasma. Samples were fixed in 0.1M sodium cacodylate (Sigma-Aldrich) at pH 7.4 with cacodylate buffer (2.5% glutaraldehyde (Merck, Germany), washed with cacodylate buffer, and dehydrated in ascending ethanol series (40) as described previously.

Membrane penetration test

By dissolving the lipid in chloroform, followed by N₂Evaporation under flow followed by standing overnight in vacuo formed a dry lipid film of the e.coli polar lipid extract in the round bottom flask wall. The lipid membrane was resuspended in 100mM 5(6) -carboxyfluorescein in 10mM Tris in water (pH 7.4 at 37 ℃) with stirring (E.coli) for 30 minutes at 55 ℃. The suspension was then vortexed and then repeatedly squeezed through a 100nm polycarbonate membrane mounted in a LipoFast mini-extruder (Avanti Polar Lipids) in order to reduce the multilayer structure and polydispersity. The uncaptured carboxyfluorescein was removed by gel filtration on a Sephadex PD-10 column (GE Healthcare, small Charles volts, UK). The efflux of carboxyfluorescein from liposomes to an external low concentration environment resulted in a loss of self-quenching and an increase in the fluorescence signal at excitation and emission wavelengths of 492nm and 517nm, respectively, and the permeability of the membrane was measured by monitoring this process. Fluorescence was measured in black Nunc Delta Surface 96-well plates (Thermo Fisher Scientific, Rousbasil, Denmark) using a Varioskan Flash Multimode Reader (Thermo Fisher Scientific, Waltham, Mass.). Each well was prepared with 2-fold serial dilutions of the peptide in tris buffer, as well as a control without peptide (background) and 0.16% triton x-100 (maximum leakage). Plates were pre-warmed to incubation temperature (37 ℃) and liposome solutions were applied using a Varioskan integral dispenser to a final lipid concentration of 10 μ M in 200 μ l. The effect of each peptide concentration on the liposome system was monitored for 45 minutes, at which time the initial leak had subsided greatly. The results shown represent the average of three replicates with standard deviation and are expressed as the percentage of total leakage using triton x-100 minus baseline values. EC (EC)₅₀Values were calculated using Graphpad Prism by fitting a sigmoidal dose-response curve with variable slope to the percentage of leakage as a function of peptide concentration (log 10).

Hemolysis test

Blood from healthy individuals was aspirated into EDTA-containing Vacutainer tubes (Becton Dickinson) and centrifuged at 800x g for 10 minutes. Plasma and buffy coat were removed. Erythrocytes were washed 3 times and resuspended in phosphate buffered saline (PBS, Medicago, usa). Cells were incubated with peptide (final concentration 30 and 60 μ M) at 37 ℃ for 1 hour with inversion rotation. Cells incubated with 2% TritonX-100(Sigma-Aldrich) served as positive controls. The samples were then centrifuged at 800x g for 10 minutes. The supernatant was collected and the absorbance of hemoglobin release was measured at 540nm and expressed as% of triton x-100 induced hemolysis.

Lactate Dehydrogenase (LDH) assay

Cytotoxicity assays were performed as described previously (41). Briefly, human mononuclear THP-1 cells (American type culture Collection (ATCC), Marnsas, Va.) were cultured in 96-well plates using Dulbecco's Modified Eagle Medium (DMEM) (PAA Laboratories) supplemented with 10% fetal bovine serum. The medium was removed and the cells were subsequently washed with DMEM. Peptides (1, 5, 10, 20, 50 μ M) diluted in DMEM were added in triplicate. The LDH-based TOX-7 kit (Sigma-Aldrich) was used according to the manufacturer's instructions. The amount of LDH released from dead cells was measured at 450 nm. 2% Triton X-100 was used as a positive control.

LPS stimulation of macrophages in vitro

Murine macrophages (RAW 264.7; ATCC) were treated at 3.5X 10⁵Cells/well were seeded in 96-well plates in DMEM (PAA Laboratories without phenol red) supplemented with 10% fetal bovine serum and antibiotic-antimycotic (Invitrogen, carlsbad, ca). After incubation overnight at 37 ℃, cells were washed once with DMEM. LPS (10ng/ml) was pre-incubated with the peptide for 20 minutes at room temperature and added to the cells. The cells were then incubated at 37 ℃ for 24 hours. Griiss reagent (Sigma, st. louis, missouri) was added to the culture supernatant at a ratio of 1:1, followed by incubation in the dark for 15 minutes. The absorbance was then measured with a spectrophotometer at 550 nm. Cells with and without LPS stimulation were used as positive and negative controls for LPS induction. To determine NO production after peptide and LPS treatment, the test was performed in triplicate and the average value of each group was analyzed. Cells were incubated at 37 ℃ and 5% CO₂And then grown in a completely humidified atmosphere.

In vitro wound healing test

Keratinocyte basal medium (KBM Gold, Lonza Group AG, Switzerland) was used at 3X 10 according to the manufacturer's instructions⁵Cells/well HaCaT cells (ATCC) were cultured into 24-well plates until fusion. Cells were cultured in serum free medium for 24 hours prior to the assay. Mechanical scratch damage was performed on cell monolayers using sterile pipette tips. Detached cells were removed by washing 2 times with PBS. Peptides derived from type VI collagen (10. mu.g/ml), LL-37 (10. mu.g/ml) and type VI collagen (10. mu.g/ml) were added to the cells and incubated at 37 ℃ for 24 hours. Cells in basal medium with and without 10% FCS were used as positive and negative controls, respectively. All tests were at 5% CO₂And 37 ℃ in a humid atmosphere. Images of the damaged cell monolayer were taken using a microscope (Olympus, SC30 digital camera, tokyo, japan) at 0 and 24 hours after scratching.

Statistical analysis

Data were analyzed using Graphpad Prism 6. Student T-tests were performed to determine statistical significance. Each value was expressed as mean ± standard error and significance was determined as a p-value < 0.05.

Results and conclusions

Adhesion of peptides derived from type VI collagen to bacterial surfaces

To examine whether the collagen VI-derived peptides (see table 3) interacted with the bacterial surface, gold-labeled peptides were incubated with pseudomonas aeruginosa and staphylococcus aureus, and negative staining transmission electron microscopy was performed thereon. Electron micrographs showed that SFV33 and LL-37 were able to adhere to the bacterial surface of staphylococcus aureus and pseudomonas aeruginosa (fig. 7A), as were other peptides (data not shown). Interestingly, even the negatively charged control peptide DVN32 bound to the bacterial surface of staphylococcus aureus and pseudomonas aeruginosa.

Binding of peptides derived from type VI collagen to LPS

In the following series of experiments, to determine whether Lipopolysaccharide (LPS) of gram-negative bacteria can serve as a potential target for collagen VI-derived peptides, e.coli LPS was incubated with gold-conjugated peptides for 1 hour and negative staining transmission electron microscopy was performed on them. Both LL-37 and SFV33 bound LPS (FIG. 7B), while DVN32 bound slightly. To investigate the interaction between LPS and these peptides, their secondary structure was analyzed using Circular Dichroism (CD) analysis. LL-37 clearly adopted a helical structure in the presence of LPS (FIG. 7C), while DVN32 did not change its structure and remained linear. CD spectra show that the change in configuration of SFV3 resembles a random coil structure compared to other peptides (GVR28, FFL25, FYL25, and VTT30) showing a mixture of alpha helices and beta platelets (data not shown).

Peptides derived from type VI collagen exhibit antibacterial activity under physiological conditions

The antibacterial activity of many AMPs is inhibited in physiological environments (such as high salt concentrations or the presence of plasma proteins) (48, 49). Live bacteria enumeration tests were performed using peptides derived from type VI collagen. For this purpose, a group of gram-positive bacteria streptococcus pyogenes and staphylococcus aureus and gram-negative bacteria escherichia coli and pseudomonas aeruginosa were treated with peptides derived from type VI collagen in the presence or absence of physiological salts of 20% human plasma. For comparison, classical AMP LL-37 was used at the same concentration. Figure 8 shows that SFV33 killed pseudomonas aeruginosa and escherichia coli very efficiently in plasma, while its antimicrobial activity against streptococcus pyogenes was slightly reduced. In particular, the antimicrobial effect of SFV33 was dose-dependent under both conditions. None of the peptides exhibited antibacterial activity against staphylococcus aureus in the presence of plasma (fig. 8). Under salt conditions, SFV33 showed an almost similar effect as LL-37, while GVR28 showed a slightly reduced effect. In contrast, as expected, the negative control peptide (DVN32) did not exert antibacterial activity, even at higher concentrations.

Membrane penetration Activity of peptides derived from type VI collagen

To investigate the effect of collagen type VI derived peptides on bacterial membranes, the uptake of propidium iodide was measured. As shown in fig. 9A, SFV33 induced a significant degree of membrane penetration on pseudomonas aeruginosa and staphylococcus aureus under saline conditions. A similar effect of SFV33 on Pseudomonas aeruginosa in plasma was also detected, but LL-37 did not. Other peptides were unable to induce membrane permeation under physiological conditions, as observed on staphylococcus aureus. However, they showed membrane disruption on pseudomonas aeruginosa in the presence of salt (fig. 9A).

The effect of SFV33 on bacterial membranes was further examined by high resolution scanning electron microscopy. At the ultrastructural level, SFV33 caused the bacteria to rupture, leading to disintegration and ejection of cytoplasmic components in the presence of salts (pseudomonas aeruginosa and staphylococcus aureus) and plasma (pseudomonas aeruginosa) (fig. 9B). DVN 32-treated bacteria did not show any membrane disruption, similar to the control. These results further support the following notions: the peptide SFV33 derived from type VI collagen destroys the cell membranes of pseudomonas aeruginosa and staphylococcus aureus at physiological ionic strengths similar to those seen for LL-37.

To investigate the effect of collagen type VI derived peptides on membranes, a liposome model was used to investigate membrane permeation. Each peptide was tested for membrane leakage in a liposome model membrane system (e.coli polar lipid extract). The results show that all peptides have the ability to cause membrane permeation at physiological pH, except DVN32 (fig. 10A). Each peptide induces a concentration-dependent release of carboxyfluorescein. FYL25 and SFV33 induced the highest membrane leakage compared to VTT30 (fig. 10B). Taken together, these results strongly support the significance of leak testing, propidium iodide uptake, and scanning electron microscopy experiments: the antimicrobial activity of type VI collagen-peptides like SFV33 is likely caused by damage to the bacterial cell membrane.

Effect of the peptides on eukaryotic cells

One major side effect of some AMPs is that they not only act on the bacterial membrane, but they may also destroy and eliminate eukaryotic cells. Cytotoxic effects of different concentrations of the peptide on erythrocytes and monocytes were evaluated. 2% Triton X-100 was used as a cytotoxic agent, a positive control. The results show that each peptide did not show any toxicity against erythrocytes and monocytes at concentrations up to 30 μ M compared to LL-37 (FIGS. 11A and B). However, SFV33 exhibited toxicity against monocytes at 50 μ M (fig. 11B). However, these results indicate that the peptides derived from type VI collagen did not affect the viability of human cells at any concentration used for sterilization.

Immunomodulatory properties of peptides derived from type VI collagen

LPS is a well studied endotoxin released from the outer membrane of gram-negative bacteria and plays an important role in the pathogenesis of certain bacterial diseases. Massive release of LPS can lead to endotoxic shock and cause death (50). The immunomodulatory effects of collagen VI-derived peptides were studied. Murine macrophages were stimulated simultaneously with e.coli LPS and collagen VI-derived peptides and the amount of nitrite in the supernatant was measured using Greiss reagent. As shown in figure 12, the addition of GVR28 significantly inhibited LPS-induced nitrite production, similar to those seen after LL-37 treatment. In contrast, other peptides were unable to block LPS-induced nitrite production.

To examine the biological effect of synthetic collagen VI-derived peptides on wound healing, HaCaT cells were cultured in wells of 24-well plates; cells were scraped and incubated with either intact type VI collagen, peptides derived from type VI collagen or LL-37 (10. mu.g/ml). Cell migration was recorded by photomicrograph at 0 and 24 hours after scraping. As shown in fig. 13, the collagen VI-derived peptides showed significant wound closure capacity after 24 hours compared to control cells without supplements. SFV33 peptide did not promote wound healing.

Example C

Introduction to the design reside in

The aim of this study was to evaluate the antimicrobial effect of collagen VI-derived peptides on many conventional antibiotic resistant bacteria (52, 53). In addition, the study also evaluated the effect of type VI collagen on the membranes of resistant bacteria.

Materials and methods

Microorganisms and culture conditions

The following multidrug-resistant bacterial strains are produced by Lisa(university of Longde infectious medicine department) friendship offers: pseudomonas aeruginosa (MRSA), staphylococcus aureus (MRSA), escherichia coli (MREC), staphylococcus epidermidis (MRSE), and klebsiella pneumoniae (MRKP). All multi-drug resistant microorganisms tested were from clinical isolates from patients with bacteremia or pneumonia. All strains were grown overnight at 37 ℃ in a humidified atmosphere containing 5% in Todd-Hewitt broth (THB, Gibco, Greenland, N.Y.).

Antimicrobial Activity test

Bacteria in Todd-Hewitt broth (OD)₆₂₀0.4) to mid-log phase, collected by centrifugation at 3,500rpm for 10 minutes, and washed twice in TBS buffer. The bacterial suspension was adjusted to 2x10 per ml⁹Individual colony forming units (cfu). Bacteria were further diluted in TBS and incubated with different collagen VI or collagen VI peptide concentrations. Bacteria incubated with TBS or 3. mu.M LL-37 antimicrobial peptide (Innovagen, Longde, Sweden) were used as negative or positive controls, respectively. Samples were incubated at 37 ℃ in a solution containing 5% CO₂For 2 hours in a humid atmosphere. Serial dilutions were plated on agar plates and incubated overnight at 37 ℃ before cfu numbers were determined by counting visible colonies. The experiment was performed in triplicate.

Scanning electron microscopy

For high resolution field emission transmission electron microscopy (FESEM), the samples were fixed overnight with 2.5% glutaraldehyde in cacodylate buffer at room temperature. They were then washed with cacodylate buffer and dehydrated in ascending ethanol series from 50% (v/v) to absolute ethanol. The samples were then critical point dried with carbon dioxide and absolute ethanol was used as an intermediate solvent. Tissue samples were mounted on aluminum mounts, sputtered with 20nm palladium/gold, and examined in a Philips/FEI XL 30FESEM scanning electron microscope using an Everhart-Tornley secondary electron detector.

Results and conclusions

Type VI collagen and peptides derived from type VI collagen are antimicrobial against multi-drug resistant mammalian pathogens to assess the potential antibacterial effects of type VI collagen and its peptides, bacteria were treated with purified preparations of the protein and its peptides. Bacteria treated with TBS buffer or antimicrobial peptide LL-37 served as negative and positive controls, respectively. The results of the viable count test showed that multidrug resistant human pathogens were killed (fig. 14A-C). Treatment at 37 ℃ for 2 hours significantly inhibited bacterial growth compared to TBS-treated control bacteria. In particular, the bacterial clearance efficiency is comparable to or better than the "classical" human antimicrobial peptide LL-37.

The killing properties of type VI collagen are associated with membrane rupture in Streptococcus and Pseudomonas as determined by electron microscopy

To understand in more detail the intrinsic killing mechanism, human skin biopsies were inoculated with MRSA and MRPA (as model systems) in the presence or absence of collagen type VI and observed by scanning electron microscopy. As shown, fig. 15 illustrates the bactericidal effect of the molecule. Membrane perturbation, blebbing and leakage of cytoplasmic contents were observed in the presence of type VI collagen. Large scale membrane destabilization events ultimately lead to bacterial cell disintegration into a mixture of cell membrane vesicles and cytoplasmic discharge. Taken together, the data presented in fig. 14 and 15 show that collagen type VI and/or several portions of this molecule exert antimicrobial activity against multi-drug resistant human pathogens through mechanisms including membrane disruption. The antimicrobial effect is dose-dependent at physiological pH and salt concentration.

Example D

Introduction to the design reside in

The aim of this study was to evaluate the antimicrobial effect of amphiphilic peptides derived from the amino acid sequence of collagen VI in a mouse model of infection in vivo in bacterial infection with invasive pseudomonas aeruginosa (abbreviated as pseudomonas aeruginosa).

Materials and methods

Microorganisms and culture conditions

At 37 ℃ in a medium containing 5% CO₂In a humid atmosphere, pseudomonas aeruginosa 15159 strain was grown overnight in Todd-Hewitt broth (THB, Gibco, gland island, new york, usa).

Animal testing

Animal experiments were performed according to protocols approved by the regional ethics committee of university of longde. Animals were housed under standard light and temperature conditions and were freely accessible to standard laboratory feed and water. Pseudomonas aeruginosa 15159 bacteria were grown to mid-exponential phase (A)₆₂₀0.5), washed in PBS and diluted to 2X10⁸cfu/ml, and placed on ice until used for injection. Female BALB/c mice, 8 weeks old, were anesthetized with isoflurane and injected intraperitoneally with 100 μ L of bacterial solution, followed 15 minutes later by 100 μ L of SFV33 or GVR28 peptide (1mg/ml), or 100 μ L of PBS alone (control).

Results

Effect of treatment with collagen VI-derived peptides on survival of infected mice

The beneficial effects of SFV33 and GVR28 were shown in murine survival studies. Intraperitoneally infected mice were treated with a single dose of either SFV33 or GVR28 for 15 minutes following infection and survival was recorded. Figure 17 shows that 50% of infected mice treated with PBS died during the first 12 hours. In contrast, in the group treated with SFV33 and GVR28, 50% mortality was observed in the animals after 24 hours. Comparing the overall mortality of the SFV33/GVR28 group and the PBS group, the peptide treated animal groups all showed prolonged survival at termination of the trial after 29 hours.

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The structure of recombinant N-terminal globules of collagen type VI α 3chain and their binding to heparin and hyaluronic acid, in Specks, u. (Structure of recombinant N-terminal glucose of type VI collagen alpha 3chain and its binding to peptide and hyaluronic.) Journal of European molecular biology organization (The EMBO Journal) 11, 4281-4290

Sequence analysis of the α 1(VI) and α 2(VI) chains of human type VI collagen (1989) showed internal triplex replicating globular domains similar to domain a of von willebrand factor and two α 2(VI) chain variants with different carboxy termini. (Sequence analysis of alpha 1(VI) and alpha2(VI) chains of human type VI collagen derivatives internal simulation of spherical domains to The A domains of von Willebrand factor and two alpha2(VI) chains of which impurities are in The carbon atom tertiary.) Journal of European molecular biology (The EMBO Journal) 8, 1939-

Studded structure of the globular domain in the alpha 3chain of human type VI collagen, Chu, m.l., Zhang, r.z., Pan, t.c., Stokes, d.d., Conway, d.s., Kuo, h.j., Glanville, r.s., Mayer, U.S., man, k.s., Deutzmann, r.and et al (1990): similarity to von willebrand factor, fibronectin, actin, sialoprotein, and aprotinin protease inhibitors. (molecular Structure of viral domains in The human type VI collagen alpha 3chain: similarity to von Willebrand factor, fibrin, actin, lipid proteins and amyloid type protease inhibitors.) Journal of European molecular biology organization (The EMBO Journal 9, Journal 385 393

Collagen type VI structures 28.Chu, m.l., Pan, t.c., Conway, d., Saitta, b., Stokes, d., Kuo, h.j., Glanville, r.w., Timpl, r., man, k, and Deutzmann, R. (1990). (The Structure of type VI collagen), annual newspaper of New York Academy of Sciences (Annals of The New York Academy of Sciences) 580, 55-63

29.Lamande,S.R.，M., Adams, n.e., Selan, C. and Allen, J.M. (2006) the C5 domain of the collagen type VI α 3(VI) chain is important for extracellular microfibril formation and is present in the extracellular matrix of cultured cells. (The C5 domain of The collagen type VI alpha3(VI) a chain is crystalline for extracellular fibrous formation and is present in The extracellular matrix of cultured cells.) The Journal of biochemistry 281, 16607-

Collagen VI was looked at 30.Cescon, m., Gattazzo, f., Chen, p., and Bonaldo, p. (2015). (Collagen VI at a great.) 128, 3525-3531 in Journal of Cell Science (Journal of Cell Science)

Hileman, R.E., Fromm, J.R., Weiler, J.M., and Linhardt, R.J, (1998) chitosan-protein interactions: definition of consensus sites in the glucosamine binding protein. (Glycosaminoglycan-protein interactions: definition of consensus sites in glycoaminoglycan binding proteins.) biological analysis: news and Reviews In Molecular, Cellular and Developmental Biology (BioEssays: News and Reviews In Molecular, Cellular and Developmental Biology) 20, 156, 167

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Effect of peptide length on interaction between consensus peptide and DOPC/DOPA bilayer 33.Ringstad, l., Schmidtchen, a. and Malmsten, M. (2006). (Effect of peptide length on The interaction between peptides and DOPC/DOPA bilayers.) Langmuir-Journal of The American chemical society of surface and Colloids 22, 5042-5050

Spissinger, T with Engel, J. (1995) bovine corneal collagen type VI beaded microfibrils depolymerize at acidic pH and depolymerize and polymerize independently of hyaluronic acid. (Type VI collagen from bone corn polymerized pH, and polymerization arm not fluidized by itself.) substrate biology (Matrix Biol.) 14(6): 499-.

Bober, M., Enochsson, C., Collin, M., and(2010) collagen VI is a sub-epithelial adhesion target of human respiratory pathogens. (Collagen VI a sub-clinical additive target for human respiratory tract pathologens.) Journal of Innate Immunity (Journal of immune) 2, 160-166

Abdillahi, S.M., Balvanovic, S.A., Baumgarten, M.and(2012) collagen VI encodes an antimicrobial activity: novel innate host extracellular matrix defense properties. (Collagen VI encodes immunological activity: novel in host properties of the extracellular matrix.) Journal of Innate Immunity 4, 371-

37.Abdillahi,S.M.，Bober,M.，Nordin,S.，Hallgren,O.，Baumgarten,M.，J., Westergren-Thorsson, G., Bjermer, L., Riesbeck, K., Egesten, A. and(2015) collagen VI is up-regulated in chronic obstructive pulmonary disease and serves as both an adhesion target and bactericidal barrier for moraxella catarrhalis. (Collagen VI Is upper regulated in COPD and services Both as an additive Target and a bacterial Barrier for Moraxella catharsis.) A congenital Immunity Journal (Journal of Innate Immunity)

Differential and restricted expression of new collagen VI chains in Gara, s.k., Grumati, p., Squarzoni, s., Sabatelli, p., urcioulo, a., Bonaldo, p., Paulsson, m., and Wagener, r. (2011) mice. (Differential and regulated expression of novel collagen VI chains in mice.) < substrate biology: journal of The International Society For Matrix Biology (Matrix Biology 30), 248-257-

Cutting and oligomerization of glial proteins (Langerhans, B., Hopkins, D., Franzke, C.W., Keene, D.R., Bruckner-Tuderman, L., Greenspan, D.S., and Koch, M. (2007)) gliosis. (clean and engineering of biomedical, a transformed collegen required for node of ventilator Format.) Journal of biochemistry 282, 10647-

40.Oehmcke,S.，M. and Herwald, h. (2009) activation of neutrophil traps by the human contact system. (Activation of the human contact system on neutral immunological membranes.) Journal of Innate Immunity 1, 225-

Baschong, w. and Wrigley, n.g. (1990) small colloidal gold conjugated to Fab fragments or to immunoglobulin G as a high resolution tag for electron microscopy: technical overview. (Small colloidal gold conjugated to Fab fragments or to immunoglobulin G as high-resolution tags for electronic Microscopy.) Journal of Electron Microscopy technology (Journal of Electron Microscopy technology) 14, 313-323

The structure of collagen VI VWA domains in Becker, a.k., Mikolajek, h., Paulsson, m., Wagener, r, and Werner, J.M. (2014) shows N-and C-termini located on opposite sides of the protein. (A Structure of acyl VI VWA domain displays N and C terminal at open sites of the protein.) Structure (Structure) 22, 199-

Lehrer, R.I., Rosenman, M., Harwig, S.S., Jackson, R.and Eisenhauer, P. (1991) ultrasensitive analysis of endogenous antimicrobial polypeptides. (ultrasensive assays for endogenous Immunological polypeptides.) Journal of immunization Methods 137, 167-

Malmsten, m., Davoudi, m., and Schmidtchen, a. (2006) are sterilized by heparin-binding peptides from PRELP and thrombospondin. (Bacterial killingby heparin-binding peptides from PRELP and thrombospondin.) substrate biology: journal of The International Society for Matrix Biology (Matrix Biology 25), 294-

45.Wimley, W.C. (2010) use an interfacial activity model to illustrate the mechanism of action of antimicrobial peptides. (parsing the mechanism of antimicrobial peptide activity with the interactive activity model.) Chemical Biology of the American society of chemistry (ACS Chemical Biology) 5, 905-917

46.Kasetty,G.，Papareddy,P.，Kalle,M.，V.，Walse,B.，Svensson,B.，M., Malmsten, m., and Schmidtchen, a. (2011) the C-terminal sequences of several human serine proteases encode host defense functions. (The C-terminal sequence ofsectional human serum proteins codes host functions in Journal of Innate Immunity 3, 471-482

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Wang, y., agnberth, b., Lothgren, a., Almstedt, a. and Johansson, J. (1998) apolipoprotein a-I binds to and inhibits human antibacterial/cytotoxic peptide LL-37. (Apolipoproteins A-I bins and inhibitors The human antibiotic/cytoxic peptide LL-37.) J.Biochem.273, 33115-

Antimicrobial proteins and peptides in the host defense, Ganz, t. (2001). (Antimicrobial proteins and peptides In host discovery.) Seminars for Respiratory infection (section In Respiratory Infections) 16, 4-10

Gram-positive and gram-negative bacterial toxins in Ramachandran, g. (2014) sepsis: for a brief overview. (Gram-positive and Gram-negative bacterial toxins in mice: brief review.) viral (viral) 5, 213-

Amino acid sequence of the triple-helical domain of human collagen type VI (1988), Chu, m.l., Conway, d., Pan, T, c., Baldwin, c., man, k., Deutzmann, R, and Timpl. (Amino acid sequence of The triple-topic domain of human collagen type VI.) (The Journal of biochemical Chemistry) 263, 18601-18606

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Sequence listing

<110> Colzyx AB

<120> novel polypeptide and medical use thereof

<130> COLBA/P62348PC

<140> EPPCT/2017/051246

<141> 2017-01-20

<150> GB - 1601136.3

<151> 2016-01-21

<160> 46

<170> BiSSAP 1.3.6

<210> 1

<211> 28

<212> PRT

<213> Artificial sequence

<220>

<223> GVR28 peptide

<400> 1

Gly Val Arg Pro Asp Gly Phe Ala His Ile Arg Asp Phe Val Ser Arg

1 5 10 15

Ile Val Arg Arg Leu Asn Ile Gly Pro Ser Lys Val

20 25

<210> 2

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> FYL25 peptide

<400> 2

Phe Tyr Leu Lys Thr Tyr Arg Ser Gln Ala Pro Val Leu Asp Ala Ile

1 5 10 15

Arg Arg Leu Arg Leu Arg Gly Gly Ser

20 25

<210> 3

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> FFL25 peptide

<400> 3

Phe Phe Leu Lys Asp Phe Ser Thr Lys Arg Gln Ile Ile Asp Ala Ile

1 5 10 15

Asn Lys Val Val Tyr Lys Gly Gly Arg

20 25

<210> 4

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> VTT30 peptide

<400> 4

Val Thr Thr Glu Ile Arg Phe Ala Asp Ser Lys Arg Lys Ser Val Leu

1 5 10 15

Leu Asp Lys Ile Lys Asn Leu Gln Val Ala Leu Thr Ser Lys

20 25 30

<210> 5

<211> 33

<212> PRT

<213> Artificial sequence

<220>

<223> SFV33 peptide

<400> 5

Ser Phe Val Ala Arg Asn Thr Phe Lys Arg Val Arg Asn Gly Phe Leu

1 5 10 15

Met Arg Lys Val Ala Val Phe Phe Ser Asn Thr Pro Thr Arg Ala Ser

20 25 30

Pro

<210> 6

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> DVN32 peptide

<400> 6

Asp Val Asn Val Phe Ala Ile Gly Val Glu Asp Ala Asp Glu Gly Ala

1 5 10 15

Leu Lys Glu Ile Ala Ser Glu Pro Leu Asn Met His Met Phe Asn Leu

20 25 30

<210> 7

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> KPE20 peptide

<400> 7

Lys Pro Glu Ile Leu Asn Leu Val Lys Arg Met Lys Ile Lys Thr Gly

1 5 10 15

Lys Ala Leu Asn

20

<210> 8

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> GFA20 peptide

<400> 8

Gly Phe Ala His Ile Arg Asp Phe Val Ser Arg Ile Val Arg Arg Leu

1 5 10 15

Asn Ile Gly Pro

20

<210> 9

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> QAP20 peptide

<400> 9

Gln Ala Pro Val Leu Asp Ala Ile Arg Arg Leu Arg Leu Arg Gly Gly

1 5 10 15

Ser Pro Leu Asn

20

<210> 10

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> KGF20 peptide

<400> 10

Lys Gly Phe Glu Ser Lys Val Asp Ala Ile Leu Asn Arg Ile Ser Gln

1 5 10 15

Met His Arg Val

20

<210> 11

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> RKV20 peptide

<400> 11

Arg Lys Val Ala Val Phe Phe Ser Asn Thr Pro Thr Arg Ala Ser Pro

1 5 10 15

Gln Leu Arg Glu

20

<210> 12

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> VAA20

<400> 12

Val Ala Ala Lys Pro Val Ala Thr Lys Met Ala Val Arg Pro Pro Val

1 5 10 15

Ala Val Lys Pro

20

<210> 13

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> AAK20 peptide

<400> 13

Ala Ala Lys Pro Val Ala Thr Lys Pro Glu Val Pro Arg Pro Gln Ala

1 5 10 15

Ala Lys Pro Ala

20

<210> 14

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> TTK20 peptide

<400> 14

Thr Thr Lys Pro Val Thr Thr Thr Lys Pro Val Thr Thr Thr Thr Lys

1 5 10 15

Pro Val Thr Thr

20

<210> 15

<211> 76

<212> PRT

<213> Artificial sequence

<220>

<223> AAA76 peptide

<400> 15

Ala Ala Ala Lys Pro Ala Pro Ala Lys Pro Val Ala Ala Lys Pro Val

1 5 10 15

Ala Thr Lys Met Ala Thr Val Arg Pro Pro Val Ala Val Lys Pro Ala

20 25 30

Thr Ala Ala Lys Pro Val Ala Ala Lys Pro Ala Ala Val Arg Pro Pro

35 40 45

Ala Ala Ala Ala Ala Lys Pro Val Ala Thr Lys Pro Glu Val Pro Arg

50 55 60

Pro Gln Ala Ala Lys Pro Ala Ala Thr Lys Pro Ala

65 70 75

<210> 16

<211> 36

<212> PRT

<213> Artificial sequence

<220>

<223> TSS36 peptide

<400> 16

Thr Ser Ser Pro Thr Ser Asn Pro Val Thr Thr Thr Lys Pro Val Thr

1 5 10 15

Thr Thr Lys Pro Val Thr Thr Thr Thr Lys Pro Val Thr Thr Thr Thr

20 25 30

Lys Pro Val Thr

35

<210> 17

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> YDR20 peptides

<400> 17

Tyr Asp Arg Leu Ile Lys Glu Ser Arg Arg Gln Lys Thr Arg Val Phe

1 5 10 15

Ala Val Val Ile

20

<210> 18

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> EQN20 peptide

<400> 18

Glu Gln Asn Phe His Lys Ala Arg Arg Phe Val Glu Gln Val Ala Arg

1 5 10 15

Arg Leu Thr Leu

20

<210> 19

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> VVH20 peptide

<400> 19

Val Val His Ala Ile Asn Ala Ile Val Arg Ser Pro Arg Gly Gly Ala

1 5 10 15

Arg Arg His Ala

20

<210> 20

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> LRL20 peptide

<400> 20

Leu Arg Leu Lys Pro Tyr Gly Ala Leu Val Asp Lys Val Lys Ser Phe

1 5 10 15

Thr Lys Arg Phe

20

<210> 21

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> FTK20 peptide

<400> 21

Phe Thr Lys Arg Phe Ile Asp Asn Leu Arg Asp Arg Tyr Tyr Arg Cys

1 5 10 15

Asp Arg Asn Leu

20

<210> 22

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> RDA20 peptide

<400> 22

Arg Asp Ala Leu Lys Ser Ser Val Asp Ala Val Lys Tyr Phe Gly Lys

1 5 10 15

Gly Thr Tyr Thr

20

<210> 23

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> TKR20 peptide

<400> 23

Thr Lys Arg Phe Ala Lys Arg Leu Ala Glu Arg Phe Leu Thr Ala Gly

1 5 10 15

Arg Thr Asp Pro

20

<210> 24

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> alpha 1N (fw) primer

<400> 24

agagctagca tgccctgtgg atctattc 28

<210> 25

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> alpha 1N (rev) primer

<400> 25

gcactcgaga atcatgtcca caatggtgt 29

<210> 26

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> alpha 1C (fw) primer

<400> 26

gcagctagct gcacatgtgg acccattga 29

<210> 27

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> alpha 1C (rev) primer

<400> 27

aacctcgagg cccagtgcca ccttcct 27

<210> 28

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> alpha 2N (fw) primer

<400> 28

agagctagca aggccgactg cccagtc 27

<210> 29

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> alpha 2N (rev) primer

<400> 29

gcactcgagg accttgatga tgcggtt 27

<210> 30

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> alpha 2C (fw) primer

<400> 30

gaagctagct gtgagaagcg ctgtggt 27

<210> 31

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> alpha 2C (rev) primer

<400> 31

gcaggatcca cagatccagc ggatg 25

<210> 32

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> alpha 3N (fw) primer

<400> 32

tatctcgagc tgatggatct gctgtgaggt ta 32

<210> 33

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> alpha 3N (rev) primer

<400> 33

aggaaccagg gatcccaggg gcctgtcata catgaagcc 39

<210> 34

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> alpha 3C (fw) primer

<400> 34

aaagctagcc tggagtgccc tgtattccca ac 32

<210> 35

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> alpha 3C (rev) primer

<400> 35

tttggatcct caaactgtta actcaggact ac 32

<210> 36

<211> 37

<212> PRT

<213> Artificial sequence

<220>

<223> LL-37 peptide

<400> 36

Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu

1 5 10 15

Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val

20 25 30

Pro Arg Thr Glu Ser

35

<210> 37

<211> 185

<212> PRT

<213> Intelligent people

<220>

<223> type VI human collagen alpha 3-chain VWA Domain alpha 3_ N10

<400> 37

Ala Asp Ile Ile Phe Leu Val Asp Ser Ser Trp Thr Ile Gly Glu Glu

1 5 10 15

His Phe Gln Leu Val Arg Glu Phe Leu Tyr Asp Val Val Lys Ser Leu

20 25 30

Ala Val Gly Glu Asn Asp Phe His Phe Ala Leu Val Gln Phe Asn Gly

35 40 45

Asn Pro His Thr Glu Phe Leu Leu Asn Thr Tyr Arg Thr Lys Gln Glu

50 55 60

Val Leu Ser His Ile Ser Asn Met Ser Tyr Ile Gly Gly Thr Asn Gln

65 70 75 80

Thr Gly Lys Gly Leu Glu Tyr Ile Met Gln Ser His Leu Thr Lys Ala

85 90 95

Ala Gly Ser Arg Ala Gly Asp Gly Val Pro Gln Val Ile Val Val Leu

100 105 110

Thr Asp Gly His Ser Lys Asp Gly Leu Ala Leu Pro Ser Ala Glu Leu

115 120 125

Lys Ser Ala Asp Val Asn Val Phe Ala Ile Gly Val Glu Asp Ala Asp

130 135 140

Glu Gly Ala Leu Lys Glu Ile Ala Ser Glu Pro Leu Asn Met His Met

145 150 155 160

Phe Asn Leu Glu Asn Phe Thr Ser Leu His Asp Ile Val Gly Asn Leu

165 170 175

Val Ser Cys Val His Ser Ser Val Ser

180 185

<210> 38

<211> 188

<212> PRT

<213> Intelligent people

<220>

<223> type VI human collagen alpha 3-chain VWA Domain alpha 3_ N9

<400> 38

Thr Ala Gln Asp Ser Ala Asp Ile Ile Phe Leu Ile Asp Gly Ser Asn

1 5 10 15

Asn Thr Gly Ser Val Asn Phe Ala Val Ile Leu Asp Phe Leu Val Asn

20 25 30

Leu Leu Glu Lys Leu Pro Ile Gly Thr Gln Gln Ile Arg Val Gly Val

35 40 45

Val Gln Phe Ser Asp Glu Pro Arg Thr Met Phe Ser Leu Asp Thr Tyr

50 55 60

Ser Thr Lys Ala Gln Val Leu Gly Ala Val Lys Ala Leu Gly Phe Ala

65 70 75 80

Gly Gly Glu Leu Ala Asn Ile Gly Leu Ala Leu Asp Phe Val Val Glu

85 90 95

Asn His Phe Thr Arg Ala Gly Gly Ser Arg Val Glu Glu Gly Val Pro

100 105 110

Gln Val Leu Val Leu Ile Ser Ala Gly Pro Ser Ser Asp Glu Ile Arg

115 120 125

Tyr Gly Val Val Ala Leu Lys Gln Ala Ser Val Phe Ser Phe Gly Leu

130 135 140

Gly Ala Gln Ala Ala Ser Arg Ala Glu Leu Gln His Ile Ala Thr Asp

145 150 155 160

Asp Asn Leu Val Phe Thr Val Pro Glu Phe Arg Ser Phe Gly Asp Leu

165 170 175

Gln Glu Lys Leu Leu Pro Tyr Ile Val Gly Val Ala

180 185

<210> 39

<211> 180

<212> PRT

<213> Intelligent people

<220>

<223> type VI human collagen alpha 3-chain VWA Domain alpha 3_ N8

<400> 39

Asp Ile Val Phe Leu Val Asp Gly Ser Ser Ala Leu Gly Leu Ala Asn

1 5 10 15

Phe Asn Ala Ile Arg Asp Phe Ile Ala Lys Val Ile Gln Arg Leu Glu

20 25 30

Ile Gly Gln Asp Leu Ile Gln Val Ala Val Ala Gln Tyr Ala Asp Thr

35 40 45

Val Arg Pro Glu Phe Tyr Phe Asn Thr His Pro Thr Lys Arg Glu Val

50 55 60

Ile Thr Ala Val Arg Lys Met Lys Pro Leu Asp Gly Ser Ala Leu Tyr

65 70 75 80

Thr Gly Ser Ala Leu Asp Phe Val Arg Asn Asn Leu Phe Thr Ser Ser

85 90 95

Ala Gly Tyr Arg Ala Ala Glu Gly Ile Pro Lys Leu Leu Val Leu Ile

100 105 110

Thr Gly Gly Lys Ser Leu Asp Glu Ile Ser Gln Pro Ala Gln Glu Leu

115 120 125

Lys Arg Ser Ser Ile Met Ala Phe Ala Ile Gly Asn Lys Gly Ala Asp

130 135 140

Gln Ala Glu Leu Glu Glu Ile Ala Phe Asp Ser Ser Leu Val Phe Ile

145 150 155 160

Pro Ala Glu Phe Arg Ala Ala Pro Leu Gln Gly Met Leu Pro Gly Leu

165 170 175

Leu Ala Pro Leu

180

<210> 40

<211> 182

<212> PRT

<213> Intelligent people

<220>

<223> type VI human collagen alpha 3-chain VWA Domain alpha 3_ N7

<400> 40

Asp Ile Ile Phe Leu Leu Asp Gly Ser Ala Asn Val Gly Lys Thr Asn

1 5 10 15

Phe Pro Tyr Val Arg Asp Phe Val Met Asn Leu Val Asn Ser Leu Asp

20 25 30

Ile Gly Asn Asp Asn Ile Arg Val Gly Leu Val Gln Phe Ser Asp Thr

35 40 45

Pro Val Thr Glu Phe Ser Leu Asn Thr Tyr Gln Thr Lys Ser Asp Ile

50 55 60

Leu Gly His Leu Arg Gln Leu Gln Leu Gln Gly Gly Ser Gly Leu Asn

65 70 75 80

Thr Gly Ser Ala Leu Ser Tyr Val Tyr Ala Asn His Phe Thr Glu Ala

85 90 95

Gly Gly Ser Arg Ile Arg Glu His Val Pro Gln Leu Leu Leu Leu Leu

100 105 110

Thr Ala Gly Gln Ser Glu Asp Ser Tyr Leu Gln Ala Ala Asn Ala Leu

115 120 125

Thr Arg Ala Gly Ile Leu Thr Phe Cys Val Gly Ala Ser Gln Ala Asn

130 135 140

Lys Ala Glu Leu Glu Gln Ile Ala Phe Asn Pro Ser Leu Val Tyr Leu

145 150 155 160

Met Asp Asp Phe Ser Ser Leu Pro Ala Leu Pro Gln Gln Leu Ile Gln

165 170 175

Pro Leu Thr Thr Tyr Val

180

<210> 41

<211> 179

<212> PRT

<213> Intelligent people

<220>

<223> type VI human collagen alpha 3-chain VWA Domain alpha 3_ N6

<400> 41

Ser Lys Arg Asp Ile Leu Phe Leu Phe Asp Gly Ser Ala Asn Leu Val

1 5 10 15

Gly Gln Phe Pro Val Val Arg Asp Phe Leu Tyr Lys Ile Ile Asp Glu

20 25 30

Leu Asn Val Lys Pro Glu Gly Thr Arg Ile Ala Val Ala Gln Tyr Ser

35 40 45

Asp Asp Val Lys Val Glu Ser Arg Phe Asp Glu His Gln Ser Lys Pro

50 55 60

Glu Ile Leu Asn Leu Val Lys Arg Met Lys Ile Lys Thr Gly Lys Ala

65 70 75 80

Leu Asn Leu Gly Tyr Ala Leu Asp Tyr Ala Gln Arg Tyr Ile Phe Val

85 90 95

Lys Ser Ala Gly Ser Arg Ile Glu Asp Gly Val Leu Gln Phe Leu Val

100 105 110

Leu Leu Val Ala Gly Arg Ser Ser Asp Arg Val Asp Gly Pro Ala Ser

115 120 125

Asn Leu Lys Gln Ser Gly Val Val Pro Phe Ile Phe Gln Ala Lys Asn

130 135 140

Ala Asp Pro Ala Glu Leu Glu Gln Ile Val Leu Ser Pro Ala Phe Ile

145 150 155 160

Leu Ala Ala Glu Ser Leu Pro Lys Ile Gly Asp Leu His Pro Gln Ile

165 170 175

Val Asn Leu

<210> 42

<211> 182

<212> PRT

<213> Intelligent people

<220>

<223> type VI human collagen alpha 3-chain VWA Domain alpha 3_ N5

<400> 42

Glu Lys Asp Val Val Phe Leu Leu Asp Gly Ser Glu Gly Val Arg Ser

1 5 10 15

Gly Phe Pro Leu Leu Lys Glu Phe Val Gln Arg Val Val Glu Ser Leu

20 25 30

Asp Val Gly Gln Asp Arg Val Arg Val Ala Val Val Gln Tyr Ser Asp

35 40 45

Arg Thr Arg Pro Glu Phe Tyr Leu Asn Ser Tyr Met Asn Lys Gln Asp

50 55 60

Val Val Asn Ala Val Arg Gln Leu Thr Leu Leu Gly Gly Pro Thr Pro

65 70 75 80

Asn Thr Gly Ala Ala Leu Glu Phe Val Leu Arg Asn Ile Leu Val Ser

85 90 95

Ser Ala Gly Ser Arg Ile Thr Glu Gly Val Pro Gln Leu Leu Ile Val

100 105 110

Leu Thr Ala Asp Arg Ser Gly Asp Asp Val Arg Asn Pro Ser Val Val

115 120 125

Val Lys Arg Gly Gly Ala Val Pro Ile Gly Ile Gly Ile Gly Asn Ala

130 135 140

Asp Ile Thr Glu Met Gln Thr Ile Ser Phe Ile Pro Asp Phe Ala Val

145 150 155 160

Ala Ile Pro Thr Phe Arg Gln Leu Gly Thr Val Gln Gln Val Ile Ser

165 170 175

Glu Arg Val Thr Gln Leu

180

<210> 43

<211> 178

<212> PRT

<213> Intelligent people

<220>

<223> type VI human collagen alpha 3-chain VWA Domain alpha 3_ N4

<400> 43

Lys Arg Asp Val Val Phe Leu Ile Asp Gly Ser Gln Ser Ala Gly Pro

1 5 10 15

Glu Phe Gln Tyr Val Arg Thr Leu Ile Glu Arg Leu Val Asp Tyr Leu

20 25 30

Asp Val Gly Phe Asp Thr Thr Arg Val Ala Val Ile Gln Phe Ser Asp

35 40 45

Asp Pro Lys Ala Glu Phe Leu Leu Asn Ala His Ser Ser Lys Asp Glu

50 55 60

Val Gln Asn Ala Val Gln Arg Leu Arg Pro Lys Gly Gly Arg Gln Ile

65 70 75 80

Asn Val Gly Asn Ala Leu Glu Tyr Val Ser Arg Asn Ile Phe Lys Arg

85 90 95

Pro Leu Gly Ser Arg Ile Glu Glu Gly Val Pro Gln Phe Leu Val Leu

100 105 110

Ile Ser Ser Gly Lys Ser Asp Asp Glu Val Val Val Pro Ala Val Glu

115 120 125

Leu Lys Gln Phe Gly Val Ala Pro Phe Thr Ile Ala Arg Asn Ala Asp

130 135 140

Gln Glu Glu Leu Val Lys Ile Ser Leu Ser Pro Glu Tyr Val Phe Ser

145 150 155 160

Val Ser Thr Phe Arg Glu Leu Pro Ser Leu Glu Gln Lys Leu Leu Thr

165 170 175

Pro Ile

<210> 44

<211> 186

<212> PRT

<213> Intelligent people

<220>

<223> type VI human collagen alpha 3-chain VWA Domain alpha 3_ N3

<400> 44

Pro Pro Pro Ala Val Glu Ser Asp Ala Ala Asp Ile Val Phe Leu Ile

1 5 10 15

Asp Ser Ser Glu Gly Val Arg Pro Asp Gly Phe Ala His Ile Arg Asp

20 25 30

Phe Val Ser Arg Ile Val Arg Arg Leu Asn Ile Gly Pro Ser Lys Val

35 40 45

Arg Val Gly Val Val Gln Phe Ser Asn Asp Val Phe Pro Glu Phe Tyr

50 55 60

Leu Lys Thr Tyr Arg Ser Gln Ala Pro Val Leu Asp Ala Ile Arg Arg

65 70 75 80

Leu Arg Leu Arg Gly Gly Ser Pro Leu Asn Thr Gly Lys Ala Leu Glu

85 90 95

Phe Val Ala Arg Asn Leu Phe Val Lys Ser Ala Gly Ser Arg Ile Glu

100 105 110

Asp Gly Val Pro Gln His Leu Val Leu Val Leu Gly Gly Lys Ser Gln

115 120 125

Asp Asp Val Ser Arg Phe Ala Gln Val Ile Arg Ser Ser Gly Ile Val

130 135 140

Ser Leu Gly Val Gly Asp Arg Asn Ile Asp Arg Thr Glu Leu Gln Thr

145 150 155 160

Ile Thr Asn Asp Pro Arg Leu Val Phe Thr Val Arg Glu Phe Arg Glu

165 170 175

Leu Pro Asn Ile Glu Glu Arg Ile Met Asn

180 185

<210> 45

<211> 181

<212> PRT

<213> Intelligent people

<220>

<223> type VI human collagen alpha 3-chain VWA Domain alpha 3_ N2

<400> 45

Lys Lys Ala Asp Ile Val Phe Leu Leu Asp Gly Ser Ile Asn Phe Arg

1 5 10 15

Arg Asp Ser Phe Gln Glu Val Leu Arg Phe Val Ser Glu Ile Val Asp

20 25 30

Thr Val Tyr Glu Asp Gly Asp Ser Ile Gln Val Gly Leu Val Gln Tyr

35 40 45

Asn Ser Asp Pro Thr Asp Glu Phe Phe Leu Lys Asp Phe Ser Thr Lys

50 55 60

Arg Gln Ile Ile Asp Ala Ile Asn Lys Val Val Tyr Lys Gly Gly Arg

65 70 75 80

His Ala Asn Thr Lys Val Gly Leu Glu His Leu Arg Val Asn His Phe

85 90 95

Val Pro Glu Ala Gly Ser Arg Leu Asp Gln Arg Val Pro Gln Ile Ala

100 105 110

Phe Val Ile Thr Gly Gly Lys Ser Val Glu Asp Ala Gln Asp Val Ser

115 120 125

Leu Ala Leu Thr Gln Arg Gly Val Lys Val Phe Ala Val Gly Val Arg

130 135 140

Asn Ile Asp Ser Glu Glu Val Gly Lys Ile Ala Ser Asn Ser Ala Thr

145 150 155 160

Ala Phe Arg Val Gly Asn Val Gln Glu Leu Ser Glu Leu Ser Glu Gln

165 170 175

Val Leu Glu Thr Leu

180

<210> 46

<211> 193

<212> PRT

<213> Intelligent people

<220>

<223> type VI human collagen alpha 3-chain VWA Domain alpha 3_ C1

<400> 46

Cys Pro Val Phe Pro Thr Glu Leu Ala Phe Ala Leu Asp Thr Ser Glu

1 5 10 15

Gly Val Asn Gln Asp Thr Phe Gly Arg Met Arg Asp Val Val Leu Ser

20 25 30

Ile Val Asn Asp Leu Thr Ile Ala Glu Ser Asn Cys Pro Arg Gly Ala

35 40 45

Arg Val Ala Val Val Thr Tyr Asn Asn Glu Val Thr Thr Glu Ile Arg

50 55 60

Phe Ala Asp Ser Lys Arg Lys Ser Val Leu Leu Asp Lys Ile Lys Asn

65 70 75 80

Leu Gln Val Ala Leu Thr Ser Lys Gln Gln Ser Leu Glu Thr Ala Met

85 90 95

Ser Phe Val Ala Arg Asn Thr Phe Lys Arg Val Arg Asn Gly Phe Leu

100 105 110

Met Arg Lys Val Ala Val Phe Phe Ser Asn Thr Pro Thr Arg Ala Ser

115 120 125

Pro Gln Leu Arg Glu Ala Val Leu Lys Leu Ser Asp Ala Gly Ile Thr

130 135 140

Pro Leu Phe Leu Thr Arg Gln Glu Asp Arg Gln Leu Ile Asn Ala Leu

145 150 155 160

Gln Ile Asn Asn Thr Ala Val Gly His Ala Leu Val Leu Pro Ala Gly

165 170 175

Arg Asp Leu Thr Asp Phe Leu Glu Asn Val Leu Thr Cys His Val Cys

180 185 190

Leu