阅读说明：本技术 一种工程化组装蛋白聚糖及其制备方法和应用 (Engineered assembly proteoglycan and preparation method and application thereof ) 是由 李福川 王文爽 韩乃寒 许莹莹 石立冉 于 2019-11-21 设计创作，主要内容包括：本发明属于蛋白质工程技术领域,涉及工程化组装蛋白聚糖及其制备方法和应用。一种工程化组装蛋白聚糖,其特征在于：包括带有醛基蛋白聚糖核心蛋白,及标记有修饰基团的糖胺聚糖侧链。一种工程化组装蛋白聚糖的制备方法,包括：制备带有醛基的蛋白聚糖核心蛋白；在糖胺聚糖侧链上标记修饰基团；所述修饰基团为肼基、氨氧基、羟胺基；将带有修饰基团的糖胺聚糖侧链通过修饰基团与醛基之间的化学反应组装到蛋白聚糖核心蛋白上。本发明的工程化组装蛋白聚糖及其制备方法,首次解决了具有特定结构GAG侧链的PGs的工程化构建问题,可以在体外和体内实现具有任意组成和结构GAGs糖链与特定PGs核心蛋白的组装,为具有特定GAG侧链的PGs构效关系研究和规模化制备奠定了基础,在相关蛋白聚糖类生物制剂和医药产品开发中具有重要应用价值。(The invention belongs to the technical field of protein engineering, and relates to engineered assembled proteoglycan, a preparation method and application thereof. An engineered assembled proteoglycan, comprising: comprises an aldehyde proteoglycan core protein and a glycosaminoglycan side chain marked with a modifying group. A method of preparing an engineered assembled proteoglycan, comprising: preparing proteoglycan core protein with aldehyde group; labeling a modification group on a glycosaminoglycan side chain; the modifying group is a hydrazine group, an aminoxy group or a hydroxylamine group; and (3) assembling the glycosaminoglycan side chain with the modification group on the proteoglycan core protein through a chemical reaction between the modification group and the aldehyde group. The engineering assembly proteoglycan and the preparation method thereof solve the engineering construction problem of PGs with GAG side chains with specific structures for the first time, can realize the assembly of GAGs sugar chains with any composition and structure and specific PGs core protein in vitro and in vivo, lay a foundation for the research and large-scale preparation of the structure-activity relationship of the PGs with specific GAG side chains, and have important application value in the development of related proteoglycan biological preparations and medical products.)

1. An engineered assembled proteoglycan, comprising: comprises proteoglycan core protein with aldehyde group and glycosaminoglycan side chain marked with modification group.

2. The engineered assembled proteoglycan of claim 1, wherein: the modifying group is a hydrazine group, an aminoxy group or a hydroxylamine group.

3. A method of preparing an engineered assembled proteoglycan, comprising:

preparing proteoglycan core protein with aldehyde group;

labeling a modification group on a glycosaminoglycan side chain; the modifying group is a hydrazine group, an aminoxy group or a hydroxylamine group;

and (3) assembling the glycosaminoglycan side chain with the modification group on the proteoglycan core protein through the chemical reaction of the modification group and an aldehyde group.

4. The method of claim 3, wherein the engineered assembled proteoglycan is prepared by: the preparation method of the proteoglycan core protein with aldehyde groups comprises the following steps:

mutating glycosylation sites of proteoglycan core protein into amino acid sequences capable of being converted into aldehyde groups;

the amino acid sequence is converted to an aldehyde group by means of an fGly-forming enzyme.

5. The method of claim 4, wherein the engineered assembly proteoglycan is selected from the group consisting of: the amino acid sequence which can be converted into aldehyde group is CXPXR, wherein X is any amino acid except proline.

6. The method of claim 5, wherein the chemical reaction is a reaction between an aldehyde group and a hydrazine, aminooxy or hydroxylamine group.

7. Use of the engineered assembled proteoglycan of claim 1 or 2 for preparing a cancer diagnostic kit.

8. Use of an engineered assembled proteoglycan of claim 1 or 2 in the preparation of a medicament.

9. Use of the method of any one of claims 3-6 for the preparation of an engineered assembled proteoglycan in the preparation of a cancer diagnostic kit.

10. Use of the method of any one of claims 3-6 for the preparation of an engineered assembled proteoglycan for the preparation of a medicament.

Technical Field

The invention belongs to the technical field of protein engineering, and relates to engineered assembled proteoglycan, a preparation method and application thereof.

Background

Proteoglycans (PGs) are a class of very complex biological macromolecules formed by one or more Glycosaminoglycan (GAG) side chains covalently attached to a core protein, often present on the surface of cell membranes and in the Extracellular matrix (ECM). PGs are involved in a range of physiological and pathological processes such as cell adhesion and migration (1, 2), cell signaling (1, 3-8), cell division (9), tissue morphogenesis (10-12), inflammation (13, 14) and cancer (15). Depending on the location of PGs in a cell, they can be classified into four categories, intracellular, cell surface, cell periphery and extracellular. The most prominent intracellular proteoglycan, serglcin, is frequently found in mast cell granules, serves as a bioadhesive to help mast cell granules store most intracellular proteases, and also binds and regulates the biological activities of a variety of inflammation-related chemokines, cytokines and growth factors (16). Cell surface PGs are mainly classified into transmembrane PGs and PGs anchored to the cell membrane surface, with syndecanon, CSPG4, bataglycan and phosphonan being transmembrane PGs, and glypican being anchored to the cell membrane surface via GPI. Syndecan has diverse biological functions including histomorphogenesis, involvement in exosome uptake (17), mediation of atherosclerotic lipoprotein clearance (18), soluble Syndecan-1 promotes myeloma growth in vivo (19), etc. CSPG4 promotes tumor angiogenesis (20), and participates in the onset of severe pseudomembranous colitis (21). Bataglycan is involved in regulating multiple functions such as reproduction and fetal growth (22), and is a recognized cancer suppressor for various cancers (23). The Glypican is involved in the regeneration of blood vessels and the growth of tumors, wherein the Glypican-3 is also a marker for early diagnosis of liver cancer. Pericytoglycan Perlecan and Agrin are involved in regulating cell adhesion (24, 25), lipid metabolism (26), thrombosis and cell death (27, 28), vascular and cartilage biomechanics (29, 30), skin and endochondral bone formation (31, 32), spur formation (33). Extracellular proteoglycans mainly include the four classes of aggrecan, versican, neurocan and brevican, in which aggrecan is involved in the morphogenesis of synovial joints and articular cartilage (34) and is a biomarker of osteoarthritis (35); versican is often involved in cell adhesion, migration and inflammation (36-38); neurocan inhibit neurite outgrowth, and increase expression level at sites of mechanical injury and ischemic injury of adult central nervous system (39, 40); brevican is associated with the development of gliomas, nerve tissue damage and repair, and alzheimer's disease (41). These biological functions have made PGs play an important role in disease diagnosis and treatment.

Early studies showed that GAG chains play a key role in the biological function of PGs. GAGs are further classified into Hyaluronic Acid (HA), Heparin/Heparan Sulfate (Hep/HS), Chondroitin Sulfate/dermatan Sulfate (CS/DS), and Keratan Sulfate (KS) according to the disaccharide unit of GAG. Wherein HA, CS/DS and Hep/HS are composed of a repetition of disaccharide units formed by hexuronic acid (D-glucuronic acid or L-iduronic acid) and hexosamine (N-acetylgalactosamine or N-acetylglucosamine), and KS is composed of a repetition of disaccharide units formed by neutral N-acetylglucosamine and galactose. HA is the only group of GAGs that do not contain a sulfate group, and although not in the form of PGs, it can bind some PGs in a non-covalent fashion to form larger molecular aggregates. In addition to HA, other GAG sugar chains are often abnormally complex due to the action of various modifying enzymes, mainly expressed as the conversion of D-glucuronic acid to L-iduronic acid by the action of C5-epimerase, and hydroxyl (-OH) and amino (-NH) groups at various positions in the sugar chain₂) Sulfation of (b), acetylation of the hexosamine diamino, and the like. GAG side chain length, variety, quantity and modification degree of different, core protein molecular size and structure difference to give PGs function diversity, different structure has different function. Unlike nucleic acids and proteins, GAGs are synthesized in a template-independent manner, and GAG chains having a specific structure cannot be selectively amplified by genetic engineering means. These reasons all limit the uniformity of the structure and the functionThe preparation of the definite proteoglycan and the research of the structure-activity relationship.

Mutation of glycosyl binding sites on PGs core proteins by molecular biology is a traditional approach to study the function of PGs side chains, and this approach only elucidates the effects of the number of GAG side chains on the function of PGs, and does not explain the effects of different types or lengths of GAG chains on the action of PGs (42). Recently, research reports have been reported that GAG synthesis-related enzymes in cells are knocked out or over-expressed by means of gene editing, so as to regulate the types and modification modes of GAG synthesized by cells, but the method cannot accurately control the specific structure of GAG sugar chain synthesis, and cannot selectively and accurately regulate the composition and structure of GAG side chain of specific proteoglycan, so that the technology cannot solve the problem of influence of GAG side chain with specific structure on the biological function of specific PGs (43), and the problems of engineering preparation and application of PGs with GAG side chain with specific structure are not solved.

Formylglycine (fGly), which is a key amino acid in the active site of sulfatase, is converted from the corresponding cysteine (Cys) residue by fGly-forming enzyme (FGE) catalyzed during or after the translation of sulfatase (44, 45). It was found that GFE specifically recognizes a short consensus conserved sequence CXPXR of sulfatase (where X can be any amino acid other than proline) and oxidizes Cys residues in the sequence to an aldehyde-containing fGly, that the sequence can be recombined into a protein of interest, by co-transfection with FGE or in vitro catalysis, introducing an aldehyde-containing fGly at a specific position in the protein of interest, that highly active and rare aldehyde groups in the protein provide ideal reaction sites for point-specific modification of the protein of interest (46). Aldehyde-labeled proteins can be modified at room temperature with aminooxy, hydrazine or hydroxylamine containing molecules under mildly acidic (pH4-6) or even physiological conditions.

The aldehyde labeling technology is applied to the point-specific modification of protein, and comprises protein fluorescent labeling, biotinylation, antibody-drug conjugate preparation, protein glycosylation and the like. In these applications, the structure of the protein-modified molecule is relatively simple, and compared with the structure, the point-specific modification of aldehyde-modified protein by glycosaminoglycan molecules with high negative charge and extremely complex structure has not been reported.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a novel engineered assembly proteoglycan, and a preparation method and application thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows: engineered assembled proteoglycans include a proteoglycan core protein with aldehyde groups and glycosaminoglycan side chains labeled with a modifying group.

Further, the modifying group is a hydrazine group, an aminoxy group or a hydroxylamine group.

The invention also provides a preparation method of the engineering assembly proteoglycan for solving the technical problem, which comprises the following steps:

preparing proteoglycan core protein with aldehyde group;

labeling a modification group on a glycosaminoglycan side chain; the modifying group is a hydrazine group, an aminoxy group or a hydroxylamine group;

and (3) assembling the glycosaminoglycan side chain with the modification group on the proteoglycan core protein through a chemical reaction between the modification group and the aldehyde group.

Further, the preparation method of the proteoglycan core protein with aldehyde groups comprises the following steps:

mutating glycosylation sites of proteoglycan core protein into amino acid sequences capable of being converted into aldehyde groups;

the amino acid sequence is converted to an aldehyde group by means of an fGly-forming enzyme.

In a preferred embodiment of the present invention, the amino acid sequence convertible to an aldehyde group is CXPXR, wherein X is any amino acid except proline.

Further, the chemical reaction refers to the reaction of aldehyde group with hydrazine group, aminoxy group or hydroxylamine group.

The invention also provides application of the engineered assembled proteoglycan in preparing a cancer diagnosis kit.

The invention also provides application of the engineered assembled proteoglycan in preparing medicines.

The invention also provides application of the preparation method of the engineered assembled proteoglycan in preparing a cancer diagnosis kit.

The invention also provides application of the preparation method of the engineered assembled proteoglycan in preparing medicines.

Compared with the prior art, the engineered assembled proteoglycan, the preparation method and the application thereof have the following beneficial effects:

the method solves the engineering construction problem of the PGs with the GAG side chain with the specific structure for the first time, can realize the assembly of the GAGs sugar chain with any composition and structure and the core protein of the specific PGs in vitro and in vivo, lays a foundation for the structure-activity relationship research and the large-scale preparation of the PGs with the specific GAG side chain, and has important application value in the development of related protein polysaccharide biological preparations and medical products.

Drawings

FIG. 1: GPC3 successfully carried an aldehyde group;

FIG. 2: GAG oligosaccharide reducing end is labeled with hydrazine group;

wherein A, HA disaccharide; b, Hep disaccharide; c, mass spectrum after HA disaccharide labeling; d, mass spectrum of HA disaccharide labeled precursor; e, mass spectrum after Hep disaccharide labeling; f, mass spectrum before Hep disaccharide marking;

in FIG. 1, HA labels disaccharide; 2, HA disaccharide; 3, Hep labeling of disaccharides; 4, Hep disaccharide;

FIG. 3: GAG oligosaccharides were successfully attached to the GPC3 core protein;

FIG. 4: engineering installation of GPC3 side chain on the surface of cell membrane;

FIG. 5: the effect of sugar chain length on Wnt3A cell signaling;

FIG. 6: the effect of different GAGs on Wnt3A cell signaling;

FIG. 7: GPC3 mediates endocytosis of Wnt 3A;

FIG. 8: GPC3 was analyzed for interactions with Frizzled-7.

Detailed Description

The following examples are set forth so as to provide a thorough disclosure of some of the commonly used techniques of how the present invention may be practiced, and are not intended to limit the scope of the invention. The inventors have made the best effort to ensure accuracy with respect to various parameters (e.g., amounts, temperature, etc.) in the examples, but some experimental errors and deviations should be accounted for. Unless otherwise indicated, molecular weight in the present invention refers to weight average molecular weight and temperature is in degrees Celsius.