CN110845568B - Engineered assembled proteoglycan and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of protein engineering, and relates to engineering assembled proteoglycan and a preparation method and application thereof. An engineered assembled proteoglycan, characterized by: comprises a glycosaminoglycan side chain marked with a modification group and an aldehyde proteoglycan core protein. A method of preparing an engineered assembled proteoglycan comprising: preparing a proteoglycan core protein with aldehyde groups; labeling a modifying group on a glycosaminoglycan side chain; the modification group is hydrazino, aminoxy or hydroxylamine; the glycosaminoglycan side chains with the modification groups are assembled onto the proteoglycan core protein by chemical reaction between the modification groups and aldehyde groups. The engineering assembled proteoglycan and the preparation method thereof solve the engineering construction problem of PGs with GAG side chains of specific structures for the first time, can realize the assembly of GAGs sugar chains with arbitrary compositions and structures and specific PGs core proteins in vitro and in vivo, lay a foundation for the research of PGs structure-activity relationship with specific GAG side chains and the large-scale preparation, and have important application values in the development of related proteoglycan biological preparations and medical products.

Description

Engineered assembled proteoglycan and preparation method and application thereof

Technical Field

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

Background

Proteoglycans (PGs) are a very complex class of biological macromolecules formed by covalent attachment of one or more Glycosaminoglycan (GAG) side chains to a core protein, often found on the cell membrane surface and in the extracellular matrix (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). PGs can be classified into four types, i.e., intracellular, cell surface, pericellular and extracellular, depending on the location of PGs present in the cell. Intracellular most predominant proteoglycans, sergilysins, are often found in mast cell granules, and act as bioadhesives helping mast cell granules to store most of the intracellular proteases, and also bind and modulate the biological activity of a variety of inflammation-related chemokines, cytokines and growth factors (16). PGs on the cell surface are mainly classified into transmembrane PGs and PGs anchored on the cell membrane surface, wherein syndecan, CSPG, bata glycan and phosphacan are transmembrane PGs, and glypican is anchored on the cell membrane surface through GPI. Syndecan has a wide variety of biological functions including tissue morphogenesis, participation in exosomes uptake (17), mediation of clearance of atherosclerosis lipoproteins (18), soluble Syndecan-1 can promote growth of myeloma in vivo (19), and the like. CSPG4 promotes the generation of tumor blood vessels (20), participates in the onset of severe pseudomembranous colitis (21), and the like. Bata glycan is involved in mediating various functions such as reproduction and fetal growth (22), and is a recognized cancer suppressor for many cancers (23). Glypican is involved in angiogenesis and tumor growth, with Glypican-3 also being a marker for early diagnosis of liver cancer. Pericellular proteoglycans 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), bone spur formation (33). Extracellular proteoglycans mainly comprise four classes, aggrecan, versican, neurocan and brevcan, in which aggrecan is involved in the morphogenesis of synovial joints and articular cartilage (34), and are biomarkers of osteoarthritis (35); versican is often involved in cell adhesion, migration and inflammation (36-38); neurocan inhibits neurite outgrowth and increases expression in sites of mechanical injury and ischemic injury in the adult central nervous system (39, 40); brevcan is associated with glioma development, nerve tissue damage and repair, and alzheimer's disease (41). These biological functions described above make PGs important in disease diagnosis and treatment.

Earlier studies have shown 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/Heparan Sulfate, hep/HS), chondroitin Sulfate/Dermatan Sulfate (Chondroitin Sulfate/Dermatan Sulfate, CS/DS), keratan Sulfate (Keratan Sulfate, KS) according to the difference in GAG disaccharide units. Wherein the HA, CS/DS and Hep/HS are all composed ofThe disaccharide unit repeat formed by uronic acid (D-glucuronic acid or L-iduronic acid) and hexosamine (N-acetamido galactose or N-acetamido glucose), while KS is composed of the disaccharide unit repeat formed by neutral N-acetamido glucose and galactose. HA is the only class of GAGs that do not contain a sulfate group, and although not in the form of PGs, it can associate with some PGs in a non-covalent form to form larger polymers. In addition to HA, other GAG sugar chains are often complicated by the action of various modifying enzymes, mainly represented by the conversion of D-glucuronic acid to L-iduronic acid by the action of C5-epimerase, hydroxyl (-OH) and amino (-NH) groups at different positions in the sugar chain ₂ ) And acetylation of the amino group at the hexosamine position. The difference of the length, the type, the number and the modification degree of the GAG side chains, the size and the structure of the core protein molecules endows PGs with various functions, and the different structures have different functions. Unlike nucleic acids and proteins, GAGs are synthesized in a template-independent manner and GAG chains with specific structures cannot be selectively amplified by genetic engineering means. These reasons have limited the study of the structure-homogeneous, functionally defined proteoglycan preparation and structure-activity relationship.

Mutation of glycosyl binding sites on PGs core proteins by molecular biology means is a traditional method of studying PGs side chain function, which only elucidates the effect of the number of GAG side chains on PGs function and cannot explain the effect of different species or lengths of GAG chains on PGs action (42). Recently, studies have reported that the enzyme related to GAG synthesis in cells is knocked out or overexpressed by gene editing means, so that the type and modification mode of GAG synthesized by cells are regulated and controlled, but the method cannot accurately control the specific structure of the synthesized GAG sugar chain, and further cannot accurately regulate the composition and structure of GAG side chains of specific proteoglycans selectively, so that the technology cannot solve the problem of influence of GAG side chains of specific structures on the biological functions of specific PGs (43), and further cannot solve the engineering preparation and application problems of GAG side chains with specific structures.

Formylglycine (fGly) is a key amino acid in the sulfatase active site, and is converted from the corresponding cysteine (Cys) residue during or after sulfatase translation by the enzyme fGly-producing enzyme (fGly-generating enzyme, FGE) (44, 45). It was found that GFE can specifically recognize a short consensus conserved sequence CXPXR of sulfatase (where X can be any amino acid other than proline) and oxidize Cys residues in the sequence to aldehyde-containing fgys, which can be recombined into the protein of interest, introducing the aldehyde-bearing fgys at specific positions of the protein of interest by co-transfection or in vitro catalysis with FGE, highly active and rare aldehyde groups in the protein provide ideal reaction sites for point-specific modification of the protein of interest (46). Under weak acidic (pH 4-6) and even physiological conditions, the aldehyde-based tag proteins may be modified at room temperature with molecules containing aminooxy, hydrazino or hydroxylamine groups.

The aldehyde labeling techniques described above have been applied to site-specific modification of proteins, including fluorescent labeling and biotinylation of proteins, antibody-drug conjugate preparation, glycosylation of proteins, and the like. In these applications the structure of the protein-modifying molecule is relatively simple, as compared to highly negatively charged and structurally very complex glycosaminoglycan molecules have not been reported for the point-specific modification of aldehyde-modified proteins.

Disclosure of Invention

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

The technical scheme adopted for solving the technical problems is as follows: an engineered assembled proteoglycan comprising a proteoglycan core protein with an aldehyde group, and a glycosaminoglycan side chain labeled with a modifying group.

Further, the modification group is hydrazino, aminoxy or hydroxylamine.

The invention solves the technical problem and also provides a preparation method of the engineering assembled proteoglycan, which comprises the following steps:

preparing a proteoglycan core protein with aldehyde groups;

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

the glycosaminoglycan side chains with the modification groups are assembled onto the proteoglycan core protein by chemical reaction between the modification groups and aldehyde groups.

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

mutating the proteoglycan core protein glycosylation site into an amino acid sequence convertible to an aldehyde group;

the amino acid sequence is converted to an aldehyde group using an fGly-producing enzyme to catalyze the amino acid sequence.

As a preferred mode 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 groups with hydrazine groups, aminoxy groups or hydroxylamine groups.

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

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

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

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

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

the engineering construction problem of PGs with GAG side chains of specific structures is solved for the first time, the assembly of GAGs sugar chains with arbitrary compositions and structures and specific PGs core proteins can be realized in vitro and in vivo, a foundation is laid for the structure-activity relationship research and the large-scale preparation of PGs with specific GAG side chains, and the engineering construction method has important application value in the development of related protein polysaccharide biological agents and medical products.

Drawings

Fig. 1: GPC3 successfully carried aldehyde groups;

fig. 2: the reducing end of GAG oligosaccharide is marked with hydrazine group;

wherein A, HA disaccharide; b, hep disaccharide; c, a mass spectrogram after HA disaccharide marking; d, HA disaccharide labeling front mass spectrogram; e, hep disaccharide labeled mass spectrogram; f, hep disaccharide labeling front mass spectrum;

in figure 1, ha marks disaccharides; 2, ha disaccharide; 3, hep-labeled disaccharide; 4, hep disaccharide;

fig. 3: GAG oligosaccharides were successfully linked to GPC3 core protein;

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

fig. 5: effects of sugar chain length on Wnt3A cell signaling;

fig. 6: influence of different GAGs on Wnt3A cell signaling;

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

fig. 8: GPC3 and Frizzled-7 interaction analysis.

Detailed Description

The following examples are set forth in order to fully disclose some general techniques for how the invention may be practiced, and are not intended to limit the scope of what the invention may be used. The inventors have made best efforts to ensure accuracy with respect to numbers used in the examples (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Molecular weight in the present invention refers to weight average molecular weight and temperature is in degrees celsius unless otherwise indicated.

EXAMPLE 1 aldehyde-labeled GPC3 amino acid sequence

The amino acid sequence near the GPC3 glycosyl binding site is mutated to LCTPSR, and FGE can convert Cys to fGly. GPC3 bearing one aldehyde group was designated GPC3-S495C and GPC3-S509C, respectively, and muteins bearing two aldehyde groups were designated GPC3-O, depending on the mutation site. Mutation of the F494 to C499 position of the GPC3 amino acid sequence to LCTPSR was designated GPC3-S495C, and the amino acid sequence thereof is shown in SEQ ID NO. 1. And the mutation from the G508 site to the G513 site of the amino acid sequence to LCTPSR is named GPC3-S509C, and the amino acid sequence is shown in SEQ ID NO. 2. The amino acid sequence of GPC3-O with both the above positions mutated is shown as SEQ ID NO. 3.

SEQ ID NO.1

MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGL KWVPETPVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMEL KFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPSLTPQAFEFVGEFFTDVS LYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLK VFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTRM WYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDKYWREYILSLEELVN GMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRFA YYPEDLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFISFYSALPGYICSHSPV AENDTLCWNGQELVERYSQKAARNGMKNQFNLHELKMKGPEPVVSQIIDKL KHINQLLRTMSMPKGRVLDKNLDEEGLCTPSRGDDEDECIGGSGDGMIKVKN QLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKLLTSMAIS VVCFFFLVH-

SEQ ID NO.2

MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGL KWVPETPVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMEL KFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPSLTPQAFEFVGEFFTDVS LYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLK VFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTRM WYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDKYWREYILSLEELVN GMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRFA YYPEDLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFISFYSALPGYICSHSPV AENDTLCWNGQELVERYSQKAARNGMKNQFNLHELKMKGPEPVVSQIIDKL KHINQLLRTMSMPKGRVLDKNLDEEGFESGDCGDDEDECILCTPSRMIKVKN QLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKLLTSMAIS VVCFFFLVH-

SEQ ID NO.3

MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGL KWVPETPVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMEL KFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPSLTPQAFEFVGEFFTDVS LYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLK VFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTRM WYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDKYWREYILSLEELVN GMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRFA YYPEDLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFISFYSALPGYICSHSPV AENDTLCWNGQELVERYSQKAARNGMKNQFNLHELKMKGPEPVVSQIIDKL KHINQLLRTMSMPKGRVLDKNLDEEGLCTPSRGDDEDECILCTPSRMIKVKN QLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKLLTSMAIS VVCFFFLVH-

EXAMPLE 2 preparation of hydrazine-labeled GAG oligosaccharides and aldehyde-labeled GPC3

To verify that GPC3 was successful in carrying an aldehyde group, GPC3 mutants carrying an aldehyde group were reacted with Biotin (Biotin-LC-hydrozide) carrying a hydrazine group. The reaction product was then added to a 96-well plate coated with GPC3 antibody, left at room temperature for 1-3 hours, washed 1-5 times with PBS after incubation, and biotin on GPC3 was detected with horseradish peroxidase (HRP) or Alkaline Phosphatase (AP) -labeled avidin. As a result, GPC3 mutants co-transfected with FGE plasmid have more remarkable binding ability to avidin as shown in FIG. 1. Furthermore, GPC3-O mutants with two glycosyl binding sites have a higher avidin binding capacity than the other two mutants. This suggests that both glycosyl binding sites successfully label the active aldehyde group.

At the same time, hydrazino-tagged GAG oligosaccharide chains were prepared. Partial degradation of GAGs with the corresponding GAG cleavage enzymes gives a series of oligosaccharides, which are then collected by high performance liquid chromatography (High performance liquid chromatography, HPLC) and the separated oligosaccharide fractions are reacted with excess adipic acid dihydrazide. To determine if oligosaccharides were successfully labeled with hydrazino groups, we performed high performance liquid and mass spectrometry (Mass spectrometry, MS) analyses before and after HA and Hep disaccharide labeling. HPLC results showed that about 50% of the disaccharides were labeled with adipoyl hydrazine (fig. 2a,2 b). To further confirm that the saccharides were indeed labelled with hydrazino groups, we performed electrospray ionization mass spectrometry (ESI-MS) analysis of the two main peaks on HPLC under negative ion conditions. The two components of HA disaccharide were 534.1933 and 378.0934m/z, respectively, which is consistent with the molecular weight of HA disaccharide before and after labelling (FIGS. 2C, 2D). Two signal peaks are shown in FIG. 2F at 247.4936 and 287.4736 m/z, which can be referred to as Hep disaccharides containing two and three sulfate groups, respectively. Accordingly, the two signal peaks in FIGS. S3E, 325.5426 and 365.5227m/z, are designated as hydrazino-labeled Hep disaccharides with two and three sulfate groups. Likewise, CS and DS oligosaccharides may also be successfully labeled.

Example 3 in vitro ligation of GAG side chain to GPC3

The structure of the core protein of GPC3 is complex, and the aldehyde group is located in the middle of the amino acid sequence of GPC3, so it is necessary to determine that the oligosaccharide chain can be successfully linked to the core protein. 293T cells transfected with FGE and GPC3 mutants were lysed, and then hydrazine-labeled GAG oligosaccharide components were added to the cell lysate for 2-6 hours. Protein samples before and after labeling were detected by Western Blot, with primary antibody selected from murine monoclonal antibody against GPC3 (prepared by laboratory using conventional methods) and secondary antibody selected from anti-mouse antibodies labeled with HRP or AP. As shown in FIG. 3, for GPC3-O containing two aldehyde groups, about 50% of the proteins were labeled with one oligosaccharide chain, and 20% of GPC3 were labeled with two oligosaccharide chains, with the labeling efficiency gradually decreasing with increasing molecular weight of the oligosaccharides. This result shows that GAG oligosaccharides can be well assembled with GPC3 with aldehyde labels, which has important roles in GPC3 structure-activity relationship research, PGs-related diagnostic kits with specific activity and small side effects, and drug development.

Example 4 engineering installation of GPC3 side chain on cell membrane surface.

293T cells were seeded in 24-well plates and transfected with GPC3-O vectors and FGE vectors. Hydrazine-labeled GAG oligosaccharide chains (pH adjusted to 5-7) were added to the medium on day 2 after transfection. After 4-12 hours, cells were lysed and Western Blot was performed, with primary antibody selected from murine monoclonal antibody against GPC3 (laboratory self-made) and secondary antibody selected from HRP or AP labeled anti-mouse antibodies. As shown in FIG. 4, the cell lysate treated with the hydrazino-labeled HS oligosaccharide showed a dispersed band similar to that of wild-type GPC3, and the cell lysate was purified by a DEAE-anion exchange column to significantly enhance the content of the dispersed band. In addition, 3 bands were detected in the cell lysates treated with hydrazino-labeled HS tetradecanose, and these three bands were GPC3 containing 0, 1, and 2 HS side chains, respectively. Similarly, two GPC 3's containing HS side chains may be concentrated by DEAE-anion exchange column. This result suggests that hydrazino-labeled HS oligosaccharides can successfully bind to the aldehyde group-containing GPC3-O core protein expressed on the surface of living cells.

Example 5, the effect of assembling sugar chains of different lengths on Wnt3A cell signaling on core proteins using the method of the invention;

293T cells were seeded in 24-well plates and transfected with GPC3-O and FGE expression vectors and cell signaling-related vectors. Hydrazine labeled HS oligosaccharide chains (pH adjusted to 5-7) were added to the medium on day 2 after transfection, and left for 4-12 hours. Cells were then incubated in Wnt3A conditioned medium or control medium for an additional 24 hours. Finally, the cells are lysed and luciferase activity is measured. Each experiment was performed at least five times, three at a time in parallel. As shown in fig. 5, the ability of GPC3 to promote Wnt3A cell signaling increases with increasing assembled HS chain length, the ability to promote cell signaling reached the strongest when HS chain length reached tetradecyl sugar (DP 14), and there was no significant change in activity to promote Wnt3A cell signaling after sugar chain length was greater than tetradecyl sugar, indicating that tetradecyl sugar is the minimum size of HS side chains required by GPC3 to stimulate Wnt3A signaling for the highest activity.

Example 6, effect of different GAGs on Wnt3A cell signaling using engineered assembled proteoglycans prepared by the method of the invention;

293T cells were seeded in 24-well plates and transfected with GPC3-O and FGE expression vectors and vectors required for cell signaling. Hydrazine labeled GAG tetradecaccharide (pH value is adjusted to 5-7) is added into the culture medium on the 2 nd day after transfection, and the culture medium is left for 4-12 hours. Cells were then incubated with hydrazine labeled GAG tetradecanose and Wnt3A conditioned medium for an additional 24 hours. Finally, the cells are lysed and luciferase activity is measured. Each experiment was performed at least five times, three at a time in parallel. As shown in fig. 6, GPC3-O (except HA) assembled with GAG side chains all had a more pronounced ability to stimulate Wnt3A signaling than GPC 3A GAGs without side chains. GPC3 containing two CS and DS side chains stimulated Wnt3A similarly, approximately 30% higher than GPC3. DELTA. GAG without sugar chains. The effect of GPC3 with two HS side chains on Wnt signaling was approximately 283% of GPC3 Δgag, while the stimulatory ability of GPC3 with Hep side chains on Wnt signaling was approximately 326% of GPC3 Δgag. This result suggests that the number of sulfate groups in the GAG side chains and disaccharide composition influence the role of GPC3 in Wnt cell signaling.

Example 7 engineering assembled GPC3 mediated endocytosis of Wnt3A

Studies have shown that GPC3 stimulates the Wnt signaling pathway by interacting with Wnt3A and mediating Wnt3A endocytosis (47). To verify whether the engineered assembled GPC3 also has the same biological function as wild-type GPC3, cells were seeded on polylysine-treated slides and transfected with GPC3-O or control vectors and FGE expression vectors. Two days after transfection, biotin and hydrazine labeled HS oligosaccharide chains (bioHS) were added and left for 4-12 hours. After completion of the incubation, the incubation was performed with DMEM containing 1-5% BSA at 4deg.C for 0.5-1 hr, followed by addition of Wnt3A or control conditioned medium and incubation at the same temperature for 1-3 hr. To study GPC3-Wnt3A endocytosis, cells were washed 1-3 times with PBS, pre-warmed DMEM was added and transferred to 37 ℃ for incubation for 0-60 min. After incubation, wash 3-5 times with PBS and fix with 4% paraformaldehyde. After cell fixation, immunostaining was performed and cells were first treated with 0.1% trion X-100 for 15-60 min and re-blocked with 5% nonfat milk powder for 1 hour. And finally adding the corresponding primary antibody and the fluorescent-labeled secondary antibody. As a result, as shown in FIG. 7, GPC3 and Wnt3A endocytosed from the cell membrane to the cytoplasm and around the nucleus when these cells were transferred to 37℃for 30 or 60 minutes. The results indicate that GPC3-bioHS is capable of mediating Wnt3A internalization, thereby activating Wnt3A signaling.

EXAMPLE 8 GPC3 interaction analysis with Frizzled-7

PGs on the cell surface are known to regulate cell signaling by interacting with various growth factors, morphological factors and their receptors (48). However, it is difficult to intuitively demonstrate the interaction between PGs and target proteins, especially complex glycoproteins expressed on the cell surface (49, 50).

In this example, a new method was established to determine the interaction of PGs with its targeting protein on the surface of living cells using two strategies, metabolic glycolabelling of glycoproteins and engineering assembly of PGs. HEK293T cells were plated on 10cm cell culture plates and co-transfected with GPC3-O, FGE vector and FZD-7 vector. After one day, the culture supernatant was added with azide-labeled glucosamine to a final concentration of 20-400. Mu.M and left for 12-36 hoursWhen (1). Then 0.5-2. Mu.M of hydrazino-labeled HS oligosaccharide chains and 20-400. Mu.M of azido-labeled glucosamine were added to the new culture supernatant without FBS. After overnight incubation of the cells, the cells were incubated with a solution containing 250mM triazomethyl amine (Tris [ (1-benzyl-1H-1, 2, 3-triazol-4-yl) methyl)]amine， TBTA)、50mM CuSO ₄ 500mM sodium L-ascorbate (L-Ascorbic Acid Sodium Salt, SA) in PBS for 1-4h at room temperature. Finally, the protease inhibitor-containing RIPA lysis buffer was used for lysis on ice for 20-60 minutes. Western Blot detection is carried out on the cell lysate, a primary FZD-7 rabbit antibody is incubated with a secondary antibody marked by HRP or AP, and finally, a protein band is detected by an enhanced chemiluminescent reagent. As a result, as shown in FIG. 8, FZD-7 having an azide group was able to bind to GPC3 having an azido oligosaccharide chain with a binding efficiency of about 20% using FZD-7 antibody as a probe. GPC3 bearing an azido oligosaccharide chain is cleaved from GPC3 in FZD-7 binding products after heparinase (Hepase) III treatment. These results demonstrate an interaction between GPC3 and FZD-7.

Example 9 application of engineered assembled GPC3 in liver cancer early diagnosis kit

GPC3 is widely expressed in various tissues during embryonic development but silences due to DNA methylation after adulthood. In recent years, GPC3 has been found to be expressed exclusively in about 75% of hepatocellular carcinomas, but not in normal tissues and benign lesions of the liver, a more specific hepatocellular carcinoma biomarker molecule than fetuin (AFP). Therefore we can prepare engineered assembled GPC3 on a large scale by microorganisms and use it as a standard in liver cancer early diagnosis kit.

GPC3 specific antibody αGCN was plated on 96-well plates at a concentration of 10-50ng/ml in a range of 30-100. Mu.l, and then blocked with 1% BSA. Washing with PBS for 1-5 times after the end of the sealing, adding the engineering assembled GPC3 and patient samples dissolved in PBS for different concentrations, standing at room temperature for 1-3 hours, and washing with PBS for 1-5 times after the end of the incubation; adding GPC3 specific rabbit polyclonal antibody, standing at room temperature for 1-3 hr, and washing with PBS for 1-5 times after incubation; adding corresponding horseradish peroxidase or alkaline phosphatase labeled anti-rabbit polyclonal antibody, standing at room temperature for 10-30min, washing with PBS for 3-7 times, adding corresponding substrate, and measuring absorbance. And diagnosing early liver cancer patients through reading the absorbance value.

EXAMPLE 10 use of engineered assembled proteoglycans in drug development

Decorin (Decorin) is the simplest proteoglycan, which contains a core protein and a DS sugar chain, which plays an important role in inhibiting subconjunctival scarring in the eye. In the present invention we used engineered assembled Decorin to prepare anti-scarring gel eye drops. The method comprises the following specific steps: adding 0.8-1.2% low acyl gellan gum into deionized water at 70-90deg.C under stirring to obtain liquid gel. The gel solution temperature was then reduced to 40 ℃ and the engineered assembled Decorin solution in PBS was added to a final gellan concentration of 0.9% and 0.2-0.3 μg of engineered assembled Decorin was contained in 1ml of gel solution. Thus, the present invention provides for the first time a novel, convenient, inexpensive method of studying the interaction of PGs with glycoproteins.

Reference to the literature

1.Afratis，N.A.，Nikitovic，D.，Multhaupt，H.A.，Theocharis，A.D.，Couchman，J.R.，and Karamanos，N.K.(2017)Syndecans-key regulators of cell signaling and biological functions. The FEBS journal 284，27-41

2.Chakraborty，S.，Lakshmanan，M.，Swa，H.L.F.，Chen，J.，Zhang，X.，Ong，Y.S.，Loo，L.S.， Akincilar，S.C.，Gunaratne，J.，Tergaonkar，V.，Hui，K.M.，and Hong，W.(2015)An oncogenic role of Agrin in regulating focal adhesion integrity in hepatocellular carcinoma. Nature communications 6

3.Filmus，J.，and Capurro，M.(2014)The role of glypicans in Hedgehog signaling.Matrix biology：journal of the International Society for Matrix Biology 35，248-252

4.Kronenberg，H.M.(2003)Developmental regulation of the growth plate.Nature 423，332-336

5.Koziel，L.，Kunath，M.，Kelly，O.G.，and Vortkamp，A.(2004)Extl-dependent heparan sulfate regulates the range of Ihh signaling during endochondral ossification.Developmental cell 6，801-813

6.Chan，J.A.，Balasubramanian，S.，Witt，R.M.，Nazemi，K.J.，Choi，Y.，Pazyra-Murphy，M.F.， Walsh，C.O.，Thompson，M.，and Segal，R.A.(2009)Proteoglycan interactions with Sonic Hedgehog specify mitogenic responses.Nature Neuroscience 12，409-417

7.Arikawa-Hirasawa，E.，Wilcox，W.R.，Le，A.H.，Silverman，N.，Govindraj，P.，Hassell，J.R.， and Yamada，Y.(2001)Dyssegmental dysplasia，Silverman-Handmaker type，is caused by functional null mutations of the perlecan gene.Nature Genetics 27，431-434

8.Mahtouk，K.，Cremer，F.W.，Reme，T.，Jourdan，M.，Baudard，M.，Moreaux，J.，Requirand，G.， Fiol，G.，De Vos，J.，Moos，M.，Quittet，P.，Goldschmidt，H.，Rossi，J.F.，Hose，D.，and Klein， B.(2006)Heparan sulphate proteoglycans are essential for the myeloma cell growth activity of EGF-family ligands in multiple myeloma.Oncogene 25，7180-7191

9.Mizuguchi，S.，Uyama，T.，Kitagawa，H.，Nomura，K.H.，Dejima，K.，Gengyo-Ando，K.，Mitani，S.，Sugahara，K.，and Nomura，K.(2003)Chondroitin proteoglycans are involved in cell division of Caenorhabditis elegans.Nature 423，443-448

10.Hacker，U.，Nybakken，K.，and Perrimon，N.(2005)Heparan sulphate proteoglycans：The sweet side of development.Nature Reviews Molecular Cell Biology 6，530-541

11.Arikawa-Hirasawa，E.，Watanabe，H.，Takami，H.，Hassell，J.R.，and Yamada，Y.(1999) Perlecan is essential for cartilage and cephalic development.Nature Genetics 23，354-358

12.Bassat，E.，Mutlak，Y.E.，Genzelinakh，A.，Shadrin，I.Y.，Umansky，K.B.，Yifa，O.，Kain，D.， Rajchman，D.，Leach，J.，Bassat，D.R.，Udi，Y.，Sarig，R.，Sagi，I.，Martin，J.F.，Bursac，N.， Cohen，S.，and Tzahor，E.(2017)The extracellular matrix protein agrin promotes heart regeneration in mice.Nature 547，179-+

13.Parish，C.R.(2006)The role of heparan sulphate in inflammation.Nature Reviews Immunology 6，633-643

14.Wang，L.C.，Fuster，M.，Sriramarao，P.，and Esko，J.D.(2005)Endothelial heparan sulfate deficiency impairs L-selectin-and chemokine-mediated neutrophil trafficking during inflammatory responses.Nature Immunology 6，902-910

15.Nikitovic，D.，Berdiaki，A.，Spyridaki，I.，Krasanakis，T.，Tsatsakis，A.，and Tzanakakis，G.N. (2018)Proteoglycans-Biomarkers and Targets in Cancer Therapy.Frontiers in Endocrinology 9

16.Korpetinou，A.，Skandalis，S.S.，Labropoulou，V.T.，Smirlaki，G.，Noulas，A.，Karamanos，N. K.，and Theocharis，A.D.(2014)Serglycin：at the crossroad of inflammation and malignancy. Frontiers in oncology 3，327

17.Christianson，H.C.，and Belting，M.(2014)Heparan sulfate proteoglycan as a cell-surface endocytosis receptor.Matrix biology 35，51-55

18.Fuki，I.V.，Kuhn，K.M.，Lomazov，I.R.，Rothman，V.L.，Tuszynski，G.P.，Iozzo，R.V.， Swenson，T.L.，Fisher，E.A.，and Williams，K.J.(1997)The syndecan family of proteoglycans.Novel receptors mediating internalization of atherogenic lipoproteins in vitro. The Journal of clinical investigation 100，1611-1622

19.Yang，Y.，Yaccoby，S.，Liu，W.，Langford，J.K.，Pumphrey，C.Y.，Theus，A.，Epstein，J.，and Sanderson，R.D.(2002)Soluble syndecan-1 promotes growth of myeloma tumors in vivo. Blood 100，610-617

20.Wang，J.，Svendsen，A.，Kmiecik，J.，Immervoll，H.，Skaftnesmo，K.O.，Planaguma，J.，Reed， R.K.，Bjerkvig，R.，Miletic，H.，Enger，P.O.，Rygh，C.B.，and Chekenya，M.(2011) Targeting the NG2/CSPG4 proteoglycan retards tumour growth and angiogenesis in preclinical models of GBM and melanoma.PLoS One 6，e23062

21.Yuan，P.，Zhang，H.，Cai，C.，Zhu，S.，Zhou，Y.，Yang，X.，He，R.，Li，C.，Guo，S.，Li，S.， Huang，T.，Perez-Cordon，G.，Feng，H.，and Wei，W.(2015)Chondroitin sulfate proteoglycan 4 functions as the cellular receptor for Clostridium difficile toxin B.Cell research 25，157-168

22.Bilandzic，M.，and Stenvers，K.L.(2011)Betaglycan：a multifunctional accessory.Molecular and cellular endocrinology 339，180-189

23.Bernabeu，C.，Lopez-Novoa，J.M.，and Quintanilla，M.(2009)The emerging role of TGF-beta superfamily coreceptors in cancer.Biochimica et biophysica acta 1792，954-973

24.Whitelock，J.M.，Graham，L.D.，Melrose，J.，Murdoch，A.D.，Iozzo，R.V.，and Underwood， P.A.(1999)Human perlecan immunopurified from different endothelial cell sources has different adhesive properties for vascular cells.Matrix Biology18，163-178

25.Lord，M.S.，Chuang，C.Y.，Melrose，J.，Davies，M.J.，Iozzo，R.V.，and Whitelock，J.M. (2014)The role of vascular-derived perlecan in modulating cell adhesion，proliferation and growth factor signaling.Matrix Biology 35，112-122

26.Fuki，I.I.，Iozzo，R.V.，and Williams，K.J.(2000)Perlecan heparan sulfate proteoglycan.A novel receptor that mediates a distinct pathway for ligand catabolism.Journal of biological chemistry275，31554

27.Nugent，M.A.，Nugent，H.M.，Iozzo，R.V.，Sanchack，K.，and Edelman，E.R.(2000) Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia.Proceedings of the National Academy of Sciences of the United States of America 97,6722-6727

28.Laplante，P.，Raymond，M.A.，Labelle，A.，Abe，J.，Iozzo，R.V.，and Hebert，M.J.(2006) Perlecan proteolysis induces an alpha2betal integrin-and Src family kinase-dependent anti-apoptotic pathway in fibroblasts in the absence of focal adhesion kinase activation. Journal of biological chemistry 281，30383-30392

29.Baker，A.B.，Ettenson，D.S.，Jonas，M.，Nugent，M.A.，Iozzo，R.V.，and Edelman，E.R. (2008)Endothelial cells provide feedback control for vascular remodeling through a mechanosensitive autocrine TGF-beta signaling pathway.Circulation research 103，289-297

30.Wilusz，R.E.，Sanchez-Adams，J.，and Guilak，F.(2014)The structure and function of the pericellular matrix of articular cartilage.Matrix Biology 39，25-32

31.Sher，I.，Zisman-Rozen，S.，Eliahu，L.，Whitelock，J.M.，Maas-Szabowski，N.，Yamada，Y.， Breitkreutz，D.，Fusenig，N.E.，Arikawa-Hirasawa，E.，Iozzo，R.V.，Bergman，R.，and Ron，D. (2006)Targeting perlecan in human keratinocytes reveals novel roles for perlecan in epidermal formation.The Journal of biological chemistry 281，5178-5187

32.Ishijima，M.，Suzuki，N.，Hozumi，K.，Matsunobu，T.，Kosaki，K.，Kaneko，H.，Hassell，J.R.， Arikawa-Hirasawa，E.，and Yamada，Y.(2012)Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation.Matrix Biology 31，234-245

33.Kaneko，H.，Ishijima，M.，Futami，I.，Tomikawa-Ichikawa，N.，Kosaki，K.，Sadatsuki，R.， Yamada，Y.，Kurosawa，H.，Kaneko，K.，and Arikawa-Hirasawa，E.(2013)Synovial perlecan is required for osteophyte formation in knee osteoarthritis.Matrix Biology 32，178-187

34.Decker，R.S.，Koyama，E.，and Pacifici，M.(2014)Genesis and morphogenesis of limb synovial joints and articular cartilage.Matrix Biology 39，5-10

35.Hsueh，M.F.，Onnerfjord，P.，and Kraus，V.B.(2014)Biomarkers and proteomic analysis of osteoarthritis.Matrix Biology 39，56-66

36.Wight，T.N.，Kinsella，M.G.，Evanko，S.P.，Potter-Perigo，S.，and Merrilees，M.J.(2014) Versican and the regulation of cell phenotype in disease.Biochimica et biophysica acta 1840， 2441-2451

37.Wight，T.N.，Kang，I.，and Merrilees，M.J.(2014)Versican and the control of inflammation. Matrix Biology 35，152-161

38.Chang，M.Y.，Tanino，Y.，Vidova，V.，Kinsella，M.G.，Chan，C.K.，Johnson，P.Y.，Wight，T. N.，and Frevert，C.W.(2014)Reprint of：A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease.Matrix Biology 35，162-173

39.Liu，B.P.，Cafferty，W.B.，Budel，S.O.，and Strittmatter，S.M.(2006)Extracellular regulators of axonal growth in the adult central nervous system.Philosophical transactions of the Royal Society of London.Series B，Biological sciences 361，1593-1610

40.Davies，J.E.，Tang，X.，Denning，J.W.，Archibald，S.J.，and Davies，S.J.(2004)Decorin suppresses neurocan，brevican，phosphacan and NG2 expression and promotes axon growth across adult rat spinal cord injuries.The Europeanjournal ofneuroscience 19，1226-1242

41.Frischknecht，R.，and Seidenbecher，C.I.(2012)Brevican：a key proteoglycan in the perisynaptic extracellular matrix of the brain.The international journal of biochemistry&cell biology 44，1051-1054

42.Mii，Y.，and Yamamoto，T.(2017)Roles of two types of heparan sulfate clusters in Wnt distribution and signaling in Xenopus.8，1973

43.Chen，Y.H.，Narimatsu，Y.，Clausen，T.M.，Gomes，C.，Karlsson，R.，and Steentoft，C.(2018) The GAGOme：a cell-based library of displayed glycosaminoglycans.15，881-888

44.Rabuka，D.，Rush，J.S.，deHart，G.W.，Wu，P.，and Bertozzi，C.R.(2012)Site-specific chemical protein conjugation using genetically encoded aldehyde tags.Nature protocols 7， 1052-1067

45.Appel，M.J.，and Bertozzi，C.R.(2015)Formylglycine，a post-translationally generated residue with unique catalytic capabilities and biotechnology applications.ACS chemical biology 10，72-84

46.Wu，P.，Shui，W.，Carlson，B.L.，Hu，N.，Rabuka，D.，Lee，J.，and Bertozzi，C.R.(2009) Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag.Proceedings of the National Academy of Sciences of the United States of America 106，3000-3005

47.Capurro，M.，Wanless，I.R.，Sherman，M.，Deboer，G.，Shi，W.，Miyoshi，E.，and Filmus，J. (2003)Glypican-3：a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology 125，89-97

48.Tanaka，Y.，Tateishi，R.，and Koike，K.(2018)Proteoglycans Are Attractive Biomarkers and Therapeutic Targets in Hepatocellular Carcinoma.International Journal of Molecular Sciences19

49.Zielinska，D.F.，Gnad，F.，Schropp，K.，Wisniewski，J.R.，and Mann，M.(2012)Mapping N-glycosylation sites across seven evolutionarily distant species reveals a divergent substrate proteome despite a common core machinery.Molecular cell 46，542-548

50.Weerapana，E.，and Imperiali，B.(2006)Asparagine-linked protein glycosylation：from eukaryotic to prokaryotic systems.Glycobiology 16，91r-101r。

Claims (6)

1. An engineered assembled proteoglycan, characterized by: comprises a proteoglycan core protein with aldehyde groups and a glycosaminoglycan side chain marked with modification groups; the side chain of the glycosaminoglycan marked with the modification group is hydrazino-marked HS oligosaccharide or hydrazino-marked Hep disaccharide; the side chain of the glycosaminoglycan marked with the modification group is a hydrazino-marked HS oligosaccharide or a hydrazino-marked Hep disaccharide, and the preparation method comprises the following steps: performing partial degradation on GAG with GAG lyase to obtain a series of oligosaccharides, collecting each oligosaccharide by high performance liquid chromatography, and reacting the separated oligosaccharide component with excessive adipic acid dihydrazide; the proteoglycan core protein with aldehyde groups was linked in vitro with hydrazino-labeled HS oligosaccharides or hydrazino-labeled Hep disaccharides by the following methods: 293T cells transfected with FGE and GPC3 mutants were lysed, and then hydrazine-labeled GAG oligosaccharide components were added to the cell lysate for 2-6 hours to obtain core protein GPC3 with two HS oligosaccharide side chains attached, or with Hep disaccharide side chains having two or three sulfate groups attached.

2. A method of preparing an engineered assembled proteoglycan according to claim 1, comprising:

preparing a proteoglycan core protein with aldehyde groups;

labeling hydrazine groups on the glycosaminoglycan side chains;

the glycosaminoglycan side chains bearing hydrazine groups are assembled onto the proteoglycan core protein by chemical reaction with aldehyde groups.

3. The method for producing an engineered assembled proteoglycan according to claim 2, wherein: the preparation method of the proteoglycan core protein with aldehyde group comprises the following steps:

mutating the glycosylation site of the proteoglycan core protein into an amino acid sequence convertible to an aldehyde group;

the amino acid sequence is converted to an aldehyde group using an fGly-producing enzyme to catalyze the amino acid sequence.

4. A method of preparing an engineered assembled proteoglycan according to claim 3, wherein: the amino acid sequence which can be converted into an aldehyde group is CXPXR, wherein X is any amino acid except proline.

5. Use of the engineered assembled proteoglycan of claim 1 for the preparation of a cancer diagnostic kit.

6. Use of the engineered assembled proteoglycan of claim 1 in the preparation of a medicament.

Images