COVID-19 represents the most serious public health event in the past few decades of the 21^st century. The development of vaccines, neutralizing antibodies, and small molecule chemical agents have effectively prevented the rapid spread of COVID-19. However, the continued emergence of SARS-CoV-2 variants have weakened the efficiency of these vaccines and antibodies, which brought new challenges for searching novel anti-SARS-CoV-2 drugs and methods. In the process of SARS-CoV-2 infection, the virus firstly attaches to heparan sulphate on the cell surface of respiratory tract, then specifically binds to hACE2. The S protein of SARS-CoV-2 is a highly glycosylated protein, and glycosylation is also important for the binding of hACE2 to S protein. Furthermore, the S protein is recognized by a series of lectin receptors in host cells. These finding implies that glycosylation plays important roles in the invasion and infection of SARS-CoV-2. Based on the glycosylation pattern and glycan recognition mechanisms of SARS-CoV-2, it is possible to develop glycan inhibitors against COVID-19. Recent studies have shown that sulfated polysaccharides originated from marine sources, heparin and some other glycans display anti-SARS-CoV-2 activity. This review summarized the function of glycosylation of SARS-CoV-2, discoveries of glycan inhibitors and the underpinning molecular mechanisms, which will provide guidelines to develop glycan-based new drugs against SARS-CoV-2.
Antibodies, Neutralizing ; Glycosylation ; Heparin ; Heparitin Sulfate ; Humans ; Polysaccharides/chemistry* ; Receptors, Mitogen/metabolism* ; SARS-CoV-2 ; Spike Glycoprotein, Coronavirus/metabolism* ; COVID-19 Drug Treatment

Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.
Animals ; Carbohydrate Epimerases ; biosynthesis ; chemistry ; genetics ; Escherichia coli ; Gene Expression ; Heparin ; metabolism ; Heparitin Sulfate ; metabolism ; Iduronic Acid ; metabolism ; Zebrafish Proteins ; biosynthesis ; chemistry ; genetics

We conducted this study to investigate the synergistic effect of human urine-derived stem cells (USCs) and surface modified composite scaffold for bladder reconstruction in a rat model. The composite scaffold (Polycaprolactone/Pluronic F127/3 wt% bladder submucosa matrix) was fabricated using an immersion precipitation method, and heparin was immobilized on the surface via covalent conjugation. Basic fibroblast growth factor (bFGF) was loaded onto the heparin-immobilized scaffold by a simple dipping method. In maximal bladder capacity and compliance analysis at 8 weeks post operation, the USCs-scaffold(heparin-bFGF) group showed significant functional improvement (2.34 ± 0.25 mL and 55.09 ± 11.81 microL/cm H2O) compared to the other groups (2.60 ± 0.23 mL and 56.14 ± 9.00 microL/cm H2O for the control group, 1.46 ± 0.18 mL and 34.27 ± 4.42 microL/cm H2O for the partial cystectomy group, 1.76 ± 0.22 mL and 35.62 ± 6.69 microL/cm H2O for the scaffold group, and 1.92 ± 0.29 mL and 40.74 ± 7.88 microL/cm H2O for the scaffold(heparin-bFGF) group, respectively). In histological and immunohistochemical analysis, the USC-scaffold(heparin-bFGF) group showed pronounced, well-differentiated, and organized smooth muscle bundle formation, a multi-layered and pan-cytokeratin-positive urothelium, and high condensation of submucosal area. The USCs seeded scaffold(heparin-bFGF) exhibits significantly increased bladder capacity, compliance, regeneration of smooth muscle tissue, multi-layered urothelium, and condensed submucosa layers at the in vivo study.
Adult Stem Cells/cytology/metabolism/*transplantation ; Animals ; Biocompatible Materials/chemistry ; Cell Differentiation ; Fibroblast Growth Factor 2/administration & dosage ; Heparin/administration & dosage ; Humans ; Materials Testing ; Models, Animal ; Poloxamer ; Polyesters ; Rats ; Reconstructive Surgical Procedures ; Regeneration ; Tissue Engineering/*methods ; Tissue Scaffolds/chemistry ; Urinary Bladder/anatomy & histology/physiology/*surgery ; Urine/*cytology

Antithrombin III (AT III) is the most important anti-clotting substance. Recombinant human antithrombin III (rhAT III) expressed in transgenic goat milk attracts more and more attention. Develop an effective purification route for rhAT III is vital to its industrial production. An efficient purification method was developed for the rapid purification of rhAT III by isoelectric precipitation and heparin affinity chromatography. First, casein was effectively removed by isoelectric precipitation. rhAT III was further purified by heparin affinity chromatography. In the process of heparin affinity chromatography, the effects of pH and temperature on the stability of rhAT III were studied, and the effects of operating conditions, elution gradient, flow rate and sample loaded, on the purification efficiency were also studied. Under the optimized conditions, the protein recovery of rhAT III was about 90% with purity over 99%, while its activity recovery was about 50%. Such a purification process is very simple and effective, and it would provide a valuable reference for the further scaling-up of industrial production.
Animals ; Animals, Genetically Modified ; Antithrombin III ; biosynthesis ; Chromatography, Affinity ; Female ; Goats ; Heparin ; Humans ; Mammary Glands, Animal ; metabolism ; Milk ; chemistry ; Recombinant Proteins ; biosynthesis

OBJECTIVETo investigate the anti-cancer effect of low-molecular-weight heparin (LMWH) combined with doxorubicin and explore the mechanism.

METHODSHepatocellular cancer HepG2 cells exposed to LMWH, doxorubicin, or both were evaluated for cell viability with MTT assay and for changes in their migration ability using wound healing assay and Transwell migration assay. The changes in cellular expressions of matrix metalloproteinase-9 (MMP-9) and MMP-2 mRNA and proteins were analyzed with quantitative real-time PCR (qRT-PCR) and Western blotting, and ELISA was used to determine heparanase (HPA) concentration in the cell culture medium.

RESULTSHepG2 cells exhibited suppressed proliferation in response to LMWH and doxorubicin treatments. The combined treatment caused a significantly higher inhibition rate of cell migration than LMWH and doxorubicin alone. LMWH enhanced doxorubicin-induced down-regulation of MMP-9, MMP-2 and HPA in the cells.

CONCLUSIONSLMWH can enhance the inhibitory effect of doxorubicin on the migration of HepG2 cells, the mechanism of which may involve the down-regulation of MMP-9, MMP-2 and HPA expressions.

Cell Movement ; drug effects ; Cell Survival ; Down-Regulation ; Doxorubicin ; pharmacology ; Glucuronidase ; chemistry ; Hep G2 Cells ; Heparin, Low-Molecular-Weight ; pharmacology ; Humans ; Liver Neoplasms ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; RNA, Messenger ; Real-Time Polymerase Chain Reaction

Crystallography, X-Ray ; Escherichia coli ; metabolism ; Fibroblast Growth Factors ; chemistry ; genetics ; metabolism ; Heparin ; metabolism ; Humans ; Models, Molecular ; Protein Binding ; Protein Isoforms ; chemistry ; metabolism ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Sulfates ; chemistry ; metabolism

To express and identify fibronectin C-terminal heparin-binding domain (FNCH BD) polypeptides in Pichia pastoris expression system and study its function, the fragment of FNCHBD was amplified by PCR and inserted into pGEM-T vector. After sequenced, the fragment was inserted into pAo815SM vector, and then cloned into the expression vector pPIC9k. The recombinant plasmid was linerarized with restrict enzyme Sal I and transferred into the yeast host cell KM71 and GS115. The positive yeast clone was screened by G418 resistant, and the target protein was induced to express in the medium containing 0.5% methanol. The culture supernatant was collected and then was purified with membrane ultrafiltration and ion exchange chromatography. The purified product was analyzed with mass spectrogram, SDS-PAGE, Western blotting and heparin affinity chromatography. The results showed that the target protein was around 32 kDa and the purity of the product was above 95%. FNCHBD could be specifically recognized by fibronectin polyclonal antibody. These results suggest that FNCHBD could be expressed and purified successfully in Pichia pastoris, which provides a good strategy to further studies.
Fibronectins ; biosynthesis ; chemistry ; genetics ; Genetic Vectors ; Heparin ; metabolism ; Peptides ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

Bone morphogenetic protein-2 (BMP-2) is used to promote bone regeneration. However, the bone regeneration ability of BMP-2 relies heavily on the delivery vehicle. Previously, we have developed heparin-conjugated fibrin (HCF), a vehicle for long-term delivery of BMP-2 and demonstrated that long-term delivery of BMP-2 enhanced its osteogenic efficacy as compared to short-term delivery at an equivalent dose. The aim of this study was to compare the bone-forming ability of the BMP-2 delivered by HCF to that delivered by clinically utilized BMP-2 delivery vehicle collagen sponge. An in vitro release profile of BMP-2 showed that HCF released 80% of the loaded BMP-2 within 20 days, whereas collagen sponge released the same amount within the first 6 days. Moreover, the BMP-2 released from the HCF showed significantly higher alkaline phosphatase activity than the BMP-2 released from collagen sponge at 2 weeks in vitro. Various doses of BMP-2 were delivered with HCF or collagen sponge to mouse calvarial defects. Eight weeks after the treatment, bone regeneration was evaluated by computed tomography, histology, and histomorphometric analysis. The dose of BMP-2 delivered by HCF to achieve 100% bone formation in the defects was less than half of the BMP-2 dose delivered by collagen sponge to achieve a similar level of bone formation. Additionally, bone regenerated by the HCF-BMP-2 had higher bone density than bone regenerated by the collagen sponge-BMP-2. These data demonstrate that HCF as a BMP-2 delivery vehicle exerts better osteogenic ability of BMP-2 than collagen sponge, a clinically utilized delivery vehicle.
Alkaline Phosphatase/metabolism ; Animals ; Bone Density ; *Bone Morphogenetic Protein 2/administration & dosage/genetics ; Bone Regeneration/*genetics ; Cells, Cultured ; Collagen Type I/chemistry/metabolism ; *Fibrin/chemistry/metabolism ; *Gene Transfer Techniques ; *Heparin/chemistry/metabolism ; Mice ; Osteogenesis/genetics ; Rats ; Rats, Sprague-Dawley

To investigate effects of collagen matrices by covalent incorporation of heparin and loading with huangqi injection (HI) on anagenetic capillaries, we established the chick chorioallantois model. The collagen matrices by covalent incorporation of heparin and loading with HI were placed and then the eggs were continuously incubated for 3 days. The number of capillaries in the vicinity of samples, the hemoglobin content inside the samples, the dry weight and the macroscopic observation were evaluated. We found the heparinized matrices had comparable angiogenic effects. The number of capillaries, the hemoglobin content, the expression of CD34 increased remarkably (P < 0.01). So we concluded that HI might be considered as an alternative or addition agent to promote the acidification of capillaries.
Animals ; Blood Vessels ; drug effects ; physiology ; Capillaries ; drug effects ; metabolism ; Chick Embryo ; Collagen ; administration & dosage ; chemistry ; pharmacology ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Heparin ; administration & dosage ; chemistry ; pharmacology ; Humans ; Injections

The ionized calcium level in blood is known to be falsely decreased when self-prepared liquid heparin anticoagulant is used, due to dilution and binding effects. The effect of liquid heparin on the determination of ionized magnesium is not as well understood. We compared the effect of liquid sodium heparin on the determination of ionized calcium and magnesium in 44 clinical samples using two types of user-prepared heparin syringes which differed in the amount of residual heparin from the BD Preset(TM) reference syringe. With the type 1 syringe, the liquid heparin was expelled once or twice such that some heparin could be left in the dead space at the syringe hub, while the liquid sodium heparin was thoroughly expelled from the type 2 syringe. The ionized magnesium levels obtained with the type 1 syringe were significantly lower than the reference value (by 0.068 mmol/L) (p < 0.0001), while the value obtained with the type 2 syringe differed less from the reference, by only 0.014 mmol/L (p < 0.0001). The heparin binding effect resulted in more negative bias in ionized magnesium (-0.026 +/- 0.032 mmol/L) than in ionized calcium (-0.009 +/- 0.042 mmol/L, p < 0.0001). In conclusion, we recommend using lyophilized, calcium-balanced, heparinized syringes for the determination of ionized magnesium and ionized calcium due to the increased negative bias in ionized magnesium determinations. When user-prepared syringes are used, the thorough evacuation of heparin solution should be strictly prescribed.
Syringes ; Protein Binding ; Magnesium/*chemistry/metabolism ; Ions ; Humans ; Heparin/administration & dosage/*therapeutic use ; Calcium/*metabolism ; Blood Specimen Collection/*methods ; Blood Chemical Analysis/*methods ; Anticoagulants/therapeutic use