Enrichment And Identification Of Bovine Sialoglycoproteins And Characterization Of Their Anti-influenza A Virus Activity And Pro-healing Function On Skin Wound

Background:Sialic acid（SA）,also known as neuraminic acid,is a class of natural carbohydrate derivatives that are widely present in biological systems.More than 50%of human proteins are glycosylated,and the terminal epitopes of the glycans are usually modified by sialic acids.They play an important role in human growth and development,the occurrence and development of diseases and the infection of pathogenic microorganisms.It has been demonstrated that influenza virus uses sialic acids on the cell surfaces as a receptor.The adhesion of viral particles to the cell surface by binding to the receptor is a prerequisite for viral infection.Therefore,the use of sialoglycoproteins to neutralize the receptor binding sites on the surface of viral particles thereby block the adhesion of viral particles to host cells is an effective strategy to prevent influenza.Although it has been shown that bovine milk is rich in sialoglycoproteins,the currently available methods for enriching sialoglycoproteins with an intact structure are complicated,time-consuming and poorly yielded.In addition,studies have shown that the cell viability of skin fibroblasts decreases with human aging,resulting in skin aging or the development of aging-related diseases.Moreover,the sialylation on the surface of skin fibroblasts decreases with human aging.However,the effect of sialylation on the surface of skin fibroblasts on their cellular functions is still not fully understood.Therefore,in this study,serotonin-functionalized magnetic particles were first prepared and used to purify sialoglycoproteins from bovine milk.Then,bovine sialoglycoproteins were identified and their N-glycans were characterized.Finally,the potential of bovine sialoglycoproteins against influenza virus and its anti-skin aging and accelerated wound healing were investigated.This study will provide a theoretical and technical basis for the large-scale application of sialoglycoproteins against influenza virus and accelerating skin wound healing.Methods:1.Serotonin-functionalized magnetic particles were prepared by the reaction of the epoxy group of epoxy-coated Fe₃O₄ magnetic particles with the amino group of serotonins and used to purify the sialoglycoproteins from bovine milk.The efficiency and specificity of the method were verified by sialic acid quantification and lectin blot assays;2.Bovine sialoglycoproteins and their N-glycans were identified by LC-MS/MS and MALDI-TOF/TOF-MS,respectively.Then,the ability of bovine sialoglycoproteins against influenza virus was verified using maackia aimrensis lectin-II（MAL-II）and sambucus nigra agglutinin（SNA）as mimics of avian and human influenza virus HA,respectively;3.Bovine lactoferrin（b Lf）were treated with sodium periodate to destroy their sialylated glycans.Then,the ability of native and desialylated lactoferrin to inhibit MAL-Ⅱor SNA binding to MDCK cells was investigated to elucidate the role of sialylated glycans on b Lf against influenza virus;4.Investigation of the effects of bovine sialoglycoproteins on the proliferation,migration,collagen secretion and sialylation of normal human dermal fibroblasts（NHDF）,proving its potential for anti-skin aging and accelerating wound healing.Results:1.The infrared spectrum of the serotonin-functionalized magnetic particles has a characteristic absorption peak at 1342.39 cm^-1 for C-N bond compared to that of the epoxy-coated Fe₃O₄ magnetic particles;2.The sialic acid content of bovine sialoglycoproteins enriched by serotonin-functionalized magnetic particles was about 3 times higher than that of raw milk proteins.Bovine sialoglycoproteins were mainly distributed at 80,33,30 and 22k Da in SDS-PAGE,of which the proteins at 80k Da mainly containedα2,6-linked sialic acid,while the proteins at 33 and 30k Da mainly containedα2,3-linked sialic acid;3.A total of 91 proteins and 24 N-glycans were identified in bovine sialoglycoproteins,of which 88 proteins（96.7%）were annotated or predicted as glycoproteins,12 N-glycans containedα2,6-linked sialic acid,and 5 N-glycans containedα2,3-linked sialic acid;4.Bovine sialoglycoproteins at 5μg/m L significantly inhibited the binding of MAL-Ⅱor SNA to MDCK cells,and the inhibition was enhanced with increasing proteins concentration in the range of 100μg/m L.When the sialylated glycans on the bovine sialoglycoproteins were destroyed,its inhibitory effect on the binding of MAL-Ⅱor SNA to MDCK cells was abolished;5.Bovine lactoferrin（b Lf）at 20μg/m L was able to significantly inhibit the binding of Mal-II and SNA to MDCK cells（inhibition efficiencies of 40%and 85%,respectively）,but the inhibition was reduced（inhibition efficiencies of 30%and 60%,respectively）after b Lf was oxidized by 10 m M sodium periodate（sialic acid content decreased from 0.462%to0.037%）;6.Bovine sialoglycoproteins at 0.5 mg/m L significantly promoted NHDF cells proliferation,shorten the cell doubling time（37.9±2.7 h vs 31.1±1.0 h,p<0.05）,facilitated the cell migration,and accelerated the contraction of fibroblast-populated collagen lattice（FPCL）;7.0.5 mg/m L of bovine sialoglycoproteins significantly inhibited the expression of transforming growth factor-β1（TGF-β1）and typeⅠcollagen（COL-Ⅰ）in NHDF cells,but promoted the expression of basic fibroblast growth factor（FGF-2）;8.0.5 mg/m L of bovine sialoglycoproteins significantly upregulated the expression of ST6β-galactosideα-2,6-sialyltransferase-1（ST6GAL1）in NHDF cells and increasedα2,6sialylation on the cell surface,but had no significant effects onα2,3 sialylation.Conclusions:1.Serotonin-magnetic functionalized particles were prepared by reacting the amino group of serotonins with epoxy groups on the surface of epoxy-coated Fe₃O₄ magnetic particles,which were effective for purifying sialoglycoproteins from bovine milk;2.Bovine sialoglycoproteins are able to inhibit the adhesion of avian and human influenza virus particles to host cells,and theirα2,3/6-linked sialic acids play a major anti-adhesive role.3.Bovine lactoferrin（b Lf）possesses bothα2,3-linked andα2,6-linked sialic acids,but mainlyα2,6-linked sialic acids.Sodium periodate at concentration of 10m M was effective in destroying the sialylated glycans without changing the protein structure of b Lf.Theα2,6-linked sialic acids on b Lf were able to inhibit the adhesion of human influenza virus to host cells.4.Bovine sialoglycoproteins were able to promote the proliferation and migration of NHDF cells,accelerate the contraction of FPCL,and inhibit the secretion of COL-I,indicating bovine sialoglycoproteins have the potential to accelerate wound healing and inhibit scar formation.In addition,bovine sialoglycoproteins could also upregulate the expression of ST6GAL1 in NHDF cells and increaseα2,6 sialylation on the cell surface,suggesting bovine sialoglycoproteins may prevent or delay the reduction ofα2,6 sialylation on the surface of dermal fibroblasts during human aging and have a potential for anti-skin aging.