Analysis Of N-Glycosylation Of Soybean Glycoproteins And Study On Their Sensitization

Traditional chinese medicine theory that the soybean's taste of drug is not obvious,with high nutritional value,soybean has certain curative effect on diseases such as asthenia labor,internal injury,fever,typhoid fever and so on.However,it can cause human allergic reactions,which is one of the priorities of traditional chinese medicine and clinical safety drug usage.Most of the soybean allergens are glycoprotein,and numerous studies have shown that the glycan may be the antigenic determinant that causes allergic reactions.Currently,the research of soybean allergy is mainly focused on protein allergen,while the glycosylation of soybean allergen and its glycan structure are rarely reported.Glycosylation plays an important role in the function and structure of proteins.So,it has important practical significance for soybean allergic mechanism study on glycan structure and function,and it can lay the foundation for development of pharmacy research and utilization of soybean resources.In this paper,soybean seeds were used as an experimental material.The N-glycan structure,glycosylation sites and sensitization of soybean allergen glycoprotein are analyzed in detail.This study provided reference for study on sensitization mechanism of soybean.The main results and conclusions obtained are summarized as follows.1.The structure and quantitative distribution of N-glycans of various soybean glycoproteins were analyzed.The protein bands of soybean protein separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)were recovered for the release and labeling of N-glycans.Subsequently,the qualitative and quantitative analysis of N-glycans was carried out by ESI-MS,MS/MS and online LC-UV-MS/MS2.The results showed that 15 protein bands were obtained from SDS-PAGE gel,including 6 non-glycosylated proteins,2 glycosylated proteins mainly containing core al,3-fucosylated N-glycans associated with soybean allergy,and 7 glycosylated proteins mainly containing oligomannose-type N-glycans.This study first proved that the y-conglycinin,sucrose binding protein,?-amylase and ?-conglycinin subunit fragment were all glycosylated and first discovered that ?',? and ? subunits of ?-conglycinin all contained core?1,3-fucosylated N-glycans possibly associated with soybean allergy.2.The total protein N-glycans of mature soybean and immature soybean were qualitatively and quantitatively compared.The N-glycans of these two soybean samples released and labeled by chemical method were qualitatively and quantitatively analyzed by ESI-MS,MS/MS and online LC-ESI-MS/MS2.The results showed that both samples contained H3N2X1,H3N2X1F1,H5N2,H6N2,H7N2,H8N2 and H9N2 N-glycans,respectively.7S globulin of both samples contained H3N2X1,H3N2X1F1,H5N2,H6N2,H7N2,H8N2 and H9N2 N-glycans,respectively.None of these contains isomers.The content of al,3-fucosylated N-glycans and ?1,2-xylose N-glycans in mature soybean is significantly higher than that in immature soybean.3.Glycosylation sites of the soybean allergen glycoprotein ?-conglycinin and Gly m Bd 30K were analyzed in detail.The protein samples were hydrolyzed by Trypsin and Pepsin,and purified glycopeptide using Con A affinity chromatography,and enzymatic hydrolysed respectively by PNGase F and Endo H.The recovered peptides and glycans were analyzed in detail by ESI-MS and MS/MS.Finally,the analysis results were verified by database retrieval.These results showed that ?-conglycinin has 5 N-glycosylation sites,including the 199th and 455th Asn residues of the a subunit,the 215th and 489th Asn residues of the?' subunit and the 326th Asn residue of the ? subunit,and each glycosylation site is modified by the four high-mannose type N-glycans,including H5N2,H6N2,H7N2 and H8N2.Gly m Bd 30K has one N-glycosylation sites,at the 170th Asn,and glycosylation site is modified by the two complex type N-glycans,including H3N2F1X1 and H3N2X1.This study provided method for the identification of glycosylation sites and corresponding glycan structures of various glycoproteins,and offered theoretical basis for in-depth understanding of the specificity of soybean glycoprotein epitopes and allergic mechanism.4.The 7S globulin sensitization of mature soybean and immature soybean were compared.The 7S globulin of soybean was used as the research object,and mice allergy model was established by 7S globulin of mature soybean and immature soybean,respectively.The expression level of IgE and histamine were quantitatively compared and analyzed.Simultaneously,the specificity of IgE antibody titers of protein samples were measured by passive cutaneous anaphylaxis.The results showed that the levels of IgE and histamine of 7S globulin-sensitized mice of mature soybean,and the specificity of IgE antibody titers were significantly higher than the 7S globulin-sensitized mice of immature soybean.This indicates that the sensitization of 7S globulin of mature soybean is higher than the immature soybean.5.The activity of the N-glycans of soybean 7S globulin in allergy was analyzed.Balb/c mice allergy model was established by 7S globulin and deglycosylated 7S globulin of mature soybean,respectively.Then the corresponding mouse model was subjected to high-dose proteins treatment,respectively.Finally,the IgE antibody and histamine in the serum were quantitatively analyzed.The results showed that the levels of IgE and histamine in serum of 7S globulin-sensitized mice of mature soybean was significantly higher than the deglycosylated 7S globulin-sensitized mice(P<0.05,P<0.01).This study showed that N-glycan played an enhanced role in the process of 7S globulin in sensitization.