Effects Of Glycosyl Modification On The Allergens From Mud Crab (Scylla Paramamosain)

Food allergy has been become of increasing concern to the food safety and public health.As one of eight major allergenic foods proposed by FAO/WHO, the allergenic incidences wereraising recently. Tropomyosin (TM) and arginine kinase (AK) were important allergens fromcrab. The allergen was generally glycoprotein with acidic isoelectric point, it was reported thatglycosylation played an important role in formation of antigenic determinant. However, reportsabout the sugar characteristic of TM and AK are few. Therefore, it was important to discuss therole of glycosyl modification in TM and AK for further study to crab allergen.In the study, we selected Scylla paramamosain as material. Firstly, purified TM and AKfrom crab were needed. PAS staining, phenol-sulfuric acid and β-elimination were applied to TMand AK, and then the total sugar content of allergens from seafood was discussed. Secondly,modern biotechnology was used to analyze the IgE binding activity of TM and AK treated byenzyme and chemical deglycosylation. Thirdly, TM and AK reacted with two reducing sugar toinvestigate the effect of Maillard reaction on their IgE binding activity and digestibility.TM and AK were purified from Scylla paramamosain by isoelectric precipitation,ammonium sulfate fractionation, column chromatography, etc. TM and AK were demonstratedto be glycoproteins by PAS staining, phenol-sulfuric acid method, β-elimination and modernbiotechnology, with0.2%and1.7%of the total sugar content, respectively. TM and AK onlycontained O-glycans, respectively. Compared with TM and AK from crab and shrimp, the totalsugar content of TM from shellfish and octopus was higher, and the carbohydrate content of AKfrom shrimp was lower than that of crab. When glycans were removed by enzymaticdeglycosylation, it was found that no obviously reduce in molecular weight of TM and AK.Further study western-blotting and inhibition ELISA using sera from allergenic patients showedno elimination of IgE binding activity of both allergens. However, periodate treatment couldresult in great reduction of IgE binding activity of TM and AK. During the glycosylation byMaillard reaction, it was found that the rate of glycosylation of TM and AK with ribose wasmore quikly than with glucose. The glycosylation of TM was more rapidly than that of AK.Compared to dry method, Maillard reaction in wet method would slow down the rate ofglycosylation. The glycosylated products of Maillard reaction would lead to the decrease of IgEbinding activity of TM and AK, and enhance their digestibility of trypsin (for TM) or pepsin (for AK). Finally, the ability of AK binding to antibody was enhanced during Maillard reaction ofcrab crude reacted with glucose.In summary, the study firstly analyzed the glycoprotein properties of TM and AK fromScylla paramamosain. TM and AK were demonstrated to be glycoproteins with0.2%and1.7%of total sugar content, both with O-glycan. Enzymatic, chemical deglycosylation and Maillardreaction were applied to investigate the role of carbohydrate structure. We cleared and definedperiodate treactment was more effective to eliminate the IgE binding activity of TM and AK thanenzymatic deglycosylation. During the glycosyl modification by Maillard reaction, dry methodwould be more effective than wet method, and the glycosylation degree of TM was higher thanthat of AK. It became more resistant to pepsin digestion for glycosylated TM after Maillardreaction, and glycosylated AK was also resistant to trypsin digestion. The antigenicity of AKwas enhanced in crab crude with Maillard reaction. The results will lay the foundation for thewhole investigation of crab allergens and development of low-allergenic food and allergen.