Structural Characterization of Royal Jelly Sensitized Glycoprotein and the Effect of Glycosaminoglycan on Its Sensitization

Author:Lin Na

Supervisor:zhang hong


Degree Year:2019





Royal jelly(RJ)glycoproteins,especially major royal jelly protein families(MRJPs),contain more than 85%of the total RJ protein,and they are the most specific physiologically functional proteins in RJ.The glycan chain on the glycoprotein can largely alter the conformation of the protein and can regulate the biological function of the protein as well as affect the interaction between the protein and the protein.In view of the lack of studies on RJ glycoproteins,particularly glycan chains,glycopeptides and their glycosylation sites,this study aimed to perform a detailed structural characterization of glycoproteins in RJ.major royal jelly protein 1(MRJP1)is considered to be the major allergic glycoprotein in RJ,which can cause severe allergic reactions in susceptible populations.Therefore,it is necessary to establish a high specificity,high sensitivity and high accuracy quantitative method to limit the content of MRJP1 allergen for preventing allergic reactions after eating RJ in a susceptible population.In addition,many literatures have reported that RJ can cause immediate hypersensitivity reactions,but so far there has been no literature to study the sensitizing response of MRJP1 and its sensitization mechanism.Therefore,another goal of this study was to establish a MRJP1 sensitized mouse model,to initially explore its sensitizing mechanism and to screen related drugs that can alleviate its allergic reactions.Glycosaminoglycan(GAG)as a large molecule with many biological activities,chondroitin sulfate(CS)and dermatan(DS)have been shown to play an important role in the immune response in vivo and in vitro,and can enhance immunological activity and play a role in immune regulation.Therefore,this study preliminarily used GAG as a potential desensitizing chemical substance to study its immunomodulatory effects on MRJP1 sensitized mice,especially in terms of its role in desensitization,and to explore its mechanism of desensitization.In order to provide a theoretical basis for further research on the mechanism of food allergy and its treatment,and the development of anti-allergic drugs.The main research contents and results of this paper are as follows:Firstly,the two glycoproteins MRJP1 and MRJP2,which are the most abundant in RJ,were identified by proteomics combined with SDS-PAGE electrophoresis.The identified peptide coverages were 88.89%and 80.53%,and the identified peptide number is 298 and 126 respectively.A quantitative method was developed for the quantification of the sensitized glycoprotein MRJP1 in RJ based on a signature peptide and a stable isotope-labeled internal standard peptide by UPLC-MRM-MS/MS.The established method was then validated and finally was successfully applied to the accurate quantification of sensitized glycoproteins in various commercially available RJ samples.The protein quantification method has strong selectivity and specificity,high accuracy,good repeatability,wide linear range(5~1000 ng/mL),high recovery rate(85.33%~95.80%),low detection limit(LOD 7.77 μg/g RJ)and can be used for accurate quantification of MRJP1 in royal jelly samples,or extended to other types of glycoproteins.In addition,this method can also be used in the quantitative analysis of allergenic proteins and provide a theoretical reference for the limit detection standard of allergenic proteins.Secondly,the glycans of glycoprotein in RJ was characterized by the sugar chain released from the glycopeptide using PNGase F.Detailed structural characterization of the N-glycans released by the PNGase F and then permethylated by iodomethane was carried out using an ion trap mass spectrometer,direct injection with a needle pump,MSn scanning mode,and a CID fragmentation mode.Finally,13 N-glycans were identified,including 7 kinds of high mannose types(Man3~99GlcNAc2),5 kinds of complex types(Hex3HexNAc3,Hex4HexNAc3,Hex3HexNAc4,Hex3HexNAc5,Hex4HexNAc5)and one hybrid type(HexsHexNAc3).These N-glycans include bi-and tri-antennary structures.The sequential mass spectrometry MSn combined with CID fragmentation method can achieve a detailed analysis of the glycan structures,including its composition,molecular weight,structural branch and monosaccharide linkage.Subsequently,the glycosylation modification of the RJ glycoprotein was analyzed in detail from the glycopeptide level.The trypsin digest of RJ glycoprotein was isolated and identified using HILIC-ion trap-MSn,and the glycopeptide was characterized by automatic acquisition of MS2 and manual acquisition of MS3 mass spectrometry.Through the glycopeptide identification method established above,a total of 48 N-glycopeptides,6 glycosylation sites,and 26 N-glycans were identified in this study.These glycopeptides are mainly distributed in MRJP1 and MRJP2,and another glycopeptide belongs to bee glycoprotein 40S ribosomal protein S8(076756).Two glycosylation sites,N177 and N394,were identified in MRJP1 and three glycosylation sites,N145,N178 and N92,were identified in the glycoprotein MRJP2.All of the 26 sugar chains identified were N-glycans,and the types of glycans included high mannose type,complex type and hybrid type.We found three new glycosylation sites in the royal jelly proteins,and they were glycosylation site N92 whose corresponding peptide sequence is YDGVPSTLNVISGKTGK(MRJP2,glycan composition Man8GlcNAc2),glycosylation site N394 whose corresponding peptide sequence is M(Qxidation)VNNDFNFDDVNFR(MRJP1,glycan composition Man9GlcNAc2),and glycosylation site N84 whose corresponding peptide sequence is IIDVVYNASNNELVR(076756,glycan composition Man9GlcNAc2).These three new glycosylation sites were not predicted in the UniProt database and have not been reported in other relevant literature.Thirdly,MRJP1 polyclonal antibody was obtained by immunizing rabbit with MRJP1,and the affinity between GAGs and MRJP1 polyclonal antibody extracted in this study was investigated by ELISA.The GAGs with good affinity for MRJP1 polyclonal antibody were monkfish DS,monkfish CS and salmon CS.Subsequently,detailed structural characterization of GAGs with different affinity was performed,and the relationship between the structure of these polysaccharide molecules and their binding ability was analyzed.It was found that the affinity between the GAG molecule and the protein has a great relationship with the degree of sulfation,the type of the polysaccharide,the type and ratio of the disaccharide and the sulfation site.Lastly,the MRJP1 sensitization model of Balb/c mice and the attenuation of food allergy symptoms model of glycosaminoglycan administration were established.The allergic reaction and its alleviation in the model group and the treatment group were explored.The results showed that the allergic symptoms of the GAG and the dexamethasone positive control group were significantly relieved compared with the sensitized model control group.The monkfish CS low-and medium-dose group and the monkfish DS group can significantly reduce the total IgE,specific IgE in the serum of sensitized mice,and reduce the Th2 type cytokine IL-4 content in the supernatant of mouse spleen cells,thereby alleviating the allergies caused by MRJP1.The mechanism of GAG desensitization may be to reduce the allergic reaction by inhibiting the differentiation of antigen-specific Th2 cells.