Studise On The Structure And Immunological Activity Of N-glycan Of Royal Jelly Glycoprotein

Royal jelly(RJ)is a thick,milky-white fluid secreted by the hypopharyngeal and mandibular glands of young worker bees.RJ has been demonstrated multiple biological properties,such as antibacterial,anti-tumor,anti-allergic,anti-inflammatory and immunomodulatory effects.The immunomodulatory effects of the RJ has been attributed to protein,especially royal jelly main protein 3(MRJP3)and apalbumin 1(MRJP1).In recent years,studies have found that glycans may be involved in immune responses,so N-glycans are play an important role in the study of immunomodulation of royal jelly.In this paper,the royal jelly glycoprotein was used as the research object to study the type and structure of N-glycan in royal jelly,and the relationship between glycans and immunological activity was studied,which provided a reference for the study of royal jelly immune response.The main findings are as follows:In this paper,the ultracentrifugation method is used to super-separate the royal jelly to obtain the upper layer solution,the middle layer solution and the lower layer precipitate.The different components of the royal jelly are freeze-dried to obtain the upper,middle and lower layers lyophilized powder of royal jelly.The basic components of the upper,middle and lower lyophilized powders were analyzed.It was found that the protein contents in the lower layer precipitate was 57.44%,the total sugar content was 12.8%,and the content of 10-HDA was 8.9%.The main protein of the lower layer royal jelly was found to be MRJP1 by SDS-PAGE analysis,and the molecular weight was 55 kDa.The four protein bands of upper layer,the middle layer and the lower layer were identified by LC-MS.The 49kDa protein in the upper layer was the main royal jelly protein 2(MRJP2),the protein band of 60-70kDa was the MRJP3,the 55 kDa protein of the lower layer of royal jelly is main royal jelly protein 1(MRJP1).Study on the type and structure of N-glycans in the main protein of royal jelly.The N-glycan of main royal jelly protein 1(MRJP1)was released by peptide-F glycosidase(PNGase F),and the glycans were Permethylated.The structure of the glycan was studied by ESI-MS.The results showed that 12 N-glycan were detected in the lower layer of royal jelly.6 high mannose types,2 hyprid types,4 complex types.Some of theseglycans had isomers.The effect of the N-glycan of main protein of royal jelly on antigen immunological activity.The hydrophilicity,plasticity,?-turn and antigen linear epitope of MRJP1 were predicted by ProtScale and IEDB online software.The N-glycosylation site A28 of MRJP1 was found to be located in the epitope region.Deglycosylated proteins may have an effect on the binding of antigen antibodies.PNGase F enzyme cuts off the N-glycans in the main protein of royal jelly,and uses enzyme-linked immunosorbent assay(ELISA)to study the effect of glycans on the antigenicity of MRJP1.The indirect ELISA and competitive inhibition ELISA experiments were performed using the polyclonal antibody anti-MRJP1 serum of the prepared rabbit as an antibody,and it was confirmed that the deglycosylated MRJP1 effectively reduced the IgG binding ability of MRJP1.The immune model of Balb/c mice was established by using MRJP1 and deglycosylated MRJP1 as allergens.The level of MRJP1-specific IgE in the serum of mice was analyzed.It was found that the level of MRJP1-specific IgE in the intact MRJP1 immunized mice was significantly higher than glycosylated MRJP1 immunized mice.It is proved that the N-glycan of the MRJP1 participates in the immune response.