In-Depth N-Glycosylation Reveals Species-Specific Modifications and Functions of the Royal Jelly Protein from Western (Apis mellifera) and Eastern Honeybees (Apis cerana)

Abstract

Royal jelly (RJ), secreted by honeybee workers, plays diverse roles as nutrients and defense agents for honeybee biology and human health. Despite being reported to be glycoproteins, the glycosylation characterization and functionality of RJ proteins in different honeybee species are largely unknown. An in-depth N-glycoproteome analysis and functional assay of RJ produced by Apis mellifera lingustica (Aml) and Apis cerana cerana (Acc) were conducted. RJ produced by Aml yielded 80 nonredundant N-glycoproteins carrying 190 glycosites, of which 23 novel proteins harboring 35 glycosites were identified. For Acc, all 43 proteins glycosylated at 138 glycosites were reported for the first time. Proteins with distinct N-glycoproteomic characteristics in terms of glycoprotein species, number of N-glycosylated sites, glycosylation motif, abundance level of glycoproteins, and N-glycosites were observed in this two RJ samples. The fact that the low inhibitory efficiency of N-glycosylated major royal jelly protein 2 (MRJP2) against Paenibacillus larvae (P. larvae) and the absence of antibacterial related glycosylated apidaecin, hymenoptaecin, and peritrophic matrix in the Aml RJ compared to Acc reveal the mechanism for why the Aml larvae are susceptible to P. larvae, the causative agent of a fatal brood disease (American foulbrood, AFB). The observed antihypertension activity of N-glycosylated MRJP1 in two RJ samples and a stronger activity found in Acc than in Aml reveal that specific RJ protein and modification are potentially useful for the treatment of hypertensive disease for humans. Our data gain novel understanding that the western and eastern bees have evolved species-specific strategies of glycosylation to fine-tune protein activity for optimizing molecular function as nutrients and immune agents for the good of honeybee and influence on the health promoting activity for human as well. This serves as a valuable resource for the targeted probing of the biological functions of RJ proteins for honeybee and medical communities.

Characterizing the Structure and Oligomerization of Major Royal Jelly Protein 1 (MRJP1) by Mass Spectrometry and Complementary Biophysical Tools

“…This relatively low coverage is partially attributed to the presence of glycans in G+ samples, which precluded the detection of peptides in the vicinity of glycosylation sites N144 and N177. 24 Control experiments using trypsin yielded a sequence coverage of 64%, only slightly higher than that for pepsin (Figure S2). The sequence stretches covered by trypsin mapping resemble those detected after pepsin digestion.…”

“…The limited sequence coverage seen with both proteases is consistent with the fact that MRJP1 experiences various post-translational modifications 9,20 in addition to glycosylation. 24 Any such covalent modifications will interfere with peptide matching, which relies on comparisons with the cDNA-derived MRJP1 sequence. 9 Analyses of the apisimin HDX behavior were not possible because of the low signal intensity of the corresponding peptides.…”

“…23,24 Sugars are bound mainly at N144 and N177, although other attachment sites also exist. 24 The glycans were shown to include a unique Gal β 1–3GalNAc unit.…”

“…23,24 Sugars are bound mainly at N144 and N177, although other attachment sites also exist. 24 The glycans were shown to include a unique Gal β 1–3GalNAc unit. 23 Deglycosylation by PNGase F causes a mass shift on sodium dodecyl sulfate (SDS) gels from an apparent MW of 56 kDa to an apparent MW of 47 kDa, 25 the latter being consistent with the amino acid sequence of the mature protein.…”

Current knowledge and perspectives of Paenibacillus: a review

“…Apis mellifera lingustica’s royal jelly proteins display higher antihypertension activity than those produced by Apis cerana cerana and is active at lower levels. Interestingly, this difference in activity grants Apis mellifera lingustica optimized molecular functioning and enhanced immune activity, and so the strain is more resistant to P. larvae than is Apis cerana cerana in that larval exposure to the spores results in infection less frequently [159]. Although the strain tested was ERIC I, this may help to explain the specificity observed in P. larvae ERIC II infections.…”