Isomeric Separation and Recognition of Anionic and Zwitterionic N-glycans from Royal Jelly Glycoproteins

• Published in: Molecular & cellular proteomics Vol. 17; no. 11; pp. 2177 - 2196
• Main Authors: Hykollari, Alba, Malzl, Daniel, Eckmair, Barbara, Vanbeselaere, Jorick, Scheidl, Patrick, Jin, Chunsheng, Karlsson, Niclas G., Wilson, Iain B.H., Paschinger, Katharina
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2018; The American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Anions; Array analysis; Cattle; Cell and Molecular Biology; Cell- och molekylärbiologi; Chemical Fractionation; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Ethanolamines - metabolism; Fatty Acids - chemistry; Fucose - metabolism; glucuronic acid; Glucuronic Acid - metabolism; Glycomics; Glycoproteins - metabolism; Glycoside Hydrolases - metabolism; Glycosylation; HPLC; Isomerism; Mannose - metabolism; Mass Spectrometry; N-Glycosylation; phosphoethanolamine; Polysaccharides - chemistry; Polysaccharides - isolation & purification; Proteome - metabolism; royal jelly; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfates - metabolism; sulphate; T antigen; Tandem Mass Spectrometry; Array analysis; Glycomics; Mass Spectrometry; phosphoethanolamine; sulphate; glucuronic acid; N-Glycosylation; T antigen; HPLC; royal jelly
• ISSN: 1535-9476; 1535-9484; 1535-9484
• DOI: 10.1074/mcp.RA117.000462

Royal jelly has special properties as the sole food for queen honeybees, but is also considered to be bioactive in other animals. Using an off-line LC-MALDI-TOF MS approach, we reveal a highly complex N-glycome with novel anionic and zwitterionic N-glycans being present on specific royal jelly glycoproteins. These glycans are shown in microarray format to bind protein ligands, revealing that there exists potential for interactions with the human innate immune system. [Display omitted] Highlights •The N-glycome of honeybee royal jelly is more diverse than previously thought•Antennal phosphoethanolamine and glucuronic acid are among the special features•Anionic/zwitterionic glycans are recognized by a pentraxin and anti-HNK-1 antibody Royal jelly has received attention because of its necessity for the development of queen honeybees as well as claims of benefits on human health; this product of the hypopharyngeal glands of worker bees contains a large number of proteins, some of which have been claimed to have various biological effects only in their glycosylated state. However, although there have been glycomic and glycoproteomic analyses in the past, none of the glycan structures previously defined would appear to have potential to trigger specific biological functions. In the current study, whole royal jelly as well as single protein bands were subject to off-line LC-MALDI-TOF MS glycomic analyses, complemented by permethylation, Western blotting and arraying data. Similarly to recent in-depth studies on other insect species, previously overlooked glucuronic acid termini, sulfation of mannose residues and core β-mannosylation of the N-glycans were found; additionally, a relatively rare zwitterionic modification with phosphoethanolamine is present, in contrast to the phosphorylcholine occurring in lepidopteran species. Indicative of tissue-specific remodelling of glycans in the Golgi apparatus of hypopharyngeal gland cells, only a low amount of fucosylated or paucimannosidic glycans were detected as compared with other insect samples or even bee venom. The unusual modifications of hybrid and multiantennary structures defined here may not only have a physiological role in honeybee development, but represent epitopes recognized by pentraxins with roles in animal innate immunity.