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

Abstract

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.

Human protein paucimannosylation: cues from the eukaryotic kingdoms

“…Thus, mounting evidence supports a role for α1,3‐fucosylated PMGs in several invertebrate phyla, including nematodes and arthropods, in mediating immune processes within their hosts or predatory targets. A. mellifera , however, also secretes royal jelly comprising proteins carrying common PMGs (Glycans #2a/b, #4, #10a/b, and #12), a unique core β1,6‐mannosylated PMG (Glycan #vii) and a sulfated PMG (Glycan #xi) (Hykollari et al ., ). This unusual sulfo‐PMG (Glycan #xi) was amongst other PMGs (Glycans #4 and #12) also identified in the hair‐like ‘setae’ of the moth Hylesia metabus , which were found to mediate pro‐inflammatory immune reactions in humans (Cabrera et al ., ).…”

“…In addition, unusual sulfated PMGs (e.g. Glycan #xi) reportedly also decorated proteins expressed in High Five cells and Lymantria dispar larvae (Stanton et al ., ) and in A. mellifera royal jelly (Hykollari et al ., ). These sulfated glycans were not found elsewhere in our survey of eukaryotic PMPs but may reflect the limited number of glycomics studies on invertebrates, and analytical shortcomings in accurately detecting labile and anionic PMGs or PMG‐peptides substituted with non‐sugar modifications.…”

Anionic and zwitterionic moieties as widespread glycan modifications in non-vertebrates

“…1). In the case of molluscs (specifically one gastropod, Volvarina rubella, and one bivalve, Mytilus edulis), glucuronic acid modifies antennal fucose residues [19,20]; thus far in insects, whether mosquitoes, the fruit fly, the honeybee or moth species, glucuronic acid was found attached to galactose [21][22][23][24] to form a non-sulphated form of the so-called HNK-1 epitope, while in one filarial nematode (Dirofilaria immitis) N-acetylgalactosamine residues are glucuronylated [25]. Thereby, it is interesting that D. immitis is transmitted via mosquitoes, but it is unknown whether the 'common' Nglycan modification with glucuronic acid is relevant to the parasite's lifecycle.…”

“…Phosphorylcholine was found on core 'GalFuc' moieties of N-glycans of the gastropod Volvarina rubella [20] as well as on the antennae of a not insignificant modification of N-glycans from Lepidoptera, including cell lines used in biotechnology such as Trichoplusia ni HighFive [24]. In other insects, it is rather phosphoethanolamine that is found on N-glycans of the honeybee [23] and the O-glycans of wasps [127]; on the other hand, N-glycans from Diptera were not yet found to contain zwitterionic modifications, even though phosphoethanolamine linked to GlcNAc is a component of glycolipids from dipteran insects such as Drosophila and Calliphora [128,129] (see example in Fig. 2) or on O-glycans from mosquito larvae with the composition HexNAc 1-2 Hex 1 HexA 2 PE 1 [21].…”

Insights into the salivary N-glycome of Lutzomyia longipalpis, vector of visceral leishmaniasis

“…One of the first indications of the capacity of insects to produce complex type N-glycans came from bee venom phospholipase A2, which contains the core α1,3-fucose (an IgE epitope allergenic to humans). Anionic and zwitterionic N-glycans with up to three antennae have more recently been found in a range of insects [29][30][31][32] . Furthermore, Vandenborre et al 33 explored glycosylation differences comparing www.nature.com/scientificreports/ several economically important insects, and found glycoproteins to be involved in a broad range of biological processes such as cellular adhesion, homeostasis, communication and stress response.…”