Structural determination of the N-glycans of a lepidopteran arylphorin reveals the presence of a monoglucosylated oligosaccharide in the storage protein

The structures of the oligosaccharides attached to arylphorin from Chinese oak silkworm, Antheraea pernyi, have been determined. Arylphorin, a storage protein present in fifth larval hemolymph, contained 4.8% (w/w) of carbohydrate that was composed of Fuc:GlcNAc:Glc:Man==0.2:4.0:1.4: 13.6 moles per mole protein. Four moles of GlcNAc in oligomannose-type oligosaccharides strongly suggest that the protein contains two N-glycosylation sites. Normal-phase HPLC and mass spectrometry oligosaccharide profiles confirmed that arylphorin contained mainly oligomannose-type glycans as well as truncated mannose-type structures with or without fucosylation. Interestingly, the most abundant oligosaccharide was monoglucosylated Man 9 -GlcNAc 2 , which was characterized by normal-phase HPLC, mass spectrometry, Aspergillus saitoi α-mannosidase digestion, and 1 H 600 MHz NMR spectrometry. This glycan structure is not normally present in secreted mammalian glycoproteins; however, it has been identified in avian species. The Glc 1 Man 9 GlcNAc 2 structure was present only in arylphorin, whereas other hemolymph proteins contained only oligomannose and truncated oligosaccharides. The oligosaccharide was also detected in the arylphorin of another silkworm, Bombyx mori, suggesting a specific function for the Glc 1 Man 9 GlcNAc 2 glycan. There were no processed glucosylated oligosaccharides such as Glc 1 Man 5 - 8 GlcNAc 2 . Furthermore, Glc 1 Man 9 GlcNAc 2 was not released from arylophorin by PNGase F under nondenaturing conditions, suggesting that the N-glycosidic linkage to Asn is protected by the protein. Glc 1 Man 9 GlcNAc 2 may play a role in the folding of arylphorin or in the assembly of hexamers.