Comprehensive analysis of α 2-3-linked sialic acid specific Maackia amurensis leukagglutinin reveals differentially occupied N-glycans and C-terminal processing.

Seeds of Maackia amurensis constitutes two sialic acid specific agglutinins known as leukagglutinin and hemagglutinin. Maackia amurensis leukagglutinin (MAL) recognizes α2-3-linked sialic acid present mainly in N-glycans and composed of two disulfide linked monomers. It exhibits potential N-glycosylation sites (four PNGs) which have been assumed to undergo differential occupancy. In this study we have characterized the site specific macro- and microheterogeneity of monomers in detail by analysing N-glycopeptides and peptides through liquid chromatography coupled to ion trap mass spectrometer in MS mode (LC-MS). We observed the presence of mainly paucimannose N-glycans at Asn, Asn and Asn whereas a high mannose type with varying Man occurs at Asn. Interestingly Asn and Asn exhibited differential occupancy which was evident by the presence of non-glycosylated peptides. This has contributed to the difference in molecular mass of monomers upon SDS-PAGE. Further the presence of disulfide linked peptides confirmed the covalent linkage of monomers which also undergoes uniform C-terminal processing.