Identification of novel high mannose N-glycan isomers undescribed by current multicellular eukaryotic biosynthetic pathways

Abstract

N-linked glycosylation is one of the most important post-translational modifications of proteins. Current knowledge of multicellular eukaryote N-glycan biosynthesis suggests high mannose N-glycans are generated in the endoplasmic reticulum and Golgi apparatus through conserved biosynthetic pathways. As a part of post-translational modifications, lipid dolichol-phosphate linked oligosaccharide Glc3Man9GlcNAc2 is transferred to proteins, and glucoses and mannose are sequentially removed by various ER- and Golgi-localized glucosidases and -1,2-mannosidases. According to reported biosynthetic pathways, four Man7GlcNAc2 isomers, three Man6GlcNAc2 isomers, and one Man5GlcNAc2 isomer are generated during this process. In this study, we applied our latest mass spectrometry method, logically derived sequence tandem mass spectrometry (LODES/MSn) to re-examine high mannose N-glycans extracted from various multicellular eukaryotes. LODES/MSn identified many high mannose N-glycan isomers previously unreported in plantae, animalia, cancer cells, and fungi. Importantly, their synthesis is yet described by known biosynthetic pathways, thereby suggesting additional and unidentified pathways for these N-glycans isomers in multicellular eukaryotic cells.

A simple tandem mass spectrometry method for structural identification of pentose oligosaccharides

“…The molecular structure in the chromatogram of each composition was identified by (1) comparing the relative abundance of isomers, as measured through MS in this study and through NMR in another study, 53 and (2) using the dehydration propensity rule discovered in previous research. 50,51 For example, the relative percentages of the areas under the blue, orange, green, and pink curves in Fig. 1(Ba) are 70.8%, 22.3%, 5.4%, and 1.5%, respectively.…”

“…[43][44][45][46] LODES/MS n has been used to determine the linkage positions, anomeric configurations, and stereoisomers of three common hexoses (glucose, galactose, and mannose) and two common N-acetylhexosamines (N-acetylglucosamine and N-acetylgalactosamine) in oligosaccharides. [47][48][49][50] Because it does not require a mass spectrum library of oligosaccharide standards, LODES/MS n is particularly useful for oligosaccharides with a structure that has not been identified previously. 49,50 LODES/MS n uses intact oligosaccharides, reducing the sample loss in permethylation/ reduction and simplifying the sample preparation process.…”

“…[47][48][49][50] Because it does not require a mass spectrum library of oligosaccharide standards, LODES/MS n is particularly useful for oligosaccharides with a structure that has not been identified previously. 49,50 LODES/MS n uses intact oligosaccharides, reducing the sample loss in permethylation/ reduction and simplifying the sample preparation process. The dissociation mechanisms of Xyl, 51 Ara, and Rib 52 have been studied recently, and extending LODES/MS n to pentose oligosaccharides is a natural progression.…”

Identification of the High Mannose N-Glycan Isomers Undescribed by Conventional Multicellular Eukaryotic Biosynthetic Pathways

“…In this study, we applied LODES/MS n to determine the structures of high mannose N -glycans of various species which are not glycosylation mutants. Many N -glycan isomers newly found in plantae, animalia, cancer cells, and fungi cannot be described by the conventional biosynthetic pathways . Using the N -glycans we found, we constructed a high mannose N -glycan database consisting of the retention time and CID mass spectra of all possible Man n GlcNAc 2 ( n = 5, 6, 7) isomers.…”

“…Many N -glycan isomers newly found in plantae, animalia, cancer cells, and fungi cannot be described by the conventional biosynthetic pathways. 35 Using the N -glycans we found, we constructed a high mannose N -glycan database consisting of the retention time and CID mass spectra of all possible Man n GlcNAc 2 ( n = 5, 6, 7) isomers. Many N -glycans in this database are not found in current N -glycan libraries.…”