Structural diversity at the beginning of multicellular eukaryotic complex N-glycan biosynthesis

N-linked glycosylation is one of the most important post-translational modifications of proteins. Current knowledge of multicellular eukaryote N-glycan biosynthesis suggests that N-glycans are produced in the endoplasmic reticulum (ER) and Golgi apparatus through conserved biosynthetic pathways. According to this biosynthesis, only one isomer of GlcNAc(Man3GlcNAc2) and two isomers of GlcNAc2(Man3GlcNAc2) are generated, and all the larger complex N-glycans are generated from these isomers. In this study, we applied our latest mass spectrometry method, logically derived sequence tandem mass spectrometry (LODES/MSn) to sodium ion adducts of N-glycan to re-examine the N-glycan structures of GlcNAc(Man3GlcNAc2) and GlcNAc2(Man3GlcNAc2) extracted from various animalia and cells. Parts of the structures were cross-checked using enzyme digestion. Many isomers not predicted by the biosynthetic pathway were identified. Moreover, many samples showed that these unusual isomers were the dominant isomers, indicating the involvement of additional biosynthetic pathways in N-glycan generation. The complex N-glycans extracted from fused lobes, MGAT-II, MGAT-IVa, or MGAT-IVb knockout of Drosophila melanogaster reveals another two additional biosynthetic pathways. The results suggest that (1) additional biosynthetic pathways exist and one should be very careful in the GlcNAc(Man3GlcNAc2) and GlcNAc2(Man3GlcNAc2) N-glycan structural identification by using the current biosynthetic pathways and (2) enzyme β-N-acetylglucosaminidase in higher eukaryotes may not only locate in lysosome.