N-linked glycosylation represents a critical post-translational modification that influences protein structure and function. Traditional model of N-glycan biosynthesis in multicellular eukaryotes propose that these glycans are assembled within the endoplasmic reticulum and Golgi apparatus via highly conserved pathways. According to this model, biosynthesis yields a single structural variant of GlcNAc(Man3GlcNAc2) and two variants of GlcNAc2(Man3GlcNAc2), which then serve as the foundational precursors for all more complex N-glycan forms. In the present work, we employed mass spectrometry and enzymatic digestion to elucidate the structures of GlcNAc(Man3GlcNAc2) and GlcNAc2(Man3GlcNAc2) isolated from a range of animal species and cell types. Structural assignments were independently validated through enzymatic synthesis approaches. Our analyses uncovered numerous glycan isomers that are not accounted for by the conventional biosynthetic model of mammalian cells, with several of these unexpected structures appearing as the predominant forms in multiple samples, indicating unusual enzymatic activities occur in some organisms and cell types, which are not universal but specialized. Examination of complex N-glycans obtained from fused lobes as well as MGAT-II, MGAT-IVa, and MGAT-IVb knockout strains of Drosophila melanogaster agrees to the two additional biosynthetic routes suggested in previous study. Collectively, these findings highlight the need for caution when assigning structures to GlcNAc(Man3GlcNAc2) and GlcNAc2(Man3GlcNAc2) glycans.
