Sighting of unusual N-glycans by mass spectrometry

All glycoproteomic investigation methods are based on the knowledge of the synthetic process in the cells. In particular, N-glycosylation is a highly ordered, sequential process that encompasses different cellular compartments. The nascent protein is glycosylated in the endoplasmic reticulum, where the precursor oligosaccharide Glc3Man9GlcNAc2 is first transferred en bloc to the polypeptide chain, and then processed in the Golgi. All the resulting N-linked glycans share the common pentasaccharide core structure of Man3-GlcNAc2 and are distinguished in complex, hybrid, and highmannose types. The huge variability of N-glycan structures basically relies on the type and position of attached sugars and branching. Basing on this biosynthetic process and on the knowledge of the enzymes present in the different organisms, the N-glycan analysis by mass spectrometry is thus less complex, so that it is often sufficient to acquire the molecular mass to delineate the structure. Here we will present three examples dealing with the first description of unconventional N-glycan structures synthesized by totally or partially different biosynthetic pathways. Primarily, it will be presented the N-glycans characterization of Paramecium Bursaria Chlorella Virus 1 (PBCV-1) major capsid protein (MCP) Vp54. Chloroviruses have a long evolutionary history, probably forgoing the eukaryotes development, thus it has been hypothesized that they could own a different glycosylation machinery. The structures of the four N-linked glycans attached to PBCV-1 MCP consist of a set of oligosaccharides not previously found in all the three domains of life. Very interestingly, these glycan structures are not located in a typical N-X-(T/S) consensus site. A second example will regard the characterization of serum N-glycans of a patient with ALG12 deficiency (ALG-12 CDG), a CDG type 1 defect. Intact serum transferrin showed, as expected, underoccupancy of Nglycosylation site. Surprisingly, total serum proteins and IgG N-glycans showed some peculiar alterations, consisting in accumulating amount of specific and unusual high-mannose and hybrid structures. Finally, it will be described very strange case of a patient with multiple genetic mutations that, perhaps interacting, lead to generation of hypersialylated N-glycans structures, never reported before in human