Structural Characterisation of Sulfolobus Glycoproteins by Mass Spectrometry

A detailed structural understanding of archaeal glycoproteins and the genes involved in their biosynthesis is important to our understanding of the function and integrity cell surface structures. We are using mass spectrometry as a tool for glycan structural determination of archaeal glycoproteins and applying glycoproteomic strategies and technologies that have evolved for the functional analysis of bacterial glycosylation and optimized on studies of the S-layer glycoprotein from the archaea Haloferax volcanii (Abu-Qarn et al., J Mol Biol (2007) 374:1224–36). Currently we are undertaking glycoproteomic studies on glycoproteins from Sulfolobus species, which phylogenetically are closer to the eukarya than other archaea. In contrast to the glycans found on the S-layer of H. volcanii, which like other previously characterized archaeal glycoproteins, contain linear glycan sequences which bear no similarity to eukaryotic glycans, Sulfolobus S-layer glycoproteins have been found to carry chitosbiose linked, mannose containing N-glycans. Initial mapping strategies looking at proteolytic digest products from the S-layer glycoprotein of S. acidocaldarius yielded particularly rich data on the C-terminal domain and identified a heterogeneous family of glycans, with the largest having a composition Glc1Man2GlcNAc2 plus 6-sulfoquinovose (QuiS). Remarkably all the consensus sequences that have been mapped have been found to be glycosylated (Peyfoon et al., Archaea (2009) 29;754101–11). Given the mature protein contains 31 predicted glycosylation sites, it is likely that there is a dense coat of glycans that blanket the cell surface of S. acidocaldarius. Currently we are exploring the biosynthetic pathways involved in S-layer glycosylation and here we will present data from the functional analysis of the N-glycan biosynthetic pathway. In complementary work, we are looking at S. solfataricus and S. islandicus, where we have discovered very similar glycan decorations and like S. acidocaldarius, it appears that the majority, if not all, of the consensus sequence sites are occupied.