Investigating the glycosylation reaction catalyzed by the Neisseria meningitids O-Oligosaccharyltransferase

MATÍAS A. MUSUMECI; MARIO F. FELDMAN

Banff

Simposio; 9th Annual Carbohydrate Symposium; 2013

Alberta Glycomics Centre

Oligosaccharyltransferases (OTases) constitute a family of glycosyltransferases that catalyse the transfer of an oligosaccharide from undecaprelol lipid carrier to an acceptor molecule, commonly a protein. These enzymes can transfer a variety of glycan structures, including polysaccharides, to different protein acceptors. This property confers the OTases with great biotechnological potential as these enzymes could be applied to produce glycoconjugates relevant to the pharmaceutical industry. Furthermore, OTases are thought to be involved in pathogenesis mechanisms. The OTases are classified as N-OTases and O-OTases, depending on the nature of the glycosylation reaction. The N-OTases catalyse the glycan transfer to amine groups in asparagines in a reaction named N-linked glycosylation. The O-OTases catalyse the O-linked glycosylation reaction, which involves the attachment of glycans to hydroxyl groups of serine or threonine residues. The aim of this work is to characterize PglL, which is the O-OTase from Neisseria meningitidis and it has been employed as prototype to study the O-OTase enzyme family. PglL is responsible for the glycosylation of multiple proteins. The first protein known to be recognized by this enzyme is the type IV pilin, encoded by the pilE gene. Type IV pilins form a pilus structure at the bacterial surface and are important for host cell adhesion and virulence. Previously was demonstrated that PglL can transfer different glycan portions from the lipid carrier undecaprenol pyrophosphate to the PilE protein acceptor. However, the influence of glycan structure on the efficiency of the reaction is unknown. We have investigated pilin glycosylation by PglL from substrates with different glycan portions, employing in vitro glycosylation assays and quantitative western-blot analysis. The role of conserved amino acids within all O-OTase family members on the activity of PglL was also analyzed by site directed mutagenesis. Through In vivo and In vitro glycosylation assays we identified amino acids that appear to be involved directly in the catalysis and other which are essential to the structural integrity of PglL. Experiments of limited proteolysis suggest a conformational rearrangement of the C-terminal domain of PglL upon interaction with glycan substrate. These experiments combined with MS-MS analysis allow us to identify a domain PglL likely involved in the interaction with glycan substrates. Our results provide novel insights about PglL functionality, which could serve as basis for future improvements concerning the production of glycoconjugates of biotechnological interest.