EVALUATING THE ROLE OF CONSERVED AMINO ACIDS IN BACTERIAL O-OLIGOSACCHARYLTRANSFERASES BY IN VIVO, IN VITRO, AND LIMITED PROTEOLYSIS ASSAYS

MATIAS A. MUSUMECI; AMIRREZA FARIDMOAYER; YASUHARU WATANABE; MARIO F. FELDMAN

GLYCOBIOLOGY

OXFORD UNIV PRESS INC

Lugar: Oxford; Año: 2014 p. 39 - 50

0959-6658

Bacterial O-Oligosaccharyltransferases (O-OTases) constitute a growing family of enzymes that catalyse the transfer of a glycan from a lipid carrier to protein acceptors. O-OTases are inner membrane proteins that display limited sequence similarity, except for the Wzy_C signature domain also present in a predicted periplasmic loop of the WaaL ligase, the enzyme responsible for transferring the O antigen to the lipid A core. The mechanism of O-OTase-dependent glycosylation is poorly understood. In this work, conserved amino acid residues in the O-OTases were replaced with alanine in PglL, the O-OTase of Neisseria meningitidis. The activities of wild-type PglL and its mutant derivatives were analysed in vivo in engineered E. coli cells, and in in vitro assays. We identified two additional sites of pilin glycosylated exclusively by PglL in E. coli. Both sites are modified with phosphoglycerol (PG) by different enzymes in N. gonorrhoeae and N. meningitidis. Limited proteolysis experiments revealed a conformational change that is triggered upon interaction of the C-terminal region of PglL with the lipid-linked oligosaccharide (LLO) substrate. These experiments showed that Q178 and Y405 are required for optimal function, whereas H349 is essential for activity and performs a critical role in the interaction with LLO. The equivalent His residue is also essential for WaaL activity, which suggests a common mechanism for both enzymes, and supports the hypothesis that O-glycosylation and LPS synthesis are evolutionarily related. These results contribute to the elucidation of the mechanism of O-OTases, which are promising targets for novel antibiotics and present an enormous potential for glycoengineering novel vaccines and therapeutics.