Discovery and characterization of a new class of O-linking oligosaccharyltransferase from bacteria in the Moraxellaceae family

ABSTRACT: Bacterial protein glycosylation can be mediated by oligosaccharyltransferases (OTases) that transfer a preassembled lipid-linked oligosaccharide or polysaccharide en bloc to acceptor proteins. O-linking OTases transfer O-antigen or capsular polysaccharides to the side chains of serine or threonine residues. Three major families of bacterial O-linking OTases have been described so far: PglL, PglS, and TfpO. TfpO enzymes are limited to transferring only short glycans whereas there are no clear upper limits for the other two families. Herein, we describe the discovery of a novel family of bacterial O-linking OTases from Moraxellacea bacteria that are similar in size and sequence to TfpO enzymes but can transfer long-chain bacterial glycans to acceptor proteins. Bioinformatic analyses show that these enzymes cluster in different clades than known bacterial OTases. Using a representative enzyme from Moraxella osloensis termed TfpMMo, we determine that the enzyme glycosylates the C-terminal amino acid side chain of a pilin protein and find that pilin fragments as short as three amino acids are substrates for the OTase. The ability of TfpMMo to transfer long-chain polysaccharide shows that this ability is not limited to the PglS and PglL families. TfpMMo is also shown to have broad substrate specificity and can transfer diverse glycans including those with glucose, galactose, or 2-N-acetyl sugars at the reducing end. The glycan substrate promiscuity of TfpMMo could allow this enzyme to be used to produce bacterial glycoconjugate vaccines. The discovery of a new class of O-linking OTase furthers our understanding of the mechanisms that underly glycan specificity by these and other O-linking OTases and enables more comparative studies of this important enzyme family.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Moraxella Osloensis

SUBMITTER: Nichollas Scott  

LAB HEAD: Christian M. Harding

PROVIDER: PXD033468 | Pride | 2022-10-13

REPOSITORIES: Pride