Abstract
Oligosaccharyltransferases (OTases) are enzymes that catalyze the transfer of an oligosaccharide from a lipid carrier to an acceptor molecule, commonly a protein. OTases are classified as N-OTases and O-OTases, depending on the nature of the glycosylation reaction. The N-OTases catalyze the glycan transfer to amide groups in asparagines in a reaction named N-linked glycosylation. The O-OTases are responsible for protein O-linked glycosylation, which involves the attachment of glycans to hydroxyl groups of serine or threonine residues. These enzymes exhibit a relaxed specificity and are able to transfer a variety of glycan structures to different protein acceptors. This property confers OTases with great biotechnological potential as these enzymes can produce glycoconjugates relevant to the pharmaceutical industry. Furthermore, OTases are thought to be involved in pathogenesis mechanisms. Several aspects of the functionality of OTases are not fully understood. In this work, we developed a novel approach to perform kinetic studies on PglL, the O-OTase from Neisseria meningitidis. We investigated the importance of the acyl moiety of the lipid glycan donor substrate on the functionality of PglL by testing the efficiency of glycosylation reactions using synthetic substrates carrying the same glycan structure but different acyl moieties. We found that PglL can function with many lipids as glycan donors, although the length and the conformation of the lipid moiety significantly influenced the catalytic efficiency. Interestingly, PglL was also able to transfer a monosaccharide employing its nucleotide-activated form, acting as a Leloir glycosyltransferase. These results provide new insights on the function and the evolution of oligosaccharyltransferases.
Highlights
PglL-like enzymes catalyze protein O-linked glycosylation in many bacteria
We investigated the importance of the acyl moiety of the lipid glycan donor substrate on the functionality of PglL by testing the efficiency of glycosylation reactions using synthetic substrates carrying the same glycan structure but different acyl moieties
We found that PglL can function with many lipids as glycan donors, the length and the conformation of the lipid moiety significantly influenced the catalytic efficiency
Summary
PglL-like enzymes catalyze protein O-linked glycosylation in many bacteria. Results: The catalytic efficiency of this enzyme correlates with the length and conformation of the acyl chain of glycan donors. N- and O-linked protein glycosylation take place in the bacterial cytoplasm preceding the export of the glycoproteins to the surface In this case, cytosolic Leloir (nucleotide-dependent) glycosyltransferases transfer the sugars from nucleotide-activated donors to the protein acceptors. PglL shows a remarkable lack of glycan specificity and is able to transfer virtually any glycan from the undecaprenylpyrophosphate carrier to proteins [19] This important feature makes this enzyme a promising tool for glycoengineering novel glycan-based vaccines, therapeutics, and diagnostics tools [20, 21]. We applied this method to assay the relevance of the aliphatic chains present in the glycan donor carriers in the glycosylation reaction. PglL can act as a Leloir glycosyltransferase, being able to use a nucleotide-activated monosaccharide as substrate
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