Abstract
N-Linked glycosylation is a frequent protein modification that occurs in all three domains of life. This process involves the transfer of a preassembled oligosaccharide from a lipid donor to asparagine side chains of polypeptides and is catalyzed by the membrane-bound oligosaccharyltransferase (OST). We characterized an alternative bacterial pathway wherein a cytoplasmic N-glycosyltransferase uses nucleotide-activated monosaccharides as donors to modify asparagine residues of peptides and proteins. N-Glycosyltransferase is an inverting glycosyltransferase and recognizes the NX(S/T) consensus sequence. It therefore exhibits similar acceptor site specificity as eukaryotic OST, despite the unrelated predicted structural architecture and the apparently different catalytic mechanism. The identification of an enzyme that integrates some of the features of OST in a cytoplasmic pathway defines a novel class of N-linked protein glycosylation found in pathogenic bacteria.
Highlights
A recent report demonstrated that an HMW1C homolog from Actinobacillus pleuropneumoniae mediates N-linked glycosylation of the H. influenzae HMW1A protein [9]
We show that the HMW1C homolog from A. pleuropneumoniae is an inverting NGT that transfers a glucose or galactose moiety to asparagine, but it does not further elongate the N-linked monosaccharide
When we extended the search to retrieve low score hits, we found that HMW1C of H. influenzae displays low similarity to the C-terminal region of the product of the XCC0866 gene from X. campestris (24% identity within a 236-amino acids sequence)
Summary
The pioneering work of St Geme and co-workers [7, 8] subsequently showed that the modified asparagine residues are found within the same consensus sequence recognized by OST (i.e. NX(S/T)) and that the enzyme responsible for this modification is the HMW1C protein, capable of transferring Glc or Gal from nucleotide-activated substrates. Glycosylation Analysis of Synthetic Peptides—Enzymatic activity using different sugar donors or peptide acceptors was assessed with 1.4 g (0.46 M) of NGT and/or ␣6GlcT in a 50-l final volume of 25 mM Tris buffer (pH 7.2).
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