MgCl 2 and DNase I (Roche Applied Science) were added to final concentrations of 5 m m and 0.1 mg/ml, respectively. After 4 h of incubation at 37 ☌, cells were harvested by centrifugation, resuspended in 30 m m Tris (pH 8) and 300 m m NaCl supplemented with 1 m m EDTA and 1 g/liter lysozyme, and incubated for 1 h at 4 ☌. When cultures reached A 600 = 0.5, 0.2% arabinose or 1 m m isopropyl β- d-thiogalactopyranoside was added for induction of protein expression. Ampicillin (100 mg/liter) or chloramphenicol (25 mg/liter) was added to the medium as needed. coli DH5α cells harboring a plasmid for expression of a relevant protein were grown in volumes of 1 liter at 37 ☌ in LB medium. We concluded that NGT integrates polypeptide recognition common to the OST-based modification into a novel framework, resulting in a general N-glycosylation system that operates in the cytoplasm. We compared the acceptor substrate range of NGT and OST and observed a highly similar specificity of the two different enzymes. Instead, we found that another glucosyltransferase was able to elaborate the N-linked glucose. pleuropneumoniae is an inverting NGT that transfers a glucose or galactose moiety to asparagine, but it does not further elongate the N-linked monosaccharide. We adopted MS and gel electrophoresis for detection of glycosyltransferase activity using peptides and proteins as substrates and performed NMR studies to characterize the reaction adducts. To characterize accurately the mechanisms of this alternative N-glycosylation pathway and to gain insight into the specificity of the key enzyme N-glycosyltransferase (NGT), we established a platform suitable for in vitro glycosylation and undertook a detailed analysis of the reaction products. 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. It therefore exhibits similar acceptor site specificity as eukaryotic OST, despite the unrelated predicted structural architecture and the apparently different catalytic mechanism. N-Glycosyltransferase is an inverting glycosyltransferase and recognizes the N X(S/T) consensus sequence. 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. 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). N-Linked glycosylation is a frequent protein modification that occurs in all three domains of life.
Glycobiology and Extracellular Matrices.