Investigation of the active site of oligosaccharyltransferase from pig liver using synthetic tripeptides as tools

Abstract

Oligosaccharyltransferase (OST), an integral component of the endoplasmic-reticulum membrane, catalyses the transfer of dolichyl diphosphate-linked oligosaccharides to specific asparagine residues forming part of the Asn-Xaa-Thr/Ser sequence. We have studied the binding and catalytic properties of the enzyme from pig liver using peptide analogues derived from the acceptor peptide N-benzoyl-Asn-Gly-Thr-NHCH3 by replacing either asparagine or threonine with amino acids differing in size, stereochemistry, polarity and ionic properties. Acceptor studies showed that analogues of asparagine and threonine with bulkier side chains impaired recognition by OST. Reduction of the beta-amide carbonyl group of asparagine yielded a derivative that, although not glycosylated, was strongly inhibitory (50% inhibition at approximately 140 microM). This inhibition may be due to ion-pair formation involving the NH3+ group and a negatively charged base at the active site. Hydroxylation of asparagine at the beta-C position increased Km and decreased Vmax, indicating an effect on both binding and catalysis. The threo configuration at the beta-C atom of the hydroxyamino acid was essential for substrate binding. A peptide derivative obtained by replacement of the threonine beta-hydroxy group with an NH2 group was found to display acceptor activity. This shows that the primary amine is able to mimic the hydroxy group during transglycosylation. The pH optimum with this derivative is shifted by approximately 1 pH unit towards the basic region, indicating that the neutral NH2 group is the reactive species. The various data are discussed in terms of the catalytic mechanism of OST, particular emphasis being placed on the role of threonine/serine in increasing the nucleophilicity of the beta-amide of asparagine through hydrogen-binding.

Asparagine-linked Oligosaccharides Present on a Non-consensus Amino Acid Sequence in the CH1 Domain of Human Antibodies

“…When the Ser residue at position 163 is eliminated, the nearest C-terminal potential hydrogen acceptor is a threonine residue at position 167. It has previously been reported that N-glycosylation could still occur if the side-chain hydroxyl group on the Ser/Thr amino acid in the ϩ2 position was replaced with an amide (13). Although Trp residues have a nitrogen atom in the indole ring, the Trp residue at position 161 is an unlikely candidate as a hydrogen acceptor, because the electron pair of the indole nitrogen is delocalized and participates in the aromatic system.…”

“…It was subsequently determined, through the use of synthetic peptides containing Thr analogues in the ϩ2 position, that introduction of charge or movement of the side-chain methyl group was not tolerated by the OST complex (13,14). The outcomes of these studies suggest the presence of a hydrophobic binding pocket in the active site of the OST complex, which results in preferential glycosylation of NXT relative to NXS due to the presence of the side-chain methyl group of Thr (15).…”

Unexpected reactivity and mechanism of carboxamide activation in bacterial N-linked protein glycosylation

“…Replacing the acceptor Asn in the sequon by the corresponding primary amine results in the residue 2,4-diaminobutanoic acid (Dab), which was reported to have inhibitory effects on eukaryotic OST 14,24 . We determined a K d of 10±0.25 mM for a synthetic peptide containing Dab at the acceptor position (Table 1, compound 9, Fig.…”

Glutamine-linked and Non-consensus Asparagine-linked Oligosaccharides Present in Human Recombinant Antibodies Define Novel Protein Glycosylation Motifs

“…Mechanistic studies involving mutation of the ϩ2 amino acid following the Asn residue have shown that a hydrogen acceptor is necessary to render the Asn residue sufficiently nucleophilic to displace the GlcNAc 2 Man 9 Glc 3 oligosaccharide from the dolichol donor (3,4). Further insight into the mechanism of N-glycosylation was obtained by replacing the ϩ2 amino acid in the consensus sequon with threonine analogues (5,6). The results from these studies indicated that the OST enzyme complex did not tolerate changes in the position of the threonine methyl group nor the introduction of charge at the ϩ2 residue.…”