¹³C- and ¹⁵N-Labeled Peptide Substrates as Mechanistic Probes of Oligosaccharyltransferase

Abstract

The carboxamide moiety that links the carbohydrate and protein moieties in N-linked glycoproteins has been unambiguously determined to arise intact from asparagine by the use of chemically synthesized Bz-[4-13C, 15N]Asn-Leu-Thr-NH2 as an oligosaccharyltransferase (OST) substrate. Bz-[4-13C]Asn-Leu-Thr-NH2 was also synthesized and used to evaluate a proposed mechanism of OST catalysis similar to that of glutamine-dependent amidotransferases using 15NH4OAc as a potential external nucleophile. Analysis of NMR and MS spectra of the isotopically labeled peptides and the resulting biosynthesized glycopeptides indicates that free 15NH3 is not lost from the doubly labeled substrate during catalysis nor can exogenous 15NH3 intercept any of several postulated enzyme-bound species. These results indicate that OST-catalyzed glycosylation does not follow a mechanism involving the transient generation of exchangeable "NH3". Thus, in contrast to several glutamine-dependent amidotransferases, OST catalysis does not lead to transient scission of the asparagine beta-carboxamide C-N bond. Together with previously published results, these data argue against nucleophilic activation of the asparagine beta-carboxamide moiety being the underlying chemical mechanism for OST-catalyzed glycosylation of peptides.

Synthesis and Evaluation of Tripeptides Containing Asparagine Analogues as Potential Substrates or Inhibitors of Oligosaccharyltransferase

“…Amide nitrogens are poor nucleophiles, and a reasonable N-glycosylation mechanism must explain the enhanced reactivity of the Asn β-carboxamide. ,− Various N-glycosylation mechanisms have been proposed, all focusing on an OST-mediated activation of the Asn β-carboxamide. While our previous research has ruled out several mechanisms based on analogy to glutamine-dependent amidotransferases involving fully formed reactive intermediates, ,, other mechanisms involving more transient species are plausible. ,, …”

A Biotin Capture Assay for Oligosaccharyltransferase

“…After 18 h the reaction mixture was diluted with EtOAc (30 ml) and extracted with 5% citric acid (30 ml), sat aq NaHCO 3 (30 ml), and brine. The organic layer was dried, filtered, and concentrated to obtain product (6, (7). TFA (7.5 ml) was added to a solution of N ␣ -Boc-L-Lys(N ⑀ -Cbz)-L-Thr-NH 2 (6, 0.39 g, 0.8 mmol) in CH 2 Cl 2 (5 ml) and stirred at room temperature.…”

Chemoenzymatic Methods for the Synthesis of Glycoproteins

“…In the biosynthesis of eukaryotic N-glycoproteins, the oligosaccharyltransferase (OST) is the sole enzyme responsible for transferring a large N-glycan precursor (Glc 3 Man 9 GlcNAc 2 ) en bloc from a dolichol-phosphate glycolipid to a consensus sequon NXS/T on the nascent unfolded protein. − Eukaryotic OST is in fact a multisubunit protein complex consisting of at least 9 subunits, and all of them are transmembrane proteins . Some early in vitro enzymatic studies from Coward, Imperiali, and several other laboratories have shown that OST is able to transfer truncated N-glycan core from the corresponding glycosyl-phosphate dolichol to synthetic peptides containing the consensus NXS/T sequence. − However, a practical application of the eukaryotic OST for in vitro glycoprotein synthesis has not been fulfilled. Major hurdles include the instability of the eukaryotic enzyme complex, the complexity of the membrane-bound multiple protein subunits that are required for catalytic activity of the STT3 subunit, and the difficulty to obtain the dolichol-associated glycolipids as the substrates.…”