Selective Control of Oligosaccharide Transfer Efficiency for the N-Glycosylation Sequon by a Point Mutation in Oligosaccharyltransferase

Abstract

Asn-linked glycosylation is the most ubiquitous posttranslational protein modification in eukaryotes and archaea, and in some eubacteria. Oligosaccharyltransferase (OST) catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. Inefficient oligosaccharide transfer results in glycoprotein heterogeneity, which is particularly bothersome in pharmaceutical glycoprotein production. Amino acid variation at the X position of the Asn-XSer/Thr sequon is known to modulate the glycosylation efficiency. The best amino acid at X is valine, for an archaeal Pyrococcus furiosus OST. We performed a systematic alanine mutagenesis study of the archaeal OST to identify the essential and dispensable amino acid residues in the three catalytic motifs. We then investigated the effects of the dispensable mutations on the amino acid preference in the N-glycosylation sequon. One residue position was found to selectively affect the amino acid preference at the X position. This residue is located within the recently identified DXXKXXX(M/I) motif, suggesting the involvement of this motif in N-glycosylation sequon recognition. In applications, mutations at this position may facilitate the design of OST variants adapted to particular N-glycosylation sites to reduce the heterogeneity of glycan occupancy. In fact, a mutation at this position led to 9-fold higher activity relative to the wild-type enzyme, toward a peptide containing arginine at X in place of valine. This mutational approach is potentially applicable to eukaryotic and eubacterial OSTs for the production of homogenous glycoproteins in engineered mammalian and Escherichia coli cells.N-linked glycosylation is the most ubiquitous posttranslational protein modification in eukaryotes and archaea, and in some eubacteria (1-4). It plays an important role in determining the biological activities of glycoproteins as well as the medicinal efficacy of many pharmaceutical recombinant glycoproteins. The covalent attachment of an oligosaccharide occurs on the side chain of an asparagine residue in a polypeptide chain, within the Asn-X-Ser and Asn-X-Thr sequences, where X is any amino acid except for proline (5, 6). About one-third of the potential N-glycosylation sequons are not glycosylated in extracellular proteins (7-9). This fact indicates that the presence of a consensus sequence is essential but not sufficient for N-glycosylation. The percentage of proteins modified at a particular sequon is referred to as "site occupancy." A recent comprehensive proteome analysis of mouse tissues and blood plasma revealed that more than 98% of N-linked glycosylated peptides derived from glycoproteins were not found in their unmodified form, indicating the high occupancy of N-glycosylation sites (10). Thus, for most N-glycosylation sequons in vivo, the N-glycan attachment is robust, resulting in a homogeneously glycosylated (100% occupancy) or unglycosylated (0% occupancy) site.In contrast, for some recombinant glycoproteins produced in cultur...

N-Linked Glycosylation in Archaea: a Structural, Functional, and Genetic Analysis

“…The recently reported full-length structure of AglB-L shows the lysinetype pocket (198). Site-directed mutation of the lysine in the DK motif to alanine (D574A) was also found to affect the amino acid preference at the X position of the sequon (201). This mutational change led to an enhanced preference for arginine and lysine at the X position.…”

“…Since the E. coli-produced versions are functional, no archaeon-specific PTM is necessary for activity, nor is the presence of any other archaeon-specific subunit (201).…”

“…AglBs from P. furiosus (30,201), A. fulgidus (185), and M. voltae (110) have been expressed and purified from E. coli and were shown in vitro to catalyze glycan transfer to target acceptor peptides. Since the E. coli-produced versions are functional, no archaeon-specific PTM is necessary for activity, nor is the presence of any other archaeon-specific subunit (201).…”

“…For instance, a novel DK motif (DXXKXXX[M/I], where X is any amino acid) is found in a kinked helix adjacent to the WWDYG motif in the tertiary structure of AglB from P. furiosus (30). The functional importance of this novel motif was demonstrated by mutating the parallel region in yeast Stt3 (30) and, later, in AglBs from A. fulgi-dus (185) and P. furiosus (201). While the DK motif is found in eukaryotic Stt3 and most AglB proteins, other AglBs and bacterial PglBs contain a so-called MI motif (MXXIXXX[I/V/W]) at the corresponding location.…”

Comparative Analysis of Archaeal Lipid-linked Oligosaccharides That Serve as Oligosaccharide Donors for Asn Glycosylation

“…Oligosaccharyl Transfer Assay-The recombinant AglB enzymes of the long paralog of P. furiosus (PfAglB-L) and the long paralog and one of the two short paralogs of A. fulgidus (AfAglB-L and AfAglB-S1) were expressed in Escherichia coli and purified to homogeneity in the presence of 1% (w/v) n-dodecyl ␤-D-maltoside (7,28,29). As alternatives to the recombinant AglB enzymes of P. calidifontis and S. solfataricus, the LLO-depleted membrane fractions that contained the endogenous AglB proteins were prepared for the oligosaccharyl transfer assay.…”