Selective control of oligosaccharide transfer efficiency for the N-glycosylation sequon by a point mutation in oligosaccharyltransferase

Mayumi Igura, Daisuke Kohda

    Research output: Contribution to journalArticle

    13 Citations (Scopus)

    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-X-Ser/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 ofOSTvariants adapted to particularN-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.

    Original languageEnglish
    Pages (from-to)13255-13260
    Number of pages6
    JournalJournal of Biological Chemistry
    Volume286
    Issue number15
    DOIs
    Publication statusPublished - Apr 15 2011

    All Science Journal Classification (ASJC) codes

    • Biochemistry
    • Cell Biology
    • Molecular Biology

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