Quantitative assessment of the preferences for the amino acid residues flanking archaeal N-linked glycosylation sites

Mayumi Igura, Daisuke Kohda

    Research output: Contribution to journalArticle

    16 Citations (Scopus)

    Abstract

    Oligosaccharyltransferase (OST) catalyzes the transfer of an oligosaccharide to an asparagine residue in polypeptide chains. Using positional scanning peptide libraries, we assessed the effects of amino acid variations on the in vitro glycosylation efficiency within and adjacent to an N-glycosylation consensus, Asn-X-Ser/Thr, with an archaeal OST from Pyrococcus furiosus. The amino acid variations at the X -2, X -1 and X +1 positions in the sequence X -2-X -1-Asn-X-Ser/Thr-X +1 strongly influenced the glycosylation efficiency to a similar extent at position X. The rank orders of the amino acid preferences were unique at each site. We experimentally confirmed that the archaeal OST does not require an acidic residue at the -2 position, unlike the eubacterial OSTs. Pro was disfavored at the -1 and +1 positions, although the exclusion was not as strict as that at X, whereas Pro was the most favored amino acid residue among those studied at the -2 position. The overall amino acid preferences are correlated with a conformational propensity to extend around the sequon. The results of the library experiments revealed that the optimal acceptor sequence was PYNVTK, with a Km of 10 M. The heat-stable, single-subunit OST of P. furiosus is a potential candidate enzyme for the production of recombinant glycoproteins in bacterial cells. Quantitative assessment of the amino acid preferences of the OST enzyme will facilitate the proper design of a production system.

    Original languageEnglish
    Pages (from-to)575-583
    Number of pages9
    JournalGlycobiology
    Volume21
    Issue number5
    DOIs
    Publication statusPublished - May 1 2011

    Fingerprint

    Glycosylation
    Amino Acids
    Pyrococcus furiosus
    Peptide Library
    Asparagine
    Enzymes
    Oligosaccharides
    Libraries
    Glycoproteins
    Hot Temperature
    dolichyl-diphosphooligosaccharide - protein glycotransferase
    Scanning
    Peptides

    All Science Journal Classification (ASJC) codes

    • Biochemistry

    Cite this

    Quantitative assessment of the preferences for the amino acid residues flanking archaeal N-linked glycosylation sites. / Igura, Mayumi; Kohda, Daisuke.

    In: Glycobiology, Vol. 21, No. 5, 01.05.2011, p. 575-583.

    Research output: Contribution to journalArticle

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