Enzymatic transfer of oligosaccharide from oligosaccharide-lipids to an Asn-Ala-Thr containing heptapeptide

Enzymatic N‐glycosylation of synthetic ASN‐X‐THR containing peptides

“…The data we report here extend our work [4] showing that thyroid rough microsomes catalyze the transfer of oligosaccharide from [Mrfa-r4C] oligosaccliaride-lipids to a synthetic human thyroglobulin heptapeptide and further to its d~itropl~enylated derivative. Our aim has been to further investigate specificity requirements of the oligosaccharide transferase, using as tools the following series of synthetic peptides: (I) H-Ala-Leu-Glu-Asn-Ala-Thr-Arg-NH, _~_._ (II) DNP-Ala-Leu-GluAsn-Ala-Thr-Arg-NH,…”

“…Dinitrophenylation was as before. Results of amino acid analysis are given in table 1. were as in [4]. Incubation assays (with -250 /rg pig thyroid rough microsomal protein in 75 ~1 final vol.)…”

“…Incubation assays (with -250 /rg pig thyroid rough microsomal protein in 75 ~1 final vol.) were as in [4], except for the following points: (i) Final concentration of DMSO in incubates was increased from 5% to 10%; (ii) Each assay contained 3-6 X lo3 cpm of were washed twice with 10% trichloracetic acid before solubilization and counting. When reduced and alkylated bovine a-lactalbumin was tested as acceptor the entire incubation was stopped and precipitated by 10% trichloracetic acid-5% phosphotungstic acid.…”

“…In [4] we have commented upon the increased efficiency conferred on the thyroglobulin heptapeptide by attachment of a lipophilic DNP-group: it probably results from a facilitated access to the membranous enzyme. In peptide VI the aromatic tyrosine residue might somewhat similarly favour the final binding to the transferase.…”

“…Recently lipid pyrophospho-oligosaccharides, postulated as necessary intermediates to initiate N-glycosylation, were shown, when supplied to microsomes, to be effective donors to proteinic acceptors presenting at least one vacant Asn-X-zk sequence: unfolded forms of proteins [Z], polypeptide fragments from CNBr cleavages [3] or a synthetic heptapeptide [4]. It was proposed [S] on the grounds of structural examination of numerous glycoproteins, that this basic tripeptide sequence was a necessary, although probably not sufficient, requirement for an asparagine residue to be N-glycosylated.…”