Enzymatic N-glycosylation of synthetic Asn--X--Thr containing peptides.

The role of polyprenol sugar derivatives in the biosynthesis of certain eukaryotic glycoproteins has been partly described [ 11. 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. 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, (III) H Asn-Ala-Thr-NH, (IV) DNPAsn-Ala-Thr-NH, (V) DNP-Ala-Let.-Glu Asn-Pro-Thr-Arg-NH, __~_._ (VI) H-Tyr-Gln-SerAsn-Ser-~r-~et-NH~ ~--