Synthetic substrates for thyroid oligosaccharide transferase. Effects of peptide chain length and modifications in the Asn-Xaa-Thr-region.

The N-glycosylation of proteins is initiated by transfer of preassembled oligosaccharides from lipid carriers to an asparagine residue in the sequence Asn-Xaa-Ser(Thr). Various synthetic peptides were previously shown to act as effective acceptors when supplied to microsomal membranes. The present work was undertaken to delineate further within such peptides the structural determinants required for the N-glycosylation process. Several new peptides were synthesized to evaluate the effects of the chain length and of the modification on the asparagine, Xaa or threonine residues: asparagine was replaced by glutamine, Xaa by proline or N-methylated alanine, threonine by an homoserine residue. Furthermore, both side chains of asparagine and threonine were simultaneously replaced by those of glutamine and homoserine respectively to restore a putative hydrogen bonding between the carbonyl and hydroxyl groups. The assays were performed with a solubilized form of the oligosaccharide transferase and the acceptor capacities of the peptides expressed by the ratio V/Km of their apparent kinetic parameters V and Km. Results showed that N-acetyl-tripeptide amides are excellent substrates for the enzyme. All modifications in the -Asn-Xaa-Thr- region resulted in a loss of acceptor capacity. The importance of the conformational aspect is discussed.

Transfer of glucose in the biosynthesis of thyroid glycoproteins. II. Possibility of a direct transfer of glucose from UDP-glucose to proteins.

When thyroid rough microsomes are incubated with labeled UDPglucose, extensive labeling occurs on endogenous protein. The present study reports various time-course experiments of glucose incorporation from UDPglucose either into endogenous acceptors or into exogenous synthetic peptide, whether or not conditions are used which depress dolichol-P-glucose synthesis. The results are not compatible with the presently accepted idea of oligosaccharide lipids as sole donors of labeled glucose to proteins. Some structural properties of glucose-labeled oligosaccharides released from proteins by endo-beta-N-acetylglucosaminidase H were also investigated. They appeared to be of a single species and less degradable by alpha-mannosidase than their lipid-linked counterpart. Since glucose-labeled glycopeptides were found susceptible to a beta-glucosidase, it is proposed that some labeled glucose residues are involved in beta-linkages. The possibility is raised that glucose might be directly transferred from UDPglucose to endogenous glycoproteins.

Transfer of glucose in the biosynthesis of thyroid glycoproteins. I. Inhibition of glucose transfer to oligosaccharide lipids by GDP-mannose.

Thyroid rough microsomes catalyzed the synthesis of glucose-containing oligosaccharide lipids which were compared to those extracted from labeled thyroid cells and were found to be largely similar. Glucose transfer to these oligosaccharide lipids in the microsomal system was shown to be markedly depressed by an addition of GDPmannose. This sugar nucleotide, already at 1 microM, blocked dolichol-P-glucose synthesis, thus restraining further glucosylation of oligosaccharide lipids. Using this concentration of radioactive GDPmannose in the incubation medium lead to the detection of three glucose containing mannose-labeled oligosaccharide lipids. Double labeling experiments suggested a precursor-product relationship between them. Previously labeled oligosaccharide lipids, containing glucose or not were compared in their efficiency to act as donors of their oligosaccharide chain to an exogenous synthetic Asn-X-Thr containing peptide. It was found that the presence of glucose did not significantly influence the transfer. Free glucose was released during the reaction when using the glucose-labeled oligosaccharide lipid.