In vivo N-glycosylation and fate of Asn-X-Ser/Thr tripeptides.

The minimum primary structural requirement for a tripeptide to serve as a substrate for oligosaccharyl transferase is the sequence -Asn-X-Ser/Thr-. In the present study the activities of three structurally different tripeptides containing acceptor sequences for oligosaccharyl transferase were compared in three systems: Xenopus oocytes, in which they were introduced into the cytoplasm by microinjection, cultured mammalian cells, and isolated rat liver microsomes. In the last two systems, the peptides were added exogenously to the culture or to the incubation medium, respectively. On the basis of lectin column and paper chromatographic analysis it was established that the microinjected acceptor tripeptides were glycosylated in Xenopus oocytes. However, lectin column analysis and retention of sensitivity to endoglycosidase H revealed that none of the three glycopeptides was processed to complex oligosaccharide chains and none was subsequently secreted. Rather, over a 24-h period the glycopeptides were degraded. Chloroquine was found to block this degradation process, but even under these conditions, the glycopeptides were not secreted into the medium. In the isolated microsomes the glycosylation of the acceptor tripeptides was time-dependent and the tripeptide with an iodotyrosine residue in the X position was found to be a poor substrate. When added to cultured mammalian cells, all three of the tripeptides were taken up, glycosylated, and subsequently secreted. These results are discussed in the context of the wide differences in glycosylation of the three peptides and their lack of secretion after glycosylation in Xenopus oocytes.

Glycosylation site binding protein, a component of oligosaccharyl transferase, is highly similar to three other 57 kd luminal proteins of the ER.

A 57 kd component of oligosaccharyl transferase, termed glycosylation site binding protein, specifically recognizes a photoaffinity probe containing the N-glycosylation site sequence Asn-Lys-Thr. It is present in the lumen of the ER (endoplasmic reticulum) and its release from this compartment results in a loss of N-glycosylation. Antibodies against this protein were used to identify cDNA clones from a lambda gt11 expression library. Analysis of its cDNA sequence reveals high sequence similarity to three other 57 kd luminal endoplasmic reticulum proteins: protein disulfide isomerase, the beta-subunit of prolyl hydroxylase, and thyroid hormone binding protein. This finding suggests that the capacity to recognize multiple polypeptide domains may reside in a single luminal protein that participates in co- and/or posttranslational modifications of newly synthesized proteins.

Subcellular localization of the synthesis and glycosylation of chondroitin sulfate proteoglycan core protein.

In order to study the temporal and topological events involved in the processing and assembly of chondroitin sulfate proteoglycan, a fractionation scheme involving differential centrifugation and discontinuous sucrose density gradient centrifugation was developed for homogenates of chick embryo sternal chondrocytes. The precursors in these subcellular fractions were examined by a series of pulse, pulse-chase, and continuous labeling experiments. When chondrocytes were pulsed for 20 min with [35S]methionine, an immunoprecipitable core protein precursor with an approximate molecular size of 376,000 Da was localized to the rough endoplasmic reticulum fractions. Further incubation under chase conditions showed the presence of the 376,000-Da species as well as two additional polypeptides of higher molecular masses in the smooth membrane-enriched fractions within the next 2 h. This translocation did not occur in the presence of the energy transfer inhibitor carbonyl cyanide-m-chlorophenylhydrazone. The labeling pattern of the newly synthesized core protein precursor with either [3H] mannose or [3H]glucosamine showed that N-linked oligosaccharide addition was found on the earliest synthesized product in the rough endoplasmic reticulum, and the addition of this oligosaccharide was inhibited by co-incubation with tunicamycin. Furthermore, the high mannose oligosaccharide was susceptible to cleavage by endo-beta-N-acetylglucosaminidase H, while upon chase approximately 56 and 31% of the glucosamine- and mannose-labeled oligosaccharides, respectively, were processed to resistant forms, presumably in the Golgi complex. Both direct assay of glycosyl- and sulfotransferases requisite for addition of chondroitin sulfate chains and sensitivity of intracellular precursors to chondroitinase, keratanase , and endoglycosidase H suggest that only the N-linked oligosaccharides are added in the rough endoplasmic reticulum and glycosaminoglycan chain addition occurs predominantly in smooth membranes.

Synthesis and mobilization of flagellar glycoproteins during regeneration in Euglena.

Flagellar glycoprotein synthesis and mobilization of flagellar glycoprotein pools have been followed during flagellar regeneration in Euglena. The glycosylation inhibitor tunicamycin has little effect on either regeneration kinetics or the complement of flagellar peptides as seen in SDS acrylamide gels, but tunicamycin totally inhibits incorporation of exogenously supplied [14C]xylose into flagellar glycoproteins. Moreover, deflagellated cells pulsed with tunicamycin for 0 min or more, regenerated for 180 min, and then redeflagellated are completely or partially inhibited from undergoing a second regeneration even when tunicamycin is no longer present. These facts are interpreted as indicating that Euglena retains sufficient glycoprotein pool for one complete flagellar assembly. Some of this pool is present on the cell surface since [125I]-labeled surface peptides can be chased into the regenerating flagellum. Glycosylation may also be taking place in the flagellum directly because [14C]xylose has been found in three flagellar fractions: glycoprotein and two others, which are lipophilic and have properties similar to those described for lipid-carrier glycoprotein intermediates in other systems. Pulse-chase experiments also suggest a precursor-product relationship between the presumptive lipid carriers and flagellar glycoproteins. From these results a model is postulated in which Euglena is visualized as retaining sufficient pool of glycoprotein for one complete flagellar regeneration, but the pool is normally supplemented by active xylosylation in situ during regeneration.