RELEASE OF OLIGOSACCHARIDE-PHOSPHATE DURING THE N-GLYCOSYLATION PROCESS : A ”BY-PASS” IN THE DOLICHOL CYCLE

Abstract

We have previously described that N-glycosylation of proteins was accompanied by the release of oligosaccharide-phosphates and neutral oligosaccharides, both originating from lipid intermediates. In order to get a better understanding of the metabolic relationships between these oligosaccharide species, we have examined the synthesis and fate of lipid intermediates ina simpler biological system: a glycosylation mutant of CHO cells which does not synthesize Man-P-Dol (B3F7 cell line). B3F7 mutant CHO cells were incubated with tritiated mannose at low glucose concentration and in the presence of glucosidase and mannosidase inhibitors (castanospermine and deoxymanno jirimycin respectively). After extraction and purification, the glycan moieties of glycoproteins, lipid intermediates, oligosaccharidephosphates and neutral oligosaccharides were analyzed by HPLC. It iinteresting to note that oligosaccharide-phosphates were not glucosylated as was the pool of oligosaccharide-PP-Dol and that, in contrast, neutral oligosaccharides were glucosylated as were the glycan moieties of newly glycosylated proteins. These results indicate that oligosaccharide-phosphates originate from the cleavage of the pyrophosphate bond of non-glucosylated oligosacharide-PP-Dol. The glucosylation of lipid intermediates channels them through the glycosylation of proteins and through the formation of neutral oligosaccharides which could be, at some steps, related to the protein glycosylation process itself, Thus, the cleavage of non glucosylated lipid intermediates into oligosaccharide-phosphates represents a “by-pass” which allow direct regeneration of P-Dol. This “by-pass” may control the availability and the structural suitability of lipid intermediates for protein glycosylation.