Requirement of the Lec35 gene for all known classes of monosaccharide-P-dolichol-dependent glycosyltransferase reactions in mammals

The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosacch arides (LLOs) and glycosylphosphatidylinositols, which are important for fu nctions such as protein folding and membrane anchoring, respectively. The h amster Lec35 gene is shown to encode the previously identified cDNA SL15, w hich corrects the Lec35 mutant phenotype and predicts a novel endoplasmic r eticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 a re characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinosito l intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-doli chol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35 p. In addition, the SL15-encoded protein was selective for MPD compared wit h GPD, suggesting that an additional GPD-selective Lec35 gene product remai ns to be identified. The predicted amino acid sequence of Lec35p does not s uggest an obvious function or mechanism. By testing the water-soluble MPD a nalog mannose-beta -1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, i t was determined that Lec35p is not directly required for the enzymatic tra nsfer of mannose from the donor to the acceptor substrate. These results sh ow that Lec35p has an essential role for all known classes of monosaccharid e-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.