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Vol. 12, Issue 2, 487-501, February 2001

Requirement of the Lec35 Gene for All Known Classes of Monosaccharide-P-Dolichol-dependent Glycosyltransferase Reactions in Mammals

Monika Anand,^* Jeffrey S. Rush, Sutapa Ray,^*§ Marie-Agnes Doucey,^¶ Jennifer Weik,^* Felecia E. Ware,^* Jan Hofsteenge, Charles J. Waechter, and Mark A. Lehrman^*#

 ^*Department of Pharmacology, UT-Southwestern Medical Center, Dallas, Texas 75390-9041;  Department of Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0084; and  Friedrich Miescher-Institut, CH-4002, Basel, Switzerland

The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are 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 glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose--1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-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.

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Present addresses: Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, Piscataway, NJ, 08854; ^§University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1060; ^¶Ludwig Institute for Cancer Research, Chemin des Boveresses 1555, CH1066 Epalinges, Switzerland.

^# Corresponding author. E-mail address: mlehrm{at}mednet.swmed.edu.

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Molecular Biology of the Cell
Vol. 12, 487-501, February 2001
Copyright © 2001 by The American Society for Cell Biology

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