dolichol-monophosphate-mannose and Thymoma

Dolichol-phosphate-mannose (Dol-P-Man) serves as a donor of mannosyl residues in major eukaryotic glycoconjugates. It donates four mannosyl residues in the N-linked oligosaccharide precursor and all three mannosyl residues in the core of the glycosylphosphatidylinositol anchor. In yeasts it also donates one mannose to the O-linked oligosaccharide. The yeast DPM1 gene encodes a Dol-P-Man synthase that is a transmembrane protein expressed in the endoplasmic reticulum. We cloned human and mouse homologues of DPM1, termed hDPM1 and mDPM1, respectively, both of which encode proteins of 260 amino acids, having 30% amino acid identity with yeast Dpm1 protein but lacking a hydrophobic transmembrane domain, which exists in the yeast synthase. Human and mouse DPM1 cDNA restored Dol-P-Man synthesis in mouse Thy-1-deficient mutant class E cells. Mouse class E mutant cells had an inactivating mutation in the mDPM1 gene, indicating that mDPM1 is the gene for class E mutant. In contrast, hDPM1 and mDPM1 cDNA did not complement another Dol-P-Man synthesis mutant, hamster Lec15 cells, whereas yeast DPM1 restored both mutants. Therefore, in contrast to yeast, mammalian cells require hDPM1/mDPM1 protein and a product of another gene that is defective in Lec15 mutant cells for synthesis of Dol-P-Man.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; CHO Cells; Cricetinae; Dolichol Monophosphate Mannose; Genetic Complementation Test; Glycosylphosphatidylinositols; Humans; Kinetics; Mammals; Mannosyltransferases; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Saccharomyces cerevisiae; Sequence Alignment; Sequence Homology, Amino Acid; Thymoma; Thymus Neoplasms; Transfection; Tumor Cells, Cultured

Many eukaryotic cell surface proteins are bound to the membrane via the glycosylphosphatidylinositol (GPI) anchor that is covalently linked to their carboxy-terminus. The GPI anchor precursor is synthesized in the endoplasmic reticulum (ER) and post-translationally linked to protein. We cloned a human gene termed PIG-B (phosphatidylinositol glycan of complementation class B) that is involved in transferring the third mannose. PIG-B encodes a 554 amino acid, ER transmembrane protein with an amino-terminal portion of approximately 60 amino acids on the cytoplasmic side and a large carboxy-terminal portion of 470 amino acids within the ER lumen. A mutant PIG-B lacking the cytoplasmic portion remains active, indicating that the functional site of PIG-B resides on the lumenal side of the ER membrane. The PIG-B gene was localized to chromosome 15 at q21-q22. This autosomal location would explain why PIG-B is not involved in the defective GPI anchor synthesis in paroxysmal nocturnal hemoglobinuria, which is always caused by a somatic mutation of the X-linked PIG-A gene.

Topics: Amino Acid Sequence; Animals; Base Sequence; Chromosome Mapping; Chromosomes, Human, Pair 15; Cloning, Molecular; DNA, Complementary; Dolichol Monophosphate Mannose; Endoplasmic Reticulum; Genes; Glycosylphosphatidylinositols; Humans; Mannose; Mannosyltransferases; Mice; Molecular Sequence Data; Recombinant Fusion Proteins; Sequence Deletion; Thymoma; Thymus Neoplasms