dolichol-monophosphate-mannose and Lymphoma

A review is presented of research carried out in this laboratory on two aspects of the dolichol pathway that may have regulatory influences on these events. (i) The validity of the phenomenon of the activation of the biosynthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol by dolichol-P-mannose is supported by experiments carried out on the Thy-1-negative mouse lymphoma cell. While this cell cannot synthesize the activating compound, this capacity was retained and revealed upon the addition of exogenous dolichol-P-mannose. (ii) The topographical orientation of the GlcNAc-transferases that catalyze the biosynthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol was investigated in microsomes from the liver of the embryonic chick using dolichol phosphate liposomes as an exogenous substrate. The formation of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol was inhibited by trypsinization under conditions where the native orientation of the microsome was maintained, as indicated by the latency of mannose-6-phosphatase. Both GlcNAc-lipids were detected on free liposomes after incubation with intact microsomes, and in the same proportions as found on the microsome. From these and other studies, evidence was obtained indicating the cytoplasmic orientation of the GlcNAc-transferases that catalyze the synthesis of the first two intermediates of the dolichol pathway.

Topics: Animals; Antigens, Surface; Carbohydrate Sequence; Cell Membrane; Chick Embryo; Dolichol Monophosphate Mannose; Glycolipids; Glycosylation; Liposomes; Lymphoma; Membrane Glycoproteins; Microsomes, Liver; Molecular Sequence Data; N-Acetylglucosaminyltransferases; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Oligosaccharides; Thy-1 Antigens; Tumor Cells, Cultured; Tunicamycin

Deficient expression of glycoinositol phospholipid (GPI) anchored proteins in affected paroxysmal nocturnal hemoglobinuria (PNH) cells has been traced to a defect in GPI anchor assembly. In a previous study (Schubert, J., Schmidt, R. E., and Medof, M. E. (1993) J. Biol. Chem., in press) we characterized the biosynthesis of putative Man-containing GPI anchor precursors in normal peripheral blood lymphocytes and investigated assembly of these intracellular GPI intermediates in CD48- affected and CD48+ unaffected T and natural killer cell lines of PNH patients. We found that affected T cells from five patients exhibited a uniform defect in which dolichol-phosphoryl-Man was synthesized but no GPI mannolipids were expressed. In this study, membranes of patients' affected T cells were labeled with UDP-[3H]GlcNAc to evaluate earlier steps in GPI synthesis, and intact cells were fused to Thy-1- murine lymphoma mutants harboring different defects in early GPI assembly to test for the presence of corresponding or complementary lesions. In all cases, affected cell membranes failed to assemble GlcNAc-inositol phospholipid, the initial precursor of GPI anchor structures, and the intact cells failed to complement class A mutants while complementing other classes. Affected polymorphonuclear leukocytes from three additional patients of different origin were then labeled with [3H]Man and the labeling patterns found to correspond to those obtained with the T lymphocytes. Taken together the data indicate that the genetic lesion in PNH cells resides in a DNA element which: 1) encodes a product required for the synthesis of GlcNAc-inositol phospholipid, 2) corresponds to that altered in class A Thy-1- murine lymphoma mutants, and 3) is commonly affected in different patients.

Topics: Acetylglucosamine; Animals; Antigens, CD; CD48 Antigen; Cell Membrane; Dolichol Monophosphate Mannose; Glycosylphosphatidylinositols; Hemoglobinuria, Paroxysmal; Humans; Killer Cells, Natural; Lymphoma; Mannose; Mice; Mutation; Neutrophils; Trypanosoma brucei brucei; Uridine Diphosphate N-Acetylglucosamine

Dolichol phosphate-mannose has been shown previously to stimulate the biosynthesis of N-acetylglucosaminyl-diphosphate-dolichol (E. L. Kean (1985) J. Biol. Chem. 260, 12561-12571). Although the class E Thy-1-negative mutant mouse lymphoma cells are unable to synthesize dolichol phosphate-mannose, the addition of this compound exogenously to membranes from the mutant cells brought about a stimulation of N-acetylglucosaminyl-lipid synthesis similar to that obtained with membranes from wild type cells. The retention of this activity by the mutant cells supports the suggestion of a regulatory role for dolichol phosphate-mannose as an intrinsic property of the glucosaminyltransferase which catalyzes the initial reaction of the dolichol pathway.

Topics: Acetylglucosamine; Animals; Cell Line; Dolichol Monophosphate Mannose; Glucosamine; Glycolipids; Lymphoma; Membrane Lipids; Mice; Mutation; Polyisoprenyl Phosphate Sugars; Pyrophosphatases

Topics: Animals; Carbohydrate Conformation; Carbohydrate Sequence; Cell Line; Cell Membrane; Dolichol Monophosphate Mannose; Glycolipids; Guanosine Diphosphate Mannose; Kinetics; Lymphoma; Mice; Nucleoside Diphosphate Sugars; Oligosaccharides; Polyisoprenyl Phosphate Sugars

Topics: Animals; Carbohydrate Sequence; Cell Line; Dolichol Monophosphate Mannose; Kinetics; Lymphoma; Mannose; Mice; Mutation; Neoplasms, Experimental; Oligosaccharides; Polyisoprenyl Phosphate Sugars; Tritium