A new procedure for the preparation of GDP-[U-14C]mannose

Braell, William A.; Tyo, M. A.; Krag, Sharon S.; Robbins, Phillips W.

doi:10.1016/0003-2697(76)90229-3

Cited by 35 publications

(6 citation statements)

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“…[2-3H]Mannose (10-15 Ci/mmol) and UDP-[3H]glucose (3.1 Cilmmol) were obtained from Amersham Corp. (Arlington Heights, IL). mannose (approximately 100 mCi/mmol) was prepared as previously described (Braell et al, 1976). UDP-N-acetyl-D-[3H]glucosamine was synthesized as described by Owada and Neufeld (19821, using D-[6-3H]glucosamine hydrochloride (20-40 Ci/mmol) purchased from Amersham Corp.…”

Section: Methodsmentioning

confidence: 99%

A clonal derivative of tunicamycin‐resistant Chinese hamster ovary cells with increased N‐acetylglucosamine‐phosphate transferase activity has altered asparagine‐linked glycosylation

Waldman¹,

Oliver²,

Krag³

1987

Journal Cellular Physiology

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A population of Chinese hamster ovary (CHO) cells resistant to the antibiotic tunicamycin (TM) had previously been isolated (Criscuolo, B.A., and Krag, S.S. (1982) J. Cell Biol. 94:586-591) by a stepwise selection procedure using progressive increments of TM added to the medium. TM inhibits asparagine-linked glycoprotein biosynthesis by blocking the transfer of N-acetylglucosamine-1-phosphate from the sugar nucleotide UDP-N-acetylglucosamine to the isoprenoid lipid carrier, dolichyl phosphate. Four clonal derivatives were isolated from the TM-resistant population in the presence of 27 micrograms TM/ml and were found to overproduce the N-acetylglucosamine-phosphate transferase activity to the same extent (approximately 15-fold compared to wild-type cells). One of these clones, 3E11, was greater than 550-fold more resistant to TM than wild-type cells. The resistance phenotype remained during at least 2.5 months of growth in the absence of TM. 3E11 cells exhibited chromosomal translocations, but no homogeneously staining regions (HSR) or double minute chromosomes. The N-acetylglucosamine-phosphate transferase activity in 3E11 cells was membrane-associated and was inhibited by TM. A 140,000-dalton membrane protein and at least four other membrane proteins were enriched in 3E11 cells. Mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities were not elevated in membranes prepared from 3E11 cells. Asparagine-linked glycosylation was altered such that 3E11 cells synthesized primarily a truncated oligosaccharide, Man5GlcNAc2, perhaps due to the reduced amount of mannosylphosphoryldolichol relative to wild-type cells.

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Section: Methodsmentioning

confidence: 99%

A clonal derivative of tunicamycin‐resistant Chinese hamster ovary cells with increased N‐acetylglucosamine‐phosphate transferase activity has altered asparagine‐linked glycosylation

Waldman¹,

Oliver²,

Krag³

1987

Journal Cellular Physiology

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“…The synthetic oligosaccharide acceptor L2 (Gal␤1-4Man␣1-PO 4 -(CH 2 ) 8 CHϭCH 2 ) was generously provided by Professor Michael Ferguson (University of Dundee). GDP-[ 3 H]Man was prepared using previously described methods (26,27). Briefly, [2-3 H]Man-6-PO 4 was synthesized enzymatically from [2-3 H]Man (NEN Life Science Products) using yeast hexose kinase and then converted to the sugar nucleotide using a mixture of yeast proteins supplemented with snake venom pyrophosphatase and glucose-1,6-diphosphate.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a Novel GDP-mannose:Serine-protein Mannose-1-phosphotransferase from Leishmania mexicana

Moss

Reid

Mullin

et al. 1999

Journal of Biological Chemistry

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Protozoan parasites of the genus Leishmania secrete a number of glycoproteins and mucin-like proteoglycans that appear to be important parasite virulence factors. We have previously proposed that the polypeptide backbones of these molecules are extensively modified with a complex array of phosphoglycan chains that are linked to Ser/Thr-rich domains via a common Man␣1-PO 4 -Ser linkage (Ilg, T., Overath, P., Ferguson, M. A. J., Rutherford, T., Campbell, D. G., and McConville, M. J. (1994) J. Biol. Chem. 269, 24073-24081). In this study, we show that Leishmania mexicana promastigotes contain a peptide-specific mannose-1-phosphotransferase (pep-MPT) activity that adds Man␣1-P to serine residues in a range of defined peptides. The presence and location of the Man␣1-PO 4 -Ser linkage in these peptides were determined by electrospray ionization mass spectrometry and chemical and enzymatic treatments. The pep-MPT activity was solubilized in non-ionic detergents, was dependent on Mn 2؉ , utilized GDP-Man as the mannose donor, and was expressed in all developmental stages of the parasite. The pep-MPT activity was maximal against peptides containing Ser/Thr-rich domains of the endogenous acceptors and, based on competition assays with oligosaccharide acceptors, was distinct from other leishmanial MPTs involved in the initiation and elongation of lipid-linked phosphoglycan chains. In subcellular fractionation experiments, pep-MPT was resolved from the endoplasmic reticulum marker BiP, but had an overlapping distribution with the cis-Golgi marker Rab1. Although Man-PO 4 residues in the mature secreted glycoproteins are extensively modified with mannose oligosaccharides and phosphoglycan chains, similar modifications were not added to peptide-linked Man-PO 4 residues in the in vitro assays. Similarly, Man-PO 4 residues on endogenous polypeptide acceptors were also poorly extended, although the elongating enzymes were still active, suggesting that the pep-MPT activity and elongating enzymes may be present in separate subcellular compartments.Parasitic protozoa of the genus Leishmania cause a spectrum of human and animal diseases that are transmitted by a sand fly vector. During their development in the digestive tract of the sand fly, these parasites differentiate from non-infective procyclic promastigotes to infective metacyclic promastigotes that target mammalian macrophages when introduced into the host during the insect's blood meal. Following their internalization into the macrophage phagolysosome, metacyclic promastigotes differentiate into a replicative amastigote stage and eventually rupture the host cell and perpetuate disease by infecting other host cells. A number of cell-surface and secreted virulence factors are thought to be crucial for the survival of these different developmental stages in their respective host environments. These include an abundant glycosylphosphatidylinositol-anchored lipophosphoglycan (LPG) 1 (1-3) and a number of secreted glycoproteins and proteophosphoglycans (PPGs) (reviewed in Refs. 4 ...

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“…The cultures were checked routinely for mycoplasma (12) and were found to be negative. (-100 mCi/mmol) was prepared as described (13).…”

Section: Methodsmentioning

confidence: 99%

Mutant of Chinese hamster ovary cells with altered mannose 6-phosphate receptor activity is unable to synthesize mannosylphosphoryldolichol.

Stoll¹,

Robbins²,

Krag³

1982

Proc. Natl. Acad. Sci. U.S.A.

100

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B4-2-1 is a Chinese hamster ovary cell mutant previously isolated and characterized as deficient in mannose 6-phosphate receptor activity. We show here that B4-2-1 is a pleiotropic mutant, defective in biosynthesis of asparagine-linked oligosaccharides. B4-2-1 is unable to synthesize mannosylphosphoryldolichol; the consequences of this defect on glycosylation are (i) biosynthesis of one major lipid-linked oligosaccharide, characterized by its resistance to endoglycosidase H and decreased size; this oligosaccharide is similar to a minor species of lipid-linked oligosaccharide found in parental cells; (ii) transfer of this oligosaccharide to newly synthesized proteins; and (iii) absence of normal "high-mannose" oligosaccharides on mature glycoproteins isolated from B4-2-1; glycoproteins from the mutant contain complex oligosaccharides as well as endoglycosidase H-resistant, amannosidase-sensitive species. While the glycosylation defect may alter adversely the function ofseveral glycoproteins in the mutant, including that of the mannose 6-phosphate receptor, it appears to have no effect on the formation or function ofthe mannose 6-phosphate recognition marker on acid hydrolases of B4-2-1.The uptake of acid hydrolases into human fibroblasts and Chinese hamster ovary (CHO) cells involves binding of a mannose 6-phosphate recognition marker on the enzymes to a receptor for mannose 6-phosphate on the cell surface (1)(2)(3). This receptor appears to be similar in the two cell types with respect to both structure (3) and function (4).The mannose 6-phosphate recognition marker and mannose 6-phosphate receptor appear to be involved not only in enzyme uptake but also in translocation of newly synthesized endogenous acid hydrolases to the lysosomes (2, 5). Most of the receptor has been shown to exist intracellularly (2). Human fibroblasts defective in mannose phosphorylation (6-9) fail to compartmentalize acid hydrolases but instead secrete those enzymes. Aberrant compartmentalization was also observed in CHO cell mutants defective in mannose 6-phosphate receptor activity (10).One ofthe receptor-defective CHO cell mutants, B4-2-1, was found to synthesize acid hydrolases ofincreased electrophoretic mobility (10). Since the receptor and the hydrolases are all glycoproteins, we speculated that this mutant might have a pleiotropic defect in glycosylation. We show here that B4-2-1 cells synthesize altered asparagine-linked oligosaccharides due to a defect in the biosynthesis of mannosylphosphoryldolichol. MATERIALS AND METHODSCells. Isolation and culture conditions for the CHO cell lines N211-1-8 and B4-2-1 (4) and WTB (11) have been described. All media contained 5% fetal bovine serum (Armour, Kankakee, IL); (North American Biologicals, Miami, FL). The cultures were checked routinely for mycoplasma (12) and were found to be negative. (-100 mCi/mmol) was prepared as described (13). Reagents. endo-p-N-acetylglucosaminidase H (endo H)Incubation of Cells with [2-3H]Mannose. Cells were labeled with 1 ml of a-modified minima...

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A new procedure for the preparation of GDP-[U-14C]mannose

Cited by 35 publications

References 3 publications

A clonal derivative of tunicamycin‐resistant Chinese hamster ovary cells with increased N‐acetylglucosamine‐phosphate transferase activity has altered asparagine‐linked glycosylation

A clonal derivative of tunicamycin‐resistant Chinese hamster ovary cells with increased N‐acetylglucosamine‐phosphate transferase activity has altered asparagine‐linked glycosylation

Characterization of a Novel GDP-mannose:Serine-protein Mannose-1-phosphotransferase from Leishmania mexicana

Mutant of Chinese hamster ovary cells with altered mannose 6-phosphate receptor activity is unable to synthesize mannosylphosphoryldolichol.

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