RESUMEN

Parental Chinese hamster ovary (CHO) cells were mutagenized and subjected first to a mannose suicide selection technique and second to a screen of individual colonies grown on polyester discs for reduced mannose incorporation into protein. The incorporation of radioactivity for the selection and the screen was conducted at 41.5 degrees C instead of the normal growth temperature of 34 degrees C in order to allow for the isolation of temperature-sensitive lesions. This selection/screening procedure resulted in the isolation of M15-4 cells, which had three- to five-fold lower incorporation of [2-3H]mannose into mannose 6-phosphate, mannose 1-phosphate, GDP-mannose, oligosaccharide-lipid, and glycoprotein at 41.5 degrees C. We detected no difference in the qualitative pattern of mannose-labeled lipid-linked oligosaccharides compared to parental cells. M15-4 cells synthesized dolichol. The defect of M15-4 cells was determined to be in hexokinase activity; crude cytosolic extracts were eight- to nine-fold lower in hexokinase activity in M15-4 cells compared to parental cells. As a result of this defect, incorporation of labeled mannose from the medium was significantly decreased. However, the level of GDP-mannose in M15-4 cells was 70% of normal. The phenotype of M15-4 was a lower specific activity of labeled GDP-mannose, not a substantial reduction in the level of GDP-mannose. Consistent with these results, no alterations in the glycosylation of a model glycoprotein, G protein of vesicular stomatitis virus, were observed. These cells grew slower than parental cells, especially in low-glucose medium.

Asunto(s)

RESUMEN

A key step in the assembly of oligosaccharide-lipid intermediates in N-linked glycosylation is the transfer of N-acetylglucosamine 1-phosphate to dolichyl phosphate, catalyzed by the enzyme UDP-N-acetylglucosaminyl:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase (L-G1PT). Comparison of the amino acid sequences of L-G1PT from five diverse species showed 75 amino acids identical in all five proteins. Using site-directed mutagenesis, we analyzed the importance of a number of these conserved residues to the enzymatic activity of L-G1PT using a plasmid shuffling procedure in Schizosaccharomyces pombe. S. pombe cells containing a chromosomal deletion of the essential gpt+ gene are rescued by a plasmid containing the S. pombe gpt open reading frame. Replacement of that plasmid by a plasmid encoding a mutated hamster L-G1PT cDNA sequence indicated that the mutated protein provided sufficient enzyme activity to permit cell growth. Mutations of aspartic acid 252 and asparagine 185 did not allow plasmid shuffling, indicating these residues were essential for activity. A combination of mutations at asparagine 182 and tryptophan 122 did not allow plasmid shuffling, although the single mutations did. Overexpression of the mutant proteins in S. pombe conferred tunicamycin (TM) resistance, indicating that the mutant proteins had a conformation necessary for binding TM, a substrate analog. The mutant proteins were also detected in Western blots and were correctly localized to the membrane fractions. However, the overexpressed proteins did not increase the endogenous level of enzymatic activity in these cells, indicating they were enzymatically inactive.

Asunto(s)

RESUMEN

The gene gpt encoding uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetylglucosaminylphosphoryltransferase (L-G1PT) was isolated by screening a Schizosaccharomyces pombe genomic DNA library in lambda phage under low-stringency hybridization using the Saccharomyces cerevisiae gene ALG7 as probe. Sequencing 2.4 kb of S. pombe DNA revealed a 1338-bp open reading frame (ORF) encoding a hydrophobic protein of 446 amino acids with a predicted molecular weight of 49,852. The S. pombe protein was 50% identical to the S. cerevisiae protein and 43% identical to the protein from Chinese hamster ovary (CHO) cells. Overexpression of the gpt gene in S. pombe cells increased resistance to tunicamycin 25-fold and increased the specific activity of the enzyme in isolated cell membranes 13-fold. This was accompanied by a 50-fold increase in poly(A)+ RNA hybridizing to the gpt probe. Northern analysis indicated a single 1.8-kb message is transcribed from the gpt gene. The gpt gene is essential for viability of S. pombe. Cells containing a disrupted ORF could be rescued by an expression plasmid containing either the intact S. pombe gpt ORF or the CHO L-G1PT cDNA. The S. pombe gpt gene was mapped to chromosome 2 near top1 and ade1.

Asunto(s)

RESUMEN

The hamster gene for uridine diphosphate N-acetyl-D-glucosamine:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase (L-G1PT) was found to extend over 6.5 kb and to contain nine exons. The exons ranged in size from 63 to 214 bp, encoding the 408 amino acid protein. The introns ranged from 85 bp to 1.4 kb. Upstream 5' sequences included two possible TATA boxes, one possible CCAAT box and at least two potential GC boxes. Heterologous expression was successful in Schizosaccharomyces pombe, and resulted in cells that were tunicamycin resistant and had 12-fold more L-G1PT activity than wild-type cells. Antiserum prepared to a hydrophilic peptide (residues 300-320) of the L-G1PT protein reacted with a 35-36 kDa protein in membrane samples from Chinese hamster ovary (CHO) cells and S. pombe cells that had increased levels of L-G1PT activity. In both cases, antigenic peptide competed with the 35-36 kDa protein detected by the antiserum.

Asunto(s)

RESUMEN

There are large increases in the rates of Glc3-Man9GlcNAc2-P-P-Dol (Oligo-P-P-Dol) biosynthesis and protein N-glycosylation during the proliferative response of murine B lymphocytes (B cells) to bacterial lipopolysaccharide (LPS). To learn more about the regulation of dolichyl-saccharide biosynthesis, the possible relationships between developmental changes in specific steps in dolichyl phosphate (Dol-P) and N-acetyl-glucosaminylpyrophosphoryldolichol (GlcNAc-P-P-Dol) biosynthesis and the induction of Oligo-P-P-Dol biosynthesis were investigated. These studies describe an impressive induction of long chain cis-isoprenyltransferase (cis-IPTase) activity, an enzyme system required for the chain elongation stage in de novo Dol-P synthesis, which corresponded to the striking increase in the rate of Oligo-P-P-Dol biosynthesis in LPS-activated B cells. The cellular level and specific activity of cis-IPTase increase 15-fold in LPS-treated cells with relatively unaltered affinity for isopentenyl pyrophosphate. The rates of Dol-P and Oligo-P-P-Dol synthesis increased substantially when cis-IPTase activity was induced, suggesting a regulatory relationship between the level of cis-IPTase activity and lipid intermediate synthesis. Distinctly different developmental patterns were observed for cis-IPTase and HMG-CoA reductase activity, and when sterol biosynthesis was drastically inhibited by lovastatin, the rate of synthesis of Dol-P was slightly higher in the presence of the drug. Modest elevations in the cellular levels of dolichol kinase, Dol-P phosphatase, and UDP-GlcNAc:Dol-P N-acetylglucosaminylphosphoryltransferase (L-G1PT) activities were also observed, but these changes were relatively small compared with the increases in cis-IPTase activity and the rates of Dol-P, Gl-cNAc-P-P-Dol, and Oligo-P-P-Dol synthesis. The expression of the L-G1PT gene is an early event in the developmental program for Oligo-P-P-Dol synthesis, but GlcNAc-P-P-Dol formation is apparently not rate-limiting. In summary, large increases in cis-IPTase activity and the rate of Dol-P biosynthesis appear to play a key regulatory role in the induction of Oligo-P-P-Dol biosynthesis during the proliferative response of B cells to LPS, and the biosynthetic pathways for Dol-P and cholesterol are regulated independently in dividing B cells.

Asunto(s)

RESUMEN

We have isolated a portion of the uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetyl-glucosamine-1-phosphate transferase gene (GTR2) from the genome of a tunicamycin-resistant clonal Chinese hamster ovary cell line, 3E11. The genomic fragment was selected by its hybridization to the yeast ALG-7 gene at low stringency. A 2.46-kilobase cDNA was isolated from a library prepared from 3E11 mRNA and probed with GTR2. The cDNA contained an open reading frame that encodes a protein of 408 amino acids with a molecular mass of 44.9 kDa. This protein was 43% identical in amino acid sequence to the protein of 448 amino acids encoded by the ALG-7 gene. The GTR2 gene fragment contained sequences for four exons coding for the carboxyl-terminal half of the protein. Transferase DNA sequences in 3E11 cells were 12-fold elevated over wild-type cells and 25-fold elevated when 3E11 cells were grown in the presence of tunicamycin. Transferase RNA levels in 3E11 cells were also elevated over wild-type levels but appeared unchanged by the presence of tunicamycin in the medium.

Asunto(s)

RESUMEN

Restriction digests of genomic DNA from tunicamycin-resistant Chinese hamster ovary cells, 3E11, were probed with the yeast transferase gene, ALG7. The data presented suggest moderate amplification of the N-acetylglucosaminyl-1-phosphate transferase gene occurred in these cells, consistent with the previously observed chromosomal translocations and increased enzymatic activity. This is the first example of gene amplification as a mechanism for aberrations in N-linked glycosylation.

Asunto(s)

RESUMEN

Sialyl conjugates which accumulate in sialidase deficient fibroblasts were made radioactive in the sialic acid moiety by culturing the cells in the presence of [3H]N-acetylmannosamine. The accumulated radioactive compounds were fractionated by anion exchange chromatography and gel filtration. At least nine chromatographically distinct sialyl conjugates were obtained. Neutral and amino sugar analysis of these compounds indicated that all were sialyloligosaccharides of the type which could be derived from glycoproteins containing complex, N-asparagine-linked carbohydrate groups.

Asunto(s)

RESUMEN

A mixture of hexosaminitols obtained by reducing N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine with sodium borohydride was resolved by borate complex anion-exchange chromatography. This procedure yielded a complete separation of N-acetylglucosaminitol, N-acetylgalactosaminitol, and N-acetylmannosaminitol and provided a rapid and accurate means for identifying and measuring N-acetylhexosamines in biological samples. This method was applied to studies on N-acetylneuraminic acid metabolism in human skin fibroblasts. It was used to identify reaction products in two enzymatic reactions: the conversion of UDP-N-acetylglucosamine to N-acetylmannosamine and UDP by UDP-N-acetylglucosamine 2-epimerase and the conversion of N-acetylneuraminic acid to N-acetylmannosamine and pyruvate by N-acetylneuraminate pyruvate-lyase. It was also used to identify the free 3H-labeled N-acetylhexosamines found in fibroblasts cultured in the presence of N-[3H]acetylmannosamine.

Asunto(s)

Asunto(s)

Asunto(s)