Genetic defect in N-acetylglucosaminyltransferase I gene of a ricin-resistant baby hamster kidney mutant

The analysis of mutations associated with glycosylation-defective cell lines has the potential for identifying critical residues associated with the activities of enzymes involved in the biosynthesis of glycoconjugates. A ricin-resistant (RicR) baby hamster kidney (BHK) cell mutant, clone RicR14, has a deficiency in N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and as a consequence is unable to synthesize complex and hybrid N-glycans. Here we show that RicR14 cells transfected with wild-type GlcNAc-TI regained the ability to synthesize complex N-glycans, demonstrating that the glycosylation defect of RicR14 cells is due solely to the lack of GlcNAc-TI activity. With the use of specific antibodies to GlcNAc-TI, RicR14 cells were shown to synthesize an inactive GlcNAc-TI protein that is correctly localized to the Golgi apparatus. We have cloned and sequenced the open reading frame of GlcNAc-TI from parental BHK and RicR14 cells. A comparison of several RicR14 cDNA clones with the parental BHK GlcNAc-TI sequence indicated the presence of two different RicR14 cDNA species. One contained a premature stop codon at position +81, whereas the second contained a point mutation in the catalytic domain of GlcNAc-TI resulting in the amino acid substitution Gly320 → Asp. The introduction of a Gly320 → Asp mutation into wild-type rabbit GlcNAc-TI resulted in a complete loss of activity; the GlcNAc-TI mutant was correctly localized to the Golgi, indicating that the inactive GlcNAc-TI protein was transport-competent. Gly320 is conserved in GlcNAc-TI from all species so far examined. Overall these results demonstrate that Gly320 is a critical residue for GlcNAc-TI activity. The nucleotide sequence data reported will appear in DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers AF087456 and AF087457.

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Sequence, catalytic properties and expression of chicken glutathione-dependent prostaglandin D2 synthase, a novel class Sigma glutathione S-transferase

Biochemical Journal ◽

10.1042/bj3330317 ◽

1998 ◽

Vol 333 (2) ◽

pp. 317-325 ◽

Cited By ~ 53

Author(s):

Anne M. THOMSON ◽

David J. MEYER ◽

John D. HAYES

Keyword(s):

Amino Acid ◽

Nucleotide Sequence ◽

Expressed Sequence Tag ◽

Sequence Data ◽

Cdna Clones ◽

Prostaglandin D2 ◽

Glutathione S Transferase ◽

Nucleotide Sequence Data ◽

Chicken Spleen ◽

Wide Range

The Expressed Sequence Tag database has been screened for cDNA clones encoding prostaglandin D2 synthases (PGDSs) by using a BLAST search with the N-terminal amino acid sequence of rat GSH-dependent PGDS, a class Sigma glutathione S-transferase (GST). This resulted in the identification of a cDNA from chicken spleen containing an insert of approx. 950 bp that encodes a protein of 199 amino acid residues with a predicted molecular mass of 22732 Da. The deduced primary structure of the chicken protein was not only found to possess 70% sequence identity with rat PGDS but it also demonstrated more than 35% identity with class Sigma GSTs from a range of invertebrates. The open reading frame of the chicken cDNA was expressed in Escherichia coli and the purified protein was found to display high PGDS activity. It also catalysed the conjugation of glutathione with a wide range of aryl halides, organic isothiocyanates and α,β-unsaturated carbonyls, and exhibited glutathione peroxidase activity towards cumene hydroperoxide. Like other GSTs, chicken PGDS was found to be inhibited by non-substrate ligands such as Cibacron Blue, haematin and organotin compounds. Western blotting experiments showed that among the organs studied, the expression of PGDS in the female chicken is highest in liver, kidney and intestine, with only small amounts of the enzyme being found in chicken spleen; in contrast, the rat has highest levels of PGDS in the spleen. Collectively, these results show that the structure and function, but not the expression, of the GSH-requiring PGDS is conserved between chicken and rat. The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank, GSDB and DDBJ Nucleotide Sequence Databases under the accession number AJ006405.

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Molecular Mechanism of Antifolate Transport-Deficiency in a Methotrexate-Resistant MOLT-3 Human Leukemia Cell Line

Blood ◽

10.1182/blood.v89.7.2494 ◽

1997 ◽

Vol 89 (7) ◽

pp. 2494-2499 ◽

Cited By ~ 42

Author(s):

Maokai Gong ◽

James Yess ◽

Tatiana Connolly ◽

S. Percy Ivy ◽

Takao Ohnuma ◽

...

Keyword(s):

Nucleotide Sequence ◽

Cell Line ◽

Cell Lines ◽

Stop Codon ◽

Leukemia Cell ◽

Premature Stop Codon ◽

Wild Type ◽

Wild Type Sequence ◽

Type Sequence

Abstract Ohnuma et al reported a series of methotrexate-resistant MOLT-3 human T-cell acute lymphoblastic leukemia cell lines that showed decreasing methotrexate (MTX) uptake as the sublines acquired increasing MTX resistance (Cancer Res 45:1815, 1985). The alteration of MTX uptake kinetics in these cells, the intermediately resistant MOLT-3/MTX200 and the highly resistant MOLT-3/MTX10,000 cell lines, was attributed to a change in Vmax for methotrexate transport, without an apparent change in affinity of the transporter for MTX. We studied these cell lines to determine whether alteration of transcription or translation of the recently isolated reduced folate carrier gene (RFC1) was the cause of MTX transport deficiency in these cell lines. Reconstitution of RFC activity in MOLT-3/MTX10,000 cells by transduction with a murine RFC retroviral vector reversed MTX resistance and trimetrexate sensitivity. Although RFC1 RNA levels were unchanged in the resistant cell lines, FACS analysis using a polyclonal anti-RFCl antibody showed no detectable RFCl protein in the MOLT-3/MTX10,000 cells. Determination of the nucleotide sequence of RFC1 genes from MOLT-3/MTX10,000 cells revealed that this cell line contained 3 RFC1 alleles: a wild-type allele, an allele containing the premature stop codon at codon 40 and a third allele containing another mutation, which resulted in a premature stop codon at codon 25. We examined the relative expression of these alleles by determining the nucleotide sequence of 24 RFC1 cDNA subclones from MOLT-3/MTX10,000 cells and found that only one-third of these clones contained the wild-type sequence. Determination of the genomic sequence of RFC1 in MOLT-3/MTX200 cells demonstrated that these cells were heterozygous for a mutation at codon 40, but were homozygous for the wild-type sequence at codon 25. Thus, the acquisition of MTX transport-deficiency in MOLT-3/MTX10,000 cells results from inactivating mutations of RFC1 gene alleles.

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