2 Department of Cell Biology and Anatomy, Unviversity of Miami School of Medicine, Miami, FL 33101, USA, and 3 Department of Biochemistry, University of Georgia, Athens, GA, USA
Received on November 29, 2001; revised on February 4, 2002; accepted on February 5, 2002
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Abstract |
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Key words: glycosylation/glycosyltransferase/mRNA/mutant
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Introduction |
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UDP-N-acetylglucosamine:-D-mannoside ß-1,6-N-acetylglucosaminyltransferase V or GlcNAc-TV (EC.4.1.155) plays a pivotal role in the synthesis of complex N-linked glycans. This enzyme catalyzes the addition of an N-acetylglucosamine to the
1,6 mannosyl core in a ß1,6 linkage and forms the ß1,6 branch on tri- and tetraantennary N-linked oligosaccharide structures. This branch provides the preferred substrate for the enzymatic subsequent synthesis of polylactosamine chains (van den Eijnden et al., 1988
) and their terminal modifications including the Lewis antigens. GlcNAc-TV is expressed in a variety of tissues at considerably different levels (Dennis and Laferte, 1989
) as well as during embryogenesis (Granovsky et al., 1995
). In some cases (i.e., brain), the level of the mRNA does not appear to correlate with the level of enzyme activity, indicating that regulation of GlcNAc-TV expression is complex with both transcriptional and translational controls.
Elevated GlcNAc-TV expression has been correlated with a number of different tumors including breast and colon (Fernandes et al., 1991) and virally transformed cells (Pierce et al., 1997
). Several studies have shown that the GlcNAc-TV gene transcription can be induced by the oncogenes, including ras (Yamashita et al., 1984
) src (Buckhaults et al., 1997
), and neu (Chen et al., 1998
). GlcNAc-TV activity is also highly associated with metastatic potential. This has recently been demonstrated by the observation that Mgat5 knockout mice lacking the GlcNAc-TV activity show reduced tumor growth and have a 20-fold decrease in metastasis to the lungs (Granovsky et al., 2000
). In addition to showing reduced metastasis, these Mgat5 mice showed increased susceptibility to autoimmune disease, due to altered T cell receptor aggregation and signaling (Demetriou et al., 2001
). The activity of this GlcNAc-TV and the presence of its ß1,6 branch product are associated with a number of cellular properties that might promote cancer progression. Correlations of GlcNAc-TV expression have also been reported for cellular adhesiveness, migration, proliferation, and apoptosis (Demetriou et al., 1995
). Thus, proper regulation of this gene appears to be significant for proper cellular behavior.
The oligosaccharide product of GlcNAc-TV forms the binding site for the lectin L-Pha. The toxicity of this lectin after binding to GlcNAc-TV-expressing cells has been used as a selective scheme to isolate several cell lines with mutations in the GlcNAc-TV gene. Two lines, Lec4 and Lec4a, were derived from Chinese hamster ovary (CHO) cells with the Lec4 having no detectable GlcNAc-TV activity and the Lec4a having enzymatic activity that is improperly localized outside the Golgi. The mRNAs for these mutants have been sequenced and show that the Lec4 mRNA has two insertions, resulting in a frame shift and premature termination. The Lec4a mRNA has a single point mutation that changes amino acid 188 from a leucine to an arginine. This amino acid change is presumably responsible for the mislocalization of the protein (Weinstein et al., 1996) and altered glycosylation. A third cell line, PhaR2.1, is a set of mouse lymphoma cells derived from mutagenized BW5147 cells (Cummings et al., 1982
). This line has no detectable GlcNAc-TV enzyme activity but does transcribe a GlcNAc-TV mRNA that is slightly larger than the 7.5-kb mRNA observed in the parental BW5147 cells. In this report, we have determined the coding and 3' untranslated sequences of the mouse GlcNAc-TV mRNA and the genetic lesion in the sequence of the mutant PhaR2.1 GlcNAc-TV mRNA.
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Results |
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In an effort to ensure the homogeneity of the PhaR2.1 cells prior to RNA isolation, they were tested for their L-Pha-binding ability. This was done using two methods. For the first method, 10-ml cultures of BW5147 and PhaR2.1 cells were grown and used to prepare crude membrane fractions. The proteins in these crude membrane preparations were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) and blotted to nylon membranes. The samples were probed for the presence of ß1,6 branched oligosaccharides with horseradish peroxidaseconjugated L-Pha. The results of this lectin blot are shown in Figure 2. These data show that there is little (if any) specific L-Pha binding to membrane proteins from PhaR2.1 cells, and the membrane proteins from the parental BW5147 cells demonstrate very strong binding. To further confirm the absence of L-Pha binding to the PhaR2.1 cells, the cells were treated with fluorescein isothiocyanate (FITC)-labeled-L-Pha and analyzed on a fluorescence-assisted cell sorting (FACS) scan. This data also in Figure 2 shows again that the PhaR2.1 cells do not express ß1.6-branched oligosaccharides, L-Pha binding sites, therefore, these cells could be used for mRNA isolation without fear of contamination with BW5147 cells.
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Test for complementation by the PhaR2.1 amplimers
To test for mutation events in each of the individual 5' and 3' amplification products of the PhaR2.1 GlcNAc-TV mRNA, they were substituted for their normal counterparts in an expression plasmid that contained a functional, full-length, wild-type mouse coding sequence driven by the mouse mammary tumor virus promoter. The replacements were made using a Cla I restriction endonuclease site that is 1147 bases (about 47%) into the reading frame and was present in the overlap between the 3' and 5' amplification products. Specifically, the 5' PhaR2.1 amplification product was excised from the pGEM-Teasy vector by digesting with EcoRI (site in the vector) and ClaI and substituted for the wild-type sequence cut with the same enzymes. This plasmid is referred to as pPhaR5'. Similarly the 3' PhaR2.1 amplimer was isolated from the pGEM-Teasy vector by digestion with Cla I and Not I (site in the vector) to created a plasmid with 5' end of the PhaR2.1 message sequence and the 3' end of the normal mouse sequence, and one plasmid with the 5' end of the normal sequence and the 3' end of the PhaR2.1 sequence (pPhaR3'). These plasmids were used to transfect the GlcNAc-T V deficient cell line CHOP4 (Heffernan and Dennis, 1991) and assayed for the presence of ß1,6 branched N-linked oligosaccharides by FITC-conjugated L-Pha binding and FACS scan analysis. The results of these transfections and scans are shown in Figure 3. These experiments show that the 3' portion of the PhaR2.1 GlcNAc-TV mRNA encodes the proper amino acid sequence to produce a functional GlcNAc-TV enzyme and confirm that the 5' portion of the PhaR2.1 GlcNAc-T V mRNA contains the mutation(s) responsible for the lack of GlcNAc-T V activity in the mutant cells. This is most likely to be due to the inserted sequence we have detected.
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Discussion |
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As would be expected from high sequence identity of the mRNAs, the amino acid sequences are also remarkably similar. The mouse and rat sequences have only two differences out of the entire 740-amino-acid length. The hamster sequence has 8 amino acid differences with the mouse protein, and the human protein has the greatest difference with 20 amino acid substitutions. The human protein also has one additional difference to the rodent proteins, because it has an additional alanine residue at position 108, giving it 741 amino acids to the 740 amino acids in the rodent proteins. Most of the amino acid differences are conserved as shown in Table I.
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Materials and methods |
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RNA isolation, library construction, and cloning
RNAs were isolated from cultured cells using RNAzol (Tel Test) according to manufacturers recommendations. Poly A containing RNA was purified from total RNA by binding to and eluting from oligo(dT)-cellulose. The BW5147 cDNA Library was constructed in pcDNA1 using the Librarian II system (Invitrogen) and oligo(dT)-primed cDNA. The NIH 3T3 cell cDNA library was constructed using random hexanucleotide primed cDNA, EcoRI linker addition, and ligation into the 8Gem vector. The libraries were screened using 32P-labeled fragments of the rat cDNA using the random prime labeling method (Roche Diagnostics).
The remaining 5' portion of the mouse mRNA sequence was obtained by RT-PCR using AMV reverse transcriptase and Tfl DNA polymerase (Promega). The 5' most portion of the BW5147 cell mRNA was amplified using a 5' primer with a sequence found in the 5' untranslated region of the rat mRNA (5'-CCCGTCGACGAGAGCCAAGGGAATGGTA-3') and a 3' primer complimentary to a sequence near 5' end of the longest cDNA clone (5'-CAAAGGAGTCTAGCACCC-3'). This amplification product was cloned into the pGEM-Teasy vector (Promega). A complete cDNA clone was constructed by sequentially adding the fragments into the pcDNA1/BW5147 cDNA clone.
The full-length PhaR2.1 GlcNAc T-V cDNA was constructed using two RT/PCR steps, one for the 5' half of the mRNA and the second for the 3' half. The primers for the 5' portion were the same as described for the cloning of the BW5147 5' portion. The 3' coding portion was amplified using the primers 5'-GAGTCTCCGGACTTTTAT-3' and 5' ATCTACCTGGATATCATTC. These amplification products were cloned into the pGEM-Teasy system (Promega) and sequenced either at the University of Miami DNA Sequencing Facility or the University of Georgia, Athens, DNA sequencing facility.
Crude membrane preparations and western blotting
Cells were collected scraping and washed in cold HBS (20 mM HEPES, pH 7.4, 8 g/L NaCl, 0.4 g/L KCl , 0.35 g/L NaHCO3, 1 g/L D-glucose, 100 µM ethylenediamine tetra-acetic acid [EDTA]) then incubated in HBS on ice for 10 min. About 107 cells were centrifuged and resuspended in 1 ml harvest buffer (20 mM sodium borate, pH 8, 200 µM EDTA) and kept on ice for 10 min. The cell suspension was then homogenized with 30 strokes in a dounce homogenizer. Unlysed cells and large particles were removed by centrifugation at 5000 x g for 5 min. The cell membranes were then pelleted by centrifugation of the clarified supernatant by centrifugation in a Beckman Table Top Ultracentrifuge at 100,000 x g for 30 min. Pellets were resuspended phosphate buffered saline (PBS) containing 0.1% SDS.
Protein concentrations were determined using the BCA Protein Assay Reagent (Pierce). Equivalent amounts of the protein samples were separated by SDSPAGE using 8% acrylamide gels and transferred to nylon membranes (Immobilon, Millipore). ß1,6 Branched N-linked oligosaccharides were detected using peroxidase-labeled L-Pha (E-M Laboratories) and chemiluminescence (Renaissance, NEN Life Science).
Transfection and FACS analysis
Cells were transfected using ExGen 500 (MBI Fermentas) gene delivery reagent according to the manufacturers recommendations. Briefly, CHOP4 cells were plated at 150,000 cells per 35-mm dish 1 day before transfection. Plasmid DNAs used for transfection were prepared using the endotoxin-free protocol (Qiagen). Eight micrograms of plasmid DNA were mixed with 26.4 µl (six equivalents) of ExGen and used to transfect 35-mm dish of CHOP4 cells for 56 h, at which time the cells were fed fresh medium and allowed to grow for about 60 h. The cells were washed two times with PBS (Ca2+ Mg2+ free) and removed from the dish using enzyme-free cell dissociation buffer (Gibco/BRL). The cells were washed twice in PBS (Ca2+ Mg2+ free) + 1% bovine serum albumin (BSA). FITC-labeled L-Pha (Vector Labs) was added to a final concentration of 2.5 µg/ml in PBS-BSA, and the cells were incubated in this mixture for 30 min at 4°C. The cells were centrifuged and resuspended in 1 ml PBS-BSA and analyzed using a Bectin-Dickenson FACS scan at the Sylvester Cancer Center FACS Facility.
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Abbreviations |
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Acknowledgments |
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Footnotes |
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References |
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