Minimal catalytic domain of N-acetylglucosaminyltransferase V

Bozena Korczak1, Thuyanh Le, Sabine Elowe, Alessandro Datti and James W. Dennis2

GlycoDesign Inc., 480 University Avenue, Suite 900, Toronto, Ontario, Canada

Received on September 29, 1999; revised on November 17, 1999; accepted on November 24, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
UDP-GlcNAc: Man{alpha}1–6Manß-R ß1–6 N-acetylglucosaminyltransferase V (EC 2.4.1.155, GlcNAc-TV) is a Golgi enzyme that substitutes the trimannosyl core in the biosynthetic pathway for complex-type N-linked glycans. GlcNAc-TV activity is regulated by oncogenes frequently activated in cancer cells (ras, src, and her2/neu) and by activators of T lymphocytes. Overexpression of GlcNAc-TV in epithelial cells results in morphological transformation, while tumor cell mutants selected for loss of GlcNAc-TV products show diminished malignant potential in mice. In this report, we have expressed and characterized a series of N- and C-terminal deletions of GlcNAc-TV. Portions of GlcNAc-TV sequence were fused at the N-terminal domain to IgG-binding domains of staphylococcal Protein A and expressed in CHOP cells. The secreted fusion proteins were purified by IgG Sepharose affinity chromatography and assayed for enzyme activities. The peptide sequence S213–740 of GlcNAc-TV was determined to be essential for the catalytic activity, the remaining amino acids comprising a 183 amino acid stem region, a 17 amino acid transmembrane domain and a 12 amino acid cytosolic moiety. Further deletion of 5 amino acids to produce peptide R218–740 reduced enzyme activity by 20-fold. Similar Km and Vmax values for donor and acceptor were observed for peptide S213–740, the minimal catalytic domain, and peptide Q39–740, which also included the stem region. Truncation of five amino acids from the C-terminus also resulted in a 20-fold loss of catalytic activity. Secondary structure predictions suggest a high frequency of turns in the stem region, and more contiguous stretches of {alpha}-helix found in the catalytic domain.

Key words: deletion mutant/glycosyltransferase/structure–function analysis


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
GlcNAc-TV catalyzes the addition of ß1–6GlcNAc to the trimannosyl core in the biosynthetic pathway for branched complex-type N-linked oligosaccharides found on some cell surface and secreted glycoproteins (Schachter, 1986Go). The ß1–6GlcNAc-branched product of GlcNAc-TV is the preferred antenna and rate limiting substrate in the pathway for addition of terminal polylactosamine sequences which affect cell–cell and cell–substratum interactions (van den Eijnden et al., 1988Go; Yousefi et al., 1991Go; Heffernan et al., 1993Go). The rat (Shoreibah et al., 1993Go) and human (Saito et al., 1994Go) GlcNAc-TV sequences are highly homologous, predicting a 740 amino acid type II transmembrane glycoprotein, but share no primary sequence homology with other known glycosyltransferases.

The human GlcNAc-TV gene is located on chromosome 2q21, having 17 exons and spanning 155 kb (Saito et al., 1995Go). The putative promoter region of the GlcNAc-TV gene has binding sites for AP1 and PEA3/Ets, and is responsive to the src and her-2/neu signaling pathways (Buckhaults et al., 1997Go; Chen et al., 1998Go). Oncogenic transformation of rodent fibroblasts with polyoma virus, v-src, H-ras, or v-fps/yes leads to increased GlcNAc-TV expression (Yamashita et al., 1985Go; Pierce and Arango, 1986Go; Dennis et al., 1987Go). The GlcNAc-TV message is also subject to increased frequency of alternate splicing in tumors cells, thereby giving rise to a peptide encoded by an intron sequence of the GlcNAc-TV gene which has been identified as a widely occurring "tumor-associated antigen" (Guilloux et al., 1996Go). Fifty percent of tested human melanoma tumors were found to express this antigen, unlike normal tissues. In a rat model of heritable liver cancer, GlcNAc-TV transcript levels are elevated in primary tumors and lymph node metastases (Miyoshi et al., 1993Go). In addition, topical expression of GlcNAc-TV results in morphological transformation and tumorigenesis in epithelial cells (Demetriou et al., 1995Go) and causes an increase in metastatic potential in mouse mammary cancer cells (Seberger and Chaney, 1999Go). Tumor cell mutants selected for loss of GlcNAc-TV activity show reduced malignant potential in vivo (Dennis et al., 1987Go; Lu et al., 1994Go).

Human tumors, including breast, colon, skin, head, and neck, and Sezary syndrome all showed increased levels of ß1–6GlcNAc-branched structures as detected by L-PHA, a plant lectin that binds these moieties (Dennis and Laferte, 1989Go; Takano et al., 1990Go; Fernandes et al., 1991Go; Derappe et al., 1996Go; Seelentag et al., 1997Go). Moreover, L-PHA staining intensity was shown to be an independent prognostic marker for disease-free survival in colorectal cancer patients (Seelentag et al., 1998Go).

This relationship between increased expression of GlcNAc-TV and the malignant phenotype has prompted a search for inhibitors of GlcNAc-TV as possible anti-cancer agents. In this study, we have expressed and purified truncated forms of GlcNAc-TV fused at the N-terminal domain to Protein A for use in structural analysis and high-throughput screening for inhibitors. The stem region and minimal catalytic domains of GlcNAc-TV were identified.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Soluble ProtA-GlcNAc-TV fusion protein
The rat cDNA encoding the luminal domain of GlcNAc-TV was subcloned into pProtA vector for expression as a secreted protein fused at the amino end to the IgG-binding domains of staphylococcal Protein A. CHO cells were cotransfected with pProtA-Q39–740 and pSV2neo, and following selection in G418, clones were screened for stable expression of secreted GlcNAc-TV activity. CHO clones secreting GlcNAc-TV protein were selected for enzyme production, and grown in hollow-fiber filter cartridges or spinner flasks. The fusion protein ProtA-Q39–740 produced from stable CHO transfectants was purified by IgG Sepharose affinity chromatography; the yield was ~0.5 mg/l of culture medium and the specific activity was in the range of 30–60 nmol/mg/min. The purified recombinant ProtA-Q39–740 GlcNAc-TV enzyme showed linear accumulation of product over 15 h and was stable for more than 2 months when stored at –20°C or 4°C (data not shown). Assays performed at 42°C showed a 2-fold increase in activity compared to those carried out at 37°C, and product accumulated in a linear manner over 4 h, suggesting that the catalytic domain of GlcNAc-TV is relatively stable (data not shown). The recombinant enzyme had a pH optimum of 6.0 to 6.7 using 0.1 M MES buffers, similar to that previously reported for native GlcNAc-TV from rat kidney (Palcic et al., 1990Go), while Na2PO4 buffers showed a wider pH optimum (data not shown). GlcNAc-TV activity was not affected by either 5 mM EDTA or 10 mM ß-mercaptoethanol.

GlcNAc-TV minimal catalytic domain
A series of GlcNAc-TV constructs containing various N-terminal deletions were used to determine the minimal size of the GlcNAc-TV catalytic domain. CHOP cells were transiently transfected with different cDNA constructs subcloned into pProtA, and 3 days later recombinant proteins were purified by IgG-Sepharose chromatography from cell culture medium. Western blot analyses for IgG reactive proteins showed major bands corresponding to the expected molecular weight for each of the recombinant proteins (Figure 1). GlcNAc-TV deletions: Q39–740, N114–740, S127–740, C155–740, and S213–740 displayed similar catalytic activities (Table I). Deletion of five additional N-terminal amino acids to produce R218–740 resulted in a 20-fold loss of catalytic activity (Table I). Further N-terminal deletions (R238–740 and L273–740) as well as truncation at the carboxyl terminus (S213–732 and S213–736) produced inactive proteins. Enzyme kinetics was performed using ProtA-Q39–740 and ProtA-S213–740 recombinant proteins. As shown in Figure 2, the Km values for the donor (1.53 mM versus 2.29 mM) and acceptor (136 µM versus 217 µM) substrates were comparable between the two constructs. These findings indicate that the deletion of GlcNAc-TV’s stem region (183 amino acids) scarcely affects the affinity for either donor or acceptor. The donor and acceptor Km values described here for ProtA-GlcNAc-TV fusion proteins are similar to those measured for purified rat kidney enzyme (Shoreibah et al., 1992Go), purified human enzyme from lung cancer cells (Gu et al., 1993Go), and enzyme in BHK cell lysates (Palcic et al., 1990Go).



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Fig. 1. Detection of secreted ProtA-GlcNAc-TV deletion proteins by Western Blot analysis. CHOP cells were transiently transfected with the following constructs: Q39–740 (lane 1); N114–740 (lane 2); S127–740 (lane 3); C155–740 (lane 4); S213–740 (lane 5); R218–740 (lane 6); R238–740 (lane 7); L273–740 (lane 8); S213–736 (lane 9); and S213–732 (lane 10). After 3 days of culture, recombinant proteins in the culture medium were purified by IgG-Sepharose chromatography, separated on 8% SDS–PAGE and analyzed by immunoblotting using antibody recognizing Protein A.

 

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Table I. Activity of ProtA-GlcNAc-TV deletion enzymes
 


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Fig. 2. (A) Determination of UDP-GlcNAc donor Km values for GlcNAc-TV deletion mutants ProtA-Q39–740 and ProtA-S213–740. GlcNAc-TV activities of ProtA-Q39–740 and ProtA-S213–740 were measured at different concentrations of UDP-GlcNAc using 1 mM synthetic trisaccharide acceptor. (B) Determination of acceptor Km values for GlcNAc-TV deletion mutants ProtA-Q39–740 and ProtA-S213–740. GlcNAc-TV activities of ProtA-Q39–740 and ProtA-S213–740 were measured at different concentrations of trisaccharide acceptor at 3.3 mM UDP-GlcNAc.

 
Previous analyses of predicted amino acid sequence of GlcNAc-TV identified six consensus sites for N-linked glycosylation, N109, N114, N117, N333, N432, and N446 (Shoreibah et al., 1993Go). The Q39–740 truncation contains all N-linked glycosylation sites, N114–740 has five sites and all of the other constructs have only three N-glycosylation sites. We observed similar specific enzyme activities in all of the active GlcNAc-TV constructs, suggesting that first three N-glycosylation sites are not essential for catalytic activity. This result contrasts with the property of recombinant core 2 GlcNAc-T which has two N-glycosylation sites near the transmembrane domain whose presence seems to be required for activity (Toki et al., 1997Go). Moreover, the three N-glycosylation sites in GlcNAc-T III at positions 243, 261, and 399 also appear to be required for enzyme localization in the Golgi and full enzyme activity (Nagai et al., 1997Go).

The predicted secondary structure of GlcNAc-TV was examined using the Chou-Fasman algorithm (Figure 3). Amino acid S213, which marks the shortest active N-terminal deletion, is followed by a long stretch of {alpha} helix, and preceded by a series of turns characteristic of a less structured stem domain. The 183 amino acid stem region spanning H31 to S213 of GlcNAc-TV is longer than that of several other glycosyltransferases, as is the 527 amino acid catalytic domain. New World monkey {alpha}1–3Gal-T has a 67 amino acid stem region, and a minimal catalytic domain of 285 amino acids (Henion et al., 1994Go), while ß1–4Gal-T has a 85 amino acid long stem region and 273 amino acid catalytic domain (Boeggeman et al., 1993Go). While the Golgi localization signal has not been clearly defined for most glycosyltransferases, a CHO cell glycosylation mutant (Lec4A) has recently been shown to have GlcNAc-TV protein mislocalized to endoplasmic reticulum instead of Golgi apparatus (Weinstein et al., 1996Go). A single point mutation (T to G) in the GlcNAc-TV gene, which results in a change from Leu to Arg at position 188 was responsible for this misclocalization. This L188 to R188 mutation is 30 amino acids prior to the start of the catalytic domain, suggesting that a targeting or Golgi retention signal is located in this region of the stem.



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Fig. 3. Chou-Fasman analysis of GlcNAc-TV secondary structure. The arrow denotes the minimal catalytic domain.

 
In summary, the minimal catalytic domain of GlcNAc-TV is S213–740, preceded by a 183 amino acid stem region, which does not appreciably affect the catalytic activity of the soluble enzyme expressed as a ProtA-fusion protein. Expression of concise functional domains is often more amenable to protein crystallization and structural analysis than full-length proteins.


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Chemicals
UDP-[3H]-N-acetylglucosamine (16.0 Ci/mmol) was purchased from Toronto Research Chemicals. The synthetic GlcNAc-TV acceptor, GlcNAcß1,2 Man{alpha}1,6 Glcß1-O-(CH2)7 CH3 was purchased from Chemica Alta Chemicals, Edmonton. The nonlabeled UDP-6-N-acetylglucosamine was obtained from Sigma Chemicals.

Recombinant GlcNAc-TV
A cDNA clone encoding rat GlcNAc-TV (Shoreibah et al., 1993Go) with an artificial EcoRI site inserted after the stop codon was used as a template for PCR reactions. Specific sets of primers were synthesized (Life Technologies) in order to generate truncated GlcNAc-TV cDNA molecules. PCR reactions were carried out with 50 ng of GlcNAc-TV cDNA in the presence of Pfu DNA polymerase (Promega) according to the method recommended by the manufacturer. Thirty cycles of PCR were performed, each cycle consisting of denaturation (95°C, 1 min), annealing (55°C, 1 min) and extension (72°C, 1 min). PCR products were digested with appropriate restriction enzymes and ligated with GlcNAc-TV cDNA fragments. All sense primers used for generation of N-terminal deletions had an additional sequence at 5' that included EcoRI restriction site. The Q39 deletion was generated with sense primer 5'-TCAGCCTGAGAGCAGCTCCAT-3' and antisense primer 5'-GCTGGGGACAGCCGTGGTGGA-3'. The PCR product was digested with EcoRI and ligated to EcoRI- digested GlcNAc-TV cDNA (342–2227 bp). The following sets of primers were used to generate N-terminal deletions: S127 (sense 5'-ACTGGAGAAAATTAATGTGGC-3' and antisense 5'-GA­GCAAGTCCCACAATATCG-3'), C155 (sense 5'-CTGCGAG­GGGAAAATCAAGTG and antisense 5'- GAGCAAGTCCC­ACAATATCG-3'), S213 (sense 5'-CTCATTGGCAGAAATCC­GCAC-3' and antisense 5'-GAGCAAGTCCCACAAT­ATCG-3'), R218 (sense 5'- CCGCACGGATTTTAACATTCT-3' and antisense 5'- GAGCAAGTCCCACAATATCG-3'), R238 (sense 5'-GAGACTTCGGATCCGCGAATG-3' and antisense 5'-GAG­CAAGTCCCACAATATCG-3') and L273 (sense 5'-CCTGGG­GCTCCTGACCAAGGA-3' and antisense 5'-GAGCAAGTCCCACAATATCG-3'). PCR products generated with these primers were digested with EcoRI and ClaI and ligated to ClaI-EcoRI GlcNAc-TV cDNA (1041–2227 bp). Two sets of primers were used to create the C-terminal deletions: G732 (sense 5' –GCCCTCCTTCTACCCCAGGAG-3' and antisense 5'-GCCCTTGATGAAGTCCCGGCA-3') and L736 (sense 5'-GCCCTCCTTCTACCCCAGGAG-3' and antisense 5'-GAGGGCCACTTGGCCCTTGAT-3'). The PCR products amplified with these primers were digested with DraIII and EcoRI and ligated to a EcoRI-DraIII fragment (2–2103 bp) of the S213–740 construct. All antisense primers used for generation of C-terminal deletions had an additional sequence at 5' that included stop codon and EcoRI restriction site. The construct N114–740 was generated by EcoRI digest of GlcNAc-TV cDNA (342–2227 bp). The truncated cDNAs were cloned in-frame into EcoRI-digested, dephosphorylated pProtA vector (Sanchez-Lopez et al., 1988Go) for expression as secreted protein A fusion proteins. Transient transfections were performed with lipofectamine reagent (Life Technologies) as recommended by the manufacturer. After 72 h of cell culture, secreted recombinant enzyme was purified from culture medium with IgG-Sepharose as described below.

In order to create a stable cell line, the expression vector encoding Q39-740 peptide was cotransfected into CHO cells with pSV2neo, in a 10:1 molar ratio, using lipofectamine. Cells were cultured in the presence of 800 µg/ml of G418 (Life Technologies), and resistant clones were selected and tested for GlcNAc-TV activity in culture medium. A representative clone showing stable expression of GlcNAc-TV activity was routinely propagated in {alpha} MEM medium (Life Technologies) containing 5% fetal bovine serum (Life Technologies) and G418 (0.2 mg/ml) in culture dishes, spinner flasks or hollow-fiber cartridges (Spectrum Laboratories).

Recombinant enzyme was isolated from cell culture medium using IgG-Sepharose Fast Flow beads (Pharmacia Biotech). 5 µl of 50% bead slurry, 2.5 µl of 2 M Tris–HCl (pH 8.0), and 5 µl of 10% Tween-20 were added per ml of culture medium. Following incubation on a rocking platform at 4°C for 20 h, the beads were collected by centrifugation, washed with 10 volumes of TST buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) and 2 volumes of 5 mM NH4Ac, pH 5.0. The recombinant ProtA- GlcNAc-TV was then eluted with one volume of 0.5 M acetic acid, pH 3.4, and resuspended in three volumes of 0.5 M MES, pH 7.5 (Calbiochem) and stored at either 4°C or –20°C.

GlcNAc-TV assay
The reaction contained 30 mM MES buffer, pH 6.5, 1 mM UDP-GlcNAc, 0.5 µCi UDP- [3H]-GlcNAc, 0.5 mM GlcNAcß1,2Man{alpha}1,6Glcß1-O-(CH2)7CH3 acceptor and purified recombinant ProtA-GlcNAc-TV. Reactions in a total volume of 30 µl were incubated at 37°C for up to 15 h and stopped by adding 0.5 ml cold water. Tubes were processed immediately or stored at –20°C. Mixtures were diluted to 5 ml in water, applied to Sep-Pak C-18 columns (Waters) and washed with 20 ml of H2O. Product was eluted with 5 ml 100% methanol and counted in a ß-scintillation counter. The reactions were linear with time of incubation under the conditions used in each assay. Protein concentrations were determined with the BCA protein assay (Pierce Chemicals) using bovine serum albumin (BSA) as the standard.

Western blot analysis
Samples containing 100 ng of purified ProtA-GlcNAc-TV recombinant protein were resuspended in SDS–PAGE loading buffer, boiled for 5 min and size fractionated on 8% SDS–PAGE. Proteins were then transferred by electroblotting to Immobilon-PVDF (Millipore) membrane filters. The membranes were treated with 5% fraction V BSA (Sigma) in TST (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) followed by incubation with primary antibody (rabbit IgG) in TST containing 1% BSA (TBS-T), washing with TBS-T, incubation with horseradish peroxidase–conjugated secondary antibody and final wash with TBS-T. Blots were developed with enhanced chemiluminescence substrate solution (ECL, Amersham Corp.).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Drs. Michael Pierce and Nevis Fregien for the rat GlcNAc-TV cDNA, and Dr. Charles Warren for helpful discussion. J.W.Dennis is a NCI of Canada Terry Fox Scientist.


    Abbreviations
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
GlcNAc, D-N-acetylglucosamine; FBS, fetal bovine serum; GlcNAc-TV, UDP-GlcNAc, Man{alpha}1–6Manß-R ß1–6N-acetylglucosaminyltransferase V (EC 2.4.1.155); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; bp, base pair; CHO, Chinese hamster ovary; CHOP, Chinese hamster ovary expressing polyoma virus large T antigen; BSA, bovine serum albumin.


    Footnotes
 
1 To whom correspondence should be addressed Back

2 Present addresses: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada, and Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada Back


    References
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 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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