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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: deletion mutant/glycosyltransferase/structurefunction analysis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The human GlcNAc-TV gene is located on chromosome 2q21, having 17 exons and spanning 155 kb (Saito et al., 1995). 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., 1997
; Chen et al., 1998
). 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., 1985
; Pierce and Arango, 1986
; Dennis et al., 1987
). 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., 1996
). 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., 1993
). In addition, topical expression of GlcNAc-TV results in morphological transformation and tumorigenesis in epithelial cells (Demetriou et al., 1995
) and causes an increase in metastatic potential in mouse mammary cancer cells (Seberger and Chaney, 1999
). Tumor cell mutants selected for loss of GlcNAc-TV activity show reduced malignant potential in vivo (Dennis et al., 1987
; Lu et al., 1994
).
Human tumors, including breast, colon, skin, head, and neck, and Sezary syndrome all showed increased levels of ß16GlcNAc-branched structures as detected by L-PHA, a plant lectin that binds these moieties (Dennis and Laferte, 1989; Takano et al., 1990
; Fernandes et al., 1991
; Derappe et al., 1996
; Seelentag et al., 1997
). Moreover, L-PHA staining intensity was shown to be an independent prognostic marker for disease-free survival in colorectal cancer patients (Seelentag et al., 1998
).
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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: Q39740, N114740, S127740, C155740, and S213740 displayed similar catalytic activities (Table I). Deletion of five additional N-terminal amino acids to produce R218740 resulted in a 20-fold loss of catalytic activity (Table I). Further N-terminal deletions (R238740 and L273740) as well as truncation at the carboxyl terminus (S213732 and S213736) produced inactive proteins. Enzyme kinetics was performed using ProtA-Q39740 and ProtA-S213740 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-TVs 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., 1992), purified human enzyme from lung cancer cells (Gu et al., 1993
), and enzyme in BHK cell lysates (Palcic et al., 1990
).
|
|
|
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 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
13Gal-T has a 67 amino acid stem region, and a minimal catalytic domain of 285 amino acids (Henion et al., 1994
), while ß14Gal-T has a 85 amino acid long stem region and 273 amino acid catalytic domain (Boeggeman et al., 1993
). 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., 1996
). 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.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recombinant GlcNAc-TV
A cDNA clone encoding rat GlcNAc-TV (Shoreibah et al., 1993) 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 (3422227 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 (10412227 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 (22103 bp) of the S213740 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 N114740 was generated by EcoRI digest of GlcNAc-TV cDNA (3422227 bp). The truncated cDNAs were cloned in-frame into EcoRI-digested, dephosphorylated pProtA vector (Sanchez-Lopez et al., 1988
) 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 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 TrisHCl (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 TrisHCl, 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 GlcNAcß1,2Man1,6Glcß1-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 SDSPAGE loading buffer, boiled for 5 min and size fractionated on 8% SDSPAGE. 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 TrisHCl, 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 peroxidaseconjugated secondary antibody and final wash with TBS-T. Blots were developed with enhanced chemiluminescence substrate solution (ECL, Amersham Corp.).
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
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
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Buckhaults,P., Chen,L., Fregien,N. and Pierce,M. (1997) Transcriptional regulation of N-acetylglucosaminyltransferase V by the src oncogene. J. Biol. Chem., 272, 1957519581.
Chen,L., Zhang,W., Fregien,N. and Pierce,M. (1998) The her-2/neu oncogene stimulates the transcription of N-acetylglucosaminyltransferase V and expression of its cell surface oligosaccharide products. Oncogene, 17, 208793.[ISI][Medline]
Demetriou,M., Nabi,I.R., Coppolino,M., Dedhar,S. and Dennis,J.W. (1995) Reduced contact-inhibition and substratum adhesion in epithelial cells expressing GlcNAc-transferase V. J. Cell Biol., 130, 383392.[Abstract]
Dennis,J.W. and Laferte,S. (1989) Oncodevelopmental expression of GlcNAc ß16Man 16Man ß1-branched asparagine-linked oligosaccharides in murine tissues and human breast carcinomas. Cancer Res., 49, 945950.[Abstract]
Dennis,J.W., Laferte,S., Waghorne,C., Breitman,M.L. and Kerbel,R.S. (1987) ß 16 branching of Asn-linked oligosaccharides is directly associated with metastasis. Science, 236, 582585.[ISI][Medline]
Derappe,C., Haentjens,G., Lemaire,S., Feugeas,J.P., Lebbe,C., Pasqualetto,V., Bussel,A., Aubery,M. and Neel,D. (1996) Circulating malignant lymphocytes from Sezary syndrome express high level of glycoproteins carrying ß (16)N-acetylglucosamine-branched N-linked oligosaccharides. Leukemia, 10, 138141.[ISI][Medline]
Fernandes,B., Sagman,U., Auger,M., Demetriou,M. and Dennis,J.W. (1991) ß16 branched oligosaccharides as a marker of tumor progression in human breast and colon neoplasia. Cancer Res., 51, 718723.[Abstract]
Gu,J., Nishikawa,A., Tsuruoka,N., Ohno,M., Yamaguchi,N., Kangawa,K. and Taniguchi,N. (1993) Purification and characterization of UDP-N-acetylglucosamine: -6-D-mannoside ß16N-acetylglucosaminytransferase (N-acetylglucosaminyltransferase V) from human lung cancer cell line. J. Biochem. (Tokyo), 113, 614619.[Abstract]
Guilloux,Y., Lucas,S., Brichard,V.G., Van Pel,A., Viret,C., De Plaen,E., Brasseur,F., Lethe,B., Jotereau,F. and Boon,T. (1996) A peptide recognized by human cytolytic T lymphocytes on HLA-A2 melanomas is incoded by an intron sequence of the N-acetylglucosaminyltransferase V gene. J. Exp. Med., 183, 11731183.[Abstract]
Heffernan,M., Lotan,R., Amos,B., Palcic,M., Takano,Y. and Dennis,J.W. (1993) Branching ß16N-acetylglucosaminyltransferases and polylactosamine expression in mouse F9 teratocarcinoma cells and differentiated counterparts. J. Biol. Chem., 268, 12421251.
Henion,T.R., Macher,B.A., Anaraki,F. and Galili,U. (1994) Defining the minimal size of catalytically active primate 1,3 galactosyltransferase: structurefunction studies on the recombinant truncated enzyme. Glycobiology, 4, 193201.[Abstract]
Lu,Y., Pelling,J.C. and Chaney,W.G. (1994) Tumor cell surface ß16 branched oligosaccharides and lung metastasis. Clin. Exp. Metastasis, 12, 4754.[ISI][Medline]
Miyoshi,E., Nishikawa,A., Ihara,Y., Gu,J., Sugiyama,T., Hayashi,N., Fusamoto,H., Kamada,T. and Taniguchi,N. (1993) N-acetylglucosaminyltransferase III and V messenger RNA levels in LEC rats during hepatocarcinogenesis. Cancer Res., 53, 38993902.[Abstract]
Nagai,K., Ihara,Y., Wada,Y. and Taniguchi,N. (1997) N-glycosylation is required for the enzymatic activity and Golgi retention of N-acetylglucosaminyltransferase III. Glycobiology, 7, 769776.[Abstract]
Palcic,M.M., Ripka,J., Kaur,K.J., Shoreibah,M., Hindsgaul,O. and Pierce,M. (1990) Regulation of N-acetylglucosaminyltransferase V activity. J. Biol. Chem., 265, 67596769.
Pierce,M. and Arango,J. (1986) Rous sarcoma virus-transformed baby hamster kidney cells express higher levels of asparagine-linked tri- and tetraantennary glycopeptides containing [GlcNAc-ß (1,6)Man- (1,6)Man] and poly-N-acetyllactosamine sequences than baby hamster kidney cells. J. Biol. Chem., 261, 1077210777.
Saito,H., Nishikawa,A., Gu,J., Ihara,Y., Soejima,H., Wada,Y., Sekiya,C., Niikawa,N. and Taniguchi,N. (1994) cDNA cloning and chromosomal mapping of human N-acetylglucosaminyltransferase V. Biochem. Biophys. Res. Commun., 198, 318327.[ISI][Medline]
Saito,H., Gu,J., Nishikawa,A., Ihara,Y., Fijii,J., Kohgo,Y. and Taniguchi,N. (1995) Organization of the human N-acetylglucosaminyltransferase V gene. Eur. J. Biochem., 233, 1826.[Abstract]
Sanchez-Lopez,R., Nicholson,R., Gesnel,M-C., Matrisian,L. and Breathnach,R. (1988) Structure-function relationships in the collagenase family member transin. J. Biol. Chem., 263, 1189211899.
Schachter,H. (1986). Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides. Biochem. Cell Biol.., 64, 163181.[ISI][Medline]
Seberger,P.J. and Chaney,W.G. (1999) Control of metastasis by Asn-linked, ß16 branched oligosaccharides in mouse mammary cancer cells. Glycobiology, 3, 235241.
Seelentag,W.K., Boni,R., Gunthert,U., Futo,E., Burg,G., Heitz,P.U. and Roth,J. (1997) Expression of CD44 isoforms and ß1,6-branched oligosaccharides in human malignant melanoma is correlated with tumor progression but not with metastatic potential. J. Cutan. Pathol., 24, 206211.[ISI][Medline]
Seelentag,W.K., Li,W.P., Schmitz,S.F., Metzger,U., Aeberhard,P., Heitz,P.U. and Roth,J. (1998) Prognostic value of ß1,6-branched oligosaccharides in human colorectal carcinoma. Cancer Res., 58, 55595564.[Abstract]
Shoreibah,M.G., Hindsgaul,O. and Pierce,M. (1992) Purification and characterization of rat kidney UDP-N-acetylglucosamine: -6-D-mannoside ß-1,6-N-acetylglucosaminyltransferase. J. Biol. Chem., 267, 29202927.
Shoreibah,M.G, Perng,G., Adler,B., Weinstein,J.,Basu,R., Cupples,R., Wen,D., Browne,J.K., Buckhaults,P., Fregien,N. and Pierce,M. (1993) Isolation, characterization and expression of cDNA encoding N-acetylglucosaminyltransferase V. J. Biol. Chem., 268, 1538115385.
Takano,R., Nose,M., Nishihira,T. and Kyogoku,M. (1990) Increase of ß16-branched oligosaccharides in human esophageal carcinomas invasive against surrounding tissue in vivo and in vitro. Am. J. Pathol., 137, 10071011.[Abstract]
Toki,D., Sarkar,M., Yip,B., Reck,F., Joziasse,D., Fukuda,M., Schachter,H. and Brockhausen,I. (1997) Expression of stable human O-glycan core 2 ß-1,6-N-acetylglucosaminyltransferase in Sf9 insect cells. Biochem. J., 325, 6369.[ISI][Medline]
van den Eijnden,D.H., Koenderman,A.H.L. and Schiphorst,W.E.C.M. (1988) Biosynthesis of blood group i-active polylactosaminoglycans. J. Biol. Chem., 263, 1246112465.
Weinstein,J., Sundaram,S., Wang,X., Delgado,D., Basu,R. and Stanley,P. (1996) A point mutation causes mistargeting of Golgi GlcNAc-TV in the Lec4A Chinese hamster ovary glycosylation mutant. J. Biol. Chem., 271, 2746227469.
Yamashita,K., Tachibana,Y., Ohkura,T. and Kobata,A. (1985) Enzymatic basis for the structural changes of asparagine-linked sugar chains of membrane glycoproteins of baby hamster kidney cells induced by polyoma transformation. J. Biol. Chem., 260, 39633969.[Abstract]
Yousefi,S., Higgins,E., Doaling,Z., Hindsgaul,O., Pollex-Kruger,A. and Dennis,J.W. (1991) Increased UDP-GlcNAc:Galß13GalNAc-R (GlcNAc to GalNAc) ß16 N-acetylglucosaminyltransferase activity in transformed and metastatic murine tumor cell lines: control of polylactosamine synthesis. J. Biol. Chem., 266, 17721783.