The Putative Tumor Suppressors EXT1 and EXT2 Are Glycosyltransferases Required for the Biosynthesis of Heparan Sulfate
Hereditary multiple exostoses, characterized by multiple cartilaginous tumors, is ascribed to mutations at three distinct loci, denoted EXT1–3. Here, we report the purification of a protein from bovine serum that harbored thed-glucuronyl (GlcA) and N-acetyl-d-glucosaminyl (GlcNAc) transferase activities required for biosynthesis of the glycosaminoglycan, heparan sulfate (HS). This protein was identified as EXT2. Expression of EXT2 yielded a protein with both glycosyltransferase activities. Moreover, EXT1, previously found to rescue defective HS biosynthesis (McCormick, C., Leduc, Y., Martindale, D., Mattison, K., Esford, L. E., Dyer, A. P., and Tufaro, F. (1998) Nat. Genet. 19, 158–161), was shown to elevate the low GlcA and GlcNAc transferase levels of mutant cells. Thus at least two members of the EXT family of tumor suppressors encode glycosyltransferases involved in the chain elongation step of HS biosynthesis. Hereditary multiple exostoses, characterized by multiple cartilaginous tumors, is ascribed to mutations at three distinct loci, denoted EXT1–3. Here, we report the purification of a protein from bovine serum that harbored thed-glucuronyl (GlcA) and N-acetyl-d-glucosaminyl (GlcNAc) transferase activities required for biosynthesis of the glycosaminoglycan, heparan sulfate (HS). This protein was identified as EXT2. Expression of EXT2 yielded a protein with both glycosyltransferase activities. Moreover, EXT1, previously found to rescue defective HS biosynthesis (McCormick, C., Leduc, Y., Martindale, D., Mattison, K., Esford, L. E., Dyer, A. P., and Tufaro, F. (1998) Nat. Genet. 19, 158–161), was shown to elevate the low GlcA and GlcNAc transferase levels of mutant cells. Thus at least two members of the EXT family of tumor suppressors encode glycosyltransferases involved in the chain elongation step of HS biosynthesis. heparan sulfate GlcA transferase GlcNAc transferase HS-polymerase hereditary multiple exostoses polyacrylamide gel electrophoresis base pair(s) phosphate-buffered saline 3-(cyclohexylamino)propanesulfonic acid. Heparan sulfate (HS)1proteoglycans, ubiquitously distributed on cell surfaces and in the extracellular matrix, consist of sulfated glycosaminoglycan chains that are covalently bound to various core proteins. HS polysaccharide, increasingly implicated in physiological processes such as cell adhesion, cytokine action, and regulation of enzymic catalysis, owes its biological properties to interactions with various proteins, mediated by specific saccharide sequences. Biosynthesis of HS chains involves the formation of an initial, simple polysaccharide, composed of alternating d-glucuronic acid (GlcA) and N-acetyl-d-glucosamine (GlcNAc) units, joined by 1→4 linkages. This polymer is subsequently modified through a series of reactions, which involves partial N-deacetylation and N-sulfation of GlcNAc units, C-5 epimerization of GlcA tol-iduronic acid residues, and O-sulfation at various positions (1Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (396) Google Scholar). The GlcA transferase (GlcA-T) and GlcNAc transferase (GlcNAc-T) reactions required to generate the initial HS polysaccharide precursor have been associated with a single protein (2Lidholt K. Weinke J.L. Kiser C.S. Lugemwa F.N. Bame K.J. Cheifetz S. Massagué J. Lindahl U. Esko J.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2267-2271Crossref PubMed Scopus (236) Google Scholar), hereafter referred to as “HS-polymerase” (HS-POL). Partial purification of proteins from bovine serum revealed a ∼70-kDa component with both activities (3Lind T. Lindahl U. Lidholt K. J. Biol. Chem. 1993; 268: 20705-20708Abstract Full Text PDF PubMed Google Scholar). We now report the molecular cloning of this protein and demonstrate that it is 94% identical to human EXT2, a member of the EXT family of tumor suppressors. Also EXT1, another member of the same family (4Cook A. Raskind W. Blanton S.H. Pauli R.M. Gregg R.G. Francomano C.A. Puffenberger E. Conrad E.U. Schmale G. Schellenberg G. Wijsman E. Hecht J.T. Wells D. Wagner M.J. Am. J. Hum. Genet. 1993; 53: 71-79PubMed Google Scholar, 5LeMerrer M. Legeai-Mallet L. Jeannin P.M. Horsthemke B. Schinzel A. Plauchu H. Toutain A. Achard F. Munnich A. Maroteaux P. Hum. Mol. Genet. 1994; 3: 717-722Crossref PubMed Scopus (189) Google Scholar, 6Wu Y.Q. Heutink P. deVries B.B.A. Sandkuijl L.A. van den Ouweland A.M.W. Niermeijer M.F. Galjaard H. Reyneirs E. Willems P.J. Halley D.J.J. Hum. Mol. Genet. 1994; 3: 167-171Crossref PubMed Scopus (149) Google Scholar, 7Ahn J. Lüdecke H.-J. Lindow S. Horton W.A. Lee B. Wagner M.J. Horsthemke B. Wells D.E. Nat. Genet. 1995; 11: 137-143Crossref PubMed Scopus (374) Google Scholar), is implicated with similar catalytic activities. Mutations of EXT genes have been associated with the development of hereditary multiple exostoses (HME), the most frequent of all skeletal dysplasias. These findings suggest that alterations in the formation of the HS precursor polysaccharide may be involved in tumor formation and further point to an important role for HS in control of bone growth. The polymerase was isolated from bovine serum using an extension of the protocol described previously (3Lind T. Lindahl U. Lidholt K. J. Biol. Chem. 1993; 268: 20705-20708Abstract Full Text PDF PubMed Google Scholar). Briefly, the procedure involved successive chromatographies through the following matrices: Red-Sepharose, concanavalin A-Sepharose connected to Red-Sepharose (recirculation for 48 h), phenyl-Sepharose, Superdex 200 (gel chromatography), UDP-Sepharose, Mono Q (anion-exchange chromatography), and Mono P (chromatofocusing). The product was finally separated by preparative SDS-PAGE and stained with Coomassie Blue. The implicated ∼70-kDa component was digested with trypsin in the gel, and the resultant peptides were separated and sequenced as described (8Rosenfeld J. Capdevielle J. Guillemont J.C. Ferrara P. Anal. Biochem. 1992; 203: 173-179Crossref PubMed Scopus (1130) Google Scholar). The cDNA probe used for screening was derived from a human EST clone (826 bp) from Soares fetal liver spleen library (GenBank accession no. U13869(IMAGE Consortium)). The clone was excised from vector pT7T3D (Amersham Pharmacia Biotech) with PacI and EcoRI and was labeled with [α-32P]dCTP, using a random-priming kit (Boehringer Mannheim). Nitrocellulose replicas of plaques from the bacteriophage λgt10 bovine kidney cDNA library (catalog no. BL3001a; CLONTECH) were hybridized with the labeled probe according to the instructions of the manufacturers. The nucleotide sequences of cDNAs were determined by repeated sequencing of both strands of alkaline-denatured plasmid DNA using the Cy5 AutoRead sequencing kit (Amersham Pharmacia Biotech). Nucleotide sequences were labeled using a Cy5-dATP labeling mix, and the sequencing reactions were performed using T7 DNA polymerase. Sequences were determined on an ALFexpress system (Amersham Pharmacia Biotech) and analyzed using the DNA-Star (DNASTAR Inc., Wisconsin) program. The nucleotide and protein sequences were applied to data base screening using BLAST search, NCBI (Internet address:http://www.ncbi.nlm.nih.gov/). The 2884-bp cDNA insert recovered from the bovine kidney cDNA library was cleaved with restriction enzyme BseRI to generate a 2256-bp fragment (corresponding to nucleotides 185–2441), which was then treated with Klenow fragment to generate blunt ends. This product was ligated into a pcDNA3 expression vector (Invitrogen), modified to introduce a His/FLAG (MGGSHHHHHHDYKDDDDK-) tag at the N terminus. COS-7 cells were cultured in Dulbecco's modified Eagle's medium-F12 (catalog no. 31330-038, Life Technologies, Inc.) supplemented with 50 units/ml penicillin, 50 μg/ml streptomycin, and 10% (v/v) heat-inactivated (56 °C, 30 min) fetal calf serum at 37 °C and 7.5% CO2. For electrotransfection 70% confluent cells in a 175-cm2 flask were trypsinized and washed with PBS supplemented with 10 mm Hepes, 2 mmMgCl2, pH 7.2. The cells were resuspended in 500 μl of washing buffer, and 30 μg of plasmid cDNA was added along with 50 μg of fish sperm carrier DNA (Boehringer Mannheim). Electrotransfection was carried out in a 0.4-cm cuvette (BTX) at 360 V and 500 microfarads. Following transfection the cells were resuspended in culture medium containing 2% Me2SO, transferred to a 10-cm culture dish, left at room temperature for 20 min, and finally incubated at 37 °C for 72 h. Protein was analyzed on 10% polyacrylamide gel in SDS, on a Bio-Rad MiniProtean unit, according to the manufacturer's instruction. For Western detection, separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore) in a Bio-Rad Trans-Blot semidry electroblot system, using 10 mm CAPS, 5% MeOH, pH 11, as transfer buffer at 6 V for 40 min. The membrane was blocked with PBS, 0.1% Tween 20, and 15% bovine serum and was then incubated with the anti-FLAG M2 antibody (Kodak) in the same solution. After washing, the His/FLAG-tagged HS-POL was detected using a chemiluminescence kit (ECL; Amersham Pharmacia Biotech), and the signal was recorded on a Bio-Rad G525 phosphoimaging device. Cell lines analyzed for GlcA-T and GlcNAc-T activities included clone 1D from Lmtk− mouse fibroblasts, mutant gro2C derived from the same cells (9Gruenheid S. Gatzke L. Meadows H. Tufaro F. J. Virol. 1993; 67: 93-100Crossref PubMed Google Scholar), COS-7 cells, and transfected variants as indicated. After washing with PBS cells were scraped off in 50 mmHepes, 0.15 m NaCl, 1% Triton X-100, pH 7.2, and lysed by incubation with gentle agitation at 4 °C for 1 h. The lysates were centrifuged at 16,000 × g for 10 min, and supernatants were subjected to glycosyltransferase assays as described before (3Lind T. Lindahl U. Lidholt K. J. Biol. Chem. 1993; 268: 20705-20708Abstract Full Text PDF PubMed Google Scholar). Briefly, GlcA-T activity was measured by incubating lysates with UDP-[14C]GlcA and a GlcNAc-[GlcA-GlcNAc]noligosaccharide acceptor (nonreducing terminal GlcNAc unit), whereas GlcNAc-T was assayed by similar incubation with UDP-[3H]GlcNAc and a [GlcA-GlcNAc]n acceptor (nonreducing terminal GlcA unit). Labeled oligosaccharides were isolated and quantified by scintillation counting. The putative HS-POL isolated previously from bovine serum (3Lind T. Lindahl U. Lidholt K. J. Biol. Chem. 1993; 268: 20705-20708Abstract Full Text PDF PubMed Google Scholar) was subjected to further purification through a series of chromatography steps (see “Materials and Methods”). The GlcA- and GlcNAc-T activities remained associated throughout this procedure. Final separation by SDS-PAGE yielded a ∼70-kDa protein, which was isolated, and four tryptic peptides were sequenced. One of the peptides, residues 129–147 in Fig. 1, matched a human EST cDNA containing a 257-amino acid residue open reading frame. The EST clone was used to screen a bovine kidney cDNA library, which yielded a 2884-bp cDNA with a coding region of 2154 bp, corresponding to a protein of 718 amino acids (Fig. 1). This cDNA was identified as EXT2. Sequence analysis of the predicted protein suggested that it adopts a type II configuration typical of glycosyltransferases (10Paulson J.C. Colley K.J. J. Biol. Chem. 1989; 264: 17615-17618Abstract Full Text PDF PubMed Google Scholar) with a short N-terminal cytoplasmic tail, a transmembrane region, and a large lumenal domain with two potential N-glycosylation sites, as has been shown for EXT1 (11McCormick C. Leduc Y. Martindale D. Mattison K. Esford L.E. Dyer A.P. Tufaro F. Nat. Genet. 1998; 19: 158-161Crossref PubMed Scopus (326) Google Scholar). The calculatedM r is 81,900, somewhat larger than the apparentM r of the purified protein. It appears that the purified bovine HS-POL is a truncated form that has lost its transmembrane domain and been subsequently released from the cell, as is well established for glycosyltransferases (10Paulson J.C. Colley K.J. J. Biol. Chem. 1989; 264: 17615-17618Abstract Full Text PDF PubMed Google Scholar). Direct evidence that the cloned bovine HS-POL cDNA encodes a protein with both GlcA- and GlcNAc-T activities was obtained by expressing the His/FLAG fusion protein in COS-7 cells. Western blots of cell lysates using anti-FLAG antibodies showed a protein product of the appropriate size (Fig. 2). The fusion protein was recovered on an anti-FLAG affinity gel and assayed for GlcA- and GlcNAc-T activities. The apparent activities were ∼4- and ∼10-fold elevated, respectively, compared with mock-transfected controls (Fig. 3). The transferase activities displayed by control cells were probably because of endogenous enzymes committed to HS biosynthesis.Figure 3Assay of GlcNAc-T and GlcA-T activities following transfection of COS-7 cells with bovine HS-POL-His/FLAG fusion protein (see “Materials and Methods”). Expressed protein was bound to anti-FLAG M2 monoclonal antibody immobilized to agarose (Kodak) in PBS, 0.1% Triton X-100 by batch incubation at room temperature for 1 h and eluted with 0.14 mm FLAG octapeptide (DYKDDDDK) in the same buffer. Glycosyltransferase assays were performed as described under “Materials and Methods.” Thebars indicate values derived from two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A recent report shows that defective HS biosynthesis in a mutant mouse fibroblast cell line (gro2C) could be partially rescued by transfection with EXT1 (11McCormick C. Leduc Y. Martindale D. Mattison K. Esford L.E. Dyer A.P. Tufaro F. Nat. Genet. 1998; 19: 158-161Crossref PubMed Scopus (326) Google Scholar). We therefore decided to analyze the gro2C cells as well as the corresponding wild-type L cells for the two glycosyltransferase activities, before and after transfection with EXT1. Both activities were 5–10-fold lower in the mutant than in the wild-type cell lysates (Table I), indicating that the abrogated HS biosynthesis in gro2C cells is probably caused by defects in HS-POL. Transfection of gro2C cells with EXT1 showed a ∼2-fold increase in both enzymatic activities (Table I), which suggested that EXT1, like EXT2, harbors HS-POL activities.Table IGlcNAc-T and GlcA-T activities in gro2C cells and L cells, with or without overexpression of EXT1CellGlcNAc-T activityGlcA-T activitycpm Hcpm CHS mutant gro2C180 ± 20200 ± 40 gro2C EXT1310 ± 40370 ± 110Wild type L890 ± 801900 ± 200 L EXT1570 ± 1001100 ± 100Cells were grown and lysed as described under “Materials and Methods,” and GlcA-T and GlcNAc-T activities were determined. Values indicate counts/min of [3H]GlcNAc and [14C]GlcA incorporated per mg of protein per min and represent means ± S.D. of four independent assays. Open table in a new tab Cells were grown and lysed as described under “Materials and Methods,” and GlcA-T and GlcNAc-T activities were determined. Values indicate counts/min of [3H]GlcNAc and [14C]GlcA incorporated per mg of protein per min and represent means ± S.D. of four independent assays. By contrast, transfection of control L cells with EXT1 led to a decrease in HS-POL activities (Table I). A similar decrease in polymerase activities was noted after transfection of COS-7 cells with bovine HS-POL/EXT2, thus explaining why expression of recombinant enzyme was detectable in terms of catalytic activity only after recovery of fusion protein by immunoabsorption. These results suggested that overexpression of either EXT1 or EXT2 proteins interfered with the apparent activity in normal cells. The HS produced by such transfected cells showed a somewhat lower apparent negative charge density than corresponding control HS, as demonstrated by ion-exchange chromatography of HS from control and EXT1-transfected L cells (Fig. 2 in Ref. 11McCormick C. Leduc Y. Martindale D. Mattison K. Esford L.E. Dyer A.P. Tufaro F. Nat. Genet. 1998; 19: 158-161Crossref PubMed Scopus (326) Google Scholar) and HS from control and HS-POL-transfected human kidney epithelial 293 cells (Fig. 4). 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Genet. 1996; 5: 1547-1557Crossref PubMed Scopus (167) Google Scholar). Recently, several novel genes have been identified that share significant sequence homology with the EXT genes (19Wise C.A. Clines G.A. Massa H. Trask B.J. Lovett M. Genome Res. PubMed Scopus Google Scholar, W. Van Hul W. Hendrickx J. F. Wauters J. De Boulle K. Van Van T. Bossuyt P. Willems P.J. J. Hum. Genet. 5: PubMed Scopus Google Scholar, Hul W. W. Hendrickx J. F. Wauters J. De Boulle K. Van Bossuyt P. Willems P.J. 1998; PubMed Scopus Google Scholar). of have been with suggest with of The findings in this report that EXT1 and EXT2 both encode a HS-POL. It is to that members of the EXT family are involved in the biosynthesis of The in HS biosynthesis in HME is of HS-POL activities in of the Partial of activity lead to the formation of it that the polymerase with or of the enzymes that the various reactions through which the precursor polysaccharide is into the sulfated product (1Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (396) Google Scholar, K. Lindahl U. Biochem. J. 1992; Scopus Google Scholar). A in the appropriate EXT protein such as well as the initial with on the of the of tumors are associated with distinct in glycosaminoglycan, HS, D.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, G. G. Biochem. J. 1987; PubMed Scopus Google Scholar, H. K. K. K. T. A. M. Biochem. J. 1992; PubMed Scopus Google Scholar, M. C. J.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, F. Lindahl U. M. J. Biochem. 1998; PubMed Scopus Google Scholar). the are the alterations may be to interactions with a of proteins that are involved in of such proteins a of growth that may be on HS S. M. B.B. Lindahl U. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar), growth extracellular A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and I. M. M. T. 1995; 14: Scholar). of EXT1 was implicated with the of a in development Y. The I. 1998; PubMed Scopus Google Scholar). The findings the of EXT type and the of either EXT1 or EXT2 that enzymes are to for We that several of the further in the have been found to in distinct U. M. L. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). It has been that HS chains with for interactions with may be through the appropriate of such in U. M. L. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. L. J. PubMed Scopus Google Scholar). EXT proteins at the are from the of HS-POL activities because of transfection of cells with either EXT1 or EXT2. the role of HS biosynthesis in to and of analysis of the expression of in cells and as well as of with of the HS