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. 2000 Sep;1(3):282-6.
doi: 10.1093/embo-reports/kvd045.

The EXT1/EXT2 tumor suppressors: catalytic activities and role in heparan sulfate biosynthesis

C Senay  1 , T Lind, K Muguruma, Y Tone, H Kitagawa, K Sugahara, K Lidholt, U Lindahl, M Kusche-Gullberg
Affiliations

The EXT1/EXT2 tumor suppressors: catalytic activities and role in heparan sulfate biosynthesis

C Senay et al. EMBO Rep. 2000 Sep.

Abstract

The D-glucuronyltransferase and N-acetyl-D-glucosaminyltransferase reactions in heparan sulfate biosynthesis have been associated with two genes, EXT1 and EXT2, which are also implicated in the inherited bone disorder, multiple exostoses. Since the cell systems used to express recombinant EXT proteins synthesize endogenous heparan sulfate, and the EXT proteins tend to associate, it has not been possible to define the functional roles of the individual protein species. We therefore expressed EXT1 and EXT2 in yeast, which does not synthesize heparan sulfate. The recombinant EXT1 and EXT2 were both found to catalyze both glycosyltransferase reactions in vitro. Coexpression of the two proteins, but not mixing of separately expressed recombinant EXT1 and EXT2, yields hetero-oligomeric complexes in yeast and mammalian cells, with augmented glycosyltransferase activities. This stimulation does not depend on the membrane-bound state of the proteins.

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Figures

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Fig. 1. Scheme of EXT constructs. (A) With transmembrane domain (TM, black box). For expression in yeast, EXT1 and EXT2 cDNA constructs were inserted into the pPICZB vector (Invitrogen). For expression in COS-1 cells, EXT1 and EXT2 were ligated into the pcDNA3.1 expression vector (Invitrogen). (B) Without transmembrane domain (SP, signal peptide, open box). EXT constructs (sEXT) lacking the first N-terminal 32 (sEXT1-GFP) or 48 (sEXT2myc-his) amino acid residues, respectively, were ligated into the pPICZαC vector (Invitrogen). sEXT1protA and sEXT2protA constructs lacking the first N-terminal 43 or 55 amino acid residues, respectively, were subcloned into pGIR201protA (Kitagawa and Paulson, 1994) and subsequently inserted into the expression vector pEF-BOS. N and C, N- and C-terminus, respectively.
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Fig. 2. Glycosyltransferase assays of yeast transformed with EXT proteins. The GlcNAc-T activities of yeast cell extracts were determined by incubating 70 µg lysate protein with 0.125 µCi of UDP-[14C]GlcNAc and 50 µg of [GlcA-GlcNAc]n oligosaccharide (the reducing-terminal sugar is modified to an 2,5-anhydromannitol unit) under the conditions described (Lind et al., 1993). After 1 h at 37°C, the mixtures were applied to columns (28 × 0.5 cm) of Sephadex G-25 in 0.5 M NaCl, 50 mM Tris–HCl, 0.1% Triton X-100, pH 7.4. Effluent fractions were analyzed by scintillation counting. The various samples were derived from yeast transformed with empty plasmid (mock, filled diamond), EXT1 (filled circle), EXT2 (open circle) and EXT1/2 (cotransformation, open diamond). GlcA-T activities were assayed in similar fashion, except that UDP-[3H]GlcA was used as a sugar donor, and GlcNAc-[GlcA-GlcNAc]n oligosaccharides were used as acceptor. The resultant gel chromatograms (not shown) were highly similar to the GlcNAc-T assays shown in the figure.
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Fig. 3. Endo H treatment of EXT-proteins expressed in Pichia. Membrane fractions from yeast transformed separately with myc-his tagged EXT1 or EXT2 or cotransformed with EXT1 and EXT2 (EXT1/2) were incubated in the absence or presence of Endo H and analyzed by 7.5% SDS–PAGE followed by western blotting with an antibody to the polyhistidine-tag. Transformed clones are identified at the top. The kilodalton values of protein molecular mass markers are indicated. Mock, transformed with vector alone.
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Fig. 4. Hetero-oligomerization of EXT1 and EXT2. Proteins from EXT-transformed yeast or transfected COS-1 cells were analyzed by SDS–PAGE, either directly or following immunopurification as indicated (see Methods). (A) Pichia proteins. Microsomal proteins from yeast cells transformed with the indicated EXT constructs were immunoprecipitated with anti-GFP antibodies. (B) COS-1 cell proteins. Samples were immunopurified by chromatography on anti-flag antibody-agarose. All samples (A and B) were immunoblotted using anti-myc antibodies. Sample designation: mock, empty vector; EXT1 or EXT2 alone, single protein expression; EXT1/EXT2, coexpression; EXT1 + EXT2, proteins expressed in separate cultures, then mixed.
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