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Tumor Suppressor EXT Gene Family Members Encode Glycosyltransferases that Synthesize Heparan Sulfate/Heparin

EXT1 and EXT2 genes are associated with the development of hereditary multiple exostoses (HME), an autosomal dominant disorder characterized by aberrant bone formation most commonly originating from the juxtaepiphyseal regions of the long bones (1). HME results from mutations in one of two homologous genes, EXT1 and EXT2. EXT1, EXT2 and three additional members, designated EXTL1, EXTL2 and EXTL3, form an EXT gene family (2) (Fig. 1). The EXT and EXTL proteins have sequence homology especially in their C-terminal regions. However, there is no evidence that defects in the EXTL genes result in HME, and hence they have been designated as EXT-like. EXT1 and EXT2 appear to act as tumor suppressors because loss of heterozygosity of these genes has been observed in exostoses-derived chondrosarcomas and osteosarcomas. Chromosomal locations of three EXT-like genes imply that they might also be tumor suppressors.
Fig. 1. Comparison of the five cloned members of the EXT gene family. All the members encode glycosyltransferase activities possibly involved in HS/Hep biosynthesis. The putative membrane spanning domains are indicated by green bars. Conserved or highly conserved regions are indicated by blue or red bars, respectively. The protein shows significant homology with the carboxy termini of the other members of the family, and the variation in size is due to differences in the length of the amino terminal side of the proteins.
Two independent studies revealed the connection between heparan sulfate (HS) synthesizing glycosyltransferases and an EXT gene family. EXT1 was identified as the gene that is capable of restoring HS synthesis in HS-deficient mutant cells (3), and the direct peptide sequencing of HS co-polymerase purified from bovine serum identified the enzyme as an EXT2 homolog (4). At around this time, the Drosophila gene tout velu (ttv), an EXT1 ortholog, was also implicated with the diffusion of Hedgehog, a presumably glycosaminoglycan-dependent process (5), which was later found to be an HS-dependent process, in embryonic development. More recent studies have firmly established that recombinant forms of EXT1 and EXT2 have indeed both alpha1,4GlcNAc (GlcNAcT-II) and beta1,4GlcA transferase (GlcAT-II) activities (6), which are required for HS chain elongation and distinct from GlcNAcT-I and GlcAT-I that transfer the first GlcNAc and GlcA residue, respectively, to the protein linkage region (Fig. 2). It should be noted that deficiency of either EXT1 or EXT2 causes HME, indicating that both EXT1 and EXT2 are essential glycosyltransferases for HS biosynthesis.
Fig. 2. Overlapping yet distinct glycosyltransferase activities of the five EXT gene family members involved in HS biosynthesis. Following completion of the glycosaminoglycan-protein tetrasaccharide linkage region (GlcA-Gal-Gal-Xyl-Ser) by the action of GlcAT-I, which does not belong to the EXT gene family, a HS/Hep chain is synthesized by the EXT family proteins on this linkage region. EXT1 and EXT2 are putative co-polymerases with both GlcAT-II and GlcNAcT-II activities. EXTL1 shows GlcNAcT-II activity for chain elongation, whereas EXTL2 shows GlcNAcT-I activity for chain initiation. EXTL3 possesses both GlcNAcTI and GlcNAcT-II activities.
EXTL1, EXTL2 and EXTL3 also encode GlcNAc transferases, which are likely involved in HS biosynthesis (7,8). Truncated forms of EXTL1, EXTL2 and EXTL3, lacking the putative NH2-terminal transmembrane and cytoplasmic domains, were transiently expressed in COS-1 cells, and found to harbor alpha-GlcNAc transferase activities. EXTL2 protein transfers alpha1,4GlcNAc to GlcAbeta1-3Galbeta1-O-naphtalenmethanol (7), which is an artificial analog of the glycosaminoglycan-protein linkage region (GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser) and is a strong candidate for the key enzyme GlcNAcT-I that initiates the synthesis of HS and heparin (Hep), segregating it from the synthesis of chondroitin sulfate and dermatan sulfate, which are formed on the same linkage region structure. Truncated EXTL1 transfers alpha1,4GlcNAc to N-acetylheparosan oligosaccharides GlcAbeta1-4GlcNAcalpha1-(4GlcAbeta1-4GlcNAcalpha1-)n that represent growing HS chains, thus has GlcNAcT-II activity (8). Truncated EXTL3 utilizes not only N-acetylheparosan oligosaccharides but also GlcAbeta1-3Galbeta1-O-C2H4NHCbz, another synthetic substrate for alpha-GlcNAc transferase I for the initiation of the HS/Hep synthesis, thus has both GlcNAcT-I and GlcNAcT-II activities (8). Neither EXTL1 nor EXTL3 shows any glucuronyltransferase activity. Hence, EXTL3 is most likely involved in both chain initiation and elongation, whereas EXTL1 is possibly involved only in the chain elongation of HS and maybe Hep as well. Thus, the acceptor specificities of the five family members overlap, but are distinct from each other except for EXT1 and EXT2 with the same specificity. In other words, all five cloned human EXT gene family proteins harbor glycosyltransferase activities which probably contribute to the synthesis of HS and Hep.

A possible explanation for the existence of the two distinct GlcNAcT-I molecular species, EXTL2 and EXTL3, for the key enzyme activity critical for the selective assembly of HS/Hep chains may be that they initiate HS and/or Hep chains on different core proteins by discriminating the amino acid sequences. It remains to be clarified how the GlcNAcT-II activity of EXTL1 or the similar GlcNAcT-II activity of EXTL3 play their respective roles in chain polymerization of HS/Hep, where it requires a glucuronyltransferase (GlcAT-II) as a partner. It is unknown which one of EXT1 or EXT2 co-operates with EXTL1 and/or EXTL3 for the synthesis of the repeating disaccharide region or chain polymerization. Human EXT1 and EXT2 form a stable complex that accumulates in the Golgi apparatus (6,9,10) to exhibit substantially higher glycosyltransferase activities than EXT1 or EXT2 alone and appears to be a biologically relevant enzyme form (6,9). It should be remembered that HS chain polymerizing enzyme activity has not been demonstrated in vitro for an EXT1/EXT2 hetero-oligomeric complex. Therefore, possibility of multimeric complex formation involving other proteins also exists. Alternatively, the single GlcNAc transfer catalyzed by EXTL1 and EXTL3 possibly breaks up the elongation process and, in effect, serves as a chain termination mechanism.

The EXT and EXTL proteins are highly conserved from Caenorhabditis elegans and Drosophila melanogaster to higher vertebrates, and recent results from Drosophila genetics strongly indicate the essential roles of HS-synthesizing enzymes in developmental processes (11). Targeted disruption of EXT1 in mice results in embryonic lethality (12). HS has indeed been demonstrated in C. elegans (13,14) and Drosophila (14). Ttv is a protein of 760 amino acids and is 56% identical to the human EXT1 protein (5). The Hedgehog, but not FGF or Wingless, signaling, is selectively affected in the ttv mutant, suggesting that other EXT genes exist (5). Although ttv has been presumed to encode a putative HS polymerase, such catalytic activities of ttv or other Drosophila EXT proteins have not been reported. Two EXT genes designated rib-1 and rib-2 exist in C. elegans, and the rib-2 protein consisted of 814 amino acids has been shown to harbor GlcNAcT-I and GlcNAc-T-II activities, and rib-2 is an EXTL3 ortholog (15). No glucuronyltransferase activity involved in HS chain polymerization has been demonstrated for either protein.
Kazuyuki Sugahara (Kobe Pharmaceutical University)
References(1)McCormick C, Tufaro F : New perspectives on the molecular basis of hereditary bone tumours. Molecular Medicine Today 5, 481-486, 1999
(2) Sugahara K, Kitagawa H : Recent advances in the study of the biosynthesis and functions of sulfated glycosaminoglycans. Curr. Opin. Struct. Biol. 10, 518-527, 2000
(3) McCormick C, Leduc Y, Martindale D, Mattison K, Esford LE, Dyer AP, Tufaro F : The putative tumour suppressor EXT1 alters the expression of cell-surface heparan sulfate. Nat. Genet. 19, 158-161, 1998
(4) Lind T, Tufaro F, McCormick C, Lindahl U, Lidholt K : The putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesis of heparan sulfate. J. Biol. Chem. 273, 26265-26268, 1998
(5) Bellaiche Y, The I, Perrimon N : Tout-velu is a Drosophila homoloue of the putative tumor suppressor EXT-1 and is needed for Hh diffusion. Nature 394, 85-88, 1998
(6) Senay C, Lind T, Muguruma K, Tone Y, Kitagawa H, Sugahara K, Lidholt K, Lindahl U, Kusche-Gullberg M : Association of the EXT1 and EXT2 proteins in heparan sulfate biosynthesis. EMBO reports 1, 282-286, 2000
(7) Kitagawa H, Shimakawa H, Sugahara K : The tumor suppressor EXT-like gene EXTL2 encodes an alpha1,4-N-acetylhexosaminyltransferase that transfers N-acetylgalactosamine and N-acetylglucosamine to the common glycosaminoglycan-protein linkage region. The key enzyme for the chain initiation of heparan sulfate. J. Biol. Chem. 274, 13933-13937, 1999
(8) Kim B-T, Kitagawa H, Tamura J, Saito T, Kusche-Gullberg M, Lindahl U, Sugahara, K : The human tumor suppressor EXT gene family members, EXTL1 and EXTL3, encode alpha1,4-N-acetylglucosaminyltransferases involved in heparan sulfate/heparin biosynthesis. Proc. Natl. Acad. Sci. USA, in press, 2001
(9) McCormick C, Duncan G, Goutsos KT, Tufaro F : The putative tumor suppressors EXT1 and EXT2 form a stable complex that accumulates in the Golgi apparatus and catalyzes the synthesis of heparan sulfate. Proc. Natl. Acad. Sci. USA 97, 668-673, 2000
(10) Kobayashi S, Morimoto K, Shimizu T, Takahashi M, Kurosawa H, Shirasawa T : Association of EXT1 and EXT2, hereditary multiple exostoses gene products, in Golgi apparatus. Biochem. Biophys. Res. Commun. 268, 860-867, 2000
(11) Perrimon N, Bernfield M : Specificities of heparan sulfate proteoglycans in developmental processes. Nature 404, 725-728, 2000
(12) Lin X, Wei G, Shi Z, Dryer L, Esko JD, Wells DE, Matzuk MM : Disruption of gastrulation and heparan sulfate biosynthesis in EXT1-deficient mice. Dev. Biol. 22, 299-311, 2000
(13) Yamada Y, Van Die I, Van den Eijnden DH, Yokota A, Kitagawa H, Sugahara K : Demonstration of glycosaminoglycans in Caenorhabditis elegans. FEBS Letters 459, 327-331, 1999
(14) Toyoda H, Kinoshita-Toyoda A, Selleck SB : Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo. J Biol Chem 275, 2269-2275, 2000
(15) Kitagawa H, Egusa N, Tamura J, Kusche-Gullberg M, Lindahl U, Sugahara K : rib-2, a Caenorhabditis elegans homolog of the human tumor suppressor EXT genes encodes a novel alpha1,4-N-acetylglucosaminyltransferase involved in the biosynthetic initiation and elongation of heparan sulfate. J. Biol. Chem. 276, 4834-4388, 2001
Jun. 15, 2001
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