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

Senay, Claire; Lind, Thomas; Muguruma, Kumi; Tone, Yuko; Kitagawa, Hiroshi; Sugahara, Kazuyuki; Lidholt, Kerstin; Lindahl, Ulf; Kusche-Gullberg, Marion

doi:10.1093/embo-reports/kvd045

Cited by 154 publications

(130 citation statements)

References 14 publications

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“…As already mentioned, co-expressed EXT1 and EXT2 proteins in mammalian and yeast cells form a hetero-oligomeric complex and colocate to the Golgi apparatus (47)(48)(49), with augmented individual GlcNAc and GlcA transferase activities (49). Nevertheless, in vitro polymerization of a heparan chain remains to be demonstrated.…”

Section: Interactions Between Hs-synthesizing Enzymes To Form Functiomentioning

confidence: 98%

Heparin and Heparan Sulfate Biosynthesis

2002

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SummaryHeparan sulfate is one of the most informationally rich biopolymers in Nature. Its simple sugar backbone is variously modified to different degrees depending on the cellular conditions. Thus, it matures to have an enormously complicated structure, which most likely exhibits a considerable number of unique overlapping sequences with peculiar sulfation profiles. Such sequences are recognized by specific complementary proteins, which form a huge group of "heparin-binding proteins," and the sugar sequences in turn support unique functions of the respective proteins through specific interactions. The heparan sulfate sequences are not directly encoded by genes, but are created by elaborate biosynthetic mechanisms, which ensure the generation of these indispensable sequences. In heparan sulfate biosynthesis, the tetrasaccharide sequence (GlcAGal-Gal-Xyl-), designated the protein linkage region, is first assembled on a specific Ser residue at the glycosaminoglycan attachment site of a core protein. A heparan sulfate chain is then polymerized on this fragment by alternate additions of GlcNAc and GlcA through the actions of glycosyltransferases with overlapping specificities encoded by the tumor suppressor EXT family genes. Then follow various modifications by N-deacetylation and N-sulfation of glucosamine, C5-epimerization of GlcA and multiple O-sulfations of the component sugars. Recent studies have achieved purification of several, and molecular cloning of most, of the enzymes responsible for these reactions. Some of these enzymes are bifunctional. The availability of cDNA probes has facilitated elucidation of the crystal structures for two of the biosynthetic enzymes, demonstration of their intracellular location, and their occurrence in complexes to achieve rapid and efficient synthesis of complex sugar sequences. Genomic structure and transcript analysis have shown the existence of multiple isoforms for most of the sulfotransferases. Many aspects of the heparan sulfate biosynthetic scheme are shared by the structural analog heparin, which is synthesized in mast cells and some other mammalian cells and is several-fold higher degree of polymerization and more extensive modification than heparan sulfate.

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Section: Interactions Between Hs-synthesizing Enzymes To Form Functiomentioning

confidence: 98%

Heparin and Heparan Sulfate Biosynthesis

2002

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“…Biochemical studies have shown that the vertebrate EXT1 and EXT2 can associate to form biologically functional hetero-oligomeric complex that exhibits glycosyltransferase activity in which both subunits are essential for full activity (Kobayashi et al 2000;McCormick et al 2000;Senay et al 2000;Wei et al 2000;Zak et al 2002). Han et al (2004b) have shown that Ttv and Sotv behave similarly in Drosophila.…”

Section: Ttv Protein Interacts With Hext1mentioning

confidence: 99%

Functional conservation of the human EXT1 tumor suppressor gene and its Drosophila homolog tout velu

et al. 2007

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Heparan sulfate proteoglycans play a vital role in signaling of various growth factors in both Drosophila and vertebrates. In Drosophila, mutations in the tout velu (ttv) gene, a homolog of the mammalian EXT1 tumor suppressor gene, leads to abrogation of glycosaminoglycan (GAG) biosynthesis. This impairs distribution and signaling activities of various morphogens such as Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp). Mutations in members of the exostosin (EXT) gene family lead to hereditary multiple exostosis in humans leading to bone outgrowths and tumors. In this study, we provide genetic and biochemical evidence that the human EXT1 (hEXT1) gene is conserved through species and can functionally complement the ttv mutation in Drosophila. The hEXT1 gene was able to rescue a ttv null mutant to adulthood and restore GAG biosynthesis.

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“…A truncated form of human EXT1 (amino acids 29 -746) was amplified by PCR from a previously constructed plasmid (18), with primers 5Ј-ATATGCGGCCGCTAGGGCATCGAGGA-3Ј (forward) and 5Ј-GCCT-GGATCCAAGTCGCTCAATGTCTCG-3 (reverse). All of the PCRs were carried out using the Expand High Fidelity PCR System (Roche Applied Science).…”

Section: Construction Of Expression Plasmids-truncated Forms Of Humanmentioning

confidence: 99%

“…Glycosyltransferase Assays-GlcNAc and GlcA transferase activities were measured essentially as described (18,21) by incubating anti-FLAG agarose-bound enzyme protein preparations with 0.125 Ci of 14 C-labeled UDP sugars (62.5 Ci/ mol; prepared by mixing radiolabeled and unlabeled UDP sugars) and 45 g of oligosaccharide acceptor at 37°C for 2 h. Oligosaccharide acceptors were generated from K5 polysaccharide, as described (22). Labeled products were isolated by gel chromatography and quantified by scintillation counting.…”

Section: Construction Of Expression Plasmids-truncated Forms Of Humanmentioning

confidence: 99%

“…Although solubilized microsomal enzymes as well as purified EXT proteins can catalyze the transfer of single GlcA or GlcNAc residues to exogenous substrates in vitro, actual polymerization on exogenous substrates has not been demonstrated (15)(16)(17)(18)(19). The previous results suggest that the biologically functional unit in HS polymerization is a complex containing EXT1 and EXT2 (18,19). In this study, we examined the ability of EXT1 and EXT2 and of the EXT1/2 complex to elongate the HS backbone in vitro.…”

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In Vitro Polymerization of Heparan Sulfate Backbone by the EXT Proteins

Busse

Kusche-Gullberg

2003

Journal of Biological Chemistry

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Multiple exosotoses is a dominantly inherited bone disorder caused by defects in EXT1 and EXT2, genes encoding glycosyltransferases involved in heparan sulfate chain elongation. Heparan sulfate polymerization occurs by the alternating addition of glucuronic acid and N-acetylglucosamine units to the nonreducing end of the polysaccharide. EXT1 and EXT2 are suggested to be dual glucuronyl/N-acetylglucosaminyltransferases, and a heterooligomeric complex of EXT1 and EXT2 (EXT1/2) is considered to be the biological functional polymerization unit. Here, we have investigated the in vitro polymerization capacities of recombinant soluble EXT1, EXT2, and EXT1/2 complex on exogenous oligosaccharide acceptors derived from Escherichia coli K5 capsular polysaccharide. Incubations of recombinant EXT1 or EXT1/2 complex with 3 H-labeled oligosaccharide acceptors and the appropriate nucleotide sugars resulted in conversion of the acceptors to higher molecular weight compounds but with different efficacies for EXT1 and EXT1/2. In contrast, incubations with recombinant EXT2 resulted in the addition of a single glucuronic acid but no further polymerization. These results indicate that EXT1 alone and the EXT1/2 heterocomplex can act as heparan sulfate polymerases in vitro without the addition of additional auxiliary proteins.

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

Cited by 154 publications

References 14 publications

Heparin and Heparan Sulfate Biosynthesis

Heparin and Heparan Sulfate Biosynthesis

Functional conservation of the human EXT1 tumor suppressor gene and its Drosophila homolog tout velu

In Vitro Polymerization of Heparan Sulfate Backbone by the EXT Proteins

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