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.
We have devised a sensitive method for the isolation and structural analysis of glycosaminoglycans from two genetically tractable model organisms, the fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. We detected chondroitin/chondroitin sulfate- and heparan sulfate-derived disaccharides in both organisms. Chondroitinase digestion of glycosaminoglycans from adult Drosophila produced both nonsulfated and 4-O-sulfated unsaturated disaccharides, whereas only unsulfated forms were detected in C. elegans. Heparin lyases released disaccharides bearing N-, 2-O-, and 6-O-sulfated species, including mono-, di-, and trisulfated forms. We observed tissue- and stage-specific differences in both chondroitin sulfate and heparan sulfate composition in Drosophila. We have also applied these methods toward the analysis of tout-velu, an EXT-related gene in Drosophila that controls the tissue distribution of the growth factor Hedgehog. The proteins encoded by the vertebrate tumor suppressor genes EXT1 and 2, show heparan sulfate co-polymerase activity, and it has been proposed that tout-velu affects Hedgehog activity via its role in heparan sulfate biosynthesis. Analysis of total glycosaminoglycans from tout-velu mutant larvae show marked reductions in heparan sulfate but not chondroitin sulfate, consistent with its proposed function as a heparan sulfate co-polymerase.
Toyoda
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Department of Molecular Biology, University of Arizona, Tucson, Arizona 85721, USA.
Kinoshita-Toyoda
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Selleck
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D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism*; D002236:Carbohydrate Conformation; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004330:Drosophila / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029721:Drosophila Proteins; D016147:Genes, Tumor Suppressor; D006497:Heparan Sulfate / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics*
D029721:Drosophila Proteins; D006497:Heparan Sulfate; D008565:Membrane Proteins; C113073:TTV protein, Drosophila