in Drosophila melanogaster

 Proteoglycans consisting of core proteins with glycosaminoglycans are abundant molecules, found both in the extracellular matrix and on the cell surface. Until recently, glycosaminoglycans were studied principally in vertebrate systems. However, genetic experiments in the Drosophila melanogaster established that glycosaminoglycans are required for normal development of this invertebrate model organism (1).

We have reported methods for structural analysis of glycosaminoglycans in Drosophila melanogaster and C. elegans using HPLC separation of disaccharides generated specifically by enzymatic digestion (2). In both organisms, chondroitin sulfate and heparan sulfate-derived disaccharides were detected, but Di-HA was not detectable in this experiment. Chondroitinase digestion of glycosaminoglycans from Drosophila produced both nonsulfated and 4-O-sulfated unsaturated disaccharides, whereas only nonsulfated forms were detected in C. elegans. This is in contrast to chondroitin sulfate found in cartilage from a wide range of animals, in which the vast majority of disaccharide units are sulfated at either the 4 or 6-O position (Fig. 1). Furthermore, equivalent release of unsaturated disaccharides with chondroitinase ABC and ACII, or ACII digestion alone, suggested that dermatan sulfate was either not found, or represented at very modest levels in Drosophila and C. elegans. It is very interesting that the profiles of disaccharides generated by chondroitinase treatment of Drosophila material resemble those generated from treatment of human bikunin, a blood plasma protein component of the inter--trypsin inhibitor family (Fig. 1).
Fig. 1 Chromatograms of unsaturated disaccharides from chondroitin sulfate.
Heparin lyase treatment of glycosaminoglycans form Drosophila and C. elegans released unsaturated disaccharides bearing N-, 2-O- and 6-O-sulfated species, including mono, di and tri sulfated forms like vertebrates (Fig. 2). One of the most striking features of glycosaminoglycans is their structural diversity, with discrete structural variants found in different tissues. To determine whether Drosophila could provide a model system for exploring the function of heparan sulfate structural variants, we examined heparan sulfate from different developmental stages and tissues (2). Indeed, Drosophila showed tissue- and stage- specific modifications of heparan sulfate (Fig. 3). For example, levels of UA-GlcNS6S were relatively higher in embryos compared with larvae and adults. GlcN 6-O-sulfate groups were potentially important in regulating growth factor signaling throughout development (3).
Fig. 2 Chromatograms of unsaturated disaccharides from heparan sulfate.
Fig.3 Comparisons of Drosophila heparan sulfate from different tissues and developmental stages.
The linkage region oligosaccharides from glycosaminoglycans were isolated from Drosophila melanogaster after chondroitinase digestion and were then derivatized with 2-aminobenzamide (4). The linkage tetrasaccharide from chondroitin sulfate was a uniform structure of –GlcA-Gal-Gal-Xyl(2-O-phosphate)-. In contrast, the unmodified and phosphorylated forms were demonstrated in heparan sulfate of adult flies at a molar ratio of 73: 27.

Three genes were recently demonstrated to affect signaling by members of the Wnt, TGF-, Hedgehog and fibroblast growth factor families in Drosophila encode proteins with homology to vertebrate enzymes involved in the glycosaminoglycan synthesis (1). Detailed analysis of glycosaminoglycans from flies bearing mutations in those three genes was carried out by a method using enzymatic digestion and following HPLC separation (5). It was found that mutations in sugarless, which encodes a protein with homology to UDP-glucose dehydrogenase, compromise the synthesis of both chondroitin and heparan sulfate, as would be predicted from a defect in UDP-glucronate production. Defects in sulfateless, a gene encoding a protein with similarity to vertebrate N-deacetylase/N-sulfotransferases, did not affect chondroitin sulfate levels or composition but altered the composition of unsaturated disaccharides from heparan sulfate. N-, 6-O- and 2-O-sulfated disaccharide units were absent and replaced entirely with an unsulfated disaccharide. A mutation in tout-velu, a gene related to the vertebrate Exostoses 1 heparan sulfate co-polymerase (B08 of this series), likewise did not affect chondroitin sulfate synthesis but reduced all forms of heparan sulfate to below the limit of detection. These findings demonstrate the utility of Drosophila as a model organism for studying the function of glycosaminoglycans in vivo.
Hidenao Toyoda (Chiba University, Graduate School of Pharmaceutical Sciences)
References (1) Selleck, S.B. (2000) Trends Genet. 16, 206-212
(2) Toyoda, H., Kinoshita-Toyoda, A., and Selleck, S.B. (2000) J. Biol. Chem. 275, 2269-2275
(3) Nakato, H., and Kimata K. (2002) Biochim. Biophys. Acta 1573, 312-318
(4) Yamada, S., Okada, Y., Ueno, M., Iwata, S., Deepa, S.S., Nishimura, S., Fujita, M., Die, I.V., Hirabayashi, Y., and Sugahara, K. (2002) J. Biol. Chem. 277, 31877-31886
(5) Toyoda, H., Kinoshita-Toyoda, A., Fox, B., and Selleck, S.B. (2000) J. Biol. Chem. 275, 21856-21861
Dec.19, 2002

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