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Glycosaminoglycans
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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). |
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| 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). |
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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. |
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| Hidenao Toyoda (Chiba University, Graduate
School of Pharmaceutical Sciences) |
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| Dec.19, 2002 | |||||||||||||||||
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