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J Biol Chem, Vol. 275, Issue 4, 2269-2275, January 28, 2000
,
From the Department of Molecular and Cellular Biology, University
of Arizona, Tucson, Arizona 85721 and the Faculty of
Pharmaceutical Sciences, Chiba University, 1-33 Yayoi, Inage-ku,
Chiba 263-8522, Japan
 |
ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
Proteoglycans consisting of core proteins with
glycosaminoglycan chains are abundant molecules, found both in the
extracellular matrix and on the cell surface. These diverse molecules
serve a wide range of functions, from affecting the compressive
properties of cartilage to growth factor reception. Until recently,
proteoglycans were studied principally in vertebrate systems. However,
genetic experiments in the fruit fly, Drosophila
melanogaster, established that proteoglycans, and their associated
glycosaminoglycans, are required for normal development of this
invertebrate model organism (reviewed in Ref. 1). A
Drosophila member of the glypican family, division
abnormally delayed
(dally)1 (2, 3),
affects signaling mediated by two conserved growth factors, Wingless, a
member of the Wnt family, and Decapentaplegic, a transforming growth
factor- Mutations affecting genes encoding proteins related to known
glycosaminoglycan biosynthetic enzymes have also been described in
Drosophila. sugarless shows striking homology to UDP-glucose dehydrogenase (7-9) and affects signaling mediated by multiple growth
factors, including Wingless, Decapentaplegic, and the fibroblast growth
factor receptor-related proteins Heartless and Breathless (10).
sulfateless encodes a protein similar to
N-deacetylase/N-sulfotransferase and is also
required for Wingless-mediated and fibroblast growth factor receptor
signaling (10, 11). Both sugarless and
sulfateless mutations disrupt glycosaminoglycan-modification
of Dally in vivo, supporting their assignment as
glycosaminoglycan biosynthetic enzymes (3, 11). pipe, a gene
required for establishing the embryonic dorsal-ventral axis, encodes a
protein with significant homology to heparan sulfate
2-O-sulfotransferase genes in vertebrates (12). Finally,
tout-velu (ttv), a gene related to the tumor suppressor genes, (EXT1 and 2) has been shown to
affect events directed by Hedgehog, a Drosophila homolog of
Sonic Hedgehog and Indian Hedgehog (13). It is not known, however,
whether tout-velu, like the vertebrate EXT1 and
2 genes, encodes an enzyme with heparan sulfate
co-polymerase activity (14).
Biochemical studies of proteoglycans from both Drosophila
and C. elegans have been reported. In addition to the
glypican, Dally, a Drosophila syndecan has been identified
and shown to be heparan sulfate-modified (15). Heparan sulfate and
chondroitin sulfate polymers have been detected in extracellular matrix
preparations of Drosophila, material that could be
radiolabeled with 35SO4 and degraded with
either chondroitinase ABC or nitrous acid (16). Other proteoglycan or
proteoglycan-like molecules have been described in
Drosophila, including DROP-1 (17), Papilin (18), and
macrophage-derived proteoglycan-1 (a hemocyte/macrophage-derived protein of the extracellular matrix) (19). A gene encoding a protein
related to perlecan, unc-52, has been studied in some detail
in C. elegans and shown to affect muscle attachment and sarcomere organization (20, 21).
Although these findings collectively show that proteoglycans exist in
Drosophila and C. elegans, performing critical
functions during development, very little is known about the different
glycosaminoglycan structures found in these organisms. A detailed
understanding of proteoglycan and glycosaminoglycan functions in these
systems will require structural information that can be used in
conjunction with genetic and molecular data. We therefore devised a
method for structural analysis of glycosaminoglycan-derived
disaccharides suitable for the relatively small samples that can be
easily obtained from these animals. We have applied these methods
toward identifying tissue-specific and developmental stage-specific
distributions of glycosaminoglycans, as well as to characterize
glycosaminoglycans in animals bearing mutations in ttv, a
gene proposed to affect heparan sulfate biosynthesis.
Materials--
The following standard unsaturated disaccharides
from heparan sulfate were purchased from Sigma:
2-acetamido-2-deoxy-4-O-(4-deoxy-
The following standard unsaturated disaccharides from chondroitin
sulfate were obtained from Seikagaku America (Falmouth, MA):
2-acetamido-2-deoxy-3-O-( Apparatus--
The chromatographic equipment included a gradient
pump (L-7000), a chromato-integrator (D-7500) from Hitachi Instruments
(San Jose, CA), a double plunger pump for the reagent solution
(AA-100-S, Eldex Laboratories, Napa, CA), a sample injector with a
20-µl loop (model 7125, Reodyne, Rohnert Park, CA), a fluorescence
spectrophotometer (RF-10AXL, Shimadzu Scientific, Columbia,
MD), a column heater (CH-30), a dry reaction bath (FH-40), and a
thermocontroller (TC-55) from Brinkman Instruments (Westbury, NY).
Determination of Unsaturated Disaccharides from Heparan Sulfate
and Chondroitin Sulfate--
Unsaturated disaccharides produced
enzymatically from heparan sulfate and chondroitin sulfate were
determined by a reversed-phase ion-pair chromatography with sensitive
and specific postcolumn detection (23). A gradient was applied at a
flow rate of 1.1 ml/min on a Senshu Pak Docosil (4.6 × 150 mm) at
55 °C. The eluents used were as follows: A, H2O; B, 0.2 M sodium chloride; C, 10 mM
tetra-n-butylammonium hydrogen sulfate; D, 50%
acetonitrile. The gradient program was as follows: 0-10 min, 1-4%
eluent B; 10-11 min, 4-15% eluent B; 11-20 min, 15-25% eluent B;
20-22 min, 25-53% eluent B; 22-29 min, 53% eluent B; equilibration
with 1% B for 20 min. The proportions of eluent C and D were constant at 12 and 17%, respectively. To the effluent were added aqueous 0.5%(w/v) 2-cyanoacetamide solution and 0.25 M sodium
hydroxide at the same flow rate of 0.35 ml/min by using a double
plunger pump. The mixture passed through a reaction coil (internal
diameter, 0.5 mm; length, 10 m) set in a dry reaction temperature
controlled bath at 125 °C and a following cooling coil (internal
diameter, 0.25 mm; length, 3 m). The effluent was monitored
fluorometrically (excitation, 346 nm; emission, 410 nm).
Preparation and Enzymatic Digestion of Chondroitin
Sulfate--
Adult worms were obtained by collection from agar plates,
washed, and separated from E. coli on a 0.25% Ficoll
gradient as described (24). For the analysis of both C. elegans and Drosophila, whole animals or tissues were
first lyophilized to dryness. Approximately 20 mg of lyophilized sample
was then homogenized with 1.0 ml of acetone. The homogenate was washed
with acetone and dried. The pellet was extracted in 1.0 ml of 0.5%
SDS, 0.1 M NaOH, 0.8% NaBH4 for 16 h at
room temperature with constant stirring. Two hundred µl of 1.0 M sodium acetate and 300 µl of 1 M HCl were
then added, the solution was filtered, and 200 µl of 1 M
HCl was added to the filtrate. Insoluble material was removed by
centrifugation at 2500 × g for 10 min at 4 °C.
Seven ml of ethanol was added to the supernatant and chilled for 2 h at 0 °C, and the crude glycosaminoglycan fraction collected by
centrifugation at 2500 × g for 10 min at 4 °C. The
resulting precipitate was dissolved in 250 µl of water. A 20-µl
portion of the crude glycosaminoglycan solution was diluted to 100 µl
with water and used for the determination of chondroitin sulfate. For
chondroitinase digestion, 5 µl of 0.2 M Tris-acetate
buffer (pH 8.0) and 10 µl of an aqueous solution containing
chondroitinase ABC (50 mIU) and chondroitinase ACII (50 mIU) were added
to a 20-µl portion of the sample solution and incubated at 37 °C
for 3 h. An 8-µl portion of this mixture was loaded onto the
high performance liquid chromatograph.
Preparation and Enzymatic Digestion of Heparan Sulfate--
To
230 µl of crude glycosaminoglycan sample, 50 µl of 0.3 M sodium phosphate buffer (pH 6.0) was added, and the
solution was applied on an Ultrafree-MC DEAE membrane, which had been
equilibrated with sodium phosphate buffer (pH 6.0) containing 0.15 M NaCl. The fractions eluted with 1.0 M NaCl in
the same buffer were collected, desalted with Biomax-5, evaporated, and
resuspended in 12 µl of water in preparation of heparin lyase
digestion. For the analysis of Drosophila heparan sulfate, 5 µl of 0.1 M acetate buffer (pH 7.0) with 10 mM calcium acetate and 15 µl of an aqueous solution containing heparin lyase mixture (Seikagaku America), heparin lyase I
(1 mIU), heparin lyase II (1 mIU), and heparin lyase III (1 mIU) were
added to a 5-µl portion of sample. The mixture was incubated at
37 °C for 16 h, and an 8-µl aliquot was loaded onto the high
performance liquid chromatograph. For the analysis of heparan sulfate
from C. elegans, chondroitin was removed from the crude
glycosaminoglycan solution prior to heparin lyase digestions by
chondroitinase treatment followed by separation with Ultrafree-MC DEAE
membrane. Enzymatic digestion with a heparin lyase mixture was then
carried out as described above, with the exception that Sigma enzymes
were used.
Genetic Analysis of tout-velu--
We used
tout-velul(2)00681 mutants for our analysis, the
only allele for which genetic studies have been described (13).
ttv/CyO, P[wt GFP] green fluorescent protein animals were
self-crossed, and ttv/ttv third instar larvae were
identified by the lack of green fluorescence derived from the green
fluorescent protein marked CyO balancer chromosome.
Sample Preparation and HPLC for the Determination of
Glycosaminoglycans--
We established a protocol for highly
reproducible and sensitive HPLC analysis of unsaturated disaccharides
from chondroitin sulfate and heparan sulfate in C. elegans
and Drosophila. The coefficient of variation for each
unsaturated disaccharide was less that 5% (adult
Drosophila, n = 5). The lower determination limits of the HPLC for chondroitin sulfate and heparan sulfate were
approximately 0.5 and 1.5 ng, respectively. All results described below
were reproduced in at least duplicate experiments.
Analysis of Glycosaminoglycans in C. elegans--
The complete
digestion of material from C. elegans with both
chondroitinase ABC and ACII released disaccharides that
co-chromatographed with
Digestion of the glycosaminoglycans isolated from C. elegans
with a heparin lyase mixture generated disaccharides represented in
vertebrate samples such as bovine kidney (Fig.
2B). The disaccharide profile
of worm heparan sulfate we observed is relatively simple, with
Analysis of Glycosaminoglycans from Drosophila--
As for the
analysis of glycosaminoglycan from C. elegans, the
identities of Drosophila disaccharides were established by
comparison with standards using two distinct separation methods,
reverse phase ion-pair (23) and graphitized carbon chromatography (25). Digestion of unfractionated glycosaminoglycans from adult flies with
chondroitinases yields disaccharides that co-chromatograph with
Heparan sulfate disaccharides are also found in glycosaminoglycan
preparations from adult flies (Fig. 2C). Fly heparan sulfate shows a greater degree of complexity than samples from C. elegans, with Analysis of Drosophila Tissues and Developmental Stages--
In
vertebrates, structural variants of glycosaminoglycans show
tissue-specific distributions (28, 29). To determine whether Drosophila tissues also show reproducible differences in
glycosaminoglycans we examined different tissues and developmental
stages in parallel samples from whole adult bodies, ovaries, embryos,
and third instar larvae. We detected reproducible differences in the
disaccharide profiles of heparan sulfates isolated from
Drosophila (Fig. 3 and Table
III). For example, third instar larvae
show a reduced percentage of Analysis of tout-velu, a Gene Related to the Vertebrate Heparan
Sulfate Co-polymerases EXT1 and EXT2--
One of the utilities of
structural analyses of glycosaminoglycans in Drosophila and
C. elegans is to examine the effects of removing specific
gene functions on the sugar polymers synthesized in an intact animal.
We have used the analytical methods we developed to examine
glycosaminoglycans in animals bearing mutations in ttv, a
gene with 56% amino acid identity to the vertebrate tumor suppressor
gene, EXT1 (13). Both EXT1 and 2 have been shown to encode enzymes with heparan sulfate
co-polymerase activity, suggesting that ttv may also affect
the synthesis of heparan sulfate (14). We examined glycosaminoglycans
in third instar larvae homozygous for a null allele of ttv.
ttv mutant larvae show a marked reduction in heparan sulfate, to
levels at least 10-fold less than wild type (Fig.
4 and Tables III and IV). The
chromatograph shown in Fig. 4 represents material greater than 5000 Da
that dissociates from DEAE membranes between 0.15 and 1.0 M
NaCl. Heparan sulfate glycosaminoglycans in this fraction or with
molecular mass less than 5000 Da (data not shown) are below the
detection limits of our methods. Animals heterozygous for
ttv show a slightly reduced amount of heparan sulfate with
the same disaccharide composition as wild type. Chondroitin sulfate,
however, is unaffected by ttv, consistent with
ttv encoding a heparan sulfate-specific co-polymerase.
Drosophila and C. elegans as Model Organisms for Studying
Proteoglycan and Glycosaminoglycan Functions--
Many studies in
Drosophila have documented the important role of
proteoglycans in developmental patterning. Yet at the structural level,
little is known about glycosaminoglycans in Drosophila or
C. elegans, another model organism that offers a powerful
array of genetic and molecular tools. We describe here a sensitive
method for analysis of disaccharides derived from chondroitin and
heparan sulfates in these model organisms.
Analysis of Chondroitin Sulfate--
Chondroitin polymers are
found in adult Drosophila and C. elegans.
Chondroitin-derived disaccharides in C. elegans were not sulfated, and only
It is interesting that the profiles of disaccharides generated by
chondroitinase treatment of C. elegans and
Drosophila material resemble those generated from treatment
of human bikunin (26), an abundant serum protein component of the
inter- Analysis of Heparan Sulfate--
Heparin lyase treatment of
glycosaminoglycans from Drosophila and C. elegans
releases disaccharides found in vertebrates. Our analysis provides the
first direct evidence for disaccharides bearing N-,
2-O-, and 6-O-sulfations in these organisms. We
did not detect
Our findings show that enzymes required for biosynthesis and
modification of heparan sulfate must exist in Drosophila and C. elegans. Indeed, genes encoding proteins with significant
homology to EXT1 (13),
N-deacetylase/N-sulfotransferase (10), C5
glucuronyl epimerase (GenBankTM accession number P46555),
and heparan-sulfate sulfotransferase (12, 32) enzymes from vertebrates
are represented in Drosophila and C. elegans. Our
analysis of ttv (see below) indicates that like its
vertebrate homolog, EXT1, Ttv affects heparan sulfate biosynthesis.
Structural studies of glycosaminoglycans from animals bearing mutations
in these genes will show which genes are required for the generation of
specific glycosaminoglycan forms in vivo.
One of the most striking features of glycosaminoglycans is their
structural diversity, with discrete structural variants found in
different tissues (28, 33). Specific forms of heparan sulfate are also
associated with different disease states and ages (29, 34). These
findings suggest that different forms of heparan sulfate are important
for influencing the biological function of the associated proteoglycan.
To determine whether Drosophila could provide a model system
for exploring the function of different heparan sulfate structural
variants, we examined glycosaminoglycans from different developmental
stages and tissues. Indeed, Drosophila does show tissue- and
stage-specific modifications of both heparan and chondroitin sulfate.
For example, levels of
The differences in 2-O-sulfated disaccharides in the ovary
compared with other tissues and stages is worthy of note. Recently it
has been shown that pipe, a gene required for establishing dorsal-ventral polarity in the embryo, encodes a protein related to
heparan sulfate 2-O-sulfotransferase (12). pipe
is expressed only in the ventral follicle cells of the ovary that
surround the developing oocyte, and is required for the proteolytic
activation of the protein ligand, Spätzle. Spätzle in turn
activates Toll, a Drosophila homologue of the vertebrate
interleukin 1 receptor that specifies ventral cell fates in the future
embryo (reviewed in Ref. 38). We demonstrate here that
2-O-sulfated heparan sulfate disaccharides are found in the
ovary, in a proportion distinct from that found in embryos, whole
adults, or larvae. These findings show that a heparan sulfate
2-O-sulfotransferase must exist in Drosophila,
and the homology of pipe to vertebrate proteins with this
activity suggests that the pipe could provide this function.
Analysis of ttv, an EXT-related Gene in Drosophila--
One of the
utilities of organisms like Drosophila and C. elegans is that they provide the means of determining the effects of specific genes on glycosaminoglycan biosynthesis in whole animals. Using the methods we devised for disaccharide analysis in
Drosophila, we examined glycosaminoglycans in animals
bearing mutations in ttv, a gene encoding a protein with
56% amino acid identity to EXT1, a tumor suppressor with heparan
sulfate co-polymerase activity (14). We isolated glycosaminoglycans
from third instar larvae homozygous for a null allele of
ttv. These animals showed a normal level of
chondroitinase-sensitive disaccharides but markedly reduced levels of
heparan sulfate-derived disaccharides. The heparan sulfate fraction
that elutes from DEAE between 0.15 and 1.0 M NaCl and has a
molecular mass of greater than 5000 Da was reduced to below detectable
limits in ttv. Nor could we detect any material in the same
DEAE fraction that passed through a 5000 Da filter (data not shown).
Further study using larger quantities of purified glycosaminoglycans
will be required to characterize exactly what heparan sulfate forms, if
any, remain in ttv mutants. Our findings clearly show that
the methods of microdetermination of heparan sulfate-derived
disaccharides that we developed can be applied to the analysis of
mutants, and demonstrate directly that ttv+ is
required for normal heparan sulfate levels in larvae. These data
support the proposal that this gene does indeed encode a heparan
sulfate glycosyltransferase (13).
This finding has implications toward understanding the biological
functions of EXT genes in vertebrates. Although it is clear that loss of EXT gene function promotes cartilaginous tumor
formation, the molecular mechanism of EXT-mediated tumor suppression in
not known. Based on the finding that ttv affects the
distribution of Hedgehog in the developing wing, it has been
hypothesized that loss of EXT function affects the levels of Indian
Hedgehog at the growth plate (13). Indian Hedgehog limits the rate of
chondrocyte differentiation, and loss of EXT could disrupt cartilage
formation via abnormalities in Indian Hedgehog distribution (39-41).
Our finding that ttv affects heparan sulfate levels provides
a link between EXT functions in vertebrates and Drosophila.
Recently, analysis of ttv mutants using a monoclonal
antibody directed against an epitope generated by heparinase III
digestion supports the conclusion that these animals are defective in
heparan sulfate biosynthesis (35).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/bone morphogenetic protein-related protein (3, 4). Wnts and
transforming growth factor-/bone morphogenetic proteins are
important patterning molecules in vertebrate and invertebrate species,
and studies of Drosophila and Caenorhabditis elegans have identified many of the evolutionarily conserved
components of these signaling systems (5, 6).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-L-threo-hex-enepyranosyluronic acid)-D-glucose (
UA-GlcNAc),
2-deoxy-2-sulfamido-4-O-(4-deoxy--L-threo-hex-enepyranosyluronic acid)-D-glucose (UA-GlcNS),
2-acetamido-2-deoxy-4-O-(4-deoxy--L-threo-hex-enepyranosyluronic acid)-6-O-sulfo-D-glucose
(UA-GlcNAc6S),
2-acetamido-2-deoxy-4-O-(4-deoxy-2-O-sulfo--L-threo-hex-enepyranosyluronic acid)-D-glucose (UA2S-GlcNAc),
2-deoxy-2-sulfamido-4-O-(4-deoxy-2-O-sulfo--L-threo-hex-enepyranosyluronic acid)-6-O-sulfo-D-glucose (UA-GlcNS6S),
2-deoxy-2-sulfamido-4-O-(4-deoxy-2-O-sulfo--L-threo-hex-enepyranosyluronic acid)-D-glucose (UA2S-GlcNS),
2-acetamido-2-deoxy-4-O-(4-deoxy-2-O-sulfo--L-threo-hex-enepyranosyluronic acid)-6-O-sulfo-D-glucose (UA2S-GlcNAc6S),
2-deoxy-2-sulfamido-4-O-(4-deoxy-2-O-sulfo--L-threo-hex-enepyranosyluronic acid)-6-O-sulfo-D-glucose (UA2S-GlcNS6S), and
heparan sulfate (from bovine kidney).
-D-gluco-4-enepyranosyluronic
acid)-D-glucose (Di-HA),
2-acetamido-2-deoxy-3-O-(-D-gluco-4-enepyranosyluronic acid)-D-galactose (Di-0S),
2-acetamido-2-deoxy-3-O-(-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (Di-4S),
2-acetamido-2-deoxy-3-O-(-D-gluco-4-enepyranosyluronic acid)-6-sulfo-D-galactose (Di-6S),
2-acetamido-2-deoxy-3-O-(2-O-sulfo--D-gluco-4-enepyranosyluronic acid)-D-galactose (Di-UA2S),
2-acetamido-2-deoxy-3-O-(2-O-sulfo--D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose
(Di-diSB),
2-acetamido-2-deoxy-3-O-(2-O-sulfo--D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose
(Di-diSD) and
2-acetamido-2-deoxy-3-O-(-D-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-D-galactose
(Di-diSE),
2-acetamido-2-deoxy-3-O-(2-O-sulfo--D-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-D-galactose (Di-triS),
chondroitinase ABC (EC 4.2.2.4), chondroitinase ACII (EC 4.2.2.5), and
chondro-4-sulfatase (EC 3.1.6.9). Heparin lyase I (EC 4.2.2.7,
heparinase I, heparinase), heparin lyase II (heparinase II,
heparitinase II), and heparin lyase III (EC 4.2.2.8, heparinase III,
heparitinase I) were obtained from Sigma and Seikagaku America. Senshu
Pak Docosil (4.6 × 150 mm; particle size, 5 µm) was obtained
from Senshu Scientific (Tokyo, Japan). Ultrafree-MC DEAE and Biomax-5
(5000 normal molecular weight limit) were obtained from Millipore Corp.
(Bedford, MA). All other chemicals used were of analytical reagent
grade. Human urinary bikunin was purified as described previously
(22).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Di-0S using reverse phase ion-pair
chromatography (Fig. 1B). The
identity of Di-0S was established using graphitized carbon
chromatography, which can resolve Di-0S and Di-HA, showing that
chondroitin is found in this invertebrate organism (data not shown)
(25). Digestion with chondroitinase ACII alone generated the same
chromatographic profile obtained with ACII plus ABC, indicating that
the majority of chondroitin contains glucuronic acid and not iduronic
acid (data not shown). We did not detect Di-4S, Di-6S, and other
over-sulfated disaccharides found in vertebrate chondroitin sulfate in
these unfractionated glycosaminoglycan preparations from C. elegans, nor were we able to detect Di-HA in our preparations.
The compositions of unsaturated disaccharides produced from chondroitin
in worms is listed in Table I.
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Fig. 1.
Chromatograms of unsaturated disaccharides
from chondroitin sulfate in human urinary bikunin, adult C. elegans, and adult Drosophila.
A, analysis of chondroitin sulfate in human urinary bikunin.
A 20-µl portion of human urinary bikunin (50 units/ml) was digested
with chondroitinase ABC and ACII as described in the text. An 8-µl
aliquot of this mixture was loaded onto the high performance liquid
chromatograph. B, analysis of chondroitin sulfate in adult
C. elegans. C, analysis of chondroitin sulfate in
adult Drosophila. The inset in C shows
the chromatogram after chondro-4-sulfatase digestion. Other conditions
were as described in the text.
Compositions of unsaturated disaccharides derived from chondroitin
sulfate in human urinary bikunin, adult C. elegans, and Drosophila
UA-GlcNS, UA2S-GlcNS, and UA2S-GlcNS6S species in nearly equal
amounts and nonsulfated UA-GlcNAc representing about 50% of the
total. The composition of disaccharides produced from heparan sulfates
in worms, compared with that derived from bovine kidney, is provided in
Table II. The identities of all
disaccharides have been confirmed by comparison with standards using
two HPLC separation methods, reversed phase ion-pair (23) and
graphitized carbon column chromatography (25).
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Fig. 2.
Chromatograms of unsaturated disaccharides
from heparan sulfate in bovine kidney, adult C. elegans,
and adult Drosophila. A,
analysis of heparan sulfate from bovine kidney. B, analysis
of heparan sulfate from adult C. elegans. C,
analysis of heparan sulfate from adult Drosophila. Other
conditions were as described in the text.
Compositions of unsaturated disaccharides produced from heparan sulfate
in bovine kidney, adult C. elegans, and adult Drosophila
Di-0S and Di-4S (Fig. 1C). As we observed for material from C. elegans, the equivalent release of disaccharides
with chondroitinase ACII compared with digestion with both ACII and ABC
indicates that the majority of polymers are glucuronate containing and
hence derived form chondroitin sulfate, not dermatan sulfate (data not
shown). The identity of the 4S species was confirmed by its conversion
to Di-0S with chondro-4-sulfatase. Chondroitin sulfate from adult
flies has a relatively low degree of sulfation, and as in the
human serum protein bikunin, only 4-O-sulfated disaccharides are represented (26). Di-HA from Drosophila was not
detectable using these methodologies. The disaccharide composition of
adult Drosophila chondroitin sulfate is listed in Table
I.
UA-GlcNAc, UA-GlcNS, UA-GlcNAc6S,
UA-GlcNS6S, UA2S-GlcNS, and UA2S-GlcNS6S species generated by
digestion with a mixture of heparin lyases I, II, and III. To determine
whether the material we detected in Drosophila is a typical
heparan sulfate polymer, we identified the disaccharides released by
treatment with heparin lyase I or III. Heparin lyase I generated
UA-GlcNS6S, UA2S-GlcNS disulfated disaccharides, and
UA2S-GlcNS6S trisulfated disaccharide (data not shown). Heparin
lyase III generated UA-GlcNAc, UA-GlcNS, UA-GlcNAc6S, and
UA-GlcNS6S disaccharides (data not shown). These findings
demonstrate that material from Drosophila has heparin lyase
disaccharide profiles typical of heparan sulfate and is not the unusual
glycosaminoglycan found in the snail Achatina futica that is
resistant to heparin lyase I and III digestion (27). Overall, the
proportion of sulfated disaccharides is high, representing about 69%
of total compared with 47% in bovine kidney. The composition of
unsaturated disaccharides produced from adult Drosophila
heparan sulfates is given in Table II.
UA-GlcNS, compared with embryos and
adults. Embryos show higher relative amounts of UA-GlcNS6S compared
with larvae, adults, or ovaries. Ovaries show a significantly higher
proportion of UA2S-GlcNS. The ratios of heparan sulfate to
chondroitin sulfate also vary widely in Drosophila (Table
VI). The ovary showed the greatest
proportion of heparan sulfate (heparan sulfate:chondroitin sulfate,
0.74), with larvae showing the lowest (0.06). The degree of
4-O-sulfation of chondroitin sulfate also differed among
tissues, ranging from 36% in the ovary to 11% in whole adult flies
(Table I).
View larger version (26K):
[in a new window]
Fig. 3.
A comparison of Drosophila
heparan sulfate from different tissues and developmental stages.
A, chromatograms of a, ovaries; b,
embryos; c, larvae and d, adults. Other
conditions were as described for Fig. 2. B, percentages of
unsaturated disaccharides from Drosophila heparan sulfates:
different tissues and developmental stages. Row a, ovaries;
row b, embryos; row c, larvae; row d,
adults. Open boxes, UA-GlcNAc; dotted boxes,
UA-GlcNS; light striped boxes, UA-GlcNAc6S;
medium striped boxes, UA-GlcNS6S; heavy striped
boxes, UA2S-GlcNS; filled boxes,
UA2S-GlcNS6S.
Compositions of unsaturated disaccharides produced from heparan
sulfates in Drosophila
Ratios of heparan sulfate to chondroitin sulfate in Drosophila
View larger version (11K):
[in a new window]
Fig. 4.
Analysis of heparan sulfate from
Drosophila bearing mutations in
ttv. A, wild type; B,
ttv/CyO; C, ttv/ttv. Other conditions
were as described for Fig. 2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Di-4S was detected in Drosophila. This
is in contrast to chondroitin sulfate found in cartilage from a wide range of animals, including the squid, in which the vast majority of
disaccharides released by chondroitinase treatment are sulfated at
either the 4 or 6-O position (30). Equivalent release of disaccharides with chondroitinase ABC and ACII, or ACII digestion alone, suggests that dermatan sulfate is either not found, or represented at very modest levels in Drosophila and C. elegans.
-trypsin inhibitor family of protease inhibitors (reviewed in
Ref. 31). This suggests that the chondroitin-modified proteins in these
invertebrates may include protease regulators. In fact, a gene with
striking homology to mouse bikunin is found in C. elegans,
showing greater than 40% amino acid identity over a stretch of 100 amino acids (GenBankTM accession number U64857). The
C. elegans gene encodes a protein most similar to tissue
factor pathway inhibitor, a member of the bovine pancreatic trypsin
inhibitor/Kunitz family of protease inhibitors.
UA2S-GlcNAc or UA2S-GlcNAc6S in either of these
organisms using the methods that we developed for microdetermination of glycosaminoglycans, but it remains possible that these forms exist, albeit at levels below our current detection limits. Our methods, using
heparin lyase digestion of small quantities of crude
glycosaminoglycans, is not suitable for detection of 3-O-S
sequences. We plan further characterization of heparan sulfate from
Drosophila and C. elegans using larger scale
preparation of purified material and NMR spectroscopy.
UA-GlcNS6S are relatively higher in embryos
compared with larvae and adults. Given the importance of GlcN
6-O-sulfate groups for binding several growth factors
(reviewed in Ref. 33) the levels of these disaccharides are potentially
important in regulating growth factor signaling throughout development
(36, 37).
| |
ACKNOWLEDGEMENTS |
|---|
We thank Bethany Fox for assistance with analysis of ttv and Sam Ward for providing expertise on C. elegans.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grant GM-54832 and a March of Dimes grant (to S. B. S.). National Institutes of Health Grant GM-25243 (to S. Ward) provided shared equipment used in this study.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Molecular & Cellular Biology, University of Arizona, 1007 E. Lowell St., Tucson, AZ 85721-0106. Tel.: 520-621-8663; Fax: 520-621-3709; E-mail: selleck@u.arizona.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: dally, division abnormally delayed; ttv, tout-velu; HPLC, high performance liquid chromatography; EXT, exostosin.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES
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