32 A. Costa-Filho et al. Insect Biochemistry and Molecular Biology 31 2001 31–40
position of isolated ovaries of the blood-sucking hemip- teran Rhodnius prolixus. Another aim of this study was
to determine the relative contribution of the ovarian tissue and of the oocytes to the synthesis and accumu-
lation of the sulfated glycosaminoglycans.
Our results show that ovaries of R. prolixus produce sulfated compounds corresponding to sulfated glycosam-
inoglycans identified as heparan sulfate and chondroitin 4–sulfate. In addition, we have demonstrated by bio-
chemical and histochemical analysis that these molecules are concentrated in the ovarian tissue. Besides this, we
provided preliminary evidence to the presence of another class of sulfated compounds distinguished from sulfated
glycosaminoglycans within the ovaries of these insects. To our knowledge, this is the first detailed description
of the presence of sulfated compounds, in particular, sul- fated glycosaminoglycans in ovaries of a blood-feed-
ing insect.
2. Materials and methods
2.1. Materials Chondroitin 4–sulfate from whale cartilage, chondro-
itin 6–sulfate from shark cartilage, dermatan sulfate from pig skin, and twice-crystallized papain 15 Umg protein
were purchased from Sigma Chemical Co. St. Louis, MO, USA. Chondroitin AC lyase EC 4.2.2.5 from
Arthrobacter aurescens and chondroitin ABC lyase EC 4.2.2.4 from Proteus vulgaris were both purchased from
Seikagaku American Inc. Rockville, MD, USA. Radi- olabeled carrier-free
35
S–Na
2
SO
4
was obtained from Instituto de Pesquisas Energe´ticas e Nucleares Sa˜o
Paulo, SP, Brazil. Standard disaccharides for analysis of chondroitin sulfate composition:
α –
DGlcUA–1→3– GalNAc4SO
4
, and α
– DGlcUA–1→3–GalNAc6SO
4
, were
purchased from
Seikagaku American
Inc. Rockville, MD.
2.2. Insects Normal mated females fed with blood at 3-week inter-
vals were taken from a colony of Rhodnius prolixus and maintained at 28
° C and 70–80 relative humidity.
2.3. In vivo metabolic labeling of ovarian sulfated glycosaminoglycans
Two days after a blood meal, insects received an injection of 20
µ Ci of Na
2 35
SO
4
at the thorax Oliveira et al., 1986, and were kept at 28
° C and 70–80 relative
humidity for 24 h. At the end of the labeling period, the insects were sacrificed and the ovaries were collected,
rinsed in PBS and subsequently incubated with 5 vol of acetone for 24 h at 4
° C and dried.
2.4. Isolation of
35
S–sulfated glycosaminoglycans from isolated ovaries
Sulfated glycosaminoglycans were isolated from
35
S– labeled ovaries following the previously described
method Silva et al., 1989. Briefly, dried ovaries were suspended in sodium acetate buffer, pH 5.5, containing
40 mg papain in the presence of 5 mM EDTA and 5 mM cysteine at 60
° C for 24 h. The suspension was cen-
trifuged at 2000g for 10 min at room temperature and the supernatant, which contained the ovarian glycosami-
noglycans, was then applied to a DEAE–cellulose col- umn 3.5
× 2.5 cm, equilibrated with 0.05 M sodium
acetate pH 5.0. The column was washed with 100 ml of the same buffer and then eluted step-wise with 25
ml of 1.0 M NaCl in the same acetate buffer. The
35
S– glycosaminoglycans eluted from the column were
exhaustively dialyzed against distilled water, lyophilized and dissolved in 0.2 ml of distilled water.
2.5. Characterization of the
35
S–labeled glycosaminoglycans
Radioactive
35
S–glycosaminoglycans were charac- terized by anion exchange chromatography on Mono Q–
FPLC, agarose gel electrophoresis, digestion with chon- droitin lyases and deaminative cleavage with nitrous acid
Garcia-Abreu et al., 1996; Werneck et al., 1999, as described below.
2.6. Anion-exchange chromatography on mono Q– FPLC
Radiolabeled glycosaminoglycans
extracted from
ovaries were applied to a Mono Q–FPLC column, equi- librated with 20 mM Tris–HCl pH 8.0. The column
was developed by a linear gradient of 0–1.5 M NaCl in the same buffer. The flow rate of the column was 0.5
mlmin, and fractions of 0.5 ml were collected. The radioactive material was detected by scintillation coun-
ting. Two peaks of
35
S–labeled compounds were eluted from the column. The first, Peak F1, eluted with 0.5 M
NaCl and the second, Peak F2, eluted with 1.0 M NaCl see Fig. 1. Fractions corresponding to Peaks F1 and
F2 were separately pooled, exhaustively dialyzed against distilled water, freeze dried, and stored at
220 °
C. 2.7. Agarose gel electrophoresis
Agarose gel electrophoresis was carried out as pre- viously described Silva et al., 1992a,b. Approximately
10,000 cpm of
35
S–materials from Peaks F1 and F2 see above, before and after chondroitin lyase digestion or
deaminative cleavage with nitrous acid, as well as a mix- ture of standard chondroitin 4–sulfate, dermatan sulfate
and heparan sulfate 10 µ
g of each were applied to 0.5
33 A. Costa-Filho et al. Insect Biochemistry and Molecular Biology 31 2001 31–40
Fig. 1. Purification of sulfated compounds from isolated R. prolixus
ovaries on a Mono Q–FPLC. The DEAE–cellulose–purified sulfated compounds were applied to a Mono Q–FPLC and purified as described
under “Material and Methods”. Fractions were monitored by scintil- lation counting I. The NaCl concentration in the fractions - - - was
determined by measuring the conductivity. The fractions correspond- ing to the unidentified sulfated compound F1 or sulfated GAGs F2
[cross-hatched peak], as indicated by horizontal bars, were pooled, dialyzed against distilled water and lyophilized. GAGs, glycosaminog-
lycans.
agarose gels in 0.05 M 1,3–diaminopropane:acetate pH 9.0. After electrophoresis, glycosaminoglycans were
fixed in the gel with 0.1 N–cetyl–N,N,N–trimethylam- monium bromide in water, and stained with 0.1 tol-
uidine blue in acetic acid:ethanol:water 0.1:5:5, vv. The
35
S–labeled glycoconjugates were visualized by autoradiography of the stained gels. The radioactive
bands having identical electrophoretic migration as stan- dard glycosaminoglycans were carefully scraped into 10
ml of 0.5 PPOtoluene solution and counted in a liquid scintillation counter.
2.8. Enzymatic and nitrous acid depolymerization of the sulfated compounds
2.8.1. Digestion with chondroitin lyases Digestions with chondroitin AC or ABC lyases were
carried out according to Saito et al. 1968. Approxi- mately 10,000 cpm of
35
S–labeled compounds were incubated with 0.3 units of chondroitin AC lyase or
chondroitin ABC lyase for 8 h at 37 °
C in 100 µ
l of 50 mM Tris–HCl pH 8.0 containing 5 mM EDTA and 15
mM sodium acetate. 2.8.2. Deamination with nitrous acid
Deamination by nitrous acid at pH 1.5, was performed as described by Shively and Conrad 1976. Briefly,
approximately 10,000 cpm of
35
S–compounds were incubated with 200
µ l of fresh generated HNO
2
at room temperature for 10 min. The reaction mixtures were then
neutralized with 1.0 M Na
2
CO
3
. 2.9. Analysis of the
35
S–disaccharides formed by enzymatic depolymerization of ovarian
35
S–chondroitin sulfate
Purified radiolabeled
ovarian glycosaminoglycan
chains present in Peak F2 obtained on Mono Q–FPLC see above were submitted to exhaustive digestion with
chondroitin AC lyase. Disaccharides and chondroitin AC lyase-resistant glycosaminoglycans composed of intact
heparan sulfate chains were recovered by a Superdex peptide-column Amersham Pharmacia Biotech linked
to a HPLC system from Shimadzu Tokyo, Japan. The column was eluted with distilled water:acetonitrile:tri-
fluoroacetic acid 80:20:0.1, vv at a flow rate of 0.5 mlmin. Fractions of 0.25 ml were collected, monitored
for UV absorbance at 232 nm and the radioactivity was counted in a liquid scintillation counter. Fractions corre-
sponding to disaccharides and to the chondroitin AC lyase-resistant glycosaminoglycans eluted at the void
volume were pooled, freeze dried, and stored at
220 °
C. The lyase-derived radiolabeled disaccharides and stan-
dard compounds were subjected to a SAX–HPLC ana- lytical column 250
× 4.6 mm, Sigma–Aldrich, as fol-
lows. After equilibration in the mobile phase distilled water adjusted to pH 3.5 with HCl at 0.5 mlmin,
samples were injected and disaccharides eluted with a linear gradient of NaCl from 0 to 1.0 M over 45 min in
the same mobile phase. The eluant was collected in 0.5 ml fractions and monitored for
35
S–labeled disaccharide content for comparison with lyase derived disaccharide
standards. 2.10. Extraction and analysis of native protein-linked
35
S–sulfated compounds from ovaries of R. prolixus As an attempt to isolate native protein-linked
35
S–sul- fated compounds, fresh collected
35
S–labeled ovaries were homogenized in 5 ml of 0.15 M NaCl containing
0.05 mgml each of soybean trypsin inhibitor, leupeptin, lima bean trypsin inhibitor and antipain, and 1 mM
benzamidine. The homogenate was centrifuged at room temperature
for 5
min at
10,000g. Pellet
homogenization-resistant material
and supernatant
ovary extract were separated. The former was incu- bated with 5 vol of acetone for 24 h at 4
° C.
35
S–sulfated compounds were extracted from the dried acetone pow-
der by papain digestion as described above.
35
S–sulfated compounds present in the supernatant and those
extracted from the pellet were analyzed by anion- exchange chromatography on Mono Q–FPLC, followed
by agarose gel electrophoresis, as described above.
2.11. Distribution of
35
S–sulfated compounds between ovary tissue and oocytes
Fresh-collected
35
S–labeled ovaries were dissected in order to separate chorionated oocytes from the ovarian
34 A. Costa-Filho et al. Insect Biochemistry and Molecular Biology 31 2001 31–40
tissue. Chorionated oocytes were dissected out of ovarian tissues, washed extensively in saline and homo-
genized. The remaining tissue was considered ovarian tissue, but it also contained non-chorionated oocytes,
which were difficult to remove.
35
S–sulfated compounds were extracted from the two fractions by papain diges-
tion and analyzed by anion exchange chromatography on Mono Q–FPLC and agarose gel electrophoresis, as
described above.
2.12. Histochemical detection of sulfated compounds in ovaries of R. prolixus
Ovaries and the female reproductive tract from two animals were dissected and fixed in 4 paraformal-
dehyde in Sorensen phosphate buffer 0.1 M, pH 7.4 at 4
° C overnight. After fixation and washing, the tissues
were dehydrated in ethanol and embedded in parafin. The sections obtained were stained with the cationic dye
1,9–dimethylmethylene blue Farndale et al., 1986 in 0.1 N HCl, containing 0.04 mM glycine and 0.04 NaCl,
according to Pava˜o et al. 1994.
3. Results
