Báo cáo khóa học: Acharan sulfate, the new glycosaminoglycan fromAchatina fulica Bowdich 1822 Structural heterogeneity, metabolic labeling and localization in the body, mucus and the organic shell matrix docx

Acharan sulfate, the new glycosaminoglycan from
Achatina fulica
Bowdich 1822
Structural heterogeneity, metabolic labeling and localization in the body, mucus
and the organic shell matrix
Tuane C. R. G. Vieira
1,2
, Adilson Costa-Filho
1,2
, Norma C. Salgado
3
, Silvana Allodi
4
, Ana-Paula Valente
2,5
,
Luiz E. Nasciutti
4
and Luiz-Claudio F. Silva
1,2
1
Laborato
´
rio de Tecido Conjuntivo, Hospital Universita
´
rio Clementino Fraga Filho,
2
Departamento de Bioquı
´
mica Me
´

dica,
3
Laborato
´
rio de Malacologia, Museu Nacional,
4
Departamento de Histologia e Embriologia and
5
Centro Nacional de Ressona
ˆ
ncia
Magne
´
tica Nuclear de Macromole
´
culas, Universidade Federal do Rio de Janeiro, Brazil
Acharan sulfate, a recently discovered glycosaminoglycan
isolated from Achatina fulica, has a major disaccharide
repeating unit of fi4)-2-acetyl,2-deoxy-a-
D
-glucopyra-
nose(1fi4)-2-sulfo-a-
L
-idopyranosyluronic acid (1fi,mak-
ing it structurally related to both heparin and heparan
sulfate. It has been suggested that this glycosaminoglycan is
polydisperse, with an average molecular mass of 29 kDa
and known minor disaccharide sequence variants containing
unsulfated iduronic acid. Acharan sulfate was found to be
located in the body of this species using alcian blue staining

and it was suggested to be the main constituent of the mucus.
In the present work, we provide further information on the
structure and compartmental distribution of acharan sulfate
in the snail body. Different populations of acharan sulfate
presenting charge and/or molecular mass heterogeneities
were isolated from the whole body, as well as from mucus
and from the organic shell matrix. A minor glycosamino-
glycan fraction susceptible to degradation by nitrous acid was
also purified from the snail body, suggesting the presence of
N-sulfated glycosaminoglycan molecules. In addition, we
demonstrate the in vivo metabolic labeling of acharan sulfate
in the snail body after a meal supplemented with [
35
S]free
sulfate. This simple approach might be applied to the study
of acharan sulfate biosynthesis. Finally, we developed histo-
chemical assays to localize acharan sulfate in the snail body
by metachromatic staining and by histoautoradiography
following metabolic radiolabeling with [
35
S]sulfate. Our
results show that acharan sulfate is widely distributed among
several organs.
Keywords: acharan sulfate; Achatina fulica; glycosamino-
glycans; mucus; organic shell matrix; snail.
Glycosaminoglycans (GAGs) consist of hexosamine and
either hexuronic acid or galactose units that are arranged in
alternating unbranched sequence, and carry sulfate substit-
uents in various positions. The common GAGs include
galactosaminoglycans (chondroitin sulfate and dermatan

sulfate) and glucosaminoglycans (heparan sulfate, heparin,
keratan sulfate and hyaluronic acid). Owing to the vari-
ability in sulfate substitution, all GAGs display considerable
sequence heterogeneity. Their strategic location and highly
charged nature make them important biological players in
the cell–cell and cell–matrix interactions that take place
during normal and pathological events related to cell
recognition, adhesion, migration and growth [1–4].
The new GAG, acharan sulfate, was first isolated and
characterized by Kim et al. in 1996 [5], as a pure GAG from
the giant African snail, Achatina fulica. Acharan sulfate
occurs in large amounts and has a unique structure. This
GAG is polydisperse with an average molecular mass of
29 kDa and has a major disaccharide repeating unit of
fi4)-2-acetyl,2-deoxy-a-
D
-glucopyranose(1fi4)-2-sulfo-a-
L
-
idopyranosyluronic acid (1fi, thereby relating it structurally
both to heparin and heparan sulfate, although it is distinctly
different from all known members of these classes of
GAGs [5].
Kim et al. reported structural heterogeneity when they
determined the structure of oligosaccharides prepared from
acharan sulfate [6]. They identified two series of oligosac-
charides, one derived from acharan sulfate’s major repeat-
ing unit and a second minor group of undersulfated
oligosaccharides. Oligosaccharides containing N-sulfated
units were not detected [6].

A great number of biological roles were suggested for this
molecule in the snail, but as this GAG was newly discovered
it has only been evaluated on the basis of structural
similarities to heparin and heparan sulfate, and the biological
functions of GAGs. Therefore, intact or chemically modified
acharan sulfate has been shown to present several in vitro
biological activities, such as: inhibition of angiogenesis due to
Correspondence to L C. F. Silva, Departamento de Bioquı
´
mica
Me
´
dica, Centro de Cieˆ ncias da Sau´ de, Universidade Federal do Rio de
Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brazil.
Fax: + 55 21 2562 2090, E-mail: lclaudio@hucff.ufrj.br
Abbreviations: GAG, glycosaminoglycan; HSQC, heteronuclear single
quantum coherence.
Enzymes: chondroitin AC lyase (EC 4.2.2.5) from Arthrobacter
aurescens; chondroitin ABC lyase (EC 4.2.2.4) from Proteus vulgaris.
(Received 17 November 2003, accepted 12 January 2004)
Eur. J. Biochem. 271, 845–854 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03989.x
inhibition of vascular endothelial growth factor or the
decrease of the mitogenic activity of fibroblast growth factor
and other pharmacological activities [7–10].
In a recent study, Jeong et al. localized acharan sulfate in
the snail body by alcian blue staining and showed that it is
mainly secreted into the outer surface of the body as a
mucous material from internal granules [11]. The mucus
secreted on the body surfaces of mollusks is known to play
crucial roles in locomotion, feeding, osmoregulation, repro-

duction and protection of epithelial surfaces [12].
Achatina fulica is an intermediate host to Angiostrongylus
cantonensis [13], the etiological agent of meningoencephalic
angiostrongiliasis [14], and may act as a major source of
human infection in places where it is commonly eaten by
people, such as Taiwan [13]. The occurrence of Achatina
fulica in Rio de Janeiro, Brazil, was recently reported [15]. In
the localities visited, snails were found living freely, and
larvae of Angiostrongylus cantonensis were not seen in any of
them. The finding of Achatina fulica in the area may be
related to its commercialization as a food item [15].
The giant African snail is considered a major pest in
many parts of the world and as acharan sulfate is an
important constituent of the snail body tissues, it might be
involved in important biological roles for the survival of this
animal. Therefore, a complete understanding of both
biochemistry and compartmental distribution of this mole-
cule might be important to develop methods of controlling
this pest. Here, we provide new information on the
biochemistry and properties of this interesting GAG
molecule.
Experimental procedures
Materials
Giant snails (Achatina (Lissachatina) fulica Bowdich 1822)
were collected in Rio de Janeiro, Brazil. Animals were
maintained in an air-conditioned laboratory at 25–28 °C
under a 12-h light : 12-h dark cycle (natural light) in plastic
tanks, and fed a vegetable diet. Chondroitin 4-sulfate,
chondroitin 6-sulfate, dermatan sulfate, heparan sulfate and
twice-crystallized papain (15 UÆmg

)1
protein) were pur-
chased from Sigma Chemical Co. Chondroitin AC lyase
(EC 4.2.2.5) from Arthrobacter aurescens and chondroitin
ABC lyase (EC 4.2.2.4) from Proteus vulgaris were pur-
chased from Seikagaku American Inc. (Rockville, MD,
USA). Radiolabeled carrier-free [
35
S]Na
2
SO
4
was obtained
from Instituto de Pesquisas Energe
´
ticas e Nucleares (Sa
˜
o
Paulo, SP, Brazil).
Preparation of GAGs from the soft body
GAGs were isolated from the soft snail body following a
previously described method, with modifications [5]. Briefly,
the shell of the giant snail was removed, and the whole soft
body was defatted using three 24 h extractions with acetone.
The fat-free, dried snail was cut into very small pieces that
were suspended in sodium acetate buffer (pH 5.5) containing
40 mg papain in the presence of 5 m
M
EDTA and 5 m
M

cysteine, and incubated at 60 °C for 24 h. The suspension
was centrifuged at 3000 g for 20 min at room temperature
and the supernatant, which contained the GAGs, was
applied into a DEAE-cellulose column (34.0 cm · 2.5 cm;
Sigma Chemical Co.), equilibrated with 0.05
M
sodium
acetate (pH 5.0). The column was washed with 200 mL of
the same buffer and then eluted stepwise with 800 mL of
3.0
M
NaCl in the same acetate buffer. The GAGs eluted
from the column were exhaustively dialyzed against distilled
water, lyophilized and dissolved in 10.0 mL of distilled water.
The partially purified GAGs were applied to a Mono Q-
FPLC column (HR 5/5; Pharmacia Biotech Inc., Uppsala,
Sweden), equilibrated with 20 m
M
Tris/HCl (pH 8.0). The
column was washed with 10 mL of the same buffer. Then,
the column-bound GAGs were eluted in a stepwise gradient
from 0.2
M
NaCl to 3.0
M
NaCl at intervals of 0.2
M
NaCl
in Tris buffer. Fractions of 0.5 mL were collected and the
elution was monitored by the metachromatic property of

the fractions using 1,9-dimethylmethylene blue [16]. The
GAGs eluted from the column were exhaustively dialyzed
against distilled water, lyophilized and dissolved in 1.0 mL
of distilled water.
Collection and analysis of mucus GAGs
Mucus was collected from the surface of live snails. The
collected mucus was mixed with three volumes of sodium
acetate buffer (pH 5.5) containing 40 mg papain in the
presence of 5 m
M
EDTA and 5 m
M
cysteine, and incubated
at 60 °C for 24 h. GAGs were isolated and purified from the
papain-digested mucus by sequential anion-exchange chro-
matograpy firstly in DEAE-cellulose column followed by
fractionation on a Mono Q-FPLC column, as described
above.
Extraction of GAGs from the organic shell matrix
The preparation of an EDTA-free extraction of organic
matrix was performed as described previously, with modi-
fications [17]. Briefly, clean, dry, powdered shells were
decalcified during 24 h at 4 °C in 200 mL of 1
M
HCl. The
solution was stirred constantly. After centrifugation at
3000 g for 20 min at room temperature, the supernatant
was dialyzed against water for four days and then lyo-
philized. This material was named soluble organic shell
matrix. GAGs were isolated from the soluble matrix and

purified as described above for the mucous material. A
purified preparation of acharan sulfate isolated from the
whole body was submitted to decalcification by the same
protocol described above, and analyzed by the same
methodology used to characterize acharan sulfate from
the other snail tissues.
In vivo
metabolic
35
S-labeling of snail body GAGs
We performed a simple method for the metabolic labeling of
snail GAGs by wetting small pieces of leaves of lettuce with
1.48 mBq [
35
S]Na
2
SO
4
, leaving it to dry, mixing it with
unlabeled lettuce and offering it as food to snails that were
1 day without food.
Isolation of the radiolabeled GAGs
Twenty four hours after a meal containing the radioactive
precursor, the snails were killed, the shell was removed and
846 T. C. R. G. Vieira et al. (Eur. J. Biochem. 271) Ó FEBS 2004
GAGs were isolated and purified as described above for the
unlabeled snails. The
35
S-labeled GAGs were detected in the
Mono Q-FPLC fractions by scintillation counting, exhaust-

ively dialyzed against distilled water and re-suspended in
1.0 mL of distilled water.
Identification of the snail GAGs
GAGs were characterized by a set of current biochemical
methods that included: agarose gel electrophoresis, diges-
tion with chondroitin lyases, heparin lyase I and deamin-
ative cleavage with nitrous acid, PAGE and NMR
spectroscopy [18,19], as described below.
Agarose gel electrophoresis
Agarose gel electrophoresis was carried out as previously
described [20]. Approximately 10 lg of GAGs, before and
after chondroitin lyase or heparin lyase I digestions or
deaminative cleavage with nitrous acid (see below), as well as
a mixture of standard chondroitin4-sulfate, dermatan sulfate
and heparan sulfate (10 lg of each) were applied to 0.5%
agarose gels in 0.05
M
1,3-diaminopropane/acetate buffer
(pH 9.0). Following electrophoresis, GAGs were fixed in the
gel with 0.1% N-cetyl-N,N,N-trimethylammonium bromide
in water, and stained with 0.1% toluidine blue in acetic acid/
ethanol/water (0.1 : 5 : 5, v/v/v). The
35
S-labeled GAGs
were visualized by autoradiography of the stained gels.
Digestion with chondroitin lyases
Digestions with chondroitin ABC lyase were performed
according to Saito et al. [21]. Approximately 100 lgofsnail
GAGs were incubated with 0.3 units of chondroitin ABC
lyase for 8 h at 37 °Cin100lLof50m

M
Tris/HCl
(pH 8.0), containing 5 m
M
EDTA and 15 m
M
sodium
acetate.
Digestion with heparin lyase I
Enzymatic digestion with heparin lyase I was performed
with addition of the enzyme (1 mU) in 100 lL of 100 m
M
sodium acetate and 10 m
M
calcium acetate (pH 7.0), over a
36hperiodat37°C[22].
Deamination with nitrous acid
Deamination by nitrous acid at pH 1.5, was performed as
described by Shively & Conrad [23]. Briefly,  100 lgof
snail GAGs were incubated with 200 lL 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
.

N-acetylation of purified GAG fractions
N-acetylation of purified snail GAG fractions was per-
formed by the addition of 0.1 mL of acetic anhydride [5].
Polyacrylamide gel electrophoresis
Fractionated GAGs from both mucus and the whole snail
body (see above for methodology) were applied to a 1 mm
thick, 6% polyacrylamide slab gel. Following electrophor-
esis at 100 V for  1 h in 0.06
M
sodium barbital (pH 8.6),
the gel was stained with 0.1% toluidine blue in 1% acetic
acid. After staining, the gel was washed for  6hin1%
acetic acid [24].
Superose-12 HPLC gel filtration chromatography
Intact, b-eliminated or papain-digested total GAGs
obtained from mucus were applied separately into a
Superose-12 (HR 10/30) column linked to a HPLC system
(Shimadzu, Tokyo, Japan). Ammonium bicarbonate 0.2
M
(pH 5.0) was the buffer used. The column was eluted with
the same solution, at a flow rate of 0.5 mLÆmin
)1
,and
fractions of 0.5 mL were collected and assayed for meta-
chromasia or for the protein content, measured by auto-
matically recording the absorption at 280 nm.
b-elimination of GAGs from mucus
b-elimination was performed by the incubation of GAGs in
0.1
M

NaOH at 37 °C for 12 h.
NMR spectroscopic analysis
1
Hand
13
C spectra were recorded using a Bruker DRX 600
with a triple resonance probe (Bruker, Germany). About
2 mg each of purified mucus and soft body GAGs were
dissolved in 0.5 mL of 99.9% D
2
O (CIL). All spectra were
recorded at 60 °C with HOD suppression by presaturation.
1
H/
13
C heteronuclear single quantum coherence (HSQC)
spectra were run with 1024 · 300 points and globally
optimized alternating phase rectangular pulses for decou-
pling. All chemical shifts were relative to external trimethyl-
silylpropionic and [
13
C]methanol.
Radioautographic and metachromatic detection
of sulfated GAGs
After the in vivo metabolic labeling of snail GAGs, the snails
were killed, the shell was removed and several organs were
dissected and fixed in 4% formaldehyde (freshly prepared
from paraformaldehyde) in Sorensen phosphate buffer
(0.1
M

,pH7.4)at4°C overnight. After fixation and
washing, the tissues were dehydrated in ethanol and
embedded in paraffin. Tissue sections (7 lm) were obtained
and collected on gelatin-coated slides. These were then
immersed in NTB
2
liquid emulsion (Kodak) and left in a
dark box for 2 weeks at 4 °C. They were developed, washed
several times, and stained with cationic dye 1,9-dimethyl-
methylene blue [16] in 0.1
M
HCl, containing 0.04 m
M
glycine and 0.04 m
M
NaCl [25]. The sections were then
examined and photographed using a light microscope
(Zeiss, Axioskop 2).
Results
Heterogenic forms of acharan sulfate are present
in the soft body, mucus and the organic shell matrix
The GAG component of the soft body, mucus and the
organic shell matrix of the giant African snail was isolated
Ó FEBS 2004 Acharan sulfate structure and distribution (Eur. J. Biochem. 271) 847
and purified by anion-exchange chromatography on a Mono
Q-FPLC column (Fig. 1A–C, respectively). Five metachro-
matic peaks were eluted from 0.2 to 1.0
M
NaCl at intervals
of 0.2

M
and designated as peaks R1 to R5 (Fig. 1A–C).
Differences in the proportion and occurrence of R-peaks
were observed among the three sources of snail GAGs. The
most drastic differences were observed in the profile obtained
for GAGs from the organic shell matrix when compared to
the other profiles, where peaks R3 and R4 predominate over
the other R-peaks, which are either present in a very small
amount or are lacking (Fig. 1C). In order to verify whether
the conditions used to dissolve the shell would alter the
structure of acharan sulfate, a control experiment using
acharan sulfate isolated from the whole body was carried out
to show that it was stable under these conditions. Similar
patterns of elution of snail body acharan sulfate subpopu-
lations, regardless of incubation with or without the presence
of the decalcifying agents, were obtained in a Mono Q-FPLC
column using the same conditions described in the legend of
Fig. 1 (data not shown).
GAGs present in the R-peaks were first analyzed by
agarose gel electrophoresis (Fig. 1D–F). Peaks R1 to R5
presented a major metachromatic band with an electro-
phoretic migration similar to that of the dermatan sulfate
standard. This band was not detected in peak R1 from the
organic shell matrix (Fig. 1F). As acharan sulfate was
shown to be the only GAG species expressed by the giant
African snail [5], we assigned this band as acharan sulfate
in Fig. 1D–F. However, a second minor band, with an
electrophoretic migration similar to that of the heparan
sulfate standard, was present only in peak R1 from the snail
body (Fig. 1D).

Fig. 1. Fractionation of snail GAGs. Purification of GAGs from whole body (A), mucus (B) and the organic shell matrix (C) of Achatina fulica on a
Mono Q-FPLC column. The DEAE-cellulose-purified GAGs were applied to a Mono Q-FPLC and purified as described. Fractions were
monitored by the metachromatic property (k). The NaCl concentration in the fractions was determined by measuring the conductivity. The
fractions corresponding to GAGs (peaks R1 to R5) as indicated by horizontal bars, were pooled, dialyzed against distilled water and lyophilized.
Peaks R1 to R5 from whole body (D), mucus (E) and the organic shell matrix (F) were analyzed by agarose gel electrophoresis. HS, heparan sulfate;
DS, dermatan sulfate; CS, chondroitin 4/6 sulfate. The major band in (D) to (F) was assigned as acharan sulfate (see text).
848 T. C. R. G. Vieira et al. (Eur. J. Biochem. 271) Ó FEBS 2004
GAGs present in peaks R1 to R5 were further analyzed by
agarose gel electrophoresis, before and after incubation with
condroitin ABC lyase (which degrades both chondroitin
and dermatan sulfate), or deaminative cleavage with nitrous
acid (which degrades N-sulfated GAGs such as heparan
sulfate and heparin). The major electrophoretic band in the
R-peaks from the whole body was resistant to both
treatments (Fig. 2A). This is as expected for acharan
sulfate, as this GAG was previously shown to be substan-
tially degraded only by heparin lyase II, thus it is resistant to
all other enzymatic treatments that degrade GAGs [5]. The
same pattern was obtained for snail GAGs in the R-peaks
from both mucus and the organic shell matrix (data not
show). Interestingly, the minor band detected only in peak
R1 from whole body (Fig. 1D) was resistant to treatment
with chondroitin ABC lyase, but was surprisingly degraded
by nitrous acid (Fig. 2A). This result suggests the existence
of either N-sulfated or free amino groups in the minor GAG
fraction of peak R1. In order to address this issue, the R1
fraction was chemically N-acetylated and subsequently
analyzed by agarose gel electrophoresis before and after
nitrous acid treatment. The N-acetylation procedure did not
affect the electrophoretic migration of GAGs in R1, but the

minor GAG band remained susceptible to the nitrous acid
treatment (Fig. 2B), indicating that it presents N-sulfated
groups. Additionally, this GAG fraction was also suscept-
ible to degradation with heparin lyase I, as expected for
N-sulfated GAGs (Fig. 2B).
The spread of elution observed to snail GAGs with
increasing salt concentration in the Mono Q-FPLC chro-
matographies (Fig. 1A–C) may be explained by increasing
molecular mass. In order to address this issue, we evaluated
their molecular mass distribution by polyacrylamide gel
electrophoresis. The obtained results revealed marked
differences between the mobility of GAGs from the R-peaks
of the polysaccharides extracted from the whole body
compared to mucus. In the snail body fraction, we found
four different metachromatic bands with increasing mole-
cular mass, from R2 to R5 (Fig. 3A), showing that acharan
sulfate is composed of heterogeneous and polydisperse
GAG chains. GAGs from R1 of the snail body were not
analyzed due to the presence of a contaminating brown
pigment that caused distortions in the metachromatic
bands. An additional incubation of GAGs present in peaks
R2 to R5 with papain did not modify the patterns of
electrophoretic mobility on polyacrylamide gel (not shown).
Less pronounced differences were observed among the
GAGs present in peaks R1 to R5 from mucus (Fig. 3B).
This analysis was not performed for GAGs present in the
R-peaks from the organic shell matrix because sufficient
material was lacking. Another interesting detail revealed by
these experiments is that the molecular masses of GAGs
from both body and mucus (Fig. 3A,B) correspond to those

of chondroitin 4- and 6-sulfate standards (S2 and S3 in
Fig. 3C, respectively) in this system (molecular mass range
of 40–60 kDa) (compare Fig. 3A and C).
NMR analysis confirms the structural identity of snail
acharan sulfate
The
1
H one-dimensional NMR spectra of the major GAG
fractions from the snail whole body and from mucus
(Fig. 4A and B, respectively) and interpretations of
1
H/
13
C
HSQC spectra of whole body GAG (Fig. 5), confirm the
identification of these GAG fractions as the previously
Fig. 2. Characterization of GAGs from the snail body. (A) Agarose gel
electrophoresis of GAGs present in peaks R1 to R5 from whole body,
before (–) and after (+) chondroitin ABC lyase digestion or deamin-
ative cleavage by nitrous acid. After enzymatic or chemical incubation,
the GAGs were applied to 0.5% agarose gel and electrophoresis was
carried out as described. (B) Samples of peak R1 were subjected to an
N-acetylation protocol and subsequently analyzed by agarose gel
electrophoresis, before (–) and after (+) deaminative cleavage by
nitrous acid. An intact sample of peak R1 was also analyzed by gel
electrophoresis after incubation with heparin lyase 1. CS, chondroitin
4/6-sulfate; DS, dermatan sulfate; HS, heparan sulfate.
Fig. 3. Polyacrylamide gel electrophoresis of GAGs present in peaks R1
to R5 from whole body (A), mucus (B) and standards (C). Standard
sulfated polysaccharides are: S1, high molecular mass dextran sulfate

(500 kDa); S2, chondroitin 6-sulfate (60 kDa); S3, chondroitin 4-sul-
fate (40 kDa); S4, low molecular mass dextran sulfate (8 kDa). Peaks
R1 to R5 were obtained as described for Fig. 1.
Ó FEBS 2004 Acharan sulfate structure and distribution (Eur. J. Biochem. 271) 849
described acharan sulfate [5]. The
1
H chemical shifts of the
residue found in the major snail body GAG fraction,
characterized as acharan sulfate, are presented in Table 1,
and are based on interpretations of
1
H/
13
CHSQC(Fig.5).
The assignment was performed based on comparison with
the previously described acharan sulfate (Table 1). The
values obtained here are in agreement with a sulfated H2 of
the a-
L
-iduronic acid residue as the major disaccharide unit.
This conclusion was reinforced by data from literature,
which show that a-
L
-iduronic acid residues from the two
types of disaccharide units composed of glucosamine linked
to either 2-O-sulfated or nonsulfated a-
L
-iduronic acid
residues, have approximately the same chemical shifts for
H4 and H5, but that the H2 is 0.69 p.p.m. downfield in the

2-O-sulfated unit (Table 1).
NMR analyses of GAGs derived from the organic shell
matrix were not performed due to the scarce amount of
purified material. In view of the very small amount of
Fig. 4.
1
H NMR spectra at 600 MHz of snail GAG from whole body
(A) and mucus (B). The samples were dissolved in 500 lLofD
2
O 100%
and the spectra recorded at 60 °C with suppression of residual HOD
signal by presaturation.
Fig. 5.
1
H/
13
C HSQC spectrum of snail GAG from whole body. The
assignment was based on comparison with the previously described
acharan sulfate. The spectrum was acquired with 1024 · 300 points
and 128 scans.
Table 1. Proton chemical shifts for acharan sulfate (from whole body), heparin and modified heparins. Chemical shifts are referenced to internal
trimethylsilylpropionic acid at 0 p.p.m. Protons designated ÔIÕ refer to those of a-
L
-iduronic acid residues, whereas those of a-
D
-glucosamine are
designed as ÔAÕ.
Proton
Chemical shift (p.p.m.)
Acharan sulfate

This work
Acharan sulfate
Kim et al. [5]
IdoAp2SGlcNpS(6S)
Mulloy et al. [29]
IdoApGlcNpS(6S)
Jaseja et al. [30]
IdoApGlcNpAc
Mulloy et al. [29]
I-1 5.20 5.19 5.22 5.00 4.89
I-2 4.34
a
4.34
a
4.35
a
3.80 3.65
I-3 4.19 4.28 4.20 4.10 3.83
I-4 4.05 4.03 4.10 4.10 4.04
I-5 5.08 4.93 4.81 4.80 4.68
A-1 5.07 5.11 5.39 5.30 5.11
A-2 3.97 4.02 3.29 3.30 4.00
A-3 3.70 3.74 3.67 3.70 3.73
A-4 3.70 3.74 3.77 3.80 3.73
A-5 3.71 3.87 4.03 4.00 4.00
A-6 3.82 3.87 4.27
a
4.20
a
3.85

Acetyl 2.04 2.08 – – 2.04
a
Values indicate positions bearing sulfate ester.
850 T. C. R. G. Vieira et al. (Eur. J. Biochem. 271) Ó FEBS 2004
purified nitrous acid-sensitive snail GAG and the difficulty
in separating it from acharan sulfate (Figs 1A,D and 2), we
did not further characterize this GAG by NMR analysis.
Acharan sulfate appears to exist as a protein-free
polysaccharide in mucus
Unfractionated mucus samples were analyzed by poly-
acrylamide gel electrophoresis before or after incubation
with papain, a protease of broad specificity (Fig. 6A), in
order to investigate whether mucus acharan sulfate is
covalently linked to proteins as a proteoglycan. No
changes on the electrophoretic migration of both intact
and papain-digested mucus were observed (Fig. 6A).
Additionally, intact, b-eliminated or papain-digested
GAGs from mucus were analyzed by gel filtration
chromatography (Fig. 6B). No significant differences were
observed in the metachromatic pattern of eluted GAG
among the three samples (Fig. 6Ba–c). The pattern of
protein elution was only modified in the papain-digested
sample (Fig. 6Bd–f). Together, these experiments suggest
that acharan sulfate in mucus is present as a protein-free
polysaccharide, because no modifications occurred on
either the metachromatic or the protein patterns of
elution after b-elimination (a procedure that separates
the GAG chain from the core protein) or papain
digestion (except by the change in the profile of protein
elution after treatment with this enzyme, as expected).

However, we cannot exclude the possibility that these
molecules may be synthesized as proteoglycans in the
snail body tissue and that protein-free GAG chains would
be produced from proteoglycans by strong protease
activities present in the mucus.
In vivo
metabolic labeling of acharan sulfate
Snails were fed with leaves of lettuce coated with 1.48 mBq
[
35
S]Na
2
SO
4
. After the labeling period, snails were killed
and GAGs were extracted and purified from the soft body,
as described in Experimental procedures. The labeled GAG
component of the soft body of Achatina fulica was purified
by anion-exchange chromatography on a Mono Q-FPLC
column (Fig. 7A). In a similar situation to that found for
the unlabeled snail GAGs (Fig. 1A), five radiolabeled peaks
were eluted from 0.2
M
to 1.0
M
NaCl at intervals of 0.2
M
andreferredtoaspeaksR1toR5(Fig.7A).However,
unlike that observed for the unlabeled GAGs, R3 and R4
were the predominant peaks (compare Figs 1A and 7A).

Agarose gel electrophoresis followed by autoradiography of
the stained gels revealed a similar electrophoretic pattern to
both metachromatic and radiolabeled bands (Fig. 7B,C,
respectively). These results show that all GAG species in the
snail body were metabolically labeled through assimilation
of labeled free sulfate from the food by the digestion
process.
Acharan sulfate is widely distributed in the snail body
Radioautography and metachromatic staining were per-
formed to evaluate the distribution of GAGs in internal
Fig. 6. Evaluation of the molecular mass of acharan sulfate from mucus. Polyacrylamide gel electrophoresis (A) of GAGs present in unfractionated
snail mucus, before (–) and after (+) papain digestion. After enzymatic incubation, the GAGs were applied to polyacrylamide gel and electro-
phoresis was carried out as described. Standard sulfated polysaccharides (Standards) are: S1, high molecular mass dextran sulfate (500 kDa); S2,
chondroitin 6-sulfate (60 kDa); S3, chondroitin 4-sulfate (40 kDa); S4, low molecular mass dextran sulfate (8 kDa). (B) The chromatographic
profiles of intact (a and d), b-eliminated (b and e) or papain-digested (c and f) GAGs from mucus samples. GAGs were detected by the
metachromatic property of the fractions (a to c), while proteins were detected by the absorption at 280 nm of the fractions (d–f).
Ó FEBS 2004 Acharan sulfate structure and distribution (Eur. J. Biochem. 271) 851
and external sites of the snail body (Fig. 8). The
metachromatic staining (purple) showed the presence of
sulfated compounds in several organs: kidney, salivary
glands, esophagus, stomach, albumen gland, feet and
spermatheca. Light micrographs for stained feet and
kidney are shown (Fig. 8A; feet and Fig. 8B,C; kidney).
Radioautographic silver grains were mostly observed on
the purple-stained structures indicating an efficient incor-
poration of [
35
S]sulfate.
Discussion
Acharan sulfate was first isolated from the body of the giant

African snail, Achatina fulica,byKimet al.[5]bymeansof
protease digestion of the dried fat-free snail tissues. The
GAG was precipitated initially by ethanol, re-dissolved and
subsequently precipitated by a quaternary ammonium salt,
cetylpyrydinium chloride, and characterized as a new GAG
species. Based on the pattern of susceptibility of this GAG
to degradation by specific mucopolysaccharidases, disac-
charide analysis by capillary electrophoresis and NMR
spectrometry [5], the authors showed that acharan sulfate
has an average molecular mass of 29 kDa and a major
disaccharide repeating unit of fi4)-2-acetyl,2-deoxy-a-
D
-
glucopyranose(1fi4)-2-sulfo-a-
L
-idopyranosyluronic acid
(1fi, making it structurally related to both heparin and
heparan sulfate but distinctly different from all known
members of these classes of GAGs. Acharan sulfate also
presents minor disaccharide sequence variants containing
unsulfated iduronic acid [6].
Here, using a different protocol to isolate and purify
GAGs from the soft body of Achatina,wewereabletoshow
that acharan sulfate from the snail body presents a great
Fig. 8. Light micrographs of the Achatina feet and kidney stained with
1,9-dimethylmethylene blue dye and labeled with silver grains obtained by
autoradiography. (A) Feet displaying metachromatic purple color on
the epithelium surface (arrowhead) and on glands with secretory
portions deeply located (asterisks). The radioautography technique
strongly labeled the connective tissue associated to the glandular

compartment (arrows). (B) Low magnification of the kidney showing
metachromatic material distributed by the organ. (C) High magnifi-
cation of the kidney. In this micrograph silver grains are clearly seen
concentrated on the metachromatic structures. Scale bar in C is 90 lm
(A); 25 lm(B);5.5lm(C).
Fig. 7. Incorporation of [
35
S]sulfate into GAGs from the snail body. (A)
Purification of
35
S-labeled GAGs from snail whole body on a Mono
Q-FPLC column. Fractions were monitored by scintillation counting (k).
The NaCl concentration in the fractions (–) was determined by measuring
the conductivity. The fractions corresponding to the
35
S-labeled GAGs,
as indicated by horizontal bars, were pooled, dialyzed against distilled
water and lyophilized. Toluidin blue stained (B) and autoradiogram (C)
of agarose gel electrophoresis of GAGs present in peaks R1 to R5. HS,
heparan sulfate; DS, dermatan sulfate; CS, chondroitin 4/6 sulfate. The
majorbandin(B)and(C)wasassignedasacharansulfate(seetext).
852 T. C. R. G. Vieira et al. (Eur. J. Biochem. 271) Ó FEBS 2004
degree of heterogeneity regarding negative net charge
density and/or molecular mass (Figs 1A and 3A, respect-
ively). In addition, we identified the presence of a GAG
species, not described before, that occurs in minor amounts
and that was susceptible to deamination by nitrous acid,
even after it had been subjected to an N-acetylation
protocol. This GAG species was also sensitive to degrada-
tion by the action of heparin lyase I, indicating that this

GAG presents N-sulfate groups at the glucosamine residue.
Kim et al. also commented in their previous paper, which
described the snail body GAG composition [5], that at least
95% of the GAG that was purified corresponded structur-
ally to acharan sulfate, whereas the remaining 5% either
corresponded, with minor structural heterogeneity, to this
GAG, or was due to a small amount of a contaminant
GAG of a different structure [5]. These findings raise the
interesting question of whether the balance between acharan
sulfate and this other GAG species remains unchanged
from birth to the adult life of the snail.
The presence of this unique GAG, acharan sulfate, with
its simple but unusual sequence poses interesting questions
about its biosynthesis. Our results, which establish that it is
possible to label GAGs in the snail body by supplementing
the food with [
35
S]sulfate (Experimental procedures and
Fig. 7), may, in the near future, turn out to be an important
tool in developing experiments to follow the biosynthesis of
acharan sulfate.
Jeong et al. conducted a histochemical analysis of the
distribution of GAGs in tissue sections of the snail body
[11]. They analyzed the alcian blue staining of GAGs from
tissue samples of exterior sites and interior spaces of the
snail body. Positive staining was visualized only on the
exterior sites, and GAGs were primarily located inside
granules and were secreted onto the surface as a mucous
material [11]. Here, we extend their basic findings by
analyzing the distribution of GAGs, by means of both

metachromatic staining and autoradiography, of several
internal organs and also over the exterior surface of the snail
body. We found that sulfated compounds are widely
distributed among the tissues examined (Fig. 8). This
finding adds new possible biological roles for snail GAGs,
especially acharan sulfate, according to the source of the
tissue, which included the digestive tract, kidney, albumen
gland, feet and spermatheca.
GAGs are reported to be present in the mollusk shell
organic matrix [17,26,27] and these molecules may be
responsible for fixation of calcium in the shell. Marxen &
Becker [16] recently suggested that a material that was
isolated from the organic shell matrix of the freshwater
snail, Biomphalaria glabrata, which was stainable with
alcian blue, would probably be sulfated proteoglycans or
GAGs. Alcian blue stainable compounds were also
identified in aqueous [26] or acidic [27] extracts isolated
from the nacreous layer of the shell from the pearl oyster,
Pinctada maxima. Although these and other reports
suggest the presence of GAGs or proteoglycans in the
mollusk shell organic matrix, the composition and
structure of these compounds have never been reported.
Here, we were able to extract significant amounts of
acharan sulfate from the organic shell matrix of Achatina
fulica (Fig. 1). Therefore, our results are the first
biochemical description of the identity of GAGs from a
mollusk shell organic matrix. The location of acharan
sulfate in the organic shell matrix of Achatina fulica
suggests that it may be involved in the promotion of shell
mineralization.

As we can see, acharan sulfate is widely distributed in the
body of Achatina fulica suggesting that this GAG may be
extremely important to the physiology of this gastropod.
Altogether, our findings open interesting possibilities to
investigate the biological roles of acharan sulfate by
analyzing the effects of GAG-modifying agents, such as
chlorate or selenate (sulfation inhibitors), on the physiology,
shell formation, mucus production and growth rates of
individuals at different ages. In this case, the metabolic
labeling protocol would be of interest to assess the alteration
in the degree of sulfation of GAGs synthesized under the
influence of the inhibitors.
Finally, Achatina fulica is an intermediate host to
Angiostrongylus cantonensis [13], the etiological agent of
meningoencephalic angiostrongiliasis [14], and may act as a
major source of human infection in places where people
commonly eat it. Interestingly, heparin-binding adhesion
proteins were reported to be expressed in adult forms of
Strongyloides venezuelensis [28]. As Achatina fulica may act
as an intermediate host for some species of Strongyloides
and also express acharan sulfate (a heparin-related GAG)
throughout the body, it is tempting to speculate that this
GAG might be involved in the processes of interaction
between the parasite and the snail.
Acknowledgements
We would like to thank Selma Pacheco Ramos and Jorge Luis da Silva
for technical assistance. This work was supported by Conselho
Nacional de Desenvolvimento Cientı
´
fico e Tecnolo

´
gico (CNPq:
PADCT and PRONEX), Fundac¸ a
˜
odeAmparoa
`
Pesquisa do Estado
do Rio de Janeiro (FAPERJ), Financiadora de Estudos e Projetos
(FINEP) and Fundac¸ a
˜
oUniversita
´
ria Jose Bonifa
´
cio (FUJB-UFRJ).
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