(Received for publication, December 19, 1995; and in revised form, February 16, 1996)
From the
A new glycosaminoglycan has been isolated from the giant African
snail Achatina fulica. This polysaccharide had a molecular
weight of 29,000, calculated based on the viscometry, and a uniform
repeating disaccharide structure of
4)-2-acetyl,2-deoxy-
-D-glucopyranose
(1
4)-2-sulfo-
-L-idopyranosyluronic acid (1
.
This polysaccharide represents a new, previously undescribed
glycosaminoglycan. It is related to the heparin and heparan sulfate
families of glycosaminoglycans but is distinctly different from all
known members of these classes of glycosaminoglycans. The structure of
this polysaccharide, with adjacent N-acetylglucosamine and
2-sulfo-iduronic acid residues, also poses interesting questions about
how it is made in light of our current understanding of the
biosynthesis of heparin and heparan sulfate. This glycosaminoglycan
represents 3-5% of the dry weight of this snail's soft body
tissues, suggesting important biological roles for the survival of this
organism, and may offer new means to control this pest. Snail
glycosaminoglycan tightly binds divalent cations, such as copper(II),
suggesting a primary role in metal uptake in the snail. Finally, this
new polysaccharide might be applied, like the Escherichia coli K5 capsular polysaccharide, to the study of glycosaminoglycan
biosynthesis and to the semisynthesis of new glycosaminoglycan analogs
having important biological activities.
Glycosaminoglycans (GAGs) ()are a family of linear
anionic polysaccharides that are typically isolated as proteoglycans
linked to a protein core. The biological functions of proteoglycans,
including the regulation of cell growth, result, in large part, through
the interaction of the GAG chains in proteoglycans with proteins, such
as growth factors and their receptors(1) . There are two major
classes of GAGs: 1) glucosaminoglycans, including heparin, heparan
sulfate, hyaluronic acid, and keratan sulfate; and 2)
galactosaminoglycans, including chondroitin and dermatan sulfates (1) .
Heparin and heparan sulfate have been the subject of
intensive study because of their well recognized ability to bind many
different proteins that regulate a variety of important biological
processes(2) . Heparin and heparan sulfate GAGs are comprised
of alternating 14 linked glucosamine and uronic acid residues.
Heparan sulfate is composed primarily of monosulfated disaccharides of N-acetyl-D-glucosamine and D-glucuronic
acid, while heparin is composed mainly of trisulfated disaccharides of N-sulfoyl-D-glucosamine and L-iduronic
acid(2) .
GAGs have been isolated from various tissues obtained from a large number of animal species including both vertebrates and invertebrates (3, 4) . An exhaustive assessment showed that while a large number of invertebrate species contain GAGs, mollusks are a particularly rich source of these sulfated polysaccharides(4) . Invertebrates were first shown to contain a heparin or heparan sulfate type GAG by Burson and co-workers in 1956(5) . Heparin has only been found in one invertebrate phylum, the Mollusca, and it often corresponds to up to 90% of the total GAG content of these organisms. While the heparins isolated from various mollusks are structurally different from human heparin (6) and pharmaceutical heparins(7, 8) , mollusk heparins contain antithrombin-dependent anticoagulant activity associated with the presence of the unique 3-O-sulfated glucosamine residue found in the antithrombin pentasaccharide binding site common to all anticoagulant heparins(5, 8, 9, 10, 11) .
While pursuing our long term study of heparin's structure, we
isolated a pure GAG in large amounts from the giant African snail Achatina fulica having a unique structure. This GAG is neither
heparin nor heparan sulfate, but instead represents a new type of
14 linked GAG. A number of biological roles are likely for this
molecule in the snail.
Gradient polyacrylamide gel electrophoresis (PAGE) was performed on a 20-cm vertical slab gel (Protean(TM)II, equipped with a model 1420B power source from Bio-Rad. Capillary electrophoresis (CE) was performed using a Dionex Capillary Electrophoresis system with advanced computer interface, model I, equipped with high voltage power supply capable of constant or gradient voltage control using a fused silica capillary from Dionex Corporation (Sunnyvale, CA). The Cannon-Ubbelohde semimicro capillary viscometer was from Cannon Instruments (State College, PA). A Perkin-Elmer (Ueberlingen, Germany) model 141 polarimeter was used to determine optical rotations.
Compositional analysis by GC (16) was performed using a capillary column AT-1, 0.53 mm
30-m (1.5-µm thickness), from Alltech Associates
(Deerfield, IL) on a Shimadzu (Kyoto, Japan) gas chromatograph, model
GC-14A, with a flame ionization detector, equipped with Shimadzu
Chromatopac CR501 integrating recorder. The injection port temperature
and the detector temperature were 270 and 280 °C, respectively. For
the analysis of mixtures of monosaccharides, the oven temperature was
programmed 120-260 °C at 10 °C/min.
Two-dimensional double
quantum-filtered COSY and multiple relayed COSY spectra were recorded
using the phase-sensitive mode. All two-dimensional spectra were
recorded with 512 2048 data points and a spectral width of 3200
Hz. The water resonance was suppressed by selective irradiation during
the relaxation delay. A total of 128-256 scans were accumulated
for each t
, with a relaxation delay of 2 s. The
digital resolution was 1.6 Hz/point in both dimensions with
zero-filling in the t
dimension. A phase-shifted
sine function was applied for both t
and t
dimensions in the case of double
quantum-filtered COSY, and a Lorentz-Gauss function was applied in all
other cases.
The GAG component of the soft body tissue of the giant
African snail was isolated by proteolysis of defatted tissue and
purified by fractional precipitation. Carbazole assay (19) of
the polysaccharide component showed that it contained uronic acid, and
Azure A dye binding assay (24) demonstrated the presence of
sulfate groups, consistent with it being a GAG. The M of snail GAG showed it to have a molecular weight of 29,000,
significantly higher than the values of 12,000 and 15,500 measured for
porcine mucosal heparin and heparan sulfate,
respectively(12, 23) . The optical rotation
[
]
of the snail GAG was +44°.
Monosaccharide compositional analysis of the polysaccharide isolated
from giant African snail using GC showed it to be composed of 47%
IdoAp, 3% GlcAp and 50% GlcNpAc(16) . The uronic acid component
of the original GAG might have been sulfated, since O-sulfoyl
groups are labile and lost on acidic methanolysis(25) . While
GlcNp and GlcNp2S residues might have been present in the original
polysaccharide and subsequently converted to GlcNpAc following
methanolysis and re-N-acetylation, it is well known that the
glycosidic linkage to these residues is resistant to acidic
methanolysis. Since no disaccharide peaks were observed in the GC
chromatogram obtained from the snail GAG (several such peaks are
detected in the acidic methanolysis of heparin) these data are
consistent with a GAG made up of IdoAp:GlcNpAc at approximately a 1:1
composition.
H NMR analysis of the intact polysaccharide
demonstrated the presence of two anomeric protons having chemical
shifts corresponding to the H-1 of GlcNpAc
1
at
5.1 and
H-1 of IdoAp2S
1
at
5.2, respectively. The H-1 of
GlcNpAc is detected at
5.4 in the spectrum of heparan
sulfate(12) . The observation of this upfield shift caused on
only the anomeric proton of GlcNpAc appeared to be attributable to the
unusual sequence GlcNpAc
IdoAp2S (Fig. 1). Complete
assignment of the
H NMR spectrum of snail GAG were obtained
using two-dimensional NMR spectroscopy ( Fig. 2and Table 1).
Figure 1: Structure of snail GAG.
Figure 2:
H NMR spectrum of snail GAG.
Assignment of each signal is shown in Table 1.
Snail GAG was examined by gradient PAGE with Alcian
blue staining (Fig. 3). Intact GAG showed a pattern of discrete
banding consistent with a polysaccharide of a relatively homogenous
structure with the greatest staining intensity at a degree of
polymerization of 64 (Fig. 3, lane a)
corresponding to a molecular weight of
29,000(23) . Next,
the sensitivity of the snail GAG toward heparin lyases I, II, and III
was examined. No degradation when the polysaccharide was treated with
heparin lyase I (Fig. 3, lane b), little degradation on
treatment with heparin lyase III (Fig. 3, lane d) and
substantial degradation on treatment with heparin lyase II (Fig. 3, lane c) was found. Products having a low level
of negative charge (<-3) do not stain, so that a disaccharide
having a single sulfate group would not be visualized. Analysis of
snail GAG treated with heparin lyase II, on Sephadex G-50 (21) , demonstrated that >90% of the weight of product
mixture corresponded to disaccharide. CE analysis confirmed that the
polysaccharide was converted by heparin lyase II into a single product (Fig. 4b). This product was prepared in milligram
amounts using semipreparative strong anion exchange HPLC (21) ,
and on CE analysis it co-migrated with a disaccharide standard having
the structure,
UAp2S(1
4)-D-GlcNpAc
,
. CE
analyses also demonstrated that the snail GAG was only very slightly
degraded by heparin lyase III and gave no products on treatment with
heparin lyase I (Fig. 4). The spectrum of the disaccharide
obtained by heparin lyase II treatment of snail GAG confirmed its
structure to be
UAp2S(1
4)-D-GlcNpAc
,
( Fig. 5and Table 2), consistent with that reported by
Yamada and co-workers(26) .
Figure 3:
Gradient PAGE analysis of snail GAG.
Analysis was on a 12-22% linear gradient gel with visualization
using Alcian blue staining. Lane a, snail GAG untreated (the
degree of polymerization of 64 corresponds to a molecular weight of
29,000); lane b, snail GAG treated with heparin lyase I; lane c, snail GAG treated with heparin lyase II; lane
d, snail GAG treated with heparin lyase III; lane e,
heparin oligosaccharide standards (degree of polymerization is as
marked).
Figure 4:
CE
analysis of snail GAG on treatment with heparin lyases. Electropherogram a, heparin lyase I; electropherogram
b, heparin lyase II; electropherogram c, heparin lyase
III. The peak observed at 11 min in electropherogram b co-migrates with UA2S(1
4)-D-GlcNAc
,
. Intact polysaccharide (control, no heparin lyase treatment
(not shown) and electropherogram a) has no chromophore
absorbing at 232 nm and shows no peak. The peaks in electropherogram c migrating at <11 min correspond to
oligosaccharide products that have >1 sulfate per disaccharide
residue, and those migrating at >11 min correspond to
oligosaccharides that have <1 sulfate per disaccharide
residue.
Figure 5:
Two-dimensional COSY spectrum of
disaccharide obtained from snail GAG on treatment with heparin lyase
II. Cross-peak 1, H-1/H-3 of UAp2S; Cross-peak
2, H-1/H-3 long range coupling of
UAp2S; Cross-peak
3, H-1/H-2 of
UAp2S; Cross-peak 4, H-1/H-2 of
GlcNpAc
; Cross-peak 5, H-1/H-2 of GlcNpAc
; Cross-peak 6, H-2/H-3 of
UAp2S.
The absorbance spectrum of the
copper(II) complex polysaccharide shows a at 240
nm, confirming the presence of sulfate and carboxylate groups in the
polysaccharide(27) . CE analysis of snail GAG in copper(II)
sulfate solution gave a response identical to low molecular
heparin(27) . The addition of copper(II) sulfate (5
mM) to snail GAG (5 mg/ml) afforded an insoluble blue
precipitate. Neither heparin nor heparan sulfate precipitate under
similar conditions.
Heparin has been prepared from a number of different species
ranging from humans (6) to
clams(5, 8, 9, 10, 11) .
While structural differences have been observed between heparins
obtained from different species and tissues (8) all heparins
characteristically exhibit a molecular weight of 10,000-25,000
and contain a high content of IdoAp2S and GlcNpS (6S or 6OH)
residues(8) . Also common to heparin is the presence of a
unique pentasaccharide sequence containing a central GlcNpS3S6S residue
that interacts specifically with antithrombin and is primarily
responsible for heparin's anticoagulant activity(28) .
The heparin isolated from clams, a member of the Mollusca phylum, is
similar to heparins obtained from higher
organisms(8, 11) . Disaccharide and oligosaccharide
analyses show that clam heparin and porcine mucosal heparin contain
4)
-D-GlcNp2S6S(1
4)
-L-IdoAp2S(1
as their major sequence, corresponding to 70 and 87%, respectively (Table 3) and have saccharide compositions falling in the ranges
shown in Table 3. Indeed, the oligosaccharide map of clam heparin
shows it to have a somewhat more complex structure than heparins
obtained from vertebrates(8) . No peptidoglycan was detected in
the snail polysaccharide preparation using an amine-specific
fluorescent reagent(29) . Thus, it is unclear whether it is
biosynthesized as a proteoglycan. Additional studies aimed at the
extraction of intact proteoglycan from the defatted snail body will be
required to clarify this question.
Heparan sulfate has a distinctly
different structure from that of heparin ( Table 3and Table 4). Heparan sulfate GAGs have molecular weights of
8,000-35,000 (30) and are comprised primarily of
sequences of
4)
-D-GlcNpAc(1
4)
-D-GlcAp(1
(45-73%) and to a lesser extent
4)-
-D-GlcNp2S(1
4)-
-D-GlcAp(1
and
4)-
-D-GlcNpAc6S(1
4)-
-D-GlcAp
(1
. Analyses of heparan sulfate isolated from various species and
tissues have been compared with those obtained on
heparin(31, 32) .
A plot of N-sulfate as a function of total O-sulfate or 2-O-sulfate for various heparin and heparan sulfate samples demonstrated that these GAGs were structurally distinct (31) (Fig. 6). Heparin, having 2.4-2.7 sulfates/disaccharide unit, is also considerably more highly sulfated than heparan sulfate, having 0.6-1.0 sulfates/disaccharide unit.
Figure 6:
Comparison of structural features of snail
GAG, heparin, and heparan sulfate. A plot of N-sulfate
groups/100 disaccharide units versus O-sulfate groups/100
disaccharide units is shown for heparins () from a variety of
tissues and species(8) , heparan sulfates (
)(32) ,
and snail GAG (
).
Extraction of the soft body tissue of
the giant African snail and subsequent purification of its GAGs showed
that this tissue contained a large amount of GAG, corresponding to
nearly 30-50 mg of GAG/g of dry defatted tissue. This GAG was
sulfated, had a higher molecular weight (M 29,000)
than either heparin or heparan sulfate, and exhibited an optical
rotation [
]
of +44 °, similar to
porcine mucosal heparin's value of +53° but
significantly below the [
]
of +75°
observed for heparan sulfate(12) . Monosaccharide analysis
showed snail GAG to have a saccharide backbone comprised of an equal
amount of IdoAp and GlcNpAc. The resistance of this polysaccharide to
heparin lyase I and III suggested that this polysaccharide was neither
heparin nor heparan sulfate(33) . No heparin has been observed
that is completely resistant to heparin lyase I, since this enzyme acts
at the major repeating disaccharide sequence
4)
-D-GlcNpS6S (or 6OH) (1
4)
-
-L-IdoAp2S (1
found in all heparins (Table 3). No heparan sulfate has been found to be completely
resistant to heparin lyase III, which primarily acts at
4)-
-D-GlcNpAc (or 2S) 6S (or 6OH)
(1
4)
-D-GlcAp (1
sequence found in all heparan
sulfates (Table 3). Snail GAG is broken down by heparin lyase II
into
UA2S(1
4)-D-GlcNpAc
,
as confirmed by
co-migration on CE (not shown) and
H NMR spectroscopy.
H NMR of the intact polymer unequivocally establishes the
structure of the snail GAG as
4)-
-D-GlcNpAc
(1
4)-
-L-IdoAp2S(1
. This sequence represents
at least 95% of the polymer structure. The small amounts of GlcAp
observed in the monosaccharide analysis (and resulting in its slight
sensitivity to heparin lyase III) and unsulfated IdoAp observed in the
H NMR may either be associated with minor structural
heterogeneity in this GAG or be due to a small amount of a
contaminating GAG of different structure.
The high molecular weight, sequence, and structural homogeneity of this GAG are inconsistent with its classification as either heparin or heparan sulfate. Additionally, the current pathway proposed for heparin/heparan sulfate biosynthesis requires N-deacetylation and N-sulfation of glucosamine residue prior to the C-5 epimerization and 2-O-sulfation of adjacent uronic acid residues(28, 34, 35) . Thus, the snail polysaccharide represents a new GAG that displays different and unique structural properties than either the heparin or heparan sulfate GAGs.
The presence of such a polysaccharide with a high molecular weight and a simple but unique sequence raises questions about its biosynthesis, including whether or not it is synthesized as a proteoglycan. While snails are known to synthesize neutral polysaccharides and glycoproteins, very little is known about their biosynthesis of acidic polysaccharides, such as GAGs(36) . Uridine diphosphate precursors, such as UDP-N-acetylglucosamine have been identified in the tissues of snails. Thus, the biosynthesis of the GAG isolated from snail probably proceeds through the polymerization of nucleotides of uronic acid and N-acetylglucosamine. Snails contain basophilic cells in their digestive glands, that bind divalent metals (37) . These cells are similar to mast cells that synthesize heparin in vertebrates and may play some role in snail GAG biosynthesis. Snails also biosynthesize glycoproteins that contain N-linked and O-linked glycans composed of mannose, fucose, xylose, and N-acetylglucosamine(38, 39) . The O-linked glycans from snails are believed to be mucins containing both sialic acids and sulfate groups (40) and are linked to threonine(41) . Thus, all the biosynthetic machinery appears to be in place to permit the biosynthesis of proteoglycans and GAGs in the snail.
The large amounts of this GAG found in snail also raise some interesting questions about its biological function(s). Many roles can be proposed for this GAG including 1) binding, uptake, and transport of divalent cations(42, 43) ; 2) an anti-desiccant(40, 44) ; 3) a molecule linked to snail mobility(45, 46) ; and 4) an antibiotic or antipredator molecule. The most likely role of snail GAG is its involvement in cation binding. A. fulica is a very large gastropod that requires substantial quantities of calcium for its shell(37, 47) . In addition, other divalent ions are critical components of their diets (37) . The blood of snails is blue, as hemocyanin is the copper-based carrier of oxygen in these animals(47) . This study shows that snail GAG binds copper(II) much more tightly than heparan sulfate and with about the same avidity of heparin, which has a 3-fold higher level of sulfation. GAGs are known to organize and hold water(48) . Since snails are particularly prone to dehydration, this suggests a second role for this polysaccharide. Snails move on a mucus slime through wave-like undulations of their foot muscle(46, 47) . This high molecular polysaccharide is extremely viscous and may represent a component of this slime. Antibiotic properties have been reported for heparin(49) , and A. fulica is known to make a bactericidal glycoprotein that is found in its mucus(50) , suggesting that snail GAG may have a protective role. Further studies are required to define the precise role(s) of the high concentration of this GAG in A. fulica.
The discovery of the new GAG poses interesting new questions about its biosynthesis and that of the related GAGs, heparin and heparan sulfate. Are the biosynthetic enzymes for all three GAGs similar, and if so, how is glucuronic acid epimerized to iduronic acid in the presence of adjacent N-acetylglucosamine residues? This GAG is easily prepared and purified and affords a valuable source of a potentially useful polysaccharide for the study of heparin/heparan sulfate biosynthesis and biological activities. A similar bacterial polysaccharide, K5, has proved very useful in such studies(51) . Chemical modification of snail polysaccharide using relatively simple methods, i.e. de-N-acetylation and re-N-sulfation, should lead to a structurally homogenous polysaccharide with the minimum structural features for binding fibroblast growth factors(52) . Finally, the giant African snail is considered a major pest in many parts of the world(47) . The use of heparin lyase II or F. heparinum (a soil isolate) capable of degrading its major polysaccharide might provide a biological means for controlling A. fulica.(53) .