- Email: [email protected]
Glycosaminoglycans in two mollusks, Aplysia californica and Helix aspersa, and in the leech, Nephelopsis obscura
Comparative Biochemistry and Physiology Part B 119 (1998) 691 – 696
Glycosaminoglycans in two mollusks, Aplysia californica and Helix aspersa, and in the leech, Nephelopsis obscura Peter Hovingh a, Alfred Linker b,* b
a Department of Biochemistry and Pathology, Uni6ersity of Utah, Salt lake City, UT 84132, USA Veterans Affairs Medical Center, Research Ser6ice (151E), 500 Foothill, Salt Lake City, UT 84148, USA
Received 1 October 1997; accepted 20 January 1998
Abstract The presence of glycosaminoglycans was examined in two mollusks (Pulmonates): the terrestrial garden snail, Helix aspersa, and the opishtobranchian sea slug, Aplysia californica and also in the leech (Hirudinea, Erpobdellidae, Nephelopsis obscura). Organs in the garden snail contained predominately chondroitin sulfate and heparan sulfate as a lesser component. The ctenidium of the sea slug contained mainly chondroitin sulfate and a compound which migrated on electrophoresis as heparin but additional data indicated that it could also represent a highly sulfated form of heparan sulfate. The foregut contained only the heparin-like polymer. No standard glycosaminoglycan could be identified in the leech although a polydispersed polysaccharide containing uronic acid, hexosamine and sulfate was shown to be present. A detailed analysis of the heparan sulfate isolated from the garden snail is also given. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Invertebrates; Glycosaminoglycans; Leech; Gastropoda; Mollusks; Hirudinea; Aplysia; Helix; Nephelopsis; Heparan sulfate; Heparin
1. Introduction The glycosaminoglycans (GAGs) constitute a family of closely related, strongly acidic polysaccharides which includes hyaluronic acid, the chondroitin sulfates, dermatan sulfate, heparan sulfate and heparin. They have been well identified and characterized over a period of many years. Though they are widely distributed in the animal kingdom in a variety of organisms from fairly simple to complex, their precise biological function often remains unclear. Comparative biochemistry should be able to provide improved insight into biological activity by examining cells, tissues and organs for
* Corresponding author. Tel.: + 1 801 5821565, ext. 1495; fax: +1 801 5839624. 0305-0491/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0305-0491(98)00044-3
GAG content and location in a variety of organisms not usually investigated. The chondroitin sulfates and heparan sulfates appear to be widely distributed in invertebrates as well as vertebrates, but heparin has been definitively identified only in terrestrial vertebrates and in the class Bivalvia of mollusks [1,2,4,10,11,21,23], suggesting that the biological function of heparin, as such, may not be essential in aquatic vertebrates and most invertebrates. The pioneering work of Rahemtulla and Lovtrup [22,23] and of Dietrich et al. [2,17,18,21] and Mauro et al. [19,20], has greatly contributed to the study of GAG evolution. In this paper we have extended their studies by examining two mollusks (Gastropoda): the snail (Helix aspersa Muller) and the sea slug (Aplysia californica Cooper) and the leech (Hirudinea): Nephelopsis obscura Verrill for the presence of GAGs in general and heparan sulfate and heparin in particular.
692
P. Ho6ingh, A. Linker / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 691–696
Table 1 Yield of glycosaminoglycans in mollusks and leech Starting material (g dry weight)
Mg 100 g−1 dry weight Heparin
Helix aspersa Organ complexes Head – foot Mantle – heart – kidney Liver Organ Isolates Heart Mantle Kidney Liver Aplysia californica Organ complexes Gills – ctenidium Foregut – crop – gizzard Digestive gland – hepatopancreas Albumen – mucous gland Head – foot Nephelopsis obscure Whole animal
Heparan sulfate
Chondroitin sulfate
nd nd nd
nd 7 B0.5
B2 22 B0.5
nd nd nd nd
36 13 5 nd
4 15 71 0.6 163
25 66 B2 nd nd
nd nd nd nd nd
29
nd
64 158 185 0.06 3.95 5.64 37.6
[nd ‘HS’]a
77 72 25 0.02
100 nd nd nd 8 nd
nd, Not detected. Heparan sulfate-like GAG, see Section 3.
a
2. Methods and material
2.1. Materials Pronase was obtained from Calbiochem; papain, cetylpyridinium chloride, nucleases, trichloroacetic acid, testicular hyaluronidase and chondroitinase ABC from Sigma; Sephadex G-150 from Pharmacia; ion-exchange resin Ag1 ×2 chloride from BioRad; cellulose acetate electrophoretic strips from Gelman; and crude Flavobacter enzymes, heparitinase I and heparinase were prepared as previously described [13]. The introduced garden snail, Helix aspersa came from Salt Lake City, UT, the leech, Nephelopsis obscura from the Uinta Mountains, UT, and the sea slug Aplysia californica from the Gulf of California, Sonora, Mexico.
2.2. Analytical procedures Analytical methods have been previously described [14] for carbazole and orcinol uronic acid determinations and for Ehrlichs and indole hexosamine determinations. Sulfate analysis was done by the methods of Terho and Hartiala [28]. Cellulose acetate electrophoresis was adapted from Seno et al. [25], using 0.2 M calcium acetate and Matthews [16], using pyridineformic acid buffer. Identification of specific glycosaminoglycans was obtained by the use of chondroitinase ABC and testicular hyaluronidase
(Sigma), heparinase, heparitinase and crude Flavobacter enzymes [14] (which consists of numerous glycosaminoglycan degrading enzymes) and by nitrous acid deaminution under low pH conditions as described by Shively and Conrad [26]. Molecular size was determined from elution patterns from columns of Sephadex G-150 by Wasteson [31] and Constantopoulos et al. [6].
2.3. Isolation of glycosaminoglycans Glycosaminoglycans were isolated by a general protocol that was previously described [14]. Briefly, the tissue was acetone dried, treated sequentially with papain and pronase, fractionated with cetylpyridinium chloride and treated with trichloroacetic acid and Benedicts reagent [5] and chromatographed on an anion-exchange column Ag 1 ×2, chloride (BioRad). Nuclease was used to remove contaminating nucleic acid as needed. Chondroitin sulfates and hyaluronic acid were removed by chondroitinase ABC and testicular hyaluronidase. The final heparan sulfate/heparin preparation was fractionated with ethanol. This general procedure was followed only if sufficient material was obtained at each step and was shortened if sufficient material was lacking. Two separate preparations were made from Helix aspersa. In the first preparation, the snail was divided into convenient organ systems: (1) The head–foot; (2) the mantle, heart and kidney, also containing portions
P. Ho6ingh, A. Linker / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 691–696
693
Table 2 Analysis of heparan sulfate from the snail organ–mantle–kidney – heart complex Fraction
1.3 1.3 1.5 1.5 1.7
Mc (0 – 30%)d M (30 – 50%) M (0 – 30%) M (30 – 50%) M (30 – 50%)
Yield mg 100 g−1
1 2 3 1 2
Uronic acid %
Sulfate %
By carbazole
By orcinol
29 53 28 36 22
25 36 17 22 15
7 10 9 9 8
Hexosamine %
MW Kav
By Ehrlichsa
By indoleb
19 30 20 24 21
11 8 10 2 4
0.28 –0.65 0.14 –0.64 0.03 –0.41 0.01 –0.23 0.01
Fractions are from salt elution of ion-exchange and fractionation with ethanol. Total. b N-sulfated. c Molarity of N2Cl at which fraction was eluted from BioRad anion exchange column. d % EtOH at which fraction was precipitated. a
of the reproductive and digestive ducts; and (3) the liver, also containing the gonads. In the second preparation, the heart, mantle, kidney and liver were dissected and analyzed separately. Organ groups were examined in the Aplysia and the whole animal was used in the Nephelopsis. The criteria for identification of chondroitin sulfate was the disappearance of this component by testicular hyaluronidase treatment as determined by electrophoretic mobility. Heparan sulfate was identified by its elution from ion-exchange in low molar salts and by disappearance with heparitinase I, the use of crude Flavobacter enzymes after testicular hyaluronidase treatment and by nitrous acid, all as followed by electrophoretic mobility. Heparin was identified by elution from ion-exchange in high molar salts (1.7 – 3.0 M), the use of crude Flavobacter enzymes after testicular hyaluronidase or chondroitinase ABC, and by nitrous acid as followed by electrophoretic mobility. However, ‘heparin’ from the Aplysia was not degraded by heparinase as determined by electrophoretic mobility. Under the conditions used here in cellulose acetate electrophoresis, heparin has a faster mobility than heparan sulfate and both can be distinguished from chondroitin sulfate. If an unknown GAG, as found in the Nephelopsis preparation, was present, analysis would be confounded and, even if present, small amounts of glycosaminoglycans could remain undetected.
3. Results As seen in Table 1 in the garden snail, chondroitin sulfate was shown to be the major GAG component distributed in the heart, mantle and kidney, while heparan sulfate was a minor component present mainly in the heart and mantle. The heparan sulfate was characterized further. Analytical data are shown in Table 2. The spread of elution with increasing salt concentration can be best explained by increasing molecular weight as
determined by Sephadex G-50 chromatography, as the sulfate values as such are not sufficiently different to account for the elution pattern. The heparan sulfate from the mantle, kidney, and heart organ complex contained fairly low total sulfate values. The ratio of sulfate to the total hexosamine varied from 0.35 to 0.43 and the N-sulfated hexosamine content was also low as indicated by the ratios of 0.1 to 0.6 for the indole to Ehrlich’s hexosamine analysis. Both of these criteria identify this GAG as heparan sulfate rather than heparin. The 1.3 and 1.5 M salt fraction (30–50% EtOH subfraction) were degraded by heparitinase but not by nitrous acid. Fig. 1 shows a representative electrophoresis pattern. The 0–30% EtOH subfraction of these two ion exchange chromatography derived isolates showed two heparan sulfate bands on electrophoresis, both of which were sensitive to degradations by heparitinase but only the faster component was degraded by nitrous acid (Fig. 1). The analytical data (Table 2) shows that the 0–30% EtOH fraction contained most of the N-sulfated material as determined by the indole reaction, supporting the nitrous acid data, while the resistance to nitrous acid of the material in the slower band and in the 30–50% EtOH subfraction indicates the presence of hexosamine that is mainly N-acetylated. This shows that the hexosamine component of snail heparan sulfate is mainly N-acetylated unlike the vertebrate GAG where the bulk of the polysaccharide contains 50% N-acetylated and 50% N-sulfated glucosamine. In Aplysia ‘heparin’ was the major GAG in the foregut while chondroitin sulfate was the major component and ‘heparin’ the minor component in the ctenidium (Table 1). We chose the term ‘heparin’ as this compound migrated as heparin on electrophoresis and was completely degraded by HNO2, however, it was not well degraded by heparinase (Fig. 2). It could therefore be a highly sulfated heparan sulfate or be similar to an unusual GAG from an African snail [12] or lobster [9].
694
P. Ho6ingh, A. Linker / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 691–696
Fig. 1. Cellulose acetate electrophoresis patterns of the 1.3–1.5 M NaCl fraction of GAGs isolated from snail mantle. Carried out in 0.2 M Ca acetate and stained with Alcian Blue reagent. Left panel: 30– 50% ethanol subfraction. Lane 1, standards of heparan sulfate, dermatan sulfate, chondroitin 6 sulfate (bottom to top). Lane 2, control GAG fraction; Lane 3, GAG plus hyaluronidase; Lane 4, GAG plus ABCase; Lane 5, GAG plus heparitinase; Lane 6, GAG plus HNO2; Lane 7, standards as in Lane 1. Right panel: 0 – 30% ethanol fraction. Lanes 1 – 7 as above.
From Nephelopsis, a polysaccharide containing uronic acid, hexosamine and sulfate was isolated (Tables 1 and 3). On electrophoresis, this polymer migrated in the area between heparan sulfate and dermatan sulfate. No chondroitin sulfate or heparin appeared to be present. This material was fractionated by ion exchange chromatography and further purified by removing any potential dermatan sulfate by precipitation with Benedict reagent. Analysis of the fractions, which is shown in Table 3, indicated that the polysaccharide is a GAG. However, data obtained with heparitinase are not easily interpretable. As shown in Fig. 2, the 1.3–1.5 combined fractions from the ion exchange procedure appears to be resistant to the enzyme, while the 1.7–3.5 combined fractions showed a change in the migration distance, indicating at least some degradation, implying the presence of a heparan sulfate similar to, but appar-
ently different from, the standard vertebrate polymer but perhaps comparable to lobster HS.
4. Discussion In general, GAGs appear to serve a variety of functions in vertebrates from structural and organizing components of connective tissues to cell-growth factor, cell–cell and cell–matrix interactions. That is, they affect growth, migration, metastasis and functional properties of cells. In addition, heparin and related polymers play a role in anticoagulation and angiogenesis. One question that arises is whether this apparent multiplicity of roles is real or in a variety of cases, at least, is due to experimental constructs rather than biological events.
Fig. 2. Electrophoresis patterns of GAGs isolated from Aplysia gut and from Nephelopsis. Left panel: Aplysia GAG; carried out in Pyridine-formic acid buffer and stained with Alcian Blue reagent. Lane 1, standard heparin; Lane 2, standards of heparan sulfate, dermatan sulfate, and chondroitin 6-sulfate (as in Fig. 1); Lane 3, control GAG fraction; Lane 4, GAG plus heparinase; Lane 5, GAG plus heparitinase; Lane 6, GAG plus HNO2; Lane 7, standard heparin; Lane 8, standards as in Lane 2. Right panel: Nephelopsis polysaccharide; carried out in 0.2 M Ca acetate, stained with Alcian Blue reagent. Lane 1, standards of heparan sulfate, dermatan sulfate, chondroitin 6-sulfate (as in left panel); Lane 2, 0.5–1.0 M NaCl fraction of polysacharide; Lane 3, 0.5–1.0 M NaCl fraction plus heparitinase; Lane 4, 1.3 – 1.5 M salt fraction of polysaccharide; Lane 5, 1.3– 1.5 M NaCl fraction plus heparitinase; Lane 6, 1.7–3.5 M NaCl fraction of polysaccharide plus heparitinase; Lane 7, 1.7 – 3.5 M NaCl fraction; Lane 8, standards as in Lane 1.
P. Ho6ingh, A. Linker / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 691–696
695
Table 3 Analysis of the leech ‘GAG’ fractions Fraction
0.5–1.0 M 1.3–1.5 M 1.7–3.5 M
Percent Uronic acid by Carbazole
Hexosamine by Ehrlichs
Sulfate
Hexose by anthrone
14 23 18
21 19 10
0 6.5 1.5
9.6 6 4
It is, therefore, tempting to use a comparative approach to study biological functions in organisms such as invertebrates to arrive at a better understanding of a basic and consistent role that these ubiquitous polysaccharides or their proteoglycans play in biological systems. GAGs are present in a few species of bacteria [3,27,29] and as a group are widely distributed in invertebrates and vertebrates. Chondroitin sulfates have been shown to be present in crustaceans [2,9], sea cucumbers [30] and various mollusks [2,17,18]. Dermatan sulfate has been isolated from an ascidian [19] and a sea urchin [15]. Heparan sulfate appears to be present in most of the organisms mentioned above, while heparin occurs much more rarely in invertebrates, mainly in mollusks [1,4,21]. Even in vertebrates, its distribution is fairly limited [10]. GAGs with characteristics which make it difficult to assign them to a specific member of the well-defined vertebrate family of these polysaccharides have been described for lobsters [9], mussels [11], an African snail [12] and others. In addition, invertebrates that lack GAGs synthesize sulfated polysaccharides such as spirulan from bivalve mulluscs [1], horatin from annelids [22], sulfated Lgalactans from Styela [20] and Kakelokelose, a sulfated mannan from another ascidian [24]. These polysaccharides, showing some structural features similar to GAGs, may well carry out the same or similar critical biological functions that GAGs do. We have shown here that chondroitin sulfate is the major GAG present in heart, mantle and kidney of Helix aspersa. Heparan sulfate is present as a minor component mainly in heart and mantle. This heparan sulfate differs somewhat from the major mammalian GAG, i.e. the total sulfate content is lower and there is considerably less N-sulfated and more N-acetylated hexoxamine, as shown by the analytical data and resistance to HNO2 treatment of the major component. In Aplysia ‘heparin’ appeared to be the major GAG in the foregut and a minor component in the ctenidium. The resistance of this heparin-like polymer to heparinase, however, indicates that it may be a highly sulfated heparan sulfate or belong to the category of heparin– heparan sulfate-like GAGs mentioned above. Chondroitin sulfate was found to be the major GAG component in the ctenidium.
This variable distribution of the polymers discussed in the different organ systems may imply some specificity of function. The polysaccharide isolated from the leech appears to be a GAG as indicated by the analytical data (Table 3) and behavior on electrophoresis (Fig. 2). However, sulfate content was fairly low; it was not degraded by chondroitinase ABC and degradation by heparitinase seemed uncertain. Therefore, it does not represent ‘standard’ GAG but may belong to the group of unusual invertebrate polymers difficult to classify. Not enough material for further characterization was available. These results extend the basic findings of Rahemtulla and Lovtrup in which no standard heparan sulfate or heparin was found in annelids and no chondroitin sulphate in Oligochaeta and Hirudinea. Dietrich et al. [2] had suggested, however, that the heparan sulfate is found in Pheretima hawayana, an annelid. By use of 185 ribosomal RNA sequences, Field et al. [7] re-examined the phylogeny of the animal kingdom and drew preliminary conclusions that annelids and mollusks were closely related and more distant from echinaderms chordates and arthropods. From the data available on GAGs, in particular heparin and heparan sulfate, we would suggest that these two polymers are associated with organisms of greater complexity, as represented by the presence of skeletons and complex circulatory and digestive systems. This may explain the presence of heparan sulfate and heparin in arthropods and mollusks. In general, the wide though inconsistent distribution of GAGs and related polymers in invertebrates indicates an essential biological role related to their common structural features, in particular their highly anionic properties and their stability. Several potential functions have been suggested, such as: sequestration of calcium or inactivation of toxic amines [11], a role in inflammatory processes [21], cation binding, in particular copper, antidessicant and antibacterial activities [12], coping with high salinity in the environment [17] and neurite outgrowth [8]. As far as potential relations to vertebrate functions is concerned, it should be noted that the invertebrate polymers, even when the characteristic structural features of vertebrate GAGs are present, a variety of modifications do occur. This is the case for chondroitin
696
P. Ho6ingh, A. Linker / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 691–696
sulfate, where the chains are substituted with fucose moieties [30], for dermatan sulfate showing differences in sulfation patterns [20], for the unusual heparin-like compound of African snails [12], and even for clam heparin which contains an unusual disaccharide unit [21]. Therefore, though there may be a common pattern of biological activity in invertebrates, one may have to concede that by and large this may be quite distinct from function in vertebrates.
Acknowledgements This work was supported in part by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.
References [1] Burson SL, Fahrenbach MJ, Frommhagen LH, Riccardi BA, Brown RA, Brockman JA, Lewry HV, Stokstad ELR. Isolation and purification of mactins, heparin-like anticoagulants from Mollusca. J Am Chem Soc 1956;78:5874–8. [2] Cassaro CMF, Dietrich CP. Distribution of sulfated mucopolysaccharides in invertebrates. J Biol Chem 1977;252:2254 – 61. [3] Chen JC-R, Stephens RS. Trachoma and LGV biovars of Chlamydia trachomatis share the same glycosaminoglycan-dependent mechanism for infection of eukaryotic cells. Mol Microbiol 1994;11:501 – 7. [4] Cifonelli JA, Mathews MD. A comparison of the chemical structures of mactins with mammalian heparins. Connect Tissue Res 1972;1:121 – 30. [5] Cifonelli JA, Roden L. Dermatan sulfates. Biochem Prep 1968;12:5 – 12. [6] Constantopoulos G, DeKaban AS, Carroll WR. Determination of molecular weight distribution of acid mucopolysaccharides by Sephadex gel filtration. Anal Biochem 1969;31:59–70. [7] Field KG, Olsen GJ, Lane DJ, Giovannoni SJ, Ghiselin MT, Raff EC, Pace NR, Raff RA. Molecular phylogeny of the animal kingdom. Science 1988;238:748–53. [8] Har-El R, Tanzer ML. Evolution of the extracellular matrix in invertebrates. FASEB J 1993;7:1115–23. [9] Hovingh P, Linker A. An unusual heparan sulfate isolated from lobsters (Homarus Americanus). J Biol Chem 1982;257:9840 – 1. [10] Hovingh P, Linker A. Biological implications of the structural, antithrombin affinity and anticoagulant activity relationships among vertebrate heparins and heparan sulfates. Biochem J 1986;237:573 – 81. [11] Hovingh P, Linker A. Glycosaminoglycans in Anodonta californiensis, a fresh water mussel. Biol Bull 1993;185:263– 76. [12] Kim YS, Jo YY, Chang IM, Toshihiko T, Youmie P, Linhardt RJ. A new glycosaminoglycan from the giant African snail Achatina fulica. J Biol Chem 1996;271:11750–5. [13] Linker A, Hovingh P. Heparinase and heparitinase from flavobacteria. Methods Enzymol 1972;28:902–11.
.
[14] Linker A, Hovingh P. The heparitin sulfates (heparan sulfates). Carbohydr Res 1973;29:41 – 62. [15] Manouras A, Karamanos NK, Tsegenidis T, Antonopoulos CA. Isolation and chemical characterization of two acid carbohydrates from the sea urchin shell, extraction and fractionation of their protein complexes. Comp Biochem Physiol 1991;99B:119– 24. [16] Mathews MB. Acid strength of carboxyl groups in isomeric chondroitin sulfates. Biochim Biophys Acta 1961;48:402–3. [17] Nader HB, Medeiros MGL, Paiva JF, Paiva VMP, Jeronimo SMB, Ferreira TMPC, Dietrich CP. A correlation between the sulfated glycosaminoglycan concentration and degree of salinity of the habitat in fifteen species of the classes crustacea, pelecypoda and gastropoda. Comp Biochem Physiol 1983;76B:433–6. [18] Nader HB, Ferreira TMFC, Paive JF, Medeiros MGL, Jeronimo SMB, Paiva VMP, Dietrich CP. Isolation and structural studies of heparan sulfates and chondroitin sulfates from three species of mollusks. J Biol Chem 1984;259:1431– 5. [19] Pavao MSG, Mourao PAS, Mulloy B, Tollefson MD. A unique dermatan sulfate-like glycosaminoglycan from Ascidian. J Biol Chem 1995;270:21027– 31036. [20] Pavao MSG, Rodrigues AM, Mourao PAS. Acidic polysaccharides of the Ascidian styeloplicata. Biosynthetic studies of the sulfated L-galactans of the tunic and preliminary characterization of a dermatan sulfate-like polymer in body tissues. Biochem Biophys Acta 1994;1199:229– 37. [21] Pealer G, Danielsson A, Bjork I, Lindahl U, Nader HB, Dietrich CP. Structure and antithrombin-binding properties of heparin isolated from the clam Anomalacardia brasiliana and Ti6ela mactroides. J Biol Chem 1987;262:11413– 21. [22] Rahemtulla F, Lovtrup S. The comparative biochemistry of invertebrate mucopolysaccharides-II Nematoda annelida. Comp Biochem Physiol 1974;49B:639 – 46. [23] Rahemtulla F, Lovtrup S. The comparative biochemistry of invertebrate mucopolysaccharides-III Oligochaete and Hirudinea. Comp Biochem Physiol 1975;50B:627 – 9. [24] Riccio R, Kinnel RB, Bifulco G, Scheuer PJ. Kakelokelose a sulfated mannose polysaccharide with anti-HIV activity from the Pacific tunicate didemnum molle. Tetrahedron Lett 1996;37:1979 – 82. [25] Seno N, Anno K, Nagase K, Saito S. Improved methods for electrophoretic separation and rapid quantitation of isomeric chondroitin sulfates on cellulose acetate strips. Anal Biochem 1970;37:192 – 202. [26] Shively JE, Conrad KE. Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry 1976;15:3932 – 42. [27] Stoolmiller AC, Dorfman A. The biosynthesis of hyaluronic acid by streptococcus. J Biol Chem 1969;244:236 – 46. [28] Terho TT, Hartiola K. Method for determination of the sulfate content of glycosamino-glycans. Anal Biochem 1971;41:471–6. [29] Vann WF, Schmidt MA, Jann B, Jann K. The structure of the capsular polysaccharide (K5 antigen) of urinary tract infective Escherichia coli 010:K5:H4. A polymer similar to desulfo-heparin. Eur J Biochem 1981;116:359 – 64. [30] Vieira RP, Mulloy B, Maourao PAS. Structure of a fucosebranched chondroitin sulfate from sea cucumber. J Biol Chem 1991;266:13530– 6. [31] Wasteson A. A method for the determination of the molecular weight and molecular weight distribution of chondroitin sulfate. J Chromatogr 1971;59:87 – 97.
Glycosaminoglycans in two mollusks, Aplysia californica and Helix aspersa, and in the leech, Nephelopsis obscura Peter Hovingh a, Alfred Linker b,* b
a Department of Biochemistry and Pathology, Uni6ersity of Utah, Salt lake City, UT 84132, USA Veterans Affairs Medical Center, Research Ser6ice (151E), 500 Foothill, Salt Lake City, UT 84148, USA
Received 1 October 1997; accepted 20 January 1998
Abstract The presence of glycosaminoglycans was examined in two mollusks (Pulmonates): the terrestrial garden snail, Helix aspersa, and the opishtobranchian sea slug, Aplysia californica and also in the leech (Hirudinea, Erpobdellidae, Nephelopsis obscura). Organs in the garden snail contained predominately chondroitin sulfate and heparan sulfate as a lesser component. The ctenidium of the sea slug contained mainly chondroitin sulfate and a compound which migrated on electrophoresis as heparin but additional data indicated that it could also represent a highly sulfated form of heparan sulfate. The foregut contained only the heparin-like polymer. No standard glycosaminoglycan could be identified in the leech although a polydispersed polysaccharide containing uronic acid, hexosamine and sulfate was shown to be present. A detailed analysis of the heparan sulfate isolated from the garden snail is also given. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Invertebrates; Glycosaminoglycans; Leech; Gastropoda; Mollusks; Hirudinea; Aplysia; Helix; Nephelopsis; Heparan sulfate; Heparin
1. Introduction The glycosaminoglycans (GAGs) constitute a family of closely related, strongly acidic polysaccharides which includes hyaluronic acid, the chondroitin sulfates, dermatan sulfate, heparan sulfate and heparin. They have been well identified and characterized over a period of many years. Though they are widely distributed in the animal kingdom in a variety of organisms from fairly simple to complex, their precise biological function often remains unclear. Comparative biochemistry should be able to provide improved insight into biological activity by examining cells, tissues and organs for
* Corresponding author. Tel.: + 1 801 5821565, ext. 1495; fax: +1 801 5839624. 0305-0491/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0305-0491(98)00044-3
GAG content and location in a variety of organisms not usually investigated. The chondroitin sulfates and heparan sulfates appear to be widely distributed in invertebrates as well as vertebrates, but heparin has been definitively identified only in terrestrial vertebrates and in the class Bivalvia of mollusks [1,2,4,10,11,21,23], suggesting that the biological function of heparin, as such, may not be essential in aquatic vertebrates and most invertebrates. The pioneering work of Rahemtulla and Lovtrup [22,23] and of Dietrich et al. [2,17,18,21] and Mauro et al. [19,20], has greatly contributed to the study of GAG evolution. In this paper we have extended their studies by examining two mollusks (Gastropoda): the snail (Helix aspersa Muller) and the sea slug (Aplysia californica Cooper) and the leech (Hirudinea): Nephelopsis obscura Verrill for the presence of GAGs in general and heparan sulfate and heparin in particular.
692
P. Ho6ingh, A. Linker / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 691–696
Table 1 Yield of glycosaminoglycans in mollusks and leech Starting material (g dry weight)
Mg 100 g−1 dry weight Heparin
Helix aspersa Organ complexes Head – foot Mantle – heart – kidney Liver Organ Isolates Heart Mantle Kidney Liver Aplysia californica Organ complexes Gills – ctenidium Foregut – crop – gizzard Digestive gland – hepatopancreas Albumen – mucous gland Head – foot Nephelopsis obscure Whole animal
Heparan sulfate
Chondroitin sulfate
nd nd nd
nd 7 B0.5
B2 22 B0.5
nd nd nd nd
36 13 5 nd
4 15 71 0.6 163
25 66 B2 nd nd
nd nd nd nd nd
29
nd
64 158 185 0.06 3.95 5.64 37.6
[nd ‘HS’]a
77 72 25 0.02
100 nd nd nd 8 nd
nd, Not detected. Heparan sulfate-like GAG, see Section 3.
a
2. Methods and material
2.1. Materials Pronase was obtained from Calbiochem; papain, cetylpyridinium chloride, nucleases, trichloroacetic acid, testicular hyaluronidase and chondroitinase ABC from Sigma; Sephadex G-150 from Pharmacia; ion-exchange resin Ag1 ×2 chloride from BioRad; cellulose acetate electrophoretic strips from Gelman; and crude Flavobacter enzymes, heparitinase I and heparinase were prepared as previously described [13]. The introduced garden snail, Helix aspersa came from Salt Lake City, UT, the leech, Nephelopsis obscura from the Uinta Mountains, UT, and the sea slug Aplysia californica from the Gulf of California, Sonora, Mexico.
2.2. Analytical procedures Analytical methods have been previously described [14] for carbazole and orcinol uronic acid determinations and for Ehrlichs and indole hexosamine determinations. Sulfate analysis was done by the methods of Terho and Hartiala [28]. Cellulose acetate electrophoresis was adapted from Seno et al. [25], using 0.2 M calcium acetate and Matthews [16], using pyridineformic acid buffer. Identification of specific glycosaminoglycans was obtained by the use of chondroitinase ABC and testicular hyaluronidase
(Sigma), heparinase, heparitinase and crude Flavobacter enzymes [14] (which consists of numerous glycosaminoglycan degrading enzymes) and by nitrous acid deaminution under low pH conditions as described by Shively and Conrad [26]. Molecular size was determined from elution patterns from columns of Sephadex G-150 by Wasteson [31] and Constantopoulos et al. [6].
2.3. Isolation of glycosaminoglycans Glycosaminoglycans were isolated by a general protocol that was previously described [14]. Briefly, the tissue was acetone dried, treated sequentially with papain and pronase, fractionated with cetylpyridinium chloride and treated with trichloroacetic acid and Benedicts reagent [5] and chromatographed on an anion-exchange column Ag 1 ×2, chloride (BioRad). Nuclease was used to remove contaminating nucleic acid as needed. Chondroitin sulfates and hyaluronic acid were removed by chondroitinase ABC and testicular hyaluronidase. The final heparan sulfate/heparin preparation was fractionated with ethanol. This general procedure was followed only if sufficient material was obtained at each step and was shortened if sufficient material was lacking. Two separate preparations were made from Helix aspersa. In the first preparation, the snail was divided into convenient organ systems: (1) The head–foot; (2) the mantle, heart and kidney, also containing portions
P. Ho6ingh, A. Linker / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 691–696
693
Table 2 Analysis of heparan sulfate from the snail organ–mantle–kidney – heart complex Fraction
1.3 1.3 1.5 1.5 1.7
Mc (0 – 30%)d M (30 – 50%) M (0 – 30%) M (30 – 50%) M (30 – 50%)
Yield mg 100 g−1
1 2 3 1 2
Uronic acid %
Sulfate %
By carbazole
By orcinol
29 53 28 36 22
25 36 17 22 15
7 10 9 9 8
Hexosamine %
MW Kav
By Ehrlichsa
By indoleb
19 30 20 24 21
11 8 10 2 4
0.28 –0.65 0.14 –0.64 0.03 –0.41 0.01 –0.23 0.01
Fractions are from salt elution of ion-exchange and fractionation with ethanol. Total. b N-sulfated. c Molarity of N2Cl at which fraction was eluted from BioRad anion exchange column. d % EtOH at which fraction was precipitated. a
of the reproductive and digestive ducts; and (3) the liver, also containing the gonads. In the second preparation, the heart, mantle, kidney and liver were dissected and analyzed separately. Organ groups were examined in the Aplysia and the whole animal was used in the Nephelopsis. The criteria for identification of chondroitin sulfate was the disappearance of this component by testicular hyaluronidase treatment as determined by electrophoretic mobility. Heparan sulfate was identified by its elution from ion-exchange in low molar salts and by disappearance with heparitinase I, the use of crude Flavobacter enzymes after testicular hyaluronidase treatment and by nitrous acid, all as followed by electrophoretic mobility. Heparin was identified by elution from ion-exchange in high molar salts (1.7 – 3.0 M), the use of crude Flavobacter enzymes after testicular hyaluronidase or chondroitinase ABC, and by nitrous acid as followed by electrophoretic mobility. However, ‘heparin’ from the Aplysia was not degraded by heparinase as determined by electrophoretic mobility. Under the conditions used here in cellulose acetate electrophoresis, heparin has a faster mobility than heparan sulfate and both can be distinguished from chondroitin sulfate. If an unknown GAG, as found in the Nephelopsis preparation, was present, analysis would be confounded and, even if present, small amounts of glycosaminoglycans could remain undetected.
3. Results As seen in Table 1 in the garden snail, chondroitin sulfate was shown to be the major GAG component distributed in the heart, mantle and kidney, while heparan sulfate was a minor component present mainly in the heart and mantle. The heparan sulfate was characterized further. Analytical data are shown in Table 2. The spread of elution with increasing salt concentration can be best explained by increasing molecular weight as
determined by Sephadex G-50 chromatography, as the sulfate values as such are not sufficiently different to account for the elution pattern. The heparan sulfate from the mantle, kidney, and heart organ complex contained fairly low total sulfate values. The ratio of sulfate to the total hexosamine varied from 0.35 to 0.43 and the N-sulfated hexosamine content was also low as indicated by the ratios of 0.1 to 0.6 for the indole to Ehrlich’s hexosamine analysis. Both of these criteria identify this GAG as heparan sulfate rather than heparin. The 1.3 and 1.5 M salt fraction (30–50% EtOH subfraction) were degraded by heparitinase but not by nitrous acid. Fig. 1 shows a representative electrophoresis pattern. The 0–30% EtOH subfraction of these two ion exchange chromatography derived isolates showed two heparan sulfate bands on electrophoresis, both of which were sensitive to degradations by heparitinase but only the faster component was degraded by nitrous acid (Fig. 1). The analytical data (Table 2) shows that the 0–30% EtOH fraction contained most of the N-sulfated material as determined by the indole reaction, supporting the nitrous acid data, while the resistance to nitrous acid of the material in the slower band and in the 30–50% EtOH subfraction indicates the presence of hexosamine that is mainly N-acetylated. This shows that the hexosamine component of snail heparan sulfate is mainly N-acetylated unlike the vertebrate GAG where the bulk of the polysaccharide contains 50% N-acetylated and 50% N-sulfated glucosamine. In Aplysia ‘heparin’ was the major GAG in the foregut while chondroitin sulfate was the major component and ‘heparin’ the minor component in the ctenidium (Table 1). We chose the term ‘heparin’ as this compound migrated as heparin on electrophoresis and was completely degraded by HNO2, however, it was not well degraded by heparinase (Fig. 2). It could therefore be a highly sulfated heparan sulfate or be similar to an unusual GAG from an African snail [12] or lobster [9].
694
P. Ho6ingh, A. Linker / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 691–696
Fig. 1. Cellulose acetate electrophoresis patterns of the 1.3–1.5 M NaCl fraction of GAGs isolated from snail mantle. Carried out in 0.2 M Ca acetate and stained with Alcian Blue reagent. Left panel: 30– 50% ethanol subfraction. Lane 1, standards of heparan sulfate, dermatan sulfate, chondroitin 6 sulfate (bottom to top). Lane 2, control GAG fraction; Lane 3, GAG plus hyaluronidase; Lane 4, GAG plus ABCase; Lane 5, GAG plus heparitinase; Lane 6, GAG plus HNO2; Lane 7, standards as in Lane 1. Right panel: 0 – 30% ethanol fraction. Lanes 1 – 7 as above.
From Nephelopsis, a polysaccharide containing uronic acid, hexosamine and sulfate was isolated (Tables 1 and 3). On electrophoresis, this polymer migrated in the area between heparan sulfate and dermatan sulfate. No chondroitin sulfate or heparin appeared to be present. This material was fractionated by ion exchange chromatography and further purified by removing any potential dermatan sulfate by precipitation with Benedict reagent. Analysis of the fractions, which is shown in Table 3, indicated that the polysaccharide is a GAG. However, data obtained with heparitinase are not easily interpretable. As shown in Fig. 2, the 1.3–1.5 combined fractions from the ion exchange procedure appears to be resistant to the enzyme, while the 1.7–3.5 combined fractions showed a change in the migration distance, indicating at least some degradation, implying the presence of a heparan sulfate similar to, but appar-
ently different from, the standard vertebrate polymer but perhaps comparable to lobster HS.
4. Discussion In general, GAGs appear to serve a variety of functions in vertebrates from structural and organizing components of connective tissues to cell-growth factor, cell–cell and cell–matrix interactions. That is, they affect growth, migration, metastasis and functional properties of cells. In addition, heparin and related polymers play a role in anticoagulation and angiogenesis. One question that arises is whether this apparent multiplicity of roles is real or in a variety of cases, at least, is due to experimental constructs rather than biological events.
Fig. 2. Electrophoresis patterns of GAGs isolated from Aplysia gut and from Nephelopsis. Left panel: Aplysia GAG; carried out in Pyridine-formic acid buffer and stained with Alcian Blue reagent. Lane 1, standard heparin; Lane 2, standards of heparan sulfate, dermatan sulfate, and chondroitin 6-sulfate (as in Fig. 1); Lane 3, control GAG fraction; Lane 4, GAG plus heparinase; Lane 5, GAG plus heparitinase; Lane 6, GAG plus HNO2; Lane 7, standard heparin; Lane 8, standards as in Lane 2. Right panel: Nephelopsis polysaccharide; carried out in 0.2 M Ca acetate, stained with Alcian Blue reagent. Lane 1, standards of heparan sulfate, dermatan sulfate, chondroitin 6-sulfate (as in left panel); Lane 2, 0.5–1.0 M NaCl fraction of polysacharide; Lane 3, 0.5–1.0 M NaCl fraction plus heparitinase; Lane 4, 1.3 – 1.5 M salt fraction of polysaccharide; Lane 5, 1.3– 1.5 M NaCl fraction plus heparitinase; Lane 6, 1.7–3.5 M NaCl fraction of polysaccharide plus heparitinase; Lane 7, 1.7 – 3.5 M NaCl fraction; Lane 8, standards as in Lane 1.
P. Ho6ingh, A. Linker / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 691–696
695
Table 3 Analysis of the leech ‘GAG’ fractions Fraction
0.5–1.0 M 1.3–1.5 M 1.7–3.5 M
Percent Uronic acid by Carbazole
Hexosamine by Ehrlichs
Sulfate
Hexose by anthrone
14 23 18
21 19 10
0 6.5 1.5
9.6 6 4
It is, therefore, tempting to use a comparative approach to study biological functions in organisms such as invertebrates to arrive at a better understanding of a basic and consistent role that these ubiquitous polysaccharides or their proteoglycans play in biological systems. GAGs are present in a few species of bacteria [3,27,29] and as a group are widely distributed in invertebrates and vertebrates. Chondroitin sulfates have been shown to be present in crustaceans [2,9], sea cucumbers [30] and various mollusks [2,17,18]. Dermatan sulfate has been isolated from an ascidian [19] and a sea urchin [15]. Heparan sulfate appears to be present in most of the organisms mentioned above, while heparin occurs much more rarely in invertebrates, mainly in mollusks [1,4,21]. Even in vertebrates, its distribution is fairly limited [10]. GAGs with characteristics which make it difficult to assign them to a specific member of the well-defined vertebrate family of these polysaccharides have been described for lobsters [9], mussels [11], an African snail [12] and others. In addition, invertebrates that lack GAGs synthesize sulfated polysaccharides such as spirulan from bivalve mulluscs [1], horatin from annelids [22], sulfated Lgalactans from Styela [20] and Kakelokelose, a sulfated mannan from another ascidian [24]. These polysaccharides, showing some structural features similar to GAGs, may well carry out the same or similar critical biological functions that GAGs do. We have shown here that chondroitin sulfate is the major GAG present in heart, mantle and kidney of Helix aspersa. Heparan sulfate is present as a minor component mainly in heart and mantle. This heparan sulfate differs somewhat from the major mammalian GAG, i.e. the total sulfate content is lower and there is considerably less N-sulfated and more N-acetylated hexoxamine, as shown by the analytical data and resistance to HNO2 treatment of the major component. In Aplysia ‘heparin’ appeared to be the major GAG in the foregut and a minor component in the ctenidium. The resistance of this heparin-like polymer to heparinase, however, indicates that it may be a highly sulfated heparan sulfate or belong to the category of heparin– heparan sulfate-like GAGs mentioned above. Chondroitin sulfate was found to be the major GAG component in the ctenidium.
This variable distribution of the polymers discussed in the different organ systems may imply some specificity of function. The polysaccharide isolated from the leech appears to be a GAG as indicated by the analytical data (Table 3) and behavior on electrophoresis (Fig. 2). However, sulfate content was fairly low; it was not degraded by chondroitinase ABC and degradation by heparitinase seemed uncertain. Therefore, it does not represent ‘standard’ GAG but may belong to the group of unusual invertebrate polymers difficult to classify. Not enough material for further characterization was available. These results extend the basic findings of Rahemtulla and Lovtrup in which no standard heparan sulfate or heparin was found in annelids and no chondroitin sulphate in Oligochaeta and Hirudinea. Dietrich et al. [2] had suggested, however, that the heparan sulfate is found in Pheretima hawayana, an annelid. By use of 185 ribosomal RNA sequences, Field et al. [7] re-examined the phylogeny of the animal kingdom and drew preliminary conclusions that annelids and mollusks were closely related and more distant from echinaderms chordates and arthropods. From the data available on GAGs, in particular heparin and heparan sulfate, we would suggest that these two polymers are associated with organisms of greater complexity, as represented by the presence of skeletons and complex circulatory and digestive systems. This may explain the presence of heparan sulfate and heparin in arthropods and mollusks. In general, the wide though inconsistent distribution of GAGs and related polymers in invertebrates indicates an essential biological role related to their common structural features, in particular their highly anionic properties and their stability. Several potential functions have been suggested, such as: sequestration of calcium or inactivation of toxic amines [11], a role in inflammatory processes [21], cation binding, in particular copper, antidessicant and antibacterial activities [12], coping with high salinity in the environment [17] and neurite outgrowth [8]. As far as potential relations to vertebrate functions is concerned, it should be noted that the invertebrate polymers, even when the characteristic structural features of vertebrate GAGs are present, a variety of modifications do occur. This is the case for chondroitin
696
P. Ho6ingh, A. Linker / Comparati6e Biochemistry and Physiology, Part B 119 (1998) 691–696
sulfate, where the chains are substituted with fucose moieties [30], for dermatan sulfate showing differences in sulfation patterns [20], for the unusual heparin-like compound of African snails [12], and even for clam heparin which contains an unusual disaccharide unit [21]. Therefore, though there may be a common pattern of biological activity in invertebrates, one may have to concede that by and large this may be quite distinct from function in vertebrates.
Acknowledgements This work was supported in part by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.
References [1] Burson SL, Fahrenbach MJ, Frommhagen LH, Riccardi BA, Brown RA, Brockman JA, Lewry HV, Stokstad ELR. Isolation and purification of mactins, heparin-like anticoagulants from Mollusca. J Am Chem Soc 1956;78:5874–8. [2] Cassaro CMF, Dietrich CP. Distribution of sulfated mucopolysaccharides in invertebrates. J Biol Chem 1977;252:2254 – 61. [3] Chen JC-R, Stephens RS. Trachoma and LGV biovars of Chlamydia trachomatis share the same glycosaminoglycan-dependent mechanism for infection of eukaryotic cells. Mol Microbiol 1994;11:501 – 7. [4] Cifonelli JA, Mathews MD. A comparison of the chemical structures of mactins with mammalian heparins. Connect Tissue Res 1972;1:121 – 30. [5] Cifonelli JA, Roden L. Dermatan sulfates. Biochem Prep 1968;12:5 – 12. [6] Constantopoulos G, DeKaban AS, Carroll WR. Determination of molecular weight distribution of acid mucopolysaccharides by Sephadex gel filtration. Anal Biochem 1969;31:59–70. [7] Field KG, Olsen GJ, Lane DJ, Giovannoni SJ, Ghiselin MT, Raff EC, Pace NR, Raff RA. Molecular phylogeny of the animal kingdom. Science 1988;238:748–53. [8] Har-El R, Tanzer ML. Evolution of the extracellular matrix in invertebrates. FASEB J 1993;7:1115–23. [9] Hovingh P, Linker A. An unusual heparan sulfate isolated from lobsters (Homarus Americanus). J Biol Chem 1982;257:9840 – 1. [10] Hovingh P, Linker A. Biological implications of the structural, antithrombin affinity and anticoagulant activity relationships among vertebrate heparins and heparan sulfates. Biochem J 1986;237:573 – 81. [11] Hovingh P, Linker A. Glycosaminoglycans in Anodonta californiensis, a fresh water mussel. Biol Bull 1993;185:263– 76. [12] Kim YS, Jo YY, Chang IM, Toshihiko T, Youmie P, Linhardt RJ. A new glycosaminoglycan from the giant African snail Achatina fulica. J Biol Chem 1996;271:11750–5. [13] Linker A, Hovingh P. Heparinase and heparitinase from flavobacteria. Methods Enzymol 1972;28:902–11.
.
[14] Linker A, Hovingh P. The heparitin sulfates (heparan sulfates). Carbohydr Res 1973;29:41 – 62. [15] Manouras A, Karamanos NK, Tsegenidis T, Antonopoulos CA. Isolation and chemical characterization of two acid carbohydrates from the sea urchin shell, extraction and fractionation of their protein complexes. Comp Biochem Physiol 1991;99B:119– 24. [16] Mathews MB. Acid strength of carboxyl groups in isomeric chondroitin sulfates. Biochim Biophys Acta 1961;48:402–3. [17] Nader HB, Medeiros MGL, Paiva JF, Paiva VMP, Jeronimo SMB, Ferreira TMPC, Dietrich CP. A correlation between the sulfated glycosaminoglycan concentration and degree of salinity of the habitat in fifteen species of the classes crustacea, pelecypoda and gastropoda. Comp Biochem Physiol 1983;76B:433–6. [18] Nader HB, Ferreira TMFC, Paive JF, Medeiros MGL, Jeronimo SMB, Paiva VMP, Dietrich CP. Isolation and structural studies of heparan sulfates and chondroitin sulfates from three species of mollusks. J Biol Chem 1984;259:1431– 5. [19] Pavao MSG, Mourao PAS, Mulloy B, Tollefson MD. A unique dermatan sulfate-like glycosaminoglycan from Ascidian. J Biol Chem 1995;270:21027– 31036. [20] Pavao MSG, Rodrigues AM, Mourao PAS. Acidic polysaccharides of the Ascidian styeloplicata. Biosynthetic studies of the sulfated L-galactans of the tunic and preliminary characterization of a dermatan sulfate-like polymer in body tissues. Biochem Biophys Acta 1994;1199:229– 37. [21] Pealer G, Danielsson A, Bjork I, Lindahl U, Nader HB, Dietrich CP. Structure and antithrombin-binding properties of heparin isolated from the clam Anomalacardia brasiliana and Ti6ela mactroides. J Biol Chem 1987;262:11413– 21. [22] Rahemtulla F, Lovtrup S. The comparative biochemistry of invertebrate mucopolysaccharides-II Nematoda annelida. Comp Biochem Physiol 1974;49B:639 – 46. [23] Rahemtulla F, Lovtrup S. The comparative biochemistry of invertebrate mucopolysaccharides-III Oligochaete and Hirudinea. Comp Biochem Physiol 1975;50B:627 – 9. [24] Riccio R, Kinnel RB, Bifulco G, Scheuer PJ. Kakelokelose a sulfated mannose polysaccharide with anti-HIV activity from the Pacific tunicate didemnum molle. Tetrahedron Lett 1996;37:1979 – 82. [25] Seno N, Anno K, Nagase K, Saito S. Improved methods for electrophoretic separation and rapid quantitation of isomeric chondroitin sulfates on cellulose acetate strips. Anal Biochem 1970;37:192 – 202. [26] Shively JE, Conrad KE. Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry 1976;15:3932 – 42. [27] Stoolmiller AC, Dorfman A. The biosynthesis of hyaluronic acid by streptococcus. J Biol Chem 1969;244:236 – 46. [28] Terho TT, Hartiola K. Method for determination of the sulfate content of glycosamino-glycans. Anal Biochem 1971;41:471–6. [29] Vann WF, Schmidt MA, Jann B, Jann K. The structure of the capsular polysaccharide (K5 antigen) of urinary tract infective Escherichia coli 010:K5:H4. A polymer similar to desulfo-heparin. Eur J Biochem 1981;116:359 – 64. [30] Vieira RP, Mulloy B, Maourao PAS. Structure of a fucosebranched chondroitin sulfate from sea cucumber. J Biol Chem 1991;266:13530– 6. [31] Wasteson A. A method for the determination of the molecular weight and molecular weight distribution of chondroitin sulfate. J Chromatogr 1971;59:87 – 97.