Evidence for glycosaminoglycans as a major component of trail mucus from the terrestrial slug, Arion ater L.

Comp. Biochem. Physiol. Vol. 104B,No. 3, pp. 455--468,1993

0305-0491/93$6.00+ 0.00 PergamonPress Ltd

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EVIDENCE FOR GLYCOSAMINOGLYCANS AS A MAJOR COMPONENT OF TRAIL MUCUS FROM THE TERRESTRIAL SLUG, A R I O N A TER L. JANET M. COTTRELL,*1" IAN F. HENDERSON,~JOHN A. PICKETT§ and DENISJ. WRIGHT'~ fDepartment of Biology, Imperial College at Silwood Park, Ascot, Berkshire SL5 7PY, U.K.; ~tDepartment of Entomology and Nematology, and §Department of Insecticides and Fungicides, Institute of Arable Crops Research, Rothamsted Experimental Research Station, Harpenden, Hertfordshire AL5 2JQ, U.K. (Received 8 September 1992; accepted 16 October 1992) Abstract--l. SDS-PAGE of trail mucus from seven species of terrestrial mollusc revealed few similarities

in the pattern of protein or carbohydrate-containing bands. 2. The trail mucus of the black slug, Arion ater L., contained 1-2% solids (9% protein, 21% hexose, 31% uronic acid, 39% hexosamine, 1% sulphate and < 1% sialic acid). 3. The major carbohydrate fraction (50-100 kDa) after borohydride reduction of A. ater mucus, was shown by PAGE and FAB-MS to consist of long chains of a repeating disaccharide, probably HexNAc-HexUA. The linkage sugar between carbohydrate and protein was a hexosamine, GIcN(Ac) or possibly GalN(Ac). 4. The carbohydrate fraction and whole A. ater mucus were susceptible to digestion with heparinase III, but not heparinase I, heparinase II, hyaluronidase or chondroitin sulphate ABC lyase, suggesting a structure similar to that of heparan sulphate.

INTRODUCTION Slugs are increasingly important pests of agriculture and horticulture (Martin and Kelly, 1986). In contrast with insect pests, control measures are unreliable and depend on toxic baits containing either metaldehyde or methiocarb. These edible formulations are also damaging to wildlife and to beneficial arthropods (Kendall et al., 1986). The failure to develop better chemical control methods is due to a number of factors, including the lack of fundamental research. Gastropod molluscs secrete mucus over their entire body surface. These mucous secretions are essential to a range of functions including locomotion, prevention of desiccation and removal of foreign or excreted particles from the body surface. The precise rheological properties of the mucous secretions are critical in enabling the secretions to fulfil these functions. During locomotion, the mucus under the foot behaves both as an elastic solid capable of resisting force beneath the stationary points of the foot, and

*To whom correspondence should be addressed. N-acetylhexosamine; HexUA: hexuronic acid; GlcN-ol: glucosaminitol; GalN-ol: galactosaminitol; GAG: glycosaminoglycan; Con A: concanavalin A (from Canavalia enisformis); SBA: soyabean agglutinin (from Glycine max); DBA: Dolichos biflorus agglutinin; PNA: peanut agglutinin (from Arachis hypogaea); UEAI: Ulex europaeus agglutinin 1; WGA: wheat germ agglutinin (from Triticum vulgaris).

Abbreviations--HexNAc:

as a fluid to minimize the forces of forward movement under the moving portions of the foot. Hence by a series of muscular waves along the foot, the mollusc is able to slide forward. The biomechanics of molluscan adhesion and locomotion have been the subject of several studies (Jones, 1973; Denny and Gosline, 1980; Denny, 1983). The rapid, alternate switching of the mucus from solid to fluid as the pedal wave moves along the foot is attributed to components within the pedal mucus which is secreted from the pedal gland located at the anterior end of the foot. Substances which interfere with these properties may be potentially useful in the search for novel molluscicidal agents. Analysis of pedal gland mucus from several species of terrestrial mollusc has been conducted (Taylor, 1963; Pancake and Karnovsky, 1971; Watanabe, 1976; Deyrup-Olsen et al., 1983). All samples contained high molecular weight material with both carbohydrate and protein present, but the precise biochemical composition of molluscan pedal gland mucus and the way in which the individual components are linked to form the final structure remain to be established. The aim of this study was to compare pedal gland (trail) mucus from several species of terrestrial mollusc in an attempt to identify common components, which could then be purified and characterized. When no common component was identified attention was focused on the biochemical composition of trail mucus from the black slug, Arion ater, to determine

455

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JANETM. COTTRELLet al.

the major structural component(s) responsible for its unique rheological properties.

MATERIALS AND M E T H O D S

With the exception of the Giant African land snail, Achatina fulica, slugs and snails were collected from the environs of IACR Rothamsted Experimental Station. They were housed at 10°C on damp blotting paper and fed a diet of lettuce, carrots and cucumber. Specimens of A. fulica (reared at IACR Rothamsted Experimental Station) were fed the same diet but were housed at 20°C. All snails were also provided with cuttlebone as a source of calcium for their shells. Slugs and snails were starved for 3 days prior to collection of mucus to minimize faecal contamination. They were placed on a glass sheet located within a Perspex chamber held at 10°C and lined with damp blotting paper to maintain high humidity. The trail mucus was scraped up as it was secreted and transferred to a pre-weighed vial. Materials

The following compounds and materials were obtained commercially: Bio-Gel P4 and all reagents and materials for S D S - P A G E and Western blotting, Bio-Rad (Hemel Hempstead, Herts, U.K.), standard sugars, glycoproteins, proteoglycans and protein markers for gel permeation chromatography and SDS-PAGE, Sigma (Poole, Dorset, U.K.), silica gel 60 TLC plates and all solvents for TLC and HPLC and Cocktail EX scintillant, Merck (Poole, Dorset, U.K.), Sepharose CL4B, Pharmacia (Milton Keynes, Bucks, U.K.), NaB[3H]4 (23Ci/mmol), Amersham International (Amersham, Bucks, U.K.), sodium borohydride, glucuronic acid, galacturonic acid and trifluoracetic acid, Aldrich Chemical Co. (Gillingham, Dorset, U.K.), Coomassie Protein Assay Reagent and Teflon-lined screw-capped glass vials, Pierce Life Sciences Laboratories (Luton, Beds, U.K.). All other reagents were from suppliers as described in the methods or were from Sigma or Merck (chemicals). Solubilization o f mucus

Samples of mucus were solubilized by stirring at 10°C in deionized water containing 1 mM phenylmethanosulphonyl fluoride (PMSF) and 0.02% NaN3 (4 ml/g wet weight mucus). After 2 days the material was centrifuged (5 min at 1200 rpm) and the supernatant removed. The pellet was resuspended in 2 ml deionized water containing 1 mM PMSF and 0.02% NaN3 and stirred for a further 2 days. After centrifugation (5min at 1200 rpm) the supernatant was removed and combined with that already stored. The pellet was analysed for protein and hexose before being discarded. For A. ater the pellet typically contained 12-20% of the total protein and hexose of the collected mucus.

Analysis o f solubilized mucus

Protein was measured using the Coomassie Protein Microassay with bovine serum albumin as standard. Hexose was measured by the phenol sulphuric acid assay (Dubois et aL, 1956), using o-glucose as standard. Interference by D-galacturonic acid was 27%, and by GalNAc was 2%. Hexosamine was measured by a modified Elson-Morgan reaction (Reissig et al., 1955) after hydrolysis in 4 M HCI and re-N-acetylation using acetic anhydride. The standard curve obtained using GalNAc produced absorbance values greater than twice those obtained when GIcNAc was used as standard. Sialic acid was measured by the thiobarbituric acid assay of Warren (1959) after hydrolysis for 1 hr at 80°C in 0.05 M H2SO 4, using N-acetylneuraminic acid as standard. Uronic acid was measured by the method of Blumenkrautz and Asboe-Hansen (1973) using o-galacturonic acid as standard. Sulphate was measured in hydrolysed samples by the method of Dodgson (1961) using K2SO 4 as standard or by HPLC on a Waters ionexchange chromatography system. Negatively charged macromolecules were quantitated relative to chondroitin sulphate A using an Alcian Blue binding assay (Whiteman, 1973). For dry weight measurement, samples of mucus direct from collection or samples solubilized as described were dried in pre-weighed vials in an oven until no further reduction in weight was observed. The vials were then reweighed. S D S - P A G E and Western blotting

Samples of solubilized or native mucus direct from collection were boiled up with sample buffer and subjected to discontinuous SDS-PAGE (Laemmli, 1970). Gels were cast (10 × 10 cm) with 3% stacking gels and 7.5-20% gradient separating gels, using a Mini-gel system (Bio-Rad). Gels were run in Tris-glycine buffer (pH 8.3) at 175 V at room temperature until the Bromophenol Blue marker reached the bottom of the gel (approx. 55 min). At the end of the run, gels were either fixed and stained for protein using an ammoniacal silver-staining procedure or were blotted onto 0.2 #m pore nitrocellulose using a Bio-Rad Mini-blot cell (100 V for 1 hr cooled with an ice pack). After blotting, the gels were fixed and silver-stained to monitor the extent of the protein transfer. Nitrocellulose blots were stained for protein using the Bio-Rad colloidal gold stain or were analysed for the presence of carbohydrate using a Glycan Detection kit (Boehringer, method B) or lectin overlays. Nitrocellulose blots were overlaid with biotinylated lectins which were detected using the Vectastain Elite ABC kit (Vector Laboratories, Peterborough, Cambs, U.K.). Biotinylated standards were run on SDS-PAGE since these transferred onto the nitrocellulose and reacted in the Vectastain kit to produce visible bands against which the lectin-stained bands could be compared.

Glycosaminoglycans in slug trail mucus

PAGE with combined Alcian Blue~silver staining of carbohydrate Standard proteoglycans, glycoproteins, whole A. ater mucus or mucus fractions were analysed by continuous PAGE in 10% (w/v) acrylamide with 0.1 M Tris-0.1 M borate-0.001 M EDTA buffer (pH 8.1) on 16 x 14 ×0.15cm gels (Min and Cowman, 1986). Samples (before and after enzymatic digestion) were diluted with 2 M sucrose in gel buffer (4 parts sample:l part buffer). Electrophoresis was continued for 3.5 hr at 175V at room temperature. Gels were rinsed in distilled water and then stained for 30 min in the dark in 0.5% AIcian Blue in 2% acetic acid. After destaining in 2% acetic acid, gels were silver-stained as for SDS-PAGE.

Gel permeation chromatography Solubilized mucus was chromatographed on a column of Sepharose CL4B (92 x 2.5 cm i.d.) at 18.5ml/hr in 0.1 M Tris-HC! (pH 7.5) containing 0.02% NaN3 at 10°C. Fractions were collected every 20 rain and analysed for protein and hexose. Fractions were pooled as appropriate and then dialysed extensively against distilled water and freeze-dried or concentrated by ultrafiltration in a 10ml-capacity Chemlab stirred-cell fitted with a 10,000tool. wt cut-off PLGC membrane (Millipore). Samples were applied to a Bio-Gel P4 column (62 x 1.6 cm i.d.) equilibrated in pyridine-acetic acid buffer (pH 5.7) at 12 ml/hr. Fractions were collected every 20 min and assayed for hexose and protein (samples and standards in buffer diluted 1/2 with water since the pyridine-acetic acid buffer interferes in the Coomassie Protein assay). Pooled fractions were freeze-dried.

Alkaline borohydride reduction of A. ater trail mucus Freeze-dried mucus (20 mg) was reconstituted in 4ml deionized water and incubated for 22 hr at 45°C with 1 M NaBH4 in 0.05 M NaOH. On some occasions 10 mCi NaB[3H]4 (23 Ci/mmol) was also added to the mixture. At the end of the incubation, 1.5 ml methanol containing a drop of acetic acid was added to the mixture and the sample dried down under NA:. This process was repeated twice and finally using 2 ml methanol containing no acetic acid. The residue was dissolved in 5.8 ml pyridine-acetic acid buffer (pH 5.7), centrifuged to remove insoluble particles and then applied to the Bio-Gel P4 column. Fractions were assayed for protein and hexose and in samples where the radiolabel was incorporated, aliquots (50/~1) were also counted for [3H] in 10ml Cocktail EX scintillant. Appropriate fractions were pooled and freeze-dried. A series of sugars (GIcN, GAIN, GicNAc, GalNAc and Man) was treated in a similar manner, but on a small scale to produce 3H-labelled sugar-alcohols for use as standards.

457

Hydrolysis of mucus and standard proteoglycans Freeze-dried samples of mucus or standard proteoglycans (I mg each in 1.5 ml-capacity glass vials with Teflon-lined screw caps) were hydrolysed under N 2 in 400 #1 2 M trifluoroacetic acid (TFA) or 400 #1 6 M HC1 for 4 hr in a boiling water bath. Samples were dried under vacuum over sodium hydroxide pellets and then reconstituted in deionized water for analysis by TLC, HPLC or assay.

Enzymatic digestion of A. ater mucus, standard glycoproteins and proteoglycans A. ater mucus, transferrin, bovine submaxillary mucin and fetuin (50#g each) were digested with 2.5 units N-Glycosidase F (Boehringer) overnight at 37°C after prior denaturation for 5 min at 100°C in 50raM phosphate buffer (pH 7.3), 25mM Na 2EDTA, 0.1% (w/v) SDS, 1% (w/v) n-octylglucoside and 1% (v/v) fl-mercaptoethanol. These samples were analysed by SDS-PAGE. Standard proteoglycans, A. ater mucus and M 1 fraction (10 mg each) were digested with 1140 units bovine testicular hyaluronidase (EC 3.2.1.35, type IV-S) in 5 ml 0.1 M sodium acetate buffer (pH 5.0) containing 0.15 M NaC1 at 37°C. Gelatin solution (1% w/v in buffer) was added to each sample and aliquots (0.5 ml) of the mixture removed at time intervals and boiled for 3 min prior to storage at -20°C. Standard proteoglycans, A. ater mucus and MI fraction (0.5 mg) were digested with 0.1 unit chondroitin sulphate AC lyase (EC 4.2.2.5, from Flavobacterium heparinum) in 0.1 M sodium acetate/0.1 M Tris-HC1 (pH 7.4) at 37°C. Aliquots (100/d) were removed at time intervals and boiled as described above. Samples were digested with chondroitin sulphate ABC lyase (EC 4.2.2.4, from Proteus vulgaris) as described for the chondroitin sulphate AC lyase digestion, except that the buffer was adjusted to pH 8.0. Standard proteoglycans, A. ater mucus and M 1 fraction (250 # g) were digested with 1.0unit heparinase I (EC 4.2.2.7, from F. heparinum), heparinase II (from F. heparinure) or heparinase III (EC 4.2.2.8, from F. heparinum) in 250#1 0.1 M Tris-HCl (pH 7.4) at room temperature for periods of up to 10 days. Aliquots (40 gl) were removed at time intervals and boiled for 3 rain prior to storage at -20°C. Analysis of hydrolysed samples by HPLC The Dionex ~ HPLC system, fitted with a CarboPac 1 column and pulsed amperometric detector was eluted at a flow rate of I ml/min. For the analysis of neutral sugars and hexosamines, the column was eluted with 16 mM NaOH. For the analysis of uronic acids and larger oligosaccharides, the column was eluted with 100mM NaOH containing 150mM NaOAc. Standards (10 nmoi each) or samples were applied in 100gl and the detector was set to a sensitivity of 3 knA. In experiments where [3H]labelled samples were analysed, fractions were

458

JANETM. COTTRELLet al.

collected every 0.5 min and aliquots (50/~1) counted for [3H] in 10 ml Cocktail EX scintillant. Analysis o f hydrolysed samples by T L C

Hydrolysed samples or standard sugars ( > 10/~g) were applied to 20 x 20cm silica gel 60 plates. The plates were developed in isopropanol:ethylacetate :water (83:11:6) for 4 h r or ethylacetate: pyridine: acetic acid: water (5: 5 : 1: 3) for 4.5 hr. Plates were stained for carbohydrate with alkaline silver nitrate. Where radiolabelled samples were run, lanes were scraped and sections counted for [3H] in Cocktail EX scintillant. RESULTS S D S - P , 4 G E and lectin staining o f Western blots

The trail mucus from seven species of terrestrial mollusc (five slugs: A. ater, ,4rion hortensis, Limax flavus, Milax budapestensis, Deroceras reticulatum; and two snails: Helix aspersa, ,4. fulica) was analysed by S D S - P A G E under reducing conditions (Fig. 1). With all species, a proportion of the material remained at the top of the separating gel, but there was no stained material in the stacking gel. Each species produced a large number of stained protein bands, the majority of which were of molecular weight < 100 kDa. There was little similarity between species with regard to the pattern of silver-stained bands. When nitrocellulose blots were stained using the Glycan Detection kit, most species showed one or two intense bands near to the top of the gel

( > 100 kDa). The snails Achatina and Helix produced the most intense staining (Fig. 2). The sample of ,4. ater treated with N-Glycosidase F produced the same pattern as the sample of A. ater which had not been digested (results not shown). In control experiments (not shown), bovine submaxillary mucin showed intense staining with the Glycan Detection kit, particularly at the very top of the gel (>200 kDa), but there were also several distinct bands between 55 and 75 kDa. This staining was unaffected by prior treatment of the bovine submaxillary mucin with N-Glycosidase F. Transferrin produced a single stained band at approximately 80 kDa which was not present in the transferrin sample which had been pre-treated with N-Glycosidase F. Fetuin produced three stained bands between 50 and 80 kDa. These bands shifted position to stained bands of lower molecular weight (45-60 kDa) after treatment with N-Glycosidase F. Nitrocellulose blots of S D S - P A G E gels were analysed for the presence of specific carbohydrates by overlaying with biotinylated lectins and then using an antibody-enzyme complex (Vectastain Elite ABC kit) to detect the biotin. Figure 3 is an example of the typical patterns produced by the lectin reactions against mucus components separated by SDS-PAGE. Each lectin produced quite a different pattern of binding. Concanavalin A (Con A) stained most species and throughout the whole molecular weight range, whereas wheat germ agglutinin (WGA) stained bands mainly towards the top of the gel and Ulex europaeus 1 agglutinin (UEA 1) stained bands mainly towards the bottom of the gel. In control experiments

Fig. 1. SDS-PAGE analysis of trail mucus from terrestrial molluscs. Samples of solubilized mucus (ca. 50 #g dry weight each sample) were analysed by SDS-PAGE on a 10 x 10 cm gel with 3% stacking gel and 7.5-20% gradient separating gel. Gels were run at 175 V for 55 min in Tris-glycine buffer (pH 8.3). The proteins were fixed and visualised by silver-staining. Arrows indicate the position of the protein standards: myosin (205 kDa); phosphorylase b sub unit (97 kDa); bovine serum albumin (66 kDa); ovalbumin (45 kDa); glyceraldehyde-3-phosphate dehydrogenase (36 kDa); carbonic anhydrase (29 kDa); soyabean trypsin inhibitor (20 kDa); lactalbumin (14 kDa). 1. Arion ater; 2. Arion hortensis; 3. Limax flavus; 4. Milax budapestensis; 5. Deroceras reticulatum; 6. Helix aspersa; 7. Achatina fulica.

Glycosaminoglycans in slug trail mucus

459

Fig. 2. Localization of total carbohydrate on nitrocellulose blots using the Boehringer Glycan Detection kit. Samples of trail mucus were analysed by SDS-PAGE (7.5-20% acrylamide) and then transferred onto nitrocellulose using a Bio-Rad Mini-blot cell (1 hr at 100 V). The nitrocellulose blots were treated with a Glycan Detection kit (Boehringer) using Method B of the manufacturer's instructions. I. Transferrin standard; 2. Arion ater; 3. Arion hortensis; 4. Limax fluvus; 5. Milax budapestensis; 6. Deroceras reticulatum; 7. Helix aspersa; 8. Achatina fulica.

where the lectin was omitted (not shown), only the biotinylated standards produced stained bands on the nitrocellulose.

Table 1 is a summary of the data from four replicate lectin experiments. Each gel was divided into three molecular weight regions; > 60 kDa, 60-20 kDa

Fig. 3. Localization of specific carbohydrates in mucus samples through lectin binding to nitrocellulose blots. Samples of trail mucus from seven species of terrestrial mollusc were analysed on replicate mini gels by SDS-PAGE (7.5-20% acrylamide). After blotting of the samples onto nitrocellulose, each piece of nitrocellulose was overlaid with a biotinylated lectin and the specific binding detected by an antibody-enzyme complex in the Vectastain Elite ABC kit. (a) Concanavalin A, (b) Wheat germ agglutinin, (e) Soyabean agglutinin, (d) Ulex europaeus agglutinin 1. For each blot the lanes are as follows: 1. Biotinylated standards: phosphorylase b sub unit (97 kDa); catalase sub unit (58 kDa); alcohol dehydrogenase (39 kDa); carbonic anhydrase (29 kDa); soyabean trypsin inhibitor (20 kDa); lysozyme (14 kDa); 2. Achatina fulica; 3. Helix aspersa; 4. Deroceras reticulatum; 5. Milax budapestensis; 6. Limax flavus; 7. Arion hortensis; 8. Arion ater. , CBPB 1 0 4 / ~

460

JANETM. COTTRELLet al.

T a b l e 1. Binding o f lectins to trail mucus immobilized on nitrocellose blots. Samples o f trail mucus from seven species o f terrestrial mollusc were separated by S D S - - P A G E a nd blotted onto nitrocellulose. Each blot was overlaid with a biotinylated lectin a n d the specific binding detected using a Vectastain Elite A B C kit. T h e table summarizes the intensity o f staining o f the bands for each species with each lectin used in the study. + + + : intense staining; + + : m e d i u m staining; + : light staining; ( + ) : faint staining; - : no staining. T h e staining has been divided into three molecular weight regions on each blot: ( > 60 k D a , 6 0 - 2 0 k D a , < 20 k D a ) . Staining has not been a t t r i b u t e d to specific bands Lectin Con A

S BA

DBA

PNA

Arion ater

Arion hortensis

+++ ++ + -. + +++ +

+++ ++ ++ +

WGA

. . + +++ + .

-

. .

.

Helix aspersa

+++ + +++ -

++ -

.

.

. ++ +

-++

+

-

+

-

+ + +++ + .

. .

Analysis o f the trail mucus o f A. ater

Figure 4(a) shows a typical profile of solubilized A. ater trail mucus chromatographed on Sepharose

CIAB. A proportion of the material (between 15 and 30% of the protein and hexose depending on the sample) eluted in the void volume, but on all occasions there was a spread of protein and carbohydrate-containing material throughout the included

+++ ++ ++ +++

>60 60-20 <20 >60 60-20 <20 >60 60-20 <20 >60 60-20

+ + +

--

. .

+ +

and < 20 kDa. The intensity of staining with each lectin for each species was graded within those molecular weight regions. The classification is fairly arbitrary in that intense staining may represent several bands of medium/heavy staining clustered together or just one or two very intensely stained bands. The results do show that (1) the staining pattern for each species varied considerably with the lectin used, and (2) the staining pattern with one lectin varied considerably between different species• The intense Con A staining could be almost completely blocked by inclusion of 200mM or-methyl glucoside and 200 mM ,t-methyl mannoside during the incubation with Con A. Similarly, soyabean lectin (SBA) binding could be totally blocked by inclusion of 200 mM GalNAc during the incubation with SBA and peanut lectin (PNA) binding could be blocked by inclusion of 200 mM Gal during the incubation with the PNA. The presence of 500 mM GlcNAc was insufficient to completely inhibit binding of WGA to the mucus components, although the intense staining was reduced over certain individual bands as compared with others• Since there were no major bands common to all species of which could be selected for subsequent purification, attention was focused on one species, A. ater, which was the largest slug in the study and produced relatively large quantities of trail mucus.

M o l wt range

.

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Achatina fulica

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++ . .

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Deroceras reticulatum

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.

+

.

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+++ ++ + + + ++ -

++ . ++ +

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+++ + -

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Fig. 4. Gel permeation chromatography of Arion ater trail mucus on Sepharose CLAB. A. ater trail mucus was applied to a Sepharose CIAB column (92 x 2.5 cm i.d.) and eluted in 0.I M Tris-HCl (pH7.5) containing 0.02% NaN3 at 18.5ml/hr. Fractions were collected every 20rain and assayed for hexose and protein. (a) Arion ater trail mucus (0.8 g) solubilized in 11.5ml deionized water containing 1 mM PMSF and 0.02% NAN3. Recovery of protein was 89%, recoveryof hexose was 93%. (b) Arion ater trail mucus (12.2 g) spontaneously solubilized; 10 ml applied. Recovery of protein was 84%; recovery of hexose was 93%. The elution position of molecular weight standards is indicated by the arrows: DB, dextran blue (2000kDa); TG, thyroglobulin (669 kDa); AF, apoferritin (443 kDa); BA, /~amylase (200 kDa); AD, alcohol dehydrogenase (150 kDa); BSA, bovine serum albumin (66 kDa).

Glyeosaminoglycansin slug trail mucus volume. When A. ater mucus was run on the same column, but with 0.22M potassium thiocyanate included in the buffer, the hexose profile was fairly similar to that given in Fig. 4(a), but the protein eluted over a lower molecular weight range with two peaks at 324 and 373 ml, respectively. Only 7*/0 of the protein was in the void volume fractions. When A. ater mucus was run on the same column but using a buffer of 2 M guanidine hydrocbloride in 0.5 M sodium acetate (pH 6.5), the profile was almost identical to that in Fig. 4(a) of the solubilized mucus in 0.1 M Tris-HCl. One sample of A. ater trail mucus (12.2 g gel) was collected and left at 10°C for 2 days (without solubilization in deionized water containing 0.02% NaN3 and 1 mM PMSF). The gel solubilized spontaneously to give a clear liquid with only a small quantity of insoluble material. The profile of the spontaneously solubilized sample chromatograpbed on Sepharose CL4B is given in Fig. 4(b). In this case only 4% protein and 10% hexose eluted in the void volume. The biochemical composition of A. ater trail mucus as determined by colorimetric assay is given in Table 2. The protein:carbohydrate ratio is approximately 10:90. The ratio differs from that found in typical vertebrate mucus giycoproteins where protein: carbohydrate is commonly 35: 65. Alkaline borohydride reduction o f A. ater mucus

Solubilized A. ater trail mucus was fractionated on a Bio-Gel P4 column in pyridine-acetic acid buffer (pH 5.7). Results are given in Fig. 5(a). The void volume material (fractions 5-9, which contained all the protein and virtually all the hexose) was freezedried and a portion was subjected to treatment with alkaline sodium borohydride containing 10mCi NaB[3H]4 as described in the methods. The protein became insoluble after this treatment and was removed by centrifugation. The supernatant was re-applied to the Bio-Gel P4 column. The profile is given in Fig. 5(b).

461

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F i g . 5. G e l p e r m e a t i o n c h r o m a t o g r a p h y o f Arian ater trail mucus on Bio-G¢l P4 before and after treatment with a l k a l i n e b o r o h y d r i d e . (a) S o l u b i l i z e d A . ater t r a i l m u c u s w a s applied to a Bio-Gel 24 column (62 x 1.6 cm i.d.) and eluted

with pyridine-acetic acid buffer (pH 5.7) at 12 ml/hr. Fractions were collected every 20 rain and assayed for protein and hexose. The void volume fractions (5-9) were pooled and freeze-dried. Co) A sample of the freeze-dried material (20 mg) was subjected to alkaline bar,hydride reduction using NaB[3I-I]4as described in the methods. The supernatant (6.5 ml) following centrifugation was applied to the same Bio-Gel P4 column and fractions collected as for Fig. 5(a). These fractions were assayed for protein and hexose and counted for tritium. They were pooled as shown in Fig. 5(b).

The majority of the carbohydrate-containing material eluted from the column with a molecular weight > 4 k D a (void volume). These results suggested that most of the carbohydrate existed as Table 2. Biochemicalcomposition of Arian ater trail mucus as oligosaecharide chains of at least 20 sugars in length determined by calorimetricassays. Dry weight was 1-2% of the (assuming an average molecular weight of 200 Da per collected wet weight. AlcianBlue-precipitablematerial(relativeto chondroitin sulphate A) was 446pg/ml (ranging from 320 to monosaccharide). This void volume fraction (M1) 615p g/ml).Withthe exceptionof hexosamineand sialicacidassays contained a small quantity of the radiolabel, prob(whereonlyone samplewasassayedbut on severaloccasions),values ably incorporated into the sugar which had been givenare the averagevaluesdeterminedfrom3 to 5 individualmucus samplescollectedseparately.The rangeof valuesis givenin paren- linked to the protein prior to the bar,hydride theses. Sulphateby (a) gelatin-bariumchloridemethodof Dodgson reduction. Some carbohydrate-containing material (1961), (b) by HPLC eluted in the included volume of the column and also Component Standard % D r y weight contained [3HI suggesting that these fractions (M2 Protein BSA 9 (6-10) and M3) may represent shorter, perhaps typical Hexose* Glucose 21 (17-24) O-linked, oligosaccharides. The M4 pool contained Hexosamine GalNAc 161 predominantly bar,hydride reaction products, but a GIcNAc 39 Uronic a c i d Galacturonic acid 31 (24-45) small quantity of free monosaccharide may also have Sialic acid NANA
462

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fraction eluted over a broad molecular weight range with the peak between 50 and 100kDa. This suggested that the oligosaccharide chains of M1 contained approximately 250-500 monosaccharides (assuming an average monosaceharide weight of 200kDa, but not allowing for sulphation). The 50-100 kDa material contained 93% of the hexose and 99% of the [3HI eluting from the column. In addition there was a small peak in the void volume which contained 7% of the hexose and 0.5% of the [3H] which was of considerably higher molecular weight. Analysis of the carbohydrate fractions produced by alkaline borohydride reduction o f A. ater trail mucus Fractions M1, M2 and M3 from NaB[3H]4 treatment of A. ater trail mucus and untreated A. ater trail mucus were hydrolysed in 2 M TFA. Samples were analysed by Dionex ® anion HPLC for neutral sugars, hexosamines and uronic acids. With the exception of a diminished galactose content for whole A. ater mucus (not shown) the profiles of the whole mucus and MI were fairly similar (as expected since M1 was the major carbohydrate fraction of the mucus). M2 and M3 contained a larger proportion of sugaralcohols which eluted at 1.9-2.1 min (Fig. 7) and proportionally more galactose relative to M1. From the Bio-Gel P4 fractionation of NaBH4-treated A. ater mucus there was approximately 10 times from M1 produced than either M2 or M3. The major sugar in fraction M1 was glucosamine. When the samples were analysed for uronic acids (the column was eluted in 100 mM NaOH containing 150mM NaOAc), none of the fractions MI, M2 or M3 contained galacturonic acid or glucuronic acid. Both M1 and the whole A. ater mucus contained a peak which eluted in the same position as an iduronic acid standard (prepared as described by Fischer and Schmidt (1959) by epimerization from glucuronic

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acid). Neither M2 nor M3 contained the iduronic acid peak. "Standard" proteoglycans of known structure were hydrolysed in 2 M TFA and analysed by the Dionex® anion HPLC. Heparan sulphate, heparin and chondroitin sulphate B, but not chondroitin sulphate A, chondroitin sulphate C or hyaluronic acid, produced elution profiles with peaks in similar positions to those found when M1 was analysed under the same conditions (Fig. 8). The profile of M1 was most like that of heparan sulphate. Hydrolysed fractions M1, M2 and M3, standard sugars and [3H]-labelled sugar-alcohol standards were applied to silica 60 TLC plates and chromatographed using two different solvent systems as described in

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the methods. After staining of the plates for carbohydrate, lanes were scraped and counted for [3H]. The results are illustrated in Fig. 9, which shows the radiolabelled profile of M 1 compared with standards run in the ethylacetate: pyridine: acetic acid :water solvent system. M I (and M2 and M3) contained some radiolabelled material which remained at the origin (perhaps due to incomplete hydrolysis of the sample). The radiolabel in M1 (and M2 and M3) moved with an Rf of 0.19-0.25, eliminating the possibility of it being due to galactitol, sorbitol, mannitol, xylitol or arabitol (Rf values of 0.38-0.49). Since the tritiated peaks obtained after counting lane M1 (and M2 and M3) were fairly broad (Fig. 9), it was not possible to determine whether the radiolabel was due to [3H]GIcN-ol or [3H]GalN-ol, although the results indicated that it was more likely [3H]GlcN-ol. These

sugars may have been N-acetylated in the native material since acetyl groups would have been removed during hydrolysis. Fractions MI, M2 and M3 were analysed by F A B - M S by Professor A. Dell (Imperial College). After permethylation, F A B - M S did not reveal the presence of typical O-linked oligosaccharides in any of the fractions. Contamination of fraction M3 with salts and possibly amino acids prevented identification of carbohydrate material which may have been present. Analysis of fraction M2 suggested the presence of small olignsaccharides containing hexoses and hexosamines, but in atypical combinations. Fraction M1 produced strong signals for molecular ions at 478, 941, 1404 and 1867 corresponding to a repeating disaccharide of HexNAc-HexUA similar to that found in the glycosaminoglycan chains of

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The degradative enzymes digested the standard proteoglycans in accordance with their known specificities. Chondroitin sulphate AC lyase digested chondroitin sulphate A and chondroitin sulphate C, but not chondroitin sulphate B, whereas chondroitin sulphate ABC lyase digested chondroitin sulphate A, B and C. Hyaluronidase digested hyaluronic acid, chondroitin sulphate A and chondroitin sulphate C, but not chondroitin sulphate B. Heparinase I digested heparin, but not heparan sulphate. Heparinase II did not digest heparin or heparan sulphate. Heparinase III digested heparan sulphate, but not heparin. Whole A. ater mucus, MI fraction and bovine submaxillary mucin (a typical vertebrate mucus) were unaffected by digestion with hyaluronidase, chondroitin sulphate AC lyase or chondroitin sulphate ABC lyase (Fig. 10 and Fig. 11). Whole A. ater and M I fraction were resistant to digestion with heparinase I and heparinase II (even after 10 days digestion), but both are completely digested with heparinase III after 24 hr (Fig. 12). With the exception of a band corresponding to that of the heparinase III, there was no residual staining in the M 1 lane after heparinase III digestion. There were a series of stained bands at regular repeats remaining in the lane corresponding to the A. ater sample after digestion with heparinase III, probably due to residual protein. Rheological properties o f A. ater trail mucus

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Fig. 9. Analysis of hydrolysed [3H]MI by TLC. Following hydrolysis of [3I-I]M1 in 2 M TFA, the dried sample was reconstituted in deionized water and 10#1 applied to a 20 × 20era silica gel 60 plate. Standards sugars (20/~g) and tritiated sugar alcohols were applied in a similar manner. The plate was developed for 4.5hr in ethylacetate:pyridine:acetic acid: water (5: 5:1 : 3). Carbohydrate spots were visualised through staining with alkaline silver nitrate. Where radiolabelled samples were run, each lane was divided into 0.5 cm sections, sections scraped into scintillation vials, eluted in 1 ml dcionized water and then counted for tritium in 10mi Cocktail EX scintillant. Figure 9 shows the migration of the radiolab¢l along the TLC lanes for each standard and M I. The spread of the xylitol spot stained with alkaline silver nitrate is shown on the [3H]GalN-ol profile.

proteoglyeans. Since MI is the major fraction of A. ater it is likely that the mucus is formed predominantly of long glycosaminoglyean chains of repeating HexNAc-HexUA structure linked to a central

Samples of A. ater collected from individual slugs were analysed within 5 hr of collection on a parallel plate Carri-Med Rheometer in the Department of Professor C. Marriott (King's College, London). The gap between the plates was set to 150/~m. Samples were tested over a frequency range of 0.5-30 Hz at 10°C at an applied stress of 10 or 20 N/m 2. Results were consistent with those reported previously (Grenon and Walker, 1980; Denny, 1983). At low frequencies all mucus samples behaved as elastic solids with stress in phase with strain and G' ~> G". At higher frequencies the stress became increasingly out of phase with the strain until at a certain frequency (14-20 Hz, depending on the sample) maximum displacement was achieved and the samples behaved as viscous fluids. This effect was exhibited at applied stresses of both 10 and 20 N/m 2. The frequency at which this solid/fluid transition occurred varied not only between samples from different slugs, but also within a sample taken from the same slug, indicating the heterogeneous nature of the trail mucus collected from individual specimens. The characteristics of the collected mucus also changed with time, becoming more fluid, even when samples were maintained on ice.

Glycosaminoglycans in slug trail mucus

Fig. 10. P A G E of A. ater mucus, M1 fraction and BSM following digestion with degradative enzymes. Samples were run in 10% acrylamide gels (16 x 14 cm) in Tris-borate-EDTA buffer and then stained with a combined Alcian Blue-ammoniacal silver stain. Lanes (from left to right; lanes 2-10--samples digested with hyaluronidase; lanes 12-15--samples digested with chondroitin sulphate AC lyase): 1. Hyaluronidase control; 2. M1 non-digested; 3. M1 0.5 hr digestion; 4. M1 4 h r digestion; 5. MI 2 4 h r digestion; 6. A. ater mucus 0 hr digestion; 7. A. ater mucus 0.5 hr digestion; 8. A. ater mucus 4 hr digestion; 9. BSM 0 hr digestion; 10. BSM 24 hr digestion; 11. D N A digest ( x 174 Hae III, 2 gg; Sigma); 12. M i 0 hr digestion; 13. MI 0.5hr digestion; 14. M1 4 h r digestion; 15. M1 2 4 h r digestion.

Fig. 11. P A G E of A. ater mucus, MI and BSM as Fig. 10. Samples digested with chondroitin sulphate AC lyase (lanes 1-5) or chondroitin sulphate ABC lyase (lanes 7-15). Lanes (from left to fight): 1. A. ater mucus 0 hr digestion; 2. A. ater mucus 0.5 hr digestion; 3. A. ater mucus 4 hr digestion; 4. BSM 0 hr digestion; 5. BSM 24 hr digestion; 6. Chondroitin sulphate ABC lyase control; 7. M 1 0 hr digestion; 8. M 1 0.5 hr digestion; 9. M 1 4 hr digestion; 10. M 1 24 hr digestion; 1 I. A. ater mucus 0 hr digestion; 12. A. ater mucus 0.5 hr digestion; 13. A. ater mucus 4 hr digestion, 14. BSM 0 hr digestion, 15. BSM 24 hr digestion.

465

466

JANETM. COTTRELLet al.

Fig. 12. PAGE of mucus and M1 as Fig. 10. Lanes (from left to right; samples digested with Heparinase I, II or III): 1. M1 non-digested; 2. MI 10 days digestion (I); 3. M1 10 days digestion (II); 4. M1 4hr digestion (III); 5. M1 24 hr digestion (III); 6. M1 I0 days digestion (III); 7. A. ater mucus non-digested; 8. A. ater mucus 10 days digestion (I); 9. A. ater mucus 10 days digestion (II); I0. A. ater mucus 4 hr digestion (III), 11. A. ater mucus 24hr digestion (III), 12. A. ater mucus 10 days digestion (III); 13, 14. Insoluble material left after solubilization of A. ater mucus; 15. DNA digest (Fig. 10). DISCUSSION When examined by SDS-PAGE, there was little similarity between trail mucus from different species, either in the pattern of silver-stained protein bands or in the distribution of the carbohydrates. Glycosylated components were predominantly of high molecular weight ( > 100 kDa); in contrast to the distribution of silver-stained protein bands, most of which were < 1 0 0 k D a . Comparative studies between species using S D S - P A G E analysis and lectin binding to nitrocellulose blots may be subject to certain limitations. High molecular weight components group together at the top of the gel or may not enter the gel at all, so are difficult to compare. Only a c c e s s i b l e sugars will be detected through lectin binding, i.e. those on the outer edges of the glycosylated structure, not masked by other sugars. A single protein, glycosylated to varying degrees, may give rise to a series of bands on the gel, possibly with different lectinbinding properties. Heavily glycosylated proteins often stain poorly with protein-specific stains and may be overlooked in a comparison between the protein bands of different species. In this study where trail mucus has been compared from different species many protein bands observed on the gels, particularly those of low molecular weight, may originate from materials excreted by the molluscs, since the body wall is a major site for the removal of waste materials. Molecules as large as 68 kDa have been shown to

pass across the body wall (Simkiss and Wilbur, 1977). The carbohydrate-containing fractions produced by alkaline borohydride reduction of A . a t e r trail mucus did not stain at all on the S D S - P A G E gels, confirming that all protein had been removed (results not shown). Unlike vertebrate mucus where such treatment would result in a protein "core" of 100-200kDa and numerous oligosaccharide chains < 4 k D a , the major A . a t e r carbohydrate fraction (M1) eluted in the void volume on Bio-Gel P4 ( > 4 kDa) and when chromatograpbed on Sepharose CL4B, eluted in the range 50-100 kDa. The precipitated protein did not produce distinct bands on SDS-PAGE, thus preventing an estimation of the molecular weight of the protein "core". Using Dionex ~ anion HPLC the major components of M 1 were identified as galactose, glucosamine and iduronic acid, but there were additional unidentified peaks possibly disaccharides from incomplete hydrolysis. F A B - M S analysis of permethylated M1 suggested that M1 was a repeating disaccharide of N-acetylhexosamine-hexuronic acid. The presence of radiolabelled GlcN-ol and/or GalN-ol in a sample of [3H]M1 analysed by TLC indicated that the long carbohydrate chains (50-100kDa) were linked to protein through hexosamine in the native mucus structure. When A . a t e r mucus and its carbohydrate fraction M1 were analysed by PAGE with combined Alcian

467

Glycosaminoglycansin slug trail mucus Blue and silver staining, both samples produced staining patterns indicative of a proteoglycan-type structure. Both were resistant to digestion with all the enzymes examined except heparinase III. The pattern of digestion with the heparinasc enzymes suggested A. ater mucus has a proteogiycan structure similar to that of heparan sulphate. These results are generally in good agreement with two earlier studies on mucus secreted by terrestrial snails (Pancake and Karnovsky, 1971; Watanabe, 1976), but differ in the reported sulphate content. The earlier studies suggested that sulphate was a major component of the molluscan mucus. The composition of Otella lactea mucus was reported by Pancake and Karnovsky (1971) to be glucosamine:iduronic acid: acetyl :sulphate (1 : 1.5:1 : 1) and that of Succinea lauta mucus was reported by Watanabe (1976) to be N-acetylglucosamine:iduronic acid :ester sulphate (1 : 1: 1). The sulphate content of A. ater trail mucus in the current study was approximately 1% of the dry weight and could not account therefore for the high negative charge of the mucus as demonstrated by Alcian Blue binding, binding to DEAE-Sephacel and migration in isoelectric focusing studies (results not given). Since the sialic acid content of the samples was equally low ( < 1%), it is likely that the uronic acid confers most of the negative charge. From the results it is proposed that the trail mucus of Arion ater has a proteoglycan-type structure. The mucus has a small protein core (ca. 10% dry weight) to which are attached long chains of repeating GIcN(Ac)-HexUA (IdUA?) disaccharides up to approximately 500 monosaccharides in length, possibly with a small proportion of the monosaccharide being sulphated. These long chains may be interspersed with short oligosaccharide chains (<20 monosaccharide units) also attached to the protein core, containing predominantly galactose. Results from this study suggest that the sugar involved in the linkage to protein is GIcN(Ac) or GaIN(At), atypical of uronic acid-containing proteoglycans. Molluscan mucus may contain a novel protein-polysaccharide linkage. Individual molecules as described above may be linked directly to one another through noncovalent bonds or may be linked through other molecules (e.g. glycoproteins acting as lectins). The latter model would be consistent with the theory of Fountain (1982) who proposed that multivalent lectins interact with specific giycosyl residues on polysaccharide or glycoprotein moieties to form the macromolecular structures found in biological mucilages and mucins. In such a case, the small oligosaccharides detected in this study (M2 and M3) may have originated from glycoprotein lectin(s) linking adjacent proteogiycan chains. A role for noncovalent bonds was implied when both potassium thiocyanate-treated mucus and mucus which had solubilized "spontaneously" on standing eluted at a much lower molecular weight on Sepharose CL4B than did mucus solubilized in Tris-HC1 buffer.

Acknowledgements--We wish to thank Prof. A. Dell for the

FAB--MS analysis, Prof. C. Marriott for the rheological measurements, Dr A. Keys for the use of his Dionex~ anion HPLC system, Dr A. Mudd for preparation of the iduronic add standard, Dr N. Ainsworth for analysis of sulphate by HPLC and Dr G. Newman for advice on silver staining of polyacrylamide gels. This work was funded by Grant No. LRG/196 from the Agricultural and Food Research Couneft, Polaris House, North Star Avenue, Swindon SN2 1UH, U.K.

REFERENCES

Blumenkrautz, N. and Asboe-Hansen G. (1973) New method for quantitative determination of uronic acid. Anal. Biochem. 54, 484-489. Denny M. (1983) Molecular Biomechanics of molluscan mucous secretions. The Mollusca I. Metabolic Biochemistry and Molecular Biomechanics, pp. 431-465. Academic Press, New York. Denny M. and Gosline J. M. (1980) The physical properties of the pedal mucus of the terrestrial slug, Ariolimax columbianus. J. exp. Biol. 88, 375 393. Deyrup-Olsen I., Luchtel D. L. and Martin A. W. (1983) Components of mucus of terrestrial slugs (Gastropoda). Am. J. Physiol. 245, R448-R452. Dodgson K. S. (1961) Determination of inorganic sulphate in studies of the enzymic and non-enzymic hydrolysis of carbohydrate and other sulphate esters. Biochem. J. 78, 312-319. Dubois M., Gilles K. A., Hamilton J. K., Rebers P. A. and Smith F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350-356. Fischer F. G. and Schmidt H. (1959) Die epimerisierungder uronsauren. Chem. Ber. 92, 2184-2188. Fountain D. W. (1982) Lectin-like properties associated with mucus and mucilage of diverse biological origin. Naturwissenschaften 69, 450-451. Grenon J.-F. and Walker G. (1980) Biochemicaland rheological properties of the pedal mucus of the limpet, Patella vulgata L. Comp. Biochem. Physiol. 66B, 451-458. Jones H. D. (1973) The mechanism of locomotion of Agriolimax reticulatus (Mollusca: Gastropoda). J. Zool. Lond. 171, 489-498. Kendall D. A., Smith B. D., Chinn M. E. and Wiltshire C. W. (1986) Cultivation, straw disposal and BYDV infection in winter cereals. Br. Crop Prot. Conf.--Pests Dis. 3, 981-987. Laemmli U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685. Martin T. J. and Kelly B. R. (1986) The effect of changing agriculture on slugs as pests of cereals. Br. Crop Prot. Conf.--Pests Dis. 2, 411-424. Min H. and Cowman M. K. (1986) Combined alcian blue and silver staining of glycosaminoglycansin polyacrylamide gels: application to electrophoretic analysis of molecular weight distribution. Anal. Biochem. 155, 275-285. Pancake S. J. and Karnovsky M. L. (1971) The isolation and characterization of a mucopolysaccharide secreted by the snail, Otella lactea. J. biol. Chem. 246, 253-262. Reissig J. L., Strominger J. L. and Leloir L. F. (1955) A modified colorimetric method for the estimation o1 N-acetylamino sugars. J. biol. Chem. 217, 959 966. Simkiss K. and Wilbur K. M. (1977) The molluscan epidermis and its secretions. Symp. Zool. Soc. Lond. 39, 35-76. Taylor R. E. (1963) Mucus of the giant slug, Ariolimax columbianus. Mucus in Invertebrates Vol. 1. XVI International Congress of Zoology, Washington, DC (abstract).

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Warren L. (1959) The thiobarbituric acid assay of sialic acids. J. biol. Chem. 234, 1971-1975. Watanabe T. (1976) The comparative study of invertebrate glycosaminoglycans. 1. Isolation of a novel acidic glycosaminoglycan, succinean sulfate, from mucus of the

snail, Succinea laura Gould. Fukishima J. Med. Sci. 22, 245-265. Whiteman P. (1973) Quantitative determination of glycosaminoglycans in urine with Alcian Blue 8GX. Biochem. J. 131, 351-357.