Glycosaminoglycans in gills of an intertidal crab (Carcinus maenas): Changes in the GAG population in response to prolonged air exposure

554 M. REGNAULT AND F. DURANDTHE JOURNAL OF EXPERIMENTAL ZOOLOGY 281:554–564 (1998)

© 1998 WILEY-LISS, INC.

Glycosaminoglycans in Gills of an Intertidal Crab(Carcinus maenas): Changes in the GAG Populationin Response to Prolonged Air Exposure

MICHÈLE REGNAULT* AND FABRICE DURANDEquipe Ecophysiologie, Observatoire Océanologique de Roscoff (UPMC,CNRS, INSU), Station Biologique, BP 74, F-29682, Roscoff Cedex, France

ABSTRACT Glycosaminoglycans (GAGs) in the gills of an intertidal crab (Carcinus maenas)were determined by biochemical analysis and digestion by specific enzymes. In control (submerged)crabs, gill total GAG content was 14.58 ± 0.97 mg · g–1 of dry defatted weight of tissue (n = 12).Half of it consisted of sulfated GAGs: chondroitin sulfate (CS), keratan sulfate (KS), and heparansulfate (HS) for 12%, 15%, and 24.5% of total GAGs, respectively; the other half consisted ofnonsulfated GAGs: hyaluronan (HA) for 18% and a chondroitin-like compound for 30%.

In crabs exposed to air for up to 72 hr, a 73% increase in gill total GAG content was observed.HA was fully responsible for the GAG increase occurring within the first 24 hr of air exposure.Both HA and chondroitin contents increased during the next 24 hr, whereas the sulfated GAGsremained at their control level. Significant changes in total sulfated GAG content occurred onlyafter 48 hr of air exposure, and the final 32% increase was only attributable to CS. Increase in gillCS content was coupled with the main (50%) increase in gill chondroitin content.

The initial increase in gill HA content is discussed in relation to the GlcNAc synthesis that weobserved previously in the gills of crabs exposed to air for 24 hr. Air exposure–induced changes inthe GAG population of gills and their possible implications in gill tissue homeostasis, when crabshave to face dehydration, acidosis, and internal ammonia overload, are discussed. J. Exp. Zool.281:554–564, 1998. © 1998 Wiley-Liss, Inc.

*Correspondence to: Dr. M. Regnault, Station Biologique, BP 74,F-29682, Roscoff Cedex, France. E-mail: [email protected]

Received 24 November 1997; Accepted 24 February 1998

In the early sixties, much attention was paid toa class of acidic polysaccharides, the glycosami-noglycans (GAGs), found in the extracellular ma-trix of many tissues in all phyla (Scott, ’60; Bennett,’63; Dorfman, ’63). These molecules are linear poly-mers built of repeating disaccharide units consist-ing of hexosamine and hexuronic acid (sometimesgalactose). According to the structure of this disac-charide unit, four major families have been defined:hyaluronan (HA), chondroitin sulfates (CS, includ-ing dermatan sulfate), keratan sulfate (KS), andheparin/heparan sulfate (Hep/HS) (Hascall et al.,’94). In all families except for CS, the hexosamineis N-acetylglucosamine (GlcNAc). During polymer-ization, hexosamine incorporation into the grow-ing chain alternates with that of glucuronic acid,both being in their nucleotide form (Höök et al.,’84). Mature GAG chains, usually bound to a pro-tein core, are extruded from the Golgi apparatus(or the cell membrane for HA) into the extracel-lular matrix, where they make dense networks.The GAG chains are strongly negatively charged(one COO– group and up to 3 SO3

– groups per dis-accharide unit) (Comper and Laurent, ’78; Hascall

and Kimura, ’82), and this electrostatic potentialenvelope confers on them specific properties suchas regulation of micro-ion distribution and trans-port in the extracellular matrix and a powerfulosmotic buffering capacity (Comper and Laurent,’78). Their interaction with other components of theextracellular matrix makes them important inter-mediates between cells and their environment(Scott, ’75; Comper and Laurent, ’78, Höök et al,’84; Jackson et al., ’91)

In marine invertebrates, the first studies onGAGs were aimed at their identification and dis-tribution in a great variety of species belongingto the major phyla (Rahemtulla and Lovtrup, ’75,’76; Cassaro and Dietrich, ’77; Nader et al., ’88).In Crustacea, CS, KS, HA, and chondroitin havebeen identified in the lobster, Homarus vulgaris(Rahemtulla and Lovtrup, ’76). Specific heparansulfate and chondroitin sulfate also were charac-

GLYCOSAMINOGLYCANS IN GILLS OF AIR-EXPOSED CRABS 555

terized in H. americanus (Hovingh and Linker, ’82)and the king crab (Seno et al., ’74), respectively.In four shrimp species inhabiting fresh water andbrackish and marine water, a correlation was ob-served between their sulfated GAG content andthe environmental salinity (Nader et al., ’83). Inthese first attempts, GAGs were evidenced inwhole-body homogenates. In contrast to other in-vertebrate taxa, GAGs of Crustacea were investi-gated no more, to our knowledge. Recently, thepresence of polyanionic sites and acid muco-polysaccharides in the gill epicuticular surfacecoat of C. maenas was demonstrated cytochemi-cally (Compère and Goffinet, ’95), suggesting thatgills of crabs would contain and possibly synthe-size GAGs, as do the gills of mussels (Hovinghand Linker, ’93).

By studying nitrogen metabolism of a crab (Can-cer pagurus) in the course of a 24-hr air exposure,storage of some unexcreted ammonia in an extra-cellular compartment was made obvious by thesudden and large NH4

+ release at reimmersion;branchial water, according to its NH4

+ concentra-tion at the end of the air exposure period, ac-counted for only 6% of this output (Regnault, ’94).Besides, GlcNAc content of gill and hindgut tis-sues significantly increased in these conditions;in the absence of a free hexosamine pool, a rapidincorporation of the newly formed GlcNAc resi-dues into oligomers and more likely polymers wasproposed (Regnault, ’96). The strategic localiza-tion of GAGs in the extracellular matrix, as wellas the very peculiar properties of these poly-saccharidic chains and their high turnover rate,made them possible candidate for GlcNAc incor-poration. Our first insight into the GAG contentof C. pagurus gills indicated that papain-resistantpolyanions did increase in air-exposed crabs, buttotal sulfated GAGs did not (unpublished results).Temporal changes in the GAG population of a tis-sue in response to injury is well known in mam-mals (Garg and Lyon, ’91; Brown et al., ’95).Therefore, the long-term effect of air exposure ongill GAGs was studied in another crab species, C.maenas, which has much larger abilities to faceprolonged air exposure than C. pagurus.

In the present study, C. maenas was exposed toair for 72 hr, in a water-saturated atmosphere,and the GAG content of its gills was determinedfollowing purification and separation. Quantita-tive and qualitative changes in the gill GAG popu-lation in response to these experimental conditionswere examined. Total polyanion and sulfated GAGcontents of anterior and posterior gills of this eu-

ryhaline species also were compared, since theyare known for exhibiting different ultrastructuraland functional properties (Compère et al., ’89;Péqueux et al., ’88; Chausson and Regnault, ’95).In this case, GAGs were quantified directly in thepapain digest.

MATERIALS AND METHODSCrabs

C. maenas were collected in the mediolittoralarea around Roscoff (North Brittany) with baitedpots in early September of 1996. They were keptin running sea water at 18°C and fed on fish(Trachurus trachurus) muscle for 1 week prior toexperiment. Four batches of 12 to 16 crabs wereused for the air exposure experiment, using theexperimental procedure described by Durand andRegnault (’98). In short, crabs were settled in in-dividual boxes (one rack of 8 boxes in a 50-L tank),immersed in running sea water for 24 hr withoutfeeding. Then, except for control crabs, which re-mained submerged, the seawater level in thetanks was progressively lowered until the crabswere totally deprived of environmental water. Air-exposed crabs were covered with a 3-cm layer ofsoaked polystyrene chips for reduction of bodywater loss. They were kept under these conditions(room temperature, 18 ± 1°C, and natural photo-period) for 3 days. After every 24-hr period of airexposure, one batch of crabs was sacrificed, andthe gills were sampled. Only male and intermoultcrabs were used.

ExtractionGills were blotted on filter paper, weighed, and

cut into small pieces in 10 vol of cold acetone andfurther homogenized using a Polytron homog-enizer. Homogenate was kept at 4°C for 24 hr andcentrifuged. The pellet was defatted by several ac-etone extractions and finally air dried. For eachcrab, 20 mg of gill dry defatted weight (ddW) wasdigested extensively with 2× crystallized papain(4 mg) in 0.1 M sodium acetate buffer (pH 6.0)containing 0.3 M NaCl, 5 mM disodium EDTA,and 5 mM cysteine hydrochloride at 62°C for 18hr; papain was activated previously as recom-mended by Scott (’60). Papain digestion wasstopped by addition of iodoacetic acid (final con-centration 10 mM), and papain digest was eitherfiltered (glass fiber, pore size 1 µm) or centrifugedat low speed to clear up the digest. Subsamplesof this crude papain digest were taken for deter-mination of both total and sulfated polyanions by

556 M. REGNAULT AND F. DURAND

the Alcian blue method and the DMMB method,respectively (see below).

Polyanion purificationCetylpyridinium chloride (CPC), which makes

insoluble complexes with polyanions at a specificelectrolyte concentration, was used for purifica-tion and isolation of gill GAGs. Furthermore, CPCalso was used in excess as a deproteinizer (Scott,’60). To 500 µl of crude papain digest, 750 µl of a5% CPC solution was added in order to discardproteolytic products with supernatant; then bi-distilled water was added progressively until allpolyanions (PAs) made insoluble complexes withCPC (final volume, 3 ml, 50 mM NaCl, 1.2% CPC).After complete precipitation, the PA-CPC com-plexes were centrifuged at 3,500g and 18°C, andthe pellet was washed with 50 mM NaCl and thenbidistilled water. The PA-CPC complexes were re-dissolved with 500 µl propanol and water (2:1),and the PAs were reprecipitated by addition of3.5 ml of a saturated ethanolic sodium acetate so-lution (Scott and Bosworth, ’90). Following PA pre-cipitation, tubes were centrifuged at 10,000g, andthe pellet was washed with cold 95% ethanol andthen 100% ethanol and dried overnight at 37°C.Polyanions recovered as Na+ salts were dissolvedin bidistilled water; a 50-µl aliquot was taken fordetermination of both total and sulfated poly-anions, as indicated previously for the papain di-gest. Hyaluronan (HA) and chondroitin sulfate(CS) standard solutions in duplicate were treatedlike the gill papain digest and were used to calcu-late the net recovery of gill purified polyanions.Na+ salts of shark cartilage CS and human um-bilical chord HA were used as standards.

Polyanion separationPurified polyanions were separated into two

groups by adding Na2SO4 to 500 µl of the Na+ saltsolution to a final concentration of 150 mM. Atthis salt concentration, sulfated GAGs and nucleicacids precipitate with CPC, whereas HA andnonsulfated GAGs do not (Scott, ’60). Followingprecipitation of the sulfated as PA-CPC complexesand their isolation by centrifugation (group 1), su-pernatant containing the nonsulfated GAGs wascarefully collected, diluted with water (finalNa2SO4 concentration, 50 mM), and precipitatedwith CPC (group 2). The PA-CPC complexes ofeach group were washed with bidistilled water,dissolved with propanol and water, and recoveredas Na+ salts, as indicated previously. CS and HAstandards were treated similarly for estimation

of their recovery from the separation process. Fol-lowing this partial separation, gill total sulfatedGAGs were quantified by the DMMB method andgill total nonsulfated GAGs by the Alcian bluemethod. Then GAGs of each group were definedby using specific enzyme digestions.

Alcian blue methodTotal polyanions were quantified by the method

of Newton et al. (’74) as modified by Scott andBosworth (’90). Strips (35 × 5 mm) of celluloseacetate membrane (pore size 0.2 µm) were soakedin 50 mM sodium acetate (pH 6.0) and subse-quently blotted on filter paper before applying thesample (4 µl forming a 3-cm-long band). Eachsample was treated in triplicate and CS (or HA)standards (from 1 to 4 µg/4 µl) in duplicate; blankstrips were used as control. Strips were stainedfor 45 min in an Alcian blue solution: 0.2% (w/v)Alcian blue 8GS (Fluka) in ethanol and water (1:1)containing sodium acetate (25 mM) and MgCl2 (30mM). They were washed in several changes of thesame solution free of dye. The stained areas werecut out and dissolved in disposable spectropho-tometer cuvettes filled with 2.5 ml of DMSO con-taining sodium acetate (25 mM), MgCl2 (25 mM),and few drops of glacial acetic acid (final pH 5.6);cuvettes were covered with a lid, and total disso-lution of the cellulose membrane was achievedwith a vortex. After 30 min at 37°C, absorbanceat 678 nm (orthochromatic peak of Alcian blue)was read against the blank. Standard solutionsof CS and HA were used for calibration of totalPA and unsulfated GAGs, respectively. The onlydisadvantage of this method was to determine theappropriate dilution of a sample containing HA,since steric exclusion of the polycationic dye by HAin concentrated solutions (>1 mg/ml) could lead tosome underestimation of the HA content of a tis-sue sample (Comper and Laurent, ’78). Therefore,our samples were diluted several times before Alcianblue staining. For some crabs, the optimal valuewas obtained at the lowest dilution, whereas forother crabs a greater dilution was needed.

DMMB methodSulfated polyanions were quantified by the

method of Farndale et al. (’86). Samples were di-luted in 4 volumes of 50 mM TRIS HCl buffer(pH 8.0). Disposable spectrophotometer cuvetteswere filled with 2.5 ml of DMMB reagent (0.0016%1,9-dimethylmethylene blue, 40.6 mM NaCl, 40.5mM glycine, and 0.1 M HCl, pH 3.0); 100 µl ofbuffered sample was poured into a cuvette, which


was covered with a lid, gently mixed by inversion,and immediately read at 525 nm against the re-agent blank. CS standards (from 1 to 5 µg/100 µl)were used for calibration. According to Farndaleet al. (’86), interference of both nucleic acid andHA at this low pH and high salt concentrationbeing negligible, this assay is assumed to be spe-cific for sulfated GAGs.

Specific digestion of glycosaminoglycansFollowing separation, GAGs solutions of individual

crabs were pooled in order to obtain 3 to 4 pools foreach air exposure period. The pools were evaporatedto dryness at 60°C and redissolved with an appro-priate water volume (total GAG ≅ 1 mg/ml).

Nonsulfated GAGs (group 2)Duplicate samples of pools were digested with

hyaluronate lyase (EC 4.2.2.1) from Streptomyceshyalurolyticus at pH 6.0 in a buffer (25 mM so-dium acetate, 150 mM NaCl) containing 10 unitsof enzyme per milliliter, for 16 hr at 37°C. Thelyase digests were purified, as indicated in theprevious purification section, and the lyase-resis-tant GAGs were recovered as Na+ salts. HA stan-dards, digested and not digested (enzyme-freebuffer), were treated like the pool samples for con-trol of digestion and recovery from purification,respectively. GAG content of undigested and di-gested pools was determined by the Alcian bluemethod. Decrease in the GAG content of the poolas a result of hyaluronate lyase digestion indi-cated the relative abundance of HA in the poolsample. HA was expressed as a percentage of to-tal nonsulfated GAGs in gill tissue.

Sulfated GAGs (group 1)Duplicate samples of these pools were digested

with chondroitinase ABC (EC 4.2.2.4) from Pro-teus vulgaris, as described by Scott and Bosworth(’90). Enzyme was dissolved in 0.25 M TRIS buffer(pH 8.0) containing 0.275 M sodium acetate, 0.6M NaCl, and 0.05% BSA as stabilizer. Bufferedenzyme solution (50 µl) was added to a poolsample (50 µl), and digestion was realized at 37°Cfor 4 hr. Chondroitinase digest was purified, asindicated in the purification section, and thechondroitinase-resistant GAGs were recovered asNa+ salts. The CS content of the pool was deter-mined as the concentration difference betweeneach digest and its control lacking enzyme. Thenthe chondroitinase-resistant GAGs were digestedwith keratanase (EC 3.2.1.103) from Pseudomo-nas spp. using the same procedure as for chon-

droitinase. Samples were digested at pH 7.5 in abuffer (50 mM TRIS, 60 mM sodium acetate, 50mM NaCl, 0.01% BSA) containing 10 units of en-zyme per milliliter, at 37°C for 4 hr. The kera-tanase digest was purified as above, and theremaining GAG, resistant to both chondroitinaseand keratanase, was quantified and assumed tobe a GAG of the Hep/HS family. The KS contentof the pool was determined as the absorbance dif-ference between chondroitinase digest and kera-tanase digest and was calibrated with a KS(bovine cornea KS) standard curve. For each en-zyme digestion, duplicate specific standards (CSor KS) were either digested (digestion control) ornot (buffer lacking enzyme). Undigested controlswere used to calculate the net recovery of samples.

ChemicalsAll GAGs used as standard and digestion en-

zymes were obtained from Sigma Chemical Com-pany (St. Quentin Fallavier, France). Alcian blue8GS, DMSO, and cellulose acetate membranewere obtained from Fluka BioChemika (St. Quen-tin Fallavier, France). DMMB was from Aldrich,and other chemicals were from Merck (Nogent surMarne, France).

GAG contents were expressed as milligramequivalent of the GAG used for calibration perunit (gram) of dry defatted weight of gill tissue(mg·g–1 ddW). Mean values ± SEM (n = 12 or 11)are given, and the significance of their changeswas estimated according to the usual tests ofeither Student or Wilcoxon-Mann-Whitney (uni-lateral tests for unpaired samples). Relative abun-dance of a defined GAG in its group was expressedas mean percentage ± SE (n = 3–4).

RESULTSCrude papain digest

In the crude papain digest of a tissue, all gly-cosaminoglycans (sulfated and nonsulfated),nucleic acids, and protein residues together arerevealed by the use of Alcian blue in the Scottand Bosworth’s (’90) method. Therefore, the gen-eral term polyanion (PA) will be used for the pa-pain digest compounds quantified by this method.In contrast, interference of nucleic acids, polypep-tidic residues, and nonsulfated glycosaminogly-cans being negligible in the DMMB assay ofFarndale et al. (’86), compounds quantified by thismethod were considered as “crude sulfated GAGs.”

As a preliminary approach to the present study,anterior and posterior gills of C. maenas were


separately analysed for their total PA and sulfatedGAG contents. C. maenas collected in late Febru-ary 1996 and kept for 2 months under laboratoryconditions (running sea water and natural photo-period) were used. Some of these crabs were ex-posed to air for 48 hr. PA and sulfated GAG contentswere determined only in the crude papain digest ofgills. No significant difference between anterior andposterior gills was observed, in submerged crabs aswell as in the air-exposed ones (Table 1). There-fore, all gills of a crab were pooled.

In control C. maenas of the present study, totalPA content of the gill was 13.69 ± 1.04 mg CSequivalent per gram of dry defatted tissue weight(n = 12), and crude sulfated GAG content was 7.71± 0.35 mg · g–1 ddW. In crabs exposed to air (Fig.1A), the PA content of gill regularly increasedthroughout the whole air-exposure period (72 hr),whereas crude sulfated GAGs remained at a rela-tively constant level, exhibiting some increase onlyat the end of this period. Values previously ob-tained for C. pagurus (collected in September of1995) and exposed to air for 20 hr are reported inFig. 1A for comparison. Similar contents of eithertotal PA or crude sulfated GAGs were observed ingills of both species collected in September andsimilarly treated prior to and during the air-ex-posure experiment. In contrast, much lower GAGcontents were observed in crabs (C. maenas) thatwere collected in late February and used for thecomparative study of the two gill types, althoughchanges observed in these contents after a 48-hrair exposure (Table 1) were in agreement withthose observed at this time in the Fig. 1A.

Purified glycosaminoglycansAt the end of every methodologic step (purifica-

tion, separation, and specific digestion), values ob-tained directly from sample measurement (grossrecovery) were corrected according to the recoveryof the corresponding standard, similarly processed,for estimation of the sample net recovery. Gener-ally, standard recovery was 90% or better at theend of each step; HA recovery (95 to 98%) usuallywas better than CS recovery. Following purification

and recovery as Na+ salts, total sulfated GAGs wereobserved to make from 92% to 97% of the “crude sul-fated GAGs” directly measured in the papain digest.

Nonsulfated glycosaminoglycansThe total unsulfated GAG content of gill was 7.06

± 0.62 mg HA equivalent per gram of dry defattedtissue weight (n = 12) in submerged crabs. The larg-est increase occurred within the first 24 hr of airexposure (Fig. 1B). At this time, the observed value(10.33 ± 0.59 mg · g–1 ddW, n = 11) indicated a 46%increase (P < 0.001). In the following hours of airexposure, the increase rate was reduced to a rela-tively constant rate: a 21.6% increase (P = 0.01)from 24 to 48 hr and a 21.4% increase (P < 0.025)from 48 to 72 hr. In crabs exposed to air for 3 days,the unsulfated GAG content of gills (15.25 ± 0.79mg HA equivalent per gram ddW) was twice as highas in crabs kept submerged.

Following digestion with hyaluronate lyase, itappeared that HA accounted for 38 ± 2.0% ofunsulfated GAGs in gills of submerged crabs, 57± 8.4% and 53 ± 0.3% in the 24- and 48-hr air-exposed crabs, respectively, and finally, 42 ± 7.5%in the 72-hr air-exposed crabs. Since hyaluronatelyase from S. hyalurolyticus, unlike other hyalu-ronidases, is inactive on chondroitin (Ohya andKaneko, ’70), the other component of nonsulfatedGAGs isolated from gill was assumed to be chon-droitin or a chondroitin-like compound (Fig. 2).Estimation of both HA and “chondroitin” contentsof gill, based on the HA relative abundance, clearlyshowed that HA was fully responsible for the to-tal increase in nonsulfated GAGs observed withinthe first 24 hr of air exposure. When these condi-tions were prolonged, chondroitin was initiallymainly and then fully responsible for the observedchanges in total nonsulfated GAGs. However, bothHA and chondroitin enrichments of gill tissue dur-ing the whole experimental period (+4.0 mg · g–1

ddW and +4.5 mg · g–1, respectively) were similar.

Sulfated glycosaminoglycansThe total sulfated GAG contents of C. maenas

gills were 7.52 ± 0.51 mg CS equivalent per gram

TABLE 1. Total PA and sulfated GAGs in the crude papain digest of anterior gills (2nd to 5th pair) and posterior gills(last 3 pairs) of C. maenas (March crabs)1

Control (submerged) crabs 48-hr air-exposed crabsAnterior gills Posterior gills Anterior gills Posterior gills

Total PA 8.04 ± 0.65 8.14 ± 0.91 12.92 ± 1.05 13.12 ± 1.09Sulfated GAGs 4.76 ± 0.58 4.87 ± 0.57 5.80 ± 0.95 6.43 ± 0.931Mean value ± SEM (n = 8).


of dry defatted tissue weight in control crabs, 8.01± 0.39 and 8.36 ± 0.64 mg·g–1 in crabs exposed toair for 24 and 48 hr, respectively, and finally, 9.90± 0.37 mg CS per gram of dry defatted tissueweight at the end of experiment (Fig. 1B). Al-though a 32% increase in total sulfated GAGs wasobserved after 3 days of air exposure, only the18% increase observed between 48 and 72 hr wassignificant (P = 0.025).

Digestion of gill sulfated GAGs with chondroi-tinase ABC indicated that chondroitin sulfates(CSA, CSB, and CSC) were forming 22 ± 6.4% oftotal sulfated GAGs in gills of control crabs; thispercentage increased from 26 ± 2 to 44 ± 8%within the last 24 hr of air exposure (Fig. 2). Es-timation of the gill CS content from these per-centages showed that CS was a minor componentof gill GAGs, and although its content doubled be-tween 48 and 72 hr, it contributed moderately(+2.65 mg·g–1 ddW) to the total GAG increase.However, CS was the only sulfated GAG that wasaffected by the present experimental conditions.In control crabs, KS and HS were forming 30%

Fig. 1. Total polyanion (PA) and GAG contents of gills ofC. maenas exposed to air for 3 days; mean value ± SEM (n =12). A: PA and “crude sulfated GAGs” determined in the pa-pain digest of C. maenas gills (open symbols); previous datasimilarly obtained for gills of C. pagurus exposed to air for

20 hr (n = 12) are reported (plain symbols). B: Purified gly-cosaminoglycans, quantified after purification and separationinto two groups, in C. maenas gills. Total sulfated (mg CSequivalent per gram of gill dry defatted weight) and totalunsulfated (mg HA equivalent per gram of gill ddW).

and 48% of the gill sulfated GAGs, respectively.Although lower percentages were observed in the72-hr air-exposed crabs (i.e., 22% and 34%), thegill KS and HS contents remained constantthroughout the whole experiment.

Total glycosaminoglycans in C. maenas gillsThey were estimated by summing total sulfated

and total nonsulfated GAGs obtained after separa-tion (Fig. 2). Significant changes (P < 0.01) in gilltotal GAGs occurred within both the first and thethird day of air exposure. Finally, a 73% increase intotal GAGs was observed as a result of a 3-day airexposure of C. maenas. GAGs composed of acetyl-galactosamine (GalNAc) and GAGs composed ofacetylglucosamine (GlcNAc) contributed 60% and40%, respectively, of the total GAG increase.

DISCUSSIONGlycosaminoglycans are very ubiquitous poly-

mers found in most phyla from bacteria to mam-mals and for which the main structural featureshave been preserved during evolution. These


polysaccharidic chains are specifically character-ized by a high number of anionic groups, whichare fully ionized under physiological conditions(Comper and Laurent, ’78) and which are assumedto have constituted a shield against hydrated elec-trons, especially in the prebiotic era (Scott, ’75).According to this author, this would explain theirsurvival during evolution and their preponderancein biological systems compared with the paucityof cationic polymers. In Crustacea, glycosami-noglycans were studied with the purpose of find-ing phylogenetic implications (Rahemtulla andLovtrup, ’75, ’76; Cassaro and Dietrich, ’77; Naderet al., ’88). From these studies and a few others itappeared that all GAG types were present inCrustacea, sometimes under a specific form (Senoet al., ’74; Hovingh and Linker, ’82). However, nei-ther their localization in tissues nor their possiblerole was approached.

The present results show that sulfated GAGs(7.52 ± 0.51 mg · g–1 ddW) were as abundant asnonsulfated GAGs (7.06 ± 0.62 mg · g–1 ddW) inthe gills of the intertidal crab C. maenas. The rela-tive abundance of each GAG in these two groups,estimated by specific enzyme digestion, showedthat HS and “chondroitin” were the major sulfated

and nonsulfated GAGs, respectively. A better in-sight into the GAG population of C. maenas gillis obtained by expressing every GAG as a part ofgill total GAGs, which appeared to be composedof CS (12%), KS (15%), HA (18%), HS (24.5%), andchondroitin (30%). These percentages need furtherexplanation. In lobster, Rahemtulla and Lovtrup(’76) observed that the nonsulfated GAG fractioncontained 30% hyaluronan and 70% chondroitin(a similar ratio was observed in gills of control C.maenas). They also pointed out that KS waseluated with salt solutions of both very low andhigh ionic strength. Precipitation of the KS-CPCcomplex is known to be special (Scott, ’60), owingto a great variability in the KS sulfation degree(Oeben et al., ’87). According to Scott and Bos-worth (’90), undersulfated KS, which representedaround 10% of total KS in mammal cornea, pre-cipitated only at 50 mM NaCl. Therefore, this KSform could not be present in our group 1 and waseither lost or collected in group 2. The relativeabundance of every GAG in total GAGs was re-calculated on the basis of a possible KS loss; KS(2.5 versus 2.24 mg · g–1 ddW) would form 17%(versus 15%) of total GAGs, but the preceding val-ues for other GAGs were not affected. In the other

Fig. 2. Air exposure–induced changes ingill GAGs of the crab C. maenas. TotalGAGs (mg · g–1 gill dry defatted weight) wasthe sum of total sulfated GAGs (mg CSequivalent per gram) and unsulfated GAGs(mg HA equivalent per gram) determinedafter purification and separation; meanvalue ± SEM (n = 12). Relative abundanceof CS, HS, and KS in the sulfated groupwas determined following successive diges-tion by chondroitinase ABC and keratanase.Relative abundance of HA in the unsulfatedgroup was determined following digestionby the hyaluronate lyase. The lyase-resis-tant GAG was assumed to be chondroitin(see main text). CS: chondroitin sulfatesCSA, CSB, and CSC; KS: keratan sulfate;HS: heparan sulfate.


alternative, the presence of undersulfated KS ingroup 2 would lead to overestimation of “chon-droitin,” determined as the nonsulfated GAGresistant to hyaluronate lyase. This second hy-pothesis cannot be ruled out completely so longas this lyase-resistant fraction is no longer inves-tigated by some other method. However, on thebasis of the presumed amount of undersulfatedKS in total KS, gill chondroitin content would beslightly overestimated

When C. maenas was exposed to air for 3 days,significant changes in the GAG population wereobserved in its gills. These changes were mainlycharacterized by (1) a marked increase in theamount of nonsulfated GAGs (HA and chon-droitin), whereas the amount of sulfated GAGs(CS excepted) remained constant, and (2) the factthat the individual variations in the three GAGcontents were uncoupled in time.

The first effect of air exposure was an increasein the gill hyaluronan amount within the first 24hr. At this time, no change occurred in the amountof other GAGs. In contrast to these sulfated GAGs,which are synthesized in the Golgi apparatus, re-quiring a specific oligosaccharide as primer andbeing modified by epimerization and sulfation re-actions before extrusion into the cell and the ex-tracellular space (Höök et al., ’84; Jackson et al.,’91), HA is synthesized in the plasma membrane,does not require a primer, and is extruded directlyin the extracellular matrix without any post-polymerization modification (Prehm, ’84). A fewhours (4 to 5 hr) are necessary for the synthesisof a complete HA chain (Heldin and Pertoft, ’93;Kitchen and Cysik, ’95), and the overall turnoverrate of HA is surprisingly rapid (i.e., half-life, 12hr to a few days) (Laurent and Fraser, ’92). Fur-thermore, HA biosynthesis is activated when in-tracellular pH is decreased (Laurent and Fraser,’86). These characteristics would be in favor of amore rapid response of HA than of other GAGs toair-exposure stress. However, the main interestof the initial HA increase rests on the intrinsicproperties of HA (Comper and Laurent, ’78). Theseproperties are partly due to the large size of theHA chain and its stiffness and propensity to oc-cupy all available space at low concentration. Al-though HA has only one charged COO– group perdisaccharide unit, it behaves like other sulfatedGAGs as a cation-exchange resin and determinesthe micro-ion distribution and transport in the ex-tracellular space as a function of the electrolyteconcentration. For HA, the critical electrolyte con-centration (CEC), defined by Scott (’85), is very

low. Furthermore, by forming entangled networksin the extracellular space, the HA molecule has avery specific ability for steric exclusion of macro-molecules. The nonideal osmotic behavior of HAis also much more pronounced than that of otherGAGs at low concentration. Fluid loss from an HA-containing tissue causes a disproportionately largeincrement in the osmotic restoring force, whereasdilution causes a negligible diminution of the os-motic pressure (Comper and Laurent, ’78). Be-sides, the coil structure of the HA molecule andits amphiphilic character (opposite hydrophobicand hydrophilic clusters; Scott, ’89) contributes,by mechanically immobilizing water within thecoil, to preservation of tissue hydration. There-fore, HA has a prominent role in the water ho-meostasis of a biological system (Laurent andFraser, ’92). In air-exposed C. maenas, increasein gill HA content, which mainly occurred withinthe first 24 hr of air exposure but continues at alower rate in the next 24 hr, would favor osmoticadjustment and maintain tissue hydration. In thecrabs of this study, osmotic pressure of the extra-cellular compartment was observed to slightly butsignificantly increase only from 48 to 72 hr of airexposure (Durand and Regnault, in press).

In mammals, increased HA synthesis in re-sponse to injury and in the early phase of woundhealing is well known (Laurent and Fraser, ’92).Furthermore, the initial increase in HA is replacedby increased levels of sulfated GAGs as the woundages (Garg and Lyon, ’91; Tammi and Tammi, ’91;Brown et al., ’95). Although damage caused tocrabs by imposed air exposure might be less del-eterious, the sequence of gill individual GAG in-crease in response to air exposure presented somesimilarity with that observed in response to in-jury. Increase in gill HA content was followed byan increase in gill chondroitin content. Chon-droitin, like HA, is not sulfated; its synthesis isknown for requiring the same conditions as CSsynthesis (protein core in the Golgi), althoughchondroitin synthesis in bacterial membrane re-cently was reported (Lidholt and Fjelstad, ’97).Binding of chondroitin to a protein core neverthe-less made it a true proteoglycan in contrast toHA. In gills of control crabs, chondroitin was themajor nonsulfated GAG (4.38 versus 2.67 mg · g–1

ddW for HA). Increase in gill chondroitin contentstarted during the second day of air exposurewhen the HA synthesis rate apparently was lower.The main chondroitin increase occurred during thethird day of air exposure, when the sulfated forms(CSA, CSB, CSC) also increased. The strong effect


of CS on water molecule and microcation (Mg2+,Ca2+) distribution in a biological system, knownas a characteristic of GAGs (Comper and Laurent,’78) and clearly evidenced in vitro (Günther et al.,’97), suggests that CS could possibly work in re-lays for preservation of the tissue homeostasiswhen gill HA content is constant.

In C. maenas exposed to air for 3 days, the totalGAG amount in gills increased from 14.6 to 25.2mg · g–1 ddW. GAGs composed of GalNAc (chon-droitin, CS) contributed to 60% of this increase, andHA, composed of GlcNAc, contributed to 40%. In amore infralittoral species (C. pagurus), GlcNAc syn-thesis occurred in gills and hindgut but not inbranchiostegal epidermis in response to a 24-hr airexposure (Regnault, ’96). Furthermore, totalpolyanions, but not sulfated GAGs, increased ingills of this species following a 20-hr air exposure(see Fig. 1A), as observed in this study in gills ofC. maenas. At this time, the HA amount synthe-sized in gills of C. maenas was twice as high asthe GlcNAc amount synthesized in gills of C.pagurus. This supports our previous assumptionthat GlcNAc, synthesized in response to air expo-sure, is incorporated into macromolecules. Thesemolecules can be now defined as GAGs and moreprecisely as hyaluronan, since this is the uniqueGAG containing GlcNAc that was affected by airexposure in this space of time.

Crabs deprived of environmental water have toface dehydration, ionic adjustment, acidosis of theextracellular compartment, and internal NH4

+

overload (Truchot, ’79; Burnett, ’88; Regnault, ’94).Gills play a major role in regulation of these func-tions, and nonimpairment of epithelial cells ap-pears to be of vital importance. These cells arebathed in both haemolymph and branchial water,two fluids whose ionic composition is modified (in-crease and decrease in Na+ content, respectively)and pH is decreased during air exposure (Burnettand McMahon, ’87; Harris and Santos, ’93). Inmammals, the basal membranes of cells in con-tact with circulating fluids (endothelial cells, lungepithelium, urinary bladder, gut) are coated withan extracellular matrix containing GAGs, theseacting as a selective filter. In Crustacea, endothe-lial cells of a blood artery were observed to becoated with a similar matrix, defined as glycocalyx(Bennett, ’63). The presence of acid mucopolysac-charides and polyanionic sites also was demon-strated in both the subcuticular space overlyingthe apical membrane of hindgut cells (Holdich andRatcliffe, ’70) and the epicuticular surface coat ofgill epithelium (Compère and Goffinet, ’95). Thus,

the existence of GAGs in the extracellular coat-ing of both apical and basal membranes of thesesalt-transporting epithelia is strongly suggestedin Crustacea. Because GAGs are able to immobi-lize any kind of free microcations, they might ef-ficiently contribute to regulate salt, H+, and NH4

+

concentration in haemolymph and avoid hypercon-centration of these ions in branchial water of air-exposed crabs. Furthermore, according to theirspecific CEC, GAGs spontaneously exchange anytrapped cations with environmental ones when pHand ionic strength are increased. This would ex-plain, in the particular case of air-exposed crabs,the surprisingly sudden NH4

+ output at reim-mersion in sea water. This large ammonia outputfirst described in C. pagurus (Regnault, ’94) wassimilarly observed in both C. maenas and Necorapuber (Durand and Regnault, ’98). Crabs of thisstudy were observed to release, within the first 5min following reimmersion, an ammonia amount(85 µmol) that corresponded to a 60-fold increasein their preimmersion ammonia excretion rate(Durand and Regnault, ’98). As discussed earlier(Regnault, ’94), this ammonia output could not fig-ure a realistic metabolic rate and could only beexplained by the ammonia unloading of some ex-tracellular compartment, which was yet unde-fined. From the present results and by assumingthat a disaccharide unit of sulfated and non-sulfated GAGs has 3 and 1 negative charges, re-spectively, an estimation of the number of bindingsites in the gills of a 72-hr air-exposed C. maenaswas attempted. It appeared that 116 binding sitesper gram of dry defatted tissue weight (i.e., 21.4sites per gram of tissue wet weight) would beavailable and that around 30 µmol of ammonia(NH4

+ ions) might be trapped in the gill extracel-lular matrix at the end of the air-exposure pe-riod. This form of ammonia storage would accountfor one-third of the observed ammonia output atreimmersion. Therefore, we propose that gill ex-tracellular matrix and its GAGs play a major rolein ammonium ion storage and might possibly fig-ure the extracellular compartment that we werelooking for.

In contrast to molluscs, which are a particularlyrich source of sulfated GAGs and—as a matter offact—the more investigated invertebrate taxa,little attention has been paid to the GAGs of Crus-tacea until now. This study of GAGs in the gillsof a crab shows that enhanced synthesis of someGAGs and resulting changes in the GAG popula-tion did occur in the gills of a marine crab underimposed aerial conditions. Several possible roles


logically can be ascribed to these GAGs (tissuehydration, ionic regulation, temporary NH4

+ stor-age). Further investigations are needed to cyto-chemically localize the GAGs in gill (basal and/orapical cell membrane), to seek for their presencein gut epithelium, and to demonstrate their as-sumed function. In any case, these polyanionicpolymers appear to have a determining role inphysiological adaptation of decapod crustaceans,especially when these experience transition fromwater to land.

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Glycosaminoglycans in gills of an intertidal crab (Carcinus maenas): Changes in the GAG population in response to prolonged air exposure