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Carbodiimide-induced labelling of sea urchin surfaces (Echinoidea, Echinodermata)
Camp. Biockem. Ph~siol. Vol. I IOB, No. 3. pp. 477482. 1995 Copyright 1~: 1995 Elsevier Science Lid Printed in Great Brkain. All rights reserved 030%0491!95-$9.50 + 0.00
Pergamon
Carbodiimide-induced labelling of sea urchin surfaces (Echinoidea, Echinodermata) Irina V. Grigolava and J. Douglas McKenzie Scottish Association for Marine Science, P.O. Box 3, Oban, Argyll, Scotland, U.K. A method for radiolabelling echinoderm body surfaces, using water-soluble carbodiimide was developed. The number of carboxyl groups reacting with carbodiimide was estimated as 20 to 30 nmole g-’ dry weight of sea urchin spines. There were no phosphate groups accessible to carbodiimide on the surface coat. Several enzymes were used to release bound radiolabel from the spines labelled with carbodiimide and radioactive amino acid. Results obtained with proteases and hyaluronidase showed that radiolabel was bound to the glycoprotein and/or protein molecules on the extracellular surface of the spine epidermis. Key words: Antifouling; Cuticle; Echinoderm; Invertebrate;
Protease; Radiolabelling; Surface coat;
Water-soluble carbodiimide (EDAC). Comp. Biochem.
Physiol.
IlOB, 477482,
1995.
Introduction Holland and Nealson (1978), using TEM, showed that the epidermis of all echinoderms has a multi-layered extracellular coat, usually termed the cuticle. In a series of papers (McKenzie, 1987, 1988) it was shown that the outermost part of this cuticle consists of a fibrous layer which was termed the fuzzy coat. It was suggested that fuzzy coat is a glycocalyx, similar to those found on the extracellular coats of some other marine invertebrates (BereiterHahn et al., 1984) and on many cell surfaces (Ito, 1969). The integument, which comprises the epidermal epithelium plus the basi-epithelial nerve plexus (Holland, 1984), is known to be crucial in respiration, food absorption, adhesion and sensation. It has been suggested that the primary function of the outermost surface of the extracellular coat is antifouling due to its surface properties (McKenzie, 1987, 1988). Although the echinoderm cuticle has been studied with light and electron microscopy, _ Correspondence lo: I. V. Grigolava,
Scottish Association for Marine Science, P.O. Box 3, Oban, Argyll, PA34 4AD, U.K. Tel. 44 631 62244; Fax 44 631 65518. Received 27 April 1994; revised 22 September 1994; accepted 28 September 1994. 477
there are very few biochemical studies on its composition and structure. The difficulties in investigating the cuticle are largely due to its extreme thinness (usually less than two microns) and its close attachment to the underlying epidermis (Holland, 1984). Another difficulty is that the chemical composition of the cuticle’s layers are likely to be similar to mucus secretions released from the animal. Invertebrate mucus contains polysaccharides (mucopolysaccharides) chemically similar to surface coat glycosaminoglycans (Souza Santos and Sasso, 1968, 1970). The surface coat of eukaryotic cell membranes has been well studied and contains proteoglycans composed of glycosaminoglycan chains covalently bound to a protein core (Spiro, 1972; Kjellen and Lindahl, 1991). The purpose of our study was to develop a chemical method for labelling the various layers of the cuticle and to investigate the chemical composition and properties of this extracellular coat. For chemical labelling we have used water-soluble carbodiimide and radioactive amine. Cell membranes are usually impermeable to water-soluble carbodiimide (Kozlov and Skulachev, 1977), thus only extracellular surfaces will be modified. At acidic pH
478
I. V. Grigolava
and J. D. McKenzie
(pH 4-6) carbodiimide interacts with free carboxyl and phosphate groups. After the addition of amine, amide is formed (Noller, 1966): RN = C = NR + R’COOH + RNHCONHR
+ R2NH2 + R’CONHR’
RN=C=NR is where carbodiimide, R’ COOH is carboxyl groups on the surface of spine, RNHCONHR is urea derivative and R’ CONHR’ is amide formed on the surface.
Materials and Methods Materials Experiments were carried out on the spines from two sea urchin species: Echinus esculentus (L.) and Psammechinus miliaris (Gmelin). Both were collected from sub-littoral populations around Argyll, Scotland by dredging. Chondroitinase ABC (chondroitine ABC lyase) from Proteus vulgaris; hyaluronidase Type IV-S, bovine; lysozyme from chicken egg white; neuraminidase from Salmonella tyType VII-T, phimurium; alkaline phosphatase bovine, protease Type XIV (pronase E), bacterial; protease Type XV (dispase), bacterial; and trypsin Type IT, from porcine pancreas-all were from Sigma, St Louis, MO. 14C glycine (1184mCi mmol-‘) and ‘H glycine (20mCi mmol-‘) were from Sigma. Amino acid mixture, labelled with tritium (high specific activity mixture of five amino acids) was from Amersham, Bucks, U.K. A tissue solubilizer (TS-1) and scintillator (Aquasafe 500) were from Zinsser Analytic. All other chemicals were from Sigma. Methods Chemical radiolabelling of spines was carried out as follows: 0.1-1.0 g of spines (between 30-300 spines) were cut from sea urchins (three animals in each experiment) and then rinsed several times with (2-20 ml) buffer Nl CO.1 M 2-/N-morpholino/ethanesulfonic acid (MES), 2% NaCl and 10 mM NaN, (sodium azide), pH 5.0). An inhibitor of proteases, 50pM phenylmethylsulfonyl fluoride (James, 1978) was present in the rinsing buffer. To obtain dry weight, some spines were dried in an oven at 50°C to constant weight. The rest of the spines were incubated with 30 mM water-soluble carbodiimide-I-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC) at ambient temperature in buffer N2 (0.6 M MES, 0.5% NaCl, pH 5.0). In the controls carbodiimide was omitted. After 30 min of incubation, the spines were rinsed with buffer N3 (0.1 M 3-/N-morpholino/ propanesulfonic acid (MOPS) and 1% NaCl, pH 8.2) and then transferred into the modified
buffer N3 containing 10 mM amino acid (1 PCi ml-‘) of either 14C glycine, ‘H glycine or amino acid mixture, labelled with tritium. After 20min of incubation at ambient temperature, the spines were rinsed with buffer N4 (0.1 M unlabelled amino acid, 2% NaCl solution, 1 mM NaN, and 50 p M phenylmethylsulfonyl fluoride, pH 8.2) until no radioactivity could be detected in the rinsing buffer. The modified spines were then incubated in tissue solubiliser for 4 hr at 50°C to release all bound radioactivity. Water-soluble carbodiimide was also used for a two-step modification of free amino groups on the surface coat. 10 mM EDAC was incubated with 3 mM 14C acetic acid (1 PCi ml ’) in buffer Nl pH 4. After 20 min of incubation, the pH was adjusted to pH 8 with triethanolamine. Spines were added to this reaction mixture and incubated for another hour. In control experiments 14C acetic acid was incubated with spines at pH 8 but with EDAC omitted. The spines were rinsed with 0.1 M sodium acetate in 1% NaCl, pH 8. Finally, the spines were treated with tissue solubilizer as before. Additional buffer compositions were: M MOPS N5 for protease digestion-IO.05 and 0.9% NaCl, pH 7.5). N6 for hyaluronidase treatment--(O. 1M MES and 0.9% NaCl, pH 5.3). treatment--lo. 1M N7 for neuraminidase MES. 0.9% NaCl, 0.004 M CaClz, pH 5.5). ABC-as N5, but N8 for chondroitinase pH 8.0. phosphatase treatment-{O. 1M N9 for triethanolamine, 0.9% NaCl, 0.5 mM MgCl, and 0.2 mM ZnCl,, pH 9.7). M MES NlO for lysozyme treatment-(0.1 and 0.9% NaCl, pH 6.3}. of different enzymes with The interaction labelled echinoderm surfaces was studied. For this purpose sea urchin spines were rinsed first with buffer Nl containing protease inhibitor, and then, with an appropriate buffer for the enzyme (without a protease inhibitor and NaN,). Spines were incubated at 50°C for 1 or 2 hours with each of the enzymes. The spines were then rinsed extensively with buffer Nl and radiolabelling was carried out as described above. Control spines were treated similarly except that the enzymes were omitted in the pretreatment medium. To further study effect of enzymatic pretreatment on the labelling process, samples of spines (each 100 mg) were incubated with pronase E at 50°C and control spines were incubated without pronase, for 10 min (sample 1), 20min (sample 2), 30 min (sample 3) 60 min (sample 4) and 2 hr (sample
Radiolabelling
of sea urchin
5). Each sample was then labelled with EDAC and r4C glycine as described above (Fig. 1). The effect of pretreatment with the chelating agent, ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis(fl-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA) on the reaction of EDAC with the surface was examined. 10 mM of EDTA and 10 mM EGTA were added to the medium, N5 pH 7.5 and incubated at 50°C for 2 hr. The modification with carbodiimide was then made. Radioactivity measurements were carried out on a liquid scintillation counter (12 19 Rackbeta, LKB Wallac).
Results Modification of sea urchin spines with water soluble carbodiimide allowed a quantitative estimate of free carboxyl groups to be made. As cell membranes are impermeable to EDAC, were only extracellular carboxyl groups modified. The total bound amino acid was calculated as follows: total bound amino acid (nmole g- ’ dry weight of spines) equals the radioactivity released by TS-1 from EDAC labelled spines minus the radioactivity released by TS-1 from control spines. Twenty experiments were carried out on spines from E. esculentus. All the measurements gave the content of free carboxyl groups as 20 to 30 nmole gg’ dry weight of spines. In five experiments carried out on P. miliaris the estimated free carboxyl groups were found to vary between 20 to 30 nmole gg’ dry weight of spines. The amount of chemical labelling was affected by the presence of sodium azide. If sodium azide was omitted, the observed number of free carboxyl groups was lower, though this was mainly due to the increase of radioactivity in the control spines. In the absence of sodium azide, TS-1 released half of the label (10 to 15 nmole g-‘) in the control spines compared to the label released in EDAC-modified spines. However, in the presence of both sodium azide and protease inhibitor this figure was always less than 6 nmole gg ‘. Spines can metabolize amino Table
I. Effect of pretreatment Without enzymatic pretreatment
Added amount mg ml-’ Incubation time, h Bound label in nmole g-’ dry weight* Bound label %
acids and this was measured as bound label in the absence of sodium azide. Sodium azide as an inhibitor of transport processes prevented metabolic uptake of the label. The absence of protease inhibitor prior to modification resulted in a lower number of modified carboxyl groups. No binding of radiolabel was found during the experiments which attempted to modify amino groups on the echinoderm surface with EDAC modified radiolabelled acetic acid. The amount of radioactivity released by tissue solubiliser was similar to the controls (not shown). The stability of the label bound to carboxyl groups was studied. It was found that after incubation at 5OC for 24 hr only a small amount of label was released: from 0% to 6% of the total bound label. However, if protease inhibitor was not present in the rinsing solutions, the spontaneous release of label was higher: from 30% to 40% of the total bound label. The modification of spines with EDAC was carried out at different pH. It was found that the difference in the labelling at pH 4.5, pH 5.0 and pH 5.5 was small. The labelling at pH 5.0 was three times higher than at pH 6.0. Table 1 shows the results of enzymatic pretreatment of spines and its effect on the reaction with EDAC. Pronase E and trypsin prevented the labelling of the surface, at the same time none of the three other enzymes: hyaluronidase, phosphatase or lysozyme had any effect on the reaction. No effects of the chelating agents, EDTA and EGTA were found (not shown). Figure 1 shows the effect of pretreatment with pronase E on labelling of spines with EDAC. An incubation period of 1 hr with pronase was sufficient to totally prevent labelling of spines. Pronase E and also other proteases are widely used to release proteoglycans from the cell surfaces (Spiro, 1972; Edward and MacKie, 1989). Results for enzymatic digestion of labelled spines are given in Table 2. Pronase, dispase and trypsin released a high proportion of the bound label. Hyaluronidase released only 14% of the bound label. In the absence of the
of spines on the reaction
Pronase 0.5
E
Trypsin
0.5 2
5
3.5 17.5
1
2
1
20 100
I8 100
I
0
5
0
419
surfaces
1
with water soluble carbodiimide
Phosphatase 0.5 1 I9 95
Hyaluronidase 0.5 2
>I8 100
*Bound label, as released radioactivity by TS-I in samples treated with EDAC and amino radioactivity (by TS-I) in control spines (minus EDAC). Spines were incubated with the enzyme at 50 C, labelling was then carried out.
Lysozyme
10 2
5 2
> 18 100
>18 100
acid, subtracted
by released
480
1. V. Grigolava
04
Q
and J. D. McKenzie
x
l&Y
w
150
time, min Fig. 1. Modification of spines with EDAC and 14C glycine, pretreated with pronase E. Spines were pretreated with pronase E at SO’C for the times shown. Pronase was removed by rinsing with buffer and the spines treated with EDAC and radioactive amino acid. The bound radiolabel, nmole g-’ dry weight of spines (as released radioactivity by tissue solubilizer) was measured.
enzyme approximately 6% was released spontaneously, suggesting that hyaluronidase released 8% of the total label (n = 5). Protease digestion kinetics of radiolabelled spines at 37°C and 50°C are shown in Fig. 2. At 37°C 1.5 mg ml-’ protease released 90% of the label after 3 hr of incubation and a total of 95% after 24 hr of incubation. At 50°C 0.5 mg ml-’ protease released more than 80% of the radioactive label after 3 hr of incubation and a total 90% after 24 hr. The results presented in Figs 1 and 2, and also in Tables 1 and 2 were from a typical experiment carried out on spines from E. esculentus. Similar results on the protease digestion of labelled spines were obtained for P. rniliaris (not shown). Neither neuraminidase (0.9 units ml-‘) or chondroitinase ABC (2 units ml-‘) released the label from radiolabelled sea urchin (E. esculentus) spines after 24 hr of incubation with labelled spines at 37°C.
Discussion A method of labelling echinoderm body surfaces using water soluble carbodiimide (EDAC) was developed that requires only very
Table 2. The release of the label from spines treated Without any treatment: spontaneous release of label mg ml-’ enzyme* Released label in nmole g-’ and in %t
< I.5 <6
mild conditions. This method of labelling the echinoderm surface has been used to study the turnover rate of echinoderm surface coats (Grigolava and McKenzie, 1994). The amount of free carboxyl groups available on the extracellular surface and the lack of amino groups accessible to EDAC treated acetic acid, indicates that the echinoderm surface is acidic. Histochemical studies with light microscopy have suggested that the echinoderm cuticle contains acid polysaccharides (Souza Santos and Sasso, 1970; Engster and Brown, 1972). Acid glycoproteins have also been shown histochemically to exist on the surface of echinoderm egg coat (Sousa et al., 1993). The pH dependence of the reaction of EDAC with the surface indicates that the modified groups have a comparatively high pK value. The label is stable even at relatively high temperatures and for up to 24 hr. Protease inhibitor was needed to produce stability of the label: spontaneous release of the label occurred if protease inhibitor was absent. This could be due to the action of endoproteases. These are present in large quantities in spines (Canicatti, 1990; Canicatti and D’Ancona, 1990) and were probably released by damage to the spines. This may explain the low
with water soluble carbodiimide
TS-I 25 100
Promise 0.5 24 96
E
Dispase 5.0 24 96
and radioactive
Trypsin 0.5 22 88
amine
Hyaluronidase 0.1 3.5 14
*Enzyme was added once at the beginning of incubation and the same amount. later after 6 hr of incubation. tFigures represent the difference between released label in treated with EDAC spines and in controls (minus Spines were incubated with (or without) enzyme for 24 hr and with TS-I for 4 hr. at 50 C.
EDAC).
Radiolabelling
of sea urchin
481
surfaces
0 0
0
ldo
!zib
3!50
time, min Fig. 2. Released radioactivity by pronase E. 0, radioactivity (as released by tissue solubiliser) was radioactivity by pronase at 50°C total radioactivity dry weight
of observed modified groups. Endogenous proteases may have digested some of the surface before the modification if protease inhibitor was not present in rinsing buffers. Enzymatic pretreatment of spines before the modification of its surface (Table I) and also interaction of different enzymes with radiolabelled surfaces (Table 2) showed that carbodiimide-reactive carboxyl groups belong to protein moieties of the glycoprotein. Pretreatment with pronase E and trypsin (but not hyaluronidase) prevented labelling. Pronase E, dispase and trypsin released the label from labelled spines, but neurominidase and chondroitinase did not and hyaluronidase released only a small portion of the label. Since phosphatase had no effect on EDAC labelling it seems that there are few if any phosphate groups, associated with the extracellular surface. Lysozyme cleaves bonds between N-acetylglucosamine and N-acetylmuramic acid in mucopeptide, a proteoglycan of bacterial cell walls. As lysozyme pretreatment had no effect on labelling, it suggests that such compounds are not important in the structures of the echinoderm surface. The release of some carbodiimide-induced label by hyaluronidase indicates that there are structures such as hyaluronic acid, chondroitin or chondroitin sulphate on the surface of echinoderms. The amount of label released was, however, small. This may suggest that such structures form only a minor component of the surface. Alternatively, the enzyme digests parts of glycosaminoglycans which do not contain free carboxyl groups. It is known that in many glycosaminoglycans the carboxyl groups are heavily sulphated (Kjellen and Lindahl, 1991) and would not be available for labelling. In number
released radioactivity by pronase at 37”C, total IlOOdpm gg’ dry weight of spines. 0, released (as released by tissue solubilizer) was 1300 dpm g-’ of spines.
preliminary experiments (not shown in this paper) chondroitinase ABC digested some surface components in spines of 35S-labelled sea urchin. The lack of effect of hyaluronidase pretreatment on labelling, therefore, does not necessarily mean that polysaccharide chains, which are the substrates for this enzyme, are absent. The kinetics of radiolabel release as a result of pronase E treatment of the labelled surface indicates the speed of enzymatic digestion of the protein structures. This is being used in studies of cuticle structure and metabolism (Grigolava and McKenzie, 1994). The carbodiimide-accessible compounds on the sea urchin surface are very sensitive to proteases (endoproteases). The digestion of echinoderm surfaces with endoproteases may have a biological role in the prevention of fouling. Under certain circumstances the echinoderms may be able to “clean” their surfaces by shedding the outermost layer via the release of endoproteases. Carbodiimide-induced labelled surfaces are useful for further studying of different enzymatic reactions on the surface which will supply interesting information about structure of the surface. This labelling method will be used in future to isolate and chemically analyse glycosaminoglycans and proteins present in the surface coats of the cuticle. AcknoM,I~dgem~no-The research discussed here is part of a European project to develop novel antifouling structures and is funded by Marine Technology Directorate (SERCU.K.) and the European Commission as part of the MAST II program (MASZ-CT9l-0009). We would like to thank the Director of Dunstaffnage Marine Laboratory for the support and provision of facilities, also M. Edward, I. Kozlov. L. Newton, M. Kelly, D. Harris and D. Watt for useful discussion and suggestions.
482
1. V. Grigolava
amd J. D. McKenzie
References Bereiter-Hahn J., Matoltsy A. G. and Richards K. S. (Eds) (1984) Biology qf rhe Integument. Vol. I. Inoertehrares. Springer, Berlin, Heidelberg. Canicatti C. (1990) Protease activity in Holorhuria polii coelomic fluid and coelomocyte lysate. Comp. Biochem. Physiol. 95B, 145-148. Canicatti C. and D’Ancona G. (1990) Biological protective substances in Marthasterias glacialis (Asteroidea) epidermal secretion. J. Zool. Lond. 222, 445-454. Edward M. and MacKie R. M. (1989) Retionic acid induced inhibition of lung colonization and changes in the synthesis and properties of glycosaminoglycans of metastatic B16 melanoma cells, J. Cell Sci. 94. 537-543. Engster M. S. and Brown S. C. (1972) Histology and ultrastructure of the tube foot epithelium in the Phanerozonian starfish, Astropecten. Tiss. Cell 4, 503-518. Grigolava I. and McKenzie J. D. (1994) Biochemical studies of echinoderm surface coats using radiolabelling techniques. In Echinoderms Through Time (Echinoderms Dijon) (Edited by David B., Guille A., Firal J. P. and Roux M.), in press. Bulkema, Rotterdam, Netherlands. Holland N. D. (1984) Echinodermata: epidermal cells. In Biology of the Integument. Vol. I. Inuertebrales (Edited by Bereiter-Hahn J., Mattoltsy A. G., Richards K. S.), pp. 7566774. Springer, Berlin, Heidelberg. Holland N. D. and Nealson K. H. (1978) The fine structure of the echinoderm cuticle and subcuticular bacteria of echinoderms, Acta Zool. (Slockholm) 59, 169%185. Ito S. (1969) Structure and function of the glycocalyx. Fedn. Proc. 28, 12-25. James G. T. (1978) Inactivation of the protease inhibitor
Phenylmethylsulfonyl fluoride in buffers. Anal),/. Biothem. 86, 574-579. Kjellen L. and Lindahl U. (1991) Proteoglycans: structures and interactions. A. Reo. Biochem. 60, 443-475. Kozlov I. A. and Skulachev V. P. (1977) H+-adenosine triphosphatase and membrane energy coupling. Biochim. hiophys. Acta 463, 29-89. McKenzie J. D. (1987) The ultrastructure of the tentacles of eleven species of dendrochirote holothurians studied with special reference to the surface coats and papillae. Cell Tissue Res. 248, 187. 199. McKenzie J. D. (1988) Echinoderm surface coats: their ultrastructure, function and significance. In Echinoderm Ph. V., Biology (Edited by Burke R. D., Mladenov Lambert Ph. and Parsley R. L.). pp. 697.-706. Balkema, Rotterdam, Brookfield. Noller C. R. (1966) In Chemistr? of Organic Compound.s. and London. D. 346. W. B. Saunders. Philadeluhia Spiro R. G. (1972) Study of the carbohydrates of glycoproteins. In Methods in Etqvmolog)~ (Edited by Ginzburg V., Colowick S. P. and Kaplan N. O.), Vol. 28, Part B: 3~_43. Sousa M.. Pinto R., Moradas-Ferreira P. and Azevedo C. (1993) Histochemical studies of the jelly coat of MarlhasAsteroidea) oocytes. Biol. levias glacialis (Echinodermata, Bull. 185, 215-224. Souza Santos H. and Sasso W. S. (1968) Morphological and histochemical studies on the secretory glands of starfish tube feet. Acta anal. 69, 41-51. Souza Santos H. and Sasso W. S. (1970) Ultrastructural and histochemical studies on the epithelium revestment layer in the tube feet of the starfish Asterina stellifera. J. Morph. 130, 287 296.
Pergamon
Carbodiimide-induced labelling of sea urchin surfaces (Echinoidea, Echinodermata) Irina V. Grigolava and J. Douglas McKenzie Scottish Association for Marine Science, P.O. Box 3, Oban, Argyll, Scotland, U.K. A method for radiolabelling echinoderm body surfaces, using water-soluble carbodiimide was developed. The number of carboxyl groups reacting with carbodiimide was estimated as 20 to 30 nmole g-’ dry weight of sea urchin spines. There were no phosphate groups accessible to carbodiimide on the surface coat. Several enzymes were used to release bound radiolabel from the spines labelled with carbodiimide and radioactive amino acid. Results obtained with proteases and hyaluronidase showed that radiolabel was bound to the glycoprotein and/or protein molecules on the extracellular surface of the spine epidermis. Key words: Antifouling; Cuticle; Echinoderm; Invertebrate;
Protease; Radiolabelling; Surface coat;
Water-soluble carbodiimide (EDAC). Comp. Biochem.
Physiol.
IlOB, 477482,
1995.
Introduction Holland and Nealson (1978), using TEM, showed that the epidermis of all echinoderms has a multi-layered extracellular coat, usually termed the cuticle. In a series of papers (McKenzie, 1987, 1988) it was shown that the outermost part of this cuticle consists of a fibrous layer which was termed the fuzzy coat. It was suggested that fuzzy coat is a glycocalyx, similar to those found on the extracellular coats of some other marine invertebrates (BereiterHahn et al., 1984) and on many cell surfaces (Ito, 1969). The integument, which comprises the epidermal epithelium plus the basi-epithelial nerve plexus (Holland, 1984), is known to be crucial in respiration, food absorption, adhesion and sensation. It has been suggested that the primary function of the outermost surface of the extracellular coat is antifouling due to its surface properties (McKenzie, 1987, 1988). Although the echinoderm cuticle has been studied with light and electron microscopy, _ Correspondence lo: I. V. Grigolava,
Scottish Association for Marine Science, P.O. Box 3, Oban, Argyll, PA34 4AD, U.K. Tel. 44 631 62244; Fax 44 631 65518. Received 27 April 1994; revised 22 September 1994; accepted 28 September 1994. 477
there are very few biochemical studies on its composition and structure. The difficulties in investigating the cuticle are largely due to its extreme thinness (usually less than two microns) and its close attachment to the underlying epidermis (Holland, 1984). Another difficulty is that the chemical composition of the cuticle’s layers are likely to be similar to mucus secretions released from the animal. Invertebrate mucus contains polysaccharides (mucopolysaccharides) chemically similar to surface coat glycosaminoglycans (Souza Santos and Sasso, 1968, 1970). The surface coat of eukaryotic cell membranes has been well studied and contains proteoglycans composed of glycosaminoglycan chains covalently bound to a protein core (Spiro, 1972; Kjellen and Lindahl, 1991). The purpose of our study was to develop a chemical method for labelling the various layers of the cuticle and to investigate the chemical composition and properties of this extracellular coat. For chemical labelling we have used water-soluble carbodiimide and radioactive amine. Cell membranes are usually impermeable to water-soluble carbodiimide (Kozlov and Skulachev, 1977), thus only extracellular surfaces will be modified. At acidic pH
478
I. V. Grigolava
and J. D. McKenzie
(pH 4-6) carbodiimide interacts with free carboxyl and phosphate groups. After the addition of amine, amide is formed (Noller, 1966): RN = C = NR + R’COOH + RNHCONHR
+ R2NH2 + R’CONHR’
RN=C=NR is where carbodiimide, R’ COOH is carboxyl groups on the surface of spine, RNHCONHR is urea derivative and R’ CONHR’ is amide formed on the surface.
Materials and Methods Materials Experiments were carried out on the spines from two sea urchin species: Echinus esculentus (L.) and Psammechinus miliaris (Gmelin). Both were collected from sub-littoral populations around Argyll, Scotland by dredging. Chondroitinase ABC (chondroitine ABC lyase) from Proteus vulgaris; hyaluronidase Type IV-S, bovine; lysozyme from chicken egg white; neuraminidase from Salmonella tyType VII-T, phimurium; alkaline phosphatase bovine, protease Type XIV (pronase E), bacterial; protease Type XV (dispase), bacterial; and trypsin Type IT, from porcine pancreas-all were from Sigma, St Louis, MO. 14C glycine (1184mCi mmol-‘) and ‘H glycine (20mCi mmol-‘) were from Sigma. Amino acid mixture, labelled with tritium (high specific activity mixture of five amino acids) was from Amersham, Bucks, U.K. A tissue solubilizer (TS-1) and scintillator (Aquasafe 500) were from Zinsser Analytic. All other chemicals were from Sigma. Methods Chemical radiolabelling of spines was carried out as follows: 0.1-1.0 g of spines (between 30-300 spines) were cut from sea urchins (three animals in each experiment) and then rinsed several times with (2-20 ml) buffer Nl CO.1 M 2-/N-morpholino/ethanesulfonic acid (MES), 2% NaCl and 10 mM NaN, (sodium azide), pH 5.0). An inhibitor of proteases, 50pM phenylmethylsulfonyl fluoride (James, 1978) was present in the rinsing buffer. To obtain dry weight, some spines were dried in an oven at 50°C to constant weight. The rest of the spines were incubated with 30 mM water-soluble carbodiimide-I-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC) at ambient temperature in buffer N2 (0.6 M MES, 0.5% NaCl, pH 5.0). In the controls carbodiimide was omitted. After 30 min of incubation, the spines were rinsed with buffer N3 (0.1 M 3-/N-morpholino/ propanesulfonic acid (MOPS) and 1% NaCl, pH 8.2) and then transferred into the modified
buffer N3 containing 10 mM amino acid (1 PCi ml-‘) of either 14C glycine, ‘H glycine or amino acid mixture, labelled with tritium. After 20min of incubation at ambient temperature, the spines were rinsed with buffer N4 (0.1 M unlabelled amino acid, 2% NaCl solution, 1 mM NaN, and 50 p M phenylmethylsulfonyl fluoride, pH 8.2) until no radioactivity could be detected in the rinsing buffer. The modified spines were then incubated in tissue solubiliser for 4 hr at 50°C to release all bound radioactivity. Water-soluble carbodiimide was also used for a two-step modification of free amino groups on the surface coat. 10 mM EDAC was incubated with 3 mM 14C acetic acid (1 PCi ml ’) in buffer Nl pH 4. After 20 min of incubation, the pH was adjusted to pH 8 with triethanolamine. Spines were added to this reaction mixture and incubated for another hour. In control experiments 14C acetic acid was incubated with spines at pH 8 but with EDAC omitted. The spines were rinsed with 0.1 M sodium acetate in 1% NaCl, pH 8. Finally, the spines were treated with tissue solubilizer as before. Additional buffer compositions were: M MOPS N5 for protease digestion-IO.05 and 0.9% NaCl, pH 7.5). N6 for hyaluronidase treatment--(O. 1M MES and 0.9% NaCl, pH 5.3). treatment--lo. 1M N7 for neuraminidase MES. 0.9% NaCl, 0.004 M CaClz, pH 5.5). ABC-as N5, but N8 for chondroitinase pH 8.0. phosphatase treatment-{O. 1M N9 for triethanolamine, 0.9% NaCl, 0.5 mM MgCl, and 0.2 mM ZnCl,, pH 9.7). M MES NlO for lysozyme treatment-(0.1 and 0.9% NaCl, pH 6.3}. of different enzymes with The interaction labelled echinoderm surfaces was studied. For this purpose sea urchin spines were rinsed first with buffer Nl containing protease inhibitor, and then, with an appropriate buffer for the enzyme (without a protease inhibitor and NaN,). Spines were incubated at 50°C for 1 or 2 hours with each of the enzymes. The spines were then rinsed extensively with buffer Nl and radiolabelling was carried out as described above. Control spines were treated similarly except that the enzymes were omitted in the pretreatment medium. To further study effect of enzymatic pretreatment on the labelling process, samples of spines (each 100 mg) were incubated with pronase E at 50°C and control spines were incubated without pronase, for 10 min (sample 1), 20min (sample 2), 30 min (sample 3) 60 min (sample 4) and 2 hr (sample
Radiolabelling
of sea urchin
5). Each sample was then labelled with EDAC and r4C glycine as described above (Fig. 1). The effect of pretreatment with the chelating agent, ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis(fl-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA) on the reaction of EDAC with the surface was examined. 10 mM of EDTA and 10 mM EGTA were added to the medium, N5 pH 7.5 and incubated at 50°C for 2 hr. The modification with carbodiimide was then made. Radioactivity measurements were carried out on a liquid scintillation counter (12 19 Rackbeta, LKB Wallac).
Results Modification of sea urchin spines with water soluble carbodiimide allowed a quantitative estimate of free carboxyl groups to be made. As cell membranes are impermeable to EDAC, were only extracellular carboxyl groups modified. The total bound amino acid was calculated as follows: total bound amino acid (nmole g- ’ dry weight of spines) equals the radioactivity released by TS-1 from EDAC labelled spines minus the radioactivity released by TS-1 from control spines. Twenty experiments were carried out on spines from E. esculentus. All the measurements gave the content of free carboxyl groups as 20 to 30 nmole gg’ dry weight of spines. In five experiments carried out on P. miliaris the estimated free carboxyl groups were found to vary between 20 to 30 nmole gg’ dry weight of spines. The amount of chemical labelling was affected by the presence of sodium azide. If sodium azide was omitted, the observed number of free carboxyl groups was lower, though this was mainly due to the increase of radioactivity in the control spines. In the absence of sodium azide, TS-1 released half of the label (10 to 15 nmole g-‘) in the control spines compared to the label released in EDAC-modified spines. However, in the presence of both sodium azide and protease inhibitor this figure was always less than 6 nmole gg ‘. Spines can metabolize amino Table
I. Effect of pretreatment Without enzymatic pretreatment
Added amount mg ml-’ Incubation time, h Bound label in nmole g-’ dry weight* Bound label %
acids and this was measured as bound label in the absence of sodium azide. Sodium azide as an inhibitor of transport processes prevented metabolic uptake of the label. The absence of protease inhibitor prior to modification resulted in a lower number of modified carboxyl groups. No binding of radiolabel was found during the experiments which attempted to modify amino groups on the echinoderm surface with EDAC modified radiolabelled acetic acid. The amount of radioactivity released by tissue solubiliser was similar to the controls (not shown). The stability of the label bound to carboxyl groups was studied. It was found that after incubation at 5OC for 24 hr only a small amount of label was released: from 0% to 6% of the total bound label. However, if protease inhibitor was not present in the rinsing solutions, the spontaneous release of label was higher: from 30% to 40% of the total bound label. The modification of spines with EDAC was carried out at different pH. It was found that the difference in the labelling at pH 4.5, pH 5.0 and pH 5.5 was small. The labelling at pH 5.0 was three times higher than at pH 6.0. Table 1 shows the results of enzymatic pretreatment of spines and its effect on the reaction with EDAC. Pronase E and trypsin prevented the labelling of the surface, at the same time none of the three other enzymes: hyaluronidase, phosphatase or lysozyme had any effect on the reaction. No effects of the chelating agents, EDTA and EGTA were found (not shown). Figure 1 shows the effect of pretreatment with pronase E on labelling of spines with EDAC. An incubation period of 1 hr with pronase was sufficient to totally prevent labelling of spines. Pronase E and also other proteases are widely used to release proteoglycans from the cell surfaces (Spiro, 1972; Edward and MacKie, 1989). Results for enzymatic digestion of labelled spines are given in Table 2. Pronase, dispase and trypsin released a high proportion of the bound label. Hyaluronidase released only 14% of the bound label. In the absence of the
of spines on the reaction
Pronase 0.5
E
Trypsin
0.5 2
5
3.5 17.5
1
2
1
20 100
I8 100
I
0
5
0
419
surfaces
1
with water soluble carbodiimide
Phosphatase 0.5 1 I9 95
Hyaluronidase 0.5 2
>I8 100
*Bound label, as released radioactivity by TS-I in samples treated with EDAC and amino radioactivity (by TS-I) in control spines (minus EDAC). Spines were incubated with the enzyme at 50 C, labelling was then carried out.
Lysozyme
10 2
5 2
> 18 100
>18 100
acid, subtracted
by released
480
1. V. Grigolava
04
Q
and J. D. McKenzie
x
l&Y
w
150
time, min Fig. 1. Modification of spines with EDAC and 14C glycine, pretreated with pronase E. Spines were pretreated with pronase E at SO’C for the times shown. Pronase was removed by rinsing with buffer and the spines treated with EDAC and radioactive amino acid. The bound radiolabel, nmole g-’ dry weight of spines (as released radioactivity by tissue solubilizer) was measured.
enzyme approximately 6% was released spontaneously, suggesting that hyaluronidase released 8% of the total label (n = 5). Protease digestion kinetics of radiolabelled spines at 37°C and 50°C are shown in Fig. 2. At 37°C 1.5 mg ml-’ protease released 90% of the label after 3 hr of incubation and a total of 95% after 24 hr of incubation. At 50°C 0.5 mg ml-’ protease released more than 80% of the radioactive label after 3 hr of incubation and a total 90% after 24 hr. The results presented in Figs 1 and 2, and also in Tables 1 and 2 were from a typical experiment carried out on spines from E. esculentus. Similar results on the protease digestion of labelled spines were obtained for P. rniliaris (not shown). Neither neuraminidase (0.9 units ml-‘) or chondroitinase ABC (2 units ml-‘) released the label from radiolabelled sea urchin (E. esculentus) spines after 24 hr of incubation with labelled spines at 37°C.
Discussion A method of labelling echinoderm body surfaces using water soluble carbodiimide (EDAC) was developed that requires only very
Table 2. The release of the label from spines treated Without any treatment: spontaneous release of label mg ml-’ enzyme* Released label in nmole g-’ and in %t
< I.5 <6
mild conditions. This method of labelling the echinoderm surface has been used to study the turnover rate of echinoderm surface coats (Grigolava and McKenzie, 1994). The amount of free carboxyl groups available on the extracellular surface and the lack of amino groups accessible to EDAC treated acetic acid, indicates that the echinoderm surface is acidic. Histochemical studies with light microscopy have suggested that the echinoderm cuticle contains acid polysaccharides (Souza Santos and Sasso, 1970; Engster and Brown, 1972). Acid glycoproteins have also been shown histochemically to exist on the surface of echinoderm egg coat (Sousa et al., 1993). The pH dependence of the reaction of EDAC with the surface indicates that the modified groups have a comparatively high pK value. The label is stable even at relatively high temperatures and for up to 24 hr. Protease inhibitor was needed to produce stability of the label: spontaneous release of the label occurred if protease inhibitor was absent. This could be due to the action of endoproteases. These are present in large quantities in spines (Canicatti, 1990; Canicatti and D’Ancona, 1990) and were probably released by damage to the spines. This may explain the low
with water soluble carbodiimide
TS-I 25 100
Promise 0.5 24 96
E
Dispase 5.0 24 96
and radioactive
Trypsin 0.5 22 88
amine
Hyaluronidase 0.1 3.5 14
*Enzyme was added once at the beginning of incubation and the same amount. later after 6 hr of incubation. tFigures represent the difference between released label in treated with EDAC spines and in controls (minus Spines were incubated with (or without) enzyme for 24 hr and with TS-I for 4 hr. at 50 C.
EDAC).
Radiolabelling
of sea urchin
481
surfaces
0 0
0
ldo
!zib
3!50
time, min Fig. 2. Released radioactivity by pronase E. 0, radioactivity (as released by tissue solubiliser) was radioactivity by pronase at 50°C total radioactivity dry weight
of observed modified groups. Endogenous proteases may have digested some of the surface before the modification if protease inhibitor was not present in rinsing buffers. Enzymatic pretreatment of spines before the modification of its surface (Table I) and also interaction of different enzymes with radiolabelled surfaces (Table 2) showed that carbodiimide-reactive carboxyl groups belong to protein moieties of the glycoprotein. Pretreatment with pronase E and trypsin (but not hyaluronidase) prevented labelling. Pronase E, dispase and trypsin released the label from labelled spines, but neurominidase and chondroitinase did not and hyaluronidase released only a small portion of the label. Since phosphatase had no effect on EDAC labelling it seems that there are few if any phosphate groups, associated with the extracellular surface. Lysozyme cleaves bonds between N-acetylglucosamine and N-acetylmuramic acid in mucopeptide, a proteoglycan of bacterial cell walls. As lysozyme pretreatment had no effect on labelling, it suggests that such compounds are not important in the structures of the echinoderm surface. The release of some carbodiimide-induced label by hyaluronidase indicates that there are structures such as hyaluronic acid, chondroitin or chondroitin sulphate on the surface of echinoderms. The amount of label released was, however, small. This may suggest that such structures form only a minor component of the surface. Alternatively, the enzyme digests parts of glycosaminoglycans which do not contain free carboxyl groups. It is known that in many glycosaminoglycans the carboxyl groups are heavily sulphated (Kjellen and Lindahl, 1991) and would not be available for labelling. In number
released radioactivity by pronase at 37”C, total IlOOdpm gg’ dry weight of spines. 0, released (as released by tissue solubilizer) was 1300 dpm g-’ of spines.
preliminary experiments (not shown in this paper) chondroitinase ABC digested some surface components in spines of 35S-labelled sea urchin. The lack of effect of hyaluronidase pretreatment on labelling, therefore, does not necessarily mean that polysaccharide chains, which are the substrates for this enzyme, are absent. The kinetics of radiolabel release as a result of pronase E treatment of the labelled surface indicates the speed of enzymatic digestion of the protein structures. This is being used in studies of cuticle structure and metabolism (Grigolava and McKenzie, 1994). The carbodiimide-accessible compounds on the sea urchin surface are very sensitive to proteases (endoproteases). The digestion of echinoderm surfaces with endoproteases may have a biological role in the prevention of fouling. Under certain circumstances the echinoderms may be able to “clean” their surfaces by shedding the outermost layer via the release of endoproteases. Carbodiimide-induced labelled surfaces are useful for further studying of different enzymatic reactions on the surface which will supply interesting information about structure of the surface. This labelling method will be used in future to isolate and chemically analyse glycosaminoglycans and proteins present in the surface coats of the cuticle. AcknoM,I~dgem~no-The research discussed here is part of a European project to develop novel antifouling structures and is funded by Marine Technology Directorate (SERCU.K.) and the European Commission as part of the MAST II program (MASZ-CT9l-0009). We would like to thank the Director of Dunstaffnage Marine Laboratory for the support and provision of facilities, also M. Edward, I. Kozlov. L. Newton, M. Kelly, D. Harris and D. Watt for useful discussion and suggestions.
482
1. V. Grigolava
amd J. D. McKenzie
References Bereiter-Hahn J., Matoltsy A. G. and Richards K. S. (Eds) (1984) Biology qf rhe Integument. Vol. I. Inoertehrares. Springer, Berlin, Heidelberg. Canicatti C. (1990) Protease activity in Holorhuria polii coelomic fluid and coelomocyte lysate. Comp. Biochem. Physiol. 95B, 145-148. Canicatti C. and D’Ancona G. (1990) Biological protective substances in Marthasterias glacialis (Asteroidea) epidermal secretion. J. Zool. Lond. 222, 445-454. Edward M. and MacKie R. M. (1989) Retionic acid induced inhibition of lung colonization and changes in the synthesis and properties of glycosaminoglycans of metastatic B16 melanoma cells, J. Cell Sci. 94. 537-543. Engster M. S. and Brown S. C. (1972) Histology and ultrastructure of the tube foot epithelium in the Phanerozonian starfish, Astropecten. Tiss. Cell 4, 503-518. Grigolava I. and McKenzie J. D. (1994) Biochemical studies of echinoderm surface coats using radiolabelling techniques. In Echinoderms Through Time (Echinoderms Dijon) (Edited by David B., Guille A., Firal J. P. and Roux M.), in press. Bulkema, Rotterdam, Netherlands. Holland N. D. (1984) Echinodermata: epidermal cells. In Biology of the Integument. Vol. I. Inuertebrales (Edited by Bereiter-Hahn J., Mattoltsy A. G., Richards K. S.), pp. 7566774. Springer, Berlin, Heidelberg. Holland N. D. and Nealson K. H. (1978) The fine structure of the echinoderm cuticle and subcuticular bacteria of echinoderms, Acta Zool. (Slockholm) 59, 169%185. Ito S. (1969) Structure and function of the glycocalyx. Fedn. Proc. 28, 12-25. James G. T. (1978) Inactivation of the protease inhibitor
Phenylmethylsulfonyl fluoride in buffers. Anal),/. Biothem. 86, 574-579. Kjellen L. and Lindahl U. (1991) Proteoglycans: structures and interactions. A. Reo. Biochem. 60, 443-475. Kozlov I. A. and Skulachev V. P. (1977) H+-adenosine triphosphatase and membrane energy coupling. Biochim. hiophys. Acta 463, 29-89. McKenzie J. D. (1987) The ultrastructure of the tentacles of eleven species of dendrochirote holothurians studied with special reference to the surface coats and papillae. Cell Tissue Res. 248, 187. 199. McKenzie J. D. (1988) Echinoderm surface coats: their ultrastructure, function and significance. In Echinoderm Ph. V., Biology (Edited by Burke R. D., Mladenov Lambert Ph. and Parsley R. L.). pp. 697.-706. Balkema, Rotterdam, Brookfield. Noller C. R. (1966) In Chemistr? of Organic Compound.s. and London. D. 346. W. B. Saunders. Philadeluhia Spiro R. G. (1972) Study of the carbohydrates of glycoproteins. In Methods in Etqvmolog)~ (Edited by Ginzburg V., Colowick S. P. and Kaplan N. O.), Vol. 28, Part B: 3~_43. Sousa M.. Pinto R., Moradas-Ferreira P. and Azevedo C. (1993) Histochemical studies of the jelly coat of MarlhasAsteroidea) oocytes. Biol. levias glacialis (Echinodermata, Bull. 185, 215-224. Souza Santos H. and Sasso W. S. (1968) Morphological and histochemical studies on the secretory glands of starfish tube feet. Acta anal. 69, 41-51. Souza Santos H. and Sasso W. S. (1970) Ultrastructural and histochemical studies on the epithelium revestment layer in the tube feet of the starfish Asterina stellifera. J. Morph. 130, 287 296.