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Inhibition of the growth of calcium carbonate crystals by multiple lectins in the coelomic fluid of the acorn barnacle Megabalanus rosa
Comp. Biochem. Physiol. Vol. 107B, No. 3, pp. 401-409, 1994 © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-0491/94 $6.00 + 0.00
Pergamon
Inhibition of the growth of calcium carbonate crystals by multiple lectins in the coelomic fluid of the acorn barnacle Megabalanus rosa Koji Muramoto, Hiroshi Yako, Koji Murakami, Satoshi Odo* and Hisao Kamiya School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-01, Japan; and *Marine Biotechnology Institute, Kamaishi, Iwate 026, Japan The interaction between the multiple lectins (BRA-2, BRA-3) in the coelomic fluid of the acorn barnacle Megabalanus rosa and calcium ions was studied by ultraviolet difference spectroscopy. The association constants for BRA-2 and BRA-3 were 1.4 × 104M -t and 2.2 × 104M -t at pH8.0, respectively. Multiple lectins inhibited the crystal growth of supersaturated calcium carbonate solution at a iectin concentration of > 0.1 mg/30 mi. Destruction of the carbohydrate-recognition domains of the lectins decreased the inhibitory activities on the crystal growth. Incorporation of BRAs into the crystal was demonstrated using FITC-labeled lectins. Key words: Calcium carbonate; Lectins; Megabalanus rosa; Recognition domains.
Comp. Biochem. Physiol. I07B, 401-409, 1994.
Introduction
The present extent of our knowledge permits us to organize the known animal lectins into several categories (Drickamer, 1988). One group consists of fl-galactose-binding proteins isolated from various tissues and serum of many vertebrates, such as the electric eel, conger eel, chicken, rat, bovine and human. They are designated as S-type lectins, as they often require thiol-reducing agents to maintain their activity. The activity is independent of divalent cations. A second group consists of the calcium-dependent (C-type) animal lectins, including a mannose-binding protein isolated from mammalian liver, membranous glycoprotein receptors isolated from mammalian and avian liver, and a galactose-binding lectin isolated from rattlesnake venom. Invertebrate lectins isolated from the flesh fly (Sarcophaga peregrina) (Takahashi et al., 1985), the acorn barnacle (Megabalanus rosa) (Muramoto and Kamiya, 1986, 1990) and Correspondence to: K. Muramoto, Faculty of Agriculture, Tohoku University, Sendai 981, Japan. Fax: 81 22-2721870. Received 13 July 1993; accepted I 1 August 1993. CBPB
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the sea urchin (Anthocidaric crassispina) (Giga et al., 1987) have been found to contain a C-type carbohydrate-recognition domain. The domain is constructed with two characteristic disulfide bond loops containing homologous amino acid sequences. The C-type carbohydrate-recognition domain has also been detected in a wide variety of proteins, such as a proteoglycan core protein (Doege et al., 1991), tetranectin (Fuhlendorff et aL, 1987), lymphocyte immunoglobulin E receptor (Kikutani et al., 1986), phospholipase A2 inhibitor (Inoue et al., 1991), pulmonary surfactant apoproteins (Hawgood, 1989), coagulation factors (Takeya et al., 1992), antifreeze polypeptides (Ng and Hew, 1992; Ewart et al., 1992) and pancreatic stone protein (De Caro et al., 1987). The findings suggest that these proteins which mediate proteincarbohydrate and protein-protein interaction can be utilized in a number of quite distinct biological contexts. We have isolated and characterized multiple galactose-binding lectins from the coelomic fluid of the acorn barnacle, Megabalanus rosa (Muramoto et al., 1985). They are composed of
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three different molecular species with respect to molecular weight, designated BRA-1 (Mr 330,000), BRA-2 (Mr 140,000) and BRA-3 (Mr 64,000). BRA-1 and BRA-2 are composed of identical subunits (M r 22,000), of which the amino acid sequence shows only 25% identity with that of the BRA-3 subunit (Mr 16,000) (Muramoto and Kamiya, 1990). We showed the seasonal changes in the multiple lectin compositions in the coelomic fluid in connection with the development of ovaries (Muramoto et al., 1991). The multiple lectins are major coelomic proteins as judged by protein determination (Muramoto et al., 1994). Invertebrate lectins are commonly assumed to function as recognition molecules, both in intercellular and intracellular systems. Although the most thoroughly explored potential functions are those involved with invertebrate immunity (Kubo et al., 1984; Cooper et al., 1984), other biological functions also need to be investigated. In this paper, we studied the interaction of the multiple lectins of M . rosa with calcium ions and the effects of the lectins upon the growth of calcium carbonate crystals using a pH-monitor system. The lectins caused a marked decrease in crystal growth in a dosedependent manner.
Materials and Methods Multiple galactose-binding lectins, BRA-1, BRA-2 and BRA-3, were isolated from the coelomic fluid of M. rosa, as previously described (Muramoto et al., 1985). The modified BRAs were prepared by reaction with fluorescein isothiocyanate (FITC) (Muramoto et al., 1985), monoiodoacetoamide and cyanogen bromide as described (Muramoto and Kamiya, 1990). Enzymatic digestion of the lectins was performed with TPCK trypsin (Worthington, Freehold, NJ), chymotrypsin (P-L Biochemicals, Milwaukee, WI) or V8 proteinase (Wako Chemicals, Osaka, Japan) in 0.1 M ammonium carbonate at 37°C for 24hr at substrate/ enzyme = 100: 1. The digests were lyophilized and used for the experiment. The average pHs of the coelomic fluid of M . rosa and the seawater, where the barnacle lived, were 7.17 and 8.05, respectively. The calcium concentrations of the coelomic fluid and the seawater were measured as 10.17_+0.02mM and 9.49_+ 0.16mM (+SE, N = 3), respectively, by a inductively coupled plasma spectrometer (Shimadzu ICPS-1000III). Organic matrix preparation M. rosa shells were carefully cleaned and air-dried. One hundred grams of the shell were
crushed and decalcified with 500 ml of 5% HC1 at room temperature overnight. The solution was centrifuged to separate insoluble materials, and the supernatant was dialyzed against tap water for 2 days and then lyophilized to yield 96 mg. The insoluble fraction was further extracted with 200 ml of 5% NaOH. The extract was dialyzed against tap water for 2 days and lyophilized to yield 144 mg. The shell was also decalcified with 15% EDTA at pH 7.5. Calcium binding o f B R A s
The association constants of the calcium binding of BRAs were measured by difference spectroscopy (Doyle et al., 1975; Matsumoto et al., 1980). BRA was dissolved in 0.1 M EDTA/50mM Tris-HC1 buffer (pH8.0) at 5 mg/ml and left for 30 min at room temperature. The solution was diluted with 3 ml of 50 mM Tris-HCl buffer (pH 8.0) and then dialyzed against the same buffer at 4°C for 2 days with several changes of the buffer to remove EDTA thoroughly. After dialysis, the sample solutions were adjusted to 1 mg/ml with the buffer and filtered through 0.45#m pore diameter membranes. Calcium-binding experiments were conducted at 25°C by the addition of small aliquots of 1M or 0.1M CaCI2 to the test solution in Semimicro quartz cells of 1 cm path length. Absorbance and difference spectra were measured between 220 and 320nm using a temperature-controlled Jasco Ubest-50 spectrophotometer. It took about 10 min for the Ca 2÷induced absorbance changes to reach equilibrium. The intensities of difference spectra (peak-trough) AA were measured as a function of increasing calcium concentration ([S]). Plots according to the equation, [S]/A = (l/Amax) x [S] + 1/Am,x
x
K
(1)
were made, where K is the association constant and Areax is the maximum absorption difference. The solid line is a least-squares plot of the data to the equation. The K value was calculated from the intercept on the abscissa. Calcium carbonate crystallization assay
The inhibition assay for calcium carbonate crystallization was monitored by the change in pH of a solution supersaturated with respect to calcium and carbonate (Gunthorpe et al., 1990). The assay began with an induction period of relatively constant pH, during which time crystal nucleation occurs, and then continues with a rapid decrease in pH indicating crystal growth. The pH was continuously monitored and recorded. The incubation medium included 29.1 ml artificial seawater (ASW:
Barnacle leetins and calcium carbonate crystals
0.5 M NaC1 and 0.011 M KC1) in a glass vessel placed in a circulating water bath maintained at 20.0°C, to which 0.3 ml 1.0 M CaCIJ2H20 was added to obtain 10 mM calcium. Next, the test sample was added and stirred for 10min. Finally, 0.6 ml of 0.4 M NaHCO3 was added to yield 8 mM dissolved inorganic carbon, and the solution was titrated to a pH of 8.30 with 1 N NaOH to begin the assay.
Incorporation of FITC-BRAs into calcium carbonate crystal The amount of FITC-labeled lectins incorporated into crystals was determined by measuring the fluorescence intensity after solubilization of the crystals. Crystallization was performed in the presence of 10 or 100#g of FITC-BRA for 2hr at 20°C under the same conditions as described above. The solution was transferred to a centrifugal tube. Resulting calcium carbonate was recovered by centrifugation at 3000 rpm for 10 min at 6°C. The pellet was washed with 5 ml distilled water three times, dissolved in 0.7 ml of 0.1 M acetic acid and filtered by a centrifugal ultrafiltration membrane (30 kDa cutoffs; Tosoh, Tokyo, Japan). Recovered FITC-BRA was dissolved in 6 ml of 0.2 M Tris-HCl (pH 8.0) and measured by a JASCO FP-550A spectrofluorometer using an excitation wavelength of 490nm and an emission wavelength of 0
403
520 nm. Quantitative analysis was performed with calibration curves of corresponding FITC-BRAs. Results The difference spectrum of BRA-2 induced by the addition of 1 mM calcium at 25°C is shown in Fig. 1. The difference spectrum induced by the addition of calcium has two positive peaks. The intensity of the difference spectrum depended on the calcium concentration. The changes in AA 292-288 nm due to the binding of the calcium ion to BRA-2 and the corresponding straight line obtained by plotting [S]/AA vs IS], according to the equation, are shown in Fig. 2, where [S] is the calcium concentration and AAr~axis the maximum change in the difference absorption between 292 and 288 nm. By plotting [S]/AA as a function of [S] as in the equation, the association constant, K, was calculated. The Kvalue, 1.4 x 104 M -I, for BRA-2 is similar to the value of 2.2 x 104 M -] obtained for BRA-3 at pH 8.0. By changing the pH to 7.0 the association constants for both BRA-2 and BRA-3 decreased to 7.4 x 104 M - ' and 6.8 x 103 M -l, respectively (Table 1). Inhibitory activities of multiple lectins on the crystal growth of calcium carbonate were monitored by the drift in pH. This assay relies on the fact that the crystallization produces hydrogen ~
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ions and the rate of crystal growth is proportional to the decrease of pH in the test solution. That the downward pH shifts during the crystallization assays did indeed indicate crystal growth was verified by the presence of a pellet of crystals produced by centrifugation of the solution. It took 60 min for the pH drift to cease. The addition of BRAs at 0.1 mg/30 ml totally inhibited the crystal growth as shown in Fig. 3. The inhibition was dose-dependent; as the lectins were decreased by one-tenth, no inhibition was observed. Neither an S-type lectin isolated from conger eel (Muramoto and Kamiya, 1992) nor bovine serum albumin showed any inhibitory effect at 0.1 mg/30ml. At 1 mg/30 ml, bovine serum albumin began to show some inhibition; meanwhile, conger eel lectin did not inhibit at all. Inhibitory activity of different concentrations of poly-aspartic acid (Asp) (Sigma, St Louis, MO) was examined (Fig. 4). At 0.1 mg/30 ml, poly-Asp completely inhibited the crystal Table 1. Effectof pH on the associationconstants of BRAs to calcium ion K (M -t)
pH BRA-2 BRA-3 7.0 7.4 x 104 (293-289)* 6.9 × 103 (296-290) 8.0 1.4 x 104 (292-289) 2.2 × 104 (296-290) 9.0 1.8 x 104 (291-288) 1.4 x I04 (295-289) *Wavelength (nm) at peak-trough.
growth. Upon 10-fold dilution of poly-Asp, a significant inhibition was still observed. Polyglutamic acid (Sigma) and phosvitin (Sigma) showed similar inhibitory activities, but phosvitin had a smaller effect than the other two. BRAs lost their inhibitory activities upon the destruction of the conformation by reduction and S-carboxamidomethylation (Fig. 5). Cyanogen bromide cleavage also diminished the inhibitory activity of BRA-2. On the other hand, BRA-3 could keep its activity to some extent. This result indicates the importance of the carbohydrate-recognition domain for inhibition, because a single methionine residue contained in a BRA-3 subunit locates outside the carbohydrate-recognition domain, and the domain can stand in spite of the breaking of the subunit structure. Tryptic digestion of BRAs decreased the inhibitory activity at 0.1 mg/30ml, whereas neither chymotrypsin nor V8 proteinase digestion had any influence on the inhibition (data not shown). Modification of BRAs with FITC decreased the inhibitory activities as shown in Fig. 6. The average degrees of labeling were 22.7, 6.4, and 1.8 mol of FITC per moi BRA-1, BRA-2, and BRA-3, respectively. The agglutinating activities of BRAs against rabbit erythrocytes did not change by labeling with FITC. Incorporation of BRAs into the calcium carbonate crystals was investigated by labeling
Barnacle lectins and calcium carbonate crystals
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BRAs with FITC. The results are summarized in Table 2. Calcium carbonate crystallization was carried out in the presence of 10 or 100 # g of FITC-BRAs under the same conditions as described above. The amount of the crystal formed was 687 + 103 # g ( + S E , N = 6) in this experiment. In the presence of 10/~g of FITC-BRAs, 0.34-0.39 # g of the lectins were
incorporated to the crystal. The amount of incorporated FITC-BRAs was relatively constant. However, by increasing the amount of applied FITC-BRAs to 100#g, the incorporated amount varied from 2 . 1 6 # g for FITC-BRA-3 to 7.02/~g for FITC-BRA-1. The soluble matrix from the acorn barnacle shell inhibited calcium carbonate crystallization
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(Fig. 7). At 1 mg/30 ml, three kinds of organic matrices showed full inhibition. The activities of HCI extract and EDTA extract decreased at 0.1 rag/30 ml.
Discussion Lectins are a group of sugar-binding proteins which recognize specific carbohydrate structures
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Fig. 6. Inhibition of the crystal growth of calcium carbonate by FITC-BRAs monitored by pH-drift. A: FITC-BRA-I; B: FITC-BRA-2; C: FITC-BRA-3; D: control. Concentration of the samples was 0.1 mg/30 ml.
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Table 2. Incorporation of FITC-BRA into the calcium observed that ultraviolet difference spectra of carbonate crystal BRAs were induced by the addition of calcium Incorporated FITC-BRA as well as by specific sugars, e.g. galactose. The (/tg, _.+SE, N = 7 ) sugar-induced spectral changes, which would be A B explained by the conformational changes in FITC-BRA-1 0.34 +__0.06 7.02 + 0.40 BRAs, could not be detected unless the calciumFITC-BRA-2 0.39 + 0.06 5.70 + 0.60 free BRA had been previously bound with FITC-BRA-3 0.37 + 0.05 2.16 + 0.26 calcium ions. This is coincidental with the calA: 10#g FITC-BRA was added; B: 100#g of FITC-BRA was added. In this experiment, 687+103#g (+SE, cium ion being required for the agglutinating activity. The association constants obtained for N = 6) of calcium carbonate was formed. BRAs are comparable to those reported for phosvitin or organic matrices prepared from and are proposed to have many different mollusk shells (Wheeler and Sikes, 1984). The biological roles. The multiple lectins from the affinity against calcium ion and the high concoelomic fluid of M . r o s a are C-type lectins, tents of the lectins could explain the difference which have the characteristic carbohydrate- of calcium concentrations between the coelomic recognition domain. C-type lectins have a rather fluid and seawater. wide range of sugar-binding specificity, but Calcium carbonate and organic matrix are they always require calcium ions for the sugar two major components in the barnacle shell binding. The calcium binding sites are located (Bourget, 1987). The latter constitutes several on the carbohydrate-recognition domain. The percent by weight of the shell. These compresence of the carbohydrate-recognition do- ponents may play important roles in regulation main in a variety of proteins, including pancre- of crystal growth, as reviewed by Mann (1988) atic stone protein and cartilage proteoglycan, and Berman e t al. (1988). Regulation could and the experimental results concerning the include nucleation, rate of growth, orientation, seasonal dynamics and the high contents of the size and overall morphology of crystals, and it multiple lectins in M . r o s a allowed us to study can occur through pH of the environment, the the interaction between the lectins and the presence of inhibitors, the presence of microencalcium ion and their involvement in shell vironments, the presence and kind of nucleation formation. sites and the degree of supersaturation (Mann, The difference spectrophotometric method 1983). Organic matrices isolated from various has been used for the study of specific inter- organisms are inhibitors of calcium carbonate actions between lectins and sugars or metals crystallization when free in solution. Inhibition (Doyle e t al., 1975; Matsumoto e t al., 1980). We may be correlated to the affinity of matrix
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408
Koji Muramoto et al.
molecules for crystal surfaces and adequate coverage of growth sites on crystal surfaces. It has also been shown that the same matrices, when attached to an insoluble support, promoted crystallization (Wheeler and Sikes, 1984). We showed in this study that multiple lectins, as well as the organic matrix, from M . rosa inhibited the crystal growth of calcium carbonate. Although we do not have enough evidence for the chemical structural relationship between the multiple lectins and the organic matrix, the matrix contained components crossreacted with anti-BRA antibodies as revealed by a dotblotting assay (data not shown). Immunohistochemical study revealed that immunostaining was found in almost all M . rosa tissues and was especially prominent in the cirri (Muramoto et al., 1991). The pH dependency of association constants against the calcium ion and the decrease of inhibitory activity by modification of the conformation, indicate that the multiple lectins are among the possible regulators for calcium cabonate crystallization.
Acknowledgements--This work was supported in part by grants from New Energy and Industrial Technology Development Organization, and the Research Institute of Marine Invertebrates, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References Addadi L. and Weiner S. (1985) Interactions between acidic proteins and crystals: stereochemical requirements in biomineralization. Proc. Natn. Acad. Sci, USA 82, 4110-4114. Berman A., Addadi L. and Weiner S. (1988) Interactions of sea-urchin skeleton macromolecules with growing calcite crystals--a study of intracrystalline proteins. Nature 331, 546-548. Bourget E. (1987) Barnacle shells: composition, structure and growth. In Barnacle Biology (Edited by Southward A. J.), A. A. Balkema, Rotterdam. Cooper E. L., Stein E. A. and Wojdani A. (1984) Recognition receptors in annelids. In Recognition Proteins, Receptors, and Probes: Invertebrates (Edited by Cohen E.), pp. 43-54. Alan R. Liss, New York. De Caro A. M., Bonicel J. J., Rouimi P., De Caro J. D., Sarles H. and Rovery M. (1987) Complete amino acid sequence of an immunoreactive form of human pancreatic stone protein isolated from pancreatic juice. Eur. J. Biochem. 168, 201-207. Doege K. J., Sasaki M., Kimura T. and Yamada Y. (I 991) Complete coding sequence and deduced primary structure of the human cartilage large aggregating proteoglycan, aggrecan. J. biol. Chem. 266, 894-902. Doyle R. J., Thomasson D. L., Gray R. D. and Glew R. H. (1975) Spectral changes accompanying the interaction
between metal ligands and concanavalin A. FEBS Lett. 52, 185-187. Drickamer K. (1988) Two distinct classes of carbohydraterecognition domains in animal lectins. J. biol. Chem. 263, 9557-9560. Ewart K. V., Rubinsky B. and Fletcher G. L. (1992) Structural and functional similarity between fish antifreeze proteins and calcium-dependent lectins. Biochem. biophys. Res. Commun. 185, 335-340. Fuhlendorff J., Clemmensen I. and Magnusson S. (1987) Primary structure of tetranectin, a plasminogen kringle 4 binding plasma protein: homology with asialoglycoprotein receptors and cartilage proteoglycan core protein. Biochemistry 26, 6757~764. Giga Y., Ikai A. and Takahashi K. (1987) The complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina. J. biol. Chem. 262, 6197~5203. Gunthorpe M. E., Sikes C. S. and Wheeler A. P. (1990) Promotion and inhibition of calcium carbonate crystallization in vitro by matrix protein from blue crab exoskeleton. Biol. Bull. 179, 191-200. Hawgood S. (1989) Pulmonary surfactant apoproteins: a review of protein and genomic structure. Am. J. Physiol. 257, L13-L22. Inoue S., Kogaki H., Ikeda K., Samejima Y. and OmoriSatoh T. (1991) Amino acid sequences of the two subunits of a phospholipase A 2 inhibitor from the blood plasma of Trimeresurus flavoviridis. J. biol. Chem. 266, 1001-1007. Kikutani H., Inui S., Sato R., Barsumian E. L., Owaki H., Yamasaki K., Kaisho T., Uchibayashi N., Hardy R. R., Hirano T., Tsunasawa S., Sakiyama F., Suemura M. and Kishimoto T. (1986) Molecular structure of human lymphocyte receptor for immunoglobulin E. Cell 47, 657~565. Kubo T., Komano H., Okada M. and Natori S. (1984) Identification of hemagglutinating protein and bactericidal activity in the hemolymph of adult Sarcophaga peregrina on injury of the body wall. Devl comp. lmmunol. 8, 282-291. Mann S. (1988) Molecular recognition in biomineralization. Nature 332, 119 124. Matsumoto I., Jinbo A., Kitagaki H., GolovtchenkoMatsumoto A. M. and Seno N. (1980) Detection of lectin-sugar interaction by ultraviolet difference spectroscopy. J. Biochem. 88, 1093-1096. Muramoto K., Ogata K. and Kamiya H. (1985) Comparison of the multiple agglutinins of the acorn barnacle, Megabalanus rosa. Agric. Biol. Chem. 49, 85-93. Muramoto K. and Kamiya H. (1986) The amino acid sequence of a lectin of the acorn barnacle Megabalanus rosa. Biochim. biophys. Acta 874, 285-295. Muramoto K. and Kamiya H. (1990) The amino-acid sequence of multiple lectins of the acorn barnacle Megabalanus rosa and its homology with animal lectins. Biochim. biophys. Acta 1039, 42-51. Muramoto K., Kado R., Takei Y. and Kamiya H. (1991) Seasonal changes in the multiple lectin compositions of the acorn barnacle Megabalanus rosa as related to ovarian development. Comp. Biochem. Physiol. 98B, 603-607. Muramoto K. and Kamiya H. (1992) The amino acid sequence of a lectin from conger eel, Conger myriaster, skin mucus. Biochim. biophys. Acta 1116, 129-136. Muramoto K., Yako H. and Kamiya H. (1994) Multiple lectins as major proteins in the coelomic fluid of the acorn barnacle Megabalanus rosa. Comp. Biochem. Physiol. 107B, 395-399. Ng N. F. L. and Hew C. L. (1992) Structure of an antifreeze polypeptide from the sea raven. J. biol. Chem. 267, 16069-16075.
Barnacle lectins and calcium carbonate crystals Takahashi H., Komano H., Kawaguchi N., Kitamura N., Nakanishi, S. and Natori S. (1985) Cloning and sequencing of cDNA of Sarcophaga peregrina humoral lectin induced on injury of the body wall. J. biol. Chem. 260, 12228-12233. Takeya H., Nishida S., Miyata T., Kawada S., Saisaka Y.,
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Morita T. and lwanaga S. (1992) Coagulation factor X activating enzyme from Russell's viper venom (RVV-X). J. biol. Chem. 267, 14109-14117. Wheeler A. P. and Sikes C. S. (1984) Regulation of carbonate calcification by organic matrix. Am. Zool. 24, 933-944.
Pergamon
Inhibition of the growth of calcium carbonate crystals by multiple lectins in the coelomic fluid of the acorn barnacle Megabalanus rosa Koji Muramoto, Hiroshi Yako, Koji Murakami, Satoshi Odo* and Hisao Kamiya School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-01, Japan; and *Marine Biotechnology Institute, Kamaishi, Iwate 026, Japan The interaction between the multiple lectins (BRA-2, BRA-3) in the coelomic fluid of the acorn barnacle Megabalanus rosa and calcium ions was studied by ultraviolet difference spectroscopy. The association constants for BRA-2 and BRA-3 were 1.4 × 104M -t and 2.2 × 104M -t at pH8.0, respectively. Multiple lectins inhibited the crystal growth of supersaturated calcium carbonate solution at a iectin concentration of > 0.1 mg/30 mi. Destruction of the carbohydrate-recognition domains of the lectins decreased the inhibitory activities on the crystal growth. Incorporation of BRAs into the crystal was demonstrated using FITC-labeled lectins. Key words: Calcium carbonate; Lectins; Megabalanus rosa; Recognition domains.
Comp. Biochem. Physiol. I07B, 401-409, 1994.
Introduction
The present extent of our knowledge permits us to organize the known animal lectins into several categories (Drickamer, 1988). One group consists of fl-galactose-binding proteins isolated from various tissues and serum of many vertebrates, such as the electric eel, conger eel, chicken, rat, bovine and human. They are designated as S-type lectins, as they often require thiol-reducing agents to maintain their activity. The activity is independent of divalent cations. A second group consists of the calcium-dependent (C-type) animal lectins, including a mannose-binding protein isolated from mammalian liver, membranous glycoprotein receptors isolated from mammalian and avian liver, and a galactose-binding lectin isolated from rattlesnake venom. Invertebrate lectins isolated from the flesh fly (Sarcophaga peregrina) (Takahashi et al., 1985), the acorn barnacle (Megabalanus rosa) (Muramoto and Kamiya, 1986, 1990) and Correspondence to: K. Muramoto, Faculty of Agriculture, Tohoku University, Sendai 981, Japan. Fax: 81 22-2721870. Received 13 July 1993; accepted I 1 August 1993. CBPB
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the sea urchin (Anthocidaric crassispina) (Giga et al., 1987) have been found to contain a C-type carbohydrate-recognition domain. The domain is constructed with two characteristic disulfide bond loops containing homologous amino acid sequences. The C-type carbohydrate-recognition domain has also been detected in a wide variety of proteins, such as a proteoglycan core protein (Doege et al., 1991), tetranectin (Fuhlendorff et aL, 1987), lymphocyte immunoglobulin E receptor (Kikutani et al., 1986), phospholipase A2 inhibitor (Inoue et al., 1991), pulmonary surfactant apoproteins (Hawgood, 1989), coagulation factors (Takeya et al., 1992), antifreeze polypeptides (Ng and Hew, 1992; Ewart et al., 1992) and pancreatic stone protein (De Caro et al., 1987). The findings suggest that these proteins which mediate proteincarbohydrate and protein-protein interaction can be utilized in a number of quite distinct biological contexts. We have isolated and characterized multiple galactose-binding lectins from the coelomic fluid of the acorn barnacle, Megabalanus rosa (Muramoto et al., 1985). They are composed of
402
Koji Muramoto et al.
three different molecular species with respect to molecular weight, designated BRA-1 (Mr 330,000), BRA-2 (Mr 140,000) and BRA-3 (Mr 64,000). BRA-1 and BRA-2 are composed of identical subunits (M r 22,000), of which the amino acid sequence shows only 25% identity with that of the BRA-3 subunit (Mr 16,000) (Muramoto and Kamiya, 1990). We showed the seasonal changes in the multiple lectin compositions in the coelomic fluid in connection with the development of ovaries (Muramoto et al., 1991). The multiple lectins are major coelomic proteins as judged by protein determination (Muramoto et al., 1994). Invertebrate lectins are commonly assumed to function as recognition molecules, both in intercellular and intracellular systems. Although the most thoroughly explored potential functions are those involved with invertebrate immunity (Kubo et al., 1984; Cooper et al., 1984), other biological functions also need to be investigated. In this paper, we studied the interaction of the multiple lectins of M . rosa with calcium ions and the effects of the lectins upon the growth of calcium carbonate crystals using a pH-monitor system. The lectins caused a marked decrease in crystal growth in a dosedependent manner.
Materials and Methods Multiple galactose-binding lectins, BRA-1, BRA-2 and BRA-3, were isolated from the coelomic fluid of M. rosa, as previously described (Muramoto et al., 1985). The modified BRAs were prepared by reaction with fluorescein isothiocyanate (FITC) (Muramoto et al., 1985), monoiodoacetoamide and cyanogen bromide as described (Muramoto and Kamiya, 1990). Enzymatic digestion of the lectins was performed with TPCK trypsin (Worthington, Freehold, NJ), chymotrypsin (P-L Biochemicals, Milwaukee, WI) or V8 proteinase (Wako Chemicals, Osaka, Japan) in 0.1 M ammonium carbonate at 37°C for 24hr at substrate/ enzyme = 100: 1. The digests were lyophilized and used for the experiment. The average pHs of the coelomic fluid of M . rosa and the seawater, where the barnacle lived, were 7.17 and 8.05, respectively. The calcium concentrations of the coelomic fluid and the seawater were measured as 10.17_+0.02mM and 9.49_+ 0.16mM (+SE, N = 3), respectively, by a inductively coupled plasma spectrometer (Shimadzu ICPS-1000III). Organic matrix preparation M. rosa shells were carefully cleaned and air-dried. One hundred grams of the shell were
crushed and decalcified with 500 ml of 5% HC1 at room temperature overnight. The solution was centrifuged to separate insoluble materials, and the supernatant was dialyzed against tap water for 2 days and then lyophilized to yield 96 mg. The insoluble fraction was further extracted with 200 ml of 5% NaOH. The extract was dialyzed against tap water for 2 days and lyophilized to yield 144 mg. The shell was also decalcified with 15% EDTA at pH 7.5. Calcium binding o f B R A s
The association constants of the calcium binding of BRAs were measured by difference spectroscopy (Doyle et al., 1975; Matsumoto et al., 1980). BRA was dissolved in 0.1 M EDTA/50mM Tris-HC1 buffer (pH8.0) at 5 mg/ml and left for 30 min at room temperature. The solution was diluted with 3 ml of 50 mM Tris-HCl buffer (pH 8.0) and then dialyzed against the same buffer at 4°C for 2 days with several changes of the buffer to remove EDTA thoroughly. After dialysis, the sample solutions were adjusted to 1 mg/ml with the buffer and filtered through 0.45#m pore diameter membranes. Calcium-binding experiments were conducted at 25°C by the addition of small aliquots of 1M or 0.1M CaCI2 to the test solution in Semimicro quartz cells of 1 cm path length. Absorbance and difference spectra were measured between 220 and 320nm using a temperature-controlled Jasco Ubest-50 spectrophotometer. It took about 10 min for the Ca 2÷induced absorbance changes to reach equilibrium. The intensities of difference spectra (peak-trough) AA were measured as a function of increasing calcium concentration ([S]). Plots according to the equation, [S]/A = (l/Amax) x [S] + 1/Am,x
x
K
(1)
were made, where K is the association constant and Areax is the maximum absorption difference. The solid line is a least-squares plot of the data to the equation. The K value was calculated from the intercept on the abscissa. Calcium carbonate crystallization assay
The inhibition assay for calcium carbonate crystallization was monitored by the change in pH of a solution supersaturated with respect to calcium and carbonate (Gunthorpe et al., 1990). The assay began with an induction period of relatively constant pH, during which time crystal nucleation occurs, and then continues with a rapid decrease in pH indicating crystal growth. The pH was continuously monitored and recorded. The incubation medium included 29.1 ml artificial seawater (ASW:
Barnacle leetins and calcium carbonate crystals
0.5 M NaC1 and 0.011 M KC1) in a glass vessel placed in a circulating water bath maintained at 20.0°C, to which 0.3 ml 1.0 M CaCIJ2H20 was added to obtain 10 mM calcium. Next, the test sample was added and stirred for 10min. Finally, 0.6 ml of 0.4 M NaHCO3 was added to yield 8 mM dissolved inorganic carbon, and the solution was titrated to a pH of 8.30 with 1 N NaOH to begin the assay.
Incorporation of FITC-BRAs into calcium carbonate crystal The amount of FITC-labeled lectins incorporated into crystals was determined by measuring the fluorescence intensity after solubilization of the crystals. Crystallization was performed in the presence of 10 or 100#g of FITC-BRA for 2hr at 20°C under the same conditions as described above. The solution was transferred to a centrifugal tube. Resulting calcium carbonate was recovered by centrifugation at 3000 rpm for 10 min at 6°C. The pellet was washed with 5 ml distilled water three times, dissolved in 0.7 ml of 0.1 M acetic acid and filtered by a centrifugal ultrafiltration membrane (30 kDa cutoffs; Tosoh, Tokyo, Japan). Recovered FITC-BRA was dissolved in 6 ml of 0.2 M Tris-HCl (pH 8.0) and measured by a JASCO FP-550A spectrofluorometer using an excitation wavelength of 490nm and an emission wavelength of 0
403
520 nm. Quantitative analysis was performed with calibration curves of corresponding FITC-BRAs. Results The difference spectrum of BRA-2 induced by the addition of 1 mM calcium at 25°C is shown in Fig. 1. The difference spectrum induced by the addition of calcium has two positive peaks. The intensity of the difference spectrum depended on the calcium concentration. The changes in AA 292-288 nm due to the binding of the calcium ion to BRA-2 and the corresponding straight line obtained by plotting [S]/AA vs IS], according to the equation, are shown in Fig. 2, where [S] is the calcium concentration and AAr~axis the maximum change in the difference absorption between 292 and 288 nm. By plotting [S]/AA as a function of [S] as in the equation, the association constant, K, was calculated. The Kvalue, 1.4 x 104 M -I, for BRA-2 is similar to the value of 2.2 x 104 M -] obtained for BRA-3 at pH 8.0. By changing the pH to 7.0 the association constants for both BRA-2 and BRA-3 decreased to 7.4 x 104 M - ' and 6.8 x 103 M -l, respectively (Table 1). Inhibitory activities of multiple lectins on the crystal growth of calcium carbonate were monitored by the drift in pH. This assay relies on the fact that the crystallization produces hydrogen ~
2.0
0.01
1.0
- 0.02
I
220
I
260
I
I
3 0 0 nm
0 220
260
300 nl
Fig. 1. A: Ultraviolet difference spectrum of BRA-2 induced by 1 mM calcium chloride. BRA-2 was in 50 mM Tris-HCI (pH 8.0) at 1 mg/ml. B: Ultraviolet absorption spectra of BRA-2 without calcium and with 1 mM calcium chloride.
404
Koji Muramoto et
al.
0.015
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/
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ions and the rate of crystal growth is proportional to the decrease of pH in the test solution. That the downward pH shifts during the crystallization assays did indeed indicate crystal growth was verified by the presence of a pellet of crystals produced by centrifugation of the solution. It took 60 min for the pH drift to cease. The addition of BRAs at 0.1 mg/30 ml totally inhibited the crystal growth as shown in Fig. 3. The inhibition was dose-dependent; as the lectins were decreased by one-tenth, no inhibition was observed. Neither an S-type lectin isolated from conger eel (Muramoto and Kamiya, 1992) nor bovine serum albumin showed any inhibitory effect at 0.1 mg/30ml. At 1 mg/30 ml, bovine serum albumin began to show some inhibition; meanwhile, conger eel lectin did not inhibit at all. Inhibitory activity of different concentrations of poly-aspartic acid (Asp) (Sigma, St Louis, MO) was examined (Fig. 4). At 0.1 mg/30 ml, poly-Asp completely inhibited the crystal Table 1. Effectof pH on the associationconstants of BRAs to calcium ion K (M -t)
pH BRA-2 BRA-3 7.0 7.4 x 104 (293-289)* 6.9 × 103 (296-290) 8.0 1.4 x 104 (292-289) 2.2 × 104 (296-290) 9.0 1.8 x 104 (291-288) 1.4 x I04 (295-289) *Wavelength (nm) at peak-trough.
growth. Upon 10-fold dilution of poly-Asp, a significant inhibition was still observed. Polyglutamic acid (Sigma) and phosvitin (Sigma) showed similar inhibitory activities, but phosvitin had a smaller effect than the other two. BRAs lost their inhibitory activities upon the destruction of the conformation by reduction and S-carboxamidomethylation (Fig. 5). Cyanogen bromide cleavage also diminished the inhibitory activity of BRA-2. On the other hand, BRA-3 could keep its activity to some extent. This result indicates the importance of the carbohydrate-recognition domain for inhibition, because a single methionine residue contained in a BRA-3 subunit locates outside the carbohydrate-recognition domain, and the domain can stand in spite of the breaking of the subunit structure. Tryptic digestion of BRAs decreased the inhibitory activity at 0.1 mg/30ml, whereas neither chymotrypsin nor V8 proteinase digestion had any influence on the inhibition (data not shown). Modification of BRAs with FITC decreased the inhibitory activities as shown in Fig. 6. The average degrees of labeling were 22.7, 6.4, and 1.8 mol of FITC per moi BRA-1, BRA-2, and BRA-3, respectively. The agglutinating activities of BRAs against rabbit erythrocytes did not change by labeling with FITC. Incorporation of BRAs into the calcium carbonate crystals was investigated by labeling
Barnacle lectins and calcium carbonate crystals
....
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Fig. 3. Inhibition of the crystal growth of calcium carbonate monitored by pH drift. A: BRA-I; B: BRA-2; C: BRA-3; D: bovine serum albumin; E: control (water). Concentration of protein samples was 0.1 mg/30 ml.
BRAs with FITC. The results are summarized in Table 2. Calcium carbonate crystallization was carried out in the presence of 10 or 100 # g of FITC-BRAs under the same conditions as described above. The amount of the crystal formed was 687 + 103 # g ( + S E , N = 6) in this experiment. In the presence of 10/~g of FITC-BRAs, 0.34-0.39 # g of the lectins were
incorporated to the crystal. The amount of incorporated FITC-BRAs was relatively constant. However, by increasing the amount of applied FITC-BRAs to 100#g, the incorporated amount varied from 2 . 1 6 # g for FITC-BRA-3 to 7.02/~g for FITC-BRA-1. The soluble matrix from the acorn barnacle shell inhibited calcium carbonate crystallization
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Fig. 4. Inhibition of the crystal growth of calcium carbonate by poly-aspartic acid monitored by pH drift. A: 0.I rag/30 ml; B: 0.01 rag/30 ml; C: 0.001 mg/30 ml; D: 0 mg/30 ml.
406
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Fig. 5. Inhibition of the crystal growth of calcium carbonate by modified BRAs monitored by pH drift. A: CAM-BRA-2; B: CAM-BRA-3; C: CNBr fragment-1 of BRA-2; D: CNBr fragment-2 of BRA-2; E: CNBr fragment of BRA-3; F: control. Concentration of the samples was 0.1 rag/30 ml.
(Fig. 7). At 1 mg/30 ml, three kinds of organic matrices showed full inhibition. The activities of HCI extract and EDTA extract decreased at 0.1 rag/30 ml.
Discussion Lectins are a group of sugar-binding proteins which recognize specific carbohydrate structures
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(min)
Fig. 6. Inhibition of the crystal growth of calcium carbonate by FITC-BRAs monitored by pH-drift. A: FITC-BRA-I; B: FITC-BRA-2; C: FITC-BRA-3; D: control. Concentration of the samples was 0.1 mg/30 ml.
|
120
Barnacle lectins and calcium carbonate crystals
407
Table 2. Incorporation of FITC-BRA into the calcium observed that ultraviolet difference spectra of carbonate crystal BRAs were induced by the addition of calcium Incorporated FITC-BRA as well as by specific sugars, e.g. galactose. The (/tg, _.+SE, N = 7 ) sugar-induced spectral changes, which would be A B explained by the conformational changes in FITC-BRA-1 0.34 +__0.06 7.02 + 0.40 BRAs, could not be detected unless the calciumFITC-BRA-2 0.39 + 0.06 5.70 + 0.60 free BRA had been previously bound with FITC-BRA-3 0.37 + 0.05 2.16 + 0.26 calcium ions. This is coincidental with the calA: 10#g FITC-BRA was added; B: 100#g of FITC-BRA was added. In this experiment, 687+103#g (+SE, cium ion being required for the agglutinating activity. The association constants obtained for N = 6) of calcium carbonate was formed. BRAs are comparable to those reported for phosvitin or organic matrices prepared from and are proposed to have many different mollusk shells (Wheeler and Sikes, 1984). The biological roles. The multiple lectins from the affinity against calcium ion and the high concoelomic fluid of M . r o s a are C-type lectins, tents of the lectins could explain the difference which have the characteristic carbohydrate- of calcium concentrations between the coelomic recognition domain. C-type lectins have a rather fluid and seawater. wide range of sugar-binding specificity, but Calcium carbonate and organic matrix are they always require calcium ions for the sugar two major components in the barnacle shell binding. The calcium binding sites are located (Bourget, 1987). The latter constitutes several on the carbohydrate-recognition domain. The percent by weight of the shell. These compresence of the carbohydrate-recognition do- ponents may play important roles in regulation main in a variety of proteins, including pancre- of crystal growth, as reviewed by Mann (1988) atic stone protein and cartilage proteoglycan, and Berman e t al. (1988). Regulation could and the experimental results concerning the include nucleation, rate of growth, orientation, seasonal dynamics and the high contents of the size and overall morphology of crystals, and it multiple lectins in M . r o s a allowed us to study can occur through pH of the environment, the the interaction between the lectins and the presence of inhibitors, the presence of microencalcium ion and their involvement in shell vironments, the presence and kind of nucleation formation. sites and the degree of supersaturation (Mann, The difference spectrophotometric method 1983). Organic matrices isolated from various has been used for the study of specific inter- organisms are inhibitors of calcium carbonate actions between lectins and sugars or metals crystallization when free in solution. Inhibition (Doyle e t al., 1975; Matsumoto e t al., 1980). We may be correlated to the affinity of matrix
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Fig. 7. Inhibition of the crystal growth of calcium carbonate by organic matrices of M. rosa shell monitored by pH drift. A: HC1 extract; B: NaOH extract; C: EDTA extract; D: control. Concentration of the sample was 0. ! rag/30ml.
408
Koji Muramoto et al.
molecules for crystal surfaces and adequate coverage of growth sites on crystal surfaces. It has also been shown that the same matrices, when attached to an insoluble support, promoted crystallization (Wheeler and Sikes, 1984). We showed in this study that multiple lectins, as well as the organic matrix, from M . rosa inhibited the crystal growth of calcium carbonate. Although we do not have enough evidence for the chemical structural relationship between the multiple lectins and the organic matrix, the matrix contained components crossreacted with anti-BRA antibodies as revealed by a dotblotting assay (data not shown). Immunohistochemical study revealed that immunostaining was found in almost all M . rosa tissues and was especially prominent in the cirri (Muramoto et al., 1991). The pH dependency of association constants against the calcium ion and the decrease of inhibitory activity by modification of the conformation, indicate that the multiple lectins are among the possible regulators for calcium cabonate crystallization.
Acknowledgements--This work was supported in part by grants from New Energy and Industrial Technology Development Organization, and the Research Institute of Marine Invertebrates, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References Addadi L. and Weiner S. (1985) Interactions between acidic proteins and crystals: stereochemical requirements in biomineralization. Proc. Natn. Acad. Sci, USA 82, 4110-4114. Berman A., Addadi L. and Weiner S. (1988) Interactions of sea-urchin skeleton macromolecules with growing calcite crystals--a study of intracrystalline proteins. Nature 331, 546-548. Bourget E. (1987) Barnacle shells: composition, structure and growth. In Barnacle Biology (Edited by Southward A. J.), A. A. Balkema, Rotterdam. Cooper E. L., Stein E. A. and Wojdani A. (1984) Recognition receptors in annelids. In Recognition Proteins, Receptors, and Probes: Invertebrates (Edited by Cohen E.), pp. 43-54. Alan R. Liss, New York. De Caro A. M., Bonicel J. J., Rouimi P., De Caro J. D., Sarles H. and Rovery M. (1987) Complete amino acid sequence of an immunoreactive form of human pancreatic stone protein isolated from pancreatic juice. Eur. J. Biochem. 168, 201-207. Doege K. J., Sasaki M., Kimura T. and Yamada Y. (I 991) Complete coding sequence and deduced primary structure of the human cartilage large aggregating proteoglycan, aggrecan. J. biol. Chem. 266, 894-902. Doyle R. J., Thomasson D. L., Gray R. D. and Glew R. H. (1975) Spectral changes accompanying the interaction
between metal ligands and concanavalin A. FEBS Lett. 52, 185-187. Drickamer K. (1988) Two distinct classes of carbohydraterecognition domains in animal lectins. J. biol. Chem. 263, 9557-9560. Ewart K. V., Rubinsky B. and Fletcher G. L. (1992) Structural and functional similarity between fish antifreeze proteins and calcium-dependent lectins. Biochem. biophys. Res. Commun. 185, 335-340. Fuhlendorff J., Clemmensen I. and Magnusson S. (1987) Primary structure of tetranectin, a plasminogen kringle 4 binding plasma protein: homology with asialoglycoprotein receptors and cartilage proteoglycan core protein. Biochemistry 26, 6757~764. Giga Y., Ikai A. and Takahashi K. (1987) The complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina. J. biol. Chem. 262, 6197~5203. Gunthorpe M. E., Sikes C. S. and Wheeler A. P. (1990) Promotion and inhibition of calcium carbonate crystallization in vitro by matrix protein from blue crab exoskeleton. Biol. Bull. 179, 191-200. Hawgood S. (1989) Pulmonary surfactant apoproteins: a review of protein and genomic structure. Am. J. Physiol. 257, L13-L22. Inoue S., Kogaki H., Ikeda K., Samejima Y. and OmoriSatoh T. (1991) Amino acid sequences of the two subunits of a phospholipase A 2 inhibitor from the blood plasma of Trimeresurus flavoviridis. J. biol. Chem. 266, 1001-1007. Kikutani H., Inui S., Sato R., Barsumian E. L., Owaki H., Yamasaki K., Kaisho T., Uchibayashi N., Hardy R. R., Hirano T., Tsunasawa S., Sakiyama F., Suemura M. and Kishimoto T. (1986) Molecular structure of human lymphocyte receptor for immunoglobulin E. Cell 47, 657~565. Kubo T., Komano H., Okada M. and Natori S. (1984) Identification of hemagglutinating protein and bactericidal activity in the hemolymph of adult Sarcophaga peregrina on injury of the body wall. Devl comp. lmmunol. 8, 282-291. Mann S. (1988) Molecular recognition in biomineralization. Nature 332, 119 124. Matsumoto I., Jinbo A., Kitagaki H., GolovtchenkoMatsumoto A. M. and Seno N. (1980) Detection of lectin-sugar interaction by ultraviolet difference spectroscopy. J. Biochem. 88, 1093-1096. Muramoto K., Ogata K. and Kamiya H. (1985) Comparison of the multiple agglutinins of the acorn barnacle, Megabalanus rosa. Agric. Biol. Chem. 49, 85-93. Muramoto K. and Kamiya H. (1986) The amino acid sequence of a lectin of the acorn barnacle Megabalanus rosa. Biochim. biophys. Acta 874, 285-295. Muramoto K. and Kamiya H. (1990) The amino-acid sequence of multiple lectins of the acorn barnacle Megabalanus rosa and its homology with animal lectins. Biochim. biophys. Acta 1039, 42-51. Muramoto K., Kado R., Takei Y. and Kamiya H. (1991) Seasonal changes in the multiple lectin compositions of the acorn barnacle Megabalanus rosa as related to ovarian development. Comp. Biochem. Physiol. 98B, 603-607. Muramoto K. and Kamiya H. (1992) The amino acid sequence of a lectin from conger eel, Conger myriaster, skin mucus. Biochim. biophys. Acta 1116, 129-136. Muramoto K., Yako H. and Kamiya H. (1994) Multiple lectins as major proteins in the coelomic fluid of the acorn barnacle Megabalanus rosa. Comp. Biochem. Physiol. 107B, 395-399. Ng N. F. L. and Hew C. L. (1992) Structure of an antifreeze polypeptide from the sea raven. J. biol. Chem. 267, 16069-16075.
Barnacle lectins and calcium carbonate crystals Takahashi H., Komano H., Kawaguchi N., Kitamura N., Nakanishi, S. and Natori S. (1985) Cloning and sequencing of cDNA of Sarcophaga peregrina humoral lectin induced on injury of the body wall. J. biol. Chem. 260, 12228-12233. Takeya H., Nishida S., Miyata T., Kawada S., Saisaka Y.,
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Morita T. and lwanaga S. (1992) Coagulation factor X activating enzyme from Russell's viper venom (RVV-X). J. biol. Chem. 267, 14109-14117. Wheeler A. P. and Sikes C. S. (1984) Regulation of carbonate calcification by organic matrix. Am. Zool. 24, 933-944.