Multiple invertebrate lysozymes in blue mussel (Mytilus edulis)

Comparative Biochemistry and Physiology Part B 136 (2003) 107–115

Multiple invertebrate lysozymes in blue mussel (Mytilus edulis) Ørjan M. Olsena, Inge Waller Nilsena, Knut Slettenb, Bjørnar Myrnesa,* a

Marine Biotechnology and Fish Health, Norwegian Institute of Fisheries and Aquaculture, P.O. Box 6122, Tromsø N-9291, Norway b Department of Biochemistry, Biotechnology Centre of Oslo, University of Oslo, Oslo N-0316, Norway Received 17 April 2003; received in revised form 12 June 2003; accepted 12 June 2003

Abstract Initial analyses of lysozyme activities in individual blue mussels Mytilus edulis indicated variations in features of activity from the crystalline style to the remaining body parts (the soft body). Two separate larger scale lysozyme isolations were performed employing extracts from 1000 styles and 50 soft bodies, respectively. The soft body origin contained one, or one major, lysozyme that was purified to homogeneity. This 13 kDa protein, designated bm-lysozyme, was sequence-analysed and found to represent the product of a recently published invertebrate-type lysozyme gene from M. edulis. Three additional lysozymes were isolated from the style extract and one of them was fully purified. All four lysozymes showed different profiles of enzymatic features such as responses to pH, ionic strengths and divalent cations. From the results and the profound differences demonstrated we believe that the observed multiple forms of lysozyme activities in blue mussel reflect multiple genes instead of individual lysozyme variants and that the lysozymes serve different functions in the blue mussel. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: Invertebrate; Blue mussel; Crystalline style; Protein; Lysozyme; Purification; Characterisation; Sequence alignment

1. Introduction Lysozyme is a small antibacterial enzyme widely distributed in organisms from bacteriophages to human. This enzyme takes part in protecting organisms from the ever-present danger of bacterial infection by hydrolysing b-1,4-linked glycoside ` bonds of bacterial cell wall peptidoglycans (Jolles, 1996). The presence of lysozyme-like activity in shellfish has been known for many years and lysozyme activity has been detected in bivalve hemolymph, hemocytes and in different tissues (McDade and Tripp, 1967; McHenery et al., 1979; McHenery and Birckbeck, 1982; Chu and La *Corresponding author. Tel.: q47-776-2900; fax: q47-77629100. E-mail address: [email protected] (B. Myrnes).

Peyre, 1993; Pipe, 1990; Myrnes and Johansen, 1994). The bivalve lysozymes are believed to be involved in digestion (McHenery et al., 1979) as well as in self-defence against pathogenic bacteria (Cheng, 1983; Chu, 1988; Carballal et al., 1997; Allam and Paillard, 1998). Up to recently, two lysozyme classes (c-type and g-type) have been described in the animal ` 1996). A short N-terminal kingdom (see Jolles, sequence of a lysozyme from the starfish Asteria rubens provided the first data indicating a novel ` and class of lysozymes among invertebrates (Jolles ` 1975). Furthermore, examples of these Jolles, invertebrate lysozymes came from marine bivalves ` et al., 1996) and the first entire protein (Jolles sequence of an invertebrate lysozyme, from the bivalve Tapes japonica, was reported by Ito et al. (1999). The first gene sequence of an invertebrate

1096-4959/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S1096-4959(03)00174-X

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lysozyme came from the work on an Icelandic scallop (Chlamys islandica) lysozyme named chlamysin (Nilsen et al., 1999). The chlamysin gene was shown to be organised differently from insect c-type lysozymes, but similar to vertebrate c-type genes with respect to the number of exons (Nilsen and Myrnes, 2001). A family tree of lysozymes demonstrated a large distance between the group of invertebrate lysozymes and the group of insect and vertebrate c-type lysozymes (Nilsen and Myrnes, 2001). In a recent report putative i-type proteins from crustacean (shrimp) and from insect (Drosophila) were included in phylogenetic analysis of lysozymes and the results suggested that i-type lysozymes form a monophyletic family (Bachali et al., 2002). The work also showed that the M. edulis i-lysozyme gene is organised in five exons, in contrast to the four-exons chlamysin gene. Enzymatic properties of invertebrate lysozymes are so far best documented for the blue mussel lysozyme (McHenery and Birckbeck, 1982), the T. japonica lysozyme (Ito et al., 1999) and chlamysin (Nilsen et al., 1999). The described T. japonica and the mussel lysozymes display temperature-dependent as well as pH-dependent profiles similar to most c-type lysozymes and they also possess chitinase activity (McHenery and Birckbeck, 1982; Ito et al., 1999). In contrast, chlamysin has significant activity at 0 8C, display no chitinase activity and no lysozyme activity at neutral or higher pH (Nilsen et al., 1999). In this paper we report the separation and partial characterisation of multiple i-lysozymes from crystalline styles and other tissues of the blue mussel. The four enzymes isolated demonstrate varying dependence and responses to pH, ionic strength and divalent cations. 2. Material and methods 2.1. Materials and biological samples Wild blue mussels of shell length 40–60 mm were collected at 0–1-m depth from a site in Southern Norway. The collection consisted of 50 shells for soft tissues (from October 1999) and 1000 shells for crystalline styles (from April 2000). Tissue samples were frozen and stored at y20 8C until use. The following chromatography columns were all supplied by Amersham Pharmacia Biotech: SP

Sepharose FF, Sephacryl S200 HR, Superdex娃 75 10y30, RESOURCE䉸 S, RESOURCE娃 PHE and Mono S HR 5y5. Micrococcus lysodeikticus was purchased from Sigma (St. Louis, USA). Chlamysin used in this study was isolated earlier (Nilsen et al., 1999). All other chemicals used were of reagent grade. 2.2. Purification of mussel lysozymes 2.2.1. Tissue extract Soft bodies of 50 mussels (542 g wet weight) were thawed at 4 8C and homogenised in 2 l of 2% acetic acid using a Warring blender. The extract was then centrifuged at 16 000=g for 15 min and the supernatant containing lysozyme activity was collected. A second extraction was performed to release further lysozyme activity from the pellet using 1 l 2% acetic acid before mixing, centrifugation and collection of supernatant. The supernatants obtained were combined and used as crude extract for lysozyme purification. All further steps in isolation of lysozyme were carried out at 6–8 8C. The crude extract was fractionated by ultrafiltration on an Amicon ultrafiltration CH2A unit fitted with Amicon Hollow Fiber cartridge H1P30-20 (cut-off 30 kDa). The filtrate (permeate) was concentrated using an Amicon Hollow Fiber cartridge H1P3-20 (cut-off 3 kDa). The resulting enzyme solution was then applied to a SP Sepharose FF cation-exchange column (36 ml) equilibrated with 0.05 M sodium acetate buffer (pH 5). The column was washed with the same buffer and bound protein was eluted employing a 0–0.5 M NaCl gradient in 0.05 M sodium acetate buffer (pH 5). Fractions containing lysozyme activity and eluted at 0.1–0.25 M NaCl were combined, concentrated and subjected to gel filtration on Sephacryl S200 HR (493 ml) in 0.05 M acetate buffer (pH 5). Lysozyme was further purified by FPLC ion exchange chromatography on Mono S HR 5y5 column in 0.05 M MES buffer (pH 5.5). Finally, the mussel lysozyme was purified by gel filtration on a FPLC䉸 Superdex娃 75 10y30 column equilibrated in 0.05 M MES buffer (pH 5.5) containing 0.15 NaCl. Sample fractions showing a single protein band of ;13 kDa after SDSyPAGE were pooled and the isolated lysozyme, designated as bm-lysozyme, was concentrated by a PM 10 membrane disc (Amicon).

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2.2.2. Crystalline style extract Crystalline styles (21 g wet weight) were thawed at 4 8C and homogenised in 84 ml of 2% acetic acid using a Polytron PT 1200C homogenizer (Kinematica AG, Switzerland) and subsequently centrifuged at 16 000=g for 15 min. The supernatant containing lysozyme activity was collected before a second extraction of remaining lysozyme in pellet using 84 ml 2% acetic acid followed by mixing and centrifugation as described. The supernatants containing lysozyme activity were combined and used as crude extract. All further steps in purification of lysozyme were carried out at 6–8 8C. The crude extract was subsequently applied to a SP Sepharose FF cationexchange column (36 ml) equilibrated with 0.05 M sodium acetate buffer (pH 5). The column was further washed and subjected to a 0–1 M NaCl gradient in 0.05 M sodium acetate buffer (pH 5). Lysozyme activity was eluted at 0.2–0.7 M NaCl. Fractions containing lysozyme were pooled before concentration on a PM 10 ultrafilter (cut off 10 kDa) and diafiltration against 0.05 M MES buffer (pH 5.5), using an Amicon Diaflo stirred cell. The filtrate was then applied to an equilibrated RESOURCE䉸 S (6 ml) column connected to FPLC䉸. The column was washed with 0.05 M MES buffer (pH 5.5) and bound protein was eluted by increasing concentrations of NaCl (0–0.5 M) in the same buffer. The chromatography gave three peaks of lysozyme activity eluted at 0.2, 0.3 and 0.4 M NaCl, respectively, and these three enzyme preparations were designated as sA-, sB- and sClysozyme. The sB-lysozyme eluted at 0.3 M NaCl was dialysed against 0.02 M ammonium acetate buffer (pH 6) containing 4.5 M NaCl and applied to a RESOURCE娃 PHE column (1 ml). Lysozyme activity passing through the column was then collected and subsequently concentrated by ultrafiltration on a PM 10 ultrafilter, followed by diafiltration against 0.05M MES buffer (pH 5.5). The crystalline style sB-lysozyme was finally purified by FPLC ion exchange chromatography on a Mono S HR 5y5 column equilibrated in MES buffer. The column was washed with MES buffer and protein was eluted with a 0–0.5 M NaCl gradient in buffer. This produced a single Coomassie-stained protein band of ;22 kDa when analysed after SDS-PAGE.

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2.3. Sequence analysis The N-terminal amino acid sequence of bmlysozyme from soft bodies was analysed by automatic Edman degradation using a protein sequencer Model 477A (Applied Biosystems, Perkin Elmer) and a G1005A N-terminal sequencer (Hewlett-Packard). The protein was also reduced and alkylated with 4-vinyl-pyridine (Fridman et al., 1970), and this alkylated bm-lysozyme was cleaved by cyanogen bromide (CNBr) and the resulting polypeptides were separated by RPHPLC using a Pep-S C2yC18 column (Pharmacia) (Foss et al., 1998). Peak-fractions were collected and taken for Edman degradation. 2.4. Determination of lysozyme activity The activity of lysozyme towards lyophilised cell of M. lysodeikticus (0.2 mgyml) was measured as previously described (Myrnes and Johansen, 1994). The activity was determined from the first minute of linear decrease in absorbance at 450 nm. One unit of enzyme activity is defined as the amount of enzyme that catalyses a decrease in absorbance of 0.001 miny1. For determination of pH for optimal activity, assay-buffers of 10 or 50 mM sodium acetate and 5 or 20 mM sodium phosphate were used for pH-ranges 3.6–5.6 and 6.0–8.0, respectively, after adjusting the buffer ionic strength using NaCl. 2.5. Other methods Protein concentrations were determined by the microwell plate protocol of Pierce BCA Protein Assay (Pierce, Rockford, USA) using bovine serum albumin (BSA) as standard protein. Gel electrophoresis was performed in 10% NuPAGE Bis–Tris gel system (Novex, San Diego, USA) using a MES SDS running buffer, and the lysozyme migration length was compared to the Mark12娃 protein standard (Novex). Protein in gel was visualised by Coomassie staining. Independent signal peptide predictions were facilitated by the two programs Signal P (Nielsen et al., 1997) and PSORT II (Nakai and Kanehisa, 1992) hosted at http:yywww.cbs.dtu.dkyservicesySignalPy and http:yypsort.nibb.ac.jpy, respectively. For analyses of potential N-terminal transmembrane regions we used the SOSUI program (Hirokawa et al., 1998) hosted at http:yysosui.proteome.bio.tuat.ac.jpy.

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Fig. 1. Purification of crystalline style lysozymes on RESOURCE䉸 S. Experimental details are given under Section 2. Buffer B: 0.05 M MES buffer (pH 5.5) with 1 M NaCl (««.); absorbance at 280 nm ( ); lysozyme activity at pH 5.2 (⽧) and pH 6.8 (e). Lysozyme activities at pH 6.8 were determined only for fractions with minor activity.

3. Results 3.1. Purification of blue mussel lysozymes Lysozyme (0.5=106 units at pH 5.2 and Is 0.1) from crystalline styles of blue mussel was extracted and subjected to cation exchange chromatography on SP Sepharose followed by RESOURCE䉸 S yielding three separate lysozyme preparations; sA-, sB- and sC-lysozyme (Fig. 1). The sB-lysozyme was further purified by hydrophobic interaction chromatography followed by MonoS chromatography yielding 10 mg of protein. The purified sB-lysozyme preparation gave a single protein band of ;22 k Dalton on SDS-PAGE Fig. 2. The sA-lysozyme and sC-lysozyme preparations were concentrated and test experiments revealed heavy loss of enzyme activity on hydrophobic interaction chromatography (results not shown). These enzymes were not further purified. Lysozyme (2.8=106 units at pH 5.2 and Is 0.1) from the soft bodies of the bivalve M. edulis was extracted and subjected to cation exchange chromatography followed by gel filtration. Fractions possessing lysozyme activity were collected and further purified by FPLC ion exchange chromatography. The resulting single peak of lysozyme

eluted at 0.3 NaCl was collected and isolated by FPLC gel filtration yielding 575 mg of protein. When this purified mussel bm-lysozyme was subjected to SDS-PAGE a single band corresponding to a Mr of ;13 k Dalton was detected after Coomassie staining (Fig. 2).

Fig. 2. SDS-PAGE analyses of the purified bm-lysozyme and sB-lysozyme. Lanes: M, molecular mass markers; sB, 0.3 mg sB-lysozyme; bm, 0.3 mg bm-lysozyme.

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3.2. Characterisation of mussel lysozymes The specific activity of the isolated blue mussel lysozymes against M. luteus was 1.7=106 Uymg for bm-lysozyme and 1.9=106 Uymg for sBlysozyme when assayed at pH 5.2 and ionic strength 0.1. Enzymatic activities of the four separated mussel lysozymes were compared in buffer systems of varying pH and ionic strengths Fig. 3. The bm-lysozyme Fig. 3a showed an optimal activity at pH 6 at ionic strength 0.1 and a pHoptimum at pH 8 when the ionic strength was lowered. The crystalline style sA- and sB-lysozyme assayed at Is0.1 displayed an optimal activity at pH 4.8 and pH 5.6, respectively, Fig. 3b and c. In contrast to the other crystalline style enzymes, the sC-lysozyme showed low enzyme activity below pH 5.5 and optimal activity at neutral pH at both ionic strengths tested (Fig. 3d). The tolerance to relatively high ionic strengths were demonstrated by the bm- and sB-lysozymes displaying their highest activities at an ionic strength of 0.11 and at Is0.26 these lysozymes were still active possessing remaining activities of 11% and 7%, respectively. The partly purified sA-lysozyme had no hydrolysing activity on cell walls of M. lysodeikticus at pH 5.2 and Is0.2 (results not shown). Table 1 shows the effect of divalent cations on the activity of the mussel lysozymes and the previously isolated chlamysin from scallop. Relative to the control without cations, the presence of 5 mM calcium or magnesium increased both bm- and sBlysozyme activities five- to seven-fold. This cation concentration had no or minimal effect on chlamysin and sA-lysozyme activities. A complete inhibition of the latter enzyme activities was seen at 20 mM Mg2q or Ca2q, while 50 mM Ca2q was necessary to totally inhibit bm-lysozyme and sBlysozyme. N-terminal analysis of the bm-lysozyme exposed a sequence of the first 45 residues of the protein Fig. 4a. After treatment with 4-vinyl-pyridine, CNBr was used to cleave the reduced and alkylated protein. The following RP-HPLC revealed that this produced one minor peak and two larger, and the two protein fragments representing the two larger peaks were sequenced. The start of peptide 1 sequence was shown to correspond to position 27 in the sequence obtained for the N-terminal part of the protein. The second fragment, peptide 2 of the bm-lysozyme, gave rise to a sequence of 31 residues not found in the N-terminus Fig. 4a.

Fig. 3. The effect of pH and ionic strength on mussel lysozyme activity. Activities of mussel lysozymes were measured in sodium acetate buffers of pH 3.6 to pH 5.6 and in sodium phosphate buffers of pH 6.0 to pH 8.0 at ionic strength of 0.1 (closed symbols) and 0.03 (open symbols). Results were expressed as relative activities for each enzyme and ionic strength. (a) bm-lysozyme; (b) sA-lysozyme; (c) sB-lysozyme and (d) sC-lysozyme.

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Table 1 Effect of divalent cations on the activity of mussel lysozymes and chlamysin Relative lysozyme activity%* Agent (mM) No Ca2q

Mg2q

*

Chlamysin

sA-lysozyme

sB-lysozyme

bm-lysozyme

5 10 20 50 5 10 20

100 100 48 0 – 91 43 0

100 130 88 0 – 170 90 0

100 450 560 280 0 820 830 380

100 710 580 260 0 1047 1053 480

Relative to the activity of each enzyme against M. luteus at pH 5.2 and ionic strength of 0.01.

Aligning the obtained bm-lysozyme sequences to the previously reported lysozyme sequence from M. edulis, Bachali et al. (2002) shows that the two lysozymes are identical with the exception of five residues as shown in Fig. 4b. The sequence of the isolated and functionally active bm-lysozyme starts at position 58 in the corresponding gene-derived sequence of M. edulis indicating that a long signal peptide has been cleaved off. To test

this possibility, signal predictions were performed on the mussel lysozyme and on three bivalve lysozymes for which the N-terminal has been ` et al., 1996; Ito experimentally determined (Jolles et al., 1999; Nilsen et al., 1999). The Signal P (Nielsen et al., 1997) and PSORT II (Nakai and Kanehisa, 1992) programs predicted a 24 residue signal, as well as a corresponding cleaving signal in the blue mussel (Fig. 4b). Exported proteins

Fig. 4. Protein sequences of bm-lysozyme and sequence alignments to other bivalve lysozymes. (a): N-terminal sequence of the purified bm-lysozyme protein and of the derived peptide fragments P1 and P2 obtained by CNBr splitting. (b): Alignment of bm-lysozyme sequences to previously reported sequences of i-type lysozymes from M. edulis and Mytilus galloprovincialis (Bachali et al., 2002), C. islandica (Nilsen et al., 1999) and T. japonica (Ito et al., 1999). Stars above the sequences mark position of varying residues in the bm-lysozyme compared to the gene-derived sequence of M. edulis. Signal sequences predicted by SignalP (Nielsen et al., 1997) and PSORTII (Nakai and Kanehisa, 1992) are in grey and the first three N-terminal amino acids experimentally determined by protein sequencing are in black.

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have to cross membranes to reach their targets and signal peptides serve the transmembrane spanning functions. Consequently the blue mussel lysozyme sequence (Bachali et al., 2002) was analysed for potential transmembrane regions using the SOSUI program (Hirokawa et al., 1998) and a transmembrane region comprising residues 6–28 was predicted. Similar results as for the M. edulis protein regarding signal peptides, cleavage sites and transmembrane regions were obtained for the homologous bivalve sequences reported by Bachali et al. (2002). 4. Discussion In this paper we describe chromatographic procedures for separation of multiple blue mussel lysozymes of which two, one from the soft body and one from the crystalline style, were purified to apparent homogeneity from tissues of a large number of individuals. We also isolated two additional and distinct style-associated lysozymes from blue mussels. Our data show that the soft body of M. edulis contains a lysozyme having a molecular weight consistent to the size of the predicted protein from a recently reported blue mussel lysozyme gene (Bachali et al., 2002). Comparison of the protein sequences obtained from the bm-lysozyme isolated in the present study to the published M. edulis sequences verifies that the previously reported mussel i-lysozyme gene encodes the bmlysozyme protein. A short N-terminal sequence of a lysozyme from the Mediterranean mussel M. galloprovenci` et al. (1996). The alis was published by Jolles gene of this lysozyme was later identified and found to encode a protein very similar to the M. edulis lysozyme (Bachali et al., 2002). We performed signal peptide predictions on both lysozymes and, not surprisingly, analogous secretion signals comprising the initial 24 amino acids were proposed by two programs, and consistent signal cleavage sites as well as transmembrane regions were found for the two proteins. The M. gallo` et al., 1996) provencialis peptide sequence (Jolles matches the sequence-determined N-terminus of the bm-lysozyme in this study, showing that not only are these proteins very similar in their overall sequence but they also have identical signal peptides and identical N-terminals. The 33 amino acid region between the putative signal and the start of the active lysozyme encompasses a high

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number of acidic residues. Such polar amino acids are not unusual in the third region of signal sequences (von Heijne, 1990), but this extent of acidic residues are usually not associated with signal peptides. Likewise, signal sequence lengths of 57 residues are highly unexpected although such long signal peptides have been reported (Eichler et al., 2003). Thus, we believe that the sequence from positions 25 to 57 in the blue mussel soft-body lysozyme, and the homologous region in the Mediterranean mussel lysozyme, is not a part of the signal peptide as proposed by Bachali et al. (2002). Instead, this region may comprise a part of the protein that is cleaved off from a possibly inactive pro-form of these i-type lysozymes. We have previously discussed a similar processing in the C-terminus of an Icelandic scallop lysozyme (Nilsen et al., 1999). The bivalve lysozymes reported so far display varying enzymatic properties. The T. japonica lysozyme has an optimum range of pH 6–8 in degradation of M. luteus (Ito et al., 1999), while chlamysin from the crystalline style of scallop C. islandica has no lysozyme activity at neutral or higher pH (Nilsen et al., 1999). Furthermore, in contrast to the T. japonica lysozyme the scallop lysozyme displayed no chitinase activity. A lysozyme purified from extract prepared from the crystalline style of M. edulis (McHenery and Birkbeck, 1979) was shown to posses optimal lysozyme activity at both pH 7.1 and at pH 4.6 (McHenery and Birckbeck, 1982). The present study reveals at least three style lysozymes of which the partly purified sA-lysozyme resembles chlamysin (Nilsen et al., 1999) by pH-profile for activity and low tolerance of calcium and magnesium on activity. Low tolerances for divalent cations have also previously been reported for the style lysozyme from blue mussel (McHenery and Birckbeck, 1982). In contrast, the activity of the isolated sB-lysozyme was increased by low concentrations of divalent calcium and magnesium ions. The sC-lysozyme display pH-activity profiles different from the other mussel lysozymes and other invertebrate lysozymes previously reported. Our results for the effects of pH and ionic strength on the M. luteus cell wall degradation of isolated bm-lysozyme from the soft body, and the effects of divalent cations on the lysozyme activity, also demonstrates differences between the soft body enzyme and lysozymes present in the crystalline style. The much higher amount of lysozyme activ-

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ities in extract obtained from 50 shells resulting in the isolation of only bm-lysozyme, compared to extract from 1000 crystalline styles resulting in the isolation of the sB-lysozymes and no bm-lysozyme, shows that quantitatively the isolated bm-lysozyme is the main lysozyme in blue mussel. The isolation of the three blue mussel style lysozymes (sA-, sB- and sC-) together with the soft-body bm-lysozyme brings the number of M. edulis lysozymes up to a total of four. These separated lysozymes originate from collections of a large number of individuals and thus each lysozyme could in theory represent the natural variant carried by the single individuals. All blue mussel individuals tested so far contain a lysozyme activity characteristic for the presented bm-lysozyme activity (Myrnes and Olsen, unpublished). However, no activities corresponding to the bm-lysozyme were detected during the isolation of style lysozymes. Consequently, all blue mussels are expected to have the bm-lysozyme in addition to style lysozyme(s). The latter group of three lysozymes display distinct enzymatic profiles of activity regarding tolerance or dependence to ionic strength, pH and divalent cations. The differences in these enzyme features seem to be too profound to be attributed to individual-specific isoforms of a style lysozyme. Instead we believe that all three lysozymes may be produced in one individual animal and that they serve specific roles. The existence of multiple lysozymes in marine mollusc species has to our knowledge not previously been reported. Other invertebrates like the medicinal leech Hirudo medicinalis (Zavalova et al., 1996, 2000), the nematode Caenorhabditis elegans (Wilson et al., 1994), and the fruit-fly Drosophila melanogaster (see Bachali et al., 2002) are, however, known to carry three or more i-type lysozyme genes. This implies that invertebrates may have a battery of specialised lysozymes similar to the specialisation of c-type lysozymes in vertebrates (Irwin and Wilson, 1989). The invertebrate lysozymes are thought to be involved in digestion (McHenery et al., 1979) and to function in self-defence against pathogenic bacteria (Cheng, 1983; Chu, 1988; Carballal et al., 1997; Allam and Paillard, 1998). Lysozyme has been found localised within granular hemocytes of blue mussel (Pipe, 1990) and higher level of activity was detected in hemocytes than in serum from blue mussel (Carballal et al., 1997) and from

´ the carpet shell Ruditapes decussatus (Lopes et al., 1997). The results presented here demonstrate that blue mussel has several lysozymes, three enzymes in crystalline style and one (major) enzyme form in the other organs. This suggests that the bm-lysozyme could be a hemocyte-produced enzyme involved in antibacterial defence, while lysozymes from the digestion gland-associated crystalline style are involved in digestion. However, the bm-lysozyme has also been purified from the digestive gland (Bachali et al., 2002) and, therefore, a digestive role cannot yet be excluded. We hope to soon be in the position to conduct further studies-related to functional biological activities of the specific lysozymes to investigate this hypothesis. Acknowledgments This study was supported financially by The Marine Biotechnology Program in Tromsø. References Allam, B., Paillard, P., 1998. Defence factors in clam extrapallial fluids. Dis. Aquat. Org. 33, 123–128. ` P., Bachali, S., Jager, M., Hassanin, A., Schoentgen, F., Jolles, Fiala-Medioni, A., et al., 2002. Phylogenetic analysis of invertebrate lysozymes and the evolution of lysozyme function. J. Mol. Evol. 54, 652–664. ´ Carballal, M.J., Lopez, C., Azeevedo, C., Villalba, A., 1997. Enzymes involved in defense functions of hemocytes of mussel Mytilus galloprovincialis. J. Invertebr. Pathol. 70, 96–105. Cheng, T.C., 1983. The role of lysosomes in molluscan inflammation. Am. Zool. 23, 129–144. Chu, F.-L.E., 1988. Humoral defence factors in marine bivalves. Spec. Publ. Am. Fish. Soc. 18, 178–188. Chu, F.-L., La Peyre, J.F., 1993. Perkinsus marinus susceptibility and defense-related activities in Eastern Oysters Crassostrea virginica. Temperature effects. Dis. Aquat. Org. 16, 223–234. Eichler, R., Lens, O., Strecker, T., Garten, W., 2003. Signal peptide of Lassa virus glycoprotein GP.C exhibit an unusual length. FEBS Lett. 538, 203–206. Foss, G.S., Nilsen, R., Cornwell III, G.G., Husby, G., Sletten, K., 1998. A glycosylated Bence Jones protein and its autologous amyloid light chain containing amyloidogenic residues. Scand. J. Immunol. 47, 348–354. Fridman, M., Krull, L.H., Cavis, J.F., 1970. The chromatographic determination of cystine and cysteine residues in protein as S-b-(4-Pyridylethyl)cysteine. J. Biol. Chem. 245, 3868–3871. von Heijne, G., 1990. The signal peptide. J. Membrane Biol. 115, 195–201. Hirokawa, T., Seah, B.C., Mitaku, S., 1998. SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 14, 378–379.

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