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917 Journal of Cell Science 112, 917-925 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS0155 A cell-surface superoxide dismutase is a binding protein for peroxinectin, a cell-adhesive peroxidase in crayfish Mats W. Johansson1, Torbjörn Holmblad1,‡, Per-Ove Thörnqvist1, Matteo Cammarata1,2, Nicolò Parrinello2 and Kenneth Söderhäll1,* 1Department of Physiological Mycology, Evolutionary Biology Centre, University of Uppsala, Villavägen 6, SE-75236 Uppsala, Sweden 2Institute of Zoology, University of Palermo, via Archirafi 18, I-90123 Palermo, Italy *Author for correspondence (e-mail: [email protected]) ‡Present address: Uppsala patentbyrå AB, SE-75009 Uppsala, Sweden Accepted 13 January; published on WWW 25 February 1999 SUMMARY Peroxinectin, a cell-adhesive peroxidase (homologous to human myeloperoxidase), from the crayfish Pacifastacus leniusculus, was shown by immuno-fluorescence to bind to the surface of crayfish blood cells (haemocytes). In order to identify a cell surface receptor for peroxinectin, labelled peroxinectin was incubated with a blot of haemocyte membrane proteins. It was found to specifically bind two bands of 230 and 90 kDa; this binding was decreased in the presence of unlabelled peroxinectin. Purified 230/90 kDa complex also bound peroxinectin in the same assay. In addition, the 230 kDa band binds the crayfish β-1,3-glucanbinding protein. The 230 kDa band could be reduced to 90 kDa, thus showing that the 230 kDa is a multimer of 90 kDa units. The peroxinectin-binding protein was cloned from a haemocyte cDNA library, using immuno-screening or polymerase chain reaction based on partial amino acid sequence of the purified protein. It has a signal sequence, a domain homologous to CuZn-containing superoxide dismutases, and a basic, proline-rich, C-terminal tail, but no membrane-spanning segment. In accordance, the 90 and 230 kDa bands had superoxide dismutase activity. Immuno-fluorescence of non-permeabilized haemocytes with affinity-purified antibodies confirmed that the crayfish CuZn-superoxide dismutase is localized at the cell surface; it could be released from the membrane with high salt. It was thus concluded that the peroxinectin-binding protein is an extracellular SOD (EC-SOD) and a peripheral membrane protein, presumably kept at the cell surface via ionic interaction with its C-terminal region. This interaction with a peroxidase seems to be a novel function for an SOD. The binding of the cell surface SOD to the cell-adhesive/opsonic peroxinectin may mediate, or regulate, cell adhesion and phagocytosis; it may also be important for efficient localized production of microbicidal substances. INTRODUCTION Pacifastacus leniusculus, in particular, two such proteins have been identified, purified, and cloned, namely the 76 kDa celladhesive peroxidase, peroxinectin (Johansson and Söderhäll, 1988; Johansson et al., 1995) and the β-1,3-glucan binding protein (βGBP; Duvic and Söderhäll, 1990; Cerenius et al., 1994). βGBP stimulates phagocytosis of fungal cells (Thörnqvist et al., 1994) and, after binding β-1,3-glucan, enhances cell spreading (Barracco et al., 1991). Crayfish βGBP is synthesized in the hepatopancreas and released to the plasma. Once βGBP has been incubated with a β-1,3-glucan it binds to the haemocyte surface (Barracco et al., 1991); a specific βGBPbinding protein believed to be responsible for this binding has been identified and purified (Duvic and Söderhäll, 1992). Peroxinectin has both cell adhesion (Johansson and Söderhäll, 1988) and peroxidase activity (Johansson et al., 1995); it is homologous to animal peroxidases of the myeloperoxidase In invertebrates, the blood cells (haemocytes) participate in immunity and haemostasis by carrying out, e.g. phagocytosis, encapsulation (haemocyte adhesion to a large foreign intruder), and nodule formation (cell-cell adhesion in the presence of a high number of micro-organism) (Ratcliffe et al., 1985; Söderhäll et al., 1996). Relatively little is known about the cell adhesion molecules and receptors involved in these reactions, which include the steps of cell communication, opsonization, and adhesion, in phagocytosis followed by ingestion and destruction. In arthropods, proteins associated with the so-called prophenoloxidase (proPO) activating system have been shown to mediate cell communication, opsonization, and cell adhesion (Johansson and Söderhäll, 1989, 1996; Söderhäll et al., 1996; Söderhäll and Cerenius, 1998). In the crayfish, Key words: Superoxide dismutase, Peroxinectin, Peroxidase, Cell adhesion, Haemocyte 918 M. W. Johansson and others family (Johansson et al., 1995). From the blood of another arthropod (the insect Pseudoplusia includens) a small peptide unrelated to peroxinectin, was isolated and shown to trigger haemocytes to spread (Clark et al., 1997). Peroxinectin is, after synthesis in the semigranular or granular haemocytes, stored in the secretory granules of these cells (Liang et al., 1992), from where it is released during degranulation. Its cell adhesion and peroxidase activities are generated in the presence of lipopolysaccharides or β-1,3-glucan, when the proPO system is activated (Johansson and Söderhäll, 1988; Johansson et al., 1995). Active peroxinectin mediates adhesion of semigranular and granular haemocytes (Johansson and Söderhäll, 1988), promotes encapsulation (Kobayashi et al., 1990), and stimulates phagocytosis (Thörnqvist et al., 1994). Recently, human myeloperoxidase was shown to support adhesion of leukocytes or of differentiated HL-60 (myeloid) cells (Johansson et al., 1997). Hence, a cell-adhesive function may be a general property of peroxidases in this family. In this context, it would be interesting to identify which cell surface molecules act as adhesive receptors for peroxidases in the different systems. We have, in this study, shown binding of the cell-adhesive peroxidase, peroxinectin, to the surface of crayfish haemocytes. We have also identified the peroxinectin-binding protein, which seems to be identical to the previously reported βGBP-binding protein. Cloning of this peroxinectin-binding protein showed that it was homologous to the CuZn-containing superoxide dismutases (SODs); it was also shown to be an extracellular SOD (EC-SOD) localized at the cell surface. The binding of the cell surface SOD to the cell-adhesive/opsonic peroxinectin is a novel function for an SOD, which possibly mediates, or regulates, cell adhesion and phagocytosis in this animal and, perhaps, in other systems. MATERIALS AND METHODS Animals Freshwater crayfish, Pacifastacus leniusculus, were purchased from Berga kräftodling, Sweden, and kept in aquaria in aerated tap water at 10°C. Only intermoult animals were used. Protein purification Peroxinectin and the β-1,3-glucan-binding protein (βGBP) were purified from the freshwater crayfish, P. leniusculus as described (Johansson and Söderhäll, 1988; Duvic and Söderhäll, 1990). βGBP was treated with the β-1,3-glucan, laminarin to yield βGBP-L as described (Duvic and Söderhäll, 1992). The 230/90 kDa protein was purified first according to the method of Duvic and Söderhäll (1992). This method was then modified and improved by replacing the detergent solubilization of the haemocyte membrane pellet with the extraction of peripheral membrane proteins with 1 M NaCl; this fraction was then desalted on a PD-10 column (Pharmacia Biotech, Uppsala, Sweden) and separated on DEAEcellulose (Sigma Chemical Co., St Louis, MO, USA) in a linear gradient of 0.25 M to 1.0 M NaCl in 20 mM Tris-HCl buffer, pH 8.0. The ion-exchange fractions were concentrated using Centricon 10 (Amicon, Beverly, MA, USA). Protein concentration Protein concentration was assayed according to the method of Bradford (1976), using bovine serum albumin (BSA) as standard. Samples containing Triton X-100 were diluted to a concentration of 0.1%, which did not interfere with the Bradford assay. Antibodies Rabbit antiserum against peroxinectin (Johansson and Söderhäll, 1988) was used. To raise antibodies against the peroxinectin-binding protein, the peptide CSIVVHAGEDDLGLGG, corresponding to the initially obtained amino acid sequence (A/SIVVHAGEDDLGLGG) from a fragment of the purified EC-SOD, was synthesized (at the Department of Medical and Physiological Chemistry, University of Uppsala, Uppsala, Sweden); the cysteine was added in order to achieve a coupling reaction. The peptide was coupled to ovalbumin (Sigma) using m-maleimidobenzoyl-N-hydrosuccinimide ester (MBS; Calbiochem, La Jolla, CA, USA; Harlow and Lane, 1988). A rabbit was injected subcutaneously with 0.5 mg peptide per injection. Antibodies were affinity purified on a column of the peptide coupled to CNBr-activated Sepharose 4B (Pharmacia). Anti-crayfish integrin β cytoplasmic domain antibodies raised and purified against a synthetic peptide in an identical manner (Holmblad et al., 1997) served as control. Fixation and isolation of haemocytes One part of haemolymph (blood) was withdrawn into a syringe containing two parts of 3.7% formaldehyde in 0.15 M NaCl, supplemented with 500 µM SITS (4-acetamido-4′-isothiocyanatostilbene-2,2′-disulphonic acid, disodium salt; Fluka AG, Buchs, Switzerland), at 10°C, and incubated for 20 minutes (Kobayashi and Söderhäll, 1990). The fixed haemocytes were separated by density gradient centrifugation using the method of Söderhäll and Smith (1983), modified for freshwater crustaceans, using a 50% Percoll gradient (Pharmacia) in 0.15 M NaCl (Kobayashi and Söderhäll, 1990). Fixed granular and semigranular haemocytes were harvested using a Pasteur pipette, postfixed with four parts of the same fixative for 30 minutes, and washed by repeated centrifugation with phosphate-buffered saline (PBS: 10 mM Na2PO4, 10 mM KH2PO4, 0.15 M NaCl, 10 µM CaCl2, 10 µM MnCl2, 2.7 mM KCl, pH 6.8) three times (50 g, 2 minutes), and resuspended in PBS. These and all the following steps were performed at 20°C. Peroxinectin binding to cells Fifty µl of fixed, isolated semigranular or granular cells were incubated with 50 µl peroxinectin (1.25 µg) for 30 minutes in Eppendorf tubes. The cells were washed with PBS by centrifugation three times, as above. The washed, resuspended cells (50 µl) were incubated with 50 µl anti-peroxinectin antiserum 1:100 for 30 minutes, washed as above, and, finally, the cells (50 µl) were incubated with 50 µl FITC-conjugated swine anti-rabbit IgG 1:20 (DAKO A/S, Glostrup, Denmark) for 30 minutes. After washing with PBS, fluorescent cells were observed, using a fluorescence microscope and a micro-photography attachment. In one control, the anti-peroxinectin antiserum was substituted by either preimmune serum or a control antiserum; the peroxinectin incubation was omitted in another control. SDS-PAGE SDS-PAGE was performed in a 5-15%, 7% or 10% polyacrylamide gel. Samples (either the haemocyte membrane fraction, 40 µg protein; or purified peroxinectin-binding protein, 2 µg) were run under nonreducing, normal reducing (5 mg/ml dithiothreitol (DTT), 3 minutes, 100°C), or under strong reducing (10 mg/ml DTT, 10 minutes, 100°C) conditions. Molecular mass markers were from Sigma. Gels were stained with Coomassie blue. In order to study the mobility of each band after reduction, a gel was first run under nonreducing conditions, stained with 0.2% Coomassie in 20% methanol, 0.5% acetic acid, destained in 30% methanol, and washed by incubation in water for 24 hours. Stained bands were excised with a scalpel, incubated with DTT (10 mg/ml) first for 30 minutes at 20°C, then boiled for 10 minutes; finally, the gel pieces and the solution were run on a second gel under strong reducing conditions. SOD binds cell-adhesive peroxidase Protein iodination and binding assay Fifteen µg peroxinectin or 40 µg of βGBP-L in 10 µl of 0.05 M sodium phosphate buffer, pH 7.4, were iodinated with 0.2 mCi sodium 125I (17.4 Ci/mg; Amersham International plc, Little Chalfont, UK) and 4 µl of 2.5 mg/ml chloramine-T (Hunter and Greenwood, 1962). The reaction was stopped after 1 minute by adding 50 µl of 5 mM tyrosine. The mixture was then added to a PD-10 column equilibrated with 0.05 M sodium phosphate buffer, 0.15 M NaCl, 0.1% BSA, pH 7.4, and eluted in 1 ml fractions. Each fraction was tested using a scintillation counter and the first fraction containing detectable radioactivity was used. The specific activities of peroxinectin and βGBP-L were 3×106 cpm/µg and 1.2×106 cpm/µg, respectively. Peroxinectin binding to haemocyte membrane proteins, or to purified crayfish EC-SOD, was assayed essentially as described (Duvic and Söderhäll, 1992). After SDS-PAGE of 40 µg membrane protein, or of 2 µg peroxinectin-binding protein under nonreducing conditions, the gel was soaked in a transfer buffer (20 mM Tris, 150 mM glycine, pH 8.8) for 10 minutes; proteins were then electrotransferred (250 mA, 1 hour) to nitrocellulose in the transfer buffer in a mini transblot cell (Bio-Rad Laboratories, Hercules, CA, USA). The nitrocellulose was blocked for 1 hour with 0.25% BSA, 0.2% Tween-20, 20 mM Tris-HCl, 150 mM NaCl, 4 mM CaCl2, pH 7.4, and was then incubated overnight either with 0.68 µg 125Iperoxinectin or with 4 µg 125I-βGBP-L in 5 ml blocking buffer. Finally, the nitrocellulose sheet was extensively washed with 0.2% Tween-20, 20 mM Tris-HCl, 150 mM NaCl, 4 mM CaCl2, pH 7.4, dried, and visualized by autoradiography by exposure for 72 hours at −70°C. Amino acid sequencing The purified crayfish peroxinectin-binding protein (10 µg) was run in SDS-PAGE under strong reducing conditions. The gel was stained with 0.2% Coomassie in 20% methanol, 0.5% acetic acid, destained in 30% methanol, and washed by incubation in water for 24 hours. The 90 kDa band was cut out, degraded either by trypsin or by endoproteinase Lys-C in the gel, and the proteolytic fragments were eluted (Rosenfeld et al., 1992). They were purified by HPLC and sequenced by automated Edman degradation on a gas-phase sequencer, with an on-line phenylthiohydantion derivative analyzer. cDNA cloning and sequencing A λgt11 cDNA library from crayfish haemocytes (Johansson at al., 1994) was screened with affinity-purified anti-peroxinectin-binding protein antibodies (10 µg/ml), using standard procedures (Sambrook et al., 1989). Positive clones were detected with the ProtoBlot Immunoscreening System (Promega Corporation, Madison, WI, USA), purified, and PCR-amplified with λgt11-specific primers (Clontech Laboratories, Inc., Palo Alto, CA; Taq polymerase and PCR reagents were from US Biochemical Corporation, Cleveland, OH, USA). In a parallel approach, PCR was performed on the library as template, with degenerate oligonucleotide primers (KEBO Lab, Spånga, Sweden), corresponding to IVVHAG, from the amino acid sequencing of the peroxinectin-binding protein, and to the SOD consensus sequence G(P/D)H(F/Y)NP. Clones/PCR products were purified by QIAquick-spin PCR Purification Kit (Qiagen GmbH, Hilden, Germany), inserted into T vector, prepared from Bluescript KS+ (Stratagene, La Jolla, CA, USA) according to the method of Marchuk et al. (1990), and sequenced with the T7 Sequencing Kit (Pharmacia). The initial EC-SOD clone was 32P-labelled using the Megaprime labelling kit (Amersham) and used for further library screening. Both strands of resulting overlapping clones were sequenced; each part of the final sequence was covered by at least three clones or by independent PCR products. Sequence analysis The cDNA sequence was analyzed with the MacVector 4.1.4 software (Kodak Scientific Imaging Systems, New Haven, CT, USA). The 919 deduced amino acid sequence was compared to databases using the BLAST network server at the National Center for Biotechnological Information (Bethesda, MD, USA). The crayfish SOD sequence was aligned to other SOD sequences by, first using the Clustal W program in the SeqPup package; then minimal adjustments were made manually, to essentially fit earlier alignments of some of these sequences (Smith and Doolittle, 1992; Bordo et al., 1994). A phylogenetic tree was constructed from the adjusted alignments (disregarding signal sequences and C-terminal tails outside the SOD domain), using the neighbour joining method with pairwise gap removal and 100 bootstrap replicates in the PhyloWin program. Superoxide dismutase activity SOD activity in SDS-polyacrylamide gels was detected according to the method of Beauchamp and Fridovich (1971). Immuno-blotting of the crayfish EC-SOD SDS-PAGE was performed in a 10% gel as described above under nonreducing conditions. After electrophoresis, the gel was soaked in transfer buffer (20 mM Tris, 150 mM glycine, pH 8.8) for 10 minutes and the proteins were transferred for 1 hour at 210 mA to nitrocellulose in the transfer buffer. The filter was soaked in blocking buffer (PBS containing 2% BSA and 0.05% Triton X-100) for 2 hours, incubated with affinity-purified anti-EC-SOD antibodies (10 µg/ml) for 1 hour, washed 4× 15 minutes with blocking buffer, and incubated with sheep anti-rabbit IgG peroxidase conjugate (Sigma; 1:1000 in blocking buffer) for 1 hour. Finally, the nitrocellulose was washed with PBS 4× 15 minutes and assayed with a mixture of 6 ml of 3 mg/ml 4chloronaphthol in methanol and 100 ml of 0.03% H2O2 in PBS. Immuno-fluorescence localization of the crayfish EC-SOD Fixed, isolated (nonpermeabilized) semigranular or granular haemocytes were incubated with affinity-purified anti-EC-SOD antibodies (100 µg/ml) for 1 hour, washed with PBS and incubated with FITC-conjugated secondary antibodies, as above. In the control experiment, the haemocytes were incubated with affinity-purified anti-crayfish integrin cytoplasmic peptide antibodies. RESULTS Peroxinectin binding to the haemocyte surface and identification of a peroxinectin-binding protein After incubation of the purified cell-adhesive peroxidase, peroxinectin at µg/ml levels with isolated, fixed, nonpermeabilized, suspended semigranular and granular haemocytes (blood cells) binding was detectable by immunofluorescence (Fig. 1A,C). Blocking the cells with BSA before the peroxinectin incubation did not decrease the fluorescence, showing that the staining was not due to non-specific binding of peroxinectin; no staining was seen when the antiperoxinectin antibodies were omitted or replaced by preimmune serum (not shown). Incubation of cells with antiperoxinectin antibodies without prior incubation with peroxinectin did not result in any fluorescence, thus confirming earlier results using immunogold-conjugated antibodies: peroxinectin is absent from the haemocyte surface in (nonpreincubated) suspended cells and is only found in the secretory granules (Liang et al., 1992). In order to identify a peroxinectin receptor, iodinated peroxinectin was added to a blot of a haemocyte membrane protein preparation that had been run on SDS-PAGE under non-reducing conditions. Peroxinectin bound to two bands of 90 kDa and 230 kDa (Fig. 2, lane B). When an excess of 920 M. W. Johansson and others Fig. 1. Peroxinectin binding to isolated, fixed, non-permeabilized, suspended crayfish semigranular or granular haemocytes (blood cells) of the crayfish, Pacifastacus leniusculus. Haemocytes were separated, incubated with or without purified peroxinectin (25 µg/ml), and binding was detected by immuno-fluorescence staining with anti-peroxinectin antiserum and FITC-labelled anti-rabbit IgG. Semigranular cells treated with peroxinectin (A), semigranular cells not treated with peroxinectin (B), granular cells treated with peroxinectin (C), and granular cells not treated with peroxinectin (D). Bar, 10 µM. unlabelled peroxinectin was present, binding of iodinated peroxinectin was decreased (Fig. 2, lane D), indicating that the peroxinectin binding to the 230/90 kDa bands is specific. The 230/90 kDa band pattern was reminiscent of the previously described binding protein or receptor for the crayfish β-1,3glucan-binding protein, after it has reacted with β-1,3-glucan (βGBP-L; Duvic and Söderhäll, 1992). Accordingly, use of excess cold βGBP-L also decreased peroxinectin binding (Fig. 2, lane C) and binding of labelled βGBP-L to the 230 kDa band was detected (Fig. 2, lane A). The 230/90 kDa complex was purified (Fig. 3), using a modification of a published method (Duvic and Söderhäll, 1992). Iodinated peroxinectin could be detected to bind also to this purified complex under non-reducing conditions (not shown). Under non-reducing, or normal reducing, conditions the purified fraction contains the two bands of 230 and 90 kDa (Fig. 3, lanes B,C). Using stronger reducing conditions, (a higher concentration of DTT and an extended boiling time) only the 90 kDa band is seen (Fig. 3, lane D). Consistent with this result, cutting out the 230 kDa band and subjecting it to strong reduction brought it down to 90 kDa (Fig. 3, lane E). cDNA sequence of the peroxinectin-binding protein and similarity to other proteins A synthetic peptide, corresponding to one of the amino acid sequences obtained from sequencing fragments of the 90 kDa band of the purified peroxinectin-binding protein, was used to raise rabbit antibodies. The affinity-purified anti-peptide Fig. 2. Peroxinectin binding to haemocyte membrane proteins. A membrane fraction from crayfish haemocytes (40 µg protein per lane) was subjected to SDS-PAGE (7.5%) under non-reducing conditions and transferred to nitrocellulose. The nitrocellulose filter was incubated with iodinated peroxinectin (0.07 µg/ml; lane B), with iodinated laminarin-treated β-1,3-glucan-binding protein (βGBP-L; 0.4 µg/ml; lane A), with iodinated peroxinectin in the presence of excess unlabelled peroxinectin (10× concentration; lane D), or with iodinated peroxinectin in the presence of excess unlabelled βGBP-L (40× concentration; lane C). Molecular mass of markers to the left. Fig. 3. The peroxinectin-binding protein from crayfish. A haemocyte membrane fraction (40 µg protein) was subjected to SDS-PAGE (5-15%) under non-reducing conditions (A). Purified protein (2 µg per lane) was subjected to SDS-PAGE under nonreducing conditions (B), normal reducing conditions (5 mg/ml DTT, 3 minutes, 100°C) (C), or strong reducing conditions (10 mg/ml DTT, 10 minutes, 100°C) (D). In E, the upper, 230 kDa band from B was excised with a scalpel and run under strong reducing conditions. Molecular mass of markers to the left (M). The gel was stained with Coomassie. SOD binds cell-adhesive peroxidase antibodies immuno-blotted both the 230 and 90 kDa bands; they also immuno-precipitated the protein from haemocytes (not shown), demonstrating that the peroxinectin-binding protein is synthesized by the haemocytes. The affinity-purified antibodies were used to screen a crayfish haemocyte cDNA library. In addition, as an alternative screening approach, we used PCR with degenerate primers corresponding to the obtained amino acid sequence, which was found to be significantly similar to the CuZncontaining superoxide dismutases (CuZnSODs), and to a CuZnSOD consensus sequence. Both methods consistently gave the same cDNA. This cDNA has an open reading frame of 651 base pairs and a deduced protein sequence of 217 amino acids (Fig. 4). The first 28 amino acids form a signal sequence, according to the rules of von Heijne (1987). The mature polypeptide has an estimated predicted molecular mass of 20 kDa. Searching protein sequence databases, we confirmed that this deduced polypeptide (in agreement with the amino acid sequences obtained from the purified protein), was significantly similar to both extracellular and cytosolic CuZnSODs from other animals, e.g. vertebrates, insects, nematodes, and helminths (Fig. 5). It showed 35-51% identity and 46-67% similarity to these proteins in the SOD domain (excluding the signal sequence and the 34 amino acid long Cterminal basic, proline-rich, region of the crayfish polypeptide). It was also significantly similar to fungal cytosolic, plant cytosolic, plant chloroplast, and bacterial periplasmic CuZnSOD (Fig. 6). The crayfish sequence has all the residues necessary for metal binding and for enzyme activity (Fig. 5). 921 The peroxinectin-binding protein is an extracellular superoxide dismutase localized at the cell surface Concordant with the sequence similarity to SODs, the 230 and 90 kDa bands of the peroxinectin-binding protein had SOD activity (Fig. 7). In addition, a band of lower molecular mass (about 25 kDa, Fig. 7, lane C) had SOD activity. Consistently, in immuno-blotting the affinity-purified anti-crayfish SOD antibodies recognized the 230/90 kDa bands as well as the 25 kDa band (Fig. 8). Immuno-fluorescence with isolated, fixed, non-permeabilized, suspended cells demonstrated that this protein is present at the surface of semigranular (Fig. 9A), and granular haemocytes (Fig. 9C). The lack of staining with control antibodies to an intracellular epitope (Fig. 9B,D) confirmed that the cells were intact and that the SOD antibody staining represents a cell-surface localization. The deduced protein sequence of the crayfish peroxinectin-binding SOD does not have any transmembrane segment (Fig. 4). Consistent with this finding, it is possible to solubilize the protein by treating haemocyte membranes with 1 M NaCl. In conclusion, we have determined that the peroxinectin-binding protein can be classified as an extracellular superoxide dismutase (EC-SOD), a peripheral membrane protein localized at the haemocyte surface. DISCUSSION We have characterized the binding of the cell-adhesive peroxidase, peroxinectin, from crayfish blood to the surface of haemocytes (blood cells). We identified two bands of 230 and 90 kDa that specifically bound labelled peroxinectin in a manner that could be competed with unlabelled protein. The AGTAATTGTATTCTCAGTTATTGTGGGATTTCTCACGACTACGCTCGCTCATGGTGAACATGACTCTCCC 70 M V N M T L P -22 GGACATGCTGGTGAAGATGATGATAGTGGGGATCATGAGTTTCATGGCTCTCGCCTCGCCTCCAGCCCCG 140 D M L V K M M I V G I M S F M A L A S P P A P 2 GCCGCCGTGGTGGACCTGGTGCCTGGCAGCGACCAGATCAGTGGAAGGCTGGAGATCTACAGGAGCTATA 210 A A V V D L V P G S D Q I S G R L E I Y R S Y 25 ATGGACTCACCATCGTAGGTACGGTGAGTGGGCTGACTCCAGGAAAACATGGCTTCCATGTGCACCAGAA 280 N G L T I V G T V S G L T P G K H G F H V H Q K 49 GGGAGACCTCGGTGATGGCTGCAAGGCTGCCGGGGGCCACTTCAACCCCTTCAACAAAAATCACGGAGCT 350 G D L G D G C K A A G G H F N P F N K N H G A 72 CCCGAGGACTTGGAACGTCATGCTGGCGACTTTGGCAACGTGGTGGCGGACTACCAAGGTGTAGCTACCA 420 P E D L E R H A G D F G N V V A D Y Q G V A T 95 TTTACATTGATGACAGCCAGGTCTCGCTGGATCCCTCGTCCGAGGCTTATATCGGTGGCCTGGCCATCGT 490 I Y I D D S Q V S L D P S S E A Y I G G L A I V 119 CGTCCACGCCGGCGTTGATGACTTGGGCCGCGGGGGCAACCCAGAGAGTGCCAAGACAGGCAATGCCGGT 560 V H A G V D D L G R G G N P E S A K T G N A G 142 GCCCGCTCAGGCTGTGGCATCATTCGGGTAGTTGCACCTACCTACCAGCCCCCACAGTCTGGCTACAGGC 630 A R S G C G I I R V V A P T Y Q P P Q S G Y R 165 CACGCCGCCCCCAACACCCCAACCGCCAGCCAGGGTTTCCTCAACAGTTCCAGTACCAGAGGACGTACAA 700 P R R P Q H P N R Q P G F P Q Q F Q Y Q R T Y N 189 CTGATGTAGCTTCTGGAACAAAGATGTAGCATGCACTGTGGGTGCCTACTCTCCATGGCTATGCCATTTC 770 stop TTTGTATAATTAAAAAAAAATTTAAAGTTTATTTTACTTACGATGAAGGAAATGCAGTTAGATATTAAAT 840 GTTTAAAACATGCTAACTATTGCTAAAGTATTTCAAACATGTAGTTTAAAAGGTTTTTATCCATTAAAAT 910 ACCGAACATTGAATAAATTTTATGTAAGCCAAAAAAAAAAAAAAAAAAA 959 Fig. 4. cDNA sequence and deduced amino acid sequence of the peroxinectinbinding extracellular superoxide dismutase (EC-SOD) from the crayfish, Pacifastacus leniusculus. Nucleotides numbered from the 5′ end and amino acids from the putative amino-terminal of the mature protein. Italics, the putative signal sequence (von Heijne, 1987); bold type, the polyadenylation signal; underlining, sequences determined by sequencing of fragments of the purified protein. These sequence data are available from GenBank under accession number AF122900. 922 M. W. Johansson and others crayfish EC-SOD human Cyt-SOD human EC-SOD Drosophila Cyt-SOD C. elegans Cyt-SOD C. elegans EC-SOD Onchocerca Cyt-SOD Onchocerca EC-SOD Schistosoma Cyt-SOD Schistosoma EC-SOD mvnmt -24 mlallcsclllaagasdaWTGEDSAEPNSDSAEWIRDMY mk minsfiviflsflifinya mtvysylv lpdmlvkmmivgimsfmalasppAPAAVVDLVPGSDQ-ISGRLEIYRSYN--GLTIVGTV MATKAVCVLKGDGP-VQGIINFEQKESNGPVKVWGSI AKVTEIWQEVMQRRDDDGTLHAACQVQPSATLDAAQPRVTGVVLFRQLAPRAKLDAFFAL MVVKAVCVINGDA---KGTVFFEQESSGTPVKVSGEV MSNRAVAVLRGET--VTGTIWITQKSENDQAVIEGEI trvvlilalsvcieaasEVIRARAYIFKAEAGKIPTE-LIGTIDFDQSGS--FLKLNGSV MSTNAIAVLRGDT--VSGIIRFKQDKEGLPTTVTGEV nlvcveathvygrrshsngmhgnGARRAVAVLRGDAG-VSGIIYFQQGSGGSITTISGSV MKAVCVMTGTAG-VKGVVKFTQETDNGPVHVHAEF ilfilldnycSAYGYGYSYYHRRHFDPAIASFTKEP--YIGAVWFTQHGD--YMYVNGSV * * * + + + + SGL---TPG-KHGFHVHQKGDLGDGCKAAGGHFNPFNKNHGAPEDLERHAGDFGNVVA KGL---TEG-LHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTA EGFPTEPNSSSRAIHVHQFGDLSQGCESTGPHYNPLAVPH--P---Q-HPGDFGNFAV CGL---AKG-LHGFHVHEFGDNTNGCMSSGPHFNPYGKEHGAPVDENRHLGDLGNIEA KGL---TPG-LHGFHVHQYGDSTNGCISAGPHFNPFGKTHGGPKSEIRHVGDLGNVEA SGL---AAG-KHGFHIHEKGDTGNGCLSAGGHYNPHKLSHGAPDDSNRHIGDLGNIES KGL---TPG-LHGFHIHQYGDTTNGCISAGPHFNPYNKTHGDRTDEIRHVGDLGNIEA SGL---TPG-LHGFHVHQYGDQTNGCTSAGDHYNPFGKTHGGPNDRIKHIGDLGNIVA SGL---KAG-KHGFHVHEFGDTTNGCTSAGAHFNPTKQEHGAPEDSIRHVGDLGNVVA AGL---PPGKLLGTHVHRYGGLGNMCLEAGPHFNPFNQRHGPRHGYPRHAGDLGNIRV 34 88 * DYQGVATIYIDDSQVSLDPSSEAYIGGLAIVVHAGVDDLGRG-G--NPESAKTGNAGA 143 DKDGVADVSIEDSVISLS--GDHCIIGRTLVVHEKADDLGKG-G--NEESTKTGNAGS -RDGSLWRYRAGLAASLA--GPHSIVGRAVVVHAGEDDLGRG-G--NQASVENGNAGR TGDCPTKVKITDSKITLF--GADSIIGRTVVVHADADDLGQG-G--HELSKSTGNAGA GADGVAKIKLTDTLVTLY--GPNTVVGRSMVVHAGQDDLGEGVGDKAEESKKTGNAGA PASGDTLISVSDSLASLS--GQYSIIGRSVVIHEKTDDLGRG-T--SDQSKTTGNAGS GADGTAHISISDQHIQLL--GPNSIIGRSIVVHADQDDLGKGVGAKKDESLKTGNAGA GANGVAEVYINSYDIKLR--GPLSVIGHSLVVHANTDDLGQGTGNMREESLKTGNAGS GADGNAVYNATDKLISLN--GSHSIIGRTMVIHENEDDLGRG-G--HELSKVTGNAGG GRGGVAKFDFYVTIKGLG--PFDGFIGRALVIHANRDDLGRN-R--DEGSRTTGNSGP RSGCGIIRVVAPTYQPPQSGYRPRRPQHPNRQPGFPQQFQYQRTYN 189 RLACGVIGIAQ RLACCVVGVCGPGLWERQAREHSERKKRRRESECKAA RIGCGVIGIAKV RAACGVIALAAPQ RLACGTIGKTFTSSQLPY RVACGIVAIGAAS RLACGVIGIAAVS RLACGVIGLAAE RLACATIGFRAP crayfish EC human Cyt human EC Dros. Cyt C.e. Cyt C.e. EC Onchoc. Cyt Onchoc. EC Schist. Cyt Schist. EC 230 kDa band was a disulphide-linked multimer of the 90 kDa units. Partial amino acid sequencing and cDNA cloning showed that the peroxinectin-protein is homologous to the CuZn-containing superoxide dismutases; enzyme staining showed that the protein had SOD activity. The deduced protein sequence has a signal sequence, a CuZnSOD domain, and a basic, proline-rich, C-terminal region. The peroxinectinbinding protein is thus considered an extracellular SOD (ECSOD). Immuno-fluorescence staining showed that the EC-SOD is constitutively present at the haemocyte surface. Because it could be removed from the membrane with high salt, it is a peripheral protein. Probably, the basic, proline-rich, C-terminal tail keeps the EC-SOD at the surface via ionic interactions. Its cell-surface localization is compatible with a possible function Fig. 5. Alignment of the peroxinectin-binding extracellular superoxide dismutase (EC-SOD) from the crayfish, Pacifastacus leniusculus with extracellular and cytosolic superoxide dismutases (EC-SOD and Cyt-SOD, respectively) from some other animals. The SWISSPROT accession numbers for these sequences are P00441 (human Cyt-SOD), P08294 (human EC-SOD), P00444 (Drosophila melanogaster Cyt-SOD), P34697 (Caenorhabditis elegans Cyt-SOD), P34461 (C. elegans putative EC-SOD), P24706 (Onchocerca volvulus CytSOD), Q07449 (O. volvulus EC-SOD), Q01137 (Schistosoma mansoni Cyt-SOD), and P16026 (S. mansoni EC-SOD). Amino acids of crayfish EC-SOD numbered from the putative amino-terminal of the mature protein. Bold type, residues identical in all the aligned sequences; lower case, known or putative signal sequences; *conserved amino acid residues responsible for Cu binding; +conserved amino acid residues responsible for Zn binding. as cell-surface receptor for peroxinectin, which is released from secretory granules as a response to infection. Physical interaction with a peroxidase appears to be a novel function for an SOD. Binding between a cell-surface SOD and an adhesive/opsonic peroxidase may serve several purposes; the SOD may mediate, or regulate, cell adhesion and/or phagocytosis. By producing hydrogen peroxide, it may also provide substrate for the peroxidase, localizing an efficient microbicidal attack. As H2O2 inhibits CuZnSODs (Beauchamp and Fridovich, 1973), removal of the peroxide by the bound peroxidase may help to keep SOD active. Two unrelated families of SODs are found in eukaryotes (Fridovich, 1995): Mitochondria have Mn-containing SODs whose sequences are completely different from the CuZncontaining SODs. The cytosol of virtually all oxygen-respiring SOD binds cell-adhesive peroxidase 923 Fig. 7. Superoxide dismutase (SOD) activity of the peroxinectinbinding protein. Haemocyte proteins (20 µg per lane; the lysate was prepared in 1 M NaCl) were subjected to SDS-PAGE under nonreducing conditions and stained with Coomassie (A), under strong reducing conditions and stained with Coomassie (B), under nonreducing conditions and stained for SOD activity (C), or under strong reducing conditions and stained for SOD activity (D). Molecular mass of markers to the left. Fig. 6. Phylogenetic tree of eukaryotic CuZn-containing superoxide dismutases (SODs), including the extracellular SOD from the crayfish, Pacifastacus leniusculus, presented in this paper. Cy, cytosolic SOD; EC, extracellular SOD; Chl, chloroplastic SOD. The species and SWISS-PROT accession numbers are: medfly, Ceratitis capitata, L35494; Drosvir, Drosophila virilis, P10791; Dros, Drosophila melanogaster, P00444; XenCyone, Xenopus laevis cytosolic SOD1, P13926; XenCytwo, Xenopus laevis cytosolic SOD2, P15107; chick, Gallus gallus, Q92059; rabbit, Oryctolagus cuniculus, P09212; horse, Equus caballus, P00443; guineap, Cavia porcellus, P33431; humanCy, P00441; bovine, Bos taurus, P00442; rat, Rattus norvegicus, P07632; mouse, Mus musculus, P08228; Neurosp, Neurospora crassa, P07509; yeast, Saccharomyces cerevisiae, P00445; C-elCy, P34697; OnchoEC, Onchocerca volvulus, Q07449; OnchoCy, P24706; SchistoEC, Schistosoma mansoni, P16026; C-elEC, Caenorhabditis elegans, P34461; tomatoChl, Solanum lycopersicum, M37151; spinachChl, Spinacia oleracea, P07505; tomatoCy, M37150; spinachCy, P22233; SchistoCy, Q01137; humanEC, P08294. eukaryotes are believed to contain a CuZnSOD. In addition, extracellular CuZnSODs (which contain a CuZnSOD domain homologous to that of the cytosolic CuZnSODs plus a signal sequence) have been found only in some animal groups, e.g. mammals and some parasitic invertebrates (in the C. elegans genome project a sequence for a putative EC-SOD has also been identified). The protein presented here adds to this group and is the first sequenced arthropod EC-CuZnSOD. It is still unclear how widespread extracellular SODs are among eukaryotes. It was recently discovered that the cytosolic SOD of decapod crustaceans seems to be an exception to the general rule (Brouwer et al., 1997): in the blue crab Callinectes sapidus and in all the other decapods studied, the cytosolic SOD contains Mn instead of Cu and Zn; the crab cytosolic SOD shows sequence similarity to mitochondrial MnSODs but not to Fig. 8. Immuno-blotting of the crayfish peroxinectin-binding EC-SOD. Haemocyte proteins (40 µg, the lysate was prepared in 1 M NaCl) were subjected to SDS-PAGE under non-reducing conditions and blotting with affinity-purified anti-crayfish EC-SOD peptide antibodies. Molecular mass of markers to the left. CuZnSODs from other species. On the other hand, it was reported that the blue crab has an extracellular CuZnSOD, of high molecular mass (130 kDa; Brouwer et al., 1997); however, no sequence data for this protein has been presented. The 90 kDa crayfish EC-CuZnSOD is most probably a counterpart of this blue crab protein. It can be concluded that decapod crustaceans seem to have only one CuZnSOD, which is extracellular and has a sequence completely different from their cytosolic MnSOD, and that the CuZnSOD from crayfish described in this paper is the first sequenced representative. In parasitic nematodes, the EC-CuZnSODs are very similar to the cytosolic CuZnSOD of the same species; this finding seems to suggest that these EC-SODs have only acquired a 924 M. W. Johansson and others Fig. 9. Immuno-fluorescence localization of the crayfish peroxinectinbinding EC-SOD on the haemocyte surface. Isolated, fixed, nonpermeablilized, suspended semigranular or granular haemocytes were stained with affinity-purified anti-crayfish EC-SOD peptide antibodies: semigranular cell incubated with anti-EC-SOD antibodies (A), semigranular cell incubated with control anti-peptide antibodies (anti-crayfish β integrin cytoplasmic domain antibodies) (B), granular cell incubated with anti-EC-SOD antibodies (C), and granular cell incubated with control antibodies (D). Bar, 10 µM. signal sequence relatively recently and that they evolved from cytosolic CuZnSODs as an adaptation to a parasitic life in order to protect the parasite from the host defense (James, 1994). In contrast, both crayfish and human EC-CuZnSOD seem phylogenetically relatively distant from all other eukaryotic CuZnSODs. For instance, crayfish EC-CuZnSOD is not more similar to the insect cytosolic CuZnSODs than to cytosolic CuZnSODs from other invertebrates, vertebrates, fungi, or plants. We thus propose that many EC-SODs in non-parasitic invertebrates and vertebrates remain to be identified; that, unlike in parasitic animals, they may have arisen (once or several times) early in animal evolution and, diverged from the cytosolic CuZnSODs; and that they participate in immunity by, e.g. mediating or regulating haemocyte or leukocyte adhesion and phagocytosis. The cDNA for the crayfish EC-CuZnSOD encodes a polypeptide of a predicted molecular mass of 20 kDa. This should correspond to the 25 kDa band that is detected by the anti-peroxinectin-binding EC-SOD antibodies and the 25 kDa band with SOD activity. Hence, the 90 kDa form should be a multimer of 25 kDa polypeptides, either by disulphide linkages that are difficult to break or by unidentified covalent bonds. A recent example of a blood protein from this animal, containing covalently linked units, is the heterodimeric proteinase inhibitor, pacifastin (Liang et al., 1997). By comparison, human EC-SOD is a 130 kDa tetramer of subunits of an apparent molecular mass of 30 kDa (Marklund, 1982; 24 kDa predicted by cDNA sequence, Hjalmarsson et al., 1987). Also the human multimers are difficult to dissociate; still after strong reduction (10 mg/ml reducing agent) all human EC-SOD could not be dissociated to 30 kDa (Marklund, 1982). Because the crayfish cell-surface SOD does not have a transmembrane region, it is unlikely that it functions as a signalling receptor. Instead, ligation of the adhesive protein, peroxinectin to the cell surface may signal into the cell via an integrin. Peroxinectin has a KGD motif: a synthetic peptide containing this sequence mimics the activity of the whole protein by triggering cell spreading (Johansson et al., 1995). A crayfish haemocyte β integrin, which we recently have cloned and identified (Holmblad et al., 1997), binds to the peroxinectin-derived KGD peptide in affinity chromatography, as well as to intact peroxinectin in an ELISA-type binding assay (unpublished). This binding of peroxinectin to integrin could not, however, be detected in the overlay assay on SDSPAGE-separated proteins presented in this paper, as ligand binding to integrins is believed to require both integrin subunits. Nevertheless, the crayfish integrin does, probably participate in mediating adhesion to peroxinectin and, through its cytoplasmic domain, in signaling to the cell interior. In the same vein, human myeloperoxidase, a homologue of peroxinectin, was recently shown to support adhesion of human cells; this adhesion was inhibited by monoclonal antibodies to the αMβ2 integrin (Johansson et al., 1997). Thus, integrins seem to be involved in cell adhesion to peroxidases both in the crayfish and in the human. Whereas integrin-ligand interactions are generally of low affinity and binding of µg/ml concentrations of soluble ligands to integrins cannot, usually, be detected (Tangemann and Engel, 1997), we did detect binding of peroxinectin to the ECSOD and to the haemocyte surface at µg/ml, or lower. Thus, it is very likely that the binding of peroxinectin to the cell-surface SOD has higher affinity than its binding to integrin. The role of this relatively high-affinity binding remains to be established, but as hereby proposed, it may mediate or regulate cell adhesion. 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