Download SOD binds cell-adhesive peroxidase - Journal of Cell Science

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. It would be interesting to investigate whether this
kind of physical interaction between a cell-adhesive peroxidase
and an SOD, e.g. between myeloperoxidase and EC-SOD also
exists in mammals and in other animals.
We thank Thomas Sicheritz-Pontén and Dr Siv Andersson, Dept of
Molecular Biology, University of Uppsala, Uppsala, Sweden, for their
help with the phylogenetic analysis. This work was supported by the
Swedish Council for Agricultural and Forestry Research (K.S. and
M.W.J.) and by the Swedish Natural Science Research Council (K.S.).
REFERENCES
Barracco, M. A., Duvic, B. and Söderhäll, K. (1991). The β-1,3-glucanbinding protein from the crayfish Pacifastacus leniusculus, when reacted
with a β-1,3 glucan, induces spreading and degranulation of crayfish
granular cells. Cell Tissue Res. 266, 491-497.
Beauchamp, C. O. and Fridovich, I. (1971). Superoxide dismutase:
improved assays and an assay applicable to polyacrylamide gels. Anal.
Biochem. 44, 276-287.
Beauchamp, C. O. and Fridovich, I. (1973). Isozymes of superoxide
dismutase from wheat germ. Biochim. Biophys. Acta 317, 50-64.
Bordo, D., Djinovic, K. and Bolognesi, M. (1994). Conserved patterns in the
Cu,Zn superoxide dismutase family. J. Mol. Biol. 238, 366-386.
Bradford, M. (1976). A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal. Biochem. 72, 248-254.
SOD binds cell-adhesive peroxidase
Brouwer, M., Hoexum Brouwer, T., Grater, W., Enghild, J. J. and
Thogersen, I. B. (1997.) The paradigm that all oxygen-respiring eukaryotes
have cytosolic CuZn superoxide dismutase and that Mn-superoxide
dismutase is localized to the mitochondria does not apply to a large group
of marine arthropods. Biochemistry 36, 13381-13388.
Cerenius, L., Liang, Z., Duvic, B., Keyser, P., Hellman, U., Palva, E. T.,
Iwanaga, S. and Söderhäll, K. (1994). Structure and biological activity of
a (1,3)-β-D-glucan binding protein in crustacean blood. J. Biol. Chem. 269,
29462-29467.
Clark, K. D., Pech, L. L. and Strand, M. R. (1997). Isolation and
identification of a plasmatocyte-spreading peptide from the hemolymph of
the lepidopteran insect Pseudoplusia includens. J. Biol. Chem. 272, 2344023447.
Duvic, B. and Söderhäll, K. (1990). Purification and characterization of a β1,3-glucan binding protein from plasma of the crayfish Pacifastacus
leniusculus. J. Biol.Chem. 265, 9327-9332.
Duvic, B. and Söderhäll, K. (1992). Purification and partial characterization
of a β-1,3-glucan-binding-protein membrane receptor from blood cells of
the crayfish Pacifastacus leniusculus. Eur. J. Biochem. 207, 223-228.
Fridovich, I. (1995). Superoxide radical and superoxide dismutases. Annu.
Rev. Biochem. 64, 97-112.
Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold
Spring Harbor: Cold Spring Harbor Laboratory Press.
Hjalmarsson, K., Marklund, S. L., Engström, Å. and Edlund, T. (1987).
Isolation and sequence of complementary DNA encoding human
extracellular superoxide dismutase. Proc. Nat. Acad. Sci. USA 84, 63406344.
Holmblad, T., Thörnqvist, P.-O., Söderhäll., K. and Johansson, M. W.
(1997). Identification and cloning of an integrin β subunit from hemocytes
of the freshwater crayfish Pacifastacus lenisculus. J. Exp. Zool. 277, 255261.
Hunter, W. M., and Greenwood, F. C. (1962). Preparation of iodine-131
labeled human growth hormone of high specific activity. Nature 194, 495496.
James, E. R. (1994). Superoxide dismutase. Parasitol. Today 10, 481-484.
Johansson, M. W. and Söderhäll, K. (1988). Isolation and purification of
a cell adhesion factor from crayfish blood cells. J. Cell Biol. 106, 17951803.
Johansson, M. W. and Söderhäll, K. (1989). Cellular immunity in
crustaceans and the proPO system. Parasitol. Today 5, 171-176.
Johansson, M. W. and Söderhäll, K. (1996). The prophenoloxidase
activating system and associated proteins in invertebrates. Progr. Mol.
Subcell. Biol. 16, 46-66.
Johansson, M. W., Keyser, P. and Söderhäll, K. (1994). Purification and
cDNA cloning of a four-domain Kazal proteinase inhibitor from crayfish
blood cells. Eur. J. Biochem. 223, 389-394.
Johansson, M. W., Lind, M. I., Holmblad, T., Thörnqvist, P.-O. and
Söderhäll, K. (1995). Peroxinectin, a novel cell adhesion protein from
crayfish blood. Biochem.Biophys. Res. Commun. 216, 1079-1087.
Johansson, M. W., Patarroyo, M., Öberg, F., Siegbahn, A. and Nilsson, K.
925
(1997). Myeloperoxidase mediates leukocyte adhesion via the αMβ2 integrin
(Mac-1, CD11b/CD18). J. Cell Sci. 110, 1133-1139.
Kobayashi, M., and Söderhäll, K. (1990). Comparison of concanavalin A
reactive determinants on isolated haemocytes of parasite-infected and noninfected freshwater crayfish. Dis. Aquatic Org. 9, 141-147.
Kobayashi, M., Johansson, M. W. and Söderhäll, K. (1990). The 76 kD celladhesion factor from crayfish haemocytes promotes encapsulation in vitro.
Cell Tissue Res. 260, 13-18.
Liang, Z., Lindblad, P., Beauvais, A., Johansson, M. W., Latgé, J.-P., Hall,
M., Cerenius, L. and Söderhäll, K. (1992). Crayfish α-macroglobulin and
76 kDa protein; their biosynthesis and subcellular localization of the 76 kDa
protein. J. Insect Physiol. 38, 987-995.
Liang, Z., Sottrup-Jensen, L., Aspán, A., Hall, M. and Söderhäll, K.
(1997). Pacifastin, a novel 155-kDa heterodimeric proteinase inhibitor
containing a unique transferrin chain. Proc. Nat. Acad. Sci. USA 94, 66826687.
Marchuk, D., Drumm, M., Saulino, A. and Collins, F. S. (1990).
Construction of T vectors, a rapid and general system for direct cloning of
PCR products. Nucl. Acids Res. 19, 1154.
Marklund, S. L. (1982). Human copper-containing superoxide dismutase of
high molecular weight. Proc. Nat. Acad. Sci. USA 79, 7634-7638.
Ratcliffe, N. A., Rowley, A. F., Fitzgerald, S. W. and Rhodes, C. P. (1985).
Invertebrate immunity: basic concepts and recent advances. Int. Rev. Cytol.
97, 183-350.
Rosenfeld, J., Capdevieille, J., Guillemot, J. C. and Ferrera, P. (1992). Ingel digestion of proteins for internal sequence analysis after one-or twodimentional gel electrophoresis. Anal. Biochem. 203, 173-179.
Sambrook J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A
Laboratory Manual. 2nd ed. Cold Spring Harbor: Cold Spring Harbor
Laboratory Press.
Smith, M. W., and Doolittle, R. F. (1992). A comparison of evolutionary rates
of the two major kinds of superoxide dismutase. J. Mol. Evol. 34, 175-184.
Söderhäll, K., and Smith, V. J. (1983). Separation of the haemocyte
population of Carcinus maenas and other marine decapods, and
prophenoloxidase distribution. Dev. Comp. Immunol. 7, 229-239.
Söderhäll, K., and Cerenius, L. (1998). Role of the prophenoloxidaseactivating system in invertebrate immunity. Curr. Opin. Immunol. 10, 23-28.
Söderhäll, K., Cerenius, L. and Johansson, M. W. (1996). The
prophenoloxidase activating system in invertebrates. In New Directions in
Invertebrate Immunology (ed. K. Söderhäll, G. Vasta and S. Iwanaga), pp.
229-253. Fair Haven: SOS Publications.
Tangemann, K. and Engel, J. (1997). Binding studies of integrins with their
ligands. In Integrin-ligand Interaction. Molecular Biology Intelligence Uni.
(ed. J. A. Eble and K. Kühn), pp. 85-100. Austin: R. G. Landes Co.
Thörnqvist, P.-O., Johansson, M. W. and Söderhäll, K. (1994). Opsonic
activity of cell adhesion proteins and β-1,3-glucan binding proteins from
two crustaceans. Dev. Comp. Immunol. 18, 3-12.
von Heijne, G. (1987). Sequence Analysis in Molecular Biology, 188 pp. San
Diego: Academic Press, Inc.