From the Institute for Molecular Biophysics,
University of Mainz, D55128 Mainz, Germany and ¶ Oregon Institute
of Marine Biology, University of Oregon, Charleston, Oregon 97420
Received for publication, November 17, 2000, and in revised form, February 8, 2001
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ABSTRACT |
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Phenoloxidase, widely distributed among
animals, plants, and fungi, is involved in many biologically essential
functions including sclerotization and host defense. In chelicerates,
the oxygen carrier hemocyanin seems to function as the phenoloxidase.
Here, we show that hemocyanins from two ancient chelicerates, the
horseshoe crab Limulus polyphemus and the tarantula
Eurypelma californicum, exhibit
O-diphenoloxidase activity induced by submicellar
concentrations of SDS, a reagent frequently used to identify
phenoloxidase activity. The enzymatic activity seems to be restricted
to only a few of the heterogeneous subunits. These active subunit types
share similar topological positions in the quaternary structures as
linkers of the two tightly connected 2 × 6-mers. Because no other
phenoloxidase activity was found in the hemolymph of these animals,
their hemocyanins may act as a phenoloxidase and thus be involved in
the primary immune response and sclerotization of the cuticle. In
contrast, hemolymph of a more recent arthropod, the crab Cancer
magister, contains both hemocyanin with weak phenoloxidase
activity and another hemolymph protein with relatively strong
phenoloxidase activity. The chelicerate hemocyanin subunits showing
phenoloxidase activity may have evolved into a separate phenoloxidase
in crustaceans.
Phenoloxidases are widespread in animals, plants, fungi, and some
bacteria. They are involved in several biological functions. They
initiate the synthesis of melanin, are responsible for the browning of
fruits, vegetables, and fungi, and are crucial for sclerotization of
arthropod exoskeleton. Their central role in a common defense cascade
for Deuterostoma and Prostoma has been reviewed (1). Phenoloxidases
catalyze the O-hydroxylation of monophenols (cresolase) or
oxidation of O-diphenols to quinones (catecholoxidase) or
both (tyrosinase) (2-4). Both melanin and phenoloxidase are considered
to be integral parts of the arthropod immune system (5, 6).
Among the Arthropoda, prophenoloxidases and their activated form,
phenoloxidases, have been well characterized in insects and crustaceans
but not in chelicerates. Hemocyanin, which is closely related to
crustacean phenoloxidase based on sequence data (7-9), is the major
constituent of the hemolymph of crustaceans and chelicerates but is
absent in insects. Interestingly, the clotting system of the horseshoe
crab, Limulus polyphemus, an ancient chelicerate, bears
several functional and biochemical similarities to the prophenoloxidase
system in insects and crustacea, including activation induced by
lipopolysaccharides or Hemocyanins are large multisubunit copper proteins composed of
different subunit types and found freely dissolved in the hemolymph of
arthropods and molluscs (13-16). Their primary biological function is
the transport of oxygen. Several physicochemical properties of
hemocyanins are very similar to those of phenoloxidases (2, 15, 17,
18). Both molecules are copper proteins. Based on x-ray structures,
dioxygen is bound in a µ We demonstrate here that hemocyanins from two ancient species of
chelicerate, L. polyphemus and E. californicum,
develop O-diphenoloxidase activity in response to
submicellar concentrations of SDS, a reagent commonly used to identify
phenoloxidase activity (12, 27). The functional conversion of
hemocyanin may indicate an induced conformational change. This
activation is restricted to only a few of the various subunit types of
each hemocyanin. We compare the enzymatic activities of these
chelicerate hemocyanins with that of Cancer magister, a crustacean.
Purification Procedures--
L. polyphemus was
purchased through Carolina Biological Supply. The hemolymph was
withdrawn from the pericardial cavity and immediately centrifuged at
1000 × g for 5 min at 4 °C using a Beckman J2-HS
centrifuge. The supernatant was then centrifuged at 12,000 × g for 10 min. The supernatant was chromatographed at 4 °C
on a BioGel A5m (400-mesh) column (120 × 2.5 cm) that was
equilibrated with a 0.1 M Tris/HCl buffer, 10 mM CaCl2, 10 mM
MgCl2, and 0.1 M NaCl, pH 7.5. The purification
of hemocyanins from E. californicum and C. magister has been described elsewhere (24, 28). The purified
proteins were stored at 4 °C. The concentrations of the proteins
were determined spectroscopically using the molar extinction
coefficient at 278 nm, Polyacrylamide Gel Electrophoresis Assay of
Phenoloxidase Activity--
Native
PAGE1 of hemolymph and
hemocyanin was carried out at pH 7.4 using a 3% gel (see Ref. 29, as
modified by Ref. 28). Dissociating, non-denaturing PAGE at pH 8.9 in
EDTA was done on a 7.5% gel. Prior to pH 8.9 PAGE, hemocyanins
were dissociated into subunits by overnight dialysis at 4 °C in 0.05 M glycine/OH Kinetic Measurements--
The time course of the
phenoloxidase activity of the hemocyanins was followed by monitoring
the formation of dopachrome and its derivatives by the absorbance at
475 nm. The substrate was added to the hemocyanin solution in 0.2 M phosphate buffer, pH 7.5. The reaction was then initiated
by injection of a concentrated SDS stock solution to a final SDS
concentration of 0.085 ± 0.005% and mixing briefly. The dead
time was less than 10 s. Substrates and reaction mixtures were
maintained in the dark because of their light sensitivity. The
condition of the chemically unstable substrates was checked
spectroscopically. Substrates DL-dopa, dopamine, and catechol were purchased from Sigma.
Phenoloxidase Activity of Native Oligomeric Hemocyanins--
Two
of the four peaks of L. polyphemus hemolymph separated by
BioGel A5m chromatography (Fig. 1) showed
phenoloxidase activity when subjected to pH 7.4 PAGE and then incubated
in the presence of dopamine and SDS (Fig.
2). These same peaks, A and B, had
280/340-nm absorbance ratios of 4.2, indicative of copper
oxygen-binding proteins. Peak B corresponded to the native 8-hexamer
hemocyanin with a molecular mass of 3.6 MDa, whereas peak A was
a higher molecular mass aggregate of hemocyanin (7.3 MDa) as determined by light scattering (data not shown). The two lower molecular mass
peaks, C and D, showed neither 340-nm absorbance nor phenoloxidase activity. To determine whether other hemolymph proteins besides hemocyanin exhibit phenoloxidase activity, we tested the unpurified hemolymph with the same assay for phenoloxidase activity. Only those
bands containing hemocyanin were stained by the activity assay.
We examined whether observed phenoloxidase activity of the structurally
closely related 4 × 6-mer hemocyanin from E. californicum, another chelicerate, could also be induced by SDS.
When intact 8 × 6-mer L. polyphemus (peak B) and 4 × 6-mer
E. californicum hemocyanins were assayed by pH 7.4 PAGE,
both hemocyanins showed phenoloxidase activity (Fig. 2). As in the case
of L. polyphemus, no bands other than the hemocyanin bands
showed phenoloxidase activity in E. californicum hemolymph
(data not shown). The absence of a unique phenoloxidase protein in
chelicerate hemolymph is in agreement with the literature (10). Thus it
is unclear what enzyme participates in vivo in the essential
biological processes, sclerotization of the cuticle after molting and
the immune response, in chelicerates.
To compare the phenoloxidase activities of chelicerate hemocyanins with
other hemocyanins, we tested the activity of crustacean (C. magister) and molluscan (Helix aspersa) hemocyanins by
pH 7.4 PAGE (Fig. 2). The C. magister hemolymph contained a
slowly migrating molecule with strong phenoloxidase activity (30). Purified 25 S hemocyanin of C. magister also showed
phenoloxidase activity but only after extended incubation with
dopamine and SDS. The quaternary structure or the subunit structure of
the crustacean hemocyanin, compared with chelicerate hemocyanin, may hinder the access of substrates to the active site sterically. Other
studies on crustacean hemolymph have reported the presence of a
hemocyte prophenoloxidase (31-33). Here, we report the presence of two different hemolymph molecules with phenoloxidase activity; one
is hemocyanin. The activity of crustacean hemocyanin is rather weak in
comparison to the slowly migrating phenoloxidase (Fig. 2).
Qualitative spectroscopic studies confirmed the results of the
phenoloxidase activity staining of the PAGE (Fig.
3). Hemocyanins of L. polyphemus and E. californicum converted
DL-dopa, dopamine, and catechol after activation by SDS.
C. magister hemocyanin showed activity only with dopamine
and catechol and lacked activity with DL-dopa. No activity
was observed when the above assay was applied using
L-tyrosine as a substrate to test the monophenoloxidase activity of these hemocyanins. Thus, these hemocyanins seem to behave
like catecholoxidases when activated by SDS.
Phenoloxidase Activity of the Hemocyanin Monomers--
When the
hemocyanins were dissociated into monomers, subjected to pH 8.9 PAGE,
and tested for phenoloxidase activity, not all subunit types showed a
reaction (Fig. 4). Three of the eight bands of dissociated L. polyphemus hemocyanin appeared to be
capable of O-diphenoloxidase activity. Several authors have
detected heterogeneous subunit types of Limulus
hemocyanin (25, 34-39). Subunits LpII, IIIA,
IIIB, and IV are present in four copies; subunit I is
present in three copies; subunit V and VI are present in two copies;
and subunit IIA is present in one copy (40). Using the
nomenclature of Brenowitz et al. (38), the
Limulus hemocyanin bands showing phenoloxidase activity are
assigned to subunits LpII, II`, II" (all three belong to the same
subunit II), V, and VI. The quaternary structure of the 4 × 6-mer
hemocyanin from E. californicum is comparable with the 4 × 6-mer half-molecule of L. polyphemus hemocyanin (41). In
E. californicum hemocyanin, only subunits b and
c showed SDS-activated O-diphenol oxidase
activity (Fig. 4). The latter two subunits also showed phenoloxidase
activity after activation by limited proteolysis (24). Two of the four
subunits from L. polyphemus that exhibit phenoloxidase
activity, LpV and LpVI, and subunits b and c from
E. californicum hemocyanin have the same topological
positions within the quaternary structure and can even be exchanged
without losing the quaternary structure or the functional properties of
the oligomeric hemocyanins (Fig. 5) (42).
These subunits, located in the center of the oligomer, bridge the four
hexamers in E. californicum hemocyanin and both of the
four-hexamer half-molecules of L. polyphemus hemocyanin (43, 44).
In the dissociated 2-6-mer hemocyanin of C. magister, all
subunits exhibit some phenoloxidase activity with subunits IV and V
giving the strongest reaction. H. aspersa hemocyanin
was included as a positive control and demonstrates strong
phenoloxidase activity on both pH 7.4 and pH 8.9 PAGE. This finding
confirms the catecholoxidase activity of molluscan hemocyanin as
observed previously (45, 46).
Are Hemocyanins Phenoloxidases in Vivo?--
Could these
observations be of physiological importance for the animals?
In the case of the horseshoe crab L. polyphemus, no O-diphenoloxidase activity in hemolymph cells has been
observed so far. The defense mechanism has been described to be similar to other arthropods, however, and all parts of the immune response cascade are found in the hemolymph (1, 10). Nellaiappan and Sugumaran
(12) recently detected an O-diphenoloxidase with a molecular mass of ~70 kDa in the hemolymph of L. polyphemus. For several reasons, we suggest that hemocyanin is
responsible for this enzymatic activity. First, the observed molecular
mass is comparable with hemocyanin subunits of L. polyphemus
as determined by sequence and x-ray analysis (47). Second, we found
that the purified hemocyanin is capable of phenoloxidase activity when activated by SDS. Our hypothesis is supported by the lack of any other
protein with O-diphenoloxidase activity in the hemolymph of L. polyphemus. It is also supported by our findings that
we were not able to detect any phenoloxidase activity in L. polyphemus and E. californicum when centrifuged
hemocytes were tested with the same assay (data not shown). Therefore
we think that the O-diphenoloxidase activity of the purified
protein in L. polyphemus observed by Nellaiappan and
Sugumaran (12) is exhibited by hemocyanin. The physiological
significance of the phenoloxidase activity of chelicerate and
crustacean hemocyanins has recently been addressed. In the horseshoe
crab, T. tridentatus, the coagulation cascade has been linked to prophenoloxidase activation through non-enzymatic
interactions of specific coagulation factors with hemocyanin (26). This
leads to the functional conversion of hemocyanin to phenoloxidase, most probably because of a conformational change of the hemocyanin. In both
T. tridentatus (26) and L. polyphemus, SDS in
submicellar concentrations mimics the function of hydrophobic and/or
polar effectors by inducing conformational conversion without denaturation.
Similarities in phenoloxidase activity in the hemocyanins of
these ancient arthropods L. polyphemus, E. californicum, and T. tridentatus suggest the following
hypothetical scenario: In the hemolymph of these chelicerates, certain
subunits of the hemocyanins are still involved in the cascade of the
immune response by converting them to phenoloxidases. This is not the
case in more recent arthropods, the crustaceans, and insects. Whereas
C. magister hemocyanin shows some phenoloxidase activity,
the primary phenoloxidase is a separate protein. The sequences of
crustacean phenoloxidases are closely related to hemocyanins (7, 9,
18, 30, 32, 48). These crustacean O-diphenoloxidases may
have evolved from those hemocyanin linker subunits that exhibit
O-diphenoloxidase activity in chelicerates. Further sequence
or structural information should clarify this idea. In crustacean
hemocyanins that aggregate only to the two-hexamer level, the
interhexamer linker subunits that allow four and eight hexamer
aggregates are not present. One hypothesis is that these subunit types
evolved to O-diphenoloxidases in crustaceans.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-1,3-glucans (10, 11). A marked
difference from insects and crustaceans, however, is that L. polyphemus amoebocyte lysate supernatant lacks phenoloxidase
activity (10). Recently Nellaiappan and Sugumaran (12) reported the
presence of a prophenoloxidase in the hemolymph of L. polyphemus. Although other prophenoloxidases are often easily activated by limited proteolysis, the prophenoloxidase of L. polyphemus could be only weakly activated by proteolysis, but
activity could be induced by detergents such as SDS or cetylpyridinium
chloride (12). Based on gel electrophoresis and substrate specificities they determined that the enzyme is an O-diphenoloxidase with
a molecular mass of about 70 kDa. Our experiments suggest that this prophenoloxidase is hemocyanin, which is present in high concentrations in the hemolymph of L. polyphemus.
2:
2
side-on coordination in hemocyanins (19), which should also hold for
phenoloxidases because of the very similar spectroscopic properties of
the two proteins (2, 20, 21). The high degree of similarity between the
active sites of phenoloxidases and hemocyanins of arthropods and
molluscs is also supported by the capability of hemocyanins to exhibit
O-diphenoloxidase activity after exposure to chaotrophic
salts, SDS, or low pH values (22, 23). The hemocyanin from a
chelicerate, the tarantula Eurypelma californicum, has shown
phenoloxidase activity after proteolysis (24). E. californicum hemocyanin is sequentially and structurally closely related to the hemocyanin from L. polyphemus (25). In
another chelicerate, Tachypleus tridentatus, it has recently
been shown that the naturally occurring coagulation cascade is linked
to prophenoloxidase activation with hemocyanin functioning as a
prophenoloxidase (26).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
278 = 1.1 ml
mg
1 cm
1 for chelicerate hemocyanin.
Concentrations were expressed on a molar basis using the value of 75 kDa for an arthropod hemocyanin subunit (16).
buffer, pH 9.5, 5 mM
in EDTA. According to electrophoresis results, more than 90% of the
hemocyanin was dissociated under these conditions. Phenoloxidase
activity after electrophoresis on both pH 7.4 and pH 8.9 gels was
demonstrated using dopamine as substrate (27). The essential part of
this assay is the activation of the phenoloxidase by SDS. After the
phenoloxidase assay, the gels were photographed and then stained with
Coomassie Blue to identify the proteins.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Elution profile of hemolymph from L. polyphemus on a BioGel A5m column. Four peaks are
present; peaks A and B are hemocyanin, and peaks
C and D are non-copper-containing proteins.
Absorption at 280 nm (solid circle) indicates protein, and
absorption at 340 nm (open circle) indicates the presence of
a CuO2Cu site.
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Fig. 2.
SDS-induced phenoloxidase activity of
hemolymph proteins and hemocyanins from L. polyphemus,
E. californicum, C. magister, and
H. aspersa. A, Coomassie staining;
B, phenoloxidase activity staining with dopamine and SDS.
1, L. polyphemus hemolymph; 2,
L. polyphemus peak A; 3, L. polyphemus
peak B; 4, L. polyphemus peak C; 5,
L. polyphemus peak D; 6, E. californicum 4-6-mer hemocyanin; 7, C. magister hemolymph; 8, C. magister 2-6-mer
hemocyanin; and 9, H. aspersa hemocyanin.
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Fig. 3.
Relative time course of phenoloxidase
activity of 24-mer Eurypelma, 48-mer
Limulus, and 12-mer C. magister
hemocyanins. The production of O-quinones was
detected by monitoring the absorbance at 475 nm using
DL-dopa. Solid line, L. polyphemus; dotted line, E. californicum;
dashed line, C. magister; dot/dash
line, control (no hemocyanin).
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Fig. 4.
Phenoloxidase activity of hemocyanin
subunits. Panel A, dissociating but non-denaturing PAGE
(7.5%, pH 8.8) of E. californicum hemocyanin. Lanes
1-4, Coomassie staining; lanes 5-8, same samples in
the same gel after phenoloxidase activity staining with dopamine and
SDS; dissociated E. californicum hemocyanin (1 and 5),
purified subunits bc (2 and 6), purified subunit
d (3 and 7), and purified subunit e (4 and 8).
The diffuse broad band below the subunit bc (2 and 6) is because of known proteolytical products of subunits
b and c during the purification. Panel
B, dissociating but non-denaturing PAGE (7.5-12.5% gradient, pH
8.8) of dissociated L. polyphemus hemocyanin. 1,
Coomassie staining; 2, phenoloxidase activity staining with
dopamine and SDS. In lane 2, an 8-fold amount of protein
relative to lane 1 has been applied. The nomenclature of the
subunits was taken from Ref. 34. Note that subunit II migrates in three
different bands as II, II`, and II". Panel C,
dissociating but non-denaturing PAGE (7.5%, pH 8.9) of hemocyanin
subunits from C. magister. 1, Coomassie staining;
2, phenoloxidase activity staining with dopamine and SDS.
Lane a, hemolymph; lane b, 2-6-mer
hemocyanin. The nomenclature of the subunits was taken from Ref.
49.
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Fig. 5.
Schematic quaternary structures of the
4-6-mer hemocyanins from E. californicum
(A) and L. polyphemus
(B). The subunits with phenoloxidase
activity are outlined in black. A, the
topology of the seven subunit types of E. californicum is
taken from Markl et al. (43). The two centrally located
subunit dimers b and c bridge the two 2-6-mer half-molecules.
B, the arrangements of the subunits of 24-mer half-molecules
of the native L. polyphemus hemocyanin are shown as given by
Lamy et al. (35).
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FOOTNOTES |
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* This study was supported in part by grants from the Fonds der Chemischen Industrie (to H. D. and E. J.), by the Naturwissenschaftlich-Medizinisches-Forschungszentrum Mainz (to H. D.), and by the National Science Foundation (to N. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 49-61-31-39-23570; E-mail: decker@biophysik.biologie.uni-mainz.de.
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M010436200
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ABBREVIATIONS |
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The abbreviation used is: PAGE, polyacrylamide gel electrophoresis.
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