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Prophenoloxidase activation in the hemolymph of Sarcophaga bullata larvae.

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Archives of Insect Biochemistry and Physiology 7:91-103 (1988)
Prophenoloxidase Activation in the
Hemolymph of Sarcophaga bu//ata Larvae
Steven J. Saul and Manickam Sugumaran
Department of Biology, University of Massachusetts at Boston Harbor Campus
Phenoloxidase in the hemolymph of Sarcophaga bullata larvae is present as
an inactive proenzyme form. Localization studies indicate that the majority
of the prophenoloxidase is present in the plasma fraction whereas only a
minor fraction (about 4%) is present in the cellular compartments (hemocytes).
Inactive prophenoloxidase can be activated by zymosan, not by either
endotoxin or laminarin. This activation process is inhibited by the serine
protease inhibitors, benzamidine and p-nitrophenyl-p’-guanidobenzoate.
Exogenously added proteases, such as chymotrypsin and subtilisin, also
activated the prophenoloxidase in the whole hemolymph but failed to activate
the partially purified proenzyme. However, an activating enzyme isolated
from the larval cuticle, which exhibits trypsinlike specificity, activated the
partially purified prophenoloxidase. Inhibition studies and activity
measurements also revealed the presence of a similar activating enzyme i n
the hemolymph. Thus, the phenoloxidase system in Sarcophaga bullata larval
hemolymph seems to be comprised of a cascade of reactions. An endogenous
protease inhibitor isolated from the larvae inhibited c hymotrypsi n-med iated
prophenoloxidase activation but failed to inhibit the cuticular activating
enzyme-catalyzed activation. Based on these studies, the roles of
prophenoloxidase, endogenous activating proteases, and protease inhibitor
in insect immunity are discussed.
Key words: protease cascade, protease inhibitor, melanization, insect immunity
INTRODUCTION
Extensive studies carried out over the past few decades on the immune
response of higher animals to invading parasites and infectious microorganisms have paved the way to a better understanding of the biochemical aspects
Acknowledgments: This work was supported in part by grants from NIH (R01-AI-14753) and
the Universtiy of Massachusetts at Boston (BRSC and Healey grants). We thank Mr. Liu Bin
for excellent technical assistance.
Received August 10,1987; accepted November 25,1987.
Address reprint requests to Dr. Manickam Sugumaran, Department of Biology, University of
Massachusetts at Boston Harbor Campus, Boston, MA 02125.
0 1988 Alan R.
Liss, Inc.
92
Saul and Sugumaran
of immunology. However, the defense mechanisms in insects and other
arthropods have remained largely unexplored. Lacking the complicated immunoglobulin system of higher animals, insects have managed to differentiate self from nonself and kill the foreign organisms effectively by a plethora
of host-defense reactions [1,2]. These include cellular reactions such as nodule formation, phagocytosis, and encapsulation and humoral responses such
as agglutination, antibacterial protein secretion, and humoral encapsulation
[1,2]. Yet, several disease-causing organisms escape the immune system of
insects and thrive in their body parts before being transmitted to animals. A
clear understanding of molecular mechanisms of insect immunity not only
can unravel the survival strategy of parasites in insects, but also can lead to
the development of new vector control measures.
The immediate response of insects to the presence of foreign objects such
as parasites, microorganisms, glass beads, and nylon in their hemolymph is
the deposition of a melanin coat around the nonself matter [3,4]. Thus, it is
quite clear that melanin and the enzyme that biosynthesizes melanin, ie,
phenoloxidase, are integral parts of the immune system of insects. Phenoloxidase, (o-diphenol, oxygen oxidoreductase E.C. 1.10.3.1) is normally present
in most insects examined in an inactive proenzyme form [1,2,5-121. The
proenzymes from Bornbyx rnori [5], Munducu sextu [7l, Muscu dornesticu [12] and
Cnlliphora [13] have been purified and characterized, but detailed studies
have been carried out only with the silkworm enzyme. Following the characterization of prophenoloxidase, the Japanese group purified a serine-type
activating protease from the silkworm larval cuticle and demonstrated that
the activation caused by this enzyme accompanied the removal of a peptide
with a molecular weight of 5,000 from prophenoloxidase [14]. Further work
by Ashida’s group revealed that @-1,3-glucanscould activate the silkworm
prophenoloxidase in the whole hemolymph, indicating a direct link between
foreign matter and prophenoloxidase activation [15]. These workers have
also shown that at least two proteases are triggered by @-1,3-glucansprior to
the activation of prophenoloxidase [16]. Thus, it is apparent that prophenoloxidase activation is achieved in this organism by a cascade of reactions
[15,161.
Recent studies from our laboratory also reveal the presence of a similar
prophenoloxidase cascade in the larvae of Munducu sextu [17,18]. In addition,
we have demonstrated that several proteases could activate the prophenoloxidase system indirectly in the hemolymph of Munducu sextu [17,18]. Because
proteolytic activation is an irreversible process and is known to be finely
controlled in higher organisms by protease inhibitors, we examined this
possibility and isolated two serine protease inhibitors from Munducu larvae
and one from Sarcophugu bullutu larvae [19,20]. These inhibitors prevented the
activation of chymotrypsin-mediated prophenoloxidase activation in the
whole hemolymph [20]. One of the Munducu inhibitors also inhibited the
cuticular protease catalyzed prophenoloxidase activation, confirming our
contention that the endogenous protease inhibitor controls the prophenoloxidase activation [21]. The present study is a continuation of our work on the
prophenoloxidase cascade in Surcophugu bullutu.
Hernolymph Prophenoloxidase Activation
93
MATERIALS AND METHODS
Subtilisin (11.9 Ulmg), thermolysin (467 Ulmg), yeast alcohol dehydrogenase (280 Ulmg), E. coli lipopolysaccharides, zymosan, and laminarin were
purchased from Sigma Chemical Co. (St. Louis, MO). Chymotrypsin (67 Ul
mg) and NPGB* were procured from Worthington Cooper Biochemical Co.
(Malvern, PA). All other chemicals were of reagent grade, with the exception
of cane sugar (Domino, El Paso, Texas).
Larvae of Sarcophaga bullata were raised on a dog food diet. Last instar
larvae were used for all biochemical studies. CS-plasma was prepared by the
method outlined by Ashida [22]. The saline solution containing 110 mM KC1,
4 mM NaC1, 15 mM MgCI2, 5 mM potassium phosphate, and 20 mM cane
sugar at pH 6.5 was filtered through a 0.2 pm sterile filter. Each larva was
injected with approximately 0.1 ml of this buffer and bled approximately 20
min later into 1.5 ml Eppendorf tubes. After centrifugation for 1to 2 min, the
supernatant was used for prophenoloxidase activation studies.
Larvae were also injected with the decoagulation buffer that contained 15
mM NaC1, 100 mM glucose, 10 mM EDTA, 30 mM trisodium citrate, and 26
mM citric acid, pH 4.6. Cells were separated from the plasma as described
by Leonard et al. [8]. The plasma sample was then desalted on a Sephadex
G-25 column (1.5 x 40 cm) equilibrated with 10 mh4 cacodylate buffer, pH
7.0, containing 5 mM CaCI2 and used for further studies. Cells were processed by the method of Leonard et al. [8] prior to their use in assays.
To isolate partially purified prophenoloxidase, Sarcophagu bullata larvae
were injected with the decoagulation buffer, and the hemolymph collected
was lyophilized. After the residue was dissolved in 10 mM sodium phosphate
buffer, pH 7.0, it was chromatographed at 4°C on a Sepharose 6B column
(2.5 x 90 cm) equilibrated with the same buffer. CS-plasma that was activated
with chymotrypsin was also chromatographed on the same Sepharose column to determine the elution volume of active phenoloxidase.
The serine protease inhibitor was isolated from the larvae of Savcophaga
bullata as outlined previously [20].
Sarcophaga bullata activating enzyme was prepared as outlined previously
[lo], with the following modifications. Gut contents from each larva were
removed and the cleaned cuticle was placed in 10 mM sodium phosphate
buffer, pH 7.0, containing 0.2 M NaCl and 1mM PTU. After 12 h, cuticle was
removed by filtration and the supernatant was brought to 50% saturation
with respect to ammonium sulfate. The precipitated proteins were collected
by centrifugation, dissolved in 10 mM sodium phosphate, pH 7.5, and dialyzed against the same buffer. The dialyzed solution was applied onto a
hydroxylapatite column equilibrated with 10 mM sodium phosphate, pH 7.5.
The column was washed with the same buffer and the activating enzyme
was eluted with 200 mM sodium phosphate buffer, pH 7.5. This partially
purified fraction was used to activate prophenoloxidase.
*Abbreviations: CS-plasma = cane sugar plasma; NPGB = p-nitrophenyl-p-guanidobenzoate;
PTU = phenylthiourea.
94
Saul and Sugumaran
Phenoloxidase activity was monitored with a l-ml reaction mixture containing enzyme sample and 5 mM dopamine in 25 mM sodium phosphate
buffer, pH 6.0. The reaction was initiated by the addition of enzyme and
followed by measuring the increase in absorbance at 410 nm resulting from
dopachrome formation. For prophenoloxidase-activationstudies, the various
activators were added to plasma preparations and incubated at room temperature. At indicated time intervals, aliquots of the previously described incubation mixtures were withdrawn and assayed for phenoloxidase activity.
Estimation of esterase activity by protease in the plasma fraction was
measured essentially as described by Yoshida and Ashida [16]. The plasma
sample was prepared with the decoagulation buffer, as described earlier, and
used for the estimation of protease as well as phenoloxidase activities after
activation with zymosan. In samples used for checking protease activity, 1
mM PTU was included to inhibit phenoloxidase. An aliquot of the incubation
mixture (50 pl) was added at indicated time intervals to a reaction mixture (1
ml) containing 2 mM N-benzoyl-L-arginine ethyl ester, 1mM NAD, 0.1 mg
of alcohol dehydrogenase, and 0.2 M semicarbazide in 0.25 M Tris-HC1, pH
8.5. The increase in absorbance of the reaction mixture at 340 nm was
measured to monitor esterase activity. An extinction coefficient of 6,220 M-'
cm-I was used to determine the concentration of NADH formed in the
reaction mixture [16].
Protease inhibitor activity was assayed as described previously [20]. The
protein content of various preparations was determined by the Coomassie
dye binding assay [23].
RESULTS
Activation of Prophenoloxidase by Zymosan
In agreement with earlier results, phenoloxidase in Surcophugu bullutu larvae existed as the inactive proenzyme [17,20]. On collecting the hernolymph,
spontaneous activation usually was not observed. Various elicitors were
added to such inactive preparations to test their ability to activate prophenoloxidase. Of the various elicitors tested, only zymosan caused significant
activation of prophenoloxidase in the CS-plasma of Surcophugu bullutu (Fig.
1A). Neither laminarin nor endotoxin activated the prophenoloxidase when
tried at various concentrations. The zymosan-caused prophenoloxidase activation was inhibited specifically by NPGB and benzamidine, which are
known to inhibit serine proteases with trypsinlike specificity. As such, these
compounds do not have any effect on phenoloxidase; therefore, the observed
inhibition of prophenoloxidase activation by these compounds must be due
to the inhibition of a serine protease. Thus zymosan seems to activate one or
more serine proteases, which in turn cause the conversion of prophenoloxidase to active phenoloxidase.
Cellular Localization of Prophenoloxidase
To test the localization of the prophenoloxidase system in the hemolymph
of Surcophugu bullutu, we used the procedure outlined by Leonard et al. [8]
0.3
E;
0.2
.
Hernolymph Prophenoloxidase Activation
IA
95
,
1
“i
z
= 0.1
4
0
15
30
TIME (min)
Fig. 1. Zymosan-mediated activation of prophenoloxidase and its inhibition by NPGB and
benzamidine in the plasma of Sarcophaga bullata larvae. A Zymosan (7.5 pg) was added to
0.5 ml of CS-plasma (54 rng proteinhl) at room temperature. At the indicated time intervals a
25-pl aliquot of the incubation mixture was withdrawn and assayed for phenoloxidase
activity. For inhibition studies, NPGB (50 p M ) or benzamidine (10 mM) was added 5 min prior
to the addition of zymosan. Treatments used ( 0 )zymosan; (0)control or zymosan + NPGB
or zymosan + benzamidine. B: Plasma prepared with the decoagulation buffer as described
in the section on materials and methods was used. Zymosan (15 pg) was incubated with 1 ml
of plasma (12.7 mg proteinhl), and an aliquot (50 pl) of the incubation mixture was withdrawn
at the indicated time intervals and assayed for phenoloxidase activity. For inhibition studies,
NPGB (50 p M ) was added 5 min prior to the addition of zyrnosan. Treatments used: ( 0 )
zyrnosan; (0)
control or zymosan + NPGB.
after suitable modification of decoagulation buffer for osmolality change.
Again, zymosan activated the prophenoloxidase system in the plasma preparations most readily (Fig. lB), whereas cellular preparations contained negligible activity (less than 4% of the activity found in the plasma). Because it
is possible that the activating protease may be absent in such preparations,
we added the cuticular activating enzyme to the cellular fraction to check for
phenoloxidase. Addition of either the cuticular activating enzyme or 1.5 M
urea caused activation of cellular prophenoloxidase, but again only 2 4 % of
total phenoloxidase activity was found in the cellular fraction; the majority
remained in the plasma fraction. Thus the prophenoloxidase system in Sarcophaga bullata seems to be mainly localized in the plasma fraction.
Protease-Mediated Prophenoloxidase Activation
Because prophenoloxidase activation is achieved by partial proteolysis, we
tested the effect of various commercially available proteases on prophenoloxidase. After a short lag period, both chymotrypsin and subtilisin caused
significant activation of prophenoloxidase in CS-plasma (Fig. 2). Pronase
activated the proenzyme only marginally, whereas trypsin and thermolysin
failed to activate it. At concentrations employed (200 pg each), it is possible
that the last three proteases could have initially activated the proenzyme and
subsequently degraded the active enzyme. We therefore checked the prophenoloxidase activation with varying amounts of these proteases as well (10
to 200 p g ) . However, we failed to witness any significant prophenoloxidase
activation by these enzymes. Therefore, further studies were carried out with
chymotrypsin and subtilisin only.
96
Saul and Sugumaran
id/.----d
a
a
15
TIME ( m i d
0
30
Fig. 2. Prophenoloxidase activation by various proteases in CS-plasma of Sarcophaga bullata
larvae. Various proteases (at 200 pg/ml of plasma) were added to CS-plasma (61-80 mg protein/
ml). At the indicated time intervals a 10-pl aliquot was withdrawn from the incubation mixture
and assayed for phenoloxidase activity. Proteases used. ( W ) none; (A)trypsin or thermolysin;
(0)
chymotrypsin; (0)
pronase; ( 0 )subtilisin.
.
.
C
U
0
10
20
TIME ( m i d
Fig. 3. Time course of prophenoloxidase activation as a function of chymotrypsin concentration in CS-plasma of Sarcophaga bollata. Chymotrypsin at varying concentrations was added
to CS-plasma (80 mg proteidml) at room temperature. Aliquots (10 pl) were withdrawn at the
indicated time intervals and assayed for phenoloxidase activity. Amount of chymotrypsin
used: (A)0 pg; ( 0 )54 pg; (0)
108 pg; and ( W ) 216 pg.
Figure 3 illustrates the effect of varying amounts of chymotrypsin addition
on the activation of prophenoloxidase. Increasing amounts of chymotrypsin
caused increased phenoloxidase activation, and maximum activation was
achieved with about 220 p g of chymotrypsin. Higher concentration of chymotrypsin also shortened the lag time. Endogenous protease inhibitor
isolated from sarcophagid larvae inhibited the chymotrypsin-mediated prophenoloxidase activation in CS-plasma [20].
Evidence for Endogenous Protease Activator
Both chymotrypsin and subtilisin could activate prophenoloxidase either
directly by proteolytic cleavage or indirectly by activating endogenous pro-
Hemolymph Prophenoloxidase Activation
97
teases, which in turn would activate the prophenoloxidase. A direct activation calls for a linear response in prophenoloxidase activation, whereas the
indirect route predicts a lag in activation. Because the observed activation
shows a strong lag period, the indirect route seems to be operative for
prophenoloxidase activation. Inhibition studies also indicated this possibility.
Although subtilisin is a serine protease, it is not inhibited by NPGB. We
took advantage of this property to distinguish the two previously mentioned
possible mechanisms of prophenoloxidase activation. As shown in Fig. 4,
prophenoloxidase activation caused by subtilisin can be inhibited by increaing concentrations of NPGB. Because NPGB has no effect on either subtilisin
or phenoloxidase, the observed inhibition must be due to the inhibition of
endogenous proteases activated by subtilisin. Further support for this contention comes from the following studies.
If prophenoloxidase activation is a direct consequence of an endogenous
protease activity, then purified prophenoloxidase devoid of such protease
activity must resist activation by either chymotrypsin or subtilisin. To meet
this end, prophenoloxidase was partially purified by molecular sieve chromatography on a Sepharose 6B column; Fig. 5A shows the elution profile of
sarcophagid larval hemolymph components. The major peak appearing after
the void volume was due to arylphorin; the peak eluting at the end, to lowmolecular-weight phenols. Prophenoloxidase eluted at fraction 48 as a shoulder to the arylphorin peak and was found to be catalytically inactive. Plasma
preparations treated with chymotrypsin, upon chromatography on Sepharose 6B column, exhibited a similar chromatographicpattern (Fig. 5B). Activity
measurements revealed that fraction 48 contained the active phenoloxidase.
Although chymotrypsin activated the prophenoloxidase in the whole plasma,
it faded to activate the partially purified prophenoloxidase from fraction 48.
Similar failure was met with subtilisin. Thus, neither chymotrypsin nor
subtilisin activates the partially purified prophenoloxidase, although both
0
10
20
30
TIME fmin)
Fig. 4. Inhibition of subtilisin-catalyzed phenoloxidase activation by NPGB. Various concentrations of NPGB (20-100 pM) were incubated with 1 ml of CS-plasma (80 mg protein/ml) for 5
rnin prior to the addition of subtilisin (200 pg). At the indicated time intervals, a 10-pl aliquot
was withdrawn from the incubation mixture and assayed for phenoloxidase activity. Concentration of NPCB used: (0)
0 pM; ( 0 )20 pM; ( W ) 40 pM and (A)100 p M .
98
Saul and Sugumaran
3
A
B
2
j
0
OD
N
a
1
ccw
_ _ _ ( -
0
60
L
0
2
30
Fraction Number
Fig. 5. Sepharose 66 column chromatography of hemolymph contents of Sarcophaga bullata
larvae from control (A) and chymotrypsin activated plasma (B). Control plasma prepared with
decoagulation buffer, as described in the materials and methods section, was lyophilized and
used. It was dissolved in 10 mM phosphate buffer, p H 7.0, centrifuged and chromatographed
on a Sepharose 68 column (2.5 x 90 cm) equlibrated with the same buffer at 4OC. A flow rate
of 31.5 ml/h was maintained, and 5.8 rnl fractions were collected. Aliquots (50 PI) were
withdrawn from each fraction and assayed for phenoloxidase activity. CS-plasma activated
with chymotrypsin (100 pg/ml) for 10 rnin was lyophilized and processed as previously.
Absorbance i s at 280 nm; ( 0 )shows phenoloxidase activity.
trigger the prophenoloxidase activation in the whole plasma. From these
observations, it was clear that these proteases activated an endogenous
protease, which in turn activated the prophenoloxidase. To test this possibility, activity measurements were made in CS-plasma treated with zymosan.
Zymosan was preferred over either chymotrypsin or subtilisin because it
does not have any protease activity and it too triggered the protease. As
shown in Figure 6, zymosan addition to plasma resulted in the activation of
prophenoloxidase as well as a protease. The significant lag period observed
prior to the appearance of both these activities indicates that these enzymes
are present in the inactive proenzyme form and that zymosan treatment
causes the conversion of proform to active form. Addition of NPGB inhibited
the appearance of both protease and phenoloxidase activities, which suggests
that the protease activated is a serine type with trypsinlike specificity.
A similar protease was isolated from the cuticle by the procedure outlined
under the section on materials and methods. This cuticular activating enzyme
readily activated the partially purified prophenoloxidase. Figure 7, for instance, gives the time course of activation of prophenoloxidase caused by
cuticular activating factor. Interestingly, this activation is also inhibited by
serine protease inhibitors, NPGB, and benzamidine. These results clearly
show that the cuticular activating factor is a serine protease with trypsinlike
Hemolymph ProphenoloxidaseActivation
20
99
40
TIME ( m i d
Fig. 6. Zymosan-mediated activation of a proprotease and prophenoloxidase. Plasma (4 mg
proteinhl) prepared with decoagulation buffer containing 1 m M PTU was incubated with
zyrnosan (30 p g h l plasma). Aliquots of this mixture were withdrawn and assayed for esterase
activity as outlined in the material and methods section. In a separate reaction, plasma
solution without PTU was incubated with zymosan (30 pg/ml plasma) and assayed for the
appearance of phenoloxidase activity. Symbols used: (0)
= esterase activity; ( 0 )= phenoloxidase activity.
0.04
C
I
E
\
z
d
a 0.02
Q
0
TIME ( m i d
Fig. 7. Sarcophaga cuticular activating enzyme-mediated activation of partially purified prophenoloxidase and i t s inhibition by NPCB and benzamidine. A 100-pl aliquot Sarcophaga
cuticular activating enzyme (2.35 mg proteinlml), prepared as described in the materials and
methods section, was incubated with 0.5 ml of partially purified prophenoloxidase (1.0 mg/
ml) solution. At the indicated time intervals, a 50-pl aliquot of the incubation mixture was
withdrawn and assayed for phenoloxidase activity. For inhibition studies NPCB (6 pM),
benzamidine (3.7 mM), or endogenous Sarcophaga inhibitor (500 IU)was incubated for 5 min
with prophenoloxidase prior to the addition of activating enzyme. Treatments used were:
(k) prophenoloxidase + activating enzyme; (0)
prophenoloxidase + activating enzyme +
protease inhibitor; (m) prophenoloxidase + activating enzyme + NPGB; ( 0 )prophenoloxidase + activating enzyme + benzarnidine.
100
Saul and Sugumaran
specificity. The activation caused by the cuticular protease is unaffected by
exogenously added sarcophagid protease inhibitor.
DISCUSSION
In accordance with the published results [17,20], phenoloxidase was found
to exist in the inactive proenzyme form in the hemolymph of Surcophugu
bullutu larvae. Bacterial products, which are known to cause activation of
prophenoloxidase in other organisms, did not do so in Suvcophugu bullata.
Although initially this observation made us doubt the role of phenoloxidase
in the immune response of Surcophugu, careful examination indicated otherwise. Although most organisms tested (Bombyx mori [15], Munduca sextu [B],
Gulleriu mellonellu [9], Antheraeu pemyi [6], and Blaberus cruniifer [8]) are not
routinely exposed to bacterial contact in their natural habitat, the flesh fly
Suvcophugu lives on decomposing meat, where numerous bacteria also thrive.
Thus, sarcophagid larvae may have evolved to coexist with bacteria, whereas
other insects such as the silkworm and tobacco hornworm live in a less septic
environment and manage bacteria as foreign, disease-causing organisms.
Therefore, they seem to respond to bacterial products immunologically,
whereas Surcophuga does not. On the other hand, yeast /3-1,3-glucan (zymosan), which is foreign to Surcophagu bullatu larvae, readily caused the phenoloxidase activation, thereby confirming the important role of phenoloxidase
in the immune response. In this context, it is interesting to note that Ashida’s
group has recently identified two separate receptor sites for triggering the
prophenoloxidase system-one for p-1, 3-glucans and one for peptidoglycans-in the larval hemolymph of Bombyx mori [24]. The binding of appropriate ligands to these receptors triggers prophenoloxidase activation. Savcophagu
bullutu may lack the peptidoglycan-mediated pathway, which accounts for
the inability of bacterial products to initiate prophenoloxidase activation in
this organism.
Zymosan-triggered activation of prophenoloxidase seems to be mediated
by serine protease. The addition of NPGB, which inhibits serine proteases
with trypsinlike specificity, totally inhibited the appearance of phenoloxidase
activity in the plasma supporting the previous contention (Fig. 1).Furthermore, both chymotrypsin and subtilisin activated the prophenoloxidase system in whole plasma but failed to activate partially purified prophenoloxidase,
which indicates the absence of an activating factor in these preparations.
However, the serine protease isolated from the cuticle readily activated the
prophenoloxidase (Fig. 7). Presence of a similar protease in the plasma
therefore would account for all the observations. Accordingly, activity measurements reveal the appearance of the protease along with phenoloxidase
in response to zymosan treatment (Fig. 6). Therefore, it appears that at least
one protease must be triggered by zymosan before prophenoloxidase activation is observed. Ashida’s group has clearly established that in Bornbyx mori
two proteases are activated in sequence before prophenoloxidase activation
[16]. We demonstrated the presence of a similar system in Munducu sextu [IS].
Thus, the prophenoloxidase system in insects seems to be comprised of a
cascade of reactions.
Hemolymph Prophenoloxidase Activation
101
Considerable controversy exists regarding the localization of prophenoloxidase components in the hemolymph of insects. Because this enzyme cascade
constitutes an important component of the immune response of insects, the
controversy needs to be clarified. Earlier work by Evans [6] and Pye [9]
indicated the presence of this system in the plasma fraction of Gallevin
mellonellu and of Antherueu pemyi. Ashida’s group used a cane sugar factor to
prepare whole hemolymph from Bornbyx movi and indicated the presence of
prophenoloxidase in the plasma [22]. The isolation procedure resulted in a
90% reduction of the hemocyte population. It therefore could be argued that
fragile hemocytes may have broken and released the prophenoloxidase system into the plasma, which would account for the observed results. On the
other hand, Leonard et al. [8] used a decoagulation buffer and separated
hemocytes from plasma. With these preparations, they observed the localization of the prophenoloxidase system in the hemocytes and not in the plasma.
Because hemocytes are known to play a major role in the defense reactions
of insects [1,2,4,8], the localization of prophenoloxidase in hemocytes seems
to be reasonable and would readily account for cell-mediated melanization of
foreign objects: as soon as the hemocytes come in contact with foreign
organisms, marker molecules such as @-1,3-glucanor peptidoglycan bind to
the hemocytes and trigger the prophenoloxidase activation, which then
causes melanization response. This hypothesis calls for the localization of
prophenoloxidase cascade in the hemocyte fraction. Thus, Leonard et al. [8]
have demonstrated the presence of proteases in hemocytes obtained from
BZuberus cruniifer hemolymph, whereas Iwama and Ashida [25] have shown
that prophenoloxidase is synthesized in the oenocytoids. The latter group of
workers have also demonstrated that the prophenoloxidase synthesized by
hemocytes is released into the cell-culture medium [25], which indicates the
possible occurrence of a similar secretion in vivo. If that is the case, it would
account for the results obtained by earlier workers [6, 9, 221. One then has to
account for the inability of Leonard et al. [8] to detect prophenoloxidase in
plasma. The plasma fractions used by these workers might contain protease
inhibitors that prevented the activation of prophenoloxidase. We have reported the presence of such inhibitors in Munducu sextu as well as in Sarcophugu bullutu [19-211. The presence of similar inhibitors in the Blabems plasma
would result in the failure to detect prophenoloxidase activation. In addition,
these authors [8] used laminarin to trigger prophenoloxidase. Because laminarin does not activate prophenoloxidase directly, the lack of appropriate
activating components such as activating protease or receptors or even suboptimal conditions employed might result in the lack of prophenoloxidase
activation and the observed false localization. Finally, as shown in an earlier
publication [B],if the activated phenoloxidase is short lived it would have
escaped detection.
In reevaluating this problem, we have taken these factors into consideration. Using cuticular activating enzyme as the direct activator of prophenoloxidase, we found that nearly 98% of the total prophenoloxidase is present
in the plasma and only 2% is localized in the hemocytes of Munducu sextu
larval hemolymph [26]. In the present studies we also find the majority of
prophenoloxidase in the plasma fraction and only a minor amount in the
102
Saul and Sugumaran
hemocyte fraction of Sarcophagu butlatu. Dipterans, such as mosquitoes, are
known to use humoral encapsulation reactions [1,21. Thus, the melanization
observed in these species may be caused by plasma components of hemolymph, which would account for the observed localization.
Although we have used considerable care to isolate intact hemocytes, the
possibility of degranulation and the release of prophenoloxidase into the
plasma still exists. Because we employed the same isolation techniques used
by Leonard et al. [8], with modification of the osmolality of the decoagulation
buffer for use in our system, and because we confirmed the integrity of
hemocytes isolated by microscopic examination, such accidental release seems
unlikely. It is possible that prophenoloxidase may be localized differentially
in the hemolymph components in different organisms. However, further
studies are necessary to resolve this matter.
At present, the role of endogenous protease inhibitors isolated from Sarcophuga bullata larvae in the prophenoloxidase cascade is not clear. Earlier, we
showed that the endogenous protease inhibitor could prevent chymotrypsinmediated prophenoloxidase activation in whole hemolymph [20]. Because
the endogenous inhibitor inhibits chymotrypsin, it is difficult to determine
whether the observed inhibition of prophenoloxidase activation is to the
inhibition of chymotrypsin or to the inhibition of the endogenous protease
that causes prophenoloxidase activation. In Manduca sexta, cuticular proteasemediated prophenoloxidase activation is severely inhibited by a hemolymph
serine protease inhibitor [21]. Although we could not demonstrate such an
inhibition in Sarcophagu bullutu, this may not necessarily mean that in Sarcophaga the protease inhibitor does not have any role in the prophenoloxidase
cascade. As Yoshida and Ashida [16] have suggested, it is possible that the
hemolymph protease activator may be different from the culticular protease
activator. Further work on the characterization of hemolymph activating
factor can throw more light on this aspect.
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