(Received for publication, September 25, 1996, and in revised form, October 28, 1996)
From the Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506
Serpin gene-1 from the tobacco hornworm, Manduca sexta, encodes, through alternative exon usage, 12 reactive site variants (Jiang, H., Wang, Y. and Kanost, M. R., (1994) J. Biol. Chem. 269, 55-58; Jiang, H., Wang, Y., Huang, Y., Mulnix, A. B., Kadel, J., Cole, K., and Kanost, M. R. (1996) J. Biol. Chem. 271, 28017-28023). These 43-kDa proteins differ from each other only in their COOH-terminal 39-46 residues, which include the reactive site. To test the hypothesis that these proteins are proteinase inhibitors of diverse selectivities and to begin to elucidate their physiological functions, we expressed the 12 serpin-1 variants in Escherichia coli. Seven of the variants inhibited mammalian serine proteinases, with association rate constants comparable with those of human serpins. Serpin-1A, with a P1 Arg residue, inhibited both trypsin and plasmin. Serpin-1B (P1 Ala) and serpin-1F (P1 Val) inhibited porcine pancreatic elastase and human neutrophil elastase. Serpin-1H, -1K, and -1Z, all with a Tyr residue at the P1 position, inhibited chymotrypsin and cathepsin G. Serpin-1I (P1 Leu) inhibited both elastase and chymotrypsin. Nine of the serpin variants were active as inhibitors of microbial serine proteinases, including subtilisin Carlsberg, proteinase K, and two proteinases secreted by an entomopathogenic fungus, Metarhizium anisopliae. In addition, one of the serpin variants, serpin-1J, strongly inhibited activation of M. sexta hemolymph phenoloxidase, a pathway involving a serine proteinase cascade. This pathway is a component of the defensive response of insects to microbial infection. These results suggest that the products of M. sexta serpin gene-1 may be important in regulating both exogenous and endogenous serine proteinases in hemolymph.
The serpin1 superfamily is composed of a large number of proteins, most of which function as serine proteinase inhibitors (1). Serpins have been identified in animals, plants, and viruses (2) and therefore appear to have evolved from a common ancestor before divergence of the animal and plant kingdoms. Individual mammalian species have a large number of serpin genes, each encoding a protein with a unique reactive site sequence and physiological function (3). Members of the serpin superfamily are involved in the regulation of a wide variety of physiological processes, including blood clotting, complement activation, inflammatory responses, hormone transport, and tumor suppression (1).
Serpins are typically 370-390 amino acid residues long, with a reactive site loop near the carboxyl terminus. This loop, exposed on the surface of the molecule, is the site at which a serpin interacts with a target serine proteinase (1). During reaction with a proteinase, the P1 residue (4) in the reactive site loop forms a bond with the active site serine residue in the enzyme. The amino acid sequence and conformation of the reactive site loop largely determine the selectivity of inhibition. Altering the loop sequence, especially at the P1 position, can cause dramatic changes in the inhibitory selectivity of a serpin (3).
Serpins have been found in the hemolymph of invertebrates, including three groups of arthropods: insects, crayfish, and horseshoe crabs (5). These invertebrate serpins have 12-30% amino acid sequence identity with various mammalian serpins. Functions of invertebrate serpins are largely unknown, although they may regulate proteinases released from blood cells during inflammation-like processes and may regulate proteinase cascades that ultimately activate proteins involved in blood coagulation and melanization.
Study of serpins from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm), has revealed a novel genetic mechanism for generating diversity in the serpin reactive site loop (6, 7, 8). Analysis of more than 50 M. sexta serpin cDNAs and their corresponding genomic sequence has shown that 12 serpin variants (named serpin-1A through -1K and -1Z) are produced from a single gene. These serpins are identical in the amino-terminal 336 residues and differ in their carboxyl-terminal 39-46 residues. This variable region includes the reactive site loop. The variable region is encoded by the ninth exon of the gene, which is present in 12 alternate forms between exons 8 and 10 (8). Alternative splicing to allow only one exon 9/molecule of mature serpin-1 mRNA agrees well with observed structures of M. sexta serpin-1 cDNAs (7) and with analogous primary structures found in two silkworm serpins (9).
Because the M. sexta serpin-1 variants differ in the amino acid sequences of their reactive site loops, they were predicted to be inhibitors of diverse selectivity with the potential to regulate proteinases in various physiological processes (7). We report here the inhibitory activities of the 12 serpin-1 variants against a panel of mammalian, fungal, and bacterial proteinases as a first step toward understanding physiological functions of these serpins.
A recombinant plasmid expressing
M. sexta serpin-1B cDNA, serpin-1B/H6pQE-60 (10), was
used as a starting point in constructing plasmids for expressing the
other 11 serpin-1 variants. A cDNA fragment from the
HindIII site upstream of the variable region to another
restriction site in the vector was substituted with the corresponding
fragment from each donor plasmid. Because vectors and restriction sites
for some serpin cDNAs were different, the cloning schemes (Fig. 1)
are explained as follows. (i) Serpin-1A, -1D, -1E, -1G, -1J, -1K, and
-1Z cDNAs, directionally cloned in EcoRI-XhoI
sites of pBluescript (SK) (7), were switched into the expression
plasmid by replacement of a HindIII-PvuII
fragment. (ii) Serpin-1F and -1J(µ) cDNAs (7, 8), located in
EcoRI site of pBluescript (SK) (6), were transferred into
the expression plasmid by nondirectional HindIII cloning.
(iii) serpin-1C, -1H, and -1I cDNA fragments were isolated from a
polymerase chain reaction-derived serpin-1 cDNA mini-library (8)
and then switched into the expression plasmid by replacement of a
HindIII-PvuII fragment. Plasmid constructs were
confirmed by sequence analysis using a specific primer, 5
-AAG CTT TTA
TCG AAG TCA AC-3
, located at the HindIII site upstream of
the variable region. The fusion proteins expressed from these constructs all have an NH2-terminal sequence of
Met-His-His-His-His-His-His-Ala-Met-Ala-Gly-Glu-Thr-Asp. The first 7 residues are encoded by the vector, whereas the rest are from the
serpin-1 cDNA, including the last 3 residues of the signal peptide
(Ala-Met-Ala) and the complete mature protein sequences. Procedures for
expression and Ni2+ affinity purification of the
recombinant serpins were described previously (10).
Inhibitory Activity Assay
To measure the inhibitory
activities, 10 µl of a serine proteinase were incubated with a serpin
(5 µl, 1 µg/µl) in 0.1 M Tris-HCl, pH 8.0, in a
cuvette at room temperature for 5 min. Then 0.7 ml of an appropriate
chromogenic substrate solution (50 µM in 50 mM Tris-HCl buffer, pH 7.8, 5 mM
CaCl2, 50 mM NaCl) was added, and the residual
enzyme activity was monitored by detecting the change in
A410 with time. The enzymes and their substrates
used in the tests were bovine pancreatic trypsin (50 ng/µl)
(Sigma) and
D-Phe-L-pipecolyl-Arg-p-nitroanilide
(Sigma); human plasmin (0.1 µg/µl) (Athens
Research & Technology, Inc.) and
Z-D-Phe-Pro-Arg-p-nitroanilide (where Z stands
for benzyloxycarbonyl), a gift from Dr. J. Tomich (Microchemical Core
Laboratory, Kansas State University); bovine pancreatic
-chymotrypsin (0.1 µg/µl) (Sigma) and
N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma); human neutrophil cathepsin G (0.2 µg/µl)
(Athens Research & Technology, Inc.) and
N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma); porcine pancreatic elastase (0.1 µg/µl)
(Worthington) and
N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide
(Sigma); human neutrophil elastase (0.2 µg/µl)
(Athens Research & Technology, Inc.) and N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide
(Sigma); subtilisin Carlsberg from
Bacillus licheniformis (0.1 µg/µl)
(Sigma) and N-benzoyl-Phe-Val-Arg-p-nitroanilide
(Sigma); proteinase K from the fungus
Tritirchium album (0.1 µg/µl) (Promega) and
N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma); a partially purified chymoelastase PR1 from
pathogenic fungus M. anisopliae isolate ME1 (<0.1
µg/µl, 360 units/µl) (a gift from Dr. J. P. Gillespie, Kansas
State University), and
N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma), a trypsin-like proteinase PR2 isolated from
the same fungus (<0.1 µg/µl, 150 units/µl) and
D-Phe-L-pipecolyl-Arg-p-nitroanilide (Sigma).
Serpins (1 nmol) were incubated with proteinases (0.1 nmol) at room temperature for 5 min. Then, reactions were stopped by the addition of diisopropyl fluorophosphate (Sigma) to a final concentration of 4 mM. In control reactions, the proteinase was treated with diisopropyl fluorophosphate prior to mixing with a serpin. Trifluoroacetic acid (Pierce) was added to a final concentration of 0.1%, and the mixtures were separated by reversed phase HPLC on a Hi-PoreTM RP318 column (Bio-Rad). A 30-60% linear gradient of acetonitrile was applied for 20 min, and absorbance was monitored at 214 nm. A peak representing a peptide present in the serpin/enzyme mixture but absent from the control reaction was collected for amino acid sequence determination. Five cycles of Edman degradation were carried out as described previously (10). Alternatively, the molecular mass of the peptide sample was determined by matrix-assisted laser desorption ionization mass spectrometry on a Lasermat 2000 instrument (Finnigan MAT). Peptide samples were mixed with cyano-4-hydroxycinnamic acid (1 mg/ml) as the matrix and a peptide of known mass as an internal standard.
Measurement of Association Rate ConstantsActive site
titration of bovine trypsin was carried out using a burst titrant,
p-nitrophenyl-p-guanidinobenzoate
(Sigma) (11). The trypsin was used to titrate
1-proteinase inhibitor (Athens Research & Technology, Inc.) (12).
This
1-proteinase inhibitor was then used as a second standard to
titrate porcine pancreatic elastase (Worthington). Bovine chymotrypsin
was titrated using p-nitrophenyl acetate (13). These three
enzymes were then used to titrate the recombinant serpins to determine
the concentration of active inhibitors. All titrations were carried out
at concentrations and preincubation times that allowed complete
association of the inhibitors, as indicated by linearity of titration
curves and confirmed by the association rate constants determined
later. The substrates for inhibitory assays described above for each proteinase were used, except that
N-
-benzoyl-DL-Arg-p-nitroanilide (Sigma) was used in the titration of serpin-1A with
trypsin. Association rate constants (ka) were
determined as described previously (10).
Hemolymph from cut prolegs of six day 2 fifth instar
larvae was collected (approximately 0.5 ml/larva) into chilled
polypropylene tubes, each containing 750 µl of anticoagulation saline
(14). Hemocytes were pelleted by centrifugation at 100 × g for 10 min. The supernatants were combined, and an equal
volume of saturated ammonium sulfate (pH 7.0) was slowly added with
gentle mixing. After centrifugation at 10,000 × g for
10 min, the pellet was dissolved in 900 µl of chilled water
(one-third of the initial hemolymph volume) and stored in aliquots at
70 °C.
Ten µl of elicitor (Micrococcus lysodeikticus ATCC 4698, 1 µg/µl, Sigma) was mixed with 100 µl of 1:5 diluted PPO activation fraction. At intervals, an aliquot (10 µl) was assayed for phenoloxidase activity (15). To test for direct inhibition of phenoloxidase activity, prophenoloxidase was activated by treatment with the detergent cetylpyridinium chloride as described by Hall et al. (15) and then incubated with serpin-1J at a final concentration of 500 µg/ml for 5 min prior to assay of phenoloxidase activity.
cDNAs for all 12 exon-9 variants of Manduca serpin-1 were cloned into an expression vector, H6pQE-60 (Fig. 1). The Escherichia coli strain XL1-blue carrying the recombinant plasmids overexpressed proteins of the expected sizes, which were recognized by antiserum against the M. sexta serpin that inhibits elastase. The plasmids had expected restriction endonuclease digestion patterns, and DNA sequence analysis indicated that the variable sequences corresponding to exons 9A-9K and 9Z were correctly located between constant regions in each plasmid construct.
Under optimized conditions (10), we observed that the level of serpin expression in all 12 constructs was high (0.3 mg/ml) in this host/vector system. However, the solubility of the recombinant serpins varied widely, with the percentage of buffer-extractable serpin from sonicated E. coli ranging from approximately 40% of the total recombinant serpin protein for serpin-1B to 2% for serpin-1A. This difference seems to be extreme, considering that the sequence differences between the variants are primarily confined to about 10 residues of the reactive site loop. Coexpression of chaperones GroEL and GroES did not increase solubility of serpin-1A,2 which suggests that the solubility is affected by properties of the reactive site loop rather than by global folding failure. Factors such as expression rate or serpin polymer formation may have effects on the solubility of these recombinant serpins (16).
The soluble fusion proteins extracted from E. coli were
recovered in one step to over 80% purity by a Ni2+
affinity batch method, as illustrated for serpin-1B (Fig.
2). The amino-terminal sequences of the recombinant
serpins were determined by Edman degradation to be identical to the
expected sequences.
Inhibitory Activity of the Recombinant Serpins
To screen the
inhibitory activity of the M. sexta serpins, we tested for
the inhibition of mammalian digestive enzymes (trypsin, chymotrypsin,
and elastase) and several serine proteinases from human blood (plasmin,
cathepsin G, and neutrophil elastase). The results, summarized in Table
I, showed that seven of the serpin-1 variants can
inhibit one or more of these enzymes. Both porcine pancreatic and human
neutrophil elastases were inhibited by Manduca serpin-1B,
-1F, and -1I. Bovine pancreatic -chymotrypsin and human neutrophil
cathepsin G were inhibited by four serpins: serpin-1H, -1K, -1Z, and
-1I. Two of the elastase inhibitors, serpin-1B and -1I, also inhibited
cathepsin G. Trypsin and plasmin were inhibited only by serpin-1A.
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Some of the serpin-1 variants are active toward serine proteinases secreted by microorganisms (Table I). PR2, a fungal serine proteinase from the chymotrypsin family (17), with specificity for cleaving after Arg residues, was inhibited by serpin-1A, -1J, and -1E. PR1, proteinase K, and subtilisin are serine proteinases from a family that is unrelated in sequence to the chymotrypsin family (18). Proteinase K, favoring Phe and Tyr residues, was inhibited by serpin-1B, -1C, -1H, and -1K. Subtilisin Carlsberg, preferring Leu to aromatic residues at the P1 position, was inhibited by serpin-1B, -1G, and -1F. Serpin-1B and -1C inhibited chymoelastase PR1 from the entomopathogenic fungus M. anisopliae.
A characteristic feature of a serpin-enzyme reaction is the formation of a complex that is stable in SDS (1). We found that all of the enzyme-serpin pairs exhibiting inhibition form SDS-stable complexes (data not shown). This includes complexes with the following serine proteinases from the subtilisin family: subtilisin Carlsberg, proteinase K, and PR1.
Scissile Bond DeterminationTo identify the position of the scissile bond in Manduca serpin-1 variants, we purified the COOH-terminal peptide released from a serpin-enzyme reaction and determined the sequence of the amino-terminal 5 amino acid residues by Edman degradation (Table I). After reaction of serpin-1B with elastase, a peptide with an NH2-terminal sequence of Ser-Leu-Ile-Leu-Tyr was released. This result is consistent with that reported earlier using a different method (10). From the amino acid sequence deduced from the serpin-1B cDNA, the P1 residue of serpin-1B is an alanine at position 343, which is consistent with the specificity of elastases.
Similarly, we determined that the newly exposed NH2 termini for serpin-1H, -1K, and -1Z are Val-Glu-Ser-Ile-Asp, Ser-Phe-His-Phe-Val, and Leu-Ser-Ala-Val-Ile, respectively, after incubation with chymotrypsin. Thus, from the deduced amino acid sequences, tyrosine was identified as the P1 residue in these three chymotrypsin inhibitors at position 341, 343, and 342 of serpin-1H, -1K, and -1Z, respectively.
We determined that serpin-1I was cleaved by porcine pancreatic elastase between Leu343 and Ser344, releasing a peptide with an NH2-terminal sequence of Ser-Leu-Glu-Phe-Ser. The molecular mass of this released peptide was determined by mass spectrometry to be 4239.1 Da. The value calculated from the deduced amino acid sequence is 4248.6 Da, which is slightly larger but within experimental error. Because serpin-1I also inhibited chymotrypsin and cathepsin G, we tried to determine whether there might be more than one scissile bond in serpin-1I. The peptide resulting from the reaction of serpin-1I with cathepsin G eluted at the same retention time on HPLC as the peptide released by elastase, and it had a mass of 4255.3 Da. These data indicate that serpin-1I was cleaved at the same position between Leu343 and Ser344 by elastase and cathepsin G, an enzyme with chymotrypsin-like specificity.
The reaction of serpin-1F with human neutrophil elastase yielded a peptide of 4542.1 Da, only 7.1 Da less than the calculated mass of the peptide beginning at residue 343 and extending to the carboxyl terminus. This result indicates that the scissile bond of serpin-1F is between Val342 and Asp343.
We previously predicted that the P1 residue of serpin-1A to be Arg342 (7). To examine this directly, we purified the COOH-terminal peptide released from the reaction of serpin-1 with Metarhizium PR2 and determined its mass to be 3866.3 Da, compared with 3860.3 Da calculated for the peptide beginning at Gln343 to the carboxyl terminus. This result suggests that the P1 residue of serpin-1A is indeed Arg342.
Measurement of Association Rate ConstantsRate constants for
the association of serpins with different proteinases
(ka) are an indication of inhibitor selectivity. The
association rate constants for some enzyme-inhibitor pairs are listed
in Table II and compared with human 1-proteinase
inhibitor and
1-antichymotrypsin. The ka of
serpin-1B with porcine pancreatic elastase is about 10- and 5-fold
greater than those of serpin-1F and -1I, respectively. These three
insect serpins react faster with pancreatic elastase than does human
1-proteinase inhibitor. Serpin-1K and -1Z inhibit chymotrypsin at
rates similar to that of
1-proteinase inhibitor. Serpin-1H and -1I
react with chymotrypsin about 10 times slower than do serpin-1K or -1Z
but 10 times faster than does human
1-antichymotrypsin. With a
ka close to 106
M
1 s
1, serpin-1A functions as
an efficient trypsin inhibitor.
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Phenoloxidase is
present in insect hemolymph as a zymogen, prophenoloxidase, that is
activated by selective proteolysis upon addition of microbial cell wall
components such as peptidoglycan or -1,3-glucans. The fraction of
M. sexta hemolymph proteins precipitated by 50% saturated
ammonium sulfate (the PPO activation fraction) contains the components
necessary for activation of prophenoloxidase, whereas the majority of
hemolymph serpins are removed. This is consistent with results
previously obtained with Bombyx mori hemolymph proteins
(20). We found that phenoloxidase activity appears within 5 min after
addition of Micrococcus cells to the PPO activation fraction
(Fig. 3). The phenoloxidase activity peaked at about 40 min and gradually decreased to 90% of its maximum after 4 h.
To test whether any of the recombinant serpins affect activation of
prophenoloxidase, we incubated the PPO activation fraction with
Micrococcus and Manduca serpin-1 variants (at
final serpin concentrations of 0.2-1 mg/ml) and measured phenoloxidase
activity after 40 min. Serpin-1J and its allelic variant
serpin-1J(µ) completely blocked phenoloxidase activation at 40 min,
whereas none of the other 11 serpin-1 variants had any effect on
prophenoloxidase activation. Serpin-1J and -1J
inhibited
prophenoloxidase activation in a concentration-dependent
manner, with 50% inhibition at a concentration of 30 µg/ml (Fig.
4). The addition of serpin-1J at 500 µg/ml after
activation of prophenoloxidase had already occurred had no effect on
phenoloxidase activity (data not shown), indicating that serpin-1J does
not inhibit phenoloxidase directly, but instead inhibits activation of
its zymogen.
Study of serpins from two lepidopteran insects, M. sexta and B. mori, has revealed an assortment of serpins in their hemolymph. Twelve serpin variants have been identified in M. sexta hemolymph, all encoded by a single gene through alternate exon splicing (7). The specific proteinase targets of these serpins have not been identified. To begin the elucidation of the physiological functions of the M. sexta serpins, we expressed the 12 reactive site variants of M. sexta serpin-1 in E. coli and characterized their inhibitory activities.
Although more than 10 serpins have been purified from hemolymph of invertebrates, their physiological functions remain largely unknown (5). The only exceptions are two Limulus intracellular serpins, LICI-1 and LICI-2, which regulate serine proteinases that are components of a cascade leading to coagulation (21, 22). In insects, several biological functions for serpins have been proposed (23). Hemolymph serpins may regulate endogenous proteinases released by hemocytes or fat body in inflammation-like processes. Serpins may also play roles in regulating proteinases involved in tissue remodeling during metamorphosis. Another pathway possibly regulated by insect serpins is the establishment of dorsal-ventral polarity during embryogenesis, which involves proteins (snake and easter) homologous to serine proteinases from the horseshoe crab clotting pathway (24). However, direct experimental support for these speculated functions for insect serpins is still lacking.
We were able to demonstrate inhibitory activity against at least one proteinase for 11 of the serpin-1 variants. Each serpin variant has a unique selectivity or spectrum of inhibition of the proteinases in the panel. Each serpin's selectivity is evidently primarily defined by the P1 residue in the reactive site. We identified the P1 residues of serpin-1A, -1B, -1F, -1H, -1I, -1K, and -1Z to be Arg342, Ala343, Val342, Tyr341, Leu343, Tyr343, and Tyr342, respectively. These results are in agreement with the cleavage specificities of their target proteinases: trypsin for serpin-1A, elastases for serpin-1B, -1F, and -1I, and chymotrypsin for serpin-1H, -1K, -1I, and -1Z. Because of technical difficulties associated with low rates of reaction, impurities in proteinase samples, and secondary proteolysis of serpins in competition with the inhibition reaction, we were unable to determine the P1 position in serpin-1C, -1E, -1G, and -1J.
Of the 12 variants, serpin-1B has the broadest selectivity, inhibiting
elastases and also cathepsin G, proteinase K, the subtilisin Carlsberg, and PR1, which resemble chymotrypsin in
specificity. A site-directed mutant of serpin-1B, replacing the
P1 Ala to Phe (A343F), is a fast chymotrypsin inhibitor but
also shows slow inhibition of trypsin (10). This broad selectivity of
serpin-1B could be related to the Pro residue at the P2
site, which may affect interaction of the P1 residue with
the primary binding site of the proteinases. Human 1-proteinase
inhibitor, with a Pro at P2 and a Met at P1,
inhibits trypsin, chymotrypsin, and elastase. Human heparin cofactor 2, also with a Pro at P2 and a Leu at P1, inhibits
both thrombin and chymotrypsin (25, 26). It was also reported the
P2 residue of human antithrombin is very important in
optimal presentation of the reactive center to a cognate proteinase
(27).
Serine proteinases from the subtilisin and chymotrypsin families are
products of convergent evolution. They have entirely different overall
folding patterns but share an essentially superimposable catalytic
machinery. Although serpins are well characterized as inhibitors of
proteinases from the chymotrypsin family, reports of their activity as
inhibitors of proteinases from the subtilisin family has been limited.
We found that several of the M. sexta serpins are active
against some bacterial and fungal enzymes from the subtilisin family.
We observed inhibition of subtilisin Carlsberg, proteinase
K, and Metarhizium PR1 by some of the serpin-1 variants and
detected SDS-stable complexes between the serpins and these enzymes.
These results are consistent with a more detailed study on the
interaction of human 1-proteinase inhibitor with subtilisin Carlsberg and proteinase K (19). Our study further supports the conclusion that serpins can inhibit proteinases from both serine
proteinase families, presumably by the same mechanism.
M. anisopliae isolate ME1 is a fungal pathogen of M. sexta. To reach the hemocoel and establish mycosis, the fungus releases digestive enzymes to penetrate through the insect cuticle, which is composed of cross-linked proteins, chitin, and lipids. Proteinases released by the fungus appear to be important in this process for invasion and subsequent establishment of infection (17, 18). The observation that some of the M. sexta serpins inhibit Metarhizium PR1, PR2, and other microbial proteinases suggests that some of the insect serpins may be involved in host defense against proteinases produced by pathogenic microorganisms.
Phenoloxidase and its activation are involved in a number of insect physiological processes (28, 29). Phenoloxidase catalyzes reactions that produce quinolic substances that either cross-link proteins and chitins in cuticular sclerotization or polymerize to form melanin. Phenoloxidase is also a part of insect defensive responses to wounding and infection. A serine proteinase cascade is thought to be triggered by recognition of bacterial and fungal cell wall components, activating prophenoloxidase by limited proteolysis. Although the hemolymph proteinases in this cascade have not been purified and characterized yet, they are probably tightly regulated to prevent excess production of cytotoxic quinones by active phenoloxidase. Injection of antibodies to a mixture of the serpin-1 variants into M. sexta larvae to inactivate hemolymph serpins was shown to result in elevated phenoloxidase activity, suggesting that the prophenoloxidase activating enzyme(s) may be regulated by endogenous serpins (23).
We provide evidence here that serpin-1J and -1J(µ), but not the
other 11 serpins encoded by the same gene, can efficiently block
activation of M. sexta prophenoloxidase (Fig. 4). This
result strongly suggests that serpin-1J may be a physiological
inhibitor of one of the serine proteinases in the proposed activating
cascade. Because serpin-1J has Arg at its P1 position, we
predict that its target enzyme in hemolymph may have trypsin-like
specificity. Serpin-1J at 30 µg/ml inhibited prophenoloxidase
activation by 50%. We determined previously that total serpin-1
concentration in M. sexta hemolymph is 0.2-0.6 mg/ml,
depending on developmental stage (30). Assuming that each serpin
accounts for (null)/1;12 of the total concentration, serpin-1J may be
present at a concentration range (17-50 µg/ml) that could
effectively regulate prophenoloxidase activation in hemolymph.
The pathways for invertebrate phenoloxidase activation and hemolymph clotting in horseshoe crabs are analogous in many ways to the complement system and blood coagulation cascade in mammals, which are regulated by serpins. This resemblance might give insights and prospects for the study of invertebrate serpin functions. It may not be unrealistic to predict the existence of many serine proteinases in insect hemolymph. A serine proteinase has been purified from hemolymph of B. mori and found to be inhibited by a serpin, silkworm antitrypsin, isolated from the same insect (20). The function of this proteinase as a component of the prophenoloxidase activating cascade was excluded (20), and thus it may participate in a process that has not yet been discovered. We have recently isolated cDNAs from M. sexta hemocytes, encoding proteins similar to horseshoe crab clotting factors.2 These proteins, whose functions are not yet known, may also be targets for regulation by hemolymph serpins.
We thank Gary Radke for amino acid sequence and molecular mass determinations. We are grateful to Drs. Jeremy Gillespie, Karl Kramer, and Gerald Reeck for helpful suggestions about the manuscript.