JBC Advanced Glycation Endproducts

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M309682200 on September 9, 2003

J. Biol. Chem., Vol. 278, Issue 47, 46556-46564, November 21, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/47/46556    most recent
M309682200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhu, Y.
Right arrow Articles by Kanost, M. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhu, Y.
Right arrow Articles by Kanost, M. R.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Manduca sexta Serpin-3 Regulates Prophenoloxidase Activation in Response to Infection by Inhibiting Prophenoloxidase-activating Proteinases*

Yifei Zhu, Yang Wang{ddagger}, Maureen J. Gorman, Haobo Jiang{ddagger}, and Michael R. Kanost§

From the Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506

Received for publication, September 2, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Many serine proteinase inhibitors of the serpin superfamily have evolved in vertebrates and invertebrates to regulate serine proteinase cascades that mediate the host defense responses. We have isolated an immuneresponsive serpin from the tobacco hornworm, Manduca sexta. This inhibitor, M. sexta serpin-3, contains a reactive site loop strikingly similar to the proteolytic activation site in prophenoloxidase (pro-PO). Molecular cloning and sequence comparison indicate that serpin-3 is orthologous to Drosophila melanogaster serpin 27A, a regulator of melanization. M. sexta serpin-3 is constitutively present in hemolymph at a low concentration of 5–12 µg/ml and increases to 30–75 µg/ml after a microbial challenge. Recombinant serpin-3 efficiently blocks pro-PO activation in the hemolymph, and it forms SDS-stable acyl-enzyme complexes with purified pro-PO-activating proteinases (PAPs) from M. sexta. PAP-serpin-3 complexes were isolated by immunoaffinity chromatography from hemolymph activated by treatment with Micrococcus luteus. Kinetic analysis of PAP-serpin-3 association strongly suggests that serpin-3 is a physiological regulator of the pro-PO activation reaction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis of melanin by insects and other arthropods in response to infection results from a proteolytic cascade triggered by microbial cell wall components (1–3). In this innate immune response, microbes are recognized as foreign when soluble pattern recognition proteins bind to bacterial peptidoglycan or lipopolysaccharide or fungal {beta}-1,3-glucan, initiating activation of a series of proteinases. Phenoloxidase (PO),1 the final enzyme in this cascade, exists in hemolymph as an inactive zymogen, prophenoloxidase (pro-PO), which is activated by a specific proteinase. Active PO catalyzes the hydroxylation of monophenols to o-diphenols and oxidation of o-diphenols to quinones that can polymerize to form melanin. Cytotoxic molecules produced in this process, including quinones and reactive oxygen intermediates, may kill the invading microorganisms that are trapped by melanin (4).

Our understanding of the pro-PO activation cascade is still incomplete, and the number of proteolytic steps in the pathway is unknown. However, serine proteinases that directly activate pro-PO, known as pro-PO activating enzyme (PPAE) or pro-PO-activating proteinase (PAP), have been isolated and cloned from several arthropod species, including the silkworm, Bombyx mori (5, 6), the tobacco hornworm, Manduca sexta (7–9), a beetle, Holotrichia diomphalia (10, 11), and a crayfish, Pacifastacus leniusculus (12, 13). These enzymes are composed of a carboxyl-terminal serine proteinase domain and at least one clip domain at their amino terminus. A specific proteolytic cleavage between the domains activates the proteinase zymogens, but the two resulting chains remain covalently linked by a disulfide bond (14). The activated proteinase cleaves pro-PO at a conserved Arg-Phe bond at approximately residue 50 and leads to its activation (6–13).

Three PAPs from M. sexta have been identified and cloned as cDNAs. PAP-1 contains one clip domain (7), whereas PAP-2 and PAP-3 each have two clip domains (8, 9). PAP-1 and PAP-3 are expressed as zymogens at a low constitutive level in naive larvae. Bacterial challenge induces synthesis of all three of the pro-PAPs, whose concentrations increase dramatically in the hemolymph (7–9). PAP-2 and PAP-3 also accumulate in the hemolymph of prepupae, and PAP-1 is present in the cuticle at the same stage.

The PAPs are synthesized as inactive zymogens and require activation by as yet unidentified proteinases upstream in the pro-PO cascade pathway. Once the PAPs are active, it is likely that they are regulated by specific inhibitors, because the quinones and reactive oxygen species generated by uncontrolled PO activation would be harmful to the host insect (1, 4). Potential candidates for regulators of PAPs are members of the serpin superfamily, which include proteins known to regulate serine proteinase cascades involved in mammalian inflammatory responses, coagulation, complement activation, and fibrinolysis (15, 16). Serpins, present in hemolymph of insects, including M. sexta (17), are proteins of ~45 kDa with a solvent-exposed reactive site loop (RSL) near their carboxyl terminus. In an inhibition reaction, hydrolysis of the P1–P1' peptide bond is accompanied by formation of a stable acyl intermediate, with the P1 residue of the serpin linked to the catalytic Ser residue of the proteinase. The cleaved serpin undergoes a large conformational rearrangement, in which the RSL inserts into a {beta}-sheet in the serpin (16).

In M. sexta, two serpin genes have been identified (18, 19). The serpin-1 gene contains 10 exons, with 12 alternative forms of exon 9. Alternative mRNA splicing generates 12 serpin variants identical in their amino-terminal 336 residues, encoded by the first eight exons, and a variable carboxyl terminus encoded by one of the exon 9 forms (20). Because exon 9 encodes the reactive center region, serpin-1 variants with different reactive site sequences have different inhibitory selectivity (21). The serpin-1 gene is constitutively expressed in fat body at a high level and, to lesser degree, in the granular hemocytes (22). The serpin-1 proteins are secreted into the plasma. M. sexta serpin-2 is expressed in cytoplasm of granular hemocytes in response to bacterial infection, but its function is not yet understood (19). There is also evidence for the existence of additional serpins in M. sexta, which are possibly encoded by different genes and may regulate the cuticular pro-PO activation (23).

Here we report the molecular cloning and analysis of an immune-responsive serpin (serpin-3) from M. sexta. Serpin-3, whose expression is stimulated by microbial infection, was shown to block the pro-PO activation cascade and to inhibit M. sexta prophenoloxidase-activating proteinases.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Insects—M. sexta eggs were originally obtained from Carolina Biological Supply. Larvae were reared as described by Dunn and Drake (24).

Collection of Hemolymph, Hemocytes, and Fat Body from M. sexta Larvae—For determining the induction pattern of serpin-3 protein in plasma, day 2 fifth instar larvae were injected with 10 µl of filter-sterilized saline (0.85% NaCl), 10 µg/µl Micrococcus luteus (Sigma), 2 x 106 cells/µl Escherichia coli, or 5 x 105 cells/µl Saccharomyces cerevisiae. Hemolymph was collected into microcentrifuge tubes at 0, 1, 2, 6, 12, 24, 32, and 48 h after injection by cutting the dorsal horn. A few crystals of 1-phenyl-2-thiourea were added to each tube to prevent hemolymph melanization. Hemocytes were removed by centrifugation at 12,000 x g for 10 min at 4 °C. For total RNA preparation, hemocytes and fat body were collected from larvae at 24 h after injection as described previously (25).

Molecular Cloning and Sequence Analysis—A serpin-3 cDNA fragment, recovered from clone M5 (GenBankTM accession number BI262710 [GenBank] ) (26), was labeled with [{alpha}-32P]dCTP and used as a probe to screen a bacteria-induced M. sexta fat body cDNA library in Uni-Zap XR (Stratagene) (27). The positive plaques were purified and subcloned by in vivo excision. The nucleotide sequence of the longest cDNA insert was determined from both strands using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems). Sequences were edited with IBI PUSTELL programs. The amino acid sequence deduced from the cDNA was used to search the non-redundant protein data base with the BLAST program (28). The most similar sequences were retrieved and aligned using the PILEUP program from the GCG Sequence Analysis software package (29). The signal peptide was predicted using the program of Nielsen et al. (30).

Detection of an Intron in the serpin-3 Gene—M. sexta genomic DNA (0.2 µg) was amplified by PCR in 30 cycles of 95 °C for 30 s, 53 °C for 30 s, and 72 °C for 2 min. The forward primer (5'-TCA AAG GTG TTC CAG AGA AC-3') and reverse primer (5'-ATT GTT TTG CGT GTG GCT TTC-3') were located in the serpin-3 cDNA corresponding to 3' end of exon 8 and the 5' end of exon 10 in the serpin-1 gene, respectively. The PCR product was cloned and sequenced to reveal the sequence of the corresponding region of the serpin-3 gene.

Expression of Recombinant Serpin-3 in E. coli—The serpin-3 cDNA encoding the mature protein from residue 1 to 435 was amplified by PCR using a forward primer (5'-TCT CCC ATG GAC GAC GTC GAC-3') that contains an NcoI site and a reverse primer (5'-TGC GCT GCA GCT CGT TCT AAA T-3') that contains a PstI site. The PCR product digested with NcoI and PstI was purified by agarose gel electrophoresis and cloned into the same sites in H6pQE60 (31). Serpin-3 was expressed in a 1-liter culture of E. coli strain XL1blue carrying the recombinant plasmid. Serpin-3 was expressed in an insoluble form and therefore was purified under denaturing conditions by nickel-affinity chromatography (32). An aliquot of the purified recombinant serpin-3 (1.5 mg) was further resolved by preparative SDS-PAGE and used to produce a polyclonal rabbit antiserum.

Production of Soluble Serpin-3 in a Baculovirus Expression System— The BamHI-XhoI cDNA fragment, containing the complete open reading frame for serpin-3, was inserted into pFastBac1 (Invitrogen). A recombinant baculovirus was generated from the resulting plasmid and amplified according to the manufacturer's instructions. For producing soluble serpin-3, Spodoptera frugiperda Sf9 cells (2x 106 cells/ml) in 250 ml of Sf-900 II serum-free medium were infected with the virus at a multiplicity of infection of 4 and incubated at 27 °C with shaking at 140 rpm for 72 h. The conditioned medium was clarified by centrifugation at 500 x g for 10 min, and the supernatant was placed in dialysis tubing and concentrated to 28 ml by covering the tubing with polyethylene glycol 8000 and then dialyzed twice against 1.0 liter of buffer A (20 mM Tris-HCl, 10 mM NaCl, pH 7.5) overnight at 4 °C. The protein solution was applied to a 12-ml Q-Sepharose column and washed with 60 ml of buffer A. The bound proteins were eluted with linear gradients of 10–220 mM NaCl (150 ml) and then 220–500 mM NaCl (50 ml) in buffer A. Fractions (2 ml each) were analyzed by 10% SDS-PAGE (33) and immunoblotting using serpin-3 antiserum as the first antibody. Concentration of the purified protein was determined by SDS-PAGE analysis along with a series of diluted ovalbumin standards. Intensity of the protein bands was digitized using Kodak Digital Science one-dimensional gel analysis software. The amino-terminal sequence of serpin-3 was determined by automated Edman degradation as described before (7).

Northern Blot Analysis and RT-PCR—Total RNA samples were isolated from hemocytes or fat body using a Rapid Total RNA Isolation kit (Eppendorf-5 Prime, Inc). Northern blot analysis was performed using 20 µg of RNA for each sample (8). The expression profile of serpin-3 was examined by RT-PCR using SuperScript One-step RT-PCR kit and Platinum Taq DNA polymerase (Invitrogen). At 0.5, 1, 2, 4, 6, 8, 12, and 24 h after injection with 100 µg of M. luteus, fat body from four larvae in each group was dissected and pooled for total RNA isolation. One µg of each RNA sample was treated with 2 units of DNase I to remove trace amounts of genomic DNA. Reverse transcription was performed at 45 °C for 30 min, followed by PCR amplification with 20 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 90 s using gene-specific primers for serpin-3 (5'-GAT TCC TCG CGA TTC GAT GC-3' and 5'-CAT TTA CGT CAT TAA GTT TCA TG-3'). Primers for ribosomal protein S3 (5'-CGC GAG TTG ACT TCG GT-3' and 5'-GCC GTT CTT GCC CTG TT-3') were used in the RT-PCR as an internal control. PCR products were analyzed by agarose gel electrophoresis with detection by ethidium bromide staining.

Proteinase Inhibition Assays—M. sexta pro-PAP-1 and pro-PAP-3 produced as recombinant proteins by baculovirus expression (34) contained spontaneously activated PAP-1 and PAP-3, which were separated from the zymogen forms by anion exchange chromatography (data not shown). Concentrations of the active PAPs were determined as described above for serpin-3. The purified PAP-1 or PAP-3, each at 50 nM, was preincubated with serpin-3 at different molar ratios (0.4:1 to 4:1) in 10 µl of buffer B (0.1 M Tris-HCl, pH 8.0, 0.03% bovine serum albumin) for 10 min at room temperature. Residual amidase activity was assayed by adding 190 µl of 50 µM N-acetyl-Ile-Glu-Ala-Arg-p-nitroanilide (IEAR-pNA) in buffer C (50 mM Tris-HCl, pH 7.8, 50 mM NaCl) (8).

Determination of the Association Rate Constants (ka)—The ka value for PAP-1 and serpin-3 was determined under second order conditions according to Beatty et al. (35). Briefly, recombinant PAP-1 and serpin-3, each at 16 nM, were incubated in 18 µl of buffer B at room temperature. At each designated time (t) (15–90 s), 182 µlof50 µM IEARpNa in buffer C was added to determine the residual amidase activity (v). Half-life of the inhibition reaction (t0.5) was determined by plotting 1/v against t, and the ka was calculated according to ka = 1/t0.5x [E0] (35). The ka value for PAP-3 and serpin-3 was also determined under second order conditions with 8.8 nM PAP-3 and 38.6 nM serpin-3. The ka was calculated using the standard Equation 1 (36),

Regulation of Pro-PO Activation in Hemolymph by Serpin-3—A preparation of hemolymph proteins that contains the components of the pro-PO activation cascade (pro-PO activation fraction) was produced as described previously (21). Ten µg of M. luteus in 1-µl sterile saline was added to 100 µl of pro-PO activating fraction (diluted 1:5 in water). At different incubation times (0–60 min), 10-µl aliquots of the reaction mixture were added to 700 µl of phenoloxidase substrate solution (2 mM 3-hydroxytyramine in 50 mM sodium phosphate, pH 6.8) to measure PO activity (37). To test the inhibition of pro-PO activation, serpin-3 at final concentrations of 0–61 µg/ml was added to diluted pro-PO activation fraction (10 µl), along with 1 µg of M. luteus. PO activity was determined after incubation for 10 min at room temperature.

For detecting the formation of serpin-PAP complex, purified serpin-3 was reacted with the purified PAP-1 or PAP-3 in buffer B at a molar ratio of 1:1 or 1:2 (serpin/proteinase). In control samples, diisopropyl fluorophosphate (DFP) at a final concentration of 5 mM was mixed with PAPs prior to the addition of serpin-3. After incubation at room temperature for 10 min, the reaction mixtures were treated with SDS sample buffer at 95 °C for 5 min and resolved by 10% SDS-PAGE. Proteins transferred onto a nitrocellulose membrane were subjected to immunoblot analysis using 1:3000 diluted antisera against PAP-1 (7), PAP-3 (9), or serpin-3 as the first antibody.

Detection of Serpin-PAP Complexes in Hemolymph by Affinity Chromatography—A serpin-3 immunoaffinity column was prepared according to Harlow and Lane (38). Briefly, 2.4 ml of protein A-Sepharose beads (Sigma) were incubated with 4.8 ml of serpin-3 antiserum in 19.2 ml of phosphate-buffered saline (0.8% NaCl, 0.02% KCl, 0.144% NaH2PO4, 0.024% KH2PO4, pH 7.4) for 1 h at room temperature to allow binding of the antibodies to protein A. Following a washing step, covalent coupling of antibodies to protein A was carried out by adding 20 mM dimethylpimelimidate in 0.2 M sodium borate, pH 9.0. After incubation at room temperature for 2 h, the reaction was stopped by incubating the beads with 0.2 M ethanolamine at pH 8.0.

Hemolymph from day 2 fifth instar larvae was collected and centrifuged to remove hemocytes. The plasma was divided into 2 aliquots (2 ml each). DFP was immediately added to 1 aliquot at a final concentration of 5 mM. M. luteus (100 µg) was added to the other aliquot, which was incubated for 5 min before addition of DFP. The two samples were then separately incubated with 0.6 ml of affinity beads in 3.4 ml of phosphate-buffered saline overnight at 4 °C. The resin was packed into two columns and washed with 20 ml of 10 mM sodium phosphate, pH 8.0. The bound proteins were eluted by sequential applications of 0.3 ml of 100 mM glycine-HCl, pH 2.5, with collection of the eluents into 60 µl, 1 M sodium phosphate, pH 8.0. The fractions were subjected to immunoblot analysis as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
cDNA Cloning and Sequence of Serpin-3—From a subtractive M. sexta cDNA library representing genes expressed in larval fat body in response to bacterial challenge (24), we isolated a cDNA clone (M5, GenBankTM accession number BI262710 [GenBank] ) that encoded a partial amino acid sequence highly similar to members of the serpin superfamily. By using the 527-bp cDNA fragment as a probe, we screened a bacteria-induced M. sexta fat body cDNA library, isolated 35 positives clones from 8x 104 plaques screened, and sequenced the two longest cDNA inserts (Fig. 1). The nucleotide sequence of serpin-3A and serpin-3B cDNAs differed slightly, with several substitutions, deletions, and insertions, mostly occurring in the 3'-untranslated region. The serpin-3B cDNA (2498 bp) is 13 bp longer in the 3'-untranslated region than serpin-3A (2485 bp) (data not shown). A polyadenylation signal, AATAAA, was present 24 bases upstream of a poly(A) tail in both cDNAs. The open reading frames of the two serpin-3 cDNAs were identical in both length and their deduced amino acid sequences, because the eight substitutions in the open reading frame were silent. The encoded polypeptide is 435 residues long with a predicted signal peptide comprising the first 20 residues. Four potential N-glycosylation sites are present at Asn38, Asn188, Asn227, and Asn294. The calculated molecular mass of the mature protein, beginning at residue 21, is 48,937 Da, and the calculated isoelectric point is 5.1.



View larger version (95K):
[in this window]
[in a new window]
  		FIG. 1.
Nucleotide and amino acid sequence of M. sexta serpin-3. A, The deduced amino acid sequence is shown below the nucleotide sequence of serpin-3A (GenBankTM accession number AF413064 [GenBank] ). The one-letter codes are aligned with the second nucleotide of each codon. The 20-residue signal peptide is underlined and assigned negative numbers. Four potential N-linked glycosylation sites are marked with * after the Asn residues. The predicted P1 and P1' residues are indicated. A polyadenylation signal (AATAAA, double underlined) is near the 3' end of the cDNA. Two full-length clones were obtained with small differences in nucleotide sequence. Substitutions in the open reading frame of serpin-3B (GenBankTM accession number AF413065 [GenBank] ) are indicated with italic letters above the nucleotide sequence of serpin-3A. B, the nucleotide sequence of an intron (obtained by PCR amplification of genomic DNA) is located between nucleotides 1343 and 1344 of the cDNA (indicated with |). The two underlined nucleotide sequences in A correspond to the primers for PCR amplification of the genomic fragment containing the intron.

 
The M. sexta serpin-1 gene contains 12 alternate forms of exon 9, each of which encodes a different reactive center loop (18, 20). The serpin variants generated by alternative splicing differ in their inhibitory selectivities (21). To test whether the serpin-3 gene might have a similar structure, we used two serpin-3-specific primers corresponding to the 3' end of exon 8 and the 5' end of exon 10 in the serpin-1 gene for PCR amplification of M. sexta genomic DNA (Fig. 1). The sequence of the amplified DNA fragment contained a 339-bp intron between nucleotides 1343 and 1344 of the serpin-3 cDNA sequence. Although this intron is located at the same position as the intron between exon 8 and exon 9A of the serpin-1 gene, the serpin-3 gene does not contain any alternate exons in this region.

Sequence Comparisons—Serpin-3 was most similar in amino acid sequence to a bacteria-induced serpin from another lepidopteran insect, Hyphantria cunea (39), with 60.3% identity. The second most similar sequence was Drosophila serpin 27A (40, 41), which shares 39.6% identity with serpin-3. Serpin-3 is only 29.8% identical to M. sexta serpin-1. A multiple sequence alignment of these insect serpins indicates that M. sexta serpin-3 and H. cunea serpin contain an amino-terminal extension of 45–50 amino acid residues that is absent from M. sexta serpin-1 and most other members of the serpin superfamily (Fig. 2). Drosophila serpin 27A has a shorter amino-terminal extension of ~30 residues. The putative reactive center loop of serpin-3 is from Gly374 to Tyr397, including Asn-Lys-Phe-Gly at the predicted P2 to P2' positions. The same sequence is present in the H. cunea serpin and Drosophila serpin 27A. With a Lys residue as its putative P1 residue, serpin-3 probably inhibits serine proteinases with trypsin-like specificity.



View larger version (70K):
[in this window]
[in a new window]
  		FIG. 2.
Sequence alignment of M. sexta serpin-3 with other insect serpins. The amino acid sequence of M. sexta serpin-3 (Ms spn-3) is aligned with H. cunea serpin (Hc spn), Drosophila melanogaster serpin 27A (Dm spn27A), and M. sexta serpin-1J (Ms spn-1J), excluding the signal peptides. The residues conserved in all four sequences are shaded. The predicted RSL of serpin-3 (P15-P9') is underlined, and the putative P2-P2' site is indicated above the sequence.

 
Purification of Recombinant Serpin-3 Protein from E. coli and Insect Cells—We expressed recombinant serpin-3 with an amino-terminal hexahistidine tag in E. coli and purified it by nickel affinity chromatography under denaturing conditions (Fig. 3A). Whereas the purified serpin-3 was used as an antigen for producing a polyclonal rabbit antiserum, it was inactive in the inhibition assays even after renaturation (data not shown). In order to characterize and study the function of serpin-3, we expressed it in insect cells using a baculovirus system. Serpin-3 was secreted into the cell culture medium by utilizing its own signal peptide. Concentrated cell culture supernatant was separated by anion exchange chromatography on a Q-Sepharose column, and serpin-3 was eluted at ~150 mM NaCl at pH 7.5. The purified serpin-3 from the baculovirus expression system (Fig. 3B) was used for characterization and functional analysis in later experiments. It has an apparent molecular mass of 56.8 kDa as estimated by SDS-PAGE, which is larger than the value calculated from the deduced amino acid sequence (48.9 kDa). This difference may be a result of glycosylation. The first five amino acid residues of the purified serpin-3 were determined to be Asp-Asp-Val-Asp-Pro, confirming the prediction that the signal peptide ends at residue 20 (Fig. 1).



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 3.
SDS-PAGE and immunoblot analysis of purified recombinant serpin-3. A, serpin-3 expressed in E. coli and purified by nickel affinity chromatography. B, serpin-3 produced in the baculovirus-insect cell expression system. The recombinant protein in the cell culture medium was concentrated and purified as described under "Experimental Procedures." Both A and B show Coomassie Blue-stained gels. C, antiserum raised against purified recombinant serpin-3, produced in E. coli, was used to detect the recombinant serpin-3 produced by Sf9 cells (lane 1), serpin-3 in naive hemolymph (lane 2), and in hemolymph (lane 3) collected from larvae at 24 h after injection with E. coli.

 
Serpin-3 mRNA and Protein Expression—In Northern blot analysis, a 2.5-kb serpin-3 mRNA was detected in RNA from the fat body of insects that were challenged by bacterial injection (Fig. 4A). A band at the same position could also be detected in RNA from control fat body and hemocytes after very long autoradiography exposure (data not shown). The time course of serpin-3 mRNA accumulation in response to bacterial challenge was examined by RT-PCR (Fig. 4B). The serpin-3 mRNA level began to increase by 1 h after injection of bacteria, reached a maximum at 6–8 h, and remained elevated to 24 h.



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 4.
Expression and induction pattern of serpin-3 mRNA. A, Northern blot analysis of serpin-3 mRNA expression. Twenty µg of total RNA isolated from fat body (F) or hemocytes (H) at 24 h after injection with saline (C for control) or M. luteus (I for immunized) was separated by electrophoresis on a 1% formaldehyde agarose gel and transferred onto nylon membrane. A serpin-3 cDNA fragment was labeled with 32P to probe the RNA blot. To confirm equal loading, a duplicate blot was probed with 32P-labeled ribosomal protein S3 (rpS3) cDNA. B, RT-PCR analysis of serpin-3 mRNA induction pattern. Day 2, fifth instar larvae were injected with M. luteus or saline. Total RNA was isolated from fat body dissected at different times after the bacterial challenge (0.5–24 h, four insects for each time). In the control (0), total RNA was obtained from fat body at 24 h after saline injection. RT-PCR was performed as described under "Experimental Procedures" using gene-specific primers for serpin-3 and rpS3 as test and internal control, respectively.

 
We examined the induction pattern of serpin-3 protein in hemolymph by immunoblotting (Fig. 5). The constitutive serpin-3 concentration was 5–12 µg/ml in naive larvae. In saline-injected larvae, serpin-3 concentration was maintained at this basal level. The microorganisms tested, including Gram-positive bacteria (M. luteus), Gram-negative bacteria (E. coli), and yeast (S. cerevisiae), stimulated similar changes in serpin-3 concentration. In larvae injected with yeast or E. coli, the serpin-3 protein level decreased during the first 2 h and then increased rapidly, whereas in the M. luteus-injected larvae, the protein level decreased and remained at a low level until 12 h after injection. The immediate decrease in serpin-3 concentration after microbial injection may reflect the use of constitutively expressed serpin-3 before new protein was synthesized to compensate for the loss. Serpin-3 concentration reached a maximum at around 32 h post-infection, with 78 µg/ml for yeasttreated larvae, 54 µg/ml for E. coli-treated larvae, and 29 µg/ml for M. luteus-treated larvae.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 5.
The induction pattern of serpin-3 protein in hemolymph. Day 2, 5th instar larvae were injected with saline, Gram-positive bacteria (M. luteus), Gram-negative bacteria (E. coli), or yeast (S. cerevisiae). Plasma samples were collected at different times after the injection for SDS-PAGE and immunoblot analysis. Serpin-3 concentration was determined by densitometry (32), using known amounts of purified recombinant serpin-3 as a standard. The error bars represent the S.E. of the mean (n = 4).

 
Inhibition of Prophenoloxidase Activation—The pro-PO activation cascade is a proteolytic pathway in insect hemolymph, which is triggered by microbial cell wall components. To investigate the physiological function of serpin-3, we tested its effect on pro-PO activation. The 0–50% ammonium sulfate fraction of M. sexta larval hemolymph, containing all the necessary components for pro-PO activation (21), was activated by incubation with M. luteus. Serpin-3 inhibited the activation of pro-PO by 90% at 61 ng/µl and 50% at 17 ng/µl (Fig. 6). This result indicates that serpin-3 inhibits at least one proteinase of the pro-PO cascade.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 6.
Inhibition of pro-PO activation by serpin-3. A pro-PO activation fraction of M. sexta hemolymph was incubated with purified recombinant serpin-3 at different concentrations (0–61 ng/µl). Activation of pro-PO was then triggered by adding M. luteus to a final concentration of 0.1 µg/µl. PO activity (mean ± S.D., n = 3) was measured in a spectrophotometer after 10 min (37).

 
Inhibition of Prophenoloxidase Activating Proteinases—We tested whether inhibition of PAPs by serpin-3 may explain the observed decrease in pro-PO activation. Recombinant M. sexta PAP-1 or PAP-3 were incubated with recombinant serpin-3 at different enzyme/inhibitor molar ratios. The residual amidase activity decreased linearly as serpin-3 concentration increased, and complete inhibition occurred at a serpin/enzyme ratio of 1.6 for PAP-1 and 1.9 for PAP-3 (Fig. 7). We determined the second order association rate constants (ka) for inhibition of PAP-1 and PAP-3 by serpin-3 as an indicator of inhibitory selectivity. Serpin-3 had a ka of 7.5 x 105 M–1 s–1 for PAP-1 and 6.9 x 105 M–1 s–1 for PAP-3. Thus, serpin-3 inhibits PAP-1 and PAP-3 at rates comparable with those observed for other serpins in mammals and arthropods acting on their physiological target proteinases (Table I).



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 7.
Stoichiometry for inhibition of M. sexta PAP-1 and PAP-3 by serpin-3. Purified recombinant serpin-3 was incubated with recombinant PAP-1 (A) or PAP-3 (B) at various molar ratios (0.4:1 to 4:1) for 10 min at room temperature. The residual amidase activity was then measured using an artificial substrate, IEARpNa (8). The intersection of a line (using the first 5 data points) extrapolated to the x axis occurred at a molar ratio of 1.6:1 for PAP-1 and 1.9:1 for PAP-3.

 


View this table:
[in this window]
[in a new window]
  		TABLE I
Association rate constants of some serpin-proteinase reactions

 
In the inhibition reaction between a serpin and a susceptible proteinase, an inhibitor-enzyme complex was formed that is stable in SDS. Such complexes were detected when we incubated serpin-3 with either PAP-1 or PAP-3 (Fig. 8). The interaction of serpin-3 and PAP-1 resulted in the formation of an ~90-kDa complex under reducing conditions, which was absent from the controls lanes with only PAP-1 or serpin-3. This complex was recognized by both serpin-3 antibody and PAP-1 antibody (Fig. 8, A and B), indicating that it was composed of these two proteins. The combined apparent molecular mass of serpin-3 (residues 1–388) (51.5 kDa) and the PAP-1 catalytic domain (34 kDa) is close to the size of the complex (~90 kDa). When the molar ratio of PAP-1 to serpin-3 was increased from 1:1 to 2:1, more of the serpin-3 was converted to the complex form (Fig. 8B, lanes 2 and 3). A band corresponding to a lower molecular weight protein (51.5 kDa) was also detected by serpin antibody. Because this band was not present in the control of serpin-3 alone, it probably represents the cleaved serpin without the 5.3-kDa carboxyl terminus (residues 389–435). This is consistent with the observation that more PAP-1 led to more of the cleaved form of serpin-3. The complex formation was blocked when an irreversible serine proteinase inhibitor, DFP, was preincubated with PAP-1 before addition of serpin-3 to the reaction mixture (Fig. 8B, lane 4). This result indicates that active PAP-1 is required for forming the complex. PAP-1 is composed of an amino-terminal clip domain and a carboxyl-terminal catalytic domain, which are linked by an interchain disulfide bond (7). Under reducing conditions, the clip domain is separated from the catalytic domain. The intensity of the clip domain band recognized by PAP-1 antibodies was similar in each sample (Fig. 9A), whereas the catalytic domain band became much weaker after reaction with serpin-3, further indicating that linkage between serpin-3 and the active site of the catalytic domain resulted in complex formation. Similar results were obtained for the inhibition of PAP-3 by serpin-3. A complex at ~90 kDa was formed between serpin-3 (51.5 kDa) and the PAP-3 catalytic domain (39 kDa) (Fig. 8, C and D).



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 8.
In vitro complex formation between serpin-3 and PAPs. Purified recombinant serpin-3 was incubated with PAP-1 (A and B) or PAP-3 (C and D) for 10 min at room temperature. The mixtures were subjected to SDS-PAGE under reducing conditions, and the complex was detected by immunoblotting using antisera raised against either PAP (A and C) or serpin-3 (B and D). A, lane 1, PAP-1 only; lane 2, PAP-1 + serpin-3 at 1:1 molar ratio; lane 3, PAP-1 + serpin-3 at 2:1 molar ratio; lane 4, PAP-1 inhibited by preincubation with DFP before addition of serpin-3. B, lane 1, serpin-3 only; lanes 2–4, same as lanes 2–4 in A. C, same as A, except that PAP-1 was replaced by PAP-3. D, same as B, except that serpin-3 was incubated with PAP-3 instead, and the lane for serpin-3 only is not shown. The first antibody used is specified below each panel. Sizes of the molecular mass markers are indicated to the left of each blot.

 



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 9.
Complex formation between serpin-3 and PAPs in plasma. Plasma samples from day 2, fifth instar larvae were incubated with 5 mM DFP immediately or with 100 µg/ml M. luteus for 5 min followed by DFP addition to stop the reaction. The serpin-3-proteinase complexes were isolated by separately passing the two reaction mixtures through serpin-3 antibody affinity columns. The proteins that bound to the column were eluted and subjected to immunoblot analysis using PAP-1 (A), PAP-3 (B), or serpin-3 (C) antibodies. Lane 1, no bacteria added; lane 2, 5 min of incubation with M. luteus before adding DFP. A band recognized by the PAP antisera was found to be protein A that eluted from the immunoaffinity support.

 
Next, we tested whether serpin-3 can inhibit and form a stable complex with the PAPs under more physiological conditions. Hemolymph collected from naive M. sexta larvae was incubated for 5 min with M. luteus to stimulate pro-PO activation. Serpin-3, constitutively present at a basal level, may inhibit the activated PAPs and form serpin-proteinase complexes during this process. After 5 min, the remaining active serine proteinases were then inactivated with 5 mM DFP. These samples were applied to an immunoaffinity column made with serpin-3 antibody to isolate any complex formed (as well as free serpin-3). The proteins that bound to the affinity column were analyzed by immunoblotting using either PAP or serpin-3 antibodies. Immunoblot analysis using PAP-1 antibodies revealed an ~90-kDa band, which is present at a much lower intensity in the control, untreated sample (Fig. 9A). Similarly, PAP-3 antibodies detected a band at about the same size in M. luteus-treated hemolymph, which was hardly detected in the control (Fig. 9B). This result demonstrated that in plasma, the activated PAP-1 and PAP-3 resulting from the bacteria-triggered activation of the pro-PO cascade can be inhibited by serpin-3, with consequent formation of a stable serpin-3-PAP complex. The complex in naive hemolymph may be because of a trace amount of enzyme that was spontaneously activated during hemolymph collection. The same samples were also examined using serpin-3 antibodies, which revealed two bands at the size range of serpin-3-proteinase complexes (Fig. 9C). We have not yet determined from this experiment which band represents which proteinase complex.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we characterized the newly discovered serpin-3 from M. sexta hemolymph. It is constitutively expressed at a low level in fat body and secreted into hemolymph. After injection of bacteria or yeast, the serpin-3 mRNA and protein levels increase dramatically. We found that serpin-3 efficiently inhibits PAP-1 and PAP-3 and forms covalent complexes with the purified enzymes and with endogenous PAP-1 and PAP-3 in plasma. The in vivo half-life of PAP-1 or PAP-3, calculated as 1/ka x [I], is 6–15 s in naive larval hemolymph and 0.9–2.8 s in induced larval hemolymph, suggesting that serpin-3 could rapidly inhibit PAP-1 and PAP-3 under physiological conditions. Taken together, these data provide strong evidence that PAP-1 and PAP-3 are natural targets of serpin-3 and that serpin-3 blocks the pro-PO activation cascade by inhibiting the PAPs specifically and rapidly. Preliminary experiments suggest that PAP-2 may also be regulated by serpin-3.2 Several insect inhibitors that block the pro-PO cascade have been reported (21, 39–41, 46, 47), but the natural proteinase targets of these inhibitors have not been assigned. To our knowledge, this is the first report to provide direct evidence that an insect serpin can block the pro-PO activation cascade by specifically and rapidly inhibiting PAPs from the same species.

It is not surprising that PAP-1 and PAP-3 share a regulator because the enzymes are similar and they cleave the same substrates. They have an overall amino acid sequence identity of 42%, and many key residues are conserved. PAP-2 and PAP-3 cleave the two subunits of pro-PO at Arg51 within the sequence Asn-Arg-Phe-Gly (7, 8, 37, 48). Serpin-3 contains Asn-Lys-Phe-Gly at its predicted P2-P2' position, which is strikingly similar to the pro-PO cleavage site. This sequence similarity may explain the structural basis for the highly specific and efficient binding between the PAPs and serpin-3.

The cleavage site of pro-POs from different insect species and a crayfish are highly conserved with an occasional substitution of (P2) Asn to Ser or Thr and (P2') Gly to Ser (39). Serpins with a P2-P2' sequence similar to the pro-PO activation site, perhaps homologs of serpin-3, may be present in other insect species to regulate pro-PO activation. Indeed, an immune-responsive H. cunea serpin, which is 60% identical to serpin-3, was shown to inhibit pro-PO activation (39). Its target protease was not identified. Drosophila serpin 27A, which is 40% identical to serpin-3 and which has the same P2-P2' residues, is also immune-inducible and inhibits activation of pro-PO (40, 41). M. sexta serpin-3, H. cunea serpin, and Drosophila serpin 27A all have an extended amino-terminal sequence that is not present in most other serpins. Spn27A mutants have higher PO activity than wild type flies; they develop melanized tissues, and they react to injury with uncontrolled melanization (40, 41). The natural target of Spn27A is unknown, but the serpin inhibits a prophenoloxidase-activating enzyme (PPAE) purified from the beetle H. diomphalia, suggesting that Spn27A may inhibit a Drosophila PPAE (40). M. sexta serpin-1J is another serpin that blocks pro-PO activation (21) and forms complexes with PAP-1, -2, and -3 (9).3 The reactive site of serpin-1J is DRCC, which is less similar to the pro-PO cleavage sequence than the serpin-3 P2-P2' sequence. Once kinetic data for serpin-1J inhibition of the PAPs are available, we can compare the inhibitory efficiency and specificity of serpin-1J and serpin-3.

Serpin-3 is an immune-responsive protein. It is constitutively expressed but rapidly up-regulated at the mRNA level after bacterial injection. PAP-1 and PAP-3 are also constitutively expressed at a low level and are immune-inducible (9, 32). The similar induction patterns among serpin-3 and the PAPs are consistent with regulation of the PAPs by serpin-3. In healthy larvae, serpin-3 may function as a scavenger to inactivate any spontaneously activated PAPs to avoid unnecessary melanization and, thus, protect the host from the cytotoxic molecules produced during this process. The small amount of complex formed between serpin-3 and PAP-1 and PAP-3 in naive larvae and the fast kinetics of the PAP-serpin-3 reactions support this hypothesis. In infected larvae, inhibition of the PAPs by serpin-3 may be necessary to prevent systemic melanization. We expect that serpin-3 acts to limit the duration of the melanization reaction and restrict its location to the infection site.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF413064 [GenBank] and AF413065 [GenBank] .

* This work was supported by National Institutes of Health Grants GM41247 (to M. R. K.) and GM58634 (to H. J.). This is contribution 04-022-J from the Kansas Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Dept. of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078. Back

§ To whom correspondence should be addressed: Dept. of Biochemistry, Kansas State University, Manhattan, KS 66506. Tel.: 785-532-6964; Fax: 785-532-7278; E-mail: kanost{at}ksu.edu.

1 The abbreviations used are: PO, phenoloxidase; pro-PO, prophenoloxidase; PAP, prophenoloxidase-activating proteinase; PPAE, prophenoloxidase-activating enzyme; DFP, diisopropyl fluorophosphate; IEARpNa, acetyl-Ile-Glu-Ala-Arg-p-nitroanilide; RT, reverse transcriptase; RSL, reactive site loop. Back

2 H. Jiang and Y. Wang, unpublished results. Back

3 Y. Wang, H. Jiang, and M. R. Kanost, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Gary Radke at the Kansas State University Microchemical Laboratory for amino acid sequencing.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Söderhäll, K., and Cerenius, L. (1998) Curr. Opin. Immunol. 10, 23–28[CrossRef][Medline] [Order article via Infotrieve]
  2. Ashida, M., and Brey, P. T. (1998) Molecular Mechanisms of Immune Responses in Insects, 1st Ed., pp. 135–172, Chapman and Hall Ltd., London
  3. Kanost, M. R., Jiang, H., Wang, Y., Yu, X.-Q., Ma, C. C., and Zhu, Y. (2001) Adv. Exp. Med. Biol. 484, 319–328[Medline] [Order article via Infotrieve]
  4. Nappi, A. J., and Ottaviani, E. (2000) BioEssays 22, 469–480[CrossRef][Medline] [Order article via Infotrieve]
  5. Dohke, K. (1973) Arch. Biochem. Biophys. 157, 210–221[CrossRef][Medline] [Order article via Infotrieve]
  6. Satoh, D., Horii, A., Ochiai, M., and Ashida, M. (1999) J. Biol. Chem. 274, 7441–7453[Abstract/Free Full Text]
  7. Jiang, H., Wang, Y., and Kanost, M. R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12220–12225[Abstract/Free Full Text]
  8. Jiang, H., Wang, Y., Yu, X.-Q., and Kanost, M. R. (2003) J. Biol. Chem. 278, 3552–3561[Abstract/Free Full Text]
  9. Jiang, H., Wang, Y., Yu, X.-Q., Zhu, Y., and Kanost, M. R. (2003) Insect Biochem. Mol. Biol. 33, 1049–1060[CrossRef][Medline] [Order article via Infotrieve]
  10. Lee, S. Y., Kwon, T. H., Hyun, J. H., Choi, J. S., Kawabata, S., Iwanaga, S., and Lee, B. L. (1998) Eur. J. Biochem. 254, 50–57[Medline] [Order article via Infotrieve]
  11. Lee, S. Y., Cho, M. Y., Hyun, J. H., Lee, K. M., Homma, K.-I., Natori, S., Kawabata, S., and Lee, B. L. (1998) Eur. J. Biochem. 257, 615–621[Medline] [Order article via Infotrieve]
  12. Aspán, A., Sturevant, J., Smith, V. J., and Söderhäll, K. (1990) Insect Biochem. Mol. Biol. 20, 709–718
  13. Wang, R., Lee, S. Y., Cerenius, L., and Söderhäll, K. (2001) Eur. J. Biochem. 268, 895–902[Medline] [Order article via Infotrieve]
  14. Jiang, H., and Kanost, M. R. (2000) Insect Biochem. Mol. Biol. 30, 95–105[CrossRef][Medline] [Order article via Infotrieve]
  15. Silverman, G. A., Bird, P. I., Carrell, R. W., Church, F. C., Coughlin, P. B., Gettins, P. G., Irving, J. A., Lomas, D. A., Luke, C. J., Moyer, R. W., Pemberton, P. A., Remold-O'Donnell, E., Salvesen, G. S., Travis, J., and Whisstock, J. C. (2001) J. Biol. Chem. 276, 33293–33296[Free Full Text]
  16. Gettins, P. G. (2002) Chem. Rev. 102, 4751–4804[CrossRef][Medline] [Order article via Infotrieve]
  17. Kanost, M. R. (1999) Dev. Comp. Immunol. 23, 291–301[CrossRef][Medline] [Order article via Infotrieve]
  18. Jiang, H., Wang, Y., and Kanost, M. R. (1994) J. Biol. Chem. 269, 55–58[Abstract/Free Full Text]
  19. Gan, H., Wang, Y., Jiang, H., Mita, K., and Kanost, M. R. (2001) Insect Biochem. Mol. Biol. 31, 887–898[CrossRef][Medline] [Order article via Infotrieve]
  20. Jiang, H., Wang, Y., Huang, Y., Mulnix, A., Kadel, J., Cole, K., and Kanost, M. R. (1996) J. Biol. Chem. 271, 28017–28023[Abstract/Free Full Text]
  21. Jiang, H., and Kanost, M. R. (1997) J. Biol. Chem. 272, 1082–1087[Abstract/Free Full Text]
  22. Kanost, M. R., Prasad, S. V., Huang, Y., and Willott, E. (1995) Insect Biochem. Mol. Biol. 25, 285–291[CrossRef][Medline] [Order article via Infotrieve]
  23. Molnár, K., Borhegyi, H. H., Csikós, G., and Sass, M. (2001) J. Insect Physiol. 47, 657–689
  24. Dunn, P., and Drake D. (1983) J. Invertebr. Pathol. 41, 77–85[CrossRef]
  25. Wang, Y., Willott, E., and Kanost, M. R. (1995) Insect Mol. Biol. 4, 113–123[Medline] [Order article via Infotrieve]
  26. Zhu, Y., Johnson, T., Myers, A., and Kanost, M. R. (2003) Insect Biochem. Mol. Biol. 33, 541–559[CrossRef][Medline] [Order article via Infotrieve]
  27. Sambrook, J., and Russel, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Ed., pp. 2.90–2.100, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  28. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389–3402[Abstract/Free Full Text]
  29. Genetics Computer Group (1991) Program Manual for the GCG Package, Version 7.0, Genetics Computer Group, Madison, WI
  30. Neilsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. (1997) Protein Eng. 10, 1–6[Medline] [Order article via Infotrieve]
  31. Lee, E., Linder, M. E., and Gilman, A. G. (1994) Methods Enzymol. 236, 146–163
  32. Wang, Y., Jiang, H., and Kanost, M. R. (2001) Protein Expression Purif. 23, 328–337[CrossRef][Medline] [Order article via Infotrieve]
  33. Laemmli, U. K. (1970) Nature 227, 680–685[CrossRef][Medline] [Order article via Infotrieve]
  34. Yu, X.-Q., Jiang, H., Wang, Y., and Kanost, M. R. (2003) Insect Biochem. Mol. Biol. 33, 197–208[CrossRef][Medline] [Order article via Infotrieve]
  35. Beatty, K., Bieth, J., and Travis, J. (1980) J. Biol. Chem. 255, 3931–3934[Abstract/Free Full Text]
  36. Ehrlich, H. J., Gebbink, R. K., Keijer, J., Linders, M., Preissner, K. T., and Pannekoek, H. (1990) J. Biol. Chem. 265, 13029–13035[Abstract/Free Full Text]
  37. Hall, M., Scott, T., Sugumaran, M., Söderhäll, K., and Law, H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7764–7768[Abstract/Free Full Text]
  38. Harlow, E., and Lane, D. (1999) Using Antibodies: A Laboratory Manual, pp. 311–344, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  39. Park, D.-S., Shin, S. W., Hong, S.-D., and Park, H.-Y. (2000) Mol. Cell 10, 186–192[CrossRef]
  40. De Gregorio, E., Han, S. J., Lee, W. J., Baek, M. J., Osaki, T., Kawabata, S., Lee, B. L., Iwanaga, S., Lemaitre, B., and Brey, P. T. (2002) Dev. Cell 3, 581–592[CrossRef][Medline] [Order article via Infotrieve]
  41. Ligoxygakis, P., Pelte, N., Ji, C., Leclerc, V., Duvic, B., Belvin, M., Jiang, H., Hoffmann, J. A., and Reichhart, J. M. (2002) EMBO J. 21, 6330–6337[CrossRef][Medline] [Order article via Infotrieve]
  42. Djie, M. Z., Stone, S. R., and Le Bonniec, B. F. (1997) J. Biol. Chem. 272, 16268–16273[Abstract/Free Full Text]
  43. Olson, S. T., Swanson, R., Day, D., Verhamme, I., Kvassman, J., and Shore, J. D. (2001) Biochemistry 40, 11742–11756[CrossRef][Medline] [Order article via Infotrieve]
  44. Hermans, J. M., Jones, R., and Stone, S. R. (1994) Biochemistry 33, 5440–5444[CrossRef][Medline] [Order article via Infotrieve]
  45. Kawabata, S., Muta, T., and Iwanaga, S. (1996) New Directions in Invertebrate Immunology, 1st Ed. (K. Söderhäll, S. Iwanaga, and G. R. Vasta, eds) pp. 255–283, SOS Publications, Fair Haven, NJ
  46. Saul, S. J., and Sugumaran, M. (1986) FEBS Lett. 208, 113–116[CrossRef][Medline] [Order article via Infotrieve]
  47. Boigegrain, R.-A., Mattras, H., Brehelin, M., Paroutaud, P., and Coletti-Previero, M.-A. (1992) Biochem. Biophys. Res. Commun. 189, 790–793[CrossRef][Medline] [Order article via Infotrieve]
  48. Jiang, H., Wang, Y., Ma, C., and Kanost, M. R. (1997) Insect Biochem. Mol. Biol. 27, 835–850[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
Z. Lu, M. H. Beck, Y. Wang, H. Jiang, and M. R. Strand
The Viral Protein Egf1.0 Is a Dual Activity Inhibitor of Prophenoloxidase-activating Proteinases 1 and 3 from Manduca sexta
J. Biol. Chem., August 1, 2008; 283(31): 21325 - 21333.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. J. Gorman, Y. Wang, H. Jiang, and M. R. Kanost
Manduca sexta Hemolymph Proteinase 21 Activates Prophenoloxidase-activating Proteinase 3 in an Insect Innate Immune Response Proteinase Cascade
J. Biol. Chem., April 20, 2007; 282(16): 11742 - 11749.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
K. Michel, C. Suwanchaichinda, I. Morlais, L. Lambrechts, A. Cohuet, P. H. Awono-Ambene, F. Simard, D. Fontenille, M. R. Kanost, and F. C. Kafatos
Increased melanizing activity in Anopheles gambiae does not affect development of Plasmodium falciparum
PNAS, November 7, 2006; 103(45): 16858 - 16863.
[Abstract] [Full Text] [PDF]

Home page