Mamestra configurata serpin-1 homologues: cloning, localization and developmental regulation

Insect Biochemistry and Molecular Biology 33 (2003) 355–369 www.elsevier.com/locate/ibmb

Mamestra configurata serpin-1 homologues: cloning, localization and developmental regulation M. Chamankhah a 1, L. Braun a, S. Visal-Shah a, M. O’Grady a, D. Baldwin a, X. Shi a, S.M. Hemmingsen b, M. Alting-Mees b, D.D. Hegedus a,∗ a b

Agriculture and Agri-Food Canada, Saskatoon, SK Canada National Research Council of Canada, Saskatoon, SK Canada Received 13 September 2002; accepted 10 December 2002

Abstract A screen of a Mamestra configurata (bertha armyworm) midgut cDNA library identified three types of cDNA clones that resemble the Manduca sexta serpin-1 gene family. Two serpins, 1b and 1c, possess a common conserved serpin amino terminal scaffold domain but bear no similarity to any members of the M. sexta gene family within the reactive centre loop. These serpins differ from one another by only two amino acids in the reactive centre loop (S363→P) and serpin signature (M369→T) regions. The other member, denoted serpin-1a, is closely related to the M. sexta serpin-1Z. M. configurata serpins as a group were expressed in all insect developmental stages including eggs, larvae and adult moths. Within larvae, serpin gene expression was restricted to the early to middle instar developmental phase and mainly in the fat body and hemocytes. Stress imposed by starvation strongly induced expression in fat body and to a lesser degree in alimentary organs, nervous system and Malphigian tubules. Conversely, starvation decreased expression in hemocytes. Wounding or inoculation with bacteria did not induce serpin gene transcription but did lead to the formation of higher and lower molecular weight forms, presumably serpin-protease complexes and resultant truncated serpin, respectively. Two dimensional PAGE and western blotting analysis revealed at least 12 distinct serpins consisting primarily of neutral, but also highly acidic and basic isoforms, as well as additional high and low molecular weight immuno-reactive species. Serpins-1b/1c are the more prominent serpin isoforms and are expressed predominantly in the fat body and subsequently exported to the hemolymph as revealed by western blotting and immunolocalization. The serpin-1b/1c isoform was found only as the fully glycosylated species within the hemolymph. Hemolymph protease activity was comprised mostly of serine proteases whose overall activity increased dramatically at the onset of the molt concomitant with a sharp decline in serpin gene expression. Crown copyright  2003 Published by Elsevier Science Ltd. All rights reserved. Keywords: Mamestra configurata; Serine proteinase; Serpin; Molt

1. Introduction Serine proteases are a group of functionally related enzymes, so called because they utilize a serine residue in the active site to guide catalysis (Krem et al., 2000). Enzymes in this class have evolved a wide variety of substrate specificities and specialized biological functions. While often regarded as simple digestive enzymes, they also function as highly specific proteases triggering many essential pathways, such as blood coagulation (Di Corresponding author: Tel.: 306-956-7667; fax: +1-306-956-7247. E-mail address: [email protected] (D.D. Hegedus). 1 Present address: College of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran 91775-1365 ∗

Cera and Cantwell, 2001; Coughlin, 1999) and complement activation (Sim and Laich, 2000). Regulation of these functions is required for homeostasis and is carried out by specialized serine protease inhibitors called serpins. Serpins are irreversible inhibitors of serine proteases that regulate proteolytic activities in both physiological and pathological situations (Janciauskiene, 2001). For example, the serum serpin antithrombin inhibits coagulation whereas plasminogen activator inhibitor I inhibits fibrinolysis (Boswell and Carrell, 1988). However, other serum serpins, such as hormone-binding globulins (corticosteroid-binding globulin and thyroxinbinding globulin) (Pemberton et al., 1988), angiotensinogen (Stein et al., 1989) and ovalbumin (Gettins, 1989), are without any known inhibitory properties.

0965-1748/03/$ - see front matter Crown copyright  2003 Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0965-1748(02)00263-1

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The relationship between serpin structure and function is well understood. Serpins possess a common scaffold domain on which a highly variable and flexible peptide is displayed. This peptide, called the variable loop, is approximately 20 amino acids in length and serves as an ideal substrate for cognate serine proteases. A region of exposed residues within the peptide form the reactive centre loop (RCL) of which the P1-P1⬘ residues, using the nomenclature of Schechter and Berger (1967), are attacked by the protease. As the RCL is inserted into the protease active site, the P1-P1⬘ bond is cleaved by the protease, affixing the P1 residue to the hydroxyl group on the active site serine of the target protease (Huntington and Stein, 2001). After the P1-P1⬘ cleavage occurs, the serpin undergoes a large conformational change arising from the insertion of the reactive center loop in the central b-sheet A. This results in deactivation of the protease by distortion of its structure (Huntington et al., 2000). Numerous protease inhibitors have been isolated from invertebrates (Seemuller et al., 1977, 1980; Sasaki, 1978; Kang and Fuchs, 1980; Armstrong and Quigley, 1985; Suzuki and Natori, 1985; Hergenhahn et al., 1987, 1988; Spycher et al., 1987; Ramesh et al., 1988). Insect serpins have been the focus of recent studies due to their involvement in insect immunity as well as possible functions unrelated to proteinase regulation (Church et al., 1997). Sasaki and Kobayashi (1984), who described the isolation and characterization of two serpins from the hemolymph of the silkworm, Bombyx mori, were the first to report insect serpins similar to human proteins. These serpins are irreversible inhibitors of trypsin and chymotrypsin, respectively, forming SDS stable complexes (Sasaki, 1985; Sasaki et al., 1987). In the tobacco hornworm, Manduca sexta, 12 serpins have been isolated from hemolymph (Kanost et al., 1989; Kanost, 1990; Jiang and Kanost, 1997). All are encoded by a single gene possessing a common scaffold-encoding region that is linked to 12 versions of exon 9, each giving rise to a RCL variant via alternate mRNA splicing. Although each of the twelve exon 9 variants are synthesized in the fat body, the majority of processed transcripts encode exon 9F (Kanost and Jiang, 1997). Of the 12 M. sexta serpins, serpin-1B, also referred to as alaserpin due to the presence of alanine at the P1 position, has been studied in the greatest detail (Kanost et al., 1989). Interestingly, while the alaserpin variant transcript is expressed in the fat body, the protein itself is secreted into the hemolymph. Mamestra configurata (bertha armyworm) is a native lepidopteran of the prairie grasslands (King, 1928) and has become a serious periodic pest of canola and oilseed rapes (Brassica napus and Brassica rapa) on the Canadian prairies (Turnock, 1985; Mason et al., 1998). A common theme in developing alternate pest control strategies has been to disrupt insect midgut physiology and/or

digestive biochemistry. To provide insight into these processes, antisera were produced against proteins associated with the peritrophic matrix and the mixture of proteins secreted into the milieu of the insect lumen. These antisera were used to screen a midgut cDNA library yielding cDNAs encoding digestive proteases, peritrophic matrix (PM) structural proteins, and surprisingly three serpin variants. Here we report the characterization of two closely related serpins, namely serpins-1b and 1c, and discuss their possible involvement in developmental processes related to the molt.

2. Materials and methods 2.1. Insect rearing and isolation of midgut tissues Mamestra configurata larvae were maintained at 21°C±1°C under a 16 h light/8 h dark photoperiod and fed ad libitum on artificial diet (Bucher and Bracken, 1976). Third through fifth instar larvae were used for the experiments described in this paper. Prior to isolation of midgut tissues and peritrophic matrix, the larvae were housed individually and starved for 6–24 h on cellulose (Whatman 3M paper) to eliminate ingested food from the intestinal tract. 2.2. cDNA library construction Midgut tissues were collected after removal of the peritrophic matrix, rinsed with cold Ringer’s solution (153 mM NaCl, 2.68 mM KCl and 1.36 mM CaCl2), quickly frozen in liquid nitrogen and stored at ⫺70°C prior to use. Total midgut RNA was isolated using Trizol reagent (Gibco BRL). Midgut mRNA was isolated using the Oligotex mRNA midi kit (Qiagen Inc), according to the manufacturer’s instructions. The cDNA library was constructed from midgut mRNA using the ZAP-cDNA Gold Cloning Kit (Stratagene), cloned into the EcoRI and XhoI sites and packaged using the Gigapack III Gold packaging extract. The resultant cDNA library was amplified once at 50 000 plaques/15 cm plate in XL1Blue MRF’ E. coli host cells. 2.3. cDNA library screening Screening of the expression library was conducted using a rabbit antisera (see below) in conjunction with the ECL chemiluminescent antibody detection module (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. A total of 6 × 106 plaques were screened, of which 114 positive clones were identified after tertiary screening. The cDNA inserts within the purified immuno-positive phage were excised in vivo as pBluescript SK(⫺) phagemids using the ZAP-cDNA Gigapack Kit protocol (Stratagene).

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2.4. DNA sequencing and sequence analysis Phagemid DNA was isolated using the alkaline lysis method. After restriction enzyme analysis the cDNA inserts were sequenced by automated cycle sequencing using T3 and T7 primers, complementary to the pBluescript SK(⫺) sequences flanking the cDNA inserts. DNA sequence analysis was performed using Lasergene program (DNASTAR, Inc., Madison, WI, USA) and putative homolouges identified in the NCBI database using BLAST. Sequences were aligned using Clustal W.12 (www.accelrys.com/about/gcg.html) and visualized using GeneDoc programs. 2.5. Northern blot analysis of serpin transcripts Total RNA was isolated from eggs, whole larvae, adult moths and various tissues dissected from fifth instar M. configurata larvae. Ten micrograms of total RNA was separated on 1% agarose gels in 10 mM sodium phosphate buffer (pH 6.8) according to the procedure described by Pelle and Murphy (1993) and transferred onto Hybond-XL nylon membranes (Amersham Pharmacia Biotech) in 20×SSC using a positive pressure transfer apparatus (Stratagene) and cross-linked with UV light using a Stratalinker (Stratagene). Blots were prehybridized, hybridized and washed at 60°C according to Church and Gilbert (1984). A DNA probe corresponding to the amino-terminal region common to all serpins was derived from an EcoRI-XbaI fragment of the serpin-1b cDNA. To examine specific serpin transcripts, a 128-bp fragment corresponding to the variable carboxyl terminus of serpin-1b was amplified by PCR using oligonucleotides PMM91-1; 5⬘-CGCGTTTTACATTTCTAG-3⬘ and PMM91-2; 5⬘-CCTAAGAGTAAAGTACTC-3⬘. All probes were labeled with the Random Primers DNA Labeling System (Gibco-BRL). 2.6. Primary midgut cell culture Early fifth instar larvae were starved overnight and surface sterilized prior to dissection. Midguts were dispersed by treatment with dispase (Grade II, Boehringer Mannheim Biochemicals) followed by incubation in Graces’s Insect Medium (Gibco BRL) supplemented with 10% FBS (Gibco BRL) and gentamicin (0.25 µg/ml). Basal lamina, connective tissues and peritrophic matrix were removed and the remaining epithelial cells recovered by centrifugation at 1600 r.p.m. at 4°C for 10 min. The cells were transferred to tissue culture flasks and grown at 27°C in Grace’s with FBS and gentamicin. Aliquots were removed at time 0, 4 h, 24 h and 6 days post-dissection, the cells pelleted, and frozen at ⫺70°C.

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2.7. Expression and purification of serpins The Bac-To-Bac baculovirus expression system (Gibco-BRL) was used to express serpin-1a for use in subsequent antiserum production. The 1.5-kb serpin-1a open reading frame was amplified using Pfu Turbo DNA polymerase (Stratagene) with the M13 forward primer and gene-specific primer Ser16-5⬘; 5⬘-GCGAATTCATGAAGCTCTTCATATGC-3⬘ (EcoRI site is underlined), digested with EcoRI and XhoI and cloned into the same sites of pFastBac-HTa. This plasmid was introduced into DH10Bac cells to allow recombination between the bacmid and donor plasmid. The bacmid DNA was transfected into Sf9 cells using CellFectin (Invitrogen) and virus collected from the supernatant 72 h post-infection. The initial viral stock was amplified by infecting approximately 5 × 106 Sf9 cells, as a monolayer of 80% confluence, with the recombinant baculovirus at a multiplicity of infection of 10. Cells were harvested at 48 h post-infection, washed once with 20 mM Tris-HCl pH 8.0 and resuspended in 5 ml binding buffer (20 mM Tris–HCl pH 7.9, 500 mM NaCl, 5 mM imidazole) containing 1% NP-40. The suspension was inverted several times and then sonicated three times for 10 sec with 1 min intervals on ice. The mixture was centrifuged at 12 000 r.p.m. for 20 min and the supernatant further cleared by passage through a 0.4 µm filter. Serpin-1a was purified using the His쐌Bind Kit (Novagen) according to the manufacturer’s instructions. The purified protein was eluted using an imidazole concentration of 80– 200 mM. To provide protein for glycosylation studies and to examine antiserum specificity the protein-encoding regions of serpin-1a and -1b/-1c cDNAs were amplified and a hexa-histidine amino-terminal tag incorporated using oligonucleotides Serpin5⬘RIHIS; 5⬘-GGGAATTCCATCATCATCATCATCATATGAAGCTCTTCATA-3⬘ and either Serpin1a Xba; 5⬘-GGTCTAGACTAAGACTGGTAGACTCC-3⬘ or Serpin1b/c Xba; 5⬘GGTCTAGACTAAGAGTAAAGTACTCC-3⬘, respectively. The amplified DNA fragments were first cloned into pGEM-T Easy (Promega) and then inserted into pPICZaC (Invitrogen) that had been cleaved with EcoRI and XbaI to generate α-factor/serpin fusions. Pichia pastoris strain KM71 cells were transformed by electroporation and colonies selected on YPDS (1% yeast extract, 2% peptone, 2% dextrose, 1M sorbitol) plates containing 100 mg/ml of Zeocin for 2–3 days. Single colonies were used to inoculate 50 ml of BMGY medium [1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6.0) 1.34% yeast nitrogen base, 0.4 µ g/ml biotin] with 1% glycerol and grown at 30°C until they reached an OD600 of 1.7. To induce serpin expression, the cells were washed with BMMY medium and resuspended 10 ml BMMY with 0.5% methanol and incubated at 30°C, 150 r.p.m. for an additional 24 h. The supernatant was col-

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lected and recombinant histidine-tagged serpins purified using the His쐌Bind Kit (Novagen). Total protein concentration was determined using Bradford reagent (BioRad Laboratories). 2.8. Antisera production Peritrophic matrices were dissected and rinsed several times with cold Ringer’s solution containing a cocktail of protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany) plus 1 mM PMSF. Proteins were extracted in Ringer’s buffer containing 500 mM NaCl, 2.5% SDS, 5% b-mercaptoethanol at 100°C for 5 min with occasional mixing using a vortex. Peritrophic matrix proteins were precipitated with trichloroacetic acid (TCA), 10% final concentration, and the pellet washed with 80% acetone to remove residual TCA. Proteins associated with the midgut lumen were obtained from the rinse eluate and also precipitated with TCA. Antisera were generated by immunizing Flemish Giant Chinchilla Cross rabbits with 100 µg of total protein in Freund’s adjuvant. Rabbits were also immunized with 200 µg of baculovirus-expressed serpin-1a to generate an antiserum common to all serpins, this was denoted anti-SerW. Synthetic 40-mer peptides corresponding to the distinct carboxy-terminal sequences of serpin-1a and serpin-1b were used to generate rabbit antisera specific for each serpin variable domain. Three immunizations were performed at biweekly intervals at which time the serum was collected. Peptides were synthesized at the National Research Council of Canada. Preimmune sera were used as controls for all immunological experiments. 2.9. Electrophoretic analysis of proteins One-dimensional SDS-PAGE was conducted using 10 or 12% acrylamide gels (Laemmli, 1970) and proteins were stained with Coomassie Blue. Two-dimensional gel electrophoresis was performed using IPG strips (BioRad Laboratories). In the first dimension, isoelectric focusing was carried out using pH 3–6 and 5–8 strips and in the second dimension proteins were separated using 10% SDS-PAGE gels in duplicate. One gel was stained with Coomassie Blue and proteins in the duplicate gel transferred to nitrocellulose membranes for western blot analysis (see below). 2.10. Tissue isolation and western blotting After feeding fifth instar larvae on cellulose overnight, different tissues were carefully dissected, rinsed in cold Ringer’s solution and frozen at ⫺80°C prior to use. Hemolymph from a wounded proleg was collected into a microcentrifuge tube containing a few crystals of phenylthiourea (PTU) to prevent melanization. Hemocytes were separated from hemolymph by mild centrifugation

and analyzed separately. To prepare soluble protein extracts, tissues were homogenized in Ringer’s solution containing 500 mM NaCl, centrifuged at 4°C and 5 µg of supernatant protein separated using 10% SDS-PAGE gels. Proteins were electrophoretically transferred onto Nitro Pure membranes (Micron Separations Inc.) in Towbin transfer buffer (Towbin et al., 1979) containing 20% methanol. The membranes were blocked using 3% BSA in TBS-TT buffer (20 mM Tris–HCl (pH 7.5), 500 mM NaCl, 0.05% Tween 20, 0.2% TritonX-100) and treated with primary antiserum at a dilution of 1/7500– 1/20 000 with 3% BSA in TBS-TT overnight at 4°C. After washing three times with TBS-TT, membranes were incubated with goat anti-rabbit IgG (H+L)-HRP conjugates (Bio-Rad Laboratories) at a 1:10 000 dilution in TBS-TT with 10% skim milk powder and developed using the ECL Kit (Amersham Pharmacia Biotech). In experiments to examine the role of serpins in defense responses, larvae were wounded by puncturing the cuticle with a 25 gauge needle or injection of 1 µl of either PBS or Escherichia coli XL1-Blue (1 × 105 cells) using a Hamilton syringe. Hemolymph samples were collected from a proleg at various time points and placed on a crystal of PTU. Prior to analysis the PTU crystals were removed by centrifugation and samples pooled from five individual larvae. 2.11. Deglycosylation of hemolymph and recombinant serpins PNGase F (Bio-Rad Laboratories) was used to remove N-linked oligosaccharides from serpins. 12 µl of hemolymph or 1 µg of purified recombinant serpin were treated with PNGase F at 37°C for 18 h. Proteins were separated on 10% SDS/PAGE gels after deglycosylation and serpins detected using western blot analysis. 2.12. Immunolocalization of serpins 5 µl of fixative (2% paraformaldehyde, 0.1% glutaraldehyde in 0.1 M sodium cacodylate) was injected into third instar larvae. After 30 min, larvae were dissected into four regions (head, anterior, middle, posterior) and placed into the fixative solution for an additional 2.5 h. Tissues were rinsed o/n in 0.1 M cacodylate buffer at 4°C, dehydrated through a graded alcohol series, infiltrated with LR White resin and polymerized under UV light (Sylvania Blacklight blue A448). Thick (1 micron) and ultrathin sections were cut with a diamond knife on a Reichert Ultracut E ultramicrotome. Some 1 micron sections were stained with 1% toluidine blue in a 1% borax solution and photographed on a Nikon Optiphot microscope. Additional 1 micron sections were used for immuno-fluorescence detection of serpins. Ultrathin silver-gold sections were placed on formvar coated nickel grids.

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For immunofluorescence studies, sections were treated with PBS for 20 min followed by formaldehyde solution for 10 min to permeabilize tissues. Blocking was performed with 10% fetal bovine serum in PBS. The primary (anti-SerW or anti-Ser1b) and the secondary antibodies (anti-rabbit IgG conjugated to fluorescein isothiocyanate) were diluted 1:1000 and 1:5000, respectively. For immunogold analysis, grids were first treated with blocking buffer [1% w/v BSA, 0.1% v/v Tween20R, 10 mM Trizma hydrochloride, 10 mM Tris-HCl (pH 7.9), 150 mM NaCl in TBS]. The anti-SerW and anti-Ser1b primary antibodies and the anti-rabbit IgG conjugated to 25-nm gold secondary antibody (Electron Microscopy Science) were diluted 1:500 and 1:50, respectively. The grids were stained with 2% aqueous uranyl acetate followed by Reynold’s lead citrate. Ultrathin sections were viewed using a Phillips 410 LS transmission electron microscope.

2.13. Assay of hemolymph protease activities

Hemolymph protease isoforms were resolved using a modification of the polyacrylamide gel electrophoresis zymographic method described by Visal-Shah et al. (2001). Equivalent amounts of protein derived from hemolµmph (ca. 5–15 µg) were mixed with non-denaturing sample buffer on ice. The samples were separated on a 12% polyacrylamide gel at a constant voltage of 200 V for 1 h. At this time the proteins were transferred to a second 12% polyacrylamide gel containing 0.1% w/v gelatin using a modified western blotting procedure. The protein transfer was carried out using 25 mM Tris, 192 mM glycine buffer (pH 8.3), without methanol, at 60 V for 20 min. Proteins were renatured by incubation in 2.5% Triton X-100 for 20 min at room temperature and then activated in 0.1 M Tris-HCl (pH 8.0), 3 mM MgCl2, 0.1% Triton X-100. Subsequently, the gels were stained using Coomassie Blue and destained in a 40% v/v methanol: 10% v/v acetic acid solution to reveal bands of clearing indicative of proteolytic cleavage of the gelatin substrate. Protease activity was quantitated as follows: A typical 100 µl assay consisted of 5 µl of hemolymph, 75 µl of buffer at varying pH and 20 µl of 5% azoalbumin (Sigma). The buffers used were 0.1 M citrate-phosphate (pH 2.4–6.5), 0.1 M Tris-HCl (pH 7.0–8.0) and 0.1 M glycine-NaOH (pH 8.5–12.0). The reaction was incubated for 24 h at 37°C at which time 300 µl of 10% TCA was added, the preparation mixed thoroughly and then centrifuged at 14 000 r.p.m. for 10 min. The supernatant was transferred to a cuvette containing 300 µl of 1.0 N NaOH and the increase in absorbance at 440 nm due to the release of azo dye recorded.

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3. Results 3.1. Screening of the M. configurata midgut cDNA library and cloning of serpins The initial objective of this work was to clone genes encoding components of the M. configurata digestive system. To this end, a midgut cDNA library was screened with antisera raised against the peritrophic matrix-associated proteins or the midgut lumen contents. We found a single type of cDNA was predominant, representing ca. 80% of all immuno-positive clones. Nucleotide sequence analysis of these cDNAs revealed a common open reading frame of 1288–1291 bp encoding three closely related proteins with molecular masses of 43 kDa and pI values approximately 5.0. All three types of clones encoded serpin proteins, exhibiting 60% identity to the serpin scaffold-encoding region of the M. sexta serpin-1 gene. The 3⬘ untranslated regions and the regions encoding the serpin scaffold and were identical with the exception of minor single base changes attributable to allelic variation. Several significant features were present in the deduced amino acid sequence of the M. configurata serpins (Fig. 1A). These included a 16residue putative signal peptide, a conserved N-glycosylation sequence (NVTK) at amino acid positions 84–87, a potential N-myristoylation site (GAVLND) at positions 116–121, and the conserved serpin signature (PFFYALK) located at residues 373–379. Other recognizable motifs are located within the carboxyl terminal 40 amino acid region and include the reactive center loop (RCL) and the flanking hinge region (Fig. 1A). Only two amino acid differences exist between serpins-1b and 1c, S363→P, and M369→T, and are likely important amino acid substitutions as they reside within the RCL and serpin signature regions, respectively (Fig. 1C). These regions are responsible for the specificity of the protease inhibitory activity and such amino acid substitutions would contribute to significant differences in their respective functions. The sequence of the variable loop in serpin-1a was markedly different from serpins-1b and -1c, most notably in RCL. Serpin-1a displays the highest degree of similarity (73% identity) within the carboxyl terminal region to the M. sexta serpin-1Z (Fig. 1B) and both possess the same P1 residue, Tyr358. Serpin-1a was localized to the midgut lumen and peritrophic matrix resulting in the anti-serpin immune response. Its biological function is likely distinct from that of serpins-1b and -1c and these results will be reported elsewhere. Alignment of all the variable loop regions arising from M. sexta serpin-1 gene with those of serpins-1b and -1c revealed that while the highest degree of similarity was within the serpin signature and the hinge regions, no readily identifiable homologue was present, as was the case for serpin-1a (Fig. 1C).

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Fig. 1. Comparison of M. configurata and M. sexta serpin-1 structures. Panel A: Nucleotide and amino acid sequence of M. configurata serpin1b. The putative signal sequence (residues 1-16) is shown in bold, N-glycosylation site (NVTK) is bold-italic, N-myristylation site (GAVLND) is bold-underlined. The carboxyl-terminal inhibitor region is divided into hinge (underline), reactive centre loop (double underline) and serpin signature (triple underline) regions. The sequences have been deposited in the NCBI database under the following accession numbers; AY148483-148485. Panel B: Alignment of M. configurata serpin-1a carboxyl-terminal inhibitor domain with M. sexta serpin-1Z. The hinge, reactive centre loop and serpin signature regions are indicated. Identical residues are shown in white with black background. The P1 residue (Tyr358) in serpin-1Z denoted by an asterisk (∗). Panel C: Alignment of the carboxyl-terminal variable region of M. configurata serpins-1b and -1c with members of M. sexta serpin-1 family. The S363→P and M369→T changes between serpins-1b and -1c are indicated in bold-italic. P1 residues that have been determined for specific M. sexta serpins are underlined. Putative P1 residues for M. configurata serpins 1b and 1c are also underlined.

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3.2. Generation of common and serpin-specific antisera

3.3. Profile of serpin gene expression

In previous reports on insect serpins, tissue expression and localization studies were performed with DNA probes and antisera that were unable to discriminate the various serpin isoforms. To facilitate experiments to localize individual serpin species, with the aim of uncovering aspects of their biological function, we generated antisera to purified recombinant serpin-1a (Fig. 2A), denoted anti-SerW, and to synthetic peptides corresponding to the serpin-1a (anti-Ser1a) and serpin-1b (anti-Ser1b) variable regions. The antiserum generated against serpin-1a (anti-Ser1a) did not react with serpin1b or -1c (Fig. 2B). Likewise, the serpin-1b specific antiserum (anti-Ser1b) was unable to recognize serpin-1a but did react with both serpin-1b and -1c, whereas the antiSerW reacted with all three recombinant serpins.

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anti-Ser1b anti-Ser1a anti-SerW Fig. 2. Purification of recombinant M. configurata serpins and testing antiserum specifity. Panel A: Sf9 insect cells were infected with recombinant baculovirus carrying the serpin-1a gene and harvested after 48 h. Serpin-1a was bound to a Ni column and eluted using a 20–1000 mM imidazole gradient (indicated) and detected via Western blotting using anti-His-tag antibody. Samples from the initial cell extract (CE) and column flow through (FT) are also shown. Panel B: Western blot analysis of purified recombinant serpins expressed in Pichia pastoris. Blots were probed with antisera against whole serpin-1a (anti-SerW) or inhibitor specific variable regions (anti-Ser1a and anti-Ser1b).

We examined the developmental pattern of serpin expression using a cDNA probe common to all serpin variants. In general, serpins were expressed in all developmental stages including eggs, larvae and adult moths (data not shown). To further this observation, we analyzed the level of serpin transcripts present in various tissues derived from both starved and fed larvae to assess the effect of nutrient deprivation on serpin transcriptional regulation (Fig. 3A). In fed insects, serpin transcripts were detected in the fat body and hemocytes and to a lesser degree in the foregut and hindgut. Upon starvation, serpin gene transcripts were elevated in the fat body and foregut but reduced in the hemocytes. In addition, starvation led to appearance of serpin transcripts in other tissues including hindgut, nervous system and Malphigian tubules. Using a DNA probe specific for the serpin-1b/1c carboxyl terminus, we observed a pattern of expression that closely paralleled that observed for serpins in general; however, this transcript was not detected within hemocytes. The results indicate that serpins-1b/1c are expressed primarily in fat body and represent the major serpin isoforms in the M. configurata tissues analyzed, while other serpins are possibly less abundant. While starvation induced serpin mRNA accumulation in all tissues examined except hemocytes, only a relatively small amount of serpin transcript was detected in midgut. We believe that during construction of the midgut cDNA library, small quantities of other tissues, particularly fat bodies and hemocytes, may have been present leading to the occurrence of serpin cDNAs in the midgut library. To clarify this, we prepared a primary midgut cell culture and examined serpin expression. Under these conditions serpin transcripts could not be detected, although other genes encoding midgut proteins, such as structural proteins associated with peritrophic matrix, were expressed (data not shown). A comparative analysis of serpin protein accumulation in the same isolated tissues was conducted using western blot analysis (Fig. 3B). The anti-SerW antiserum detected serpins in several tissues including foregut, midgut, circulating hemolymph and hemocytes, and fat body, emphasizing their involvement in a variety of biological functions. However, the bulk of the serpin was associated with the hemolymph. The hemolymph also exhibited immuno-reactive species of sizes higher and lower than predicted for serpins. These bands may represent serpin-protease complexes and the resultant truncated serpin, respectively. While serpins in general were observed in many tissues, serpins-1b/1c were found only in hemolymph, suggesting their possible interaction with hemolymph serine proteases. A time course analysis was conducted to examine the role of hemolymph serpins in defense responses to

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Fig. 3. Accumulation of M. configurata serpin transcripts and localization of serpin proteins in various tissues. Panel A: Northern blot analysis of various tissues isolated from fed and starved larvae probed with common and serpins-1b/1c specific DNA probes. Lanes: whole larvae (whole); cuticle-associated remnants after removal of organs (carcass); midgut (MG); fat body (FB); hindgut (HG); foregut (FG); nervous system (NS); Malphigian tubules (MT); hemocytes (HC). Total RNA loading controls for each blot are shown in the lower panels. Panel B: Western blot analysis of tissues isolated from starved larvae and probed with antisera against a serpin common (anti-SerW) or serpin -1b/1c specific (anti-Ser1b) regions. Lanes: foregut (FG); midgut (MG); hindgut (HG); hemolymph (HL); fat body (FB); Malphigian tubules (MT). Molecular weight markers are shown in middle margin.

wounding and bacterial challenge. We found that serpin 1b/c levels increased as larvae aged; however, neither cuticle puncture, nor injection with PBS or bacteria led to an increase in either total hemolymph serpin or serpin1b/1c levels over the course of 72 h (Fig. 4A). When larger amounts of total protein were analyzed low and high molecular weight products reacting with the common serpin antiserum appeared in the hemolymph within 6 h of bacterial injection but not after mechanical wounding (Fig. 4B). The anti-serpin 1b/1c specific antiserum did not recognize these alternate forms in the bacteria-challenged larvae. 3.4. Immunolocalization of serpins Immunolocalization of all serpins and specifically serpins-1b/1c in larvae was conducted by treating fixed 1

micron sections of intact third instar larvae with antiSerW and anti-Ser1b and viewing sections under immunofluorescence. The anti-SerW antiserum detected serpins associated with the sub-cuticular layer, outside the muscle basement membrane, and within fat bodies throughout the anterior region of starved third instar larva (Fig. 5b). In contrast, serpins-1b/1c were localized almost exclusively in fat body and to a lesser degree in some areas of the cuticle (Fig. 5d). A greater level of fluorescence was seen in sections from starved versus fed third instar larvae, reflecting the general increase in serpin-1 gene expression observed earlier (Fig. 5f). For immunolocalization of serpins at the ultrastructural level, ultrathin sections from whole larvae were treated with anti-SerW and anti-Ser1b primary antibodies and anti-rabbit IgG gold-conjugated secondary antibody. In fed larvae, serpins were evident inside fat

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B Fig. 4. Effect of wounding and bacterial challenge on serpin accumulation. Panel A: Western blot analysis of hemolymph from early fourth instar larvae collected at various times (hours; indicated) after puncture with a syringe (wounded) or injection with phosphate buffered saline (PBS) or E. coli. Membranes were probed with either the common serpin (anti-SerW) or serpin-1b/1c specific (anti-Ser1b) antisera. Panel B: Western blot analysis of earlier time points using larger amounts of hemolymph/lane and probed with anti-SerW.

body( Fig. 6a), along the basement membrane surrounding both muscle (Fig. 6b) and midgut, within and along the outer edge of the sub-cuticular tissue (Fig. 6c), and on the outer edge of the tracheal tissue (Fig. 6d). Sections from starved larva treated with anti-Ser1b also had gold particles present in fat body but to a lesser extent than that seen with anti-SerW. In addition, serpins-1b/1c were also detected in an area of the haemocoel presumed to contain coagulated hemocytes/ hemolymph (Fig. 6e) and in tracheal tissue along both the outer edge and adjacent to the tracheal lumen (Fig. 6f). For immunofluorescence and immunogold localizations, corresponding sections treated with pre-bleed serum were used as controls; fluorescent and gold signals were minimal or absent (data not shown). 3.5. Hemolymph-associated serpins To more accurately determine the number of serpins present within the hemolymph, we separated total hemolymph proteins using two-dimensional PAGE followed by western blot analysis with both anti-SerW and antiSer1b antisera (Fig. 7). A cluster of approximately 25 acidic spots with pI values in the range of 3–6 were found to react with the anti-SerW antiserum, twelve of which were approximately 43 kDa molecular weight as calculated for serpin-1 gene products (Fig. 7A). Three of these immuno-reactive spots exhibited highly acidic pIs near 3.0. IPG strips in the pH 5–8 range were used

to further resolve the larger acidic cluster. The anti-SerW anti-serum will recognize the highly conserved serpin scaffold region and would be expected to cross-react with most serpin isoforms. In Manduca sexta 12 serpin isoforms derive from alternate splicing of a single transcript differing only in their respective carboxyl-terminal regions (Jiang and Kanost, 1997). If these also exist in different glycosylation states, as we have shown below, then one could expect an even larger number of crossreactive proteins. As well, higher molecular immunoreactive proteins of ~80 kDa were also detected on both gels (Fig. 7A,B). These may represent complexes of serpins in association with their target serine proteases (Kanost, 1990; Sasaki et al., 1987). Finally, upon interaction of serpin with their target protease, the P1-P1⬘ residues of the reactive center loop are cleaved leading to the formation of a truncated product with an approximate molecular weight of 40 kDa. This is consistent with a third band of proteins observed on the blots. When probed with anti-Ser1b three spots were detected at 43 kDa and could represent the native serpin1b, serpin-1c and the processed/unprocessed forms of either or both (Fig. 7C). As expected, the anti-Ser1b antiserum, specific for only the inhibitory-loop peptide, does not recognize higher and lower molecular weight forms. The anti-Ser1a anti-serum did not recognize any of the hemolymph-associated serpins but rather a serpin present in the midgut lumen, these results will be reported elsewhere.

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Fig. 5. Immunolocalization of serpins in cross sections through the anterior region of third instar larvae. In situ immunofluorescence analysis of fixed tissues using anti-SerW and anti-Ser1b antibody. The corresponding 1 micron stained section is shown immediately to the left. Panels a, b: cross section from a starved larva treated with anti-SerW; c, d: cross section from a fed larva treated with anti-Ser1b; e, f: cross section from a starved larva treated with anti-Ser1b. (왖) denotes sample position in panel 6e and (←) denotes sample position at higher magnification in panel 6f. Muscle (m), fat body (fb), cuticle (c), sub-cuticle (sc), tracheae (t) and Malpighian tubule (Mt) are indicated.

3.6. Glycosylation of hemolymph serpins A potential N-linked glycosylation site (NVTK) was found at position Asn84 in each of the M. configurata serpin types. This sequence is also conserved in all members of the M. sexta serpin-1 family and the serpins were reported to exist in two molecular weight forms in the hemolymph (Kanost and Jiang, 1997). We also observed two bands in SDS-PAGE and western blot analyses of total hemolymph serpins (Fig. 8). In order to confirm that serpins were glycosylated, both hemolymph and recombinant serpins were subjected to treatment with PNGase F to remove N-linked oligosaccharides followed by western blot analysis. Two serpin bands differing by 1–2 kDa are clearly visible in the hemolymph sample when probed with the anti-SerW antiserum, whereas after PNGase F treatment only a single, lower molecular weight, band occurs. The same observation was made for recombinant serpins-1a, -1b and -1c. The anti-Ser-

1b antiserum revealed that serpins-1b/1c associated with the hemolymph were present only as a fully glycosylated forms. While total hemolymph serpins comprised both glycosylated and non-glycosylated forms, individual species, such as serpins-1b/1c, existed in only one configuration; the biological significance of this remains to be determined. 3.7. Implication for the involvement of serpins in molting The serpin-1 gene expression pattern was examined throughout the third to fifth instar cycles including the molt. Serpin transcripts were detected in whole larvae throughout the early and middle instar phase but declined sharply as larvae entered the molt (Fig. 9A). Only after the molt was complete did serpin expression resume. Northern blot analysis with probes specific for serpin-1a and serpin-1b/1c generated the same pattern of

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Fig. 6. Immunogold localization of serpins in cross sections through the anterior region of third instar larvae. Panels a–d are cross sections of starved larva treated with anti-SerW (same insect as in 4A,B); a, fat body; b, muscle; c, cuticle/sub-cuticular regions; d, trachea. Panels e–f are a cross section from a starved larva treated with anti-Ser1b (same insect as in 4E,F); e, area near dorsal heart in hemocoel; f, trachea. Note 25 nm gold particles (circled).

expression as that with the common serpin probe. To determine if the decline in serpin expression was correlated with hemolymph protease activity samples of hemolymph were collected at different time intervals from early, mid and late instars and analyzed using an in-gel gelatin protease assay (Fig. 9B). During the early to middle instar intermolt phase hemolymph protease activity was not detected, however, in late instar larvae a dramatic increase in protease activity was observed correspondent with changes in cuticle structure and appearance and a general decline in serpin gene expression. At least four major, and several minor, protease isoforms were detected that ranged in size from approximately 26–60 kDa. The optimal pH for hemo-

lymph protease activity was found to be 8-10, which is also the optimum for serine proteases, and was completely inhibited by the serine protease inhibitor Pefobloc-SC.

4. Discussion Serpins represent an interesting class of protease inhibitors in that the amino-terminal region is highly conserved and functions as a scaffold on which a varying 30-50 amino acid carboxyl-terminal inhibitor loop is displayed. The serpins that we report in this study appear to be homologues of M. sexta serpin-1. In M. sexta, at

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pH 6.0

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Fig. 7. Two-dimensional gel electrophoresis analysis of M. configurata hemolymph serpins. Hemolymph proteins were subjected to IEF at pH 3–6 and pH 5–8 (indicated) followed by separation on 12% SDS-PAGE and Western blot analysis using either the common serpin anti-SerW (Panels A,B) or serpin-1b/1c specific anti-Ser1b (Panel C) antisera. Arrows denote location of intact serpin (~43 kDa). Immunoreactive proteins of higher (~80 kDa) and lower (~40 kDa) than expected molecular weight are also present.

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Fig. 8. Glycosylation of M. configurata hemolymph serpins. Hemolymph samples (Hemo) or recombinant serpins-1a, -1b and -1c produced in P. pastoris (indicated) were subjected to western blot analysis using either the common serpin (anti-SerW) or serpin-1b/1c specific (anti-Ser1b) antisera prior to (-) and after (+) treatment with PNGase F.

least 12 distinct serpin-1 proteins are derived from mutually exclusive differential splicing of a single mRNA yielding species that differ only in their inhibitory loops, and resultant protease specificity (Jiang et al., 1994; Jiang et al., 1996). This mechanism for generating inhibitor diversity on a single scaffold may be a general mechanism in Lepidoptera since all of the M. configurata serpins also possessed identical scaffold-encoding and 3⬘ untranslated regions. However, specific inhibitory domains appear to have undergone major alterations suggesting that their cognate protease targets have evolved as well. Using two polyclonal antisera Molnar and co-

Fig. 9. Analysis of serpin-1 gene expression and hemolymph protease activity during larval development. Panel A: Developmental Northern blot analysis of early-mid third instar larvae (inter-molt L3) at various time points post-molt (h; indicated), molting larvae (M) and middle fourth (L4) and fifth (L5) instar larvae using the common serpin-1 gene probe. Total RNA loading control is shown in the lower panel. Panel B: Hemolymph protease profile in middle instar larvae (inter-molt) and larvae immediately prior to molting (pre-molt). Dark bands represent protease activities as visualized on negative of Coomassie-stained gelatin gel overlay. Molecular weight markers are shown in upper margin.

workers (2001) reported that various tissues of M. sexta exhibited differential labeling patterns suggesting that a second serpin gene may also be present in M. sexta; this was subsequently corroborated by the discovery of a hemocyte specific serpin-2 gene (Gan et al., 2001). While the serpin-2 gene products are intracellular and are believed to function in defense responses (Gan et al., 2001), the serpin-1 products are secreted from a variety of tissues and redistributed to distant sites where their activities are required. The scissile bond and protease specificity for many of the serpin-1 variants have been identified, however, the precise in vivo target has been identified for only serpin-1J. This serpin is involved in the regulation of the phenoloxidase pathway, a key insect defense response to infecting agents, by targeting the prophenoloxidase-activating serine protease (Jiang and Kanost, 1997). Eight serine proteases have been identified amongst M. sexta hemocyte and fat body cDNAs, including two prophenoloxidase activating enzymes (Jiang et al., 1999; Kanost et al., 2001) and are probable targets for serpin regulation. In the horseshoe crab, serpins prevent rampant, delocalized hemolymph clotting by inhibiting the protease cascade leading to clotting factor activation (Argarwala et al., 1996). The tight negative regulation of anti-fungal peptide expression is also believed to be mediated by serpins (Levashina et al., 1999). We have shown that wounding or challenge with bacteria did not lead to an increase in serpin-1 transcription in any of the tissues examined, but

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did result in the formation of higher molecular weight species indicative of a serpin-protease complex. The specific serpin variants forming these complexes remain to be identified since serpins-1a, -1b and -1c did not exhibit this behavior under such conditions. It has become apparent that the spatial distribution of specific serpin variants is crucial to their function in the immune response and also in other processes such as insect development. In a cytokinin-dependant mechanism analogous to that of anti-fungal peptide expression, serpins coordinate the establishment of an activated spa¨ tzle gradient that in turn governs dorsalventral patterning during Drosophila embryogenesis (Smith et al., 1995; Levashina et al., 1999). In M. configurata the serpin-1 gene is expressed primarily in the fat body, with the bulk of the product being deposited in the hemolymph. As larvae enter the molting phase, hemolymph serine protease activity rises sharply concomitant with a dramatic decrease in serpin expression. During the larval growth phase 20-hydroxyecdysone peaks denote the end of each intermolt period; the outcome of which is determined by the presence (molt to larvae) or absence (metamorphosis) of juvenile hormone (Hiruma et al., 1999). In M. sexta an abrupt decline in serpin-1 gene expression occurs during the intermolt period and in response to injection of 20-hydroxyecdysone (Kanost et al., 1995). Serpins possess a highly stable architecture (Luo et al., 1999) and thus would be expected to turn over slowly. Shirai and co-workers (2000) have reported that after the onset of spinning in B. mori the fat body reabsorbs serpins, thus explaining the rapid disappearance of hemolymph serpins at this time. Interestingly, we found that while larvae cease to feed just prior to undergoing molting or metamorphosis, starvation did not mimic the reduction in serpin-1 gene expression but rather a general increase in most tissues. This may be a protective mechanism to prevent premature tissue destruction during non-feeding periods that arise due to lack of a suitable food source or illness. Our studies show that the fat body is a major site of serpin gene expression, but that serpins become associated with the sub-cuticular layer and the basement membrane of tissues such as muscle, midgut, Malphigian tubules and tracheae. Serpin-1b/1c isoforms were also found to penetrate the tracheal basal membrane and localize nearer the lumen. These results suggest that serpins may be intimately involved in the regulation of destructive processes involved in cuticle turnover during the molt and possibly in other developmental phenomena. For example, the transient destruction and reformation of the basal membrane is now thought to be a general process requisite for tissue remodeling (FujiiTaira et al., 2000). Collagen IV, present within the D. melanogaster basal membrane, undergoes site-specific cleavage during the ecdysone-induced imaginal disk eversion, however, the precise proteolytic activity per-

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forming this function has not been identified (Fessler et al., 1993). Other immunocytochemical studies have shown that M. sexta serpin-1B, formerly alaserpin, is associated with neurite bundles and the receptor cell layer undergoing morphogenesis to antennal olfactory neurons (Hanneman and Kanost, 1992). We observed as many as 12 43 kDa serpins in M. configurata hemolymph representing both glycosylated and non-glycosylated forms; specifically, serpins-1b and -1c were present in only the fully-glycosylated state. While the glycosylation state does not appear to affect the kinetics of protease inhibition (Nash et al., 2000), this may reflect the pathways by which they are exported from the cell. A non-glycosylated form of plasminogen activator inhibitor 2 was secreted via a pathway independent of the endoplasmic reticulum and Golgi apparatus (Ritchie and Booth, 1998). Thus, it is possible that the two glycosylation states relate to a differential sorting process for serpins analogous to the growth factordependent secretion of the mammalian lysosomal cysteine protease (MEP). MEP is mono-glycosylated and exhibits low affinity for the mannose-6-phosphate receptor that is responsible for lysosomal targeting by the endoplasmic reticulum (Dong and Sahagian, 1990). In response to growth hormone, levels of the mannose-6phosphate receptor decline until the concentration becomes limiting leading to the selective secretion of MEP (Prence et al., 1990). The effect of insect hormone levels, such as juvenile hormone and ecdysone, on mannose-6-phosphate receptor distribution or glycosylation levels has yet to be examined. Clearly, much remains to be discovered with respect to the roles that individual serpins play in disparate processes such as insect immunity, embryogenesis and morphogenesis. Serpins appear to be intimately involved in regulating a wide variety of cellular processes and as such their activity is controlled at multiple genetic, biochemical and biophysical levels. Key to this understanding will be the identification of specific protease targets. Major insect genomics initiatives in B. mori, mosquito and Drosophila (www.hgmp.mrc.ac.uk/GenomeWeb/ insect-gen-db.html) and allied projects will provide ample opportunities for such discoveries.

Acknowledgements This research was supported by a strategic grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada and the Federal Matching Investments Initiative. We wish to thank Sarah Caldwell for the excellent electron microscopy, Mina Faghih for technical assistance and Martin Erlandson for critical review of the manuscript.

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