Effect of Ancylostoma ceylanicum hookworm platelet inhibitor on platelet adhesion and peripheral blood mononuclear cell proliferation.

Ancylostoma ceylanicum is a zoonotic parasitic nematode that can cause iron-deficiency anemia and malnutrition in humans. A. ceylanicum hookworm platelet inhibitor (Ace-HPI) can inhibit platelet aggregation in the host to facilitate blood sucking, but whether it possesses platelet adhesion inhibitory activity or immunomodulatory role is yet unknown. To explore the effect of Ace-HPI on platelet adhesion, we expressed the recombinant protein in two competent cells, BL21 (DE3) and Rosetta-gami2 (DE3), and incubated this protein with canine platelets in a 96-well microplate. Ace-HPI was used to stimulate peripheral blood mononuclear cells (PBMC) in vitro to investigate the effect on PBMC proliferation and cytokine expression. Results showed that Ace-HPI expressed in Rosetta-gami2 (DE3) strain was mostly soluble. The inhibitory effect of this protein on platelet adhesion was relatively weak (7-8%). This protein stimulated the proliferation of PBMC and promoted the expression of Treg and Th2 cytokines, such as IL-10 and IL-13. These results lay a foundation for exploring the role of Ace-HPI in hookworm disease pathogenesis and as a candidate molecule for hookworm vaccines.

Hookworm-Derived Metabolites Suppress Pathology in a Mouse Model of Colitis and Inhibit Secretion of Key Inflammatory Cytokines in Primary Human Leukocytes.

Iatrogenic hookworm therapy shows promise for treating disorders that result from a dysregulated immune system, including inflammatory bowel disease (IBD). Using a murine model of trinitrobenzenesulfonic acid-induced colitis and human peripheral blood mononuclear cells, we demonstrated that low-molecular-weight metabolites derived from both somatic extracts (LMWM-SE) and excretory-secretory products (LMWM-ESP) of the hookworm, Ancylostoma caninum, display anti-inflammatory properties. Administration to mice of LMWM-ESP as well as sequentially extracted fractions of LMWM-SE using both methanol (SE-MeOH) and hexane-dichloromethane-acetonitrile (SE-HDA) resulted in significant protection against T cell-mediated immunopathology, clinical signs of colitis, and impaired histological colon architecture. To assess bioactivity in human cells, we stimulated primary human leukocytes with lipopolysaccharide in the presence of hookworm extracts and showed that SE-HDA suppressed ex vivo production of inflammatory cytokines. Gas chromatography-mass spectrometry (MS) and liquid chromatography-MS analyses revealed the presence of 46 polar metabolites, 22 fatty acids, and five short-chain fatty acids (SCFAs) in the LMWM-SE fraction and 29 polar metabolites, 13 fatty acids, and six SCFAs in the LMWM-ESP fraction. Several of these small metabolites, notably the SCFAs, have been previously reported to have anti-inflammatory properties in various disease settings, including IBD. This is the first report showing that hookworms secrete small molecules with both ex vivo and in vivo anti-inflammatory bioactivity, and this warrants further exploration as a novel approach to the development of anti-inflammatory drugs inspired by coevolution of gut-dwelling hookworms with their vertebrate hosts.

Changes in protein expression after treatment with Ancylostoma caninum excretory/secretory products in a mouse model of colitis.

Different reports have highlighted the potential use of helminths and their secretions in the treatment of inflammatory bowel disease (IBD) conditions; however, no reports have investigated their effects at a proteome level. Herein, we characterise the protein expression changes that occur in lamina propria (LP) and the intestinal epithelial cells (IEC) of mice with dextran sulfate sodium (DSS)-induced colitis treated with Ancylostoma caninum excretory/secretory (ES) products using a quantitative proteomic approach. We have shown how parasite products can significantly alter the expression of proteins involved in immune responses, cell death and with an antioxidant activity. Interestingly, significant changes in the expression levels of different mucins were observed in this study. MUC13, a mucin implicated in gastrointestinal homeostasis, was upregulated in the LP of mice with DSS-induced colitis treated with ES, while MUC2, a major component of mucus, was upregulated in the IEC. In addition, A. caninum proteins have an important effect on proteins with antioxidant functions and proteins involved in intestinal homeostasis and tissue integrity and regeneration. Understanding how parasites can ameliorate IBD pathogenesis can help us design novel treatments for autoimmune diseases.

Phenolic Metabolites of Dalea ornata Affect Both Survival and Motility of the Human Pathogenic Hookworm Ancylostoma ceylanicum.

Hookworms are ubiquitous human parasites, infecting nearly one billion people worldwide, and are the leading cause of anemia and malnutrition in resource-limited countries. Current drug treatments rely on the benzimidazole derivatives albendazole and mebendazole, but there is emerging resistance to these drugs. As part of a larger screening effort, using a hamster-based ex vivo assay, anthelmintic activity toward Ancylostoma ceylanicum was observed in the crude extract of aerial parts of Dalea ornata. These studies have led to the isolation and characterization of phenolic metabolites 1-10. The structures were determined by 1D and 2D NMR spectroscopy, and the absolute configuration of 1 was assigned using electronic circular dichroism data. The new compound, (2S)-8-(3-methylbut-2-en-1-yl)-6,7,4'-trihydroxyflavanone (1), was weakly active at 7.3 µM, with 17% reduction in survival of the hookworms after 5 days. The rotenoids deguelin (9) and tephrosin (10), predictably perhaps, were the most active, with complete worm mortality observed by day 4 (or earlier) at 6.3 and 6.0 µM, respectively. The effects of 1-10 on hookworm motility and on toxicity to hamster splenocytes were also explored as important measures of treatment potential.

Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting.

Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbß3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbß3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

The structure of hookworm platelet inhibitor (HPI), a CAP superfamily member from Ancylostoma caninum.

Secreted protein components of hookworm species include a number of representatives of the cysteine-rich/antigen 5/pathogenesis-related 1 (CAP) protein family known as Ancylostoma-secreted proteins (ASPs). Some of these have been considered as candidate antigens for the development of vaccines against hookworms. The functions of most CAP superfamily members are poorly understood, but one form, the hookworm platelet inhibitor (HPI), has been isolated as a putative antagonist of the platelet integrins αIIbß3 and α2ß1. Here, the crystal structure of HPI is described and its structural features are examined in relation to its possible function. The HPI structure is similar to those of other ASPs and shows incomplete conservation of the sequence motifs CAP1 and CAP2 that are considered to be diagnostic of CAP superfamily members. The asymmetric unit of the HPI crystal contains a dimer with an extensive interaction interface, but chromatographic measurements indicate that it is primarily monomeric in solution. In the dimeric structure, the putative active-site cleft areas from both monomers are united into a single negatively charged depression. A potential Lys-Gly-Asp disintegrin-like motif was identified in the sequence of HPI, but is not positioned at the apex of a tight turn, making it unlikely that it interacts with the integrin. Recombinant HPI produced in Escherichia coli was found not to inhibit the adhesion of human platelets to collagen or fibrinogen, despite having a native structure as shown by X-ray diffraction. This result corroborates previous analyses of recombinant HPI and suggests that it might require post-translational modification or have a different biological function.

Peroxiredoxin-1 from the human hookworm Ancylostoma ceylanicum forms a stable oxidized decamer and is covalently inhibited by conoidin A.

Hookworms are parasitic nematodes that have a devastating impact on global health, particularly in developing countries. We report a biochemical and structural analysis of a peroxiredoxin from the hookworm Ancylostoma ceylanicum, AcePrx-1. Peroxiredoxins provide antioxidant protection and act as signaling molecules and chaperones. AcePrx-1 is expressed in adult hookworms and can be inactivated by 2,3-bis(bromomethyl)quinoxaline-1,4-dioxide (conoidin A). Conoidin A inactivates AcePrx-1 by alkylating or crosslinking the catalytic cysteines, while maintaining the enzyme in the "locally unfolded" conformation. Irreversible oxidation of the resolving cysteine may contribute additional inhibitory activity. A crystal structure of oxidized AcePrx-1 reveals a disulfide-linked decamer. A helix macrodipole near the active site increases the reactivity of the catalytic cysteines to conoidin A. This work demonstrates the promise of conoidin compounds as probes to evaluate peroxiredoxins as drug targets in human parasites.

Structural conservation of ligand binding reveals a bile acid-like signaling pathway in nematodes.

Bile acid-like molecules named dafachronic acids (DAs) control the dauer formation program in Caenorhabditis elegans through the nuclear receptor DAF-12. This mechanism is conserved in parasitic nematodes to regulate their dauer-like infective larval stage, and as such, the DAF-12 ligand binding domain has been identified as an important therapeutic target in human parasitic hookworm species that infect more than 600 million people worldwide. Here, we report two x-ray crystal structures of the hookworm Ancylostoma ceylanicum DAF-12 ligand binding domain in complex with DA and cholestenoic acid (a bile acid-like metabolite), respectively. Structure analysis and functional studies reveal key residues responsible for species-specific ligand responses of DAF-12. Furthermore, DA binds to DAF-12 mechanistically and is structurally similar to bile acids binding to the mammalian bile acid receptor farnesoid X receptor. Activation of DAF-12 by cholestenoic acid and the cholestenoic acid complex structure suggest that bile acid-like signaling pathways have been conserved in nematodes and mammals. Together, these results reveal the molecular mechanism for the interplay between parasite and host, provide a structural framework for DAF-12 as a promising target in treating nematode parasitism, and provide insight into the evolution of gut parasite hormone-signaling pathways.

Ac-AP-12, a novel factor Xa anticoagulant peptide from the esophageal glands of adult Ancylostoma caninum.

Immunoscreening an Ancylostoma caninum cDNA library with canine hookworm-infected dog serum resulted in the isolation of a 461 bp cDNA encoding Ac-AP-12, a new 9.1 kDa anticoagulant peptide (100 amino acids) with 43-69% amino acid homology to other nematode anticoagulant peptides (NAPs) from Ancylostoma hookworms. Messenger RNA transcription and expression of Ac-AP-12 was unique to the adult stage of A. caninum. The yeast expressed recombinant Ac-AP-12 demonstrated potent anticoagulant activity on human blood plasma in a concentration dependent manner, and was shown to specifically inhibit human factor Xa activity. Immunolocalization with specific rabbit antiserum showed that Ac-AP-12 was exclusively located in the esophageal glands of adult hookworm. Ac-AP-12 is hypothesized to facilitate both parasite blood feeding and digestion.

Characterisation of hookworm heat shock factor binding protein (HSB-1) during heat shock and larval activation.

When hookworm infective L3s infect their mammalian host, they undergo a temperature shift from that of the ambient environment to that of their endothermic host. Additionally, L3s living in the environment can be exposed to temperature extremes associated with weather fluctuations. The heat shock response (HSR) is a conserved response to heat shock and other stress that involves the expression of protective heat shock proteins (HSPs). The HSR is controlled by heat shock factor-1 (HSF-1), a conserved transcription factor that binds to a heat shock element in the promoter of HSPs, causing their expression. HSF-1 is negatively regulated in part by a HSF binding protein (HSB-1) that binds to and removes HSF-1 trimers bound to HSP gene promoters, resulting in attenuation of the HSR. Herein we describe an HSB-1 orthologue, Ac-HSB-1, from the hookworm Ancylostoma caninum. The Ac-hsb-1 cDNA encodes a 79 amino acid protein that is 71% identical to the Caenorhabditis elegans HSB-1, and is predicted to share the characteristic coiled-coil structural motif comprised of two interacting alpha helices. Recombinant Ac-HSB-1 immunoprecipitated Ce-HSF-1 expressed in mammalian cells that had been heat shocked for 1h at 42°C, but not from cells incubated at 37°C, indicating that HSB-1 only bound to the active DNA binding form of HSF-1. Expression of Ac-hsb-1 transcripts decreased following 1h of heat shock, but increased when L3s were incubated at 37°C for 1h. Activation of hookworm L3s induces a five-sixfold increase in Ac-hsb-1 expression that peaks at 12h, coincident with L3 feeding, but that subsequently decreases to two-threefold above control at 24h. Recombinant Ac-HSB-1 immunoprecipitates greater amounts of 70 and 40kDa proteins from extracts of activated L3s than from non-activated L3s. We propose that an increase in Ac-hsb-1 levels early in activation allows feeding to resume, but that a subsequent decrease in expression permits a HSR that protects non-developing L3s at host-like temperatures. Further investigations of the HSR will clarify the role of HSB-1 and HSF-1 in hookworm infection.

An anticoagulant peptide from the human hookworm, Ancylostoma duodenale that inhibits coagulation factors Xa and XIa.

A full-length cDNA encoding an anticoagulant peptide, named AduNAP4, was cloned and identified from the human hookworm Ancylostoma duodenale. AduNAP4 has 104 amino acids including a predicted 23-residue signal peptide and shows

Proteomics analysis of the excretory/secretory component of the blood-feeding stage of the hookworm, Ancylostoma caninum.

Hookworms are blood-feeding intestinal parasites of mammalian hosts and are one of the major human ailments affecting approximately 600 million people worldwide. These parasites form an intimate association with the host and are able to avoid vigorous immune responses in many ways including skewing of the response phenotype to promote parasite survival and longevity. The primary interface between the parasite and the host is the excretory/secretory component, a complex mixture of proteins, carbohydrates, and lipids secreted from the surface or oral openings of the parasite. The composition of this complex mixture is for the most part unknown but is likely to contain proteins important for the parasitic lifestyle and hence suitable as drug or vaccine targets. Using a strategy combining the traditional technology of one-dimensional SDS-PAGE and the newer fractionation technology of OFFGEL electrophoresis we identified 105 proteins from the excretory/secretory products of the blood-feeding stage of the dog hookworm, Ancylostoma caninum. Highly represented among the identified proteins were lectins, including three C-type lectins and three beta-galactoside-specific S-type galectins, as well as a number of proteases belonging to the three major classes found in nematodes, aspartic, cysteine, and metalloproteases. Interestingly 28% of the identified proteins were homologous to activation-associated secreted proteins, a family of cysteine-rich secreted proteins belonging to the sterol carrier protein/Tpx-1/Ag5/PR-1/Sc-7 (TAPS) superfamily. Thirty-four of these proteins were identified suggesting an important role in host-parasite interactions. Other protein families identified included hyaluronidases, lysozyme-like proteins, and transthyretin-like proteins. This work identified a suite of proteins important for the parasitic lifestyle and provides new insight into the biology of hookworm infection.

Active and exo-site inhibition of human factor Xa: structure of des-Gla factor Xa inhibited by NAP5, a potent nematode anticoagulant protein from Ancylostoma caninum.

Hookworms are hematophagous nematodes capable of growth, development and subsistence in living host systems such as humans and other mammals. Approximately one billion, or one in six, people worldwide are infected by hookworms causing gastrointestinal blood loss and iron deficiency anemia. The hematophagous hookworm Ancylostoma caninum produces a family of small, disulfide-linked protein anticoagulants (75-84 amino acid residues). One of these nematode anticoagulant proteins, NAP5, inhibits the amidolytic activity of factor Xa (fXa) with K(i)=43 pM, and is the most potent natural fXa inhibitor identified thus far. The crystal structure of NAP5 bound at the active site of gamma-carboxyglutamic acid domainless factor Xa (des-fXa) has been determined at 3.1 A resolution, which indicates that Asp189 (fXa, S1 subsite) binds to Arg40 (NAP5, P1 site) in a mode similar to that of the BPTI/trypsin interaction. However, the hydroxyl group of Ser39 of NAP5 additionally forms a hydrogen bond (2.5 A) with His57 NE2 of the catalytic triad, replacing the hydrogen bond of Ser195 OG to the latter in the native structure, resulting in an interaction that has not been observed before. Furthermore, the C-terminal extension of NAP5 surprisingly interacts with the fXa exosite of a symmetry-equivalent molecule forming a short intermolecular beta-strand as observed in the structure of the NAPc2/fXa complex. This indicates that NAP5 can bind to fXa at the active site, or the exosite, and to fX at the exosite. However, unlike NAPc2, NAP5 does not inhibit fVIIa of the fVIIa/TF complex.

Molecular cloning and characterization of a C-type lectin from Ancylostoma ceylanicum: evidence for a role in hookworm reproductive physiology.

Lectins comprise a family of related proteins that mediate essential cell functions through binding to carbohydrates. Within this protein family, C-type lectins are defined by the requirement of calcium for optimal biologic activity. Using reverse transcription PCR, a cDNA corresponding to a putative C-type lectin has been amplified from the hookworm parasite Ancylostoma ceylanicum. The 550 nucleotide open reading frame of the A. ceylanicum C-type Lectin-1 (AceCTL-1) cDNA corresponds to a 167 amino acid mature protein (18,706 Da) preceded by a 17 amino acid secretory signal sequence. The recombinant protein (rAceCTL-1) was expressed in Drosophila S2 cells and purified using a combination of affinity chromatography and reverse phase HPLC. Using in vitro carbohydrate binding studies, it was determined that rAceCTL-1 binds N-acetyl-d-glucosamine, a common component of eukaryotic egg cell membranes. Using a polyclonal IgG raised against the recombinant protein, the native AceCTL-1 was identified in sperm and soluble protein extracts of adult male A. ceylanicum by immunoblot. Probing of adult hookworm sections with the polyclonal IgG demonstrated localization to the testes in males, as well as the spermatheca and developing embryos in females, consistent with its role as a sperm protein. Together, these data strongly suggest that AceCTL-1 is a male gender-specific C-type lectin with a function in hookworm reproductive physiology.

Intermolecular interactions and characterization of the novel factor Xa exosite involved in macromolecular recognition and inhibition: crystal structure of human Gla-domainless factor Xa complexed with the anticoagulant protein NAPc2 from the hematophagous nematode Ancylostoma caninum.

NAPc2, an anticoagulant protein from the hematophagous nematode Ancylostoma caninum evaluated in phase-II/IIa clinical trials, inhibits the extrinsic blood coagulation pathway by a two step mechanism, initially interacting with the hitherto uncharacterized factor Xa exosite involved in macromolecular recognition and subsequently inhibiting factor VIIa (K(i)=8.4 pM) of the factor VIIa/tissue factor complex. NAPc2 is highly flexible, becoming partially ordered and undergoing significant structural changes in the C terminus upon binding to the factor Xa exosite. In the crystal structure of the ternary factor Xa/NAPc2/selectide complex, the binding interface consists of an intermolecular antiparallel beta-sheet formed by the segment of the polypeptide chain consisting of residues 74-80 of NAPc2 with the residues 86-93 of factor Xa that is additional maintained by contacts between the short helical segment (residues 67-73) and a turn (residues 26-29) of NAPc2 with the short C-terminal helix of factor Xa (residues 233-243). This exosite is physiologically highly relevant for the recognition and inhibition of factor X/Xa by macromolecular substrates and provides a structural motif for the development of a new class of inhibitors for the treatment of deep vein thrombosis and angioplasty.

Identification of a DAF-7 ortholog from the hookworm Ancylostoma caninum.

Infective hookworm L3 encounter a host specific signal during invasion that re-activates suspended developmental pathways. Response to this cue is critical for the successful infection and completion of the life cycle in the host. In the free-living nematode Caenorhabditis elegans, recovery from the developmentally arrested dauer stage in response to environmental cues is analogous to the resumption of development in invading hookworm L3. Transforming growth factor beta (TGF-beta) and insulin-like signalling pathways mediate dauer formation and recovery. An insulin-like signalling pathway mediates L3 activation in hookworms. To determine the role of TGF-beta signalling in hookworm infection, an ortholog of the C. elegans TGF-beta signalling molecule daf-7 was cloned and characterised. Sequence from a hookworm expressed sequence tag was used to design specific primers for PCR amplification of Ac-daf-7 from Ancylostoma caninum infective L3 cDNA. Amplicons from the 5' and 3' ends were cloned, sequenced, and combined to create a full-length composite Ac-daf-7 cDNA sequence. The 1,634 nucleotide cDNA encoded a 355 amino acid open reading frame with significant homology to Ce-DAF-7 and other TGF-beta signalling molecules. The deduced amino acid sequence contained seven conserved cysteines characteristic of TGF-beta family members, as well as two additional conserved cysteines found in members of the TGF-beta/activin subfamily. Ac-DAF-7 contains a characteristic C-terminal ligand domain that is predicted to be released from a propeptide by proteolytic cleavage at a tetrabasic cleavage site. Ac-daf-7 mRNA was strongly detected by reverse transcriptase PCR in L3 and serum stimulated L3 cDNA, and weakly in cDNA from L1 and adult life cycle stages. Antiserum against Escherichia coli expressed recombinant Ac-DAF-7 detected the mature protein in L3 and adult soluble extracts, but not in excretory/secretory products from serum stimulated L3 or adults. Increased expression in arrested L3 stages suggests that Ac-daf-7 is important for developmental arrest.

Isolation and molecular cloning of a secreted hookworm platelet inhibitor from adult Ancylostoma caninum.

Hookworms, bloodfeeding intestinal nematodes, are a leading cause of iron deficiency anemia in the developing world. These parasites have evolved potent mechanisms of interfering with mammalian hemostasis, presumably for the purpose of facilitating bloodfeeding. Adult Ancylostoma caninum worm extracts contain an activity that inhibits platelet aggregation and adhesion by blocking the function of two cell surface integrin receptors, Glycoprotein IIb/IIIa and GPIa/IIa. Using rpHPLC, the hookworm platelet inhibitor activities have been purified from protein extracts of A. caninum. Because the two inhibitory activities co-purified through multiple chromatographic steps, have similar molecular masses and share identical N-terminal as well as internal amino acid sequence homology, it is likely that they represent a single gene product. A cDNA corresponding to the purified hookworm platelet inhibitor (HPI) protein has been cloned from adult A. caninum RNA, and the translated amino acid sequence shows significant homology to Neutrophil Inhibitory Factor and Ancylostoma Secreted Proteins, suggesting that these related hookworm proteins represent a novel class of integrin receptor antagonists. Polyclonal antibodies raised against the recombinant HPI protein recognize corresponding native proteins in A. caninum extracts and excretory/secretory products, and immunohistochemistry data have identified the cephalic glands as the major source of the inhibitor within the adult hookworm. These data suggest that HPI is secreted by the adult stage of the parasite at the site of intestinal attachment. As such, it may represent a viable target for a vaccine-based strategy aimed at interfering with hookworm-induced gastrointestinal hemorrhage and iron deficiency anemia.

Molecular cloning of a novel multidomain Kunitz-type proteinase inhibitor from the hookworm Ancylostoma caninum.

Degenerate oligonucleotide primers derived from conserved serine protease inhibitors were used to amplify a 90-base pair (bp) amplicon from an Ancylostoma caninum adult-stage complementary deoxyribonucleic acid (cDNA) library by polymerase chain reaction (PCR). The amplicon was labeled and used as a probe to screen the library, and a 2,300-bp cDNA clone was identified. The 5' end of the molecule was obtained from adult cDNA by 5'-RACE. The complete sequence named A. caninum Kunitz-type protease inhibitor (Ac-kpi-1) was 2,371 bp and encoded a 759-amino acid open reading frame. The deduced amino acid sequence had a calculated molecular weight of 84,886 Da and contained an amino terminal signal peptide, suggesting that the protein is secreted. Analysis of the predicted protein sequence indicates 12 highly conserved Kunitz-type serine protease inhibitor domains connected by short, conserved spacers. On the basis of sequence analysis, the first 11 domains are predicted to be active serine protease inhibitors based on the P1 amino acid. Domains 5-8 have identical amino acid sequences, and the remaining domains are 38-88% identical. Domain 12 lacks several of the conserved cysteine residues and has an atypical amino acid in the P1 position, suggesting that it is nonfunctional. Reverse transcriptase-PCR indicated that the Ac-kpi-1 messenger ribonucleic acid is present in egg, L1, L3, and adult stages but is most abundant in the adult stage. Ac-KPI-1 is most similar in domain architecture to several extracellular matrix proteins involved in cellular remodeling during insect development. In addition, there are 44 nematode proteins containing one or more Kunitz domains in GenBank, including several with multiple domains.