Induced thiacloprid insensitivity in honeybees (Apis mellifera L.) is associated with up-regulation of detoxification genes.

Honey bees, Apis mellifera, are markedly less sensitive to neonicotinoid insecticides containing a cyanoimino pharmacophore than to those with a nitroimino group. Although previous work has suggested that this results from enhanced metabolism of the former by detoxification enzymes, the specific enzyme(s) involved remain to be characterized. In this work, a pretreatment of honey bees with a sublethal dose of thiacloprid resulted in induced insensitivity to the same compound immediately following thiacloprid feeding. A longer pretreatment time resulted in no, or increased, sensitivity. Transcriptome profiling, using microarrays, identified a number of genes encoding detoxification enzymes that were over-expressed significantly in insecticide-treated bees compared with untreated controls. These included five P450s, CYP6BE1, CYP305D1, CYP6AS5, CYP315A1, CYP301A1, and a carboxyl/cholinesterase (CCE) CCE8. Four of these P450s were functionally expressed in Escherichia coli and their ability to metabolize thiacloprid examined by liquid chromatography-mass spectrometry (LC-MS) analysis.

Assessing cross-resistance within the pyrethroids in terms of their interactions with key cytochrome P450 enzymes and resistance in vector populations.

BACKGROUND: It is important to understand whether the potential impact of pyrethroid resistance on malaria control can be mitigated by switching between different pyrethroids or whether cross-resistance within this insecticide class precludes this approach. METHODS: Here we assess the relationships among pyrethroids in terms of their binding affinity to, and depletion by, key cytochrome P450 enzymes (hereafter P450s) that are known to confer metabolic pyrethroid resistance in Anopheles gambiae (s.l.) and An. funestus, in order to identify which pyrethroids may diverge from the others in their vulnerability to resistance. We then investigate whether these same pyrethroids also diverge from the others in terms of resistance in vector populations. RESULTS: We found that the type I and II pyrethroids permethrin and deltamethrin, respectively, are closely related in terms of binding affinity to key P450s, depletion by P450s and resistance within vector populations. Bifenthrin, which lacks the common structural moiety of most pyrethroids, diverged from the other pyrethroids tested in terms of both binding affinity to key P450s and depletion by P450s, but resistance to bifenthrin has rarely been tested in vector populations and was not analysed here. Etofenprox, which also lacks the common structural moiety of most pyrethroids, diverged from the more commonly deployed pyrethroids in terms of binding affinity to key P450s and resistance in vector populations, but did not diverge from these pyrethroids in terms of depletion by the P450s. The analysis of depletion by the P450s indicated that etofenprox may be more vulnerable to metabolic resistance mechanisms in vector populations. In addition, greater resistance to etofenprox was found across Aedes aegypti populations, but greater resistance to this compound was not found in any of the malaria vector species analysed. The results for pyrethroid depletion by anopheline P450s in the laboratory were largely not repeated in the findings for resistance in malaria vector populations. CONCLUSION: Importantly, the prevalence of resistance to the pyrethroids α-cypermethrin, cyfluthrin, deltamethrin, λ-cyhalothrin and permethrin was correlated across malaria vector populations, and switching between these compounds as a tool to mitigate against pyrethroid resistance is not advised without strong evidence supporting a true difference in resistance.

Sequence characterization of cytochrome P450 CYP6P9 in pyrethroid resistant and susceptible Anopheles funestus (Diptera: Culicidae).

Anopheles funestus, one of the main African malaria vectors, caused a major malaria outbreak in South Africa during 1999/2000, even though South Africa had an effective vector control program in place. The outbreak was due to pyrethroid resistant An. funestus invading KwaZulu/Natal. Increased activity of cytochrome P450 (monooxygenase) was responsible for the pyrethroid resistance in this species. A monooxygenase gene, CYP6P9, was highly overexpressed in the pyrethroid-resistant strain compared with a susceptible strain. Characterization of this gene as well as the redox partners involved in the catalytic cycle of P450s was investigated. The full length of the CYP6P9 sequence was isolated, sequenced and compared between the pyrethroid-resistant and -susceptible strains. Sequence identity between the two strains was 99.3%; the sequence differences were mainly outside of the conserved regions. The functional significance is still unknown, but it is feasible that these variations are associated with differences in insecticide metabolism. A second CYP6 gene (CYP6P13) was also isolated; it shared close similarities with CYP6P9. The putative redox partners, cytochrome b(5) (cyt b(5)) and NADPH-cytochrome P450 reductase (CPR), were isolated from An. funestus (resistant strain) and showed high levels of sequence identity to other insect cyt b(5) and CPRs. Isolation of the coding sequences CYP6P9 and its cognate redox partners enables expression of functional recombinant protein for biochemical and structural analysis.

Insights into drug metabolism by cytochromes P450 from modelling studies of CYP2D6-drug interactions.

The cytochromes P450 (CYPs) comprise a vast superfamily of enzymes found in virtually all life forms. In mammals, xenobiotic metabolizing CYPs provide crucial protection from the effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. Ideally, the information on the possible metabolism by CYPs required during drug development would be obtained from crystal structures of all the CYPs of interest. For some years only crystal structures of distantly related bacterial CYPs were available and homology modelling techniques were used to bridge the gap and produce structural models of human CYPs, and thereby obtain useful functional information. A significant step forward in the reliability of these models came seven years ago with the first crystal structure of a mammalian CYP, rabbit CYP2C5, followed by the structures of six human enzymes, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4, and a second rabbit enzyme, CYP2B4. In this review we describe as a case study the evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism. This work has led directly to the successful design of CYP2D6 mutants with novel activity-including creating a testosterone hydroxylase, converting quinidine from inhibitor to substrate, creating a diclofenac hydroxylase and creating a dextromethorphan O-demethylase. Our modelling-derived hypothesis-driven integrated interdisciplinary studies have given key insight into the molecular determinants of CYP2D6 and other important drug metabolizing enzymes.

Characterization of inhibitors and substrates of Anopheles gambiae CYP6Z2.

Three CYP6Z genes are linked to a major pyrethroid resistance locus in the mosquito Anopheles gambiae. We have expressed CYP6Z2 in Escherichia coli and produced a structural model in order to examine its role in detoxification. E. coli membranes co-expressing CYP6Z2 and An. gambiae P450 reductase (AgCPR) catalysed the dealkylation of benzyloxyresorufin with kinetic parameters K(m) = 0.13 microM; K(cat) = 1.5 min(-1). The IC(50) values of a wide range of compounds were measured. Pyrethroids cypermethrin and permethrin produced low IC(50) values, but were not metabolized. Plant flavanoids were the most potent inhibitors. Several compounds were shown to be substrates, suggesting that CYP6Z2 has broad substrate specificity and plays an important chemo-protective role during the herbivorous phase of the life-cycle.

Nitric oxide synthases catalyze the activation of redox cycling and bioreductive anticancer agents.

Nitric oxide synthases (NOSs) play a crucial role in the control of blood flow, memory formation, and the immune response. These proteins can be structurally divided into oxygenase and reductase domains. The reductase domain shares a high degree of sequence homology with P450 reductase, which is thought to be the major enzyme responsible for the one-electron reduction of foreign compounds, including bioreductive antitumor agents currently undergoing clinical trials. In view of the structural similarities between NOS and P450 reductase, we investigated the capacity of NOS to reduce the hypoxic cytotoxin tirapazamine, the antitumor agent doxorubicin, and also the redox cycling compound menadione. All three isoforms exhibited high levels of activity toward these compounds. In the case of doxorubicin and menadione, the activity of NOS II was 5-10-fold higher than the other enzymes, whereas with tirapazamine, the activities were broadly similar. NOS-mediated metabolism of tirapazamine resulted in a large increase in plasmid DNA strand breaks, demonstrating that the reduction was a bioactivation process. In addition, tirapazamine inhibited NOS activity. Because nitric oxide is implicated in maintaining tumor vascular homeostasis, it is conceivable that tirapazamine could potentiate its own toxicity by increasing the degree of hypoxia. This study suggests that the NOSs could play a key role in the therapeutic effects of tirapazamine, particularly because NOS activity is markedly increased in several human tumors. In addition, the presence of NOS in the heart indicates that these enzymes may contribute to the cardiotoxicity of redox cycling drugs, such as doxorubicin.

Gene expression in Echis carinatus (carpet viper) venom glands following milking.

RNA was extracted from the venom glands of Echis carinatus at different times after milking, and the temporal pattern and nature of mRNA transcribed during venom regeneration was examined by in vitro translation. Venom products were immunoprecipitated with E. carinatus venom polyclonal antiserum. Maximum transcriptional activity occurred 3 days after milking. Electrophoretic analysis of the translation products showed minimal differences in the banding patterns at each time interval. Analysis of the translation products from Kenyan, Nigerian and Saudi Arabian carpet vipers, however, revealed differences which suggest that the observed heterogeneity in E. carinatus venom occurs at the level of the genome.

Anopheles gambiae P450 reductase is highly expressed in oenocytes and in vivo knockdown increases permethrin susceptibility.

We describe an in vivo model for investigation of detoxification mechanisms of the mosquito Anopheles gambiae, important for the development of malaria control programmes. Cytochrome P450s are involved in metabolic insecticide resistance and require NADPH cytochrome P450 reductase (CPR) to function. Here we demonstrate that the major sites of adult mosquito CPR expression are oenocytes, mid-gut epithelia and head appendages. High CPR expression was also evident in Drosophila oenocytes indicating a general functional role in these insect cells. RNAi mediated knockdown drastically reduced CPR expression in oenocytes, and to a lesser extent in mid-gut epithelia; the head was unaffected. These flies showed enhanced sensitivity to permethrin, demonstrating a key role for abdominal/mid-gut P450s in pyrethroid metabolism, aiding the development of insecticides.

The role of bioreductive activation of doxorubicin in cytotoxic activity against leukaemia HL60-sensitive cell line and its multidrug-resistant sublines.

Clinical usefulness of doxorubicin (DOX) is limited by the occurrence of multidrug resistance (MDR) associated with the presence of membrane transporters (e.g. P-glycoprotein, MRP1) responsible for the active efflux of drugs out of resistant cells. Doxorubicin is a well-known bioreductive antitumour drug. Its ability to undergo a one-electron reduction by cellular oxidoreductases is related to the formation of an unstable semiquionone radical and followed by the production of reactive oxygen species. There is an increasing body of evidence that the activation of bioreductive drugs could result in the alkylation or crosslinking binding of DNA and lead to the significant increase in the cytotoxic activity against tumour cells. The aim of this study was to examine the role of reductive activation of DOX by the human liver NADPH cytochrome P450 reductase (CPR) in increasing its cytotoxic activity especially in regard to MDR tumour cells. It has been evidenced that, upon CPR catalysis, DOX underwent only the redox cycling (at low NADPH concentration) or a multistage chemical transformation (at high NADPH concentration). It was also found, using superoxide dismutase (SOD), that the first stage undergoing reductive activation according to the mechanism of the redox cycling had the key importance for the metabolic conversion of DOX. In the second part of this work, the ability of DOX to inhibit the growth of human promyelocytic-sensitive leukaemia HL60 cell line as well as its MDR sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX) was studied in the presence of exogenously added CPR. Our assays showed that the presence of CPR catalysing only the redox cycling of DOX had no effect in increasing its cytotoxicity against sensitive and MDR tumour cells. In contrast, an important increase in cytotoxic activity of DOX after its reductive conversion by CPR was observed against HL60 as well as HL60/VINC and HL60/DOX cells.

Cloning of metalloprotease genes in the carpet viper (Echis pyramidum leakeyi). Further members of the metalloprotease/disintegrin gene family.

The genes coding for two metalloproteases, EcH-I and EcH-II, have been cloned from an Echis pyramidum leakeyi venom gland cDNA library. The cDNA sequences predict two zymogen molecules with strong amino acid sequence similarity and the same domain structure present in other members of the viper metalloprotease/disintegrin gene family. EcH-I and EcH-II contain pro-protein, enzyme and disintegrin domains. Analysis of the cDNAs coding for EcH-I, EcH-II, jararhagin, trigramin and Ht-e reveals a strong similarity, particularly in the untranslated regions and regions coding for the pro-peptide. Comparison of EcH-I and EcH-II with venom metalloproteases, mammalian matrix-degrading metalloproteases, sperm proteins, and a potential tumour suppressor gene highlights the presence of a number of motifs with potential functional significance.

Stage specific differences in steady state levels of mRNA encoding the major surface glycoprotein of Brugia pahangi.

The glycoprotein, gp30, is the major soluble cuticular antigen of the adult lymphatic fiarial worm Brugia pahangi (Maizels et al., 1983, Devaney, 1988). Cookson et al., 1992 suggested that gp30 may function as an antioxidant enzyme protecting B. pahangi from the vertebrate host defence mechanism. In this communication we report that the gp30 transcript is present in each of the life cycle stages, including mosquito derived L3's, and that there is a 50 fold increase in the transcription of gp30 in young adults (28 days post infection) compared to mature adults. These findings suggest that gp30 performs a general function relevant throughout the B. pahangi life cycle and in particular to young adults.

Engineering of a functional human NADH-dependent cytochrome P450 system.

A functional human NADH-dependent cytochrome P450 system has been developed by altering the cofactor preference of human NADPH cytochrome P450 reductase (CPR), the redox partner for P450s. This has been achieved by a single amino acid change of the conserved aromatic amino acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in the nicotinamide-binding site. Of the mutations made, the substitution of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent enzyme, which catalyzed the reduction of cytochrome c and ferricyanide as well as facilitated the metabolism of 7-ethoxyresorufin by CYP1A2. Kinetic analysis measuring cytochrome c activity revealed that the NADH-dependent k(cat) of W676A is equivalent (90%) to the NADPH-dependent k(cat) of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent K(M)(NADPH) and K(M)(NADH) values of W676A are 80- and 150-fold decreased, respectively. In accordance with structural data, which show a bipartite binding mode of NADPH, substitution of Trp-676 does not affect 2'-AMP binding as seen by the inhibition of both wild-type CPR and the W676A mutant. Furthermore, NADPH was a potent inhibitor of the W676A NADH-dependent cytochrome c reduction and CYP1A2 activity. Overall, the results show that Trp-676 of human CPR plays a major role in cofactor discrimination, and substitution of this conserved aromatic residue with alanine results in an efficient NADH-dependent cytochrome P450 system.

Functional high level expression of cytochrome P450 CYP2D6 using baculoviral expression systems.

Cytochrome P-450 CYP2D6 plays a central role in the metabolism of many widely used therapeutic drugs including beta-adrenergic antagonists, antiarrhythmics, and tricyclic antidepressants. Recombinant baculoviruses have been constructed containing the full-length human CYP2D6 cDNA and used to express CYP2D6 in Spodoptera frugiperda (Sf9) cells. High levels of recombinant protein have been produced using either polyhedrin or basic protein promoters (0.05-0.20 nmol/mg cell protein; 0.05-0.15 nmol/liter). The enzyme is catalytically active toward CYP2D6 substrates such as bufuralol and metoprolol. In order to optimize catalytic activity human reductase was coexpressed with CYP2D6 in Sf9 cells; reductase activity was in the region of 1000-1500 units per mg cell protein, while spectrally active CY2D6 was in the range 10-20 pmol/mg cell protein. The K(m) and K(cat) values for bufuralol metabolism were estimated as 4.7 microM and 12.23 min-1, respectively. The use of the conventional very late promoters such as the polyhedrin promoter generate a large proportion of inactive CYP2D6. The problem was to a degree circumvented using the "late" basic promoter which is active earlier in the baculovirus infection cycle. The yield of functional CYP2D6 was at least as high as with very late promoters, but the proportion of inactive protein was reduced. Bufuralol hydroxylase activity could be measured directly by HPLC analysis of cell culture media supplemented with bufuralol, and we have developed a plate assay system which provides a simple method for the analysis of drug metabolism reactions using Sf9 cells. Expression using baculovirus provides a valuable source of functional CYP2D6 for work aimed at elucidating the structure and function of the enzyme.

Purification, cloning, and molecular characterization of a high molecular weight hemorrhagic metalloprotease, jararhagin, from Bothrops jararaca venom. Insights into the disintegrin gene family.

A large hemorrhagin, jararhagin, has been cloned from a Bothrops jararaca venom gland cDNA expression library. The cDNA sequence predicts a 421-amino acid residue molecule with strong amino acid sequence homology and similar domain structure to HR1B, a high molecular weight hemorrhagic metalloprotease isolated from Trimeresurus flavoviridis (Habu) venom. Like HR1B, jararhagin contains enzyme, disintegrin, and cysteine-rich carboxyl-terminal regions. In the disintegrin region, the Arg-Gly-Asp sequence is replaced by Glu-Cys-Asp, as found in non-Arg-Gly-Asp disintegrin regions of HR1B and a guinea pig sperm fusion protein PH-30 beta. The cDNA sequence of jararhagin predicts a precursor protein (proprotein) with striking similarity to cryptic regions in precursors of the disintegrin peptides trigramin and rhodostomin. Comparison of jararhagin with disintegrin precursors highlights the modular arrangement of proprotein, metalloprotease, and disintegrin domains in the metalloprotease/disintegrin family and provides an insight into their biosynthesis and evolution.

Sequence of the cDNA coding for the lethal neurotoxin Tx1 from the Brazilian "armed" spider Phoneutria nigriventer predicts the synthesis and processing of a preprotoxin.

A cDNA library was constructed from the venom glands of the Brazilian "armed" spider, Phoneutria nigriventer, and a clone coding for Tx1, a lethal toxin, was identified and sequenced. The sequence data derived from this cDNA clone combined with the previously determined amino acid sequence predict that Tx1 is initially synthesized as a preprotoxin. Four segments (comprising the signal sequence, a short, 15-amino acid, glutamate-rich sequence, the functional toxin, and 2 glycine residues) can be distinguished. The structure of the preprotoxin and the proposed processing steps required to form the mature Tx1 toxin show similarities with the synthesis and processing of omega-agatoxin IA.

The molecular cloning of a phospholipase A2 from Bothrops jararacussu snake venom: evolution of venom group II phospholipase A2's may imply gene duplications.

The sequence coding for a snake venom phospholipase A2 (PLA2), BJUPLA2, has been cloned from a Bothrops jararacussu venom gland cDNA library. The cDNA sequence predicts a precursor containing a 16-residue signal peptide followed by a molecule of 122 amino acid residues with a strong sequence similarity to group II snake venom PLA2's. A striking feature of the cDNA is the high sequence conservation of the 5' and 3' untranslated regions in cDNAs coding for PLA2's from a number of viper species. The greatest sequence variation was observed between the regions coding for the mature proteins, with most substitutions occurring in nonsynonymous sites. The phylogenetic tree constructed by alignment of the amino acid sequence of BJUPLA2 with group II PLA2's in general groups them according to current taxonomical divisions and/or functional activity. It also suggests that gene duplications may have occurred at a number of different points during the evolution of snake venom group II PLA2's.

Role of the conserved phenylalanine 181 of NADPH-cytochrome P450 oxidoreductase in FMN binding and catalytic activity.

NADPH-cytochrome P450 oxidoreductase (P450 reductase, EC 1.6.2.4) is an essential component of the P450 monooxygenase complex and binds FMN, FAD, and NADPH cofactors. Residues Tyr140 and Tyr178 are known to be involved in FMN binding. A third aromatic side chain, Phe181, is also located in the proximity of the FMN ring and is highly conserved in FMN-binding proteins, suggesting an important functional role. This role has been investigated by site-directed mutagenesis. Substitution of Phe181 with leucine or glutamine decreased the cytochrome c reductase activity of the enzyme by approximately 50%. Ferricyanide reductase activity was unaffected, indicating that the FAD domain was unperturbed. The mutant FMN domains were expressed in Escherichia coli, and the redox potentials and binding energies of their complexes with FMN were determined. The affinity for FMN was decreased approximately 50-fold in the Leu181 and Gln181 mutants. Comparison of the binding energies of the wild-type and mutant enzymes in the three redox states of FMN suggests that Phe181 stabilizes the FMN-apoprotein complex. The amide 1H and 15N resonances of the Phe181Leu FMN domain were assigned; comparison of their chemical shifts with those of the wild-type domain indicated that the effect of the substitution on FMN affinity results from perturbation of two loops which form part of the FMN binding site. The results indicate that Phe181 cooperates with Tyr140 and Tyr178 to play a major role in the binding and stability of FMN.

Expression, purification, and biochemical characterization of a human cytochrome P450 CYP2D6-NADPH cytochrome P450 reductase fusion protein.

Cytochrome P450 CYP2D6 metabolizes a wide range of pharmaceutical compounds. A CYP2D6 fusion enzyme (CYP2D6F), containing an amino-terminal human CYP2D6 sequence and a carboxyterminal human NADPH-cytochrome P450 oxidoreductase (CPR) moiety, was constructed. High levels of expression were achieved in Escherichia coli (60-100 nmol/liter) and the enzyme was catalytically active with optimal activities achieved in the presence of the antioxidant, GSH. Turnover values for bufuralol 1'-hydroxylation, metoprolol alpha-hydroxylation, O-desmethylation, and dextromethorphan O-demethylation, using membranes expressing the fusion enzyme, were 5.6, 0.4, 0.72, and 6.19 min(-1), respectively. These values were similar to E. coli membranes which coexpressed human CYP2D6 and CPR (CYP2D6/R). The K(m) and k(cat) values for bufuralol metabolism were estimated to be 10.2 microM and 4.1 min(-1), respectively. The enzyme was purified using ion-exchange chromatography, affinity chromatography (2'-5' ADP-Sepharose), and gel filtration. Estimated turnover rates for bufuralol 1'-hydroxylation, metoprolol alpha-hydroxylation, O-desmethylation, and dextromethorphan O-demethylation were 1.2, 0.52, 0.79, and 0.76 min(-1), respectively. Bufuralol 1'-hydroxylase activity by purified CYP2D6F was enhanced by phospholipids and added CPR. The CYP2D6F enzyme was able to stimulate CYP3A4 testosterone 6beta-hydroxylase activity in a reconstitution system indicating that electron transfer may be largely intermolecular. The catalytically self-sufficient CYP2D6F enzyme will facilitate investigations of P450-CPR interactions and the development of new biocatalysts.

Metabolism of MPTP by cytochrome P4502D6 and the demonstration of 2D6 mRNA in human foetal and adult brain by in situ hybridization.

1. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a thermal breakdown product of synthetic 'street' heroin, is known to cause Parkinson's Disease-like symptoms in man. 2. The mechanism of action of this neurotoxin is thought to involve activation by the monoamine oxidase B system and subsequent toxicity by inhibition of neuronal mitochondrial respiration. The manifestation of toxicity will be a balance between the rate of activation of this compound versus its rate of inactivation through metabolism by enzymes such as the cytochrome P450-dependent monooxygenases. 3. In this report we demonstrate that MPTP N-demethylation, a detoxification pathway, is catalysed by cytochrome P450 CYP2D6 and up to 40% of the hepatic metabolism is mediated by this enzyme. 4. Perhaps more importantly we also demonstrate by in situ hybridization that CYP2D6 is localized in the pigmented neurons of the substantia nigra indicating that 2D6-mediated detoxification will occur in target cells. 5. These data present evidence that CYP2D6 will be a factor in susceptibility to MPTP neuronal toxicity and provide a biochemical rationale for the genetic observations linking a polymorphism at the CYP2D6 locus with susceptibility to Parkinson's.

cDNA cloning and deduced amino acid sequence of prothrombin activator (ecarin) from Kenyan Echis carinatus venom.

The complete amino acid sequence of ecarin is deduced from the nucleotide sequence of a cDNA clone isolated by screening a venomous gland cDNA library of Kenyan Echis carinatus. The cDNA sequence with 2379 base pairs encodes an open reading frame of 616 amino acids with a remarkable sequence homology to the putative precursor protein of trigramin from Trimeresurus gramineus venom (61% identity) and a large hemorrhagin, jararhagin, from the pit viper Bothrops jararaca venom (62% identity). Thus, ecarin, as well as jararhagin and trigramin, is translated as a precursor protein, which may be processed posttranslationally. The ecarin proprotein has a "cysteine switch" motif (-Pro-Lys-Met-Cys-Gly-Val-) similar to that involved in the activation of matrix metalloproteinase zymogens. The processed mature protein consists of 426 amino acid residues (residues 191-616), showing the strongest sequence similarity with that of Russell's viper venom factor X activator (RVV-X) heavy chain (64% identity). Like RVV-X heavy chain, ecarin contains metalloproteinase, disintegrin, and cysteine-rich domains. The metalloproteinase domain has a typical zinc-chelating sequence (-His-Glu-Xaa-Xaa-His-Xaa-Xaa-Gly-Xaa-Xaa-His-), as found in crayfish astacin. In the disintegrin domain of ecarin, the Arg-Gly-Asp sequence is replaced by Arg-Asp-Asp, as found in the disintegrin domains of RVV-X heavy chain (Arg-Asp-Glu) and a guinea pig sperm fusion protein, PH-30 beta (Thr-Asp-Glu). These findings show that while there are structural and evolutionary relationships among these proteins, each has a unique functional activity.