Antivirus activity, but not thiolreductase activity, is conserved in interferon-gamma-inducible GILT protein in arthropod.

We have previously reported that gamma-interferon inducible lysosomal thiolreductase (GILT) functions as a host defense factor against retroviruses by digesting disulfide bonds on viral envelope proteins. GILT is widely conserved even in plants and fungi as well as animals. The thiolreductase active site of mammalian GILT is composed of a CXXC amino acid motif, whereas the C-terminal cysteine residue is changed to serine in arthropods including shrimps, crabs, and flies. GILT from Penaeus monodon (PmGILT) also has the CXXS motif instead of the CXXC active site. We demonstrate here that a human GILT mutant (GILT C75S) with the CXXS motif and PmGILT significantly inhibit amphotropic murine leukemia virus vector infection in human cells without alterning its expression level and lysosomal localization, showing that the C-terminal cysteine residue of the active site is not required for the antiviral activity. We have reported that human GILT suppresses HIV-1 particle production by digestion of disulfide bonds on CD63. However, GILT C75S mutant and PmGILT did not digest CD63 disulfide bonds, and had no effect on HIV-1 virion production, suggesting that they do not have thiolreductase activity. Taken together, this study found that antiviral activity, but not thiolreductase activity, is conserved in arthropod GILT proteins. This finding provides a new insight that the common function of GILT is antiviral activity in many animals.

Convergent recruitment of adamalysin-like metalloproteases in the venom of the red bark centipede (Scolopocryptops sexspinosus).

Many venom proteins have presumably been convergently recruited by taxa from diverse venomous lineages. These toxic proteins have characteristics that allow them to remain stable in solution and have a high propensity for toxic effects on prey and/or potential predators. Despite this well-established convergent toxin recruitment, some toxins seem to be lineage specific. To further investigate the toxic proteins found throughout venomous lineages, venom proteomics and venom-gland transcriptomics were performed on two individual red bark centipedes (Scolopocryptops sexspinosus). Combining the protein phenotype with the transcript genotype resulted in the first in-depth venom characterization of S. sexspinosus, including 72 venom components that were identified in both the transcriptome and proteome and 1468 nontoxin transcripts identified in the transcriptome. Ten different toxin families were represented in the venom and venom gland with the majority of the toxins belonging to metalloproteases, CAPS (cysteine-rich secretory protein, antigen 5, and pathogenesis-related 1 proteins), and ß-pore-forming toxins. Nine of these toxin families shared a similar proteomic structure to venom proteins previously identified from other centipedes. However, the most highly expressed toxin family, the adamalysin-like metalloproteases, has until now only been observed in the venom of snakes. We confirmed adamalysin-like metalloprotease activity by means of in vivo functional assays. The recruitment of an adamalysin-like metalloprotease into centipede venom represents a striking case of convergent evolution.

Cytochrome P450 diversity in the tree of life.

Sequencing in all areas of the tree of life has produced >300,000 cytochrome P450 (CYP) sequences that have been mined and collected. Nomenclature has been assigned to >41,000 CYP sequences and the majority of the remainder has been sorted by BLAST searches into clans, families and subfamilies in preparation for naming. The P450 sequence space is being systematically explored and filled in. Well-studied groups like vertebrates are covered in greater depth while new insights are being added into uncharted territories like horseshoe crab (Limulus polyphemus), tardigrades (Hypsibius dujardini), velvet worm (Euperipatoides_rowelli), and basal land plants like hornworts, liverworts and mosses. CYPs from the fungi, one of the most diverse groups, are being explored and organized as nearly 800 fungal species are now sequenced. The CYP clan structure in fungi is emerging with 805 CYP families sorting into 32 CYP clans. >3000 bacterial sequences are named, mostly from terrestrial or freshwater sources. Of 18,379 bacterial sequences downloaded from the CYPED database, all are >43% identical to named CYPs. Therefore, they fit in the 602 named P450 prokaryotic families. Diversity in this group is becoming saturated, however 25% of 3305 seawater bacterial P450s did not match known P450 families, indicating marine bacterial CYPs are not as well sampled as land/freshwater based bacterial CYPs. Future sequencing plans of the Genome 10K project, i5k and GIGA (Global Invertebrate Genomics Alliance) are expected to produce more than one million cytochrome P450 sequences by 2020. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Evolutionary ecology of beta-lactam gene clusters in animals.

Beta-lactam biosynthesis was thought to occur only in fungi and bacteria, but we recently reported the presence of isopenicillin N synthase in a soil-dwelling animal, Folsomia candida. However, it has remained unclear whether this gene is part of a larger beta-lactam biosynthesis pathway and how widespread the occurrence of penicillin biosynthesis is among animals. Here, we analysed the distribution of beta-lactam biosynthesis genes throughout the animal kingdom and identified a beta-lactam gene cluster in the genome of F. candida (Collembola), consisting of isopenicillin N synthase (IPNS), δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACVS), and two cephamycin C genes (cmcI and cmcJ) on a genomic scaffold of 0.76 Mb. All genes are transcriptionally active and are inducible by stress (heat shock). A beta-lactam compound was detected in vivo using an ELISA beta-lactam assay. The gene cluster also contains an ABC transporter which is coregulated with IPNS and ACVS after heat shock. Furthermore, we show that different combinations of beta-lactam biosynthesis genes are present in over 60% of springtail families, but they are absent from genome- and transcript libraries of other animals including close relatives of springtails (Protura, Diplura and insects). The presence of beta-lactam genes is strongly correlated with an euedaphic (soil-living) lifestyle. Beta-lactam genes IPNS and ACVS each form a phylogenetic clade in between bacteria and fungi, while cmcI and cmcJ genes cluster within bacteria. This suggests a single horizontal gene transfer event most probably from a bacterial host, followed by differential loss in more recently evolving species.

Bacterial origin of a diverse family of UDP-glycosyltransferase genes in the Tetranychus urticae genome.

UDP-glycosyltransferases (UGTs) catalyze the conjugation of a variety of small lipophilic molecules with uridine diphosphate (UDP) sugars, altering them into more water-soluble metabolites. Thereby, UGTs play an important role in the detoxification of xenobiotics and in the regulation of endobiotics. Recently, the genome sequence was reported for the two-spotted spider mite, Tetranychus urticae, a polyphagous herbivore damaging a number of agricultural crops. Although various gene families implicated in xenobiotic metabolism have been documented in T. urticae, UGTs so far have not. We identified 80 UGT genes in the T. urticae genome, the largest number of UGT genes in a metazoan species reported so far. Phylogenetic analysis revealed that lineage-specific gene expansions increased the diversity of the T. urticae UGT repertoire. Genomic distribution, intron-exon structure and structural motifs in the T. urticae UGTs were also described. In addition, expression profiling after host-plant shifts and in acaricide resistant lines supported an important role for UGT genes in xenobiotic metabolism. Expanded searches of UGTs in other arachnid species (Subphylum Chelicerata), including a spider, a scorpion, two ticks and two predatory mites, unexpectedly revealed the complete absence of UGT genes. However, a centipede (Subphylum Myriapoda) and a water flea and a crayfish (Subphylum Crustacea) contain UGT genes in their genomes similar to insect UGTs, suggesting that the UGT gene family might have been lost early in the Chelicerata lineage and subsequently re-gained in the tetranychid mites. Sequence similarity of T. urticae UGTs and bacterial UGTs and their phylogenetic reconstruction suggest that spider mites acquired UGT genes from bacteria by horizontal gene transfer. Our findings show a unique evolutionary history of the T. urticae UGT gene family among other arthropods and provide important clues to its functions in relation to detoxification and thereby host adaptation.

The crystal structure of a crustacean prophenoloxidase provides a clue to understanding the functionality of the type 3 copper proteins.

UNLABELLED: Phenoloxidase (PO), which is classified as a type 3 copper protein, catalyzes the hydroxylation of monophenol to o-diphenol and subsequent oxidation to the corresponding o-quinone. The geometry and coordination environment of the active site of the arthropod PO are very similar to those of the arthropod hemocyanin (Hc). However, unlike the POs, Hc is an oxygen carrier in crustaceans, and does not possess PO activity in general. Recently, we identified a new type of proPO from a crustacean and designated it proPOß. This enzyme has many characteristics that are rather similar to those of Hc, such as its maturation, localization, and oligomeric state. Here, we determined the crystal structure of proPOß prepared from the hemolymph of kuruma prawns (Marsupenaeus japonicus) at 1.8-Å resolution. M. japonicus proPOß forms a homohexamer rather similar to that of arthropod Hc. The geometry of the active copper site in proPOß is nearly identical to that of arthropod Hc. Furthermore, the well-characterized 'place-holder' phenylalanine is present (Phe72). However, the accessibility to the active site differs in several ways. First, another phenylalanine, which shields the active site by interacting with a copper-coordinated histidine in crustacean Hc, is replaced by valine in the proPOß structure. Second, two tyrosines, Tyr208 and Tyr209, both of which are absent in Hc, show the alternative conformations and form a pathway providing access to the reaction center. Thus, the present crystal structure clarifies the similarities and differences in the activity of two closely related proteins, PO and Hc. DATABASE: Structural data are available in the RSCB protein data bank under the accession number 3WKY. ray crystallography (View interaction).

Development and validation of a quick easily used biochemical assay for evaluating the viability of small immobile arthropods.

Quickly, accurately, and easily assessing the efficacy of treatments to control sessile arthropods (e.g., scale insects) and stationary immature life stages (e.g., eggs and pupae) is problematic because it is difficult to tell whether treated organisms are alive or dead. Current approaches usually involve either maintaining organisms in the laboratory to observe them for development, gauging their response to physical stimulation, or assessing morphological characters such as turgidity and color. These can be slow, technically difficult, or subjective, and the validity of methods other than laboratory rearing has seldom been tested. Here, we describe development and validation of a quick easily used biochemical colorimetric assay for measuring the viability of arthropods that is sufficiently sensitive to test even very small organisms such as white fly eggs. The assay was adapted from a technique for staining the enzyme hexokinase to signal the presence of adenosine triphosphate in viable specimens by reducing a tetrazolium salt to formazan. Basic laboratory facilities and skills are required for production of the stain, but no specialist equipment, expertise, or facilities are needed for its use.

Construction and expression of an antimicrobial peptide scolopin 1 from the centipede venoms of Scolopendra subspinipes mutilans in Escherichia coli using SUMO fusion partner.

Antimicrobial peptide scolopin 1 (AMP-scolopin 1) is a small cationic peptide identified from centipede venoms of Scolopendra subspinipes mutilans. It has broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as an antimicrobial agent. We first report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of cationic antimicrobial peptide AMP-scolopin 1. The fusion protein expressed in a soluble form was purified to a purity of 95% by Ni-IDA chromatography. After the SUMO-scolopin 1 fusion protein was cleaved by the SUMO protease at 30°C for 1 h, the cleaved sample was reapplied to a Ni-IDA. The recombinant scolopin1 had similar antimicrobial properties to the synthetic scolopin 1. Thus, we successfully established a system for purifying peptide of centipede, which could be used for further research.

Evolution of the salivary apyrases of blood-feeding arthropods.

Phylogenetic analyses of three families of arthropod apyrases were used to reconstruct the evolutionary relationships of salivary-expressed apyrases, which have an anti-coagulant function in blood-feeding arthropods. Members of the 5'nucleotidase family were recruited for salivary expression in blood-feeding species at least five separate times in the history of arthropods, while members of the Cimex-type apyrase family have been recruited at least twice. In spite of these independent events of recruitment for salivary function, neither of these families showed evidence of convergent amino acid sequence evolution in salivary-expressed members. On the contrary, in the 5'-nucleotide family, salivary-expressed proteins conserved ancestral amino acid residues to a significantly greater extent than related proteins without salivary function, implying parallel evolution by conservation of ancestral characters. This unusual pattern of sequence evolution suggests the hypothesis that purifying selection favoring conservation of ancestral residues is particularly strong in salivary-expressed members of the 5'-nucleotidase family of arthropods because of constraints arising from expression within the vertebrate host.

[Study on separation and analysis of anticoagulant compounds for anticoagulant activity in vitro on mixture of peptide from pepsin enzymolysis of centipede].

OBJECTIVE: To separate anticoagulant components from the pepsin enzymolysis of centipede by gel filtration and reverse-phase C18 chromatography, and to detect the distribution range of their molecular mass. METHOD: Cingula and 280 nm ultraviolet spectrometry were used to detect and collect the chromatographic solutions. The components' anticoagulant activity in vitro was detected with activated partial thromboplastin time (APTT) as the index, and the molecular mass range of the active composition was detected by MALDI-TOF-MS. RESULT: Anticoagulant active compounds were produced by gel filtration and reverse-phase C18 chromatography. The distribution range of relative molecular mass was determined to be from 597 to 1 146. CONCLUSION: Gel filtration and reverse-phase C18 chromatography are feasible for separating and purifying the pepsin enzymolysis of Centipede. The anticoagulant active compounds are oligopeptides.

Cysteine proteases from bloodfeeding arthropod ectoparasites.

Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model about the contribution of cysteine peptidases to blood digestion and how their concerted action with other tick midgut proteases leads to the absorbance of nutrients by the midgut epithelial cells.

Telomere biology in Metazoa.

In this review we present critical overview of some of the available literature on the fundamental biology of telomeres and telomerase in Metazoan. With the exception of Nematodes and Arthropods, the (TTAGGG)(n) sequence is conserved in most Metazoa. Available data show that telomerase-based end maintenance is a very ancient mechanism in unicellular and multicellular organisms. In invertebrates, fish, amphibian, and reptiles persistent telomerase activity in somatic tissues might allow the maintenance of the extensive regenerative potentials of these species. Telomerase repression among birds and many mammals suggests that, as humans, they may use replicative aging as a tumor protection mechanism.

Dual function of a bee venom serine protease: prophenoloxidase-activating factor in arthropods and fibrin(ogen)olytic enzyme in mammals.

Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles for Bi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

Platelet aggregation inhibitors from hematophagous animals.

Salivary glands from blood-sucking animals (e.g., mosquitoes, bugs, sand flies, fleas, ticks, leeches, hookworms, bats) are a rich source of bioactive molecules that counteract hemostasis in a redundant and synergistic manner. This review discusses recent progress in the identification of salivary inhibitors of platelet aggregation, their molecular characterization, and detailed mechanism of action. Diversity of inhibitors is remarkable, with distinct families of proteins characterized as apyrases that enzymatically degrade ADP or as collagen-binding proteins that prevent its interaction with vWF, or platelet integrin α2ß1 or GPVI. Molecules that bind ADP, TXA(2), epinephrine, or serotonin with high affinity have also been cloned, expressed, and their structure determined. In addition, a repertoire of antithrombins and an increasingly number of RGD and non-RGD disintegrins targeting platelet αIIbß3 have been reported. Moreover, metalloproteases with fibrinogen(olytic) activity and PAF phosphorylcholine hydrolase are enzymes that have been recruited to the salivary gland to block platelet aggregation. Platelet inhibitory prostaglandins, lysophosphatydilcholine, adenosine, and nitric oxide (NO)-carrying proteins are other notable examples of molecules from hematophagous salivary secretions (herein named sialogenins) with antihemostatic properties. Sialogenins have been employed as tools in biochemistry and cell biology and also display potential therapeutic applications.

Effects of of habitats and pesticides on aerobic capacity and survival of soil fauna.

OBJECTIVE: Faunal health is largely dependent on their soil environment and available litter quality. So the effects of different soil habitats and pesticides on citrate synthase (CS) activity of soil fauna and its population were studied. METHODS: The soil animals were collected from different pedoecosystems for habitat study. Whereas Vigna radiata based system was selected for pesticidal observations. The field was divided into five equal plots for control and treatment of gamma-BHC, quinalphos, carbaryl and cypermethrin. Soil fauna was collected by quadrat method and extracted by Tullgren funnel. Individuals of a species having similar sizes were collected for the estimation of CS activity. They were homogenized and fractions were obtained by differential centrifugation. The activity of CS was assayed spectrophotometrically. RESULTS: Citrate synthase (CS) activity of beetle (Rasphytus fregi), woodlouse (Porcellio laevis) and centipede (Scolopendra morsitans) varied significantly with respect to changes in different soil habitats. Though the CS activity of R. fregi, P. laevis, and S. morsitans differed among themselves but the highest activity of CS in these animals was in V. radiata and lowest in A. nilotica based pedoecosystem. The aerobic capacity of centipede was maximum followed by woodlouse and beetle. The treatment of gamma-BHC, quinalphos, carbaryl and cypermethrin significantly reduced the CS activity of these animals. Gamma-BHC showed maximum reduction in CS activity indicating highly toxic effect of organochlorine on aerobic metabolism of soil fauna. However, minimum reduction was observed in response to carbaryl (in beetle) or cypermethrin (in woodlouse/centipede) leading to impairment of aerobic capacity. The differences in pesticide effects might be assigned to the differences in chemical nature of pesticides and their interactions with below-ground fauna. Treatment of gamma-BHC and quinalphos reduced the population of Acari, Coleoptera, Collembola, other arthropods as well as total soil fauna. Acari was least affected by gamma-BHC and maximally affected (72%) in response to quinalphos. The effect of gamma-BHC was fairly similar on Coleoptera, Collembola, other arthropod and total soil fauna suggesting almost similar sensitivity to this pesticide. Likewise, quinalphos was similarly effective on Collemobola and other soil arthropods. Application of carbaryl decreased Acari and Coleoptera population but increased Collembola, other arthropods and total faunal populations. However, application of cypermethrin significantly reduced the population of Acari, Coleoptera, Collembola and total soil fauna and increased the population of other soil arthropods. In both the cases, acarine population was least affected. CONCLUSION: The observations show the habitat-specific variation in aerobic capacity of soil fauna. However, pesticide-dependent loss in population might be due to impairment of aerobic capacity of soil inhabiting animals in desert.

The prophenoloxidase-activating system in invertebrates.

A major innate defense system in invertebrates is the melanization of pathogens and damaged tissues. This important process is controlled by the enzyme phenoloxidase (PO) that in turn is regulated in a highly elaborate manner for avoiding unnecessary production of highly toxic and reactive compounds. Recent progress, especially in arthropods, in the elucidation of mechanisms controlling the activation of zymogenic proPO into active PO by a cascade of serine proteinases and other factors is reviewed. The proPO-activating system (proPO system) is triggered by the presence of minute amounts of compounds of microbial origins, such as beta-1,3-glucans, lipopolysaccharides, and peptidoglycans, which ensures that the system will become active in the presence of potential pathogens. The presence of specific proteinase inhibitors prevents superfluous activation. Concomitant with proPO activation, many other immune reactions will be produced, such as the generation of factors with anti-microbial, cytotoxic, opsonic, or encapsulation-promoting activities.

SDS-induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum, and Cancer magister.

Phenoloxidase, widely distributed among animals, plants, and fungi, is involved in many biologically essential functions including sclerotization and host defense. In chelicerates, the oxygen carrier hemocyanin seems to function as the phenoloxidase. Here, we show that hemocyanins from two ancient chelicerates, the horseshoe crab Limulus polyphemus and the tarantula Eurypelma californicum, exhibit O-diphenoloxidase activity induced by submicellar concentrations of SDS, a reagent frequently used to identify phenoloxidase activity. The enzymatic activity seems to be restricted to only a few of the heterogeneous subunits. These active subunit types share similar topological positions in the quaternary structures as linkers of the two tightly connected 2 x 6-mers. Because no other phenoloxidase activity was found in the hemolymph of these animals, their hemocyanins may act as a phenoloxidase and thus be involved in the primary immune response and sclerotization of the cuticle. In contrast, hemolymph of a more recent arthropod, the crab Cancer magister, contains both hemocyanin with weak phenoloxidase activity and another hemolymph protein with relatively strong phenoloxidase activity. The chelicerate hemocyanin subunits showing phenoloxidase activity may have evolved into a separate phenoloxidase in crustaceans.

Mitochondrial DNA sequences support allozyme evidence for cryptic radiation of New Zealand Peripatoides (Onychophora).

A combination of single-strand conformation polymorphism analysis (SSCP) and sequencing were used to survey cytochrome oxidase I (COI) mitochondrial DNA (mtDNA) diversity among New Zealand ovoviviparous Onychophora. Most of the sites and individuals had previously been analysed using allozyme electrophoresis. A total of 157 peripatus collected at 54 sites throughout New Zealand were screened yielding 62 different haplotypes. Comparison of 540-bp COI sequences from Peripatoides revealed mean among-clade genetic distances of up to 11. 4% using Kimura 2-parameter (K2P) analysis or 17.5% using general time-reversible (GTR + I + Gamma) analysis. Phylogenetic analysis revealed eight well-supported clades that were consistent with the allozyme analysis. Five of the six cryptic peripatus species distinguished by allozymes were confirmed by mtDNA analysis. The sixth taxon appeared to be paraphyletic, but genetic and geographical evidence suggested recent speciation. Two additional taxa were evident from the mtDNA data but neither occurred within the areas surveyed using allozymes. Among the peripatus surveyed with both mtDNA and allozymes, only one clear instance of recent introgression was evident, even though several taxa occurred in sympatry. This suggests well-developed mate recognition despite minimal morphological variation and low overall genetic diversity.

Evolution of the arthropod prophenoloxidase/hexamerin protein family.

Phylogenetic analysis of the prophenoloxidase/hexamerin family of arthropods revealed four well supported subfamilies: (1) the arylphorin subfamily, including arylphorins, storage proteins, and other proteins of uncertain function from insects; (2) the hemocyanins of branchiopod crustaceans, which are copper-binding proteins involved in oxygen transport; (3) the hemocyanins of chelicerates; and (4) the prophenoloxidases (proPO) of both insects and branchiopods, which are copper-binding molecules that play a role in sclerotization of cuticle and encapsulation of foreign particles. The phylogeny indicated that insect and branchiopod proPO constitute a monophyletic group but that branchiopod and chelicerate hemocyanins do not constitute a monophyletic group. Branchiopod hemocyanin and proPO diverged from each other prior to the divergence of insects from branchiopods and probably prior to the divergence of chelicerates from the insect-branchiopod lineage. Likewise, the insect arylphorin subfamily diverged from proPO prior to the divergence of insects from branchiopods and probably prior to the divergence of chelicerates; thus, the results did not support the hypothesis that insect arylphorins represent hemocyanins freed to assume a new function because the insect tracheal respiratory system removes the need for an oxygen-transport molecule. Nonetheless, reconstruction of ancestral sequences by the maximum parsimony method suggested that the ancestors of the arylphorin family were copper-binding. Regions corresponding to the copper-binding domains were found to have a faster rate of nonsynonymous evolution in arylphorin subfamily genes than in other hexamerin family genes; this presumably reflects a relaxation of purifying selection after the loss of copper-binding function.

Proenzyme of Manduca sexta phenol oxidase: purification, activation, substrate specificity of the active enzyme, and molecular cloning.

Phenol oxidase (PO) was isolated as a proenzyme (pro-phenol oxidase, pro-PO) from the hemolymph of Manduca sexta larvae and purified to homogeneity. Pro-PO exhibits a M(r) of 130,000 on gel filtration and two bands with an apparent M(r) of approximately 100,000 on SDS/PAGE, as well as size-exclusion HPLC. Activation of pro-PO was achieved either by specific proteolysis by a cuticular protease or by the detergent cetylpyridinium chloride at a concentration below the critical micellar concentration. A cDNA clone for M. sexta pro-PO was obtained from a larval hemocyte cDNA library. The clone encodes a polypeptide of approximately 80,000 Da that contains two copper-binding sites and shows high sequence similarity to POs, hemocyanins, and storage proteins of arthropods. The M. Sexta pro-PO, together with other arthropod pro-POs, contains a short stretch of amino acids with sequence similarity to the thiol ester region of alpha-macroglobulins and complement proteins C3 and C4.