A bi-functional anti-thrombosis protein containing both direct-acting fibrin(ogen)olytic and plasminogen-activating activities

Direct-acting fibrin(ogen)olytic agents such as plasmin have been proved to contain effective and safety thrombolytic potential. Unfortunately, plasmin is ineffective when administered by the intravenous route because it was neutralized by plasma antiplasmin. Direct-acting fibrin(ogen)olytic agents with resistance against antiplasmin will brighten the prospect of anti-thrombosis. As reported in ‘Compendium of Materia Medica’, the insect of Eupolyphaga sinensis Walker has been used as traditional anti-thrombosis medicine without bleeding risk for several hundreds years. Currently, we have identified a fibrin(ogen)olytic protein (Eupolytin1) containing both fibrin(ogen)olytic and plasminogen-activating (PA) activities from the beetle, E. sinensis. Objectives: To investigate the role of native and recombinant eupolytin1 in fibrin(ogen)olytic and plasminogen-activating processes. Methods and Results: Using thrombus animal model, eupolytin1 was proved to contain strong and rapid thrombolytic ability and safety in vivo, which are better than that of urokinase. Most importantly, no bleeding complications were appeared even the intravenous dose up to 0.12 µmol/kg body weight (3 times of tested dose which could completely lyse experimental thrombi) in rabbits. It is the first report of thrombolytic agents containing both direct-acting fibrin(ogen)olytic and plasminogen-activating activities. Conclusions: The study identified novel thrombolytic agent with prospecting clinical potential because of its bi-functional merits containing both plasmin- and PA-like activities and unique pharmacological kinetics in vivo.     

Molecular cloning and fibrin(ogen)olytic activity of a bumblebee (Bombus hypocrita sapporoensis) venom serine protease

Although several bee venom serine protease genes have been previously described, fibrin(ogen)olytic activity of these serine proteases has been reported for only two bumblebees to date, Bombus ignitus and B. terrestris. Here, we cloned venom serine proteases from the other bumblebee species, B. hypocrita sapporoensis and B. ardens ardens. The venom serine protease genes of B. h. sapporoensis and B. a. ardens consist of 358 amino acids and 357 amino acids, respectively. We compared the predicted mature protein sequences of these serine protease genes to those previously reported for other bees. A phylogenetic analysis shows that B. h. sapporoensis venom serine protease is further immediately close to B. ignitus and B. terrestris venom serine proteases, excluding the venom serine protease of B. a. ardens. Using B. h. sapporoensis venom serine protease (Bs-VSP), we identified that Bs-VSP acts as a fibrin(ogen)olytic enzyme. We also found that Bs-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. Our results further define roles for bumblebee venom serine proteases as fibrin(ogen)olytic agents.     

Structural and functional comparison of proteolytic enzymes from plant latex and snake venoms

This work describes classification, functions, location, inhibition, activation, and therapeutic applications of proteases from snake venoms and vegetables. Snake venoms and vegetables can present toxins that unchain necrosis or proteolysis due to the direct cytotoxic action of venom proteases. These proteases are potential tools in the development of drugs for the prevention and treatment of several illnesses. We report herein mainly fibrinogenolytic metallo proteases and serine proteases (“thrombin-like”). These enzymes are extensively used in the treatment and prevention of thrombotic disorders, since they serve as defibrinogenating agents. The therapeutic uses of fibrin(ogen)olytic metallo proteases hold promise for clinical application due to potential in reversing the effects of thrombosis; this has been shown to be an alternative approach to the prevention and treatment of cardiovascular disorders, which are among the most prominent causes of mortality around the world. Plant proteases can be utilized for many cellular and molecular activities, in antibacterial and anticancer therapies, and in the treatment of snakebites, inhibiting snake venom activities such as blood-clotting, defibrinogenation, and fibrin(ogen)olytic and hemorrhagic actions. These toxins also display potential for clinical use in the treatment of hemostatic disorders.     

Snake venom proteases affecting hemostasis and thrombosis

The structure and function of snake venom proteases are briefly reviewed by putting the focus on their effects on hemostasis and thrombosis and comparing with their mammalian counterparts. Up to date, more than 150 different proteases have been isolated and about one third of them structurally characterized. Those proteases are classified into serine proteases and metalloproteinases. A number of the serine proteases show fibrin(ogen)olytic (thrombin-like) activities, which are not susceptible to hirudin or heparin and perhaps to most endogenous serine protease inhibitors, and form abnormal fibrin clots. Some of them have kininogenase (kallikrein-like) activity releasing hypotensive bradykinin. A few venom serine proteases specifically activate coagulation factor V, protein C, plasminogen or platelets. The venom metalloproteinases, belonging to the metzincin family, generally show fibrin(ogen)olytic and extracellular matrix-degrading (hemorrhagic) activities. A few venom metalloproteinases show a unique substrate specificity toward coagulation factor X, platelet membrane receptors or von Willebrand factor. A number of the metalloproteinases have chimeric structures composed of several domains such as proteinase, disintegrin-like, Cys-rich and lectin-like domains. The disintegrin-like domain seems to facilitate the action of those metalloproteinases by interacting with platelet receptors. A more detailed analysis of snake venom proteases should find their usefulness for the medical and pharmacological applications in the field of thrombosis and hemostasis.     

A novel fibrin(ogen)olytic trypsin-like protease from Chinese oak silkworm (Antheraea pernyi): Purification and characterization

A novel fibrin(ogen)olytic protease from Antheraea pernyi (important economically insect), named cocoonase, was isolated by a combination of ion-exchange chromatography and gel filtration. Furthermore, the characterization of cocoonase was investigated using fibrin(ogen)olytic, thrombolysis, and hemorrhagic assays. The NH₂-terminal sequence (IVGGY SVTID KAPYQ) was established by Edman degradation. Based on the N-terminal sequencing, cocoonase cDNA has been cloned by means of RT-PCR and 5′RACE. It is composed of 261 amino acid residues and possesses the structural features of trypsin-like serine protease. The purified cocoonase showed specific esterase activity on N-β-benzoyl-l-arginine ethyl (BAEE), and the kinetic constants, Km and Vmax were 2.577 × 10⁻³ mol/L and 4.09 × 10⁻³ μmol/L/s, respectively. Cocoonase showed strong activities on both fibrin and fibrinogen, preferentially hydrolyzed Aα and Bβ chains followed by γ-chains of fibrinogen. Cocoonase exhibited a thrombolysis activity both in vitro (blood-clot lysis activity assay) and in vivo (carrageenan-induced thrombosis model). These findings indicate that A. pernyi cocoonase ia a novel fibrin(ogen)olytic enzyme and may have a potential clinical application as an antithrombotic agent.     

Antifibrinolytic role of a bee venom serine protease inhibitor that acts as a plasmin inhibitor

Bee venom is a rich source of pharmacologically active substances. In this study, we identified a bumblebee (Bombus ignitus) venom Kunitz-type serine protease inhibitor (Bi-KTI) that acts as a plasmin inhibitor. Bi-KTI showed no detectable inhibitory effect on factor Xa, thrombin, or tissue plasminogen activator. In contrast, Bi-KTI strongly inhibited plasmin, indicating that it acts as an antifibrinolytic agent; however, this inhibitory ability was two-fold weaker than that of aprotinin. The fibrin(ogen)olytic activities of B. ignitus venom serine protease (Bi-VSP) and plasmin in the presence of Bi-KTI indicate that Bi-KTI targets plasmin more specifically than Bi-VSP. These findings demonstrate a novel mechanism by which bumblebee venom affects the hemostatic system through the antifibrinolytic activity of Bi-KTI and through Bi-VSP-mediated fibrin(ogen)olytic activities, raising interest in Bi-KTI and Bi-VSP as potential clinical agents.     

Enzymatic properties and identification of a fibrinolytic serine protease purified from Bacillus subtilis DC33

A novel fibrinolytic enzyme subtilisin FS33 was purified from Bacillus subtilis DC33, isolated from a traditional flavour-rich food in China. The purified subtilisin FS33 was a single chain protein with a molecular mass of 30 kDa measured by SDS-PAGE. After activated SDS-PAGE, the enzyme band exhibited strong fibrinolytic activity on the fibrin plate. Subtilisin FS33 was temperature-stable below 60°C over the pH range 5–12, with a maximum activity at pH 8.0, but the activity completely disappeared after 10 min above 65°C. The NH₂-terminal amino acid sequence of the enzyme was different from that of other known fibrinolytic enzymes, such as NK, CK, SMCE, KA38, subtilisin E, subtilisin DFE and Katsuwokinase. The amidolytic activities of subtilisin FS33 were inhibited completely by phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI). EDTA did not affect the enzyme activity, and none of the ions tested activated the activity. Therefore, the enzyme was thought to be a subtilisin-like serine protease. The enzyme degraded the Bβ-chains of fibrin(ogen) very rapidly and then degraded the Aα-chain and at least five fragments from fibrin(ogen) were obtained after hydrolysis. Subtilisin FS33 was also able to cleave blood clots in the absence of endogenous fibrinolytic factors.     

Role of D and E domains in the migration of vascular smooth muscle cells into fibrin gels

The structure of fibrin plays an important role in the organization of thrombi, the development of atherosclerosis, and restenosis after PTCA. In this study, we examined the mechanisms of the migration of vascular smooth muscle cells (SMCs) into fibrin gels, using an in vitro assay system. Cultured SMCs from bovine fetal aortic media migrated into fibrin gels prepared with thrombin, which cleaves both fibrinopeptides A and B from fibrinogen, without other chemotactic stimuli. Both desA fibrin gels prepared with batroxobin, which cleaves only fibrinopeptide A, and desB fibrin gels prepared with Agkistrodon contortrix thrombin-like enzyme (ACTE), which cleaves only fibrinopeptide B, similarly induced the migration of SMCs compared to fibrin gels prepared with thrombin. These results suggest that the cleavage of fibrinopeptides is not necessary, but rather that the three-dimensional structure of the gel may be important for the migration of SMCs. Furthermore, gels prepared with protamine sulfate, which forms fibrin-like gels non-enzymatically, similarly induced the migration of SMCs compared to the gels prepared with thrombin. Both anti-fibrin(ogen) fragment D and anti-fibrin(ogen) E antibodies inhibited the migration of SMCs into fibrin gels, suggesting that both the D and E domains of fibrin(ogen) are involved in the migration of SMCs into fibrin gels. The addition of GRGDS, a synthetic RGD-containing peptide, but not that of GRGES, a control peptide, partially inhibited the migration of SMCs into fibrin gels, suggesting that the migration of SMCs into fibrin gels is at least in part dependent on the RGD-containing region of the alpha chain. The migration of SMCs into fibrin gels was also inhibited by a monoclonal antibody for integrin alpha v beta 3 and alpha 5 beta 1, indicating that migration is dependent on these integrins. Furthermore, both fibrin(ogen) fragments D and E inhibited the migration of SMCs into fibrin gels, suggesting that these fragments, generated during fibrino(geno)lysis, may be relevant in the regulation of SMC migration into fibrin gels.     

Computational predictions of cysteine cathepsin‐mediated fibrinogen proteolysis

Fibrin clot formation is a proteolytic cascade of events with thrombin and plasmin identified as the main proteases cleaving fibrinogen precursor, and the fibrin polymer, respectively. Other proteases may be involved directly in fibrin(ogen) cleavage, clot formation, and resolution, or in the degradation of fibrin‐based scaffolds emerging as useful tools for tissue engineered constructs. Here, cysteine cathepsins are investigated for their putative ability to hydrolyze fibrinogen, since they are potent proteases, first identified in lysosomal protein degradation and known to participate in extracellular proteolysis. To further explore this, we used two independent computational technqiues, molecular docking and bioinformatics sequence analysis (PACMANS), to predict potential binding interactions and sites of hydrolysis between cathepsins K, L, and S and fibrinogen. By comparing the results from these two objective, computational methods, it was determined that cathepsins K, L, and S do bind and cleave fibrinogen α, β, and γ chains at similar and unique sites. These differences were visualized experimentally by the unique cleaved fibrinogen banding patterns after incubation with each of the cathepsins, separately. In conclusion, human cysteine cathepsins K, L, and S are a new class of proteases that should be considered during fibrin(ogen) degradation studies both for disease processes where coagulation is a concern, and also in the implementation and design of bioengineered systems.     

Cloning of fibrinolytic enzyme gene from Bacillus subtilis isolated from Cheonggukjang and its expression in protease-deficient Bacillus subtilis strains

Bacillus subtilis CH3-5 was isolated from cheonggukjang prepared according to traditional methods. CH3-5 secreted at least four different fibrinolytic proteases (63, 47, 29, and 20 kDa) into the culture medium. A fibrinolytic enzyme gene, aprE2, encoding a 29 kDa enzyme was cloned from the genomic DNA of CH3-5, and the DNA sequence determined. aprE2 was overexpressed in heterologous B. subtilis strains deficient in extracellular proteases using a E. coli-Bacillus shuttle vector. A 29 kDa AprE2 band was observed and AprE2 seemed to exhibit higher activities towards fibrin rather than casein.     

Lack of plasminogen leads to milk stasis and premature mammary gland involution during lactation

The extracellular serine protease, plasmin, is activated from its precursor, plasminogen (Plg), by the urokinase-type and tissue-type Plg activators (uPA and tPA respectively). One of the main plasmin substrates, fibrin, is formed from fibrinogen via thrombin activity. We have previously shown that mice deficient for Plg are strikingly less able to support a litter during lactation compared to wild type mice. Here we suggest a mechanism responsible for this lactation defect. Reduced epithelial content and increased apoptosis are observed in Plg-deficient mammary glands at lactation day 7. Immunofluorescence analysis reveals the presence of fibrin(ogen) in the stroma surrounding mammary alveoli and adipocytes and identifies fibrin(ogen) as a component of breast milk in both wild type and Plg-deficient mice. Furthermore, a large accumulation of fibrin(ogen) together with apoptotic epithelial cells is observed in the lactating mammary alveoli and ducts of some Plg-deficient mice. This suggests that fibrin plays a key role in the malfunction of mammary glands in the absence of Plg, possibly through blockade of mammary ducts inducing milk stasis, inhibiting milk expulsion and thereby inducing premature apoptosis and involution.     

A new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine proteinase from Vipera lebetina snake venom

Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin-agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-l-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, Aα-chain and more slowly Bβ-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms.     

Human rhinovirus 3C protease: a cysteine protease showing the trypsin(ogen)-like fold

Viral-encoded proteases cleave precursor polyprotein(s) leading to maturation of infectious virions. Strikingly, human rhinovirus 3C protease shows the trypsin(ogen)-like serine protease fold based on two topologically equivalent six-stranded β-barrels, but displays residue Cys147 as the active site nucleophile. By contrast, papain, which is representative of most cysteine proteases, does not display the trypsin(ogen)-like fold. Remarkably, in human rhinovirus 3C cysteine protease, the catalytic residues Cys147, His40 and Glu71 are positioned as Ser195, His57 and Asp102, respectively, building up the catalytic triad of serine proteases in the chymotrypsin–trypsin–elastase family. However, as compared to trypsin-like serine proteases and their zymogens, residue His40 and the oxyanion hole of human rhinovirus 3C cysteine protease, both key structural components of the active site, are located closer to the protein core. Human rhinovirus 3C cysteine protease cleaves preferentially GlnGly peptide bonds or, less commonly, the GlnSer, GlnAla, GluSer or GluGly pairs. Finally, human rhinovirus 3C cysteine protease and the 3CD cysteine protease–polymerase covalent complex bind the 5′ non-coding region of rhinovirus genomic RNA, an essential function for replication of the viral genome.     

Evidence that catalytically-inactivated thrombin forms non-covalently linked dimers that bridge between fibrin/fibrinogen fibers and enhance fibrin polymerization

Phe-pro-arg-chloromethyl ketone-inhibited alpha-thrombin [FPR alpha-thr] retains its fibrinogen recognition site (exosite 1), augments fibrin/fibrinogen [fibrin(ogen)] polymerization, and increases the incorporation of fibrin into clots. There are two 'low-affinity' thrombin-binding sites in each central E domain of fibrin, plus a non-substrate 'high affinity' gamma' chain thrombin-binding site on heterodimeric 'fibrin(ogen) 2' molecules (gamma(A), gamma'). 'Fibrin(ogen) 1' (gamma(A), gamma(A)) containing only low-affinity thrombin-binding sites, showed concentration-dependent FPR alpha-thr enhancement of polymerization, thus indicating that low-affinity sites are sufficient for enhancing polymerization. FPR gamma-thr, whose exosite 1 is non-functional, did not enhance polymerization of either fibrin(ogen)s 1 or 2 and DNA aptamer HD-1, which binds specifically to exosite 1, blocked FPR alpha-thr enhanced polymerization of both types of fibrin(ogen) (1>2). These results showed that exosite 1 is the critical element in thrombin that mediates enhanced fibrin polymerization. Des B beta 1-42 fibrin(ogen) 1, containing defective 'low-affinity' binding sites, was subdued in its FPR alpha-thr-mediated reactivity, whereas des B beta 1-42 fibrin(ogen) 2 (gamma(A), gamma') was more reactive. Thus, the gamma' chain thrombin-binding site contributes to enhanced FPR alpha-thr mediated polymerization and acts through a site on thrombin that is different from exosite 1, possibly exosite 2. Overall, the results suggest that during fibrin clot formation, catalytically-inactivated FPR alpha-thr molecules form non-covalently linked thrombin dimers, which serve to enhance fibrin polymerization by bridging between fibrin(ogen) molecules, mainly through their low affinity sites.     

Purification and characterization of a new fibrinolytic enzyme of Bacillus licheniformis KJ-31, isolated from Korean traditional Jeot-gal

Jeot-gal is a traditional Korean fermented seafood and has long been used for seasoning. We isolated 188 strains from shrimp, anchovy, and yellow corvina Jeot-gal, and screened sixteen strains that showed strong fibrinolytic activities on a fibrin plate. Among those strains, the strain that had the largest halo zone was chosen and identified as Bacillus licheniformis by using 16S rDNA sequencing and an API CHB kit. The fibrinolytic activity of Bacillus licheniformis was characterized and designated as bpKJ-31. The active component of bpKJ-31 was identified as a 37 kDa protein, designated bacillopeptidase F, by internal peptide mapping and N-terminal sequencing. The optimum activity of bpKJ-31 was shown at pH 9 and 40 degrees C, with a chromogenic substrate for plasmin. It had high degrading activity for the Bbeta-chain and Aalpha-chain of fibrin(ogen), and also acted on thrombin, but not skim milk and casein. The amidolytic activity of bpKJ-31 was inhibited by 1 mM phenylmethanesulfonyl fluoride, but 1 mM EDTA did not affect the enzyme activity, indicating that bpKJ-31 is an alkaline serine protease, like a plasmin. The bpKJ-31 showed approximately 14.3% higher fibrinolytic activity than the plasmin. These features of bpKJ-31 make it attractive as a health-promoting biomaterial.     

Bacterial plasminogen activators and receptors

Invasive bacterial pathogens intervene at various stages and by various mechanisms with the mammalian plasminogen/plasmin system. A vast number of pathogens express plasmin(ogen) receptors that immobilize plasmin(ogen) on the bacterial surface, an event that enhances activation of plasminogen by mammalian plasminogen activators. Bacteria also influence secretion of plasminogen activators and their inhibitors from mammalian cells. The prokaryotic plasminogen activators streptokinase and staphylokinase form a complex with plasmin(ogen) and thus enhance plasminogen activation. The Pla surface protease of Yersinia pestis resembles mammalian activators in function and converts plasminogen to plasmin by limited proteolysis. In essence, plasminogen receptors and activators turn bacteria into proteolytic organisms using a host-derived system. In Gram-negative bacteria, the filamentous surface appendages fimbriae and flagella form a major group of plasminogen receptors. In Gram-positive bacteria, surface-bound enzyme molecules as well as M-protein-related structures have been identified as plasminogen receptors, the former receptor type also occurs on mammalian cells. Plasmin is a broad-spectrum serine protease that degrades fibrin and noncollagenous proteins of extracellular matrices and activates latent procollagenases. Consequently, plasmin generated on or activated by Haemophilus influenzae, Salmonella typhimurium, Streptococcus pneumoniae, Y. pestis, and Borrelia burgdorferi has been shown to degrade mammalian extracellular matrices. In a few instances plasminogen activation has been shown to enhance bacterial metastasis in vitro through reconstituted basement membrane or epithelial cell monolayers. In vivo evidence for a role of plasminogen activation in pathogenesis is limited to Y. pestis, Borrelia, and group A streptococci. Bacterial proteases may also directly activate latent procollagenases or inactivate protease inhibitors of human plasma, and thus contribute to tissue damage and bacterial spread across tissue barriers.     

Interaction of fibrin(ogen) with heparin: further characterization and localization of the heparin-binding site

The beta chain 15-42 sequence of the fibrin(ogen) E region was implicated in heparin binding [Odrljin et al. (1996) Blood 88, 2050-2061]; whether heparin binds to other fibrin(ogen) regions remains to be clarified. To address this question, we studied the interaction of heparin with fibrinogen, fibrin, and their major fragments D(1), D-D, E(1), E(3), and alphaC, which together cover the entire structure of the molecule, by ligand blotting, surface plasmon resonance, and fluorescence. All three techniques revealed that at physiological ionic conditions only fibrin(ogen) and the E(1) fragment bind heparin, indicating that the only physiologically relevant heparin-binding site of fibrin(ogen) is located in its E region. To test whether the beta15-42 sequence is sufficient to form this site or some additional sequences are also involved, we tested the interaction of heparin with a number of beta15-42-containing fragments. The synthetic beta15-42 peptide bound heparin weakly (K(d) = 44.5 microM) while the recombinant beta15-57 and beta15-64 fragments exhibited almost 7-fold higher affinity (K(d) = 6.4 and 7.1 microM, respectively), indicating that the beta43-57 region is also important for heparin binding. At the same time the recombinant dimeric disulfide-linked (beta15-66)(2) fragment which mimics the dimeric arrangement of the beta chains in fibrin bound heparin with high affinity (K(d) = 66 nM), almost 100-fold higher than that for the monomeric fragments. This affinity was similar to those determined for fibrin and the E(1) fragment (K(d) = 72 and 70 nM, respectively) suggesting that (beta15-66)(2) mimics well the heparin-binding properties of the latter two. Altogether, these results indicate that the only heparin-binding site in fibrin(ogen) is formed by NH(2)-terminal portions of the beta chains, including residues beta15-57, and that dimerization is essential for high-affinity binding.     

Identification and characterisation of the excreted/secreted serine proteases of larvae of the old world screwworm fly, Chrysomya bezziana

Serine proteases are the major proteolytic activity excreted or secreted from Chrysomya bezziana larvae as demonstrated by gelatin gel analyses and the use of specific substrates, benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Serine proteases were identified through their inhibition by 4-(2-aminoethyl)-benzene sulphonyl fluoride and classified as trypsin- and chymotrypsin-like on the basis of inhibition by tosyl-L-lysine chloromethyl ketone and tosyl-L-phenylalanine chloromethyl ketone, respectively. Like most insect serine proteases, the C. bezziana enzymes were active over broad pH range from mildly acidic to alkaline. The excreted or secreted serine proteases were purified by affinity chromatography using soybean trypsin inhibitor. A different subset of the serine proteases was isolated by salt elution from washed larval peritrophic matrices. Amino-terminal sequencing identified both trypsin and chymotrypsin-like sequences in the excreted or secreted pool with the latter being the dominant protease, whereas trypsin was the dominant species in the peritrophic matrix eluant. These results suggest that trypsin was possibly preferably adsorbed by the peritrophic matrix and may act as a final proteolytic processing stage as partially digested and ingested polypeptides pass through the peritrophic matrix. Immunoblot analysis on dissected gut tissues indicated that the anterior and posterior midguts were the main source of the serine proteases, although a novel species of 32 kDa was predominantly associated with the peritrophic matrix. Proteases are a target for a partially protective immune response and understanding the complexity of the secreted and digestive proteases is a necessary part of understanding the mechanism of the host's immunological defence against the parasite.     

Structural and functional diversities in lepidopteran serine proteases

Primary protein-digestion in Lepidopteran larvae relies on serine proteases like trypsin and chymotrypsin. Efforts toward the classification and characterization of digestive proteases have unraveled a considerable diversity in the specificity and mechanistic classes of gut proteases. Though the evolutionary significance of mutations that lead to structural diversity in serine proteases has been well characterized, detailing the resultant functional diversity has continually posed a challenge to researchers. Functional diversity can be correlated to the adaptation of insects to various host-plants as well as to exposure of insects to naturally occurring antagonistic biomolecules such as plant-derived protease inhibitors (PIs) and lectins. Current research is focused on deciphering the changes in protease specificities and activities arising from altered amino acids at the active site, specificity-determining pockets and other regions, which influence activity. Some insight has been gained through in silico modeling and simulation experiments, aided by the limited availability of characterized proteases. We examine the structurally and functionally diverse Lepidopteran serine proteases, and assess their influence on larval digestive processes and on overall insect physiology. Invited paper