Prophenoloxidase-activating proteinase-3 (PAP-3) from Manduca sexta hemolymph: a clip-domain serine proteinase regulated by serpin-1J and serine proteinase homologs

Phenoloxidase (PO) is a key enzyme implicated in several defense mechanisms in insects and crustaceans. It is converted from prophenoloxidase (proPO) through limited proteolysis by prophenoloxidase-activating proteinase (PAP). We previously isolated PAP-1 from integument and PAP-2 from hemolymph of the tobacco hornworm, Manduca sexta. Here, we report the purification, characterization, and regulation of PAP-3 from the hemolymph. Similar to M. sexta PAP-2, PAP-3 consists of two amino-terminal clip domains followed by a carboxyl-terminal catalytic domain, whereas PAP-1 contains only one clip domain at its amino-terminus. Purified PAP-3 cleaved proPO at Arg51 and generated a low level of PO activity. However, the enzyme efficiently activated proPO when M. sexta serine proteinase homolog-1 and -2 were present. These proteinase-like proteins associate with immulectin-2, a pattern-recognition receptor for lipopolysaccharide. M. sexta PAP-3 was inhibited by recombinant serpin-1J, which formed an SDS-stable complex with the enzyme. PAP-3 mRNA was detected at a low level in the fat body or hemocytes of naive larvae, but was elevated in insects that had been challenged with bacteria. These data, along with our previous results on PAP-1 and PAP-2, indicate that proPO activation by PAPs is a tightly regulated process. Individual PAPs could play different roles during immune responses and developmental processes.     

Heat stable proteinase from Thermomonospora fusca. Characterization as a serine proteinase

An extracellular proteinase secreted by the thermophilic bacteria Thermomonospora fusca YX (YX-proteinase) is a serine proteinase as shown by its inactivation by the site specific reagents, phenylmethanesulfonyl fluoride, dansyl fluoride, and carbobenzoxy-L-phenylalanine chloromethyl ketone. This conclusion is further supported by the effect of various proteinase inhibitors on its activity. The activity of the proteinase toward small synthetic ester substrates shows that the enzyme has a primary specificity for the aromatic and hydrophobic amino acids. The amino acid composition and NH2-terminal sequence, as well as its size, suggest that the enzyme is related to the chymotrypsin-like microbial proteinase, alpha-lytic protease from Myxobacter 495 and protease A and B from Streptomyces griseus.     

Purification and characterization of Manduca sexta serpin-6: a serine proteinase inhibitor that selectively inhibits prophenoloxidase-activating proteinase-3

The proteolytic activation of prophenoloxidase (proPO) is a critical defense mechanism in insects and crustaceans. We have isolated three prophenoloxidase-activating proteinases (PAPs) from cuticular extracts or hemolymph of Manduca sexta pharate pupae, which are negatively regulated by serpin-1J and serpin-3. To test if other serpins may also inhibit the PAPs, we fractionated the induced hemolymph by ammonium sulfate precipitation, gel filtration, and lectin affinity chromatography. A 47 kDa protein, designated M. sexta serpin-6, was identified in concanavalin A-bound fractions, which formed an SDS-stable complex with PAP-3. This inhibitor, not recognized by the serpin-1 or serpin-3 antibodies, was further purified on HPLC anion exchange and hydroxylapatite columns. The molecular mass and isoelectric point of serpin-6 were found to be 46,710 +/- 10 Da and 5.4. While its amino terminus was blocked, we obtained five internal peptide sequences, one of which is highly similar to M. sexta serpins-1, -2, and -3. Serpin-6 strongly inhibited PAP-3 but not PAP-1 or PAP-2, suggesting that the proPO activation by PAPs is differentially regulated by multiple serpins. When included in the reaction mixture containing proPO, PAP-3, and its cofactor, serpin-6 efficiently blocked the cleavage activation of proPO.     

Characterization of a serine proteinase mediating encystation of Acanthamoeba

Members of the genus Acanthamoeba, amphizoic protozoan parasites, are causative agents of granulomatous amoebic encephalitis and amoebic keratitis. Proteinases play a role in various biologic actions in Acanthamoeba, including host tissue destruction, pathogenesis, and digestion of phagocytosed food. Interestingly, we found that encystation of Acanthamoeba was inhibited by the serine proteinase inhibitor phenylmethanesulfonyl fluoride. In this study, we characterize a serine proteinase that is involved in mediating the encystation of Acanthamoeba. This encystation-mediating serine proteinase (EMSP) is shown to be highly expressed during encystation by real-time PCR and Western blot analysis. Chemically synthesized small interfering RNA against EMSP inhibited the expression of EMSP mRNA and significantly reduced the encystation efficiency of Acanthamoeba. An EMSP-enhanced green fluorescent protein fusion protein localized to vesicle-like structures within the amoeba. Using LysoTracker analysis, these vesicular structures were confirmed to be lysosomes. After incubation of the transfected amoeba in encystment media, small fluorescent vesicle-like structures gathered and formed ball-like structures, which were identified as colocalizing with the autophagosome. Taken together, these results indicate that EMSP plays an important role in the differentiation of Acanthamoeba by promoting autolysis.     

Prophenoloxidase-activating proteinase-2 from hemolymph of Manduca sexta. A bacteria-inducible serine proteinase containing two clip domains

Proteolytic activation of prophenoloxidase in insects is a component of the host defense system against invading pathogens and parasites. We have purified from hemolymph of the tobacco hornworm, Manduca sexta, a new serine proteinase that cleaves prophenoloxidase. This enzyme, designated prophenoloxidase-activating proteinase-2 (PAP-2), differs from another PAP, previously isolated from integuments of the same insect (PAP-1). PAP-2 contains two clip domains at its amino terminus and a catalytic domain at its carboxyl terminus, whereas PAP-1 has only one clip domain. Purified PAP-2 cleaved prophenoloxidase at Arg(51) but yielded a product that has little phenoloxidase activity. However, in the presence of two serine proteinase homologs, active phenoloxidase was generated at a much higher level, and it formed covalently linked, high molecular weight oligomers. The serine proteinase homologs associate with a bacteria-binding lectin in M. sexta hemolymph, indicating that they may be important for ensuring that the activation of prophenoloxidase occurs only in the vicinity of invading microorganisms. PAP-2 mRNA was not detected in naive larval fat body or hemocytes, but it became abundant in these tissues after the insects were injected with bacteria.     

Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530.

A serine protease from the keratin-degrading Streptomyces pactum DSM 40530 was purified by casein agarose affinity chromatography. The enzyme had a molecular weight of 30,000 and an isoelectric point of 8.5. The proteinase was optimally active in the pH range from 7 to 10 and at temperatures from 40 to 75 degrees C. The enzyme was specific for arginine and lysine at the P1 site and for phenylalanine and arginine at the P1' site. It showed a high stereoselectivity and secondary specificity with different synthetic substrates. The keratinolytic activity of the purified proteinase was examined by incubation with the insoluble substrates keratin azure, feather meal, and native and autoclaved chicken feather downs. The S. pactum proteinase was significantly more active than the various commercially available proteinases. After incubation with the purified proteinase, a rapid disintegration of whole feathers was observed. But even after several days of incubation with repeated addition of enzymes, less than 10% of the native keratin substrate was solubilized. In the presence of dithiothreitol, degradation was more than 70%.     

Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species.

The gene encoding a subtilisin-like serine proteinase in the psychrotrophic Vibrio sp. PA44 has been successfully cloned, sequenced and expressed in Escherichia coli. The gene is 1593 basepairs and encodes a precursor protein of 530 amino acid residues with a calculated molecular mass of 55.7 kDa. The enzyme is isolated, however, as an active 40.6-kDa proteinase, without a 139 amino acid residue N-terminal prosequence. Under mild conditions the enzyme undergoes a further autocatalytic cleavage to give a 29.7-kDa proteinase that retains full enzymatic activity. The deduced amino acid sequence of the enzyme has high homology to proteinases of the proteinase K family of subtilisin-like proteinases. With respect to the enzyme characteristics compared in this study the properties of the wild-type and recombinant proteinases are the same. Sequence analysis revealed that especially with respect to the thermophilic homologues, aqualysin I from Thermus aquaticus and a proteinase from Thermus strain Rt41A, the cold-adapted Vibrio-proteinase has a higher content of polar/uncharged amino acids, as well as aspartate residues. The thermophilic enzymes had a higher content of arginines, and relatively higher number of hydrophobic amino acids and a higher aliphatic index. These factors may contribute to the adaptation of these proteinases to different temperature conditions.     

Structural and functional properties of a novel serine protease inhibiting peptide family in arthropods

Recently, several arthropod peptides that belong to a new serine protease inhibitor family were discovered. Three members (HI, PMP-D2=LMCI-1 and PMP-C=LMCI-2) were isolated from the migratory locust, Locusta migratoria. Five additional members (SGPI-1-5) were identified in the desert locust Schistocerca gregaria, and a heterodimeric serine protease inhibitor (pacifastin) was isolated from the hemolymph of the crayfish Pacifastacus leniusculus. The light chain of pacifastin constitutes the inhibitory subunit that has nine cysteine-rich domains (PLDs) that are homologous with the locust inhibitors. These locust inhibitors and PLDs share a conserved array of six cysteine residues (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa(3)-Cys), which are involved in an identical disulfide bridge pattern (Cys(1)-Cys(4), Cys(2)-Cys(6), Cys(3)-Cys(5)). The solution structures of LMCI-1 and LMCI-2 showed a similar, compact, globular folding, which is unique within the group of the small 'canonical' inhibitors. Moreover, the reactive site, including the P1-P'1 bond was thoroughly investigated by means of synthetic variants. However, the biological function(s) of the locust inhibitors is (are) not fully understood. LMCI-1 and LMCI-2 were shown to inhibit the endogenous proteolytic activating cascade of prophenoloxidase. Northern blot analysis indicated that the genes encoding the SGPI precursors are differentially expressed in a time-, stage- and hormone-dependent manner.     

Matrix degrading properties of sperm serine proteinase, acrosin.

The serine proteinase acrosin plays an important role in sperm penetration of the zona pellucida. In the present study we investigated the effect of the enzyme on various matrix proteins. Acrosin degraded proteolytically fibronectin, type IV collagen and heat denatured type I collagen, whereas neither native type I collagen nor laminin were cleaved by the enzyme. The specific activity of acrosin with type IV collagen as substrate (66.6 g/h/g) was 125-fold higher than that of known type IV collagenase or stromelysin. These results suggest that acrosin may act as a matrix-degrading proteinase.     

An unusual specificity in the activation of neutrophil serine proteinase zymogens

The majority of proteinases exist as zymogens whose activation usually results from a single proteolytic event. Two notable exceptions to this generalization are the serine proteinases neutrophil elastase (HNE) and cathepsin G (cat G), proteolytic enzymes of human neutrophils that are apparently fully active in their storage granules. On the basis of amino acid sequences inferred from the gene and cDNAs encoding these enzymes, it is likely that both are synthesized as precursors containing unusual C-terminal and N-terminal peptide extensions absent from the mature proteins. We have used biosynthetic radiolabeling and radiosequencing techniques to identify the kinetics of activation of both proteinases in the promonocyte-like cell line U937. We find that both N- and C-terminal extensions are removed about 90 min after the onset of synthesis, resulting in the activation of the proteinases. HNE and cat G are, therefore, transiently present as zymogens, presumably to protect the biosynthetic machinery of the cell from adventitious proteolysis. Activation results from cleavage following a glutamic acid residue to give an activation specificity opposite to those of almost all other serine proteinase zymogens, but shared, possibly, by the "granzyme" group of related serine proteinases present in the killer granules of cytotoxic T-lymphocytes and rat mast cell proteinase II.     

Isolation and properties of serine proteinase from Aspergillus oryzae

A serine proteinase having an activity optimum at pH 6.7-8.2 has been isolated from amylorisine P-10x (a mixture of Aspergillus oryzae enzymes) by chromatography on DEAE-Sephadex A-50 and bacitracin Sepharose 4B. The proteinase is fully inactivated by phenylmethylsulfonylfluoride and diisopropylfluorophosphonate, the specific inhibitors of the enzyme, and has a pI at pH 7.5. The molecular mass of serine proteinase is 30000 Da; its amino acid composition appears as: Met2, Asp33, Thr18, Ser29, Glu21, Pro9, Glu32, Ala38, Val24, Ile16, Leu15, Tyr8, Phe8, His8, Lys18, Arg4, Trp6. The N-terminal sequence of the serine proteinase: Gly-Leu-Thr-Thr-Gln-Lys-Ser-Ala-Pro-Trp-Gly-Leu-Gly-Ser-Ile-Ser-Xaa-Lys- Gly-Gln-Gln-Ser-Thr-Asp-Tyr-Ile-Tyr, which coincides practically completely with the corresponding sequence of alkaline proteinase of A. oryzae, ATCC20386, has been determined. Similar to subtilisin, the enzyme catalyzes the condensation of leucine and alanine p-nitroanilides with N-benzyloxycarbonyl-alanyl-alanine and glycyl-alanine methyl esters.     

Characterization of a collagenolytic serine proteinase from the Atlantic cod (gadus morhua)

A collagenolytic proteinase was purified from the intestines of Atlantic cod by (NH₄)₂SO₄ fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). The proteinase has an estimated molecular weight of 24.1 (±0.5) kDa as determined by SDS-PAGE and belongs to the chymotrypsin family of serine proteinases. The enzyme cleaves native collagen types I, III, IV and V, and also readily hydrolyzes succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin, as well as succinyl-l-Ala-l-Ala-l-Pro-l-Leu-p-nitroanilide, a reported elastase substrate, but had no detectable activity towards several other substrates of these proteinases or of trypsin. The pH optimum of the enzyme was between pH 8.0 and 9.5 and it was unstable at pH values below 7. Maximal activity of the enzyme when assayed against sAAPFpna was centered between 45 and 50°C. Calcium binding stabilized the cod collagenase against thermal inactivation, but even in the presence of calcium, the enzyme was unstable at temperatures above 30°C.     

Structural and functional characteristics of plant proteinase inhibitor-II (PI-II) family

Plant proteinase inhibitor-II (PI-II) proteins are one of the promising defensive proteins that helped the plants to resist against different kinds of unfavorable conditions. Different roles for PI-II have been suggested such as regulation of endogenous proteases, modulation of plant growth and developmental processes and mediating stress responses. The basic knowledge on genetic and molecular diversity of these proteins has provided significant insight into their gene structure and evolutionary relationships in various members of this family. Phylogenetic comparisons of these family genes in different plants suggested that the high rate of retention of gene duplication and inhibitory domain multiplication may have resulted in the expansion and functional diversification of these proteins. Currently, a large number of transgenic plants expressing PI-II genes are being developed for enhancing the defensive capabilities against insects, bacteria and pathogenic fungi. Much emphasis is yet to be given to exploit this ever expanding repertoire of genes for improving abiotic stress resistance in transgenic crops. This review presents an overview about the current knowledge on PI-II family genes, their multifunctional role in plant defense and physiology with their potential applications in biotechnology.     

Poliovirus-encoded proteinase 3C: a possible evolutionary link between cellular serine and cysteine proteinase families

Here we demonstrate significant similarities between the amino acid sequences of trypsin (a serine protease) and the N-terminal piece of a specific fragment of the poliovirus polyprotein encompassing the sequence of the viral proteinase 3C, and also between cathepsin H (a cysteine protease) and the C-terminal piece of the same fragment. A coherent alignment of the sequences of the 3 proteases was obtained, in which the principal catalytically active residues occupy identical positions. A hypothesis is proposed that the viral enzyme may provide an evolutionary link between serine and cysteine protease families.     

Proapoptotic effects of NARC 1 (= PCSK9), the gene encoding a novel serine proteinase.

BACKGROUND: NARC 1/PCSK9 encodes a novel serine proteinase known to play a role in cholesterol homeostasis. NARC 1 mRNA expression in cerebellar granule neurons (CGNs) was discovered to be induced following an apoptotic injury. Coregulation of known apoptotic mediators (caspase-3 and death receptor 6) raises the possibility that NARC 1 might be involved in the propagation of apoptotic signaling in neurons. METHODS: CGNs were transfected with EGFP-fusion constructs of wild-type and mutant NARC 1, and a laser scanning cytometry-based method of scoring cell death in transfectants was applied. Use of the poly-caspase inhibitor BAF allowed assessment of the caspase-dependence of the NARC 1 proapoptotic effect. RESULTS: Wild-type NARC 1 was found to have substantial proapoptotic effects that were only partially reversible by BAF. Mutation of the active site serine or deletion of the catalytic domain resulted in a reduced level of cell death, consistent with loss of the BAF-sensitive component of cell death. NH(2)-terminal deletion constructs of NARC 1 had effects similar to wild-type, both in the absence and presence of BAF, whereas expression of COOH-terminal deletion mutants produced a rate of cell death similar to wild-type in the absence of BAF treatment, but which lacked the capacity to be reduced by treatment with BAF. CONCLUSION: The mechanism by which NARC 1-EGFP over-expression induces cell death in cultured CGNs remains unclear. Mutation analysis established a positive correlation between the presence of the Narc 1 active site serine in the transiently expressed protein and induction of the BAF-sensitive component of the cell death phenotype. A caspase-independent component proved sufficiently complex to map discretely within the Narc 1 protein.     

Conformational properties of serine proteinase inhibitors (serpins) confer multiple pathophysiological roles

Serine proteinase inhibitors (Serpins) are irreversible suicide inhibitors of proteases that regulate diverse physiological processes such as coagulation, fibrinolysis, complement activation, angiogenesis, apoptosis, inflammation, neoplasia and viral pathogenesis. The molecular structure and physical properties of serpins permit these proteins to adopt a number of variant conformations under physiological conditions including the native inhibitory form and several inactive, non-inhibitory forms, such as complexes with protease or other ligands, cleaved, polymerised and oxidised. Alterations of a serpin which affect its structure and/or secretion and thus reduce its functional levels may result in pathology. Serpin dysfunction has been implicated in thrombosis, emphysema, liver cirrhosis, immune hypersensitivity and mental disorders. The loss of inhibitory activity of serpins necessarily results in an imbalance between proteases and their inhibitors, but it may also have other physiological effects through the generation of abnormal concentrations of modified, non-inhibitory forms of serpins. Although these forms of inhibitory serpins are detected in tissues and fluids recovered from inflammatory sites, the important questions of which conditions result in generation of different molecular forms of serpins, what biological function these forms have, and which of them are directly linked to pathologies and/or may be useful markers for characterisation of disease states, remain to be answered. Elucidation of the biological activities of non-inhibitory forms of serpins may provide useful insights into the pathogenesis of diseases and suggest new therapeutic strategies.     

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     

Characterization of homogeneous atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor.

We previously reported the discovery and partial characterization of bovine atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor, which is associated with atrial granule membranes. We now report the physicochemical properties of electrophoretically homogeneous enzyme purified by a series of chromatography steps from a subcellular fraction enriched for atrial granules. The enzyme tends to associate during purification to higher molecular weight species, but SDS-PAGE analysis reveals a single polypeptide chain of molecular weight 70,000. The enzyme is activated 2-3 fold by Ca+2 and 1.5-fold by Mg+2 and is nearly 100% inhibited by Zn+2 or Co+2. Thus, the enzyme can be considered a calcium activated, neutral pH, serine proteinase. Based on the hydrolysis of numerous synthetic peptide substrates, the recognition sequence for the enzyme within the pro-hormone has been mapped to A96PRSLRR102; cleavage occurs at the Arg98-Ser99 bond yielding bioactive atrial natriuretic peptide directly from the pro-hormone. The doublet of basic amino acids is part of the recognition sequence but is not the primary cleavage site. It is our hypothesis that the processing site sequence acts as a recognition element for the endoproteinase and resides at the surface of the pro-hormone and thus contributes to the molecular basis for limited proteolysis.     

Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity.

The RNA polymerase gene of human coronavirus (HCV) 229E encodes a large polyprotein that contains domains with motifs characteristic of both papain-like cysteine proteinases and proteinases with homology to the 3C proteinase of picornaviruses. In this study, we have, first, expressed the putative HCV 229E 3C-like proteinase domain as part of a beta-galactosidase fusion protein in Escherichia coli and have shown that the expressed protein has proteolytic activity. The substitution of one amino acid within the predicted proteinase domain (His-3006-->Asp-3006) abolishes, or at least significantly reduces, this activity. Amino-terminal sequence analysis of a purified, 34-kDa cleavage product shows that the bacterial fusion protein is cleaved at the dipeptide Gln-2965-Ala-2966, which is the predicted amino-terminal end of the putative 3C-like proteinase domain. Second, we have confirmed the proteolytic activity of a bacterially expressed polypeptide with the amino acid sequence of the predicted HCV 229E 3C-like proteinase by trans cleavage of an in vitro translated polypeptide encoded within open reading frame 1b of the RNA polymerase gene. Finally, using fusion protein-specific antiserum, we have identified a 34-kDa, 3C-like proteinase polypeptide in HCV 229E-infected MRC-5 cells. This polypeptide can be detected as early as 3 to 5 h postinfection but is present in the infected cell in very low amounts. These data contribute to the characterization of the 3C-like proteinase activity of HCV 229E.     

Cloning of the cDNA of a human neutrophil bactericidal protein. Structural and functional correlations

The bactericidal permeability increasing protein (BPI) is a 50-60-kDa membrane-associated protein isolated from granules of polymorphonuclear leukocytes. A full-length cDNA clone encoding human BPI has been isolated and the derived amino acid sequence reveals a structure that is consistent with previously determined biological properties. BPI may be organized into two domains: the amino-terminal half, previously shown to contain all known antimicrobial activity, contains a large fraction of basic and hydrophilic residues. In contrast, the carboxyl-terminal half contains more acidic than basic residues and includes several potential transmembrane regions which may anchor the holoprotein in the granule membrane. The cytotoxic action of BPI is limited to many species of Gram-negative bacteria; this specificity may be explained by a strong affinity of the very basic aminoterminal half for the negatively charged lipopolysaccharides that are unique to the Gram-negative bacterial envelope. The amino-terminal end of BPI exhibits significant similarity with the sequence of a rabbit lipopolysaccharide-binding protein, suggesting that both molecules share a similar structure for binding lipopolysaccharides.