A salivary serine protease of the haematophagous reduviid Panstrongylus megistus: sequence characterization,expression pattern and characterization of proteolytic activity

A cDNA encoding a trypsin‐like protease from the salivary glands of the haematophagous reduviid Panstrongylus megistus was cloned and sequenced. The deduced protein sequence showed similarities to serine proteases of other hemipterans but with substitutions in the catalytic triad and the substrate binding site. The expression of the gene increased more than sixfold after feeding. Saliva showed the highest proteolytic activity at neutral to slightly basic pH. Substrate and inhibitor profiles and zymography indicated the presence of a trypsin‐like protease with preference for Arg and Lys at P1. Using chromatography, a fibrinolytic enzyme was purified whose sequence was identified by tandem mass spectrometry as that encoded by the cDNA.     

An estimate of the number of serine protease genes expressed in sheep blowfly larvae (Lucilia cuprina)

A large and diverse family of serine protease genes was identified in first-instar larval cDNA of the sheep blowfly (Lucilia cuprina). This complex repertoire of genes was identified via a PCR approach using highly degenerate primers based on structurally conserved regions which surround the active site His and Ser residues found in all serine proteases. PCR products from entire first-instar larval cDNA, or from third-instar larval salivary glands or cardia, generated using a microscale RT-PCR method, were cloned into a plas-mid vector. Comparison of the restriction fragment patterns of PCR products generated from the three different sources suggests a highly diverse tissue-specific pattern of serine protease expression in this organism. Detailed analysis of the restriction fragment patterns of sixty-nine randomly selected clones from entire first-instar larvae revealed forty-nine different classes of PCR product. Maximum likelihood analysis of these data indicate that between 125 and 220 different serine protease genes are expressed in first-instar larvae of L. cuprina. DNA sequence analysis of ten randomly-selected clones, derived from the three tissue sources, indicated that all ten encoded serine protease gene fragments. A frequently occurring PCR product, generated from both first-instar total cDNA and third-instar cardia cDNA, showed 73% amino acid identity to a digestive protease expressed in Droso-phila melanogaster larval gut cells.     

Excretory/secretory chymotrypsin from Lucilia cuprina: purification, enzymatic specificity and amino acid sequence deduced from mRNA

Two chymotrypsin-like proteases were purified from the secretory and excretory material of first-instar larvae of Lucilia cuprina. The hydrolysis of N-succinyl-L-phenylalanine-nitroanilide was used to monitor the purification of these proteases which was achieved by affinity chromatography on soybean trypsin inhibitor-Sepharose followed by anion exchange and hydrophobic interaction chromatographies. The enzymatic specificity of the most abundant protease {Lucilia chymotrypsin b; LCTb) was further defined by determining the amino acid sequence of peptides released from insulin B chain after incubation with LCTb. Peptide amino acid sequences obtained from LCTb were used to design degenerate oligonucleotide primers which, in conjunction with the polymerase chain reaction, enabled cDNA coding for LCTb to be cloned and sequenced. The deduced amino acid sequence of LCTb showed many of the structural features of serine proteases as well as significant amino acid sequence homology with chymotrypsins from a diverse range of species. It is probable that LCTb plays an important role in establishing the myiasis-causing larvae of L. cuprina on host skin as well as providing nutrients for the rapidly growing larvae.     

Serine proteases identified from a Costelytra zealandica (White) (Coleoptera: Scarabaeidae) midgut EST library and their expression through insect development

Costelytra zealandica larvae are pests of New Zealand pastures causing damage by feeding on the roots of grasses and clovers. The major larval protein digestive enzymes are serine proteases (SPs), which are targets for disruption in pest control. An expressed sequence tag (EST) library from healthy, third instar larval midgut tissue was constructed and analysed to determine the composition and regulation of proteases in the C. zealandica larval midgut. Gene mining identified three trypsin-like and 11 chymotrypsin-like SPs spread among four major subgroups. Representative SPs were examined by quantitative PCR and enzyme activity assayed across developmental stages. The serine protease genes examined were expressed throughout feeding stages and downregulated in nonfeeding stages. The study will improve targeting of protease inhibitors and bacterial disruptors of SP synthesis.     

Cowpea bruchid Callosobruchus maculatus uses a three-component strategy to overcome a plant defensive cysteine protease inhibitor

The soybean cysteine protease inhibitor, soyacystatin N (scN), negatively impacts growth and development of the cowpea bruchid, Callosobruchus maculatus[Koiwa et al. (1998) Plant J 14: 371-379]. However, the developmental delay and feeding inhibition caused by dietary scN occurred only during the early developmental stages (the 1st, 2nd and 3rd instars) of the cowpea bruchid. The 4th instar larvae reared on scN diet (adapted) exhibited rates of feeding and development which were comparable to those feeding on an scN-free diet (unadapted) prior to pupation. Total gut proteolytic capacity at this larval stage significantly increased in the scN-adapted insects. The elevated enzymatic activity was attributed to a differential expression of insect gut cysteine proteases (representing the major digestive enzymes), and of aspartic proteases. scN degradation by the gut extract was observed only in adapted bruchids, and this activity appeared to be a combined effect of scN-induced cysteine and aspartic proteases. Thirty cDNAs encoding cathepsin L-like cysteine proteases were isolated from insect guts, and they were differentially regulated by dietary scN. Our results suggest that the cowpea bruchid adapts to the challenge of scN by qualitative and quantitative remodelling of its digestive protease complement, and by activating scN-degrading protease activity.     

Evidence that proteases are involved in superoxide production by human polymorphonuclear leukocytes and monocytes.

The possible participation of proteases in superoxide (O2-) production by human polymorphonuclear leukocytes (PMN) and monocytes was explores using various protease inhibitors and substrates. Protease inhibitors of serine proteases and synthetic inhibitors that modify the active site of serine proteases. Substrates used were synthetic substrates of the chymotrypsin type as well as trypsin type of protease. All these inhibitors and substrates inhibited O2- oroduction by human PMN and monocytes induced by cytochalasin E and concanavalin A, though PMN were more sensitive to these inhibitors and substrates than monocytes. Inhibition appeared rapidly even when the inhibitors were added at the same time as the stimulants, during the "induction time of O2-production" or at the time of maximum O2- production, whereas much greater inhibition was observed when the cells were preincubated with the inhibitors. These observations suggest that enzymatically active serine proteases are essential for these phagocytic cells to initiate and maintain the O2- production in response to the stimuli. The inhibitory effect of the inhibitor and substrate for chymotrypsin type protease was greater than that of those substances for trypsin-type protease. Macromolecular inhibitors also inhibited the O2- production. These findings suggest that the serine proteases involved in the O2- production by human PMN and monocytes are similar to chymotrypsin rather than trypsin, and are possibly located at the cell surface membrane.     

Cloning of a family of serine protease genes from the cat flea Ctenocephalides felis

Serine protease gene fragments approximately 480 nucleotides in length were amplified from Ctenocephalides felis larval and adult cDNA libraries using degenerate oligonucleotide PCR primers. Partial clones of thirty-eight distinct serine protease encoding sequences were isolated, and nineteen different full-length cDNAs encoding mature serine proteases were subsequently cloned and sequenced. All of the mature proteases contained the histidine, aspartic acid and serine amino acids of the catalytic triad characteristic of serine proteases. The mature C. felis serine proteases had amino acid sequences that were at most 29–53% identical to those known insect and arachnid serine proteases. Two of the C. felis gene sequences had similarity with the Drosophila melanogaster developmental genes snake and stubble. mRNA expression of selected serine protease genes was examined in different life stages, tissues, genders, and in response to bloodfeeding.     

A gut-specific serine protease from the malaria vector Aanopheles gambiae is downregulated after blood ingestion

A chymotrypsin-like serine protease gene (AgChyL) was cloned from the mosquito Anopheles gambiae by a polymerase chain reaction (PCR)-based subtractive cDNA cloning strategy. AgChyL messenger RNA (mRNA) is abundant in the adult female gut prior to, and for 8 h following, a blood meal. During the peak of digestion, from 12 to 24 h following a blood meal, AgChyL mRNA abundance decreased to barely detectable levels. AgChyL mRNA was abundant again by 48 h following a blood meal. Recombinant pro-AgChyL was expressed in Escherichia coli. The pro-enzyme can be activated by trypsin. Activated AgChyL cleaves the synthetic chymotrypsin substrate succinyl-L-Ala-Ala-Pro-Phe-nitroanilide, but not two other synthetic chymotrypsin substrates or synthetic trypsin and elastase substrates. The potential role of AgChyL in the coordination of An. gambiae digestion is discussed.     

Activity and sequence characterization of two cysteine proteases in the digestive tract of the reduviid bug Triatoma infestans

Cathepsin B- and cathepsin L-like activities were identified in gut extracts of the blood-sucking bug Triatoma infestans using specific substrates and inhibitors. Activities decreased during the first 2 days after feeding but increased to a maximum value at 5 and 10 days post feeding. The deduced 332 and 328 amino acid sequences showed high levels of identity (50-60%) to other insect cathepsin B- and L-like proteases, respectively. The three amino acid residues of the catalytic domain, CHN, and the GCNGG motif were conserved in both cathepsins, but the occluding loop, characterizing B-like cathepsins, was present only in one. ERFNIN and GNFD motifs occurred in the other sequence, defining it as cathepsin L-like. The cathepsin B-like gene was expressed at low, constitutive levels in unfed and fed T. infestans.     

Molecular cloning and characterization of two digestive serine proteases from the Hessian fly, Mayetiola destructor

Full-length cDNA and genomic sequences for two genes (designated mdesprot-I and mdesprot-II) encoding digestive serine proteases in Hessian fly, Mayetiola destructor, have been cloned and characterized. The deduced amino acid sequences revealed similarity with trypsin-like digestive serine proteases from other Dipterans. Both mdesprot-I and mdesprot-II encoded proteins with secretion signal peptides at the N-terminals, indicating the proteins are secreted proteases that should function as midgut digestive proteases. A cytological analysis with fluorescent in situ hybridization revealed the cytological localization of mdesprot-I and mdesprot-II on the long arm of Autosome 2. Results are discussed in the context of the efficacy of potential protease inhibitors to develop Hessian fly resistant wheat through genetic engineering approaches.     

Cysteine protease inhibitors isolated from human malignant melanoma tissue

Cysteine protease inhibitors that specifically reacted with several cysteine proteases were found in KSCN extract of human melanoma tissue. From 30 gm of the tissue, approximately 593.5 U inhibitor was obtained. The inhibitors were adsorbed on a papain-Sepharose column and could be eluted with 10 mmol/L phosphate buffer, pH 6.0, containing NaCl or KCl, or with 20 mmol/L acetate buffer, pH 4.0, containing KSCN. They revealed a strong inhibitory activity for cysteine proteases such as ficin, papain, and cathepsin B, but did not react with cysteine protease bromelain or serine protease trypsin. No immunologic relationship was confirmed between the inhibitor and other well-known plasma inhibitors such as alpha 1-antitrypsin, alpha 2-macroglobulin, alpha 1-antichymotrypsin, antithrombin III, C1-in-activator, and alpha 2-plasmin inhibitor. With Sephadex G-100, two main peaks of molecular weight 40,000 and 10,000 were detected in the KSCN extract of the human melanoma tissue. However, the inhibitors revealed three molecular weights of 10,000, 25,000, and 80,000 when estimated by Sephadex G-100 gel filtration after papain-Sepharose affinity chromatography. On the other hand, the molecular weights of the inhibitors changed to two peaks of 25,000 and 10,000 on rechromatography with a papain-Sepharose column.     

Identification and partial purification of a (Na-K) ATPase stimulating serine protease from plasma of insulin-dependent diabetics

Plasma from insulin- dependent diabetics shows an increased ability to specifically activate the (Na-K)ATPase from different sources. Several protease inhibitors like phenyl methyl sulfonyl fluoride, trypsin inhibitor, antithrombin III and aprotinin, produced a significant dose-dependent inhibition of the stimulatory effect produced by a 1/100 final dilution of plasma on the beef heart (Na-K)ATPase activity. Serine proteases employed at scalar concentrations in the ATPase medium gave a dose-dependent stimulation of the enzyme activity as did diabetic plasma. The maximum percent stimulation of the (Na-K)ATPase activity (about 60%) was reached by 0.56 μg/ml of thrombin, 0.50 μg/ml of kallikrein and 0.55 μg/ml of trypsin. The protease-induced ATPase stimulation was significantly reduced by antithrombin III, trypsin inhibitor and by aprotinin. A partial purification of the activating plasma factor was obtained by eluting plasma on a heparin-Sepharose column. Two (Na-K)ATPase stimulating fractions were found, which eluted with 1.0 and 3.0 mol/l NaCl, respectively. Half-maximal stimulation of the enzyme occurred with 3.4 μg/ml proteins of fraction 1.0 mol/l and with 45 ng/ml proteins of fraction 3.0 mol/l, this last representing the most purified plasma fraction (about 8 890-fold purification). The proteolytic activity of both plasma and purified plasma fractions was tested on Tos-Arg-OMe substrate which was hydrolyzed to a much higher degree by the most purified plasma fraction. Like the (Na-K)ATPase stimulation, the esterolytic activity was inhibited by protease inhibitors, the most effective to this regard being antithrombin.     

Insect intestinal mucins and serine proteases associated with the peritrophic matrix from feeding,starved and moulting Mamestra configurata larvae

Insect intestinal mucins (McIIM2‐4) expressed in the midgut of feeding, starved and moulting Mamestra configurata larvae were identified. McIIM2 and McIIM4 were associated with the peritrophic matrix (PM). PMs from feeding and starved larvae were translucent and contained organized chitin bundles perpendicular to their long axis, whereas PM from moulting larvae consisted of an inner opaque mass surrounded by an outer translucent sleeve. Serine protease genes (McSP1, McSP2, McSP25 and McSP29) were also expressed in these larvae and several serine proteases were associated with the PM. Serine protease activity was also detected in the midgut of feeding, starved and moulting larvae.     

Paraphyly in Hawaiian hybrid blowfly populations and the evolutionary history of anthropophilic species

Complementary nuclear (28S rRNA) and mitochondrial (COI + II) gene markers were sequenced from the blowflies, Lucilia cuprina and Lucilia sericata, from Europe, Africa, North America, Australasia and Hawaii. Populations of the two species were phylogenetically distinct at both genes, with one exception. Hawaiian L. cuprina possessed typical L. cuprina-type rRNA, but had L. sericata-type mitochondrial (COI + II) sequences. An explanation for this pattern is that Hawaiian flies are hybrids and comparison of observed levels of sequence divergence to possible introduction events, e.g. Polynesian colonization, suggests that Hawaiian L. cuprina may be evolving rapidly. Moreover, the monophyly of these flies also suggests that the L. sericata mtDNA haplotype was apparently fixed in Hawaiian L. cuprina by lineage sorting, indicating a population bottleneck in the evolutionary history of these island flies.     

Secretory leukocyte protease inhibitor suppresses the inflammation and joint damage of bacterial cell wall-induced arthritis.

Disruption of the balance between proteases and protease inhibitors is often associated with pathologic tissue destruction. To explore the therapeutic potential of secretory leukocyte protease inhibitor (SLPI) in erosive joint diseases, we cloned, sequenced, and expressed active rat SLPI, which shares the protease-reactive site found in human SLPI. In a rat streptococcal cell wall (SCW)-induced model of inflammatory erosive polyarthritis, endogenous SLPI was unexpectedly upregulated at both mRNA and protein levels in inflamed joint tissues. Systemic delivery of purified recombinant rat SLPI inhibited joint inflammation and cartilage and bone destruction. Inflammatory pathways as reflected by circulating tumor necrosis factor alpha and nuclear factor kappaB activation and cartilage resorption detected by circulating levels of type II collagen collagenase-generated cleavage products were all diminished by SLPI treatment in acute and chronic arthritis, indicating that the action of SLPI may extend beyond inhibition of serine proteases.     

AANA journal course. Update for nurse anesthetists--part 4--life in the balance: the role of serpins in disease genesis and prevention

Complex biological systems are often shaped and maintained by opposing forces. A relevant biological example is the delicate balance between proteases and their inhibitors. Serine proteases contain a serine residue in the active site of the molecule that is essential to the activity of the enzyme. Protease inhibitors limit the activity of proteases in the body. As examples, aprotinin (Trasylol), a serine protease inhibitor, and aminocaproic acid (Amicar), a lysine protease inhibitor, are used to decrease the rate of fibrinolysis and have recently been the subject of considerable controversy in the literature regarding safety and efficacy. This AANA journal course reviews 2 common examples of protease inhibitor disorders, angioedema and a form of emphysema, that are of particular anesthetic relevance.