Phylogenetic distribution of cysteine proteinases in beetles: evidence for an evolutionary shift to an alkaline digestive strategy in Cerambycidae

We characterized the digestive proteinases of eight species of beetles to improve our understanding of the phylogenetic distribution of serine and cysteine proteinases. Serine proteinases function optimally under alkaline pH conditions, whereas cysteine proteinases require acidic pH. The phylogenetic distribution of cysteine proteinases suggests that they first appeared in an early cucujiform ancestor, however, data for some groups is patchy, and there has been speculation that they have been lost in at least one group, the long-horned beetles (Cerambycidae). The pattern we found supports the hypothesized origin of the proteinases and extends their distribution to an additional superfamily. In addition, we confirmed the presence of cysteine proteinases in some Curculionoidea. Cysteine proteinases were absent, however, from all three species of cerambycids surveyed, supporting the hypothesis that this group has reverted to the more ancestral serine (alkaline) digestive strategy. In four species we compared the pH optima for total proteolytic activity to the actual pH of the midgut and found the match between optimal and actual pH to be weaker in the cerambycids. These findings suggest that either a close correlation between midgut pH and the proteolytic pH optimum is not needed for adequate digestive efficiency, or that midgut pH is a more constrained digestive feature and there has been insufficient time for it to shift upwards to maximize serine proteinase activity.     

A fluorescamine-based sensitive method for the assay of proteinases, capable of detecting the initial cleavage steps of a protein.

The properties of the reaction of fluorescamine with proteins are the basis for the development of a sensitive, general and simple method for the assay of proteolytic activities. More importantly, the assay measures the initial step(s) of proteolytic attack, making it specially suitable for the examination of the controlling factors that regulate proteolytic degradation and/or the detection of 'specific' proteinases. The method allows the simple determination of the general parameters of enzyme action, V and Km, using proteins, i.e. the physiological substrates of the proteinases. The more appropriate proteins to be used as substrates are the N-amino-terminally blocked ones. Many proteins fulfill this requirement. If the particular protein whose degradation has to be studied lacks this modification, three different approaches can be used to study its degradation: (a) the accumulation of N-amino termini in excess over that of the intact substrate; (b) a gel filtration/continuous method and (c) the chemical blockage of its amino groups. The particular advantages of each of these approaches are discussed.     

Characterization of two hemorrhagic zinc proteinases, toxin c and toxin d, from western diamondback rattlesnake (Crotalus atrox) venom

Two hemorrhagic proteinases from Crotalus atrox venom, hemorrhagic toxin c (Ht-c) and hemorrhagic toxin d (Ht-d), were characterized and compared to one another. The two toxins are zinc metalloproteinases which both have molecular weights of 24,000. Their isoelectric points are slightly acidic, Ht-c being the more basic of the two with an isoelectric point of 6.2, whereas Ht-d has an isoelectric point of 6.1. Only minor differences were found in the amino acid compositions of the two toxins. The toxins were both demonstrated to be hemorrhagic, using an in vivo assay, and also proteolytic. Prior treatment of the hemorrhagic proteinases with ethylenediaminetetraacetic acid and o-phenanthroline eliminated both the hemorrhagic and the proteolytic activities. Aprotinin and phenylmethylsulfonyl fluoride had no effect upon these activities. The pH optimum of the proteolysis by Ht-c and Ht-d on hide powder azure as the substrate was between pH 8 and pH 9. The circular dichroism spectra for Ht-c and Ht-d appear almost identical with respect to minima positions and elipticities, indicative of very similar solution structures for the two enzymes. Antiserum raised in mice against Ht-c was assayed on double-diffusion Ouchterlony plates for cross-reactivity with other hemorrhagic toxins from C. atrox venom. From this experiment it was concluded that the two hemorrhagic proteinases Ht-c and Ht-d share identical antigenic structures. This was corroborated by tryptic mapping of the two toxins. Only one major difference was observed from the maps. In the case of Ht-c, it was determined that an aspartate was substituted by an alanine when compared to Ht-d. From these characterization studies we conclude that Ht-c and Ht-d are isoenzymes with only very minor differences in their structures.     

Proteolysis in eukaryotic cells. Identification of multiple proteolytic enzymes in yeast

A previous study led to the discovery of new proteinases in yeast (Achstetter, T., Ehmann, C., and Wolf, D. H. (1981) Arch. Biochem. Biophys. 207, 445-454). The search for proteolytic enzymes active in the neutral pH range has been extended. Studies were done on a mutant lacking four well-known proteinases involved in protein degradation, the two endoproteinases A and B and the two carboxypeptidases Y and S. Twenty-nine chromogenic peptides (amino terminally blocked peptidyl-4-nitroanilides) as well as [3H]methylcasein were used as substrates in this search. For the detection of endoproteolytic activity using chromogenic peptide substrates two versions of the assay were used. In one system the direct cleavage of the 4-nitroanilide bond was measured. In the second, the cleavage of the chromogenic peptide at some site other than the 4-nitroanilide bond was measured. Both variations led to the discovery of multiple proteinase activities. Regulation of these proteolytic activities under different growth conditions of cells was observed. Proteolytic activity on [3H]methylcasein was also found. Ion-exchange chromatography and gel filtration were used for the reproducible separation of the multiple proteolytic activities.     

A BODIPY fluorescent microplate assay for measuring activity of calpains and other proteases

The use of 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL) labeled casein in autoquenching assays of proteolytic activity has been recently described, and we have adapted this assay to measurement of calpain activity. BODIPY-FL coupled to casein at a ratio of 8 mol of BODIPY-FL/mol of casein or higher produces a BODIPY-FL-casein substrate that can be used in an autoquenching assay of calpain proteolytic activity. This assay has a number of advantages for measuring calpain activity. (1) The procedure does not require precipitation and removal of undegraded protein, so it is much faster than other procedures that require a precipitation step, and it can be used directly in kinetic assays of proteolytic activity. (2) The BODIPY-FL-casein assay is easily adapted to a microtiter plate format, so it can be used to screen large numbers of samples. (3) Casein is an inexpensive and readily available protein substrate that more closely mimics the natural substrates of endoproteinases, such as the calpains, than synthetic peptide substrates do. Casein has K(m) values for micro- and m-calpain that are similar to those of other substrates such as fodrin or MAP2 that may be "natural" substrates for the calpains, and there is no reason to believe that calpain hydrolysis of casein is inherently different from hydrolysis of fodrin or MAP2, which are much less accessible as substrates for protease assays. (4) The BODIPY-FL-casein assay is capable of detecting 10 ng ( approximately 5 nM) of calpain and is nearly as sensitive as the most sensitive calpain assay reported thus far. (5) The BODIPY-FL-casein assay is as reproducible as the FITC-casein assay, whose reproducibility is comparable to or better than the reproducibility of other methods used to assay calpain activity. The BODIPY-FL-casein assay is a general assay for proteolytic activity and can be used with any protease that cleaves casein.     

The characteristics of protein hydrolysis on the digestive-transport surfaces of the cestode Eubothrium rugosum and of the intestines of its host--the burbot]

The functioning of different proteinases hydrolysing proteins in a wide pH range, most of which display activity in the alkaline zone of pH, on the digestive-absorptive surfaces of the parasite and host has been investigated. The dynamics of desorption of these proteinases from the intestine of fishes and tegument of cestodes has been studied. It has been shown that the worms possess less proteolytic activity and less capacity for adsorption of proteinases as compared to the intestines of their hosts. The dependence of proteolytic activity of desorbed fractions on the incubation medium temperature has been noted: with the increase in temperature the enzymes, bound closely with the membranes, increase their capacity to hydrolyse proteins. The predominance in cestodes, as compared to the intestine, of easily desorbed fractions D1 and D2 (in the percent ratio of the total proteolytic activity of all fractions) has been detected.     

Inter-alpha-trypsin inhibitor. Inhibition spectrum of native and derived forms

The conversion of inter-alpha-trypsin inhibitor (I alpha I) into active, acid-stable derivatives by proteolytic degradation has been tested with 10 different proteinases. Of these, only plasma kallikrein, cathepsin G, neutrophil elastase, and the Staphylococcus aureus V-8 proteinase were found to be effective, each releasing more than 50% of this activity. However, a strong correlation between inhibitor degradation and significant release of acid-stable activity could only be found with the V-8 enzyme. Inhibition kinetics for the interaction of native I alpha I, the inhibitory fragment released by digestion with S. aureus V-8 proteinase, or the related urinary trypsin inhibitor, with seven different proteinases indicated that all had essentially identical Ki values with an individual enzyme and, where measurements were possible, nearly identical second order association rate constants. Significantly, none of the five human proteinases tested, including trypsin, chymotrypsin, plasmin, neutrophil elastase, and cathepsin G, would appear to have low enough Ki values to be physiologically relevant. Thus, the role of native I alpha I or its degradation products in controlling a specific proteolytic activity is still unknown.     

ATP-activated, high-molecular-mass proteinase-I from rat skeletal muscle is a cysteine proteinase-alpha 1-macroglobulin complex

From rat skeletal muscle tissue we have isolated and purified a proteolytic activity of molecular mass 750 kDa. The enzyme, designated 'proteinase I', which has been found to be located in capillaries of skeletal muscle tissue, catalyzes the hydrolysis of Z-Phe-Arg-MCA and [14C]methylcasein and this process is activated about 2-fold by ATP. As judged by SDS-polyacrylamide gel electrophoresis the subunit pattern of 'proteinase I' is similar to alpha-macroglobulin. Immunoelectrophoretic analyses of 'proteinase I' with antisera to rat alpha 1-macroglobulin, alpha 2-macroglobulin, and rat liver cathepsins reveal that this high-molecular-mass proteinase is a complex of alpha 1-macroglobulin and the cysteine proteinases cathepsin B, H and L. A similar 'proteinase' has been isolated from rat serum. Two ATP-activated high molecular-mass proteinases that have been previously identified in liver and heart muscle by other investigators equally show a positive immunological reaction with the antiserum raised against 'proteinase I'. From these data, together with results presented in an accompanying paper (Kuehn, L., Dahlmann, B., Gauthier, F. and Neubauer, H.-P. (1989) Biochim. Biophys. Acta 991, 263), we conclude that the ATP-stimulated high-molecular-mass proteolytic activity is partly due to the presence of a complex of alpha-macroglobulin and cysteine proteinases.     

News

An assay has been developed for protease activity which uses the gelatin on the surface of an unprocessed X-ray film as the proteolytic substrate. A positive reaction is indicated by a clear zone on the film. The method is inexpensive, rapid, simple, and useful when large numbers are to be screened for their protease inhibitory activity.     

Ixodidin, a novel antimicrobial peptide from the hemocytes of the cattle tick Boophilus microplus with inhibitory activity against serine proteinases

The presence of an effective immune response in the hemocoel of arthropods is essential for survival as it prevents the invasion of pathogens throughout the animal body. Antimicrobial peptides (AMPs) play an important role in this response by rapidly killing invading microorganisms. In this study, a novel cysteine-rich AMP has been isolated and characterized from the hemocytes of the cattle tick, Boophilus microplus. In addition to growth inhibition of Escherichia coli and Micrococcus luteus, the newly described AMP, designated ixodidin (derived from the Family Ixodidae), was found to exert proteolytic inhibitory activity against two exogenous serine proteinases, elastase and chymotrypsin. This is the first report of a molecule of an arachnid that has been shown to inhibit bacterial growth and proteinase activity.     

Internalisation and degradation of histatin 5 by Candida albicans

Histatins, salivary antimicrobial peptides, are susceptible to proteolytic degradation, often ascribed to host proteinases. In this study, we addressed the question whether proteolytic activity from microbial sources can contribute to this degradation. Candida albicans, an opportunistic yeast that is susceptible to the histatins, was used as target organism. The most potent histatin (histatin 5: sequence: DSHAKRHHGYKRKFHEKHHSHRGY), two histatin 5 fragments (dh-5: sequence: KRKFHEKHHSHRGY; P-113: sequence: AKRHHGYKRKFH) and an all-D isomer of the latter (P-113D) were used as model peptides. All L-peptides were susceptible to degradation by C. albicans. Cleavage was established at Lys5 and His19 of histatin 5, Lys11, Arg12, Phe14, Glu16, Lys17, His18 and Ser20 of dh-5 and Ala4 and Lys11 of P-113. In addition, it was found that secreted C. albicans enzymes are not involved in the degradation process and that blocking cell entry of the peptides greatly impedes degradation. Moreover, P-113D, which is biologically as active as P-113, was hardly susceptible to proteolysis. These data imply that proteolysis occurs mainly intracellularly and is not used as a protective mechanism against histatin activity. Together, our results suggest that, besides host proteinases, microbial enzymes play an important role in histatin degradation.     

Inhibition of proteolytic activity of poliovirus and rhinovirus 2A proteinases by elastase-specific inhibitors.

A polyprotein cleavage assay has been developed to assay the proteolytic activities in vitro of the 2A proteinases encoded by poliovirus and human rhinovirus 14, which are representative members of the Enterovirus and Rhinovirus genera of picornaviruses, respectively. The elastase-specific substrate-based inhibitors elastatinal and methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK) inhibited both 2A proteinases in vitro. The electrophoretic mobilities of both 2A proteinases were reduced upon incubation with elastatinal, whereas the mobility of a Cys-109-->Ala poliovirus 2Apro mutant was unchanged, an observation suggesting that this inhibitor may have formed a covalent bond with the active-site Cys-109 nucleophile. Iodoacetamide, calpain inhibitor 1, and antipain inhibited poliovirus 2Apro. MPCMK caused a reduction in the yields of the enteroviruses poliovirus type 1 and coxsackievirus A21 and of human rhinovirus 2 in infected HeLa cells but did not affect the growth of encephalomyocarditis virus, a picornavirus of the Cardiovirus genus. MPCMK abrogated the shutoff of host cell protein synthesis that is induced by enterovirus and rhinovirus infection and reduced the synthesis of virus-encoded polypeptides in infected cells. These results indicate that the determinants of substrate recognition by 2A proteinases resemble those of pancreatic and leukocyte elastases. These results may be relevant to the development of broad-range chemotherapeutic agents against entero- and rhinoviruses.     

Human cytomegalovirus proteinase: candidate glutamic acid identified as third member of putative active-site triad.

The human cytomegalovirus (HCMV) proteinase is synthesized as a 709-amino-acid precursor that undergoes at least three autoproteolytic cleavages. The mature proteinase, called assemblin, is one of the products of autoproteolysis and is composed of the first 256 amino acids of the precursor. HCMV assemblin and its homologs in other herpes group viruses contain five highly conserved domains (CD1 through CD5). An absolutely conserved serine in CD3 has been shown by site-directed mutagenesis of the simian cytomegalovirus (SCMV) and herpes simplex virus type 1 (HSV-1) enzymes and by inhibitor affinity labeling of the HSV-1 and HCMV enzymes to be the active-site nucleophile of assemblin. An absolutely conserved histidine in CD2 has also been demonstrated by site-directed mutagenesis of the SCMV and HSV-1 enzymes to be essential for proteolytic activity and has been proposed to be a second member of the catalytic triad of this serine proteinase. We report here the use of site-directed mutagenesis to investigate the active-site amino acids of HCMV assemblin. Substitutions were made for the CD3 serine and CD2 histidine residues implicated as active-site components, and for other amino acids whose influence on enzyme activity was of interest. The mutant proteinases were tested in a transient transfection assay for their ability to cleave their natural substrate, the assembly protein precursor. Results of these experiments verified that HCMV CD3 serine (Ser-132) and CD2 histidine (His-63) are essential for proteolytic activity and identified a glutamic acid (Glu-122) within CD3 that is also essential for proteolytic activity and may be conserved among all herpesvirus assemblin homologs. We suggest that CD3 Glu-122, CD3 Ser-132, and CD2 His-63 constitute the active-site triad of this serine proteinase.     

Growth kinetics, antigen profiling, and proteinase activity of Egyptian Trichomonas tenax isolates derived from patients having oral infections

The role of Trichomonas tenax as a pathogen had been clearly implicated in various pathological processes that arise outside the boundaries of the mouth. Although a relationship between the increased occurrence of this protozoan and progression of periodontal disease has been demonstrated, the ability of T. tenax in causing oral infections and the precise mechanism of tissue damage is not well known. The present study aimed to investigate different isolates of T.tenax from individuals having oral infections. Plaques and/or calculi samples were collected from 70 individuals who were diagnosed as having periodontitis and/or gingivitis, then subjected to parasitological examination and culture on modified trypticase, yeast and iron medium (TYI-S-33). Isolates successfully maintained in culture were further subjected to analysis of protein profile of lysates by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and analysis of proteinases by non-denaturing gelatin-SDS-PAGE. Comparison of growth kinetics of seven T. tenax isolates showed a wide variability in the growth characteristics. Protein profiles of the seven isolates revealed a total 53 bands ranged in molecular weight (MW) from 5 to 95kDa using 12% resolution gel. Also, T. tenax isolates were found to possess 19 proteinase bands ranged in MW from 14 to 66kDa. The proteolytic bands were intensified by a cysteine proteinase activator and totally disappeared by treatment with a cysteine proteinase inhibitor suggesting that the proteinases were of cysteine proteinases type. The high frequency of T. tenax detected (28.6%) along with the variability in protein profiling and proteolytic activity of the isolates supports the possible pathogenicity of T. tenax and clarifies a conclusion that different strains with possibility of variable pathogenic potential may exist.     

Bikunin--not just a plasma proteinase inhibitor

Bikunin is a plasma proteinase inhibitor that has received little attention in the past, probably because its activity towards various proteinases was found to be relatively weak in early work. It was recently discovered, however, that bikunin effectively inhibits a proteinase that seems to be involved in the metastasis of tumour cells--cell surface plasmin--and that a fragment of bikunin inhibits two proteinases of the coagulation pathway--factor Xa and kallikrein. Furthermore, it has been found that bikunin has other properties, such as the ability to modulate cell growth and to block cellular calcium uptake. Most of the bikunin in the blood occurs as a covalently linked subunit of the proteins pre- and inter-alpha-inhibitor. In this form bikunin lacks some of its known activities, and there is evidence that its release by partial proteolytic degradation may function as a regulatory mechanism. Although the physiological function of bikunin still remains to be established, current data suggest that this protein plays a role in inflammation. Further studies could therefore lead to results of therapeutical value.     

Inactivation of human alpha 1-proteinase inhibitor by thiol proteinases.

Human plasma alpha1 proteinase inhibitor is the body's principal modulator of serine proteinases (such as those released from phagocytic cells). Cysteine-active-site proteinases, which are not inhibited, have now been found to inactivate this important inhibitor by proteolytic cleavage of a scissile peptide bond. Papain carries out this inactivation catalytically, whereas cathepsin B1 acts stoicheiometrically. Thus thiol proteinases could easily disrupt the delicately regulated balance between serine proteinases and alpha1 proteinase inhibitor.     

Effect of pasteurization on the activity of proteinases in salmon roe

We studied the dependence of activity and stability of proteolytic enzymes in salmon roe on pH and temperature. The activity of proteolytic enzymes in roe was primarily determined by proteinases. These enzymes were active at acid pH and had an optimum of 3.6. A study of subclasses of proteolytic enzymes in salmon roe and the published data suggest that the activity of proteinases may be related to the presence of aspartyl proteinases (cathepsin D). Serine proteinases and metalloenzymes were not found in roe. The activity of cysteine proteinases was low. The proposed conditions of pasteurization favored the complete inactivation of salmon roe at pH 6.0-6.4.     

Effect of Pasteurization on the Activity of Proteinases in Salmon Roe

We studied the dependence of activity and stability of proteolytic enzymes in salmon roe on pH and temperature. The activity of proteolytic enzymes in roe was primarily determined by proteinases. These enzymes were active at acid pH and had an optimum of 3.6. A study of subclasses of proteolytic enzymes in salmon roe and the published data suggest that the activity of proteinases may be related to the presence of aspartyl proteinases (cathepsin D). Serine proteinases and metalloenzymes were not found in roe. The activity of cysteine proteinases was low. The proposed conditions of pasteurization favored the complete inactivation of salmon roe at pH 6.0–6.4.     

Aspartic proteinases in the digestive tract of marine decapod crustaceans

Decapod crustaceans synthesize highly active proteolytic enzymes in the midgut gland and release at least a part of them into the stomach where they facilitate the first step in peptide hydrolysis. The most common proteinases in the gastric fluid characterized so far are serine proteinases, that is, trypsin and chymotrypsin. These enzymes show highest activities at neutral or slightly alkaline conditions. The presence of acid proteinases, as they prevail in vertebrates, has been discussed contradictorily yet in invertebrates. In this study, we show that acid aspartic proteinases appear in the gastric fluid of several decapods. Lobsters Homarus gammarus showed the highest activity with a maximum at pH 3. These activities were almost entirely inhibited by pepstatin A, which indicates a high share of aspartic proteinases. In other species (Panulirus interruptus, Cancer pagurus, Callinectes arcuatus and Callinectes bellicosus), proteolytic activities were present at acid conditions but were distinctly lower than in H. gammarus. Zymograms at pH 3 showed in each of the studied species at least one, but mostly two-four bands of activity. The apparent molecular weight of the enzymes ranged from 17.8 to 38.6 kDa. Two distinct bands were identified which were inhibited by pepstatin A. Acid aspartic proteinases may play an important role in the process of extracellular digestion in decapod crustaceans. Activities were significantly higher in clawed lobster than in spiny lobster and three species of brachyurans. Therefore, it may be suggested that the expression of acid proteinases is favored in certain groups and reduced in others.