Ontogeny of proteolytic immunity: IgM serine proteases

We report the chemical activity of immunoglobulin micro and kappa/lambda subunits expressed on the surface of B cells and in secreted IgM antibodies (Abs) found in the preimmune repertoire. Most of the nucleophilic reactivity of B cells measured by formation of covalent adducts of a hapten amidino phosphonate diester was attributed to micro and kappa/lambda subunits of the B cell receptor. Secreted IgM Abs displayed superior nucleophilic reactivity than IgG Abs. IgM Abs catalyzed the cleavage of model peptide substrates at rates up to 344-fold greater than IgG Abs. Catalytic activities were observed in polyclonal IgM Abs from immunologically na?ve mice and humans without immunological disease, as well as monoclonal IgM Abs to unrelated antigens. Comparison of several IgM Abs indicated divergent activity levels and substrate preferences, with the common requirement of a basic residue flanking the cleavage site. Fab fragments of a monoclonal IgM Ab expressed catalytic activity, confirming the V domain location of the catalytic site. The catalytic reaction was inhibited by the covalently reactive hapten probe and diisopropylfluorophosphate, suggesting a serine protease-like mechanism. These observations indicate the existence of serine protease-like BCRs and secreted IgM Abs as innate immunity components with potential roles in B cell development and Ab effector functions.     

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     

Neutrophil serine proteases: specific regulators of inflammation

Neutrophils are essential for host defence against invading pathogens. They engulf and degrade microorganisms using an array of weapons that include reactive oxygen species, antimicrobial peptides, and proteases such as cathepsin G, neutrophil elastase and proteinase 3. As discussed in this Review, the generation of mice deficient in these proteases has established a role for these enzymes as intracellular microbicidal agents. However, I focus mainly on emerging data indicating that, after release, these proteases also contribute to the extracellular killing of microorganisms, and regulate non-infectious inflammatory processes by activating specific receptors and modulating the levels of cytokines.     

Recent developments in inhibiting cysteine and serine proteases

Some 20 years ago, affinity labelling was introduced to help gather information on active-site catalytic groups and the mechanisms of proteolytic enzymes. Now this knowledge is used to produce specific and selective inhibitors for these enzymes. The concept of "biospecific drug design" has stimulated progress in turning the inhibitors into therapeutically applicable agents. For instance, sales of antihypertensive drugs based on inhibitors of the angiotensin converting enzyme are expected to be over 2 billion US-$ in 1992. This partly illustrates the efforts made by many researchers to introduce strategies in potease inhibition for medicinal purposes. This review discusses some of the concepts.     

Buried water in homologous serine proteases.

Buried water molecules in the structurally homologous family of eukaryotic serine proteases were examined to determine whether buried waters and their protein environments are conserved in these proteins. We found 16 equivalent water sites conserved in trypsin/ogen, chymotrypsin/ogen, elastase, kallikrein, thrombin, rat tonin and rat mast cell protease, and 5 additional water sites in enzymes which share the primary specificity of trypsin. Based on an alignment of 30 serine protease sequences, it appears that the protein environments of these 21 conserved buried waters are highly conserved. The protein environments of buried waters are comprised primarily of atoms from highly conserved residues or main chain atoms from nonconserved residues. In one instance, the protein environment of a water is conserved even in the presence of an unlikely Pro/Ala substitution. We also note 3 instances in which a histidine side chain substitutes for water, suggesting that the structural role of water at these sites is satisfied by the presence of an alternative hydrogen bonding partner. Buried waters appear to be integral structural components of these proteins and should be incorporated into protein structures predicted on the basis of sequence homology to this family, including the catalytic domains of coagulation proteases.     

Subtilases: the superfamily of subtilisin-like serine proteases.

Subtilases are members of the clan (or superfamily) of subtilisin-like serine proteases. Over 200 subtilases are presently known, more than 170 of which with their complete amino acid sequence. In this update of our previous overview (Siezen RJ, de Vos WM, Leunissen JAM, Dijkstra BW, 1991, Protein Eng 4:719-731), details of more than 100 new subtilases discovered in the past five years are summarized, and amino acid sequences of their catalytic domains are compared in a multiple sequence alignment. Based on sequence homology, a subdivision into six families is proposed. Highly conserved residues of the catalytic domain are identified, as are large or unusual deletions and insertions. Predictions have been updated for Ca(2+)-binding sites, disulfide bonds, and substrate specificity, based on both sequence alignment and three-dimensional homology modeling.     

Pseudo-peptides derived from isomannide: inhibitors of serine proteases

In this paper, we describe the synthesis of a novel class of pseudo-peptides derived from isomannide and several oxazolones as potential inhibitors of serine proteases as well as preliminary pharmacological assays for hepatitis C. Hepatitis C, dengue and West Nile fever are among the most important flaviviruses that share one important serine protease enzyme. Serine proteases belong to the most studied class of proteolytic enzymes and are a primary target in the drug development field. Several pseudo-peptides were obtained in good yields from the reaction of isomannide and oxazolones, and their anti-HCV potential using the HCV replicon-based assay was shown.     

Granzymes: a family of lymphocyte granule serine proteases

Granzymes, a family of serine proteases, are expressed exclusively by cytotoxic T lymphocytes and natural killer (NK) cells, components of the immune system that protect higher organisms against viral infection and cellular transformation. Following receptor-mediated conjugate formation between a granzyme-containing cell and an infected or transformed target cell, granzymes enter the target cell via endocytosis and induce apoptosis. Granzyme B is the most powerful pro-apoptotic member of the granzyme family. Like caspases, cysteine proteases that play an important role in apoptosis, it can cleave proteins after acidic residues, especially aspartic acid. Other granzymes may serve additional functions, and some may not induce apoptosis. Granzymes have been well characterized only in human and rodents, and can be grouped into three subfamilies according to substrate specificity: members of the granzyme family that have enzymatic activity similar to the serine protease chymotrypsin are encoded by a gene cluster termed the 'chymase locus'; granzymes with trypsin-like specificities are encoded by the 'tryptase locus'; and a third subfamily cleaves after unbranched hydrophobic residues, especially methionine, and is encoded by the 'Met-ase locus'. All granzymes are synthesized as zymogens and, after clipping of the leader peptide, maximal enzymatic activity is achieved by removal of an amino-terminal dipeptide. They can all be blocked by serine protease inhibitors, and a new group of inhibitors has recently been identified - serpins, some of which are specific for granzymes. Future studies of serpins may bring insights into how cells that synthesize granzymes are protected from inadvertent cell suicide.     

食线虫真菌致病相关丝氨酸蛋白酶的研究进展

食线虫真菌是一类土壤微生物,作为线虫的天敌,它们对于维持线虫在土壤生态环境中的种群动态平衡发挥着十分重要的作用。食线虫真菌通过形成特殊的捕食器官或产生毒素等方式来捕捉和杀死线虫。丝氨酸蛋白酶是食线虫真菌侵染线虫的重要毒力因子,近年来,研究人员对不同食线虫真菌来源的致病相关丝氨酸蛋白酶进行了大量的研究,尤其在丝氨酸蛋白酶的晶体结构和分子进化方面的研究取得了较大的进展。本文对食线虫真菌致病相关丝氨酸蛋白酶的生物化学性质和功能进行了系统的总结,对丝氨酸蛋白酶的晶体结构、催化机制及分子进化等最新的进展进行了评述。     

Type II transmembrane serine proteases in development and disease

Recent advances in the mammalian genome projects have resulted in the identification of a surprisingly large number of genes encoding putative new members and even entire new families of proteolytic enzymes. In the past few years, one of these new families, type II transmembrane serine proteases (TTSPs), underwent a particularly rapid transformation from a group of predicted DNA and protein sequences into an established family of cell surface-associated proteases with important roles in the development and homeostasis of mammalian tissues such as heart, skin, inner ear, placenta, and digestive tract. Additionally, aberrant expression of TTSP genes appears to be involved in the aetiology of several human disorders, including cancer. This review presents our current knowledge of the biological functions of the individual TTSPs in mouse and human tissue development and disease.     

Transmigration of melanoma cells through the blood-brain barrier: role of endothelial tight junctions and melanoma-released serine proteases

Malignant melanoma represents the third common cause of brain metastasis, having the highest propensity to metastasize to the brain of all primary neoplasms in adults. Since the central nervous system lacks a lymphatic system, the only possibility for melanoma cells to reach the brain is via the blood stream and the blood-brain barrier. Despite the great clinical importance, mechanisms of transmigration of melanoma cells through the blood-brain barrier are incompletely understood. In order to investigate this question we have used an in vitro experimental setup based on the culture of cerebral endothelial cells (CECs) and the A2058 and B16/F10 melanoma cell lines, respectively. Melanoma cells were able to adhere to confluent brain endothelial cells, a process followed by elimination of protrusions and transmigration from the luminal to the basolateral side of the endothelial monolayers. The transmigration process of certain cells was accelerated when they were able to use the routes preformed by previously transmigrated melanoma cells. After migrating through the endothelial monolayer several melanoma cells continued their movement beneath the endothelial cell layer. Melanoma cells coming in contact with brain endothelial cells disrupted the tight and adherens junctions of CECs and used (at least partially) the paracellular transmigration pathway. During this process melanoma cells produced and released large amounts of proteolytic enzymes, mainly gelatinolytic serine proteases, including seprase. The serine protease inhibitor Pefabloc® was able to decrease to 44–55% the number of melanoma cells migrating through CECs. Our results suggest that release of serine proteases by melanoma cells and disintegration of the interendothelial junctional complex are main steps in the formation of brain metastases in malignant melanoma.     

Electrostatic recognition in substrate binding to serine proteases

Serine proteases of the Chymotrypsin family are structurally very similar but have very different substrate preferences. This study investigates a set of 9 different proteases of this family comprising proteases that prefer substrates containing positively charged amino acids, negatively charged amino acids, and uncharged amino acids with varying degree of specificity. Here, we show that differences in electrostatic substrate preferences can be predicted reliably by electrostatic molecular interaction fields employing customized GRID probes. Thus, we are able to directly link protease structures to their electrostatic substrate preferences. Additionally, we present a new metric that measures similarities in substrate preferences focusing only on electrostatics. It efficiently compares these electrostatic substrate preferences between different proteases. This new metric can be interpreted as the electrostatic part of our previously developed substrate similarity metric. Consequently, we suggest, that substrate recognition in terms of electrostatics and shape complementarity are rather orthogonal aspects of substrate recognition. This is in line with a 2‐step mechanism of protein‐protein recognition suggested in the literature.     

Theory of allosteric effects in serine proteases.

The classical Botts-Morales theory for the action of a modifier on the catalytic properties of an enzyme has been extended to deal with allosteric effects in serine proteases. The exact analytical solution derived for the linkage scheme at steady state provides a rigorous framework for the study of many biologically relevant systems, including enzymes activated by monovalent cations and cofactor-controlled protease-zymogen interactions in blood coagulation. When the enzyme obeys Michaelis-Menten kinetics, the exact solution of the kinetic linkage scheme simplifies considerably. Of particular importance for practical applications is a simple equation expressing the dependence of the specificity constant of the enzyme, kcat/Km, on the concentration of the modifier, from which the equilibrium binding constant for the formation of the enzyme-modifier complex can be estimated. Analysis of the allosteric changes in thrombin activity induced by thrombomodulin and Na+ in terms of this equation yields accurate determinations of the equilibrium binding constants for both effectors.     

Physicochemical properties of alkaline serine proteases in alcohol

The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1g/ml and that of alcalase was 48.1g/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters,l-Ala-l-Ala-(d-orl-)Pro-l-Phe-OMe andl-Ala-l-Ala-(d-orl-)Ala-l-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents.     

Vanadium inhibition of serine and cysteine proteases.

A study was made on the effect of vanadium, in both the tetravalent state in vanadyl sulphate and in the pentavalent state in sodium meta-vanadate, and ortho-vanadate, on the proteolysis of azocasein by two serine proteases, trypsin and subtilisin and two cysteine proteases bromelain and papain. Also the proteolysis of bovine azoalbumin by serine proteases was considered. An inhibitory effect was present in all cases, except meta-vanadate with subtilisin. The oxidation level of vanadium by itself did not determine the inhibition kinetics, which also depended on the type and composition of the vanadium containing molecule and on the enzyme assayed. The pattern of inhibition was similar for proteases belonging to the same class. The highest inhibition was obtained with meta-vanadate on papain and with vanadyl sulphate on bromelain.     

Active-site titration of serine proteases in organic solvents

Calculation of kinetic constants of an enzymatic reaction in organic solvents requires knowledge of the functional active-site concentration in organic solvents, and this can be significantly different than that in water. An experimental method for active-site titration of serine proteases in organic media has been developed based on the kinetics of inhibition by phenylmethanesulfonyl fluoride (PMSF), a serine-specific inhibitor (or suicide substrate). This kinetic approach is fundamentally different from other techniques that require complete titration of all accessible enzyme active sites. This active site titration method was applied to subtilisins BPN' and Carlsberg and alpha-chymotrypsin and resulted in fractions of active sites that ranged from 8 to 62% (of the fraction active in water) depending on the enzyme, the method of enzyme preparation, and the organic solvent used. The active-site concentration of subtilisin BPN' and Carlsberg increased with increasing hydrophobicity of the solvent and with increasing solvent hydration in tetrahydrofuran. The dependence of the fraction of active sites on the nature of the organic solvent appears to be governed largely by solvent-induced inactivation caused by direct interaction of a hydrophilic solvent with the enzyme. (c) 1996 John Wiley & Sons, Inc.     

Plant serine proteases: biochemical, physiological and molecular features.

In the latest two decades, the interest received by plant proteases has been on the rise. Serine proteases (EC 3.4.21)-in particular those from cucurbits, cereals and trees-share indeed a number of biochemical and physiological features, that may prove useful toward understanding of several mechanisms at the subcellular level. This critical review focuses on the characterization of most plant serine proteases, and comprehensively lists information produced by more and more sophisticated research tools that have led to the current state of the art in knowledge of these unique enzymes.     

Chymotrypsin- and trypsin-type serine proteases in rat mast cells: properties and functions

Two of the major enzymes present in and released from rat mast cells are chymotrypsin-type serine protease (chymase) and trypsin-type serine protease (tryptase), and these have been postulated to be important in the inflammatory reactions. There have been no clear data regarding the trypsin-type protease in rat mast cells. Tryptase was recently purified from rat peritoneal mast cells with an associated protein (trypstatin) that inhibited the protease activity above pH 7.5. Chymase was also purified from rat peritoneal cells by employing a one-step method involving hydrophobic chromatography on octyl-Sepharose 4B or arginine-Sepharose 4B. The properties of chymase and tryptase were described in relation to substrate specificity and their relative sensitivity to inhibitors. It was found that proteolytic activities of these enzymes were modulated by naturally occurring substances, such as phosphoglycerides, long-chain fatty acids, and trypstatin. There is as yet little evidence for the physiological roles of these enzymes in the inflammatory reaction. It has been found that the specific, low-molecular-weight inhibitor of chymase, chymostatin, and that of tryptase, leupeptin, inhibit histamine release induced by addition of anti-rat IgE to mast cells. However, the inhibitors with molecular weights of more than 6000 were found to have no effect in this process. The data suggest that chymase and tryptase in mast cell granules play a crucial or significant role in the process of degranulation.     

ClpP: a distinctive family of cylindrical energy-dependent serine proteases

Processes maintaining protein homeostasis in the cell are governed by the activities of molecular chaperones that mainly assist in the folding of polypeptide chains and by a large class of proteases that regulate protein levels through degradation. ClpP proteases define a distinctive family of cylindrical, energy-dependent serine proteases that are highly conserved throughout bacteria and eukaryota. They typically interact with ATP-dependent AAA+ chaperones that bind and unfold target substrates and then translocate them into ClpP for degradation. Structural and functional studies have provided a detailed view of the mechanism of function of this class of proteases.