Chymotrypsin Family Of Serine Proteases Research Articles

Understanding the functions of endogenous and exogenous tissue-type plasminogen activator during stroke.

Alteplase (Actilyse or Activase) is the gold standard acute treatment of ischemic stroke1 and receives attention for hematoma resolution in hemorrhagic stroke. The active principle of Alteplase is the recombinant form of an endogenous protease, tissue-type plasminogen activator (tPA). Its intravascular thrombolytic activity is well known, but less are its multifaceted functions in the central nervous system (CNS). Endogenous tPA is not only released in the blood by endothelial cells but also expressed by many cells within the brain parenchyma (online-only Data Supplement), and it can act on all cell types of the brain virtually. Endogenous tPA has been involved in an ever-increasing number of brain functions, of which several are highly relevant during and after stroke. Importantly, knowledge on the mechanisms of action of endogenous tPA may hold true for recombinant tPA. In this review, we provide an up-to-date overview of the current knowledge on the enzymatic or cytokine-like effects of action of tPA in the CNS, its various molecular substrates or receptors, focusing on the processes occurring during and after ischemic or hemorrhagic stroke, including excitotoxicity, apoptosis, blood–brain barrier breakdown, inflammation, axonal damage, and demyelination. tPA is a mosaic protease of 527 amino acids consisting of 5 distinct modules: a Finger domain, an epidermal growth factor (EGF)–like domain, 2 kringle domains (K1 and K2), and a serine protease proteolytic domain. Through these domains, tPA can interact with a variety of binding proteins and receptors in the brain parenchyma, thus extending its functions above the conversion of plasminogen into plasmin (Figure 1). Figure 1. Main cellular and molecular targets of tissue-type plasminogen activator in the central nervous system. BBB indicates blood–brain barrier, EGFR, epidermal growth factor receptor; LRP, low-density lipoprotein receptor–related protein; NMDAR, N-methyl-d-aspartate receptors; and PDGFR, platelet-derived growth factor receptor. tPA is not fibrinolytic by itself. …

A High Affinity Hepatocyte Growth Factor-binding Site in the Immunoglobulin-like Region of Met

Hepatocyte growth factor (HGF) and its high affinity receptor, the tyrosine kinase Met, play a key role in embryo development and tumor invasion. Both HGF and Met are established targets for cancer therapy. However, the mechanism of their interaction is complex and remains elusive. HGF is secreted as a monomeric precursor (pro-HGF) that binds to but does not activate Met. Mature HGF is a α/β heterodimer containing a high affinity Met-binding site in the α-chain (HGF-α) and a low affinity Met-binding site in the β-chain (HGF-β). The extracellular portion of Met contains a semaphorin (Sema) domain, a cysteine-rich hinge (plexin-semaphorin-integrin), and four immunoglobulin-like domains (immunoglobulin-like regions in plexins and transcription factors (IPT) 1-4). HGF-β binds to Sema through a low affinity contact. The domain of Met responsible for high affinity binding to HGF-α has not been identified yet. Here we show that this long sought after binding site lies in the immunoglobulin-like region of Met and more precisely in IPT 3 and 4. We also show that IPT 3 and 4 are sufficient to transmit the signal for kinase activation to the cytoplasm, although the lack of Sema makes the receptor equally sensitive to mature HGF and pro-HGF. Finally, we provide evidence that soluble Met-derived proteins containing either the low affinity or high affinity HGF-binding site antagonize HGF-induced invasive growth both in vitro and in xenografts. These data suggest that the immunoglobulin-like region of Met cooperates with the Sema domain in binding to HGF and in controlling Met kinase activity. Although the IPT-HGF-α interaction provides binding strength, the Sema-HGF-β contact confers selective sensitivity to the active form of the ligand.

Distinct Contributions of Residue 192 to the Specificity of Coagulation and Fibrinolytic Serine Proteases

Archetypal members of the chymotrypsin family of serine proteases, such as trypsin, chymotrypsin, and elastase, exhibit relatively broad substrate specificity. However, the successful development of efficient proteolytic cascades, such as the blood coagulation and fibrinolytic systems, required the evolution of proteases that displayed restricted specificity. Tissue-type plasminogen activator (t-PA), for example, possesses exquisitely stringent substrate specificity, and the molecular basis of this important biochemical property of t-PA remains obscure. Previous investigations of related serine proteases, which participate in the blood coagulation cascade, have focused attention on the residue that occupies position 192 (chymotrypsin numbering system), which plays a pivotal role in determining both the inhibitor and substrate specificity of these enzymes. Consequently, we created and characterized the kinetic properties of new variants of t-PA that contained point mutations at position 192. These studies demonstrated that, unlike in coagulation serine proteases, Gln-192 does not contribute significantly to the substrate or inhibitor specificity of t-PA in physiologically relevant reactions. Replacement of Gln-192 with a glutamic acid residue did, however, decrease the catalytic efficiency of mature, two-chain t-PA toward plasminogen in the absence of a fibrin co-factor.

Spontaneous inactivation of human tryptase involves conformational changes consistent with conversion of the active site to a zymogen-like structure.

The conformational changes accompanying spontaneous inactivation and dextran sulfate (DS) mediated reactivation of the serine protease human tryptase were investigated by analysis of (i) intrinsic fluorescence, (ii) inhibitor binding, and (iii) catalytic efficiency. Spontaneous inactivation produced a marked decrease in fluorescence emission intensity that was reversed by the addition of DS. Fluorescence decreases at high (4.0 microM) and low (0.1 microM) tryptase concentrations were similar at early times and coincided with loss of enzymatic activity but deviated significantly from activity loss at later times by showing a difference in the extent of change. The fluorescence losses were best described by a two-step kinetic model in which the major decrease correlated to activity loss (t1/2 of 4.3 min in 0.2 M NaCl, pH 6.8, 30 degrees C) and was followed by a further decrease (t1/2 approximately 60 min) whose extent differed with tryptase concentration. The ability to bind the competitive inhibitor p-aminobenzamidine was reversibly lost upon spontaneous inactivation, providing evidence for conformational changes affecting the major substrate binding site (S1-pocket). Estimation of catalytic efficiency using an active site titrant showed that the specific activity of tryptase remained unchanged upon inactivation and reactivation. Return of enzymatic activity, intrinsic fluorescence, and the S1 pocket appeared to occur in the same time frame (t1/2 approximately 3 min). These studies indicate that spontaneous inactivation involves reversible changes which convert the active site to a nonfunctional state. The association of activity loss with an intrinsic fluorescence decrease and loss of the S1-pocket is consistent with the disruption of a critical ionic bond at the active site. Formation of this ionic bond is the basis of zymogen activation for the chymotrypsin family of serine proteases.

Optimal Subsite Occupancy and Design of a Selective Inhibitor of Urokinase

Human urokinase type plasminogen activator (u-PA) is a member of the chymotrypsin family of serine proteases that can play important roles in both health and disease. We have used substrate phage display techniques to characterize the specificity of this enzyme in detail and to identify peptides that are cleaved 840-5300 times more efficiently by u-PA than peptides containing the physiological target sequence of the enzyme. In addition, unlike peptides containing the physiological target sequence, the peptide substrates selected in this study were cleaved as much as 120 times more efficiently by u-PA than by tissue type plasminogen activator (t-PA), an intimately related enzyme. Analysis of the selected peptide substrates strongly suggested that the primary sequence SGRSA, from position P3 to P2', represents optimal subsite occupancy for substrates of u-PA. Insights gained in these investigations were used to design a variant of plasminogen activator inhibitor type 1, the primary physiological inhibitor of both u-PA and t-PA, that inhibited u-PA approximately 70 times more rapidly than it inhibited t-PA. These observations provide a solid foundation for the design of highly selective, high affinity inhibitors of u-PA and, consequently, may facilitate the development of novel therapeutic agents to inhibit the initiation and/or progression of selected human tumors.

Substrate Recognition by Recombinant Serine Collagenase 1 from Uca pugilator

Uca pugilator serine collagenase 1 was cloned and sequenced from a fiddler crab hepatopancreas cDNA library. A full-length sequence encodes a 270-amino acid pre-pro-enzyme highly identical in structure to the chymotrypsin family of serine proteases. The zymogen form of the enzyme was expressed in Saccharomyces cerevisiae as a fusion with the alpha-factor signal sequence under control of the alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase promoter. Upon activation with trypsin, the recombinant collagenase possesses collagenolytic properties identical to those of the enzyme isolated from the crab hepatopancreas. The collagenase substrate binding pocket recognizes a wide range of basic, hydrophobic, and neutral polar residues. beta-Branched and acidic amino acids are poor substrates. Acylation is rate-limiting for collagenase versus peptidyl amides, rather than deacylation, as for trypsin and chymotrypsin. Correlations relating substrate volume and hydrophobicity to catalysis were found for collagenase and compared to those for chymotrypsin and elastase. Relative enzyme efficiencies on single amino acid versus tetrapeptide amide substrates show that collagenase derives less catalytic efficiency from binding of the primary substrate residue than trypsin or chymotrypsin, but compensates in binding of the extended peptidyl residues. Serine collagenase 1 is a novel member of the chymotrypsin protease family, by virtue of its amino acid sequence and multifunctional active site.

Cloning of cDNA for human granzyme 3

A serine protease gene was cloned from cDNA prepared from tissue isolated from human ascites. The gene codes for a protein of 264 amino acids, identified as human granzyme 3 by the N-terminal amino acid sequence. Granzyme 3 has the expected features of the chymotrypsin family of serine proteases, and is closely related to other human granzymes (40–45% identity). However, the closest granzyme 3 homologue is the recently characterized rat tryptase, RNK-Tryp-2 (75% identity). From Northern blots, granzyme 3 appears to be highly expressed in peripheral blood leukocytes, spleen, thymus, and lung tissues.

Ecotin: lessons on survival in a protease-filled world.

Ecotin, an Escherichia coli periplasmic protein of 142 amino acids, has been shown to be a potent inhibitor of a group of homologous serine proteases with widely differing substrate recognition. It is highly effective against a number of enzymes, including both pancreatic and neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein. Recent structural and functional studies on ecotin and its interactions with different serine proteases have clarified these initial observations and revealed the remarkable features of this protein in inhibiting a strikingly large subset of the chymotrypsin family of serine proteases. The structures of the ecotin:serine protease complexes provide the first examples of protein-protein recognition where the concept of specificity of interactions needs to be reexamined. The binding sites show a fluidity of protein contacts derived from ecotin's innate flexibility in fitting itself to proteases while strongly interfering with their function.

Probing structure-function relationships of tissue-type plasminogen activator by site-specific mutagenesis

Tissue-type plasminogen activator (t-PA), a 65 kDa member of the chymotrypsin family of serine proteases, catalyzes the rate-limiting step in the endogenous fibrinolytic cascade: conversion of the zymogen plasminogen into the active enzyme plasmin.‘-” Plasmin, a serine protease of relatively broad specificity, efficiently degrades the fibrin that forms the meshwork of a thrombus or blood clot.‘-3 The fibrinolytic cascade, therefore, provides an important counterbalance in vivo to the blood coagulation cascade. Appreciation that inappropriate thrombosis was the focal event in the initiation of acute myocardial infarction, and the demonstration that timely administration of thrombolytic agents could restore patency to occluded coronary arteries sparked great interest in thrombolytic therapy, the endogenous fibrinolytic system and, in particular, the plasminogen activators. Current thrombolytic therapy achieves lysis of stable coronary thrombi through the intravenous administration of plasminogen activators, usually either human t-PA or the bacterial protein, streptokinase.‘.” The evidence that thrombolytic therapy is capable of rapidly lysing thrombi without undue risk of hemorrhage is now convincing; numerous studies and clinical trials have confirmed both the efficacy and the safety of thrombolytic agents in the treatment of patients with an evolving myocardial infarction.4-‘2 t-PA is synthesized and secreted by vascular endothelial cells.’ 3-16 The amino acid sequence of the human t-PA precursor, originally deduced from the nucleotide sequence of cloned cDNAs”,‘~ and subsequently confirmed by amino acid sequencing of the purified proteinI consists of 563 amino acids. As this precursor is transported along the secretory pathway, oligosaccharide groups are added at three positions (Asn 117, Asn 184, and Asn 448), and both a hydrophobic signal sequence and a hydrophilic prosequence are removed by endopeptidic cleavage. ‘9-2’ The resulting 527 amino

Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity

Thrombin Quick II is one of two dysfunctional forms of thrombin derived from the previously described congenital dysprothrombin prothrombin Quick. Thrombin Quick II does not clot fibrinogen, hydrolyze p-nitroanilide substrates of thrombin, or bind N2-[5-(dimethylamino)naphthalene-1-sulfonyl]arginine N,N-(3-ethyl-1,5-pentanediyl)amide, a high-affinity competitive inhibitor of thrombin. To determine the structural alteration in thrombin Quick II, the reduced, carboxymethylated protein was hydrolyzed by a lysyl endopeptidase. A peptide not present in a parallel thrombin hydrolysate was identified by reverse-phase chromatography. The peptide was purified by rechromatography and subjected to Edman degradation which showed that Gly-558 of human prothrombin had been replaced by Val. This corresponds to a point mutation of the Gly codon GGC to GUC. This Gly residue, which is highly conserved in the chymotrypsin family of serine proteases, forms part of the substrate binding pocket for bulky aromatic and basic side chains in chymotrypsin and trypsin, respectively. However, in porcine elastase 1, the corresponding residue is threonine. Consistent with the identified structural alteration, thrombin Quick II incorporates [3H]diisopropyl fluorophosphate stoichiometrically and hydrolyzes the elastase substrate succinyl-Ala-Ala-Pro-Leu-p-nitroanilide with a relative kcat/KM of 0.14 when compared to thrombin. This results from a 3-fold increase in KM and a 2.5-fold decrease in kcat for thrombin Quick II when compared to thrombin acting on the same substrate. These results and those of other investigators studying mutant trypsins support the conclusion that the catalytic activity of serine proteases is very sensitive to structural alterations in the primary substrate binding pocket.

Characterization of the cyanogen bromide fragments of the beta chain of human haptoglobin.

Characterization of the cyanogen bromide (CNBr) fragments of the beta chain of human haptoglobin revealed five major fragments resulting from cleavage of four methionyl residues. The fragments were isolated by gel filtration in guanidine-HCl on Sepharose 6B and Bio-Gel P10 and P60. Compositional analyses of the five cyanogen bromide fragments accounted for 248-253 amino acid residues in agreement with the number of residues determined for the intact beta chain. Most of the carbohydrate was attached to CNBr II. Automated amino-terminal sequence analysis and carboxyl-terminal hydrolysis with carboxypeptidase of the haptoglobin beta chain and cyanogen bromide fragments identified 139 residues, or about 55% of the beta-chain molecule. The placement of the fragments within the beta-chain molecule was established by sequence analysis of whole beta chain and a plasmin cleavage fragment. The position of CNBr V was confirmed by the absence of homoserine or homoserine lactone. Cyanogen bromide reaction of intact haptoglobin 1-1 resulted in the isolation of a beta-chain fragment, CNBr III, covalently attached to the intact alpha1 chain by a single disulfide bond. The beta chain was shown to have primary structural similarities to the chymotrypsin family of serin eproteases. Partial sequence analysis of CNBr V established the region which is comparable to the serine-195 active-site region: /Asp-Thr-Cys-Tyr-Gly-Asp-Ala-Gly-Ser-Ala-Phe/ (residues 189-199, chymotrypsinogen A numbering). The active-site serine-195 is replaced by alanine; however, the specificity residue of the trypsin-like enzymes, Asp-189, is preserved. Several minor cyanogen bromide cleavage products were also identified in yields of up to 15%. These minor cleavage products give evidence that tryptophanyl residues in proteins, or glycoproteins, are also susceptible to cyanogen bromide cleavage.