Serine Protease Zymogen Research Articles

Clotting and immune defense in Limulidae.

The blue blood of the horseshoe crab contains a sophisticated defense system very sensitive to pathogens or foreign materials. The hemocytes circulating in the hemolymph detect trace amounts of LPS molecules on the invading microorganisms and respond quickly to release the granular components into the external milieu. The coagulation system composed of three serine protease zymogens, factor C, factor B, and proclotting enzyme, and a clottable protein, coagulogen, is activated by LPS to form insoluble coagulin gel. The coagulation system also responds to beta-(1,3) glucan through the activation of unique heterodimeric serine protease zymogen, factor G. The pathogens are, thus, engulfed in the gel and subsequently killed by antimicrobial substances with various specificities, which are also released from cells. The horseshoe crab has developed two kinds of serine protease zymogens as biological sensors, factor C and factor G, which are responsive to LPS and beta-(1,3) glucan on the surface of Gram-negative bacteria and fungi, respectively. These are possible invaders for horseshoe crabs and also for most animals including humans. This novel heterodimeric serine protease zymogen, factor G, may open a new way to develop an innovative assay system to quantitate beta-(1,3) glucans. Furthermore, these LPS and beta-(1,3) glucan sensitive factors could be utilized as a unique tool to analyze other biological reactions caused by LPS or the glucan. Although the coagulation reaction in horseshoe crabs is famous, it is not the only defense mechanism of this animal. Many agglutinins are present either in hemolymph plasma or in the cell. The hemolymph plasma also has cytolytic activity against foreign cells. These cellular and humoral defense systems, in concert, defend themselves from invading foreign organisms. Such a sophisticated defense system has allowed the horseshoe crab to survive for more than 200 million years on the earth. Horseshoe crabs are often called ¿living fossils." However, they are not fossils. They are living.

Spatial regulation of Drosophila snake protease activity in the generation of dorsal-ventral polarity.

Positional information along the dorsal-ventral axis of the Drosophila embryo is acquired through a signal transduction pathway which employs a extracellular protease cascade. The sequential activation of serine protease zymogens results in the ventrally localized production of a ligand in the perivitelline space of the embryo. Snake is one of several serine proteases which function in generating the ventralizing signal. Here, we investigate the biochemical properties of Snake in vivo and in vitro using recombinant forms of the protease. Wild-type Snake zymogen completely rescues embryos from snake null females when microinjected into the perivitelline space. Biochemical evidence for a covalently associated two-chain form of the activated protease is presented. The contribution of the activation peptide region to zymogen activation was addressed using site-directed mutagenesis. The phenotypic rescue properties of an autoactivated form of Snake reveal that the covalently associated proenzyme polypeptide chain suppresses a dominant effect associated with the activated catalytic chain alone. Recombinant active catalytic chain was produced and found to be short lived as a recombinant protein. These results suggest a model in which the proenzyme polypeptide both stabilizes and targets the Snake catalytic chain to a ventrally localized activation complex within the perivitelline space.

Variants of Tissue-type Plasminogen Activator with Substantially Enhanced Response and Selectivity toward Fibrin Co-factors

Unlike most proteases, tissue-type plasminogen activator (t-PA) is not synthesized as an inactive precursor or zymogen. Instead, the single-chain "proenzyme" form of t-PA possesses very significant catalytic activity. Recent investigations of the molecular basis of the unusually high enzymatic activity of single-chain t-PA have focused attention upon Asp-194, a residue that is invariant among chymotrypsin-like enzymes. The critical role of this residue in securing the active conformation of mature chymotrypsin-like enzymes has been discussed extensively. Subsequent work, however, has indicated that this conserved residue can also form interactions that dramatically influence the catalytic activity of serine protease zymogens. While Asp-194 forms interactions that suppress the activity of the zymogen chymotrypsinogen, it may, by contrast, directly promote the catalytically active conformation of single-chain t-PA. To test the hypothesis that Asp-194 promotes the activity of both single- and two-chain t-PA and therefore plays opposing roles in single-chain t-PA and chymotrypsinogen, and also to examine whether this invariant residue plays an essential role in the stimulation of t-PA by fibrin, we used site-directed mutagenesis to construct the following variants of t-PA: t-PA/D194E, t-PA/D194N, t-PA/R15E,D194E, and t-PA/R15E,D194N. In the absence of fibrin, the activity of enzymes carrying a mutation at position 194 was reduced by factors of 1000-2000 compared to wild type t-PA. Similar reductions of activity were observed for both single- and two-chain variants, suggesting an important role for Asp-194 in both forms of the enzyme. The mutated enzymes, however, displayed a dramatically enhanced response to fibrin monomers. While the activity of wild type t-PA was stimulated by fibrin monomers by a factor of 960, the corresponding stimulation factor for the mutated enzymes varied from 498,000-1,050,000.

Ostrich trypsinogen: Purification, kinetic properties and characterization of the pancreatic enzyme

Trypsinogen is a serine protease zymogen (EC. 3.4.21.4) which has proved to be of key significance in a family of about 20 structurally and functionally related pancreatic digestive enzymes. This study was an endeavour to isolate, purify and characterize a stable form of ostrich trypsinogen, which has thus far not yet been accomplished. Trypsinogen (anionic) was isolated and purified by alkaline extraction of pancreatic acetone powder, followed by Toyopearl DEAE 650M, hydroxylapatite and LBTI-Sepharose affinity chromatography. The enzyme was chemically, physically and kinetically characterized, using amidase and esterase activity and spectrofluorometric determinations. Effects of CaCl 2 and pH, among others, were examined. Purification of homogeneous anionic ostrich trypsinogen was achieved. Immunochemical analysis and spectrofluorometric reaction with sulphonyl-Ala-Ala-Pro-Arg-7-amino-4-methylcoumarin indicated trypsin-free ostrich trypsinogen, with an average M r of 23,016 and a pI of 4.93. N-terminal sequence data revealed an unique activation peptide sequence, VPGDADDDK. Certain concentrations of Ca 2+ enhanced trypsinogen activation, whilst others appeared to have the opposite effect. The k cat K m values obtained at different pHs, using N α -benzoyl- dl-arginine- p-nitroanilide, p-toluenesulphonyl-arginine-methylester and p-toluenesulphonyl-lysine-methylester, followed the pH profile activity trend closely, with maximum catalytic activity at about pH 8 for both ostrich and bovine activated trypsinogen. Ostrich trypsin has significantly higher amidase activity than bovine trypsin, while esterase activities of the two enzymes have an inverse ratio. Kinetic p K a values were 7.2 and 7.4 for ostrich and bovine activated trypsinogens, respectively. The existence of ostrich trypsinogen in a now homogeneous stable form, free of autocatalytic inducing impurities, together with its characterization scenario will hopefully make a significant contribution to the field of comparative biochemistry. This study also confirms that ostrich trypsinogen is closely related to its serine protease counterparts.

A Novel Type of limulus Lectin-L6. PURIFICATION, PRIMARY STRUCTURE, AND ANTIBACTERIAL ACTIVITY

Lipopolysaccharide (LPS)-binding protein(s) was first screened in the detergent extract of horseshoe crab (limulus) hemocytes using LPS-immobilized agarose. A protein, designated L6 (M(r) = 27,000), was found to bind to LPS-agarose and was eluted with EDTA or o-phenanthroline. The L6 protein, however, did not inhibit the LPS-mediated activation of a limulus serine protease zymogen factor C. L6 had an affinity to the matrix of Sepharose CL-6B itself, and it could be eluted with high concentrations of monosaccharides (0.5-1.0 M), such as glucose, mannose, and galactose, suggesting a lectin-like nature. The entire amino acid sequence of L6 was determined by sequencing peptides derived from CNBr and enzymatic cleavages. L6 contained 7 half-cystines, and 1 cysteine residue at position 201 had a free SH-group. In addition, positions of the remaining three intrachain disulfide bonds were assigned by amino acid and sequence analyses of three cystinyl peptides produced by lysyl endopeptidase digestion. These results indicated that the entire sequence of L6 consisted of 221 residues with no N-linked sugar and was composed of six tandem repeats, each consisting of 33-38 amino acid residues. Inductively coupled plasma spectrometry of L6 indicated the presence of 0.75 mol zinc/mol of protein. No significant sequence homology was observed between L6 and other proteins, including various animal lectins and LPS-binding proteins. However, L6 showed agglutinating activity on LPS-coated sheep erythrocytes and Gram-negative and Gram-positive bacteria, it inhibited the growth of Gram-negative bacteria, and thus it presumably recognizes carbohydrate components in the cell wall of bacteria.

A serine protease zymogen in insect plasma. Purification and activation by microbial cell wall components.

A protease zymogen present in the plasma fraction of the hemolymph of silkworm, Bombyx mori, was purified to homogeneity as judged by SDS/PAGE and IEF/PAGE. An activating system for the zymogen was also isolated from the plasma fraction and was shown to be triggered by zymosan (yeast cell wall polysaccharide containing beta-1,3-glucan) or peptidoglycan. Using this system, the purified zymogen was activated and the active enzyme was purified to homogeneity. The physiological function of the zymogen or its active form is not yet known, but the active form was shown to have narrower substrate specificity than trypsin. Among 33 peptide derivatives examined, Boc-Gln-Arg-Arg-NH-Mec and Boc-Val-Pro-Arg-NH-Mec (Boc = tert-butoxycarbonyl, NH-Mec = 4-methylcoumaryl-7-amide) were the best and the second best substrates, respectively. The purified zymogen was determined to be a 39-kDa protein consisting of a single polypeptide. The active form of the zymogen was labeled with [3H]diisopropylfluorophosphate and was completely inactivated by (p-amidinophenyl)methanesulfonyl fluoride. The molecular mass of the [3H]-labeled enzyme was determined to be 38 kDa in SDS/PAGE under reducing conditions. These results indicate that the 39-kDa protein purified in the present study is a zymogen of a serine-type protease and that the activation of the zymogen occurs by limited proteolysis.

A Serine Protease Zymogen in Insect Plasma. Purification and Activation by Microbial Cell Wall Components

A protease zymogen present in the plasma fraction of the hemolymph of silkworm, Bombyx mori, was purified to homogeneity as judged by SDS/PAGE and IEF/PAGE. An activating system for the zymogen was also isolated from the plasma fraction and was shown to be triggered by zymosan (yeast cell wall polysaccharide containing β-1,3-glucan) or peptidoglycan. Using this system, the purified zymogen was activated and the active enzyme was purified to homogeneity. The physiological function of the zymogen or its active form is not yet known, but the active form was shown to have narrower substrate specificity than trypsin. Among 33 peptide derivatives examined, Boc-Gln-Arg-Arg-NH-Mec and Boc-Val-Pro-Arg-NH-Mec (Boc =tert -butoxycarbonyl, NH-Mec = 4-methylcoumaryl-7-amide) were the best and the second best substrates, respectively. The purified zymogen was determined to be a 39-kDa protein consisting of a single polypeptide. The active form of the zymogen was labeled with [3H]diisopropylfluorophosphate and was completely inactivated by (p -amidinophenyl)methanesulfonyl fluoride. The molecular mass of the [3H]-labeled enzyme was determined to be 38 kDa in SDS/PAGE under reducing conditions. These results indicate that the 39-kDa protein purified in the present study is a zymogen of a serine-type protease and that the activation of the zymogen occurs by limited proteolysis.

Purified Horseshoe Crab Factor G: RECONSTITUTION AND CHARACTERIZATION OF THE (1→3)-β-D-GLUCAN-SENSITIVE SERINE PROTEASE CASCADE

Horseshoe crab hemocyte lysate responds to (1-->3)-beta-D-glucans, initiating an enzymatic cascade, which culuminates in clot formation. We have purified to homogeneity the serine protease zymogen factor G, which is directly activated by (1-->3)-beta-D-glucans and which initiates the hemolymph clotting cascade. Factor G is a heterodimeric protein composed of two noncovalently associated subunits alpha (72 kDa) and beta (37 kDa). In the presence of (1-->3)-beta-D-glucans such as curdlan and paramylon, factor G is autocatalytically activated to an active serine protease named factor G. This activation is accompanied by limited proteolysis of both subunits: the 72-kDa subunit alpha is cleaved to 55-kDa and 17-kDa fragments, and the 37-kDa subunit beta is shortened to 34 kDa. Longer incubations with (1-->3)-beta-D-glucans result in cleavage of the 55-kDa fragment to 46 kDa and the 34-kDa fragment to 32 kDa, with concomitant loss of amidase activity. Reconstitution experiments using purified proteins participating in the hemolymph clotting cascade demonstrate that factor G is capable of activating proclotting enzyme directly, resulting in the conversion of coagulogen to coagulin gel. Thus, purified factor G is shown to be the primary initiator of the (1-->3)-beta-D-glucan-sensitive coagulation pathway in the horseshoe crab hemocyte lysate.

Characteristic effects of activated human protein C on tissue thromboplastin-induced disseminated intravascular coagulation in rabbits

Protein C (PC) is the zymogen of an anticoagulant serine protease and is converted to its active form (activated protein C: APC) by thrombin in the presence of thrombomodulin. APC plays an important role in regulating thrombosis and fibrinolysis by inhibiting not only blood coagulation factors Va and Villa but also type-1 plasminogen activator inhibitor (PAI-1). In the present study we examined the effects of human APC on tissue thromboplastin-induced disseminated intravascular coagulation (DIC) in rabbits and compared them with those of heparin. Both APC (300–3000U/kg) and heparin (100–300 IU/kg) inhibited the decreases in platelet count and fibrinogen level equally. APC improved the prolonged bleeding time, but heparin aggravated bleeding with potent prolongation of activated partial thromboplastin time (APTT). Furthermore, in APC-treated animals, fibrin deposition in glomeruli was less than in heparin-treated animals. This result suggests that APC accelerated local fibrinolysis by neutralizing PAI-1. From our findings, we concluded that APC can improve both coagulation and fibrinolysis in a DIC model and should be useful for the clinical remedy of DIC without having an adverse side effect like a bleeding tendency.

The pathophysiology of venous thrombosis

HE PATHOPHYSIOLOGY of venous thrombosis directly relates to the evolutionary developments of the cardiovascular system. All members of the animal kingdom have some form of blood. In the more primitive species, this is called hemolymph. In crustacea, this material contains proteins that can coagulate with trauma but there is no enzymic clotting cascade. In more evolved species such as horseshoe crabs, a series of serine protease zymogens are present, but there is no recognizable fibrinogen. Coagulation proceeds through sticky white blood cells. In contrast, even the more primitive vertebrates, such as Lampreys, have fibrinogen, prothrombin, tissue factor, and plasminogen and, in overall organization, are analogous to the situation in humans. The presence of a coagulation system is required for the maintenance of fluid-derived bodily nutrition and, hence, is essential for life. Unlike the open low pressure circulation system of the invertebrates, in higher animals and, in particular, in mammals, an enclosed high pressure circulation exists. This situation exposes more extreme demands on the hemostatic and flow requirements of the circulatory system. The net result is the concept of a balance between the requirements to allow blood to remain fluid to provide organ nutrition yet, at the same time, to be able to rapidly form blood clots. The biologic divergence of these two competing requirements probably explains the multiple levels of activity and regulation in the hemostatic system. The simplistic approach to the regulation of thrombosis is to divide the process into discrete compartments. These are the constituents of Virchov's classical triad of causes of thrombosis that involved vessel wall factors, blood clotting, and blood flow characteristicsJ In practice, these components have evolved as a highly integrated process, but, for the purpose of discussing their functions, it is convenient to maintain the traditional format and to crossreference interactions.

Proline at the P2 position in protein C is important for calcium- mediated regulation of protein C activation and secretion

Protein C is a vitamin K-dependent plasma serine protease zymogen, which upon activation, functions as an anticoagulant. Protein C activation is catalyzed by a complex of thrombin (T) with thrombomodulin (TM). This activation is Ca(2+)-dependent, but Ca2+ inhibits protein C activation by thrombin alone. In most proteases, specificity is determined primarily by the residues that lie near the scissile bond. In protein C, the P2 position is Pro, whereas in the fibrinogen A chain, P2 is Val. We have expressed a Pro-->Val mutant of protein C (P168V) in mammalian cells. At saturating Ca2+, the P168V and wild-type proteins were activated by the T-TM complex equivalently, but half maximal rates of activation were obtained at 50 mumol/L Ca2+ for wild type and approximately 5 mmol/L Ca2+ for the P168V mutant. In the absence of TM, Ca2+ no longer inhibited the activation of the P168V mutant. These results indicate that Pro168 influences the Ca(2+)- dependent conformational changes in protein C that control activation. Recently, a patient with thrombotic complications has been identified with a Pro168-->Leu substitution. Both the P168V and the P168L mutation lead to impaired secretion caused by retention within the cell.

Proline at the P2 Position in Protein C Is Important for Calcium-Mediated Regulation of Protein C Activation and Secretion

AbstractProtein C is a vitamin K-dependent plasma serine protease zymogen, which upon activation, functions as an anticoagulant. Protein C activation is catalyzed by a complex of thrombin (T) with thrombomodulin (TM). This activation is Ca2+-dependent, but Ca2+ inhibits protein C activation by thrombin alone. In most proteases, specificity is determined primarily by the residues that lie near the scissile bond. In protein C, the P2 position is Pro, whereas in the fibrinogen A chain, P2 is Val. We have expressed a Pro → Val mutant of protein C (P168V) in mammalian cells. At saturating Ca2+ the P168V and wild-type proteins were activated by the T-TM complex equivalently, but half maximal rates of activation were obtained at 50 μmol/L Ca2+ for wild type and ≈5 mmol/L Ca2+ for the P168V mutant. In the absence of TM, Ca2+ no longer inhibited the activation of the P168V mutant. These results indicate that Pro168 influences the Ca2+-de-pendent conformational changes in protein C that control activation. Recently, a patient with thrombotic complications has been identified with a Pro168 → Leu substitution. Both the P168V and the P168L mutation lead to impaired secretion caused by retention within the cell.

Activation-induced exposure of the thrombin anion-binding exosite. Interactions of recombinant mutant prothrombins with thrombomodulin and a thrombin exosite-specific antibody.

The activation of serine protease zymogens involves conformational changes that increase the affinity of substrate binding and the activity of the catalytic center. The activation of prothrombin is particularly complex and requires several cleavages in the proenzyme region in addition to the conserved activation cleavage after Arg320. To understand how these cleavages lead to the exposure of the thrombin anion-binding exosite, a major macromolecular recognition site, interactions of recombinant human prothrombin derivatives with thrombomodulin, and an exosite-specific antibody were studied by competition binding and immunoprecipitation. By either method, the anion-binding exosite is not functional on prethrombin 2, which is cleaved after Arg271 and lacks fragment 1.2, nor on meizothrombin, which is cleaved only after Arg320. In contrast, the exosite is fully exposed on meizothrombin des-F1, which is cleaved after both Arg320 and Arg155 and therefore lacks amino-terminal fragment 1 (F1). Thus, two events are required to create the exosite. First, cleavage after Arg320 causes conformational changes that are much more extensive than those accompanying the activation of trypsinogen. Second, removal of amino-terminal F1 is necessary, perhaps to relieve steric hindrance. These results indicate that the F1 fragment regulates access to the thrombin exosite. The properties of meizothrombin des-F1 suggest that this prothrombin derivative could have a biological function.

Horseshoe crab (1,3)-beta-D-glucan-sensitive coagulation factor G. A serine protease zymogen heterodimer with similarities to beta-glucan-binding proteins.

Horseshoe crab factor G is an intracellular serine protease zymogen that initiates the (1,3)-beta-D-glucan-sensitive hemolymph clotting pathway. Unlike other known serine protease zymogens, which are composed of a single subunit, factor G consists of two distinct subunits, alpha and beta, which are autocatalytically converted to active factor G in the presence of (1,3)-beta-D-glucan. We have now cloned and sequenced cDNAs encoding both subunits of factor G. The subunits are derived from separate mRNA species and thus encoded by different genes. Subunit beta is a serine protease zymogen which consists of 278 residues with a calculated molecular mass of 30,846 Da; it exhibits homology to the serine protease domain of horseshoe crab factor B. Subunit alpha, on the other hand, is a new type of mosaic protein with intriguing features. The mature protein consists of 654 residues with a calculated molecular mass of 73,916 Da. The NH2-terminal portion of this subunit is similar to bacterial beta-1,3-glucanases. Its 126 amino acid COOH terminus exhibits a repetitive sequence having partial homology to xylanases. Between these regions are three repeating units of 47 amino acids, whose similarity to carbohydrate-binding proteins suggests that these may be the (1,3)-beta-D-glucan-binding domain(s) of factor G. Factor G, thus, is a structurally unique heterodimeric serine protease zymogen and as such may represent a new class of active defense proteins.

A Limulus intracellular coagulation inhibitor with characteristics of the serpin superfamily. Purification, characterization, and cDNA cloning.

A Limulus intracellular coagulation inhibitor, designated LICI, was isolated from hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), using three steps of chromatography, including dextran sulfate-Sepharose CL-6B, Sephacryl S-200, and Mono S. LICI is a single-chain glycoprotein with an apparent M(r) = 48,000 estimated by SDS-polyacrylamide gel electrophoresis. It blocks the amidolytic activities of Limulus lipopolysaccharide-sensitive serine protease, factor C, by forming a covalent 1:1 complex with the protease. The second-order rate constant for inhibition of factor C was 2.5 x 10(6) M-1 s-1 at 37 degrees C. LICI also inhibited human alpha-thrombin, rat salivary kallikrein, bovine plasmin, and trypsin but not Limulus clotting enzyme, Limulus factor B, bovine factor Xa, human factor XIa, human tissue plasminogen activator, human urokinase, chymotrypsin, elastase, and papain. Glycosaminoglycans such as heparin and heparan sulfate had no effect on the inhibitory activity. A cDNA coding for LICI was isolated from a hemocyte cDNA library. The open reading frame of the 1,257-base pair cDNA codes for the mature protein of 394 amino acids, of which 223 residues were confirmed by amino acid sequence analysis. LICI shows significant sequence identities to members of the serpin superfamily, such as human plasminogen activator inhibitor type 2 (40%) and human monocyte/neutrophil elastase inhibitor (39%). LICI contains a putative reactive site, -Arg-Ser-, at the corresponding position present in several inhibitors of the serpin superfamily. The subcellular localization, determined using an anti-LICI polyclonal antibody, indicated that LICI colocates with the Limulus serine protease zymogens in large granules in the hemocyte.

Horseshoe crab coagulation factor B. A unique serine protease zymogen activated by cleavage of an Ile-Ile bond.

Horseshoe crab factor B is an intracellular serine protease zymogen involved in the bacterial endotoxin-responsive hemolymph coagulation cascade. cDNAs for factor B were isolated utilizing a polymerase chain reaction product using two primers derived from the partial amino acid sequence. The cloned cDNA of 1928 base pairs encoded 400 amino acid residues of factor B precursor. The first 23 amino acid residues constitute a presumed prepropeptide that may be processed by both a signal peptidase and a processing protease, similar to mammalian vitamin K-dependent protease precursors. The mature protein consists of 377 amino acids with a calculated molecular mass of 40,570 Da. The overall structure is highly homologous to that of limulus proclotting enzyme (35.9% identity), the substrate for active factor B in the cascade. Like the proclotting enzyme, mature factor B is composed of an amino-terminal "clip"-like domain and a carboxyl-terminal serine protease domain homologous to that of human plasma prekallikrein (36.5%). Internal sequences encode a unique activation peptide. Surprisingly, the cleavage sites of the zymogen factor B for activation by limulus active factor C were found to be an Arg-Ser and an Ile-Ile bond, the latter of which has not been found in any other protease zymogens. These cleavages result in the release of the activation peptide, which consists of 21 residues with a carboxyl-terminal isoleucine. These results indicate that the intracellular clotting system of the limulus hemocyte, like mammalian plasma clotting cascade, proceeds with the sequential activation of three serine protease zymogens: factor C, factor B, and proclotting enzyme.

Specific molecular interaction sites on factor VII involved in factor X activation.

Factor VII, a serine-protease zymogen, and tissue factor, the cellular receptor/coenzyme, are the protein components of the macromolecular complex which initiates the extrinsic pathway of the coagulation cascade. Previous studies were directed to the identification of functional sites on factor VII which mediate factor X activation, employing a series of potentially inhibitory synthetic peptides representing the primary structure of factor VII and antibodies to selected peptides. The involvement of at least four high-affinity interactive regions [factor VII (44-50), (196-229), (285-305) and (376-396) peptides] on the surface of factor VII was clearly demonstrated. The minimal sequences for the expression of inhibitory activity of these four molecular recognition domains on factor VII were identified using short and overlapping peptides. The short factor VII-(206-218)-peptide (most inhibitory peptide in the sequence 196-229 on factor VII) inhibited the binding of factor VII to the tissue-factor-expressing J82 cell line. Furthermore, radiolabeled [Tyr201] factor VII-(199-221)-peptide, with a tyrosine substituted for the normal tryptophan residue, was specifically bound to J82 cells, and also the binding of the radiolabeled peptide to this cell line was specifically inhibited by a monoclonal antibody to tissue factor, confirming that the interaction site for tissue factor on factor VII is present within the peptide sequence 196-229. Kinetic analyses suggested that the regions represented by factor VII-(285-305)- and factor VII-(376-396)-peptides are involved in factor X recognition and the chemical cross-linking of the radiolabeled peptides resulted in specific binding to factor X, confirming that these two regions on factor VII represent the substrate-recognition site. Furthermore, these radiolabeled peptides specifically interact with the heavy chain of factor X, suggesting that the complementary binding region for the substrate-recognition site on factor VII are present on the heavy chain of factor X.

Conversion of glutamic acid 192 to glutamine in activated protein C changes the substrate specificity and increases reactivity toward macromolecular inhibitors.

Protein C is a vitamin K-dependent serine protease zymogen that upon activation inhibits the coagulation cascade by inactivating factors Va and VIIIa. In an attempt to improve the anticoagulant activity of activated protein C (APC), we have prepared a mutant of protein C in mammalian cells in which Glu at position 192 (chymotrypsin numbering system) has been replaced with Gln (PC E192Q). Our strategy is based on the observation that the same substitution in thrombin improves the catalytic activity toward natural and synthetic substrates that contain Asp residues at P3 and P3'. Since factor Va also has an Asp at position P3 in the APC cleavage site of the factor Va heavy chain, we hypothesized that APC E192Q would inactivate factor Va more rapidly than wild type APC. The mutant inactivated factor Va approximately 2-3-fold faster than wild type. In plasma the mutant exhibited slightly less anticoagulant activity than wild type enzyme. Further characterization revealed that APC E192Q is inhibited 280 times faster than APC by alpha 1-antitrypsin (K2 = 2.8 x 10(3) M-1S-1 versus 10 M-1 S-1), and unlike APC, APC E192Q is inhibited by antithrombin III in the presence of heparin (K2 = 1.17 x 10(3) M-1 S-1) M-1 S-1) and absence of heparin (K2 = 57 M-1 S-1). Ca2+ increased K2 more than 4-fold with or without heparin. Unlike wild type APC, APC E192Q was effectively inhibited by pancreatic trypsin inhibitor (Ki = 10.6 +/- 0.26 nM) and tissue factor pathway inhibitor (58 +/- 5 nM). Like factor Xa, APC E192Q rapidly processed factor IX to factor IX alpha. These observations suggest that even though Glu at position 192 is not an optimal residue for catalyzing factor Va inactivation, it is an evolutionary adaptation to slow inhibition by plasma protease inhibitors.

Molluscan Chymotrypsin-like Protease: Structure, Localization, and Substrate Specificity

A messenger RNA encoding a chymotrypsin-like preproprotease is expressed abundantly and specifically in the distal quarter of the intestine of the molluse Haliotis rufescens (red abalone). Consistent with this finding, a chymotrypsin-like activity was detected at highest concentration in the lumen of this segment of the intestine. Because the complexity of total protein in the distal intestinal fluid was low and the chymotrypsin-like protease was highly expressed, purification of the enzyme to near homogeneity was achieved by a single passage over an anion-exchange resin. The primary specificity of the protease, predicted from homology of the key amino acid residues (Serl89, Gly216, and Ser226) in the substrate binding site with similar residues lining the S 1 subsites of other chymotrypsin-like enzymes, was confirmed by hydrolysis of a family of tetrapeptide substrates with different P 1 amino acids. The optimal P 1 residues include those with bulky, γ-branched side chains (phenylalanine and leucine) similar to the side chain of the asparagine residue at P 1 of the activation peptide of the proenzyme. Thus, unlike zymogens of the pancreatic serine proteases, which are activated by the common tryptic mechanism, the zymogen of the molluscan enzyme appears to be activated by an autocatalytic mechanism, i.e., by cleavage with active chymotrypsin-like protease. Additional unique properties of the enzyme predicted from the primary sequence include an unpaired cysteine, with the potential for internal thiol-disulfide isomerization between two different conformers of the protease.

The limulus clotting reaction

Our biochemical studies on the hemolymph coagulation-complement system using limulus indicate that the circulating hemocytes contain at least four serine protease zymogens and one clottable protein, coagulogen, which constitute a cascade triggered by bacterial endotoxins and (1,3)-β- D-glucan. We also found several antimicrobial substances, tachyplesin peptides and anti-lipopolysaccharide factor, in the hemocytes. These clotting factors and antimicrobial substances are released into the hemolymph in response to lipopolysaccharide, where they cooperate in immobilization and killing of invading microorganisms as a host defense.