Characterization of serine proteases of Lumbriculus variegatus and their role in regeneration

Serine proteases, ubiquitous enzymes known to function in digestion and immune protection in both vertebrates and invertebrates and implicated in regeneration in some species, were investigated in the California blackworm, Lumbriculus variegatus. Several serine proteases, rather than a single enzyme with broad specificity, were present in tissue extracts from the worms. Extracts were treated with a fluorescein‐labeled peptide chloromethyl ketone that specifically binds to trypsin/thrombin‐like proteases. Denaturing gel electrophoresis of labeled extracts showed several serine proteases with their molecular weight ranging 28,000–38,000 daltons. The trypsin/thrombin‐like activity was localized, using the fluorescein‐conjugated reagent, to the pharynx and digestive tract of L. variegatus. Movement of cells labeled by the reagent into regenerating tissues suggests that some differentiated endodermal tissues were used for reformation of digestive structures during regeneration in L. variegatus. The types of serine proteases in the extracts were further characterized by inhibitor studies. Presence of plasmin‐like activity was indicated by degradation of fibrin by tissue homogenates from the worms and the inhibitory effect of aprotinin on enzymes in these extracts. The ability of L. variegatus extracts to generate clots when incubated with rabbit plasma and partial inhibition of extract activity by phenylmethylsulfonyl fluoride and hirudin indicated presence of thrombin‐like activity. Consistent with the detection of trypsin, chymotrypsin, and plasmin‐like enzymes in the extracts was partial inhibition of L. variegatus serine protease activity by aminoethyl benzenesulfonyl fluoride and soybean trypsin inhibitor. Selective inhibition of chymotrypsin‐like activity by N‐tosyl‐l ‐phenylalanine chloromethyl ketone and chymostatin as well as trypsin‐like activity by N‐tosyl‐l ‐lysine chloromethyl ketone was observed. A potential role during regeneration for serine proteases is suggested by blockage of formation of head and tail structures by aminoethyl benzenesulfonyl fluoride, an inhibitor of these proteases.     

Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays

A novel microarray-based proteolytic profiling assay enabled the rapid determination of protease substrate specificities with minimal sample and enzyme usage. A 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized and microarrayed, along with fluorescent calibration standards, in glycerol nanodroplets on microscope slides. The arrays were then activated by deposition of an aerosolized enzyme solution, followed by incubation and fluorometric scanning. The specificities of human blood serine proteases (human thrombin, factor Xa, plasmin, and urokinase plasminogen activator) were examined. The arrays provided complete maps of protease specificity for all of the substrates tested and allowed for detection of cooperative interactions between substrate subsites. The arrays were further utilized to explore the conservation of thrombin specificity across species by comparing the proteolytic fingerprints of human, bovine, and salmon thrombin. These enzymes share nearly identical specificity profiles despite approximately 390 million years of divergent evolution. Fluorogenic substrate microarrays provide a rapid way to determine protease substrate specificity information that can be used for the design of selective inhibitors and substrates, the study of evolutionary divergence, and potentially, for diagnostic applications.     

The phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors

Serine proteases are involved in many processes in the nervous system and specific inhibitors tightly control their proteolytic activity. Thrombin is thought to play a role in tissue development and homeostasis. To date, protease nexin-1 is the only known endogenous protease inhibitor that specifically interferes with thrombotic activity and is expressed in the brain. In this study, we report the detection of a novel thrombin inhibitory activity in the brain of protease nexin-1(-/-) mice. Purification and subsequent analysis by tandem mass spectrometry identified this protein as the phosphatidylethanolamine-binding protein (PEBP). We demonstrate that PEBP exerts inhibitory activity against several serine proteases including thrombin, neuropsin, and chymotrypsin, whereas trypsin, tissue type plasminogen activator, and elastase are not affected. Since PEBP does not share significant homology with other serine protease inhibitors, our results define it as the prototype of a novel class of serine protease inhibitors. PEBP immunoreactivity is found on the surface of Rat-1 fibroblast cells and although its sequence contains no secretion signal, PEBP-H(6) can be purified from the conditioned medium upon recombinant expression.     

Amino acid sequence of human D of the alternative complement pathway

The primary structure of human D, the serine protease activating the C3 convertase of the alternative complement pathway, has been deduced by sequencing peptides derived from various chemical (CNBr and o-iodosobenzoic acid) and enzymatic (trypsin, lysine protease, Staphylococcus aureus V8 protease, and chymotrypsin) cleavages. Carboxypeptidase A was also used to confirm the COOH-terminal sequence. The peptides were purified by high-pressure liquid chromatography. The proposed sequence of human D contains 222 amino acids and has a calculated molecular weight of 23 748. It exhibits a high degree of homology with other serine proteases, especially around the NH2-terminus as well as the three residues corresponding to the active-site His-57, Asp-102, and Ser-195 (chymotrypsinogen numbering). This sequence homology is highest (40%) with plasmin, intermediate (35%) with pancreatic serine proteases, such as elastase, trypsin, chymotrypsin, and kallikrein, and least (30%) with the serum enzymes thrombin and factor X. D, however, exhibits only minimal amino acid homology with the other sequenced complement serine proteases, Clr (25%) and Bb (20%). The substitution of a basic lysine for a neutral amino acid three residues NH2-terminal to the active-site serine as well as a small serine residue for a bulky aromatic amino acid at position 215 (chymotrypsinogen numbering) in the binding pocket may be important in determining the exquisite substrate specificity of D. The presence of His-40 which interacts with Asp-194 (chymotrypsinogen numbering) to stabilize other serine protease zymogens [Freer, S. T., Kraut, J., Robertus, J. D., Wright, H. T., & Xuong, N. H. (1970) Biochemistry 9, 1997] argues in favor of such a D precursor molecule.     

Functional and Structural Characterization of Vibrio cholerae Extracellular Serine Protease B,VesB

The chymotrypsin subfamily A of serine proteases consists primarily of eukaryotic proteases, including only a few proteases of bacterial origin. VesB, a newly identified serine protease that is secreted by the type II secretion system in Vibrio cholerae, belongs to this subfamily. VesB is likely produced as a zymogen because sequence alignment with trypsinogen identified a putative cleavage site for activation and a catalytic triad, His-Asp-Ser. Using synthetic peptides, VesB efficiently cleaved a trypsin substrate, but not chymotrypsin and elastase substrates. The reversible serine protease inhibitor, benzamidine, inhibited VesB and served as an immobilized ligand for VesB affinity purification, further indicating its relationship with trypsin-like enzymes. Consistent with this family of serine proteases, N-terminal sequencing implied that the propeptide is removed in the secreted form of VesB. Separate mutagenesis of the activation site and catalytic serine rendered VesB inactive, confirming the importance of these features for activity, but not for secretion. Similar to trypsin but, in contrast to thrombin and other coagulation factors, Na⁺ did not stimulate the activity of VesB, despite containing the Tyr²⁵⁰ signature. The crystal structure of catalytically inactive pro-VesB revealed that the protease domain is structurally similar to trypsinogen. The C-terminal domain of VesB was found to adopt an immunoglobulin (Ig)-fold that is structurally homologous to Ig-folds of other extracellular Vibrio proteins. Possible roles of the Ig-fold domain in stability, substrate specificity, cell surface association, and type II secretion of VesB, the first bacterial multidomain trypsin-like protease with known structure, are discussed.     

Cucumisin-like protease from the latex of Euphorbia supina

A protease has been purified from the latex of Euphorbia supina Rafin by two steps of chromatography. The Mr was estimated by SDS-PAGE to be 80 kDa. Its activity was inhibited strongly by diisopropyl fluorophosphate, but not by EDTA, pepstatin, or cysteine protease inhibitors, indicating that the enzyme is a serine protease. The specificity of the protease is broad, but the preferential cleavage sites were C-terminal sites of hydrophobic amino acid residues. The N-terminal sequence of the first fifteen residues was determined and six of the residues match those in cucumisin [EC 3.4.21.25], a protease from the sarcocarp of melon fruit (Cucumis melo L. var. Prince). The results indicate that the E. supina protease is a cucumisin-like serine protease.     

Biochemical characterization of the human complement protein C6. Association with alpha-thrombin-like enzyme and absence of serine protease activity in cytolytically active C6

Complement protein C6 has been proposed by others to be a serine protease whose activity is obligatory for complement-directed cell lysis. We separated the serine protease (Mr approximately 30,000) activity found associated with apparently homogeneous preparations of C6 from the hemolytically active C6 protein. The protease was characterized as thrombin-like based on substrate specificity, inhibitor profile, and kinetic studies. Although the proteolytic activity of C6 preparations was inhibitable by several inhibitors of serine proteases, the C6 hemolytic activity remained unaffected. Acid-induced (C(5,6)a complex formation between C5 and C6 (protease-free) was demonstrated by ion-exchange fast protein liquid chromatography, reversed-phase high performance liquid chromatography, and reactive cytolytic activity in the presence of C7, C8, and C9; but no cleavage of the alpha-chain of C5 was observed. Diisopropylphosphorofluoridate pretreatment of the components did not affect their ability to generate functionally active (C(5,6)a. Evidently, C6-associated thrombin is not required for formation of functional C(5,6)a. Thus, C6 does not function in the membrane attack pathway of complement as a serine protease. A method for the isolation of homogeneous C6 in the hemolytically fully active form is described. No free sulfhydryl group was detected in C6. The amino acid sequence of 20 amino-terminal residues was determined.     

Structural features of a snake venom thrombin-like enzyme: thrombin and trypsin on a single catalytic platform?

The Lachesis muta thrombin-like enzyme (LM-TL) is a single chain serine protease that shares 38% sequence identity with the serine protease domain of thrombin and also displays similar fibrinogen-clotting activity. In addition, the 228 amino acid residue LM-TL is 52% identical to trypsin, and cleaves chromogenic substrates with similar specificity. Herein we report a three-dimensional (3D) model validated experimentally for LM-TL based on these two homologous proteins of known 3D structure. Spatial modeling of LM-TL reveals a serine protease with a chymotrypsin fold presenting a hydrophobic pocket on its surface, involved in substrate recognition, and an important 90's loop, involved in restricting the LM-TL catalytic site cleft. Docking analysis showed that LM-TL would not form a stable complex with basic pancreatic trypsin inhibitor and wild-type ecotin since its 90's loop would restrict the access to the catalytic site. LM-TL formed acceptable interactions with fibrinopeptide A and a variant of ecotin; ecotin-TSRR/R in which both the primary and secondary binding sites are mutated Val81Thr, Thr83Ser, Met84Arg, Met85Arg and Asp70Arg. Furthermore, analysis of the primary structures of LM-TL and of the seven snake venom thrombin-like enzymes (SVTLEs) family reveals a subgroup formed by LM-TL, crotalase, and bilineobin, both closely related to thrombin. Therefore, LM-TL provides an initial point to compare SVTLEs with their counterparts, e.g. the mammalian serine proteases, and a basis for the localization of important residues within the little known SVTLEs family.     

Purification of rat urinary kallikrein: comparative studies with rat submandibular gland kallikrein-like serine protease.

A 427-fold purification of rat urinary kallikrein (RUK) was achieved in three steps involving chromatography on columns of DEAE-Sepharose CL-6B, gel filtration on Sephadex G-100 and affinity chromatography on a column of benzamidine-Sepharose. Purified enzyme showed a single band on SDS-PAGE with an estimated molecular weight of 43,000. The amino-terminal sequences of the first 25 residues of RUK resemble the reported sequence for true kallikrein and share 80% identity with rat submandibular gland (RSMG) kallikrein-like serine protease. The RUK is highly reactive towards kallikrein substrates Bz-pro-phe-arg-pNA and DL-val-leu-arg-pNA, and plasmin substrate D-val-leu-lys-pNA. RSMG enzyme is more reactive towards Bz-val-gly-arg-pNA and tosyl-gly-pro-arg-pNA, preferential chromogenic substrates for trypsin-like proteases and thrombin, respectively. Both leupeptin and aprotinin inhibit RUK strongly, but soy bean trypsin inhibitor has no effect on this enzyme. RSMG enzyme is poorly inhibited by any of these inhibitors. The data suggest that although both enzymes are members of tissue kallikrein multigene family, urinary enzyme is a true kallikrein and RSMG enzyme is a kallikrein-like serine protease with different substrate specificity.     

Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition.

Thrombin is a serine protease that plays a central role in blood coagulation. It is inhibited by hirudin, a polypeptide of 65 amino acids, through the formation of a tight, noncovalent complex. Tetragonal crystals of the complex formed between human alpha-thrombin and recombinant hirudin (variant 1) have been grown and the crystal structure of this complex has been determined to a resolution of 2.95 A. This structure shows that hirudin inhibits thrombin by a previously unobserved mechanism. In contrast to other inhibitors of serine proteases, the specificity of hirudin is not due to interaction with the primary specificity pocket of thrombin, but rather through binding at sites both close to and distant from the active site. The carboxyl tail of hirudin (residues 48-65) wraps around thrombin along the putative fibrinogen secondary binding site. This long groove extends from the active site cleft and is flanked by the thrombin loops 35-39 and 70-80. Hirudin makes a number of ionic and hydrophobic interactions with thrombin in this area. Furthermore hirudin binds with its N-terminal three residues Val, Val, Tyr to the thrombin active site cleft. Val1 occupies the position P2 and Tyr3 approximately the position P3 of the synthetic inhibitor D-Phe-Pro-ArgCH2Cl. Thus the hirudin polypeptide chain runs in a direction opposite to that expected for fibrinogen and that observed for the substrate-like inhibitor D-Phe-Pro-ArgCH2Cl.     

A collagenolytic serine protease with trypsin-like specificity from the fiddler crab Uca pugilator

A second collagenolytic serine protease has been isolated from the hepatopancreas of the fiddler crab, Uca pugilator. This enzyme cleaves the native triple helix of collagen under physiological conditions of pH, temperature, and ionic strength. In addition to its collagenolytic activity, the enzyme exhibits endopeptidase activity toward other polypeptides and small molecular weight synthetic substrates. The polypeptide bond specificity of this enzyme is similar to that of bovine trypsin as is its interaction with specific protease inhibitors. The amino-terminal sequence of this enzyme displays significant homology with other serine proteases, most notably with that of crayfish trypsin, and demonstrates that this enzyme is a member of the trypsin family of serine endopeptidases. The relatively unique action of this protease with regard to both collagenous and noncollagenous substrates has important implications concerning the specificity and mechanism of collagen degradation.     

Purification of Rat Urinary Kallikrein: Comparative Studies with Rat Submandibular Gland Kallikrein-Like Serine Protease

Abstract

A 427-fold purification of rat urinary kallikrein (RUK) was achieved in three steps involving chromatography on columns of DEAE-Sepharose CL-6B, gel filtration on Sephadex G-100 and affinity chromatography on a column of benzamidine-Sepharose. Purified enzyme showed a single band on SDS-PAGE with an estimated molecular weight of 43,000. The amino-terminal sequences of the first 25 residues of RUK resemble the reported sequence for true kallikrein and share 80% identity with rat submandibular gland (RSMG) kallikrein-like serine protease. The RUK is highly reactive towards kallikrein substrates Bz-pro-phe-arg-pNA and DL-val-leu-arg-pNA, and plasmin substrate D-val-leu-lys-pNA. RSMG enzyme is more reactive towards Bz-val-gly-arg-pNA and tosyl-gly-pro-arg-pNA, preferential chromogenic substrates for trypsin-like proteases and thrombin, respectively. Both leupeptin and aprotinin inhibit RUK strongly, but soy bean trypsin inhibitor has no effect on this enzyme. RSMG enzyme is poorly inhibited by any of these inhibitors. The data suggest that although both enzymes are members of tissue kallikrein multigene family, urinary enzyme is a true kallikrein and RSMG enzyme is a kallikrein-like serine protease with different substrate specificity.     

Evolution of Thrombin and Other Hemostatic Proteases by Survey of Protochordate, Hemichordate, and Echinoderm Genomes

Protochordate genomes enable a prevalence of hemostasis evolution. Broad searches were performed for homologs of human serine proteases of hemostasis on the genomes of Branchiostoma floridae, Saccoglossus kowalevskii, and Strongylocentrotus purpuratus. Sequences were analyzed by multiple bioinformatic tools. The survey revealed numerous homologous components. Amphioxus was rich in some serine proteases not accompanied by gamma-carboxyglutamic or kringle domains similar more to thrombin than to other coagulation factors. The serine proteases found in amphioxus exhibited the attributes similar to those of thrombin by phylogeny relationships, sequence conservation, gene synteny, spatial structure, and ligand docking. A few plasminogen- and plasminogen activators-like proteases with kringles were also present. Those serine proteases demonstrated the greatest proximity rather to plasminogen or plasminogen activators than to thrombin. Searching for homologs of serine protease hemostatic factors in acorn worm and sea urchin revealed several components similar to those found in amphioxus. Hypothetically, the common ancestor of chordates had three separate serine proteases that evolved independently into immunoglobulin-like and kringle proteases in lancelets, and prothrombin, plasminogen activators, and plasminogen in vertebrates. Ancestral proteases evolved in vertebrates into hemostasis factors after merging the proper N-terminal domains and duplications.     

丙型肝炎病毒丝氨酸蛋白酶特异性结合肽的筛选和鉴定

丙型肝炎病毒丝氨酸蛋白酶在病毒复制和包装中的重要作用使其成为特异性抗病毒药物研究的首选靶标。根据丝氨酸蛋白酶晶体结构特点,用柔性连接子连接NS3丝氨酸蛋白酶结构域和NS4A的核心序列,构建成单链丝氨酸蛋白酶基因并且在大肠杆菌中获得高水平的可溶性表达,纯化后的目的蛋白能够切割重组蛋白底物NS5ab。随后,以单链丝氨酸蛋白酶为靶分子对噬菌体展示的随机十二肽库进行了三轮淘筛,挑选的44个克隆中有37个克隆能够特异性地结合丝氨酸蛋白酶,并且这种结合作用为竞争性ELISA试验结果所支持。对13个克隆进行序列测定,得到6种序列,它们在氨基酸组成上存在明显偏性,富含组氨酸和色氨酸,缺乏酸性氨基酸;6种序列存在一个共有序列。     

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.     

SERINE PROTEASE FROM MIDGUT OF Bombus terrestris MALES

A serine protease was isolated from midguts of the bumblebee male Bombus terrestris by a combination of precipitation procedures with column chromatography. The purified enzyme exhibited two bands with molecular masses of 25 and 26 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These bands showed a proteolytic activity in zymography assay. Midgut enzymes showed optimum proteolytic activity at pH 9 and 35°C using N‐succinyl‐L‐alanyl‐L‐alanyl‐L‐prolyl‐L‐phenyl‐alanine 4‐nitroanilide as a substrate. The Michaelis constant (Km) and maximum reaction rate (Vmax) were 0.55 ± 0.042 mM and 0.714 ± 0.056 μmol p‐nitroalanine produced min^?1 mg protein^?1, respectively. Inhibition was affected by trypsin inhibitor, but not by phenylmethylsulfonyl fluoride and N‐tosyl‐L‐phenylalanine chloromethyl ketone, which indicated the trypsin‐like but not chymotrypsin‐like specificity. The identity of the serine protease was confirmed by nanoliquid‐tandem mass spectrometry. Eleven unique peptides of the B. terrestris serine protease were found. It shows high homology to a previously reported B. ignitus serine protease covering more than 65% of the protein amino acid sequence.     

Alteration of serpin specificity by a protein cofactor. Vitronectin endows plasminogen activator inhibitor 1 with thrombin inhibitory properties.

Serine protease inhibitors ("serpins") are highly homologous proteins which inhibit selected "target" serine proteases by acting as a pseudo-substrate. Their specificity is primarily determined by the amino acid sequence around the carboxyl-terminally located reactive center (P1-P1'). In addition, the association rate constant between a serpin and a serine protease can be dramatically increased by non-protein cofactors, such as heparin in the case of thrombin inhibition by antithrombin III. In an attempt to alter the specificity of PAI-1 from an inhibitor of the fibrinolytic system to an inhibitor of coagulation, we replaced P1-P1' or P3 through P3' of the reactive center of PAI-1 by the corresponding residues of antithrombin III and assessed whether the mutant proteins, purified from lysates of transformed Escherichia coli cells, had acquired thrombin inhibitory properties. The experiments were performed in the presence and absence of vitronectin, a multifunctional protein which has been shown to bind PAI-1 in plasma and in the matrix of endothelial cells. The second-order rate constants for t-PA inhibition of "wild-type" PAI-1 and PAI P1-P1'ATIII, irrespective of the presence of vitronectin, were similar, whereas replacing P3-P3' resulted in a 40-fold decrease of the second-order rate constant towards t-PA, again independent of vitronectin. In the absence of vitronectin, reactivity of PAI-1 and its "antithrombin III-like" variants towards thrombin was slow; however, PAI-1 P3-P3' ATIII had a 10-fold higher k1 than wild-type PAI-1 (1.3 x 10(4) M-1 s-1 versus 1.1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin, PAI-1 and even more rapidly PAI-1 P3-P3'ATIII were found to be effective thrombin inhibitors, with k1 values of 2.2 x 10(5) M-1s-1 and 1.8 x 10(6) M-1 s-1, respectively. Thus, in the presence of vitronectin, PAI-1 P3-P3'ATIII displays a 3-fold higher k1 with thrombin than with t-PA. It is shown that vitronectin enhances, in a dose-dependent manner, the formation of sodium dodecyl sulfate-resistant complexes between PAI-1 or mutants thereof and thrombin. Therefore, vitronectin is the first protein described to function as a cofactor for serpin specificity. PAI-1 is proposed to be a versatile inhibitor which, in the presence of vitronectin, can modulate both coagulation and fibrinolysis.     

The region of the thrombin receptor resembling hirudin binds to thrombin and alters enzyme specificity.

A thrombin receptor has recently been cloned and the sequence deduced. The sequence reveals a thrombin cleavage site that accounts for receptor activation. The receptor also has an acidic region with some similarities to the carboxyl-terminal region of the leech thrombin inhibitor, hirudin. Synthetic peptides corresponding to the receptor cleavage site (residues 38-45), the hirudin-like domain (residues 52-69), and the covalently associated domains (residues 38-64) were evaluated for their ability to bind to thrombin. Peptides 38-45 and 38-64 were competitive inhibitors of thrombin's chromogenic substrate activity (Ki = 0.96 mM and 0.6 microM, respectively. Residues 52-69 altered the chromogenic substrate specificity, resulting in accelerated cleavage of some substrates and inhibited cleavage of others. The same peptide binds to thrombin and alters the fluorescence emission intensity of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-thrombin in which the dansyl is attached directly to the active site serine (Kd = 32 +/- 7 microM). Residues 52-69 displace the carboxyl-terminal peptide of hirudin, indicating that they share a common binding site in the anion exosite of thrombin. These data suggest that the thrombin receptor has high affinity for thrombin due to the presence of the hirudin-like domain and that this domain alters the specificity of thrombin. This change in specificity may account for the ability of the receptor to serve as an excellent thrombin substrate despite the presence of an Asp residue in the P3 site, which is normally inhibitory to thrombin activity.     

Molecular cloning of dog mast cell tryptase and a related protease: structural evidence of a unique mode of serine protease activation

Mast cell tryptase is a secretory granule associated serine protease with trypsin-like specificity released extracellularly during mast cell degranulation. To determine the full primary structure of the catalytic domain and precursor forms of tryptase and to gain insight into its mode of activation, we cloned cDNAs coding for the complete amino acid sequence of dog mast cell tryptase and a second, possibly related, serine protease. Using RNA from dog mastocytoma cells, we constructed a cDNA library in lambda gt 10. Screening of the library with an oligonucleotide probe based on the N-terminal sequence of tryptase purified from the same cell source allowed us to isolate and sequence overlapping clones coding for dog mast cell tryptase. The tryptase sequence includes the essential residues of the catalytic triad and an aspartic acid at the base of the putative substrate binding pocket that confers P1 Arg and Lys specificity on tryptic serine proteases. The apparent N-terminal signal/activation peptide terminates in a glycine. A glycine in this position has not been observed previously in serine proteases and suggests a novel mode of activation. Additional screening of the library with a trypsinogen cDNA led to the isolation and sequencing of a full-length clone apparently coding for the complete sequence of a second tryptic serine protease (DMP) which is only 53.4% identical with the dog tryptase sequence but which contains an apparent signal/activation peptide also terminating in a glycine. Thus, the proteases encoded by these cloned cDNAs may share a common mode of activation from N-terminally extended precursors.     

Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin

Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Å resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC₅₀ of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases.