Human mesotrypsin is a unique digestive protease specialized for the degradation of trypsin inhibitors

Mesotrypsin is an enigmatic minor human trypsin isoform, which has been recognized for its peculiar resistance to natural trypsin inhibitors such as soybean trypsin inhibitor (SBTI) or human pancreatic secretory trypsin inhibitor (SPINK1). In search of a biological function, two conflicting theories proposed that due to its inhibitor-resistant activity mesotrypsin could prematurely activate or degrade pancreatic zymogens and thus play a pathogenic or protective role in human pancreatitis. In the present study we ruled out both theories by demonstrating that mesotrypsin was grossly defective not only in inhibitor binding, but also in the activation or degradation of pancreatic zymogens. We found that the restricted ability of mesotrypsin to bind inhibitors or to hydrolyze protein substrates was solely due to a single evolutionary mutation, which changed the serine-protease signature glycine 198 residue to arginine. Remarkably, the same mutation endowed mesotrypsin with a novel and unique function: mesotrypsin rapidly hydrolyzed the reactive-site peptide bond of the Kunitz-type trypsin inhibitor SBTI, and irreversibly degraded the Kazal-type temporary inhibitor SPINK1. The observations suggest that the biological function of human mesotrypsin is digestive degradation of trypsin inhibitors. This mechanism can facilitate the digestion of foods rich in natural trypsin inhibitors. Furthermore, the findings raise the possibility that inappropriate activation of mesotrypsinogen in the pancreas might lower protective SPINK1 levels and contribute to the development of human pancreatitis. In this regard, it is noteworthy that the well known pathological trypsinogen activator cathepsin B exhibited a preference for the activation of mesotrypsinogen of all three human trypsinogen isoforms, suggesting a biochemical mechanism for mesotrypsinogen activation in pancreatic acinar cells.     

Mesotrypsin Signature Mutation in a Chymotrypsin C (CTRC) Variant Associated with Chronic Pancreatitis

Human chymotrypsin C (CTRC) protects against pancreatitis by degrading trypsinogen and thereby curtailing harmful intra-pancreatic trypsinogen activation. Loss-of-function mutations in CTRC increase the risk for chronic pancreatitis. Here we describe functional analysis of eight previously uncharacterized natural CTRC variants tested for potential defects in secretion, proteolytic stability, and catalytic activity. We found that all variants were secreted from transfected cells normally, and none suffered proteolytic degradation by trypsin. Five variants had normal enzymatic activity, whereas variant p.R29Q was catalytically inactive due to loss of activation by trypsin and variant p.S239C exhibited impaired activity possibly caused by disulfide mispairing. Surprisingly, variant p.G214R had increased activity on a small chromogenic peptide substrate but was markedly defective in cleaving bovine β-casein or the natural CTRC substrates human cationic trypsinogen and procarboxypeptidase A1. Mutation p.G214R is analogous to the evolutionary mutation in human mesotrypsin, which rendered this trypsin isoform resistant to proteinaceous inhibitors and conferred its ability to cleave these inhibitors. Similarly to the mesotrypsin phenotype, CTRC variant p.G214R was inhibited poorly by eglin C, ecotin, or a CTRC-specific variant of SGPI-2, and it readily cleaved the reactive-site peptide bonds in eglin C and ecotin. We conclude that CTRC variants p.R29Q, p.G214R, and p.S239C are risk factors for chronic pancreatitis. Furthermore, the mesotrypsin-like CTRC variant highlights how the same natural mutation in homologous pancreatic serine proteases can evolve a new physiological role or lead to pathology, determined by the biological context of protease function.     

Human mesotrypsin exhibits restricted S1' subsite specificity with a strong preference for small polar side chains

Mesotrypsin, an inhibitor-resistant human trypsin isoform, does not activate or degrade pancreatic protease zymogens at a significant rate. These observations led to the proposal that mesotrypsin is a defective digestive protease on protein substrates. Surprisingly, the studies reported here with alpha1-antitrypsin (alpha1AT) revealed that, even though mesotrypsin was completely resistant to this serpin-type inhibitor, it selectively cleaved the Lys10-Thr11 peptide bond at the N-terminus. Analyzing a library of alpha1AT mutants in which Thr11 was mutated to various amino acids, we found that mesotrypsin hydrolyzed lysyl peptide bonds containing Thr or Ser at the P1' position with relatively high specificity (kcat/KM approximately 10(5) m(-1) x s(-1)). Compared with Thr or Ser, P1' Gly or Met inhibited cleavage 13- and 25-fold, respectively, whereas P1' Asn, Asp, Ile, Phe or Tyr resulted in 100-200-fold diminished rates of proteolysis, and Pro abolished cleavage completely. Consistent with the Ser/Thr P1' preference, mesotrypsin cleaved the Arg358-Ser359 reactive-site peptide bond of alpha1AT Pittsburgh and was rapidly inactivated by the serpin mechanism (ka approximately 10(6) m(-1) s(-1)). Taken together, the results indicate that mesotrypsin is not a defective protease on polypeptide substrates in general, but exhibits a relatively high specificity for Lys/Arg-Ser/Thr peptide bonds. This restricted, thrombin-like subsite specificity explains why mesotrypsin cannot activate pancreatic zymogens, but might activate certain proteinase-activated receptors. The observations also identify alpha1AT Pittsburgh as an effective mesotrypsin inhibitor and the serpin mechanism as a viable stratagem to overcome the inhibitor-resistance of mesotrypsin.     

Presence versus absence of hydrogen bond donor Tyr-39 influences interactions of cationic trypsin and mesotrypsin with protein protease inhibitors

Mesotrypsin displays unusual resistance to inhibition by polypeptide trypsin inhibitors and cleaves some such inhibitors as substrates, despite a high degree of conservation with other mammalian trypsins. Substitution of Arg for the generally conserved Gly-193 has been implicated as a critical determinant of the unusual behavior of mesotrypsin toward protein protease inhibitors. Another relatively conserved residue near the trypsin active site, Tyr-39, is substituted by Ser-39 in mesotrypsin. Tyr-39, but not Ser-39, forms a hydrogen bond with the main chain amide nitrogen of the P₄′ residue of a bound protease inhibitor. To investigate the role of the Tyr-39 H-bond in trypsin-inhibitor interactions, we reciprocally mutated position 39 in mesotrypsin and human cationic trypsin to Tyr-39 and Ser-39, respectively. We assessed inhibition constants and cleavage rates of canonical protease inhibitors bovine pancreatic trypsin inhibitor (BPTI) and the amyloid precursor protein Kunitz protease inhibitor domain by mesotrypsin and cationic trypsin variants, finding that the presence of Ser-39 relative to Tyr-39 results in a 4- to 13-fold poorer binding affinity and a 2- to 18-fold increase in cleavage rate. We also report the crystal structure of the mesotrypsin-S39Y•BPTI complex, in which we observe an H-bond between Tyr-39 OH and BPTI Ile-19 N. Our results indicate that the presence of Ser-39 in mesotrypsin, and corresponding absence of a single H-bond to the inhibitor backbone, makes a small but significant functional contribution to the resistance of mesotrypsin to inhibition and the ability of mesotrypsin to proteolyze inhibitors.     

The pancreatic islet β-cell-enriched transcription factor Pdx-1 regulates Slc30a8 gene transcription through an intronic enhancer

PRSS3/mesotrypsin is an atypical isoform of trypsin, the up-regulation of which has been implicated in promoting tumour progression. Mesotrypsin inhibitors could potentially provide valuable research tools and novel therapeutics, but small-molecule trypsin inhibitors have low affinity and little selectivity, whereas protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. In the present study, we use mutagenesis of a mesotrypsin substrate, APPI (amyloid precursor protein Kunitz protease inhibitor domain), and of a poor mesotrypsin inhibitor, BPTI (bovine pancreatic trypsin inhibitor), to dissect mesotrypsin specificity at the key P(2)' position. We find that bulky and charged residues strongly disfavour binding, whereas acidic residues facilitate catalysis. Crystal structures of mesotrypsin complexes with BPTI variants provide structural insights into mesotrypsin specificity and inhibition. Through optimization of the P(1) and P(2)' residues of BPTI, we generate a stable high-affinity mesotrypsin inhibitor with an equilibrium binding constant K(i) of 5.9 nM, a >2000-fold improvement in affinity over native BPTI. Using this engineered inhibitor, we demonstrate the efficacy of pharmacological inhibition of mesotrypsin in assays of breast cancer cell malignant growth and pancreatic cancer cell invasion. Although further improvements in inhibitor selectivity will be important before clinical potential can be realized, the results of the present study support the feasibility of engineering protein protease inhibitors of mesotrypsin and highlight their therapeutic potential.     

Structural basis for accelerated cleavage of bovine pancreatic trypsin inhibitor (BPTI) by human mesotrypsin

Human mesotrypsin is an isoform of trypsin that displays unusual resistance to polypeptide trypsin inhibitors and has been observed to cleave several such inhibitors as substrates. Whereas substitution of arginine for the highly conserved glycine 193 in the trypsin active site has been implicated as a critical factor in the inhibitor resistance of mesotrypsin, how this substitution leads to accelerated inhibitor cleavage is not clear. Bovine pancreatic trypsin inhibitor (BPTI) forms an extremely stable and cleavage-resistant complex with trypsin, and thus provides a rigorous challenge of mesotrypsin catalytic activity toward polypeptide inhibitors. Here, we report kinetic constants for mesotrypsin and the highly homologous (but inhibitor sensitive) human cationic trypsin, describing inhibition by, and cleavage of BPTI, as well as crystal structures of the mesotrypsin-BPTI and human cationic trypsin-BPTI complexes. We find that mesotrypsin cleaves BPTI with a rate constant accelerated 350-fold over that of human cationic trypsin and 150,000-fold over that of bovine trypsin. From the crystal structures, we see that small conformational adjustments limited to several side chains enable mesotrypsin-BPTI complex formation, surmounting the predicted steric clash introduced by Arg-193. Our results show that the mesotrypsin-BPTI interface favors catalysis through (a) electrostatic repulsion between the closely spaced mesotrypsin Arg-193 and BPTI Arg-17, and (b) elimination of two hydrogen bonds between the enzyme and the amine leaving group portion of BPTI. Our model predicts that these deleterious interactions accelerate leaving group dissociation and deacylation.     

Mesotrypsin Has Evolved Four Unique Residues to Cleave Trypsin Inhibitors as Substrates

Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. These findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.     

Sequence and Conformational Specificity in Substrate Recognition: SEVERAL HUMAN KUNITZ PROTEASE INHIBITOR DOMAINS ARE SPECIFIC SUBSTRATES OF MESOTRYPSIN*

Mesotrypsin is an isoform of trypsin that is uniquely resistant to polypeptide trypsin inhibitors and can cleave some inhibitors rapidly. Previous studies have shown that the amyloid precursor protein Kunitz protease inhibitor domain (APPI) is a specific substrate of mesotrypsin and that stabilization of the APPI cleavage site in a canonical conformation contributes to recognition by mesotrypsin. We hypothesized that other proteins possessing potential cleavage sites stabilized in a similar conformation might also be mesotrypsin substrates. Here we evaluated a series of candidate substrates, including human Kunitz protease inhibitor domains from amyloid precursor-like protein 2 (APLP2), bikunin, hepatocyte growth factor activator inhibitor type 2 (HAI2), tissue factor pathway inhibitor-1 (TFPI1), and tissue factor pathway inhibitor-2 (TFPI2), as well as E-selectin, an unrelated protein possessing a potential cleavage site displaying canonical conformation. We find that Kunitz domains within APLP2, bikunin, and HAI2 are cleaved by mesotrypsin with kinetic profiles of specific substrates. TFPI1 and TFPI2 Kunitz domains are cleaved less efficiently by mesotrypsin, and E-selectin is not cleaved at the anticipated site. Cocrystal structures of mesotrypsin with HAI2 and bikunin Kunitz domains reveal the mode of mesotrypsin interaction with its canonical substrates. Our data suggest that major determinants of mesotrypsin substrate specificity include sequence preferences at the P₁ and P′₂ positions along with conformational stabilization of the cleavage site in the canonical conformation. Mesotrypsin up-regulation has been implicated previously in cancer progression, and proteolytic clearance of Kunitz protease inhibitors offers potential mechanisms by which mesotrypsin may mediate pathological effects in cancer.     

The amino acid sequence of a Bowman-Birk type proteinase inhibitor from faba beans (Vicia faba L.).

The amino acid sequence of a Bowman-Birk type proteinase inhibitor (FBI) from seeds of faba bean (Vicia faba L.) was determined by analysis of peptide fragments generated by reduction and S-carboxymethylation of enzymatically modified inhibitors, which were obtained from native FBI by limited proteolysis with TPCK-trypsin or TLCK-chymotrypsin at pH 3.5. The established sequence showed that FBI is highly homologous with Vicia angustifolia inhibitor (VAI0 but lacks the portion corresponding to the C-terminal 9 amino acids of VAI. The trypsin reactive-site peptide bond in FBI was also indicated to be Lys(16)-Ser(17) and the chymotrypsin reactive-site peptide bond to be Tyr(42)-Ser(43) by limited proteolysis with TPCK-trypsin or TLCK-chymotrypsin and by sequence comparison with other Bowman-Birk type inhibitors.     

Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P₁ (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P′₂ favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P₁ and P′₂ substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin·APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.     

The Amyloid Precursor Protein/Protease Nexin 2 Kunitz Inhibitor Domain Is a Highly Specific Substrate of Mesotrypsin

The amyloid precursor protein (APP) is a ubiquitously expressed transmembrane adhesion protein and the progenitor of amyloid-β peptides. The major splice isoforms of APP expressed by most tissues contain a Kunitz protease inhibitor domain; secreted APP containing this domain is also known as protease nexin 2 and potently inhibits serine proteases, including trypsin and coagulation factors. The atypical human trypsin isoform mesotrypsin is resistant to inhibition by most protein protease inhibitors and cleaves some inhibitors at a substantially accelerated rate. Here, in a proteomic screen to identify potential physiological substrates of mesotrypsin, we find that APP/protease nexin 2 is selectively cleaved by mesotrypsin within the Kunitz protease inhibitor domain. In studies employing the recombinant Kunitz domain of APP (APPI), we show that mesotrypsin cleaves selectively at the Arg¹⁵-Ala¹⁶ reactive site bond, with kinetic constants approaching those of other proteases toward highly specific protein substrates. Finally, we show that cleavage of APPI compromises its inhibition of other serine proteases, including cationic trypsin and factor XIa, by 2 orders of magnitude. Because APP/protease nexin 2 and mesotrypsin are coexpressed in a number of tissues, we suggest that processing by mesotrypsin may ablate the protease inhibitory function of APP/protease nexin 2 in vivo and may also modulate other activities of APP/protease nexin 2 that involve the Kunitz domain.     

Biochemical and structural insights into mesotrypsin: an unusual human trypsin

Thirty five years ago mesotrypsin was first isolated from the human pancreas. It was described as a minor trypsin isoform with the remarkable property of near total resistance to biological trypsin inhibitors. Another unusual feature of mesotrypsin was discovered later, when it was found that mesotrypsin has defective affinity toward many protein substrates of other trypsins. As the younger sibling of the two major trypsins secreted by the pancreas, cationic and the anionic trypsin, it has been speculated to represent an evolutionary waste with no apparent function. We know now that mesotrypsin is functionally very different from the other trypsins, with novel substrate specificity that hints at distinct physiological functions. Recently, evidence has begun to emerge implicating mesotrypsin in direct involvement in cancer progression. This review will explore the biochemical characteristics of mesotrypsin and structural insights into its specificity, function, and inhibition.     

Chemical-enzymatic insertion of an amino acid residue in the reactive site of soybean trypsin inhibitor (Kunitz).

Modified (Arg63-Ile64 reactive-site peptide bond hydrolyzed) soybean trypsin inhibitor (Kunitz) with all reactive amino groups, except that of Ile64, protected was described in the preceding paper (Kowalski, D., and Laskowski, M., Jr. (1976), Biochemistry, preceding paper in this issue). Treatment of this inhibitor with tert-butyloxycarbonyl-Ala- and tert-butyloxycarbonyl-Ile-N-hydroxy-succinimide esters yields inactive endo-tert-butyloxycarbonyl-Ala63A-and endo-tert-butyloxycarbonyl-Ile63A-modified inhibitors. The tert-butyloxycarbonyl groups were removed by treatment of the proteins with trifluoroacetic acid. After renaturation and purification, the resultant endo-Ala63A- and endo-Ile63A-modified inhibitors co-electrophorese with modified inhibitor both on disc gels (pH 9.4) and sodium dodecyl sulfate gels (after reduction of disulfide bonds) and show end groups corresponding to the 63A residue. These derivatives fail to form stable complexes with trypsin, extending the previous observation (Kowalski, D., and Laskowski, M., Jr. (1972), Biochemistry 11, 3451) that acylation of the P1' residue in modified inhibitors leads to inactivation. However, the incubation of endo-Ala63A- and endo-Ile63A-modified inhibitors with trypsin at pH 6.5 leads to the synthesis of the Arg63-Ala63A and Arg63-Ile63A peptide bonds in 4% yield. This is very close to the yield anticipated from a semiquantitative theory for the value of the equilibrium constant for reactive-site peptide bond. An alternative chemical method of insertion is also described. Controlled treatment of modified inhibitor with the N-carboxyanhydride of Glu produced inactive endo-Glu63A-modified inhibitor. Incubation of this inactive derivative with trypsin at pH 6.5 leads to 16% synthesis of the Arg63-Glu63A peptide bond. The higher yield of single chain protein in this case is attributed to the influence of the negative charge of the Glu63A side chain. Thus, the insertion of an amino acid residue between the P1 and P1' residues in soybean trypsin inhibitor (Kunitz) converts a trypsin inhibitor into a trypsin substrate.     

Replacement of lysine by arginine, phenylalanine and tryptophan in the reactive site of the bovine trypsin-kallikrein inhibitor (Kunitz) and change of the inhibitory properties.

The reactive-site sequence of a proteinase inhibitor can be written as . . . -P3-P2-P1-P'1-P'2-P'3- . . . , where-P1-P'1-denotes the reactive site. Three semisynthetic homologues have been synthesized of the bovine trypsin-kallikrein inhibitor (Kunitz) with either arginine, phenylalanine or tryptophan in place of the reactive-site residue P1, lysine-15. These homologues correspond to gene products after mutation of the lysine 15 DNA codon to an arginine, phenylalanine or tryptophan DNA codon. Starting from native (virgin) inhibitor, reactive-site hydrolyzed, still active (modified) inhibitor was prepared by chemical and enzymic reactions. Modified inhibitor was then converted into inactive des-Lys15-inhibitor by reaction with carboxypeptidase B. Inactive des-Lys15-inhibitor was reactivated by enzymic replacement of the P1 residue according to Leary and Laskowski, Jr. The introduction of arginine was catalyzed by an inverse reaction with carboxypeptidase B, while phenylalanine or tryptophan were replaced by carboxypeptidase A. The reactivated semisynthetic inhibitors were trapped by complex formation with either trypsin or chymotrypsin. The enzyme - inhibitor complexes were subjected to kinetic-control dissociation, and the semisynthetic virgin inhibitors were isolated. The inhibitory properties of the semisynthetic inhibitors have been investigated against bovine trypsin and chymotrypsin and against porcine pancreatic kallikrein and plasmin. The homologues with either lysine or arginine in the P1 position are equally good inhibitors of trypsin, plasmin and kallikrein. The Arg-15-homologue is a slightly more effective kallikrein inhibitor than the Lys15-inhibitor. The semisynthetic phenylalanine and tryptophan homologues, however, are weak inhibitors of trypsin and still weaker inhibitors of kallikrein, but are excellent inhibitors of chymotrypsin. Their association constant with chymotrypsin is at least ten times higher than that of native Lys-15-inhibitor. A dramatic specificity change is observed with the phenylalanine and tryptophan homologues, which in contrast to the native inhibitor do not at all inhibit porcine plasmin. Thus, the nature of the P1 residue strongly influences the primary inhibitory specificity of the bovine inhibitor (Kunitz).     

Mesotrypsin, a brain trypsin, activates selectively proteinase-activated receptor-1, but not proteinase-activated receptor-2, in rat astrocytes

Proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, which are activated by serine proteases, such as trypsin, play pivotal roles in the CNS. Mesotrypsin (trypsin IV) has been identified as a brain-specific trypsin isoform. However, its potential physiological role concerning PAR activation in the brain is largely unknown. Here, we show for the first time that mesotrypsin, encoded by the PRSS3 (proteinase, serine) gene, evokes a transient and pronounced Ca(2+) mobilization in both primary rat astrocytes and retinal ganglion RGC-5 cells, suggesting a physiological role of mesotrypsin in brain cells. Mesotrypsin mediates Ca(2+) responses in rat astrocytes in a concentration-dependent manner, with a 50% effective concentration (EC(50)) value of 25 nm. The maximal effect of mesotrypsin on Ca(2+) mobilization in rat astrocytes is much higher than that observed in 1321N1 human astrocytoma cells, indicating that the activity of mesotrypsin is species-specific. The pre-treatment of cells with thrombin or the PAR-1-specific peptide TRag (Ala-pFluoro-Phe-Arg-Cha-HomoArg-Tyr-NH(2), synthetic thrombin receptor agonist peptide), but not the PAR-2-specific peptide, reduces significantly the mesotrypsin-induced Ca(2+) response. Treatment with the PAR-1 antagonist SCH79797 confirms that mesotrypsin selectively activates PAR-1 in rat astrocytes. Unlike mesotrypsin, the two other trypsin isoforms, cationic and anionic trypsin, activate multiple PARs in rat astrocytes. Therefore, our data suggest that brain-specific mesotrypsin, via the regulation of PAR-1, is likely to be involved in multiple physiological/pathological processes in the brain.     

Chemical-enzymatic replacement of Ile64 in the reactive site of soybeen trypsin inhibitor (Kunitz).

All the reactive amino groups in soybean trypsin inhibitor (Kunitz) were protected by guanidination of 9 out of 10 lysyl residues with O-methylisourea and by carbamoylation of the NH2 terminal Asp with potassium cyanate. This derivative was converted to modified inhibitor (Arg63-Ile64 reactive site peptide bond hydrolyzed) by incubation with trypsin at pH 3. The NH2 terminal of Ile64 was allowed to react with phenyl isothiocyanate to produce inactive phenylthiocarbamoyl-modified inhibitor. Treatment with trifluoroacetic acid formed the anilinothiazolinone of Ile64 yielding des-Ile64-modified inhibitor. After renaturation and purification, this material coelectrophoresed with modified inhibitor but did not form a stable complex with trypsin. Incubation with tert-butyloxycarbonyl-(amino acid)-N-hydroxysuccinimide esters yielded [tert-butyloxycarbonyl-(amino acid64)]-modified inhibitor. The tert-butyloxycarbonyl protective group was removed in trifluoroacetic acid. After renaturation, active [amino acid64]-modified inhibitors were obtained for Ile64, Ala64, Leu64, and Gly64 replacements. The resynthesis of the reactive-site peptide bound by kinetic control dissociation of the trypsin-inhibitor complex yielded fully active [Ala64]-virgin inhibitor. Thus, soybean trypsin inhibitor (Kunitz) has been shown to tolerate the replacement of the P1' residue with retention of activity. The importance of P1' residues in the function of protein proteinase inhibitors is discussed.     

The complete amino acid sequence of a major trypsin inhibitor from seeds of foxtail millet (Setaria italica)

The complete amino acid sequence of a major trypsin inhibitor (FMTI-II) from seeds of foxtail millet (Setaria italica) was determined by analysis of peptides derived from the reduced and S-carboxymethylated protein by digestion with TPCK-trypsin and Staphylococcus aureus V8 protease. FMTI-II consists of 67 amino acid residues, including 10 half-cystine residues which are involved in 5 disulfide bridges in the molecule. The established sequence had a high degree of homology to Bowman-Birk type inhibitors from leguminous and gramineous plants. The trypsin reactive-site peptide bond in FMTI-II also appears to be Lys (16)-Ser (17) by comparison with these sequences.     

Interaction between trypsin-like enzyme from Streptomyces erythraeus and chicken ovomucoid

Chicken ovomucoid (CO), an effective inhibitor of bovine trypsin, has a reactive site in each of three tandem domains. When CO was subjected to inhibition assay by the method of Green and Work, the second domain (CO Domain II) inhibited bovine trypsin but not TLE-Se, a trypsin-like enzyme from Streptomyces erythraeus, and the first domain (CO Domain I) inhibited neither bovine trypsin nor TLE-Se. However, when the interaction between CO and TLE-Se was analyzed by means of a Lineweaver-Burk plot, it was found that the ovomucoid exhibited competitive inhibition of the bacterial protease at pH 8.0 (Ki = 5.2 microM). In this case, the reactive-site peptide bonds of the first and second domains were specifically hydrolyzed. The isolated CO Domain I also exhibited competitive inhibition of TLE-Se (Ki = 3.1 microM), which specifically hydrolyzed its reactive-site peptide bond.     

Aprotinin derivatives with chromophoric leaving groups can be used as highly selective active-site titrants for serine proteinases and permit the determination of kinetic constants of enzyme-inhibitor complexes

This paper reports a novel and valuable approach to active-site titration. The starting substance for the preparation of the active-site titrants is aprotinin (bovine pancreatic trypsin inhibitor) in which the reactive-site peptide bond, Lys15-Ala16, is split. Two cystine disulfide bonds hold together the two peptide chains. The Lys15 of the reactive site is substituted by arginine-, phenylalanine- and valine-4-nitroanilide or by valine-7-amido-4-methylcoumarin. The different incorporated amino acid residues correspond to different specificities against serine proteinases. Serine proteinases with suitable specificity are able to remove 4-nitroaniline or 7-amino-4-methylcoumarin from these aprotinin derivatives while at the same time resynthesis of the reactive-site peptide bond occurs. The proteinase is then trapped in a stable enzyme-inhibitor complex, which prevents the proteinase from releasing further leaving groups. The quantity of 4-nitroaniline or 7-amino-4-methylcoumarin, which can be assayed spectrophotometrically or fluorometrically is equimolar to the quantity of proteinase used and trapped. The aprotinin derivatives with an incorporated Phe15 or Val15 residue are highly specific for chymotrypsin or for elastase from human leukocytes, respectively. The kinetic constants kon and koff of the enzyme-inhibitor complexes, and hence the equilibrium dissociation constants, can be calculated from the respective titration curves.     

A peptide from the eel pancreas with structural similarity to human pancreatic secretory trypsin inhibitor

The primary structure of a 61-amino-acid residue peptide from the pancreas of the European eel (Anguilla anguilla) has been established as E E K S G(5)L Y R K P(10)S C G E M(15)S A M H A(20)C P M N F(25)A P V C G(30)T D G N T(35)Y P N E C(40)S L C F Q(45)R Q N T K(50)T D I L I(55)T K D D R(60)C. There was no indication of microheterogeneity. This peptide shows structural similarity to pancreatic secretory trypsin inhibitors from several mammalian species and to a cholecystokinin-releasing peptide isolated from rat pancreatic juice. A comparison of the amino acid sequences of the peptides has identified a domain in the central region of the molecules that has been strongly conserved during evolution. In contrast, the amino acid sequence in the region corresponding to the reactive centre of the mammalian trypsin inhibitors is very poorly conserved in the eel peptide. The P1-P1' reactive site lysine-isoleucine (or arginine-isoleucine) bond in the mammalian trypsin inhibitors is replaced by a methionine-asparagine bond. This region does, however, show limited homology to the reactive centre of human alpha 1-protease inhibitor suggesting that the eel peptide may function as an inhibitor of other proteolytic enzymes in the pancreas.