Bovine pancreatic trypsin inhibitor and related isoinhibitors in bovine liver

Summary A Kunitz-type inhibitor family has been biochemically and histochemically characterized in bovine liver. This family includes the well-known pancreatic trypsin inhibitor (BPTI) and three BPTI-related molecular forms (isoinhibitors I, II and III). The purification of the inhibitors was performed by affinity chromatography on immobilized trypsin followed by fast protein liquid chromatography. The inhibitors were identical to those identified previously in bovine spleen and lung. Light immunohistochemical experiments were done by a streptavidin-biotin-peroxidase method using two different immunoglobulin preparations, which selectively discriminated between BPTI and the other isoinhibitors. BPTI-related immunoreactivity was found exclusively at the level of isolated cells, of which many were identified as mast cells by toluidine blue staining. By contrast, isoinhibitor-related immunoreactivity showed a more widespread distribution, including hepatocytes, mast cells and biliary duct epithelial cells. Finally, specific immunoreactivity was also present in plasma. These results suggest that: i) BPTI and related isoinhibitors may be involved in the regulation of the activity of some mast cell proteases, as it happens in other bovine organs (Businaro et al. 1987, 1988); ii) BPTI isoinhibitors, but not BPTI itself, may also control proteolytic activities in hepatic specific structures (hepatocytes and biliary duct epithelial cells).     

Cellular localization of bovine pancreatic trypsin inhibitor and related molecular forms in bovine lung

Summary In addition to bovine pancreatic trypsin inhibitor (BPTI), three BPTI-related molecular forms (isoinhibitors I, II and III) were isolated from bovine lung by affinity chromatography on immobilized trypsin and subsequently purified by Fast Protein Liquid Chromatography. These inhibitors are identical to the isoinhibitors previously isolated from bovine spleen. Their localization in bovine lung was studied by immunohistochemical techniques, using two different immunoglobulin preparations, selectively recognizing BPTI or the other molecular forms.BPTI-related immunoreactivity was found to be restricted to isolated cells, often identified as mast cells by Toluidine Blue staining. In contrast, isoinhibitor-related immunoreactivity, which also occurs in the mast cells, is present in a number of other cell types. These types include: (i) the smooth muscle cells of different calibre vessels, (ii) the ciliated cells of the bronchial epithelium and the related mucus, and (iii) many cells at alveolar level.Comparison of these data with previous results obtained for bovine spleen suggest multiple physiological roles for these inhibitors.     

Identification and immunohistochemical localization of various bovine pancreatic trypsin inhibitor-isoforms in bovine pituitary gland

Summary Three isoinhibitors of bovine pancreatic trypsin inhibitor (BPTI) have been identified and isolated from bovine pituitary gland. The results of the purification process by affinity chromatography on immobilized trypsin, the electrophoretic mobility in non-denaturing conditions, the antiproteolytic activity and the immunochemical reactions indicate that these inhibitors correspond to those previously isolated from bovine spleen and lung. In addition, immunohistochemical experiments show that the isoinhibitors and BPTI are exclusively localized in the mast cells, and not in the endocrine cells, of the pars intermedia and posterior lobe (neurohypophysis) of the pituitary gland. The physiological implications of these findings are discussed.     

Immunolocalisation of BPTI-like serine proteinase inhibitory proteins in mast cells, chondrocytes and intervertebral disc fibrochondrocytes of ovine and bovine connective tissues. An immunohistochemical and biochemical study

A polyclonal anti-bovine pancreatic trypsin inhibitor (BPTI) IgY was raised in chickens immunised with aprotinin. The anti-BPTI IgY was subsequently isolated from egg yolks and purified to homogeneity by affinity chromatography on immobilised aprotinin and by Superose 6 size exclusion fast protein liquid chromatography (FPLC). Immunoblotting with the chicken IgY demonstrated its specificity for BPTI; 3.9 ng BPTI could be detected by this technique. There was no crossreactivity against alpha1-proteinase inhibitor (human and sheep), inter-alpha-trypsin inhibitor (human and sheep), secretory leucocyte proteinase inhibitor or a range of serine proteinase inhibitory proteins (SPIs) isolated from plant sources (soybean and lima bean trypsin inhibitor, potato trypsin and chymotrypsin inhibitors) or serum SPIs (antithrombin-III, alpha2-macroglobulin). Immunoblotting using the anti-BPTI IgY identified the 6- to 12- and 58-kDa forms of endogenous ovine cartilage SPIs in cartilage extracts, confirming the interrelationship of the ovine cartilage SPIs with BPTI. BPTI-domain SPIs were immunolocalised within mast cells of ovine and bovine duodenum, lung and pancreas, and in ovine and bovine bronchial cartilage chondrocytes, chondrocytes of the superficial and intermediate zones of articular cartilage and in the fibrochondrocytes/chondrocytes of the nucleus     

Bovine pancreatic trypsin inhibitor and homologous polypeptide inhibitors in nephron cells.

Bovine pancreatic trypsin inhibitor (BPTI, aprotinin) is a fifty-eight amino acid polypeptide, which is present together with related molecular isoforms in various bovine organs. In the present study these protease inhibitors were isolated from bovine kidney by affinity chromatography on immobilized trypsin and a subsequent FPLC step. Due to their electrophoretic, structural, and inhibitory properties, the inhibitors were strictly similar to the polypeptides identified previously in other bovine organs. Immunohistochemical experiments showed a widespread localization of these polypeptides in nephron epithelial cells (proximal and distal tubules, loop of Henle, collecting tubules).     

Vascular localization of bovine pancreatic trypsin-inhibitor-related molecular forms in bovine spleen

Bovine spleen proteic inhibitors of serine proteases, belonging to the bovine pancreatic trypsin inhibitor (BPTI or aprotinin) family, have been localized, using immunocytochemical techniques, in the smooth muscle cells of some bovine spleen blood vessels. This vascular localization also occurs in a variety of bovine organs and differs from that of BPTI itself which is found exclusively in bovine mast cells, in agreement with previous reports. These data would be in favour of a possible involvement of one or more BPTI-type inhibitors in vascular processes by acting at the level of the smooth muscle cells, the tissue responsible for vasodilation/vasoconstriction events.     

Adaptive evolution in the snake venom Kunitz/BPTI protein family

Snake venoms are rich sources of serine proteinase inhibitors that are members of the Kunitz/BPTI (bovine pancreatic trypsin inhibitor) family. However, only a few of their gene sequences have been determined from snakes. We therefore cloned the cDNAs for the trypsin and chymotrypsin inhibitors from a Vipera ammodytes venom gland cDNA library. Phylogenetic analysis of these and other snake Kunitz/BPTI homologs shows the presence of three clusters, where sequences cluster by functional role. Analysis of the nucleotide sequences from the snake Kunitz/BPTI family shows that positive Darwinian selection was operating on the highly conserved BPTI fold, indicating that this family evolved by gene duplication and rapid diversification.     

Double-headed protease inhibitors from black-eyed peas. I. Purification of two new protease inhibitors and the endogenous protease by affinity chromatography.

Two new double-headed protease inhibitors have been isolated from black-eyed peas. The isoinhibitors can be purified to homogeneity with greater than 90% recovery in a four-step procedure by means of sequential affinity chromatography on trypsin-Sepharose and chymotrypsin-Sepharose affinity columns. The isoinhibitors both have molecular weights near 8,000 and both have the same NH1-terminal residue serine. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) has an isoelectric point of 5.1 and inhibits trypsin and chymotrypsin simultaneously. Black-eyed pea trypsin inhibitor (BEPTI) has an isoelectric point of 6.5 and inhibits 2 molecules of trypsin simultaneously. BEPTI binds to chymotrypsin-Sepharose above pH 6 but does not inhibit chymotrypsin in the standard inhibitor assay with 10-3 M substrate. These new inhibitors are distinct from the Ventura inhibitor isolated from Serido black-eyed peas. An endogenous seed protease has been isolated from black-eyed peas by affinity chromatography on soybean inhibitor-carboxymethylcellulose affinity columns. A protease-BEPCI complex has been isolated by ion exchange chromatography. A dual physiological function of inhibition and protection of the seed protease is suggested as a plausible role of seed protease inhibitors.     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.     

Kunitz-type proteinase inhibitors in sheep and ox.

1. Four protein proteinase inhibitors, belonging to the Kunitz family, were isolated and purified from several sheep organs. 2. Their structural, functional and immunological properties were determined and compared to those of similar inhibitors purified from bovine organs. 3. The Kunitz-type isoinhibitors appear differently distributed in the two species: BPTI, which is the prevailing form in bovids, is found only in minute amounts in sheep organs. 4. The presence of multiple forms of these inhibitors in sheep is discussed on the basis of the same biosynthetic and post-translational processes proposed for the molecules of bovine origin.     

Purification and characterization from tobacco (Nicotiana tabacum) leaves of six small, wound-inducible, proteinase isoinhibitors of the potato inhibitor II family.

Six small molecular mass, wound-inducible trypsin and chymotrypsin inhibitor proteins from tobacco (Nicotiana tabacum) leaves were isolated to homogeneity. The isoinhibitors, cumulatively called tobacco trypsin inhibitor (TTI), have molecular masses of approximately 5500 to 5800 D, calculated from gel filtration analysis and amino acid content. The amino acid sequence of the entire 53 residues of one isoinhibitor, TTI-1, and the sequence of 36 amino acid residues from the N terminus of a second isoinhibitor, TTI-5, were determined. The two isoinhibitors differ only at residue 11, which is threonine in TTI-1 and lysine in TTI-5. The isoinhibitors are members of the potato inhibitor II family and show considerable identity with the small molecular mass members of this family, which include the eggplant inhibitor, two small molecular mass trypsin and chymotrypsin inhibitors from potatoes, and an inhibitor from pistils of the ornamental plant Nicotiana alata. Antibodies produced against the isoinhibitors in rabbits were used in radial immunoassays to quantify both the systemic wound inducibility of TTI in tobacco leaves and its constitutive levels in flowers.     

Trichuris suis: a secretory chymotrypsin/elastase inhibitor with potential as an immunomodulator

A serine protease inhibitor, termed TsCEI, was purified from adult-stage Trichuris suis by acid precipitation, affinity chromatography (elastase-agarose), and reverse-phase HPLC. The molecular weight of TsCEI was estimated at 6.437 kDa by laser desorption mass spectrometry. TsCEI potently inhibited both chymotrypsin (K(i) = 33.4 pM) and pancreatic elastase (K(i) = 8.32 nM). Neutrophil elastase, chymase (mouse mast cell protease-1, mMCP-1), and cathepsin G were also inhibited by TsCEI, whereas trypsin, thrombin, and factor Xa were not. The cDNA-derived amino acid sequence of the mature TsCEI consisted of 58 residues including 9 cysteine residues with a molecular mass of 6.196 kDa. TsCEI displayed 48% sequence identity to a previously characterized trypsin/chymotrypsin inhibitor of T. suis, TsTCI. TsCEI showed 36% sequence identity to a protease inhibitor from the hemolymph of the honeybee Apis mellifera. Sequence similarity was also detected with the trypsin/thrombin inhibitor of the European frog Bombina bombina, the elastase isoinhibitors of the nematode Anisakis simplex, and the chymotrypsin/elastase and trypsin inhibitors of the nematode Ascaris suum. The inhibitors of T. suis, an intestinal parasite of swine, may function as components of a parasite defense mechanism by modulating intestinal mucosal mast cell-associated, protease-mediated, host immune responses.     

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.     

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.     

Purification and properties of a progesterone-induced plasmin/trypsin inhibitor from uterine secretions of pigs and its immunocytochemical localization in the pregnant uterus

The porcine uterus secretes a group of basic, low molecular weight protease inhibitors under the influence of progesterone, but not estrogen. One of these inhibitors (Mr approximately 14,500) which inhibits trypsin, plasmin, and chymotrypsin, but not other proteases tested, has been purified 10- to 15-fold from uterine secretions of pseudopregnant pigs using Sephadex G-100 chromatography, CM-cellulose ion exchange chromatography, and Sephadex G-50 or Bio-Gel P-10 chromatography. The inhibitor which is relatively heat- and pH-stable forms a 1:1 molar complex with trypsin which is not dissociated in sodium dodecyl sulfate except by boiling. Chymotrypsin appears to bind at the same site on the inhibitor as trypsin. The inhibitor is high in half-cysteine residues and basic amino acids, and appears not to be a glycoprotein. Antiserum has been raised against the purified inhibitor in rabbits and used to test its distribution in pigs using the immunoperoxidase-staining technique on tissue sections. The inhibitor is associated only with the glandular and surface epithelium of the uterus. Endometrial explants from pseudopregnant animals, cultured in presence of L-[3H]leucine, release the inhibitor in radioactive form indicating that it is a uterine product. The antiserum against the inhibitor cross-reacts with at least three other, basic, low molecular weights plasmin/trypsin inhibitors in porcine uterine secretions, suggesting that a family of isoinhibitors exists which may constitute up to 15% of the protein in porcine uterine secretions. The inhibitor(s) appears to coat and to be taken up by the trophoectoderm cells of the elongating blastocyst during pregnancy. It is suggested that the inhibitors may serve to protect the uterus from proteases released by the porcine trophoblast or to prevent degradation of essential macromolecules, such as uteroferrin, which have to be taken up by the conceptus.     

Isolation and characterization of isoinhibitors of the potato protease inhibitor I family from the latex of the rubber trees, Hevea brasiliensis

Three isoinhibitors have been isolated to homogeneity from the C-serum of the latex of the rubber tree, Hevea brasiliensis clone RRIM 600, and named HPI-1, HPI-2a and HPI-2b. The three inhibitors share the same amino acid sequence (69 residues) but the masses of the three forms were determined to be 14,893+/-10, 7757+/-5, and 7565+/-5, respectively, indicating that post-translational modifications of the protein have occurred during latex collection. One adduct could be removed by reducing agents, and was determined to be glutathione, while the other adduct could not be removed by reducing agents and has not been identified. The N-termini of the inhibitor proteins were blocked by an acetylated Ala, but the complete amino acid sequence analysis of the deblocked inhibitors by Edman degradation of fragments from endopeptidase C digestion and mass spectrometry confirmed that the three isoinhibitors were derived from a single protein. The amino acid sequence of the protein differed at two positions from the sequence deduced from a cDNA reported in GenBank. The gene coding for the inhibitor is wound-inducible and is a member of the potato inhibitor I family of protease inhibitors. The inhibitor strongly inhibited subtilisin A, weakly inhibited trypsin, and did not inhibit chymotrypsin. The amino acid residues at the reactive site P(1) and P(1)(') were determined to be Gln45 and Asp46, respectively, residues rarely reported at the reactive site in potato inhibitor I family members. Comparison of amino acid sequences revealed that the HPI isoinhibitors shared from 33% to 55% identity (50-74% similarity) to inhibitors of the potato inhibitor I family. The properties of the isoinhibitors suggest that they may play a defensive role in the latex against pathogens and/or herbivores.     

Trypsin inhibitor in the hemolymph of a solitary ascidian, Halocynthia roretzi. Purification and characterization

A purified preparation of trypsin inhibitor was obtained from the hemolymph of a solitary ascidian, Halocynthia roretzi, by a procedure including trypsin-Sepharose chromatography, DEAE-cellulose chromatography, and Sephadex G-50 gel filtration. The product was a mixture of two isoinhibitors, inhibitors I and II. They were separated from each other by high-performance liquid chromatography on an anion exchanger column, and showed almost identical amino acid compositions. They were also indistinguishable in terms of apparent specific inhibitory activity against bovine trypsin when the activity was assayed with the inhibitors at rather high concentrations (greater than 50 nM). A large difference was observed between them, however, in the inhibition constants, which correspond to the dissociation constants of the inhibitor-trypsin complexes; the inhibition constant of inhibitor I was 90 pM, whereas that of inhibitor II was 4.7 nM. The molecular weights of inhibitors I and II were estimated to be 6,000 and 4,500, respectively, by SDS-polyacrylamide gel electrophoresis, while an almost identical value, 9,000, was obtained for both of them by gel filtration. The molecular weight calculated from the amino acid compositions was 5,929 for both. The isoelectric points were also identical, that is about 5.0. Both of the inhibitors were heat-stable. Ascidian inhibitor I also inhibited other trypsin-like enzymes of mammalian origin, as well as those of ascidian origin.     

Production of native, correctly folded bovine pancreatic trypsin inhibitor by Escherichia coli

A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter. When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI. In particular, the BPTI produced by E. coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea. This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed.     

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.