Pyroglutamyl-aprotinin, a new aprotinin homologue from bovine lungs--isolation, properties, sequence analysis and characterization using 1H nuclear magnetic resonance in solution

A new Kunitz-inhibitor, which is different from aprotinin was extracted from bovine lungs with methanol, further purified by affinity chromatography on trypsin-Sepharose CL-6B and by repeated cation exchange chromatography on CM-Sephadex C-25. The inhibitor, which is less basic than aprotinin was characterized by polyacrylamide gel electrophoresis and ion-exchange HPLC. The N-terminus is blocked by pyroglutamic acid (Glu-1). After enzymatic removal of this residue with pyroglutamate aminopeptidase, complete identity with the primary structure of aprotinin was established by sequencing the inhibitor, which had been oxidized with performic acid, and by sequencing a tryptic fragment. The occurrence of the inhibitor, which can be denoted as pyroglutamyl-aprotinin or Glu-1-aprotinin, but which cannot be distinguished from aprotinin regarding its inhibitory specificity, is obviously the result of a different proteolytic processing of the bovine aprotinin precursor. By using CD and NMR-techniques it was shown that the N-terminus of the inhibitor is blocked, and that the conformation and the internal mobility correspond with those of aprotinin.     

Semisynthetic engineering of proteinase inhibitor homologues

A semisynthetic approach to modulate the inhibitory specificity of aprotinin, the Kunitz trypsin inhibitor from bovine mast cells, is described. By the use of peptide-chemical procedures a single amino acid of its reactive site can be replaced by any other coded or non-coded amino acid. Thus, a series of aprotinin homologues have been prepared which demonstrate the individual contribution of a single side chain to the inhibition of a particular target proteinase and enable specific inhibitors to be designed.     

Ala214,38]aprotinin: preparation by partial desulphurization of aprotinin by means of Raney nickel and comparison with other aprotinin derivatives

Treatment of aprotinin with Raney nickel in the presence or absence of denaturants yielded [Ala2 14,38]aprotinin. Aprotinin and [Ala2 14,38]aprotinin were separated by ion exchange chromatography at pH 8 using CM-Sepharose, fast flow. [Ala2 14,38]aprotinin is a proteinase inhibitor, but it possesses lower affinities than aprotinin, for the enzymes trypsin, alpha-chymotrypsin, pancreatic kallikrein and plasmin as reflected by higher Ki values [Ala2 14,38]aprotinin is slowly degraded by trypsin. The optical activity of [Ala2 14,38]aprotinin in different solvents is quite similar to that of aprotinin, or that of its hydrolysis products, [seco-15/16]aprotinin or [di-seco-15/16,39/40]-aprotinin. This is taken as good evidence for analogous molecular conformations of all these substances.     

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).     

Studies on the extraction of DesPro(2)-Val15-Leu17-aprotinin from the culture broth of a recombinant Saccharomyces cerevisiae.

The application of an aqueous two-phase system is described for the extraction of DesPro(2)-Val15-Leu17-aprotinin from yeast culture supernatant, using native chymotrypsin as affinity ligand. The interaction is driven by hydrophobic forces and leads to the accumulation of the aprotinin-chymotrypsin complex in the salt-rich (bottom) phase of a polyethyleglycol/salt system. The complex may be dissociated at low pH values. The separation of the recombinant aprotinin and the protease required chromatographic processes, which proved difficult to interface with the affinity extraction.     

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, VII. Determination of the amino-acid sequence of the trypsin-released inhibitor from bovine inter-alpha-trypsin inhibitor

An acid-labile proteinase inhibitor, quite similar to human inter-alpha-trypsin inhibitor, was isolated from bovine serum. An acid-resistant 30-kDa inhibitor, exhibiting properties similar to human HI-30, was also isolated. Upon limited proteolysis of both bovine inhibitors, active 14-kDa domains are released which are identical with respect to molecular mass and acid resistance. The amino-acid sequence determination of these fragments revealed a strong homology to the corresponding human inhibitor HI-14 which is characterized by two covalently linked Kunitz-type domains. The reactive-site residue is leucine in the N-terminal domain (in the human inhibitor methionine) and arginine in the C-terminal domain in both bovine and human inhibitor.     

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).     

Characterization of five new low-molecular-mass trypsin inhibitors from white mustard (Sinapis alba L.) seed.

Five new low-molecular-mass trypsin inhibitors belonging to the RTI/MTI-2 family were identified from white mustard (Sinapis alba L. ; MTI-2) seed. Purified MTI-2 consisted of a peptide mixture, displaying Ile or Arg at position 43, Trp or kynurenine (Kyn) at position 44, and C-terminal ragged ends. The occurrence of Ile or Arg at position 43 suggested that MTI-2 inhibitors originated from different genes. The presence of 5-oxo-proline (pyroglutamic acid; 5-oxoPro1) and Kyn44 reflected post-translational processing of the serine proteinase inhibitor. MTI-2 showed approximately 70% amino-acid identity with low-molecular-mass trypsin inhibitors isolated from oil rape (Brassica napus var. oleifera; RTI-III) seed and with serine proteinase inhibitors mapped in Arabidopsis thaliana chromosome II (ATTI). Furthermore, MTI-2 was homologous to brazzein, the sweet-tasting protein from Pentadiplandra brazzeana Baillon fruit ( approximately 30% amino-acid identity). Although snake-venom toxins showed a low amino-acid identity (< 20%) with MTI-2, RTI-III, and ATTI, some structurally relevant residues were conserved. The disulfide bridge pattern of MTI-2 (Cys5-Cys27, Cys18-Cys31, Cys42-Cys52, and Cys54-Cys57) corresponded to that of RTI-III and of snake-venom toxins, being different from that of brazzein. Therefore, protein similarity might be attributable to the three-dimensional arrangement rather than to the amino-acid sequence. Values of Ka for MTI-2 binding to bovine beta-trypsin (trypsin) and bovine alpha-chymotrypsin were 6.3 x 109 M-1 and 2.0 x 106 M-1, respectively, at pH 8.0 and 21.0 degrees C. Moreover, values of kon for MTI-2 binding to trypsin and of koff for the dissociation of the serine proteinase:inhibitor complex were 5.6 x 105 M-1.s-1 and 8.9 x 10-5 M-1.s-1, respectively, at pH 8.0 and 21.0 degrees C. Despite the heterogeneity of the purified inhibitor peptide mixture, the inhibition properties of the different MTI-2 inhibitors were indistinguishable.     

Genetic variants of bovine beta-lactoglobulin. A novel wild-type beta-lactoglobulin W and its primary sequence

A novel bovine beta-lactoglobulin W has been isolated and its complete primary structure is presented. It was isolated by chromatofocusing of a beta-lactoglobulin AW heterozygote and purified by recrystallization. During sequencing of the oxidized protein, it became evident that the new beta-lactoglobulin W is a subtype of variant B with a single difference at position 56. This Ile----Leu substitution, which means a shift of a methyl group from C-beta to C-gamma of the amino-acid side chain causes a change of pI of 0.007 units, which can be detected by high resolution electrophoresis. This Ile56 amino-acid residue is among the most conserved residues with the exception of kangaroo beta-LG. The structures of other bovine beta-lactoglobulins and their relationships are discussed.     

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.     

Kallikrein inhibitors.

So far the Cl inactivator, alpha 2-macroglobulin, antithrombin III (in the presence of heparin), and alpha 1-antitrypsin have been identified as inhibitors of plasma kallikrein; alpha 1-antitrypsin reacts slowly also with tissue kallikreins. Of the various naturally occurring kallikrein inhibitors the basic trypsin-kallikrein inhibitor of bovine organs, aprotinin (the active substance of Trasylol), has attained by far the most interest. This inhibitor, which is produced by mast cells, has unusual properties due to its compact tertiary structure. Additional topics of aprotinin and structurally related inhibitors discussed are the mechanism of enzyme-inhibitor complex formation, the production of chemical mutants of aprotinin, the structural basis of kallikrein inhibition, and selected aspects regarding aprotinin medication.     

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     

Purification and characterization of aprotinin from porcine lungs

Aprotinin, the most studied serine proteinase inhibitor, was isolated from porcine lung for the first time. The purified porcine aprotinin had an Mr value of ∼7 kDa. It cross-reacted with polyclonal serum anti-commercial aprotinin. About 1 μg porcine aprotinin inhibited 6 μg trypsin whereas 1 μg commercial soybean inhibitor inhibited only 1 μg trypsin. The aprotinin gene was also isolated from porcine lung: the deduced amino acid sequence showed 74% identity to bovine aprotinin.     

Preparation of chemically 'mutated' aprotinin homologues by semisynthesis. P1 substitutions change inhibitory specificity

The semisynthesis of homologues of aprotinin (BPTI) is described. The P1 amino acid residue of these homologues was substituted by other amino acids using peptide synthetic methods. The reactive-site-modified inhibitor (with the Lys15-Ala16 peptide bond hydrolyzed) was used as starting material. All carboxyl groups of the modified inhibitor were esterified with methanol, then the Lys15 methyl ester group was hydrolyzed selectively. Afterwards, Lys15 itself was split off. A new amino acid residue was incorporated by using water-soluble carbodiimide combined with an acylation catalyst. tert-Butyl-ester-protected amino acids were used for reinsertion. The method was tested by re-insertion of Lys15 to reconstitute the original inhibitor. Thirteen BPTI homologues with coded (Lys, Glu, Gly, Ala, Val, Ile, Leu) or uncoded amino acids (Abu, Ape, aIle, Ahx, tLeu, Neo) in position 15 were synthesized and the specificity of the inhibitors investigated. Amongst these, [Val15]BPTI was shown to be an excellent inhibitor for human polymorphonuclear leukocyte elastase having a complex dissociation constant of 0.11 nM. This inhibitor showed no detectable affinity to bovine pancreatic trypsin.     

Isolation of acid-resistant urinary trypsin inhibitors by high performance liquid chromatography and their characterization by N-terminal amino-acid sequence determination

Two crude fractions of acid-resistant trypsin inhibitors (apparent molecular masses 44 and 20 kDa, respectively) were prepared from human urine by gel permeation chromatography. From both preparations the pure inhibitors were isolated by high performance liquid chromatography (HPLC). Their N-terminal amino-acid sequences were determined and compared with those of HI-30 and HI-14 as isolated by reversible binding to either immobilized trypsin or immobilized chymotrypsin. The N-terminal amino-acid sequence of the high-molecular mass inhibitor UI-I isolated by HPLC was identical with those of HI-30 and UI-C-I isolated via immobilized trypsin or chymotrypsin, respectively. The low-molecular mass inhibitors UI-II and UI-C-II differ from HI-14 by the N-terminal extension Glu-Val-Thr-Lys-when obtained by HPLC or by the extension Thr-Lys-when obtained via immobilized chymotrypsin, respectively. The comparison of these N-termini with the amino-acid sequence of HI-30 (Ala1-...-Val16-Thr-Glu-Val-Thr-Lys-HI-14) defines the low molecular urinary trypsin inhibitors as proteolytic degradation products of the high-molecular urinary inhibitor. Proteolysis may occur at different bonds. The existing discrepancies in molecular architecture and in molecular masses of the urinary trypsin inhibitors are discussed.     

Determination of the sequence of the aralkyl acyl-CoA:amino acid N-acyltransferase from bovine liver mitochondria

The aralkyl acyl-CoA:amino-acid N-acyl-transferase was previously purified to homogeneity from bovine liver mitochondria. The N-terminal amino-acid sequence and sequences obtained by cyanogen bromide cleavage of the enzyme were used to design oligonucleotide probes that were used to screen a bovine liver cDNA library. Several clones were isolated and sequenced, and the sequence is given. The cDNA contains 126 bases of 5′-untranslated region and 188 bp of 3′ untranslated region. The cDNA codes for an enzyme containing 295 amino-acid residues. The sequence gives a molecular weight for the enzyme of 39,229, which is larger than previously estimated. The amino-acid composition of the enzyme, based on this sequence, is in agreement with the previously obtained amino-acid analysis on the purified kidney enzyme. © 1997 John Wiley & Sons, Inc.     

Guinea pig plasma trypsin inhibitors. Purification and characterization of two functionally distinct proteinase inhibitors.

Two trypsin inhibitors (TI-1, TI-2) were isolated from guinea pig plasma and purified to homogeneity. In amino-acid composition as well as molecular masses, TI-1 (Mr 58,000) and TI-2 (Mr 57,000) are similar to each other and to human and mouse alpha 1-proteinase inhibitors, and mouse con-trapsin. The two inhibitors form equimolar complexes with proteinases. The effectiveness of the inhibitors was characterized by association rate constants under second-order rate conditions. The inhibitory action of TI-1 was rapid for bovine trypsin, porcine pancreatic elastase and guinea pig plasma kallikrein, but slow for bovine thrombin and guinea pig plasmin and not detectable for bovine chymotrypsin and porcine pancreatic kallikrein. The inhibitory action of TI-2 was rapid for trypsin and chymotrypsin, but slow for guinea pig plasma kallikrein and not detectable for other proteinases. These results show that TI-1 and TI-2 are physicochemically similar but functionally distinct from each other and from human alpha 1-proteinase inhibitor that inhibits trypsin, chymotrypsin and elastase.     

A new inhibitor of plasmin and trypsin from porcine leukocytes.

A new intracellular inhibitor of plasmin and trypsin was isolated from porcine leukocytes by ion exchange chromatography and affinity chromatography. In dodecyl sulphate gel electrophoresis a single protein band with an apparent molecular mass of 15 kDa was found under reducing conditions. On isoelectric focusing three protein bands with isoelectric points between pH 4.0 and 4.5 were found. The association rate constants and the inhibition constants were determined for porcine plasmin and bovine trypsin. The inhibitor shows no immunologic cross-reactivity with any of the tested leukocyte inhibitors. On the basis of its N-terminal amino-acid sequence a great degree of similarity to Kunitz-type inhibitors was observed.     

Selective oxidation of methionine residues in Kunitz-type protease inhibitors

Bovine pancreatic trypsin inhibitor (BPTI, also known as aprotinin or Kunitz inhibitor, a mini-protein composed of 58 amino-acid residues, containing a single methionine residue at position 52) has been selectively oxidized by treatment with chloramine T, under mild conditions, to the methionyl sulfoxide derivative. Spleen inhibitor II (SI II, an isoform of BPTI containing two methionine residues at positions 18 and 52) has been oxidized under the same conditions. Oxidation affects the functional properties of the two inhibitors differently: the antiproteolytic activity of BPTI towards bovine trypsin and chymotrypsin, porcine kallikrein and human leukocyte elastase is not changed upon oxidation, while in the oxidized SI II, the affinity for both chymotrypsin and elastase decreases, with respect to the native protein. These results have been directly related to the oxidation of Met18 in SI II, located at the P'3 site in the contact area with the proteases.     

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.