Ascaris suum: localization by immunochemical and fluorescent probes of host proteases and parasite proteinase inhibitors in cross-sections

Cross-sections of muscle, intestine, and genital tract fluoresced in defined locations when live Ascaris suum adults were incubated in medium containing chymotrypsin liganded with fluorescein-5-isothiocyanate. This suggests that the protease, or portions of it, are assimilated by A. suum. A. suum chymotrypsin/elastase isoinhibitors were found in muscle sarcolemma, eggs, sperm, and intestine, and host chymotrypsin was localized in the same regions of these tissues by immunofluorescence and immunoperoxidase techniques. These experiments demonstrate that host chymotrypsin enters the parasite, that it is present in specific regions of Ascaris, and that it probably exists as an enzyme-inhibitor complex.     

The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: the reactive site

Five isoinhibitors of chymotrypsin/elastase present in aqueous extracts of Ascaris were isolated. The reactive site in each isoinhibitor, the peptide bond that during encounter is positioned over the catalytic site in chymotrypsin, is Leu-Met. This bond was hydrolyzed by incubating intact isoinhibitors with 5-25 mol% chymotrypsin at pH 3.2 for 4-6 days (isoinhibitor 1) or 2.5-5 weeks (isoinhibitors 2-5). The reaction under these conditions did not proceed beyond 60% modified isoinhibitor (peptide bond hydrolyzed) and 40% intact inhibitor. The Leu-Met bond, hydrolyzed in modified isoinhibitor, can be resynthesized at pH 7.6 by incubating modified inhibitor with a stoichiometric amount of chymotrypsin bound to Sepharose CL-4B and then dissociating the complex in a kinetically controlled fashion with 5% trichloroacetic acid. The product, intact inhibitor, was obtained in greater than 80% yield. The site in the isoinhibitor that is positioned over the catalytic site in elastase during encounter is the same as for encounter with chymotrypsin. The Leu-Met bond hydrolyzed during encounter with elastase can be resynthesized by chymotrypsin. Chymotrypsin and elastase bind to the inhibitor at the same site.     

Proteinase inhibitors from desert locust, Schistocerca gregaria: engineering of both P(1) and P(1)' residues converts a potent chymotrypsin inhibitor to a potent trypsin inhibitor.

Two peptides, SGCI and SGTI, that inhibited chymotrypsin and trypsin, respectively, were isolated from the haemolymph of Schistocerca gregaria. Their primary structures were found to be identical with SGP-2 and SGP-1, two of a series of peptides isolated from ovaries of the same species (A. Hamdaoui et al., FEBS Lett. 422 (1998) 74-78). All these peptides are composed of 35-36 amino acid residues and contain three homologous disulfide bridges. The residues imparting specificity to SGCI and SGTI were identified as Leu-30 and Arg-29, respectively. The peptides were synthesised by solid-phase peptide synthesis, and the synthetic ones displayed the same inhibition as the natural forms: SGCI is a strong inhibitor of chymotrypsin (K(i) = 6.2 x 10(-12) M), and SGTI is a rather weak inhibitor of trypsin (K(i) = 2.1 x 10(-7) M). The replacement of P(1) then P(1)' residues of SGCI with trypsin-specific residues increased affinity towards trypsin 3600- and 1100-fold, respectively, thus SGCI was converted to a strong trypsin inhibitor (K(i) = 5.0 x 10(-12) M) that retained some inhibitory affinity towards chymotrypsin (K(i) = 3.5 x 10(-8) M). The documented role of both P(1) and P(1)' highlights the importance of S(1)'P(1)' interactions in enzyme-inhibitor complexes.     

The effects of reactive site location on the inhibitory properties of the serpin alpha(1)-antichymotrypsin

The large size of the serpin reactive site loop (RSL) suggests that the role of the RSL in protease inhibition is more complex than that of presenting the reactive site (P1 residue) to the protease. This study examines the effect on inhibition of relocating the reactive site (Leu-358) of the serpin alpha(1)-antichymotrypsin either one residue closer (P2) or further (P1') from the base of the RSL (Glu-342). alpha(1)-Antichymotrypsin variants were produced by mutation within the P4-P2' region; the sequence ITLLSA was changed to ITLSSA to relocate the reactive site to P2 (Leu-357) and to ITITLS to relocate it to P1' (Leu-359). Inhibition of the chymotrypsin-like proteases human chymase and chymotrypsin and the non-target protease human neutrophil elastase (HNE) were analyzed. The P2 variant inhibited chymase and chymotrypsin but not HNE. Relative to P1, interaction at P2 was characterized by greater complex stability, lower inhibition rate constants, and increased stoichiometry of inhibition values. In contrast, the P1' variant inhibited HNE (stoichiometry of inhibition = 4) but not chymase or chymotrypsin. However, inhibition of HNE was by interaction with Ile-357, the P2 residue. The P1' site was recognized by all proteases as a cleavage site. Covalent-complexes resistant to SDS-PAGE were observed in all inhibitory reactions, consistent with the trapping of the protease as a serpin-acyl protease complex. The complete loss in inhibitory activity associated with lengthening the Glu-342-reactive site distance by a single residue and the enhanced stability of complexes associated with shortening this distance by a single residue are compatible with the distorted-protease model of inhibition requiring full insertion of the RSL into the body of the serpin and translocation of the linked protease to the pole opposite from that of encounter.     

The amino acid sequence and reactive site of a single-headed trypsin inhibitor from wheat endosperm

The sequence of a trypsin inhibitor, isolated from wheat endosperm, is reported. The primary structure was obtained by automatic sequence analysis of the S-alkylated protein and of purified peptides derived from chemical cleavage by cyanogen bromide and digestion withStaphylococcus aureus V8 protease. This protein, named wheat trypsin inhibitor (WTI), which is comprised of a total of 71 amino acid residues, has 12 cysteines, all involved in disulfide bridges. The primary site of interaction (reactive site) with bovine trypsin has been identified as the dipeptide arginyl-methionyl at positions 19 and 20. WTI has a high degree of sequence identity with a number of serine proteinase inhibitors isolated from both cereal and leguminous plants. On the basis of the findings presented, this protein has been classified as a single-headed trypsin inhibitor of Bowman-Birk type.     

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.     

Effect of P(2)' site tryptophan and P(20)' site deletion of Momordica charantia trypsin inhibitor II on inhibition of proteinases

Momordica charantia trypsin inhibitor II (MCTI-II) inhibits the amidolytic activity of factor Xa with a K(i) value 10-100-fold smaller than those of other squash family inhibitors. It also inhibits factor X activation mediated by factor VIIa-tissue factor complex or factor IXa. Comparison of other squash family inhibitors reveal Trp at position 7 (P(2)') and a deletion at position 25 (P(20)') are characteristics of MCTI-II. In order to elucidate the effect of these positions on the inhibitory activity, we chemically synthesized three inhibitors: S-MCTI-II whose amino acid sequence is identical to natural MCTI-II, S-MCTI-II(7L) whose P(2)'(Trp) is substituted with Leu, and S-MCTI-II(25N) whose P(20)'(deletion) is filled with Asn. The dissociation constants of the complexes of human factor Xa with S-MCTI-II, S-MCTI-II(7L), and S-MCTI-II(25N) were 1.3x10(-6) M, 2.8x10(-5) M, and 7.3x10(-6) M, respectively. They inhibited factor X activation mediated by factor VIIa with the same degree. As in the case of natural MCTI-II, S-MCTI-II suppressed factor X activation mediated by factor IXa, while S-MCTI-II(7L) and S-MCTI-II(25N) did not. Both the Trp at the P(2)' position and deletion at the P(20)' position are thus likely required for the inhibition of factor Xa, trypsin, and factor IXa, while these two positions do not affect factor X activation initiated by the factor VIIa-tissue factor complex.     

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.     

Trypsin inhibitors in guinea pig plasma: isolation and characterization of contrapsin and two isoforms of alpha-1-antiproteinase and acute phase response of four major trypsin inhibitors

Contrapsin and two isoforms, F (fast) and S (slow), of alpha-1-antiproteinase (also called alpha-1-proteinase inhibitor) were isolated in an apparently homogeneous state from plasma of inflamed guinea pigs. Contrapsin inactivated trypsin, but did not significantly affect chymotrypsin, pancreatic elastase, or pancreatic kallikrein. On the other hand, both isoforms of alpha-1-antiproteinase inhibited trypsin, chymotrypsin, and elastase, but not plasma or pancreatic kallikrein. The S isoform of alpha-1-antiproteinase was present in barely detectable amounts in healthy animals, but increased markedly when the acute-phase reaction was induced by subcutaneous injection of turpentine. On the other hand, the plasma levels of the F isoform, contrapsin, and alpha-macroglobulin showed moderate (1.5 to 2.3-fold) elevation during the acute-phase reaction. In contrast to the previous findings that rats and rabbits contain two different alpha-macroglobulins, one of which is an acute-phase reactant while the other is not, inflamed guinea pigs contained only one species of alpha-macroglobulin. Murinoglobulin, the most prominent acute-phase negative protein in both mice and rats, showed no significant change in guinea pigs. These results indicate that guinea pig plasma contains four major trypsin inhibitors, i.e., contrapsin, alpha-1-antiproteinase, alpha-macroglobulin, and murinoglobulin, the properties of which are very similar to those of the respective mouse homologues, but that the acute-phase response of these inhibitors differs greatly from that of the homologous proteins in rats or mice.     

Primary Structure of a Trypsin Inhibitor (Copaifera langsdorffii Trypsin Inhibitor-1) Obtained from C. langsdorffii Seeds

In this study, the aim was to determine the complete sequence of the Copaifera langsdorffii trypsin inhibitor (CTI)-1 using 2-dimensional (2D)-PAGE, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF), and quadrupole time-of-flight (QTOF) spectrometry. Spots A (CTI-1) and F (CTI-2) were submitted to enzymatic digestions with trypsin, SV8, and clostripain. The accurate mass of the peptide obtained from each digest was determined by mass spectrometry (MS) using MALDI-TOF. The most abundant peptides were purified and sequenced in a liquid chromatograph connected to an electrospray ionization-QTOF MS. When the purified trypsin inhibitor was submitted to 2D electrophoresis, different spots were observed, suggesting that the protein is composed of 2 subunits with microheterogeneity. Isoelectric points of 8.0, 8.5, and 9.0 were determined for the 11 kDa subunit and of 4.7, 4.6, and 4.3 for the 9 kDa subunit. The primary structure of CTI-1, determined from the mass of the peptide of the enzymatic digestions and the sequence obtained by MS, indicated 180 shared amino acid residues and a high degree of similarity with other Kunitz (KTI)-type inhibitors. The peptide also contained an Arg residue at the reactive site position. Its 3-dimensional structure revealed that this is because the structural discrepancies do not affect the canonical conformation of the reactive loop of the peptide. Results demonstrate that a detailed investigation of the structural particularities of CTI-1 could provide a better understanding of the mechanism of action of these proteins, as well as clarify its biologic function in the seeds. CTI-1 belongs to the KTI family and is composed of 2 polypeptide chains and only 1 disulfide bridge.     

Synthesis of an elastase inhibitor by monosubstitution of arginine-5 with valine at the reactive site in a trypsin inhibitor from squash seeds (CMTI III)

Using the solid-phase procedure an analog of trypsin inhibitor CMTI III containing Val5 instead of Arg5 in position P1, was synthesized. The substitution in only this one position P1 increased the affinity of synthetic inhibitor to porcine pancreatic elastase and human leukocyte elastase by the factor of 10(3) and 10(7), respectively.     

Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex

Cytochrome bc(1) is an integral membrane protein complex essential for cellular respiration and photosynthesis; it couples electron transfer from quinol to cytochrome c to proton translocation across the membrane. Specific bc(1) inhibitors have not only played crucial roles in elucidating the mechanism of bc(1) function but have also provided leads for the development of novel antibiotics. Crystal structures of bovine bc(1) in complex with the specific Q(o) site inhibitors azoxystrobin, MOAS, myxothiazol, stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole were determined. Interactions, conformational changes and possible mechanisms of resistance, specific to each inhibitor, were defined. Residues and secondary structure elements that are capable of discriminating different classes of Q(o) site inhibitors were identified for the cytochrome b subunit. Directions in the displacement of the cd1 helix of cytochrome b subunit in response to various Q(o) site inhibitors were correlated to the binary conformational switch of the extrinsic domain of the iron-sulfur protein subunit. The new structural information, together with structures previously determined, provide a basis that, combined with biophysical and mutational data, suggest a modification to the existing classification of bc(1) inhibitors. bc(1) inhibitors are grouped into three classes: class P inhibitors bind to the Q(o) site, class N inhibitors bind to the Q(i) site and the class PN inhibitors target both sites. Class P contains two subgroups, Pm and Pf, that are distinct by their ability to induce mobile or fixed conformation of iron-sulfur protein.     

Natural plant enzyme inhibitors: isolation and properties of a trypsin inhibitor from jack bean (Canavalia ensiformis)

A protease inhibitor which is equally active on bovine and porcine trypsins was isolated in a homogenous form from jack bean (Canavalia ensiformis). The preparation with a molecular weight of 18 kDa was found to be a glycoprotein with a high half cysteine content. Isoleucine and tyrosine were found to be absent. The inhibitor was heat-stable and stable at pH 2.0 and 11.0. It was ten times less active on bovine alpha-chymotrypsin and pronase than on trypsin. It displayed weak action on subtilisin BPN, porcine elastase and pepsin. The inhibitor was most effective in blocking the total proteolytic, tryptic and chymotryptic activities of rabbit pancreatic preparation. The relative ratios of inhibitions of the three activities on rabbit, bovine and human systems were respectively 1250:100:1, 600:100:1 and 46:18:1. While different substrates (except denatured serum albumin) did not significantly alter the magnitude of inhibition of bovine trypsin, the extent of inhibition of bovine alpha-chymotrypsin by the jack bean inhibitor was highly dependent on the substrate used in the assay.     

The conserved P1' Ser of Bowman-Birk-type proteinase inhibitors is not essential for the integrity of the reactive site loop

The isolated reactive site beta-hairpin loop of Bowman-Birk-type proteinase inhibitors has become a widely studied proteinomimetic because it retains the three-dimensional structure and much of the inhibitory potency of the corresponding region of the complete protein. Here we analyse the role of the P1' Ser residue which is highly conserved and intramolecularly hydrogen bonded in the complete proteins. A combined kinetic and structural analysis of variant proteinomimetic peptides demonstrates that the hydrogen-bond potential of the side-chain oxygen atom of the P1' Ser is not essential for the integrity of the reactive site loop and that it provides only a small contribution to the trypsin affinity and no apparent contribution to the stability against tryptic turnover. We conclude that the potential of the P1' side chain to engineer improved inhibition and selectivity for serine proteinases is best explored further in concert with the side chains of the P2 and P5' residues which may interact or compete for the same space.