Enzymatic cyclization of a potent Bowman-Birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide

The most potent known naturally occurring Bowman-Birk inhibitor, sunflower trypsin inhibitor-1 (SFTI-1), is a bicyclic 14-amino acid peptide from sunflower seeds comprising one disulfide bond and a cyclic backbone. At present, little is known about the cyclization mechanism of SFTI-1. We show here that an acyclic permutant of SFTI-1 open at its scissile bond, SFTI-1[ 6,5], also functions as an inhibitor of trypsin and that it can be enzymatically backbone-cyclized by incubation with bovine beta-trypsin. The resulting ratio of cyclic SFTI-1 to SFTI1[6,5] is similar to9:1 regardless of whether trypsin is incubated with SFTI-1[ 6,5] or SFTI-1. Enzymatic resynthesis of the scissile bond to form cyclic SFTI-1 is a novel mechanism of cyclization of SFTI-1[ 6,5]. Such a reaction could potentially occur on a trypsin affinity column as used in the original isolation procedure of SFTI-1. We therefore extracted SFTI-1 from sunflower seeds without a trypsin purification step and confirmed that the backbone of SFTI-1 is indeed naturally cyclic. Structural studies on SFTI-1[ 6,5] revealed high heterogeneity, and multiple species of SFTI-1[ 6,5] were identified. The main species closely resembles the structure of cyclic SFTI-1 with the broken binding loop able to rotate between a cis/trans geometry of the I7-P8 bond with the cis conformer being similar to the canonical binding loop conformation. The non-reactive loop adopts a beta-hairpin structure as in cyclic wild-type SFTI-1. Another species exhibits an isoaspartate residue at position 14 and provides implications for possible in vivo cyclization mechanisms.

Urotensin-II-converting enzyme activity of furin and trypsin in human cells in vitro

Human urotensin-II (hU-II) is processed from its prohormone (ProhU-II) at putative cleavage sites for furin and serine proteases such as trypsin. Although proteolysis is required for biological activity, the endogenous urotensin-converting enzyme (UCE) has not been investigated. The aim of this study was to investigate UCE activity in cultured human cells and in blood, comparing activity with that of furin and trypsin. In a cell-free system, hU-II was detected by high-performance liquid chromatography-mass spectrometry after coincubating 10 muM carboxyl terminal fragment (CTF)-ProhU-II with recombinant furin (2 U/ml, 3 h, 37degreesC) at pH 7.0 and pH 8.5, but not at pH 5.0, or when the incubating medium was depleted of Ca2+ ions and supplemented with 2 mM EDTA at pH 7.0. hU-II was readily detected in the superperfusate of permeabilized epicardial mesothelial cells incubated with CTF-ProhU-II (3 h, 37degreesC), but it was only weakly detected in the superperfusate of intact cells. Conversion of CTF-ProhU-II to hU-II was attenuated in permeabilized cells using conditions found to inhibit furin activity. In a cell-free system, trypsin (0.05 mg/ml) cleaved CTF-ProhU-II to hU-II, and this was inhibited with 35 muM aprotinin. hU-II was detected in blood samples incubated with CTF-ProhU-II (3 h, 37degreesC), and this was also inhibited with aprotinin. The findings revealed an intracellular UCE in human epicardial mesothelial cells with furin-like activity. Aprotinin-sensitive UCE activity was detected in blood, suggesting that an endogenous serine protease such as trypsin may also contribute to proteolysis of hU-II prohormone, if the prohormone is secreted into the circulation.

Sunflower trypsin inhibitor-1

SFTI-1 is a bicyclic 14 amino acid peptide that was originally isolated from the seeds of the sunflower Helianthus annuus. It is a potent inhibitor of trypsin, with a sub-nanomolar K, value and is homologous to the active site region of the well-known family of serine protease inhibitors known as the Bowman-Birk trypsin inhibitors. It has a cyclic backbone that is cross-braced by a single disulfide bridge and a network of hydrogen bonds that result in a well-defined structure. SFTI-1 is amenable to chemical synthesis, allowing for the creation of synthetic variants. Alterations to the structure such as linearising the backbone or removing the disulfide bridge do not reduce the potency of SFTI-1 significantly, and minimising the peptide to as few as nine residues results in only a small decrease in reactivity. The creation of linear variants of SFTI-1 also provides a tool for investigating putative linear precursor peptides. The mechanism of biosynthesis of SFTI-1 is not yet known but it seems likely that it is a gene-coded product that has arisen from a precursor protein that may be evolutionarily related to classic Bowman-Birk inhibitors.

Discovery, structural determination, and putative processing of the precursor protein that produces the cyclic trypsin inhibitor sunflower trypsin inhibitor 1

Backbone-cyclized proteins are becoming increasingly well known, although the mechanism by which they are processed from linear precursors is poorly understood. In this report the sequence and structure of the linear precursor of a cyclic trypsin inhibitor, sunflower trypsin inhibitor 1 (SFTI-1) from sunflower seeds, is described. The structure indicates that the major elements of the reactive site loop of SFTI-1 are present before processing. This may have importance for a protease-mediated cyclizing reaction as the rigidity of SFTI-1 may drive the equilibrium of the reaction catalyzed by proteolytic enzymes toward the formation of a peptide bond rather than the normal cleavage reaction. The occurrence of residues in the SFTI-1 precursor susceptible to cleavage by asparaginyl proteases strengthens theories that involve this enzyme in the processing of SFTI-1 and further implicates it in the processing of another family of plant cyclic proteins, the cyclotides. The precursor reported here also indicates that despite strong active site sequence homology, SFTI-1 has no other similarities with the Bowman-Birk trypsin inhibitors, presenting interesting evolutionary questions.

Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1

SFTI-1 is a novel 14 amino acid peptide comprised of a circular backbone constrained by three proline residues, a hydrogen-bond network, and a single disulfide bond. It is the smallest and most potent known Bowman-Birk trypsin inhibitor and the only one with a cyclic peptidic backbone. The solution structure of [ABA(3,11)]SFTI-1, a disulfide-deficient analogue of SFTI-1, has been determined by H-1 NMR spectroscopy. The lowest energy structures of native SFTI-1 and [ABA(3,11)]SFTI-1 are similar and superimpose with a root-mean-square deviation over the backbone and heavy atoms of 0.26 +/- 0.09 and 1.10 +/- 0.22 Angstrom, respectively. The disulfide bridge in SFTI-1 was found to be a minor determinant for the overall structure, but its removal resulted in a slightly weakened hydrogen-bonding network. To further investigate the role of the disulfide bridge, NMR chemical shifts for the backbone H-alpha protons of two disulfide-deficient linear analogues of SFTI-1, [ABA(3,11)]SFTI-1[6,5] and [ABA(3,11)]SFTI-1[1,14] were measured. These correspond to analogues of the cleavage product of SFTI-1 and a putative biosynthetic precursor, respectively. In contrast with the cyclic peptide, it was found that the disulfide bridge is essential for maintaining the structure of these open-chain analogues. Overall, the hydrogen-bond network appears to be a crucial determinant of the structure of SFTI-1 analogues.

Knots in rings - The circular knotted protein momordica cochinchinensis trypsin inhibitor-II folds via a stable two-disulfide intermediate

The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI-II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. For both oxidative folding and reductive unfolding, the same disulfide intermediate species was prevalent and was characterized to be a native-like two-disulfide intermediate in which the Cys(1)-Cys(18) disulfide bond was absent. Overall, the folding pathway of this head-to-tail cyclized protein was found to be similar to that of linear cystine knot proteins from the squash family of trypsin inhibitors. However, the pathway differs in an important way from that of the cyclotide kalata B1, in that the equivalent two-disulfide intermediate in that case is not a direct precursor of the native protein. The size of the embedded ring within the cystine knot motif appears to play a crucial role in the folding pathway. Larger rings contribute to the independence of disulfides and favor an on-pathway native-like intermediate that has a smaller energy barrier to cross to form the native fold. The fact that macrocyclic proteins are readily able to fold to a complex knotted structure in vitro in the absence of chaperones makes them suitable as protein engineering scaffolds that have remarkable stability.

Elevated plasma levels of human urotensin-II immunoreactivity in congestive heart failure

Human urotensin-II (hU-II) is the most potent endogenous cardiostimulant identified to date. We therefore determined whether hU-II has a possible pathological role by investigating its levels in patients with congestive heart failure (CHF). Blood samples were obtained from the aortic root, femoral artery, femoral vein, and pulmonary artery from CHF patients undergoing cardiac catheterization and the aortic root from patients undergoing investigative angiography for chest pain who were not in heart failure. Immunoreactive hU-II (hU-II-ir) levels were determined with radioimmunoassay. hU-II-ir was elevated in the aortic root of CHF patients (230.9 +/- 68.7 pg/ml, n = 21; P < 0.001) vs. patients with nonfailing hearts (22.7 +/- 6.1 pg/ml, n = 18). This increase was attributed to cardiopulmonary production of hU-II-ir because levels were lower in the pulmonary artery (38.2 +/- 6.1 pg/ml, n = 21; P < 0.001) than in the aortic root. hU-II-ir was elevated in the aortic root of CHF patients with nonischemic cardiomyopathy (142.1 +/- 51.5 pg/ml, n = 10; P < 0.05) vs. patients with nonfailing hearts without coronary artery disease (27.3 +/- 12.4 pg/ml, n = 7) and CHF patients with ischemic cardiomyopathy (311.6 +/- 120.4 pg/ml, n = 11; P < 0.001) vs. patients with nonfailing hearts and coronary artery disease (19.8 +/- 6.6 pg/ml, n = 11). hU-II-ir was significantly higher in the aortic root than in the pulmonary artery and femoral vein, with a nonsignificant trend for higher levels in the aortic root than in the femoral artery. The findings indicated that hU-II-ir is elevated in the aortic root of CHF patients and that hU-II-ir is cleared at least in part from the microcirculation.

Angiotensinogen localization and secretion in the rat pancreas

Renin and angiotensinogen have been previously found in the rat pancreas, and angiotensin receptors have been located in the apical domain of duct cells. To evaluate the possibility that angiotensin II could be generated within the duct system, we decided to determine whether angiotensinogen is present in rat pancreatic juice and the angiotensinogen-immunoreactive pancreatic cell types that could be responsible for its production. Angiotensinogen was detected in significant amounts by Western blotting in pancreatic juice collected from several individual rats. Different isoforms between plasma and pancreatic juice angiotensinogens were demonstrated by isoelectric focusing. Immunocytochemical experiments revealed angiotensinogen-immunoreactive cells at the periphery of the islets of Langerhans, and confocal microscopy demonstrated that most angiotensinogen-immunoreactive cells were glucagon-secreting cells. Secretion of angiotensinogen did not follow the regulated secretory pathway since it was absent from the glucagon-containing granules. This was confirmed by electron microscopy immunocytochemistry. Duct and acinar cells did not express angiotensinogen at an immunocytochemical detectable level. The present findings indicated an exocrine secretion of angiotensinogen by glucagon-secreting cells and suggest that one of the final targets of the local pancreatic renin-angiotensin system may be the duct epithelium.

Disulfide folding pathways of cystine knot proteins: Tying the knot within the circular backbone of the cyclotides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (LeNguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway.

Structure of Petunia hybrida Defensin 1, a Novel Plant Defensin with Five Disulfide Bonds

The structure of a novel plant defensin isolated from the flowers of Petunia hybrida has been determined by H-1 NMR spectroscopy. P. hybrida defensin 1 (PhD1) is a basic, cysteine-rich, antifungal protein of 47 residues and is the first example of a new subclass of plant defensins with five disulfide bonds whose structure has been determined. PhD1 has the fold of the cysteine-stabilized alphabeta motif, consisting of an alpha-helix and a triple-stranded antiparallel beta-sheet, except that it contains a fifth disulfide bond from the first loop to the alpha-helix. The additional disulfide bond is accommodated in PhD1 without any alteration of its tertiary structure with respect to other plant defensins. Comparison of its structure with those of classic, four-disulfide defensins has allowed us to identify a previously unrecognized hydrogen bond network that is integral to structure stabilization in the family.

Temperature dependence of the thrombin-catalyzed proteolysis of prothrombin

Measurement of the temperature-dependence of thrombin-catalyzed cleavage of the Arg(155)-Ser(156) and Arg(284)-Thr(285) peptide bonds in prothrombin and prothrombin-derived substrates has yielded Arrhenius parameters that are far too large for classical mechanistic interpretation in terms of a simple hydrolytic reaction. Such a difference from the kinetic behavior exhibited in trypsin- and chymotrypsin-catalyzed proteolysis of peptide bonds is attributed to contributions by enzyme exosite interactions as well as enzyme conformational equilibria to the magnitudes of the experimentally determined Arrhenius parameters. Although the pre-exponential factor and the energy of activation deduced from the temperature-dependence of rate constants for proteolysis by thrombin cannot be accorded the usual mechanistic significance, their evaluation serves a valuable role by highlighting the existence of contributions other than those emanating from simple peptide hydrolysis to the kinetics of proteolysis by thrombin and presumably other enzymes of the blood coagulation system. (C) 2004 Elsevier B.V. All rights reserved.

A pore-forming haemolysin from the hookworm, Ancylostoma caninum

Hookworms feed on blood, but the mechanism by which they lyse ingested erythrocytes is unknown. Here we show that Ancylostoma caninum, the common dog hookworm, expresses a detergent soluble, haemolytic factor. Activity was identified in both adult and larval stages, was heat-stable and unaffected by the addition of protease inhibitors, metal ions, chelators and reducing agents. Trypsin ablated lysis indicating that the haemolysin is a protein. A closely migrating doublet of hookworm proteins with apparent molecular weights of 60-65 kDa bound to the erythrocyte membrane after lysis of cells using both unlabeled and biotinylated detergent-solubilised hookworm extracts. In addition, separation of detergent-soluble parasite extracts using strong cation-exchange chromatography, resulted in purification of 60-65 kDa proteins with trypsin-sensitive haemolytic activity. Erythrocytes lysed with particulate, buffer-insoluble worm extracts were observed using scanning electron microscopy and appeared as red cell ghosts with approximately 100 nm diameter pores formed in the cell membranes. Red blood cell ghosts remained visible indicating that lysis was likely caused by pore formation and followed by osmotic disruption of the cell. (C) 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: The importance of the cyclic cystine knot

The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata 132, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.