Immunomodulatory activity of a chymotrypsin inhibitor from Momordica cochinchinensis seeds.

Serine protease inhibitors are widely distributed in the plant kingdom. Many of them have been purified and characterized from different species. While the physicochemical properties of these protease inhibitors have been extensively investigated, their biological effects, e.g. immunomodulatory effect, remain relatively unexplored. Recently, we isolated a chymotrypsin-specific inhibitor (MCoCI) from the seeds of Momordica cochinchinensis (Lour) Spreng (Family Cucurbitaceae), the traditional Chinese medicine known as Mubiezhi, which has been used as an antiinflammatory agent. In the present study, the effects of MCoCI on different types of cells of the immune system, including splenocytes, splenic lymphocytes, neutrophils, bone marrow cells and macrophages, were investigated. MCoCI was shown to possess immuno-enhancing and antiinflammatory effects. MCoCI could stimulate the proliferation of different cells of the immune system, e.g. splenocytes, splenic lymphocytes and bone marrow cells, in a manner comparable to that of Concanavalin A. Moreover, MCoCI could also suppress the formation of hydrogen peroxide in neutrophils and macrophages. These immunomodulatory effects may explain some of the therapeutic actions of Mubiezhi.     

Multiple trypsin inhibitors from Momordica cochinchinensis seeds, the Chinese drug mubiezhi

Five trypsin inhibitors, with N-terminal sequences demonstrating homology to each other and exhibiting a molecular weight of 5100, 4800, 4400, 4100, and 3900, respectively, were isolated from Momordica cochinchinensis seeds with a protocol involving acid extraction, ion exchange chromatography on SP-Sepharose chromatography, and RP-HPLC on a C18 column. Specific inhibitory activity against trypsin was demonstrated by the trypsin isoinhibitors with Ki values ranging from 5.3 x 10(-8) to 1.8 x 10(-6) M. None of the isoinhibitors could be cleaved by trypsin.     

First report on a potato I family chymotrypsin inhibitor from the seeds of a Cucurbitaceous plant, Momordica cochinchinensis

A 7514-Da chymotrypsin inhibitor was isolated from the seed extract of Momordica cochinchinensis (Family Cucurbitaceae) by chromatography on chymotrypsin-Sepharose 4B and subsequently by C18 reversed-phase HPLC. This inhibitor, named MCoCl, possessed remarkable thermostability and was stable from pH 2 to 12. MCoCl also inhibited subtilisin, but had at least 50-fold lower inhibitory activity towards trypsin and elastase. Amino acid sequencing of a peptide fragment of MCoCl revealed a sequence of 23 amino acids. Comparison of this sequence and the molecular mass with those of other protease inhibitors suggests that MCoCl belongs to the potato I inhibitor family.     

Diversity in the disulfide folding pathways of cystine knot peptides

Summary The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif (CCK). This unique family was discovered only recently but contains over 50 known sequences to date. Various biological activities are associated with these peptides including antimicrobial and insecticidal activity. The knotted topology and cyclic nature of the cyclotides poses interesting questions about the folding mechanisms and how the knotted arrangement of disulfide bonds is formed. Some studies have been performed on related inhibitor cystine knot (ICK) containing peptides, but little is known about the folding mechanisms of CCK molecules. We have examined the oxidative refolding and reductive unfolding of the prototypic member of the cyclotide family, kalata B1. Analysis of the rates of formation of the intermediates along the reductive unfolding pathway highlights the stability conferred by the cystine knot motif. Significant differences are observed between the folding of kalata B1 and an acyclic cystine knot protein, EETI-II, suggesting that the circular backbone has a significant influence in directing the folding pathway.     

Stable and biocompatible cystine knot peptides from the marine sponge Asteropus sp.

Two new cystine knot peptides, asteropsins F (ASPF) and G (ASPG), were isolated from the marine sponge Asteropus sp. ASPF and ASPG are composed of 33 and 32 amino acids, respectively, and contain six cysteines which are involved in three disulfide bonds. They shared the characteristic features of the asteropsin family, such as, N-terminal pyroglutamate modification, incorporation of cis prolines, and the unique anionic profile, which distinguish them from other knottin families. Tertiary structures of the peptides were determined by high resolution NMR. ASPF and ASPG were found to be remarkably resistant not only to digestive enzymes (chymotrypsin, pepsin, elastase, and trypsin) but also to thermal degradation. In addition, these peptides were pharmacologically inert; non-hemolytic to human and fish red blood cells, non-stimulatory to murine macrophage cells, and nontoxic in vitro or in vivo. These observations support their stability and biocompatibility as suitable carrier scaffolds for the design of oral peptide drug.     

Purification and properties of a new ribosome-inactivating protein with RNA N-glycosidase activity suitable for immunotoxin preparation from the seeds of Momordica cochinchinensis

A ribosome-inactivating protein similar to those already known (Stirpe and Barbieri (1986) FEBS Lett. 195, 1-8) was purified from the seeds of Momordica cochinchinensis. This protein, for which the name of momorcochin-S is proposed, is a glycoprotein, has an Mr of approx. 30,000, and an alkaline isoelectric point and can be considered as an iso-form of the previously purified momorcochin from the roots of M. cochinchinensis. Momorcochin-S inhibits protein synthesis by a rabbit-reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and alters rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Momorcochin-S was linked to a monoclonal antibody (8A) against human plasma cells, and the resulting immunotoxin was selectively toxic to target cells.     

Functional evolution of scorpion venom peptides with an inhibitor cystine knot fold

The ICK (inhibitor cystine knot) defines a large superfamily of polypeptides with high structural stability and functional diversity. Here, we describe a new scorpion venom-derived K⁺ channel toxin (named λ-MeuKTx-1) with an ICK fold through gene cloning, chemical synthesis, nuclear magnetic resonance spectroscopy, Ca²⁺ release measurements and electrophysiological recordings. λ-MeuKTx-1 was found to adopt an ICK fold that contains a three-strand anti-parallel β-sheet and a 3₁₀-helix. Functionally, this peptide selectively inhibits the Drosophila Shaker K⁺ channel but is not capable of activating skeletal-type Ca²⁺ release channels/ryanodine receptors, which is remarkably different from the previously known scorpion venom ICK peptides. The removal of two C-terminal residues of λ-MeuKTx-1 led to the loss of the inhibitory activity on the channel, whereas the C-terminal amidation resulted in the emergence of activity on four mammalian K⁺ channels accompanied by the loss of activity on the Shaker channel. A combination of structural and pharmacological data allows the recognition of three putative functional sites involved in channel blockade of λ-MeuKTx-1. The presence of a functional dyad in λ-MeuKTx-1 supports functional convergence among scorpion venom peptides with different folds. Furthermore, similarities in precursor organization, exon–intron structure, 3D-fold and function suggest that scorpion venom ICK-type K⁺ channel inhibitors and Ca²⁺ release channel activators share a common ancestor and their divergence occurs after speciation between buthidae and non-buthids. The structural and functional characterizations of the first scorpion venom ICK toxin with K⁺ channel-blocking activity sheds light on functionally divergent and convergent evolution of this conserved scaffold of ancient origin.     

Uromyces umiamensis, a new rust species on Momordica cochinchinensis in India

Uromyces umiamensis is described as a new rust fungus (Basidiomycota, Uredinales) of Momordica cochinchinensis (Cucurbitaceae) from Meghalaya, in northeastern India. The species differs from the known Uromyces species on members of Cucurbitaceae in terms of the character of the teliospores and aeciospores and its apparently demicyclic life cycle. Besides U. yakushimensis and the present species, all Uromyces species known from cucurbits occur in the New World. A key to known Uromyces species on Cucurbitaceae is provided.     

Isolation of a trypsin inhibitor with deletion of N-terminal pentapeptide from the seeds of Momordica cochinchinensis, the Chinese drug mubiezhi.

A trypsin inhibitor, MCCTI-1, with a molecular weight of 3479 Da as determined by mass spectrometry, was isolated from Momordica cochinchinensis seeds with a procedure involving extraction with 5% acetic acid, ammonium sulfate precipitation, ion exchange chromatography on CM-Sepharose and reverse-phase high performance liquid chromatography. The sequence of its first 13 N-terminal amino acid residues was ILKKCRRDSDCPG which was about 85% identical with the sequence of trypsin inhibitor MCTI-1 from Momordica charantia Linn. When compared with the sequences of most other squash family trypsin inhibitors, the sequence of MCCTI-1 was characterized by the deletion of a pentapeptide from the N-terminus. Trypsin inhibitors also existed in seeds of some hitherto uninvestigated Cucurbitaceae species.     

Grafting of thrombopoietin-mimetic peptides into cystine knot miniproteins yields high-affinity thrombopoietin antagonists and agonists

Thrombopoietin is the primary regulator of platelet production. We exploited two naturally occurring miniproteins of the inhibitor cystine knot family as stable and rigid scaffolds for the incorporation of peptide sequences that have been shown to act as high-affinity thrombopoietin antagonists. Several miniproteins that antagonistically block thrombopoietin-mediated receptor activation were identified using a microscale reporter assay. Covalent miniprotein dimerization yielded potent bivalent c-Mpl receptor agonists with EC(50) values in the low nanomolar or picomolar range. One selected miniprotein-derived thrombopoietin agonist was almost as active as natural thrombopoietin with regard to stimulation of megakaryocyte colony formation from human bone marrow mononuclear cells, and elicited doubling of platelet counts in mice. Our data suggest that dimeric cystine knot miniproteins have considerable potential for the future development of small and stable receptor agonists. This approach may provide a promising strategy for pharmaceutical interference with other receptors activated by ligand-induced dimerization.     

Antioxidative effect of a chymotrypsin inhibitor from Momordica cochinchinensis (Cucurbitaceae) seeds in a primary rat hepatocyte culture.

The antioxidative activity of a chymotrypsin-specific potato type I inhibitor from Momordica cochinchinensis (MCoCI) (Cucurbitaceae) has been investigated using the primary rat hepatocyte system. tert-Butyl hydroperoxide (t-BHP) was used to induce oxidative stress. Pretreatment of hepatocytes with MCoCI for 24 h significantly reversed t-BHP-induced cell damage, and the associated glutathione depletion and lipid peroxidation. The activities of glutathione-S-transferase and superoxide dismutase were also increased. These results suggested that MCoCI possessed antioxidative activity which may account for some of the pharmacological effects of Momordica cochinchinensis seeds, the traditional Chinese medicine known as Mubiezhi, from which MCoCI was isolated.     

Squash trypsin inhibitors from Momordica cochinchinensis exhibit an atypical macrocyclic structure

Three trypsin inhibitors (TIs), from the seeds of the squash Momordica cochinchinensis (MCo), have been isolated and purified using gel filtration, ion exchange chromatography, and reverse-phase HPLC. Their sequences could be determined only after proteolytic cleavages. In the case of MCoTI-I and -II, it was shown that their polypeptide backbones are cyclic, a structure that has never been described in squash TIs. They contain 34 amino acid residues with 3 disulfide bridges and measured molecular masses of 3453.0 and 3480.7, respectively. They are the largest known macrocyclic peptides containing disulfide bridges. Their sequences show strong homology to other squash TIs, suggesting a similar three-dimensional structure and an analogous mechanism of action. A model of MCoTI-II was constructed by analogy to the crystal structure of the complex between bovine trypsin and CMTI-I, indicating that the linker connecting the two termini is flexible and does not impose significant geometrical constraints. This flexibility allows an Asp-Gly peptide bond rearrangement to occur in this region, giving rise to two isoforms of MCoTI-II. Although the importance of cyclization is not clear, it might confer increased stability and resistance to proteolysis. A minor species, MCoTI-III, was also characterized as containing 30 amino acid residues with a molecular mass of 3379.6. This component possesses a linear backbone with a blocked N-terminus. MCoTIs represent interesting candidates for drug design, either by changing their specificity of inhibition or by using their structure as natural scaffolds bearing new binding activities.     

Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif

Several macrocyclic peptides ( approximately 30 amino acids), with diverse biological activities, have been isolated from the Rubiaceae and Violaceae plant families over recent years. We have significantly expanded the range of known macrocyclic peptides with the discovery of 16 novel peptides from extracts of Viola hederaceae, Viola odorata and Oldenlandia affinis. The Viola plants had not previously been examined for these peptides and thus represent novel species in which these unusual macrocyclic peptides are produced. Further, we have determined the three-dimensional structure of one of these novel peptides, cycloviolacin O1, using (1)H NMR spectroscopy. The structure consists of a distorted triple-stranded beta-sheet and a cystine-knot arrangement of the disulfide bonds. This structure is similar to kalata B1 and circulin A, the only two macrocyclic peptides for which a structure was available, suggesting that despite the sequence variation throughout the peptides they form a family in which the overall fold is conserved. We refer to these peptides as the cyclotide family and their embedded topology as the cyclic cystine knot (CCK) motif. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals they can be separated into two subfamilies, one of which tends to contain a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-Pro peptide bond and may conceptually be regarded as a molecular Moebius strip. Here we define the structural features of the two apparent subfamilies of the CCK peptides which may be significant for the likely defense related role of these peptides within plants.     

Isolation and characterization of an abortifacient protein, momorcochin, from root tubers of Momordica cochinchinensis (family cucurbitaceae)

A glycoprotein with a molecular weight of 32,000 as estimated by SDS-polyacrylamide gel electrophoresis, and characterized by an abundance of Asp and Glu residues and an absence of Cys residues in its amino acid analysis, was isolated from fresh root tubers of Momordica cochinchinensis using a procedure that involved acetone precipitation, ammonium sulfate precipitation, ion exchange chromatography on DEAE Sepharose CL-6B and gel filtration on Sephadex G-75. The protein was capable of inducing mid-term abortion in mice. The characteristics of this protein were compared and contrasted with those of the abortifacient proteins isolated from other plants of the Cucurbitaceae family.     

A new family of antimicrobial peptides from skin secretions of Rana pleuraden

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Guizhou region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the Yunnan frog, Rana pleuraden. Members of the new peptide family named pleurain-As are composed of 26 amino acids with a unique N-terminal sequence (SIIT) and a disulfide-bridged heptapeptide sequence (CRLYNTC). By BLAST search, pleurain-As had no significant similarity to any known peptides. Native and synthetic peptides showed antimicrobial activities against tested microorganisms including Gram-negative and Gram-positive bacteria and fungi. Twenty different cDNAs encoding pleurain-As were cloned from the skin cDNA library of R. pleuraden. The precursors of pleurain-As are composed of 69 amino acid residues including predicted signal peptides, acidic propieces, and cationic mature antimicrobial peptides. The preproregion of pleurain-A precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature pleurain-As are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. Furthermore, pleurain-As could exert antimicrobial capability against Helicobacter pylori. This is the first report of naturally occurring peptides with anti-H. pylori activity from Rana amphibians.     

A fusion protein system for the recombinant production of short disulfide bond rich cystine knot peptides using barnase as a purification handle

The inhibitor cystine knot (ICK) structural motif has been found in several small proteins and peptides from plants, insects, marine molluscs, and also in human. It is defined by a triple beta-sheet that is held together by three intramolecular disulfide bonds built by six conserved cysteine residues that generate a highly rigid and stable fold. We describe a procedure for the production of ICK peptides with correct disulfide bond connectivities via expression in Escherichia coli as fusion proteins with an enzymatically inactive variant of the Bacillus amyloliquefaciens RNAse barnase. Barnase directs the fused peptide to the culture medium and the fusion protein can be isolated by combined cation exchange/reverse-phase chromatography. The ICK peptides are released from the barnase expression and purification handle either by cyanogen bromide or by protease cleavage to give pure and correctly folded cystine knot peptides.     

FMRFamide-related peptides (FaRPs): A new family of peptides from amphibian defensive skin secretions

Amphibian defensive skin secretions are known to contain a plethora of biologically-active peptides that are often structural and functional analogues of vertebrate neuropeptides. Here we report the structures of two invertebrate neuropeptide analogues, IPPQFMRF amide (IF-8 amide) and EGDEDEFLRF amide (EF-10 amide), from the defensive skin secretions of two different species of African hyperoliid frogs, Kassina maculata and Phylictimantis verrucosus, respectively. These represent the first canonical FMRF amide-related peptides (FaRPs) from a vertebrate source. The cDNA encoding IF-8 amide was cloned from a skin secretion library and found to contain a single copy of the peptide located at the C-terminus of a 58 amino acid residue open-reading frame. These data extend the potential targets of the defensive arsenal of amphibian tegumental secretions to parasitic/predatory invertebrates and the novel peptides described may represent the first vertebrate peptidic endectocides.     

Oxidative folding of the cystine knot motif in cyclotide proteins

The cyclotides are a large family of plant proteins that have a cyclic backbone and a knotted arrangement of three conserved disulfide bonds. Despite the apparent complexity of their cystine knot motif it is possible to efficiently fold these proteins, as exemplified by oxidative folding studies on the prototypic cyclotide, kalata B1. This mini-review reports on the current understanding of the folding process in cyclotides. The synthesis and folding of these molecules paves the way for their application as stable molecular templates.     

Tying the knot: the cystine signature and molecular-recognition processes of the vascular endothelial growth factor family of angiogenic cytokines

The cystine-knot motif, made up of three intertwined disulfide bridges, is a unique feature of several toxins, cyclotides and growth factors, and occurs in a variety of species, including fungi, insects, molluscs and mammals. Growth factor molecules containing the cystine-knot motif serve as ligands for a diverse range of receptors and play an important role in extracellular signalling. This superfamily of polypeptides comprises several homodimeric and heterodimeric molecules that are central characters in both health and disease. Amongst these molecules are a group of proteins that belong to the vascular endothelial growth factor (VEGF) subfamily. The members of this family are known angiogenic factors that regulate processes leading to blood vessel formation in physiological and pathological conditions. The focus of the present review is on the structural characteristics of proteins that belong to the VEGF family and on signal-transduction pathways that become initiated via the VEGF receptors.     

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 (Le-Nguyen, 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.