Aaron G. Poth

Discovery of Cyclotides in the Fabaceae Plant Family Provides New Insights into the Cyclization, Evolution, and Distribution of Circular Proteins

Cyclotides are plant proteins whose defining structural features are a head-to-tail cyclized backbone and three interlocking disulfide bonds, which in combination are known as a cyclic cystine knot. This unique structural motif confers cyclotides with exceptional resistance to proteolysis. Their endogenous function is thought to be as plant defense agents, associated with their insecticidal and larval growth-inhibitory properties. However, in addition, an array of pharmaceutically relevant biological activities has been ascribed to cyclotides, including anti-HIV, anthelmintic, uterotonic, and antimicrobial effects. So far, >150 cyclotides have been elucidated from members of the Rubiaceae, Violaceae, and Cucurbitaceae plant families, but their wider distribution among other plant families remains unclear. Clitoria ternatea (Butterfly pea) is a member of plant family Fabaceae and through its usage in traditional medicine to aid childbirth bears similarity to Oldenlandia affinis, from which many cyclotides have been isolated. Using a combination of nanospray and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) analyses, we examined seed extracts of C. ternatea and discovered cyclotides in the Fabaceae, the third-largest family of flowering plants. We characterized 12 novel cyclotides, thus expanding knowledge of cyclotide distribution and evolution within the plant kingdom. The discovery of cyclotides containing novel sequence motifs near the in planta cyclization site has provided new insights into cyclotide biosynthesis. In particular, MS analyses of the novel cyclotides from C. ternatea suggest that Asn to Asp variants at the cyclization site are more common than previously recognized. Moreover, this study provides impetus for the examination of other economically and agriculturally significant species within Fabaceae, now the largest plant family from which cyclotides have been described.

Discovery of an unusual biosynthetic origin for circular proteins in legumes

Cyclotides are plant-derived proteins that have a unique cyclic cystine knot topology and are remarkably stable. Their natural function is host defense, but they have a diverse range of pharmaceutically important activities, including uterotonic activity and anti-HIV activity, and have also attracted recent interest as templates in drug design. Here we report an unusual biosynthetic origin of a precursor protein of a cyclotide from the butterfly pea, Clitoria ternatea, a representative member of the Fabaceae plant family. Unlike all previously reported cyclotides, the domain corresponding to the mature cyclotide from this Fabaceae plant is embedded within an albumin precursor protein. We confirmed the expression and correct processing of the cyclotide encoded by the Cter M precursor gene transcript following extraction from C. ternatea leaf and sequencing by tandem mass spectrometry. The sequence was verified by direct chemical synthesis and the peptide was found to adopt a classic knotted cyclotide fold as determined by NMR spectroscopy. Seven additional cyclotide sequences were also identified from C. ternatea leaf and flower, five of which were unique. Cter M displayed insecticidal activity against the cotton budworm Helicoverpa armigera and bound to phospholipid membranes, suggesting its activity is modulated by membrane disruption. The Fabaceae is the third largest family of flowering plants and many Fabaceous plants are of huge significance for human nutrition. Knowledge of Fabaceae cyclotide gene transcripts should enable the production of modified cyclotides in crop plants for a variety of agricultural or pharmaceutical applications, including plant-produced designer peptide drugs.

Cyclotides as grafting frameworks for protein engineering and drug design applications

Cyclotides are a family of naturally occurring backbone-cyclized macrocyclic mini-proteins from plants that have a knotted trio of intramolecular disulfide bonds. Their structural features imbue cyclotides with extraordinary stability against degradation at elevated temperatures or in the presence of proteolytic enzymes. The plasticity of their intracysteine loop sequences is exemplified by the more than 250 natural cyclotides sequenced to date, and this tolerance to sequence variation, along with their diverse bioactivities, underpins the suitability of the cyclic cystine knot motif as a valuable drug design scaffold and research tool for protein engineering studies. Here, we review the recent literature on applications of cyclotides for the stabilization of peptide epitopes and related protein engineering studies. Possible future directions in this field are also described.

Cyclotides associate with leaf vasculature and are the products of a novel precursor in petunia (Solanaceae).

Background: Cyclotides are defense-related cyclic plant peptides. Results: Petunia cyclotides are encoded by novel cyclotide genes and occur in a discrete pattern in leaf architecture. Conclusion: Novel cyclotides exist in the Solanaceae and are abundant in vascular tissues. Significance: Cyclotide localization is consistent with an anti-herbivory role. Novel Solanaceae genes provide opportunities for expressing designer cyclic peptides in major crop species. Cyclotides are a large family of plant peptides that are structurally defined by their cyclic backbone and a trifecta of disulfide bonds, collectively known as the cyclic cystine knot (CCK) motif. Structurally similar cyclotides have been isolated from plants within the Rubiaceae, Violaceae, and Fabaceae families and share the CCK motif with trypsin-inhibitory knottins from a plant in the Cucurbitaceae family. Cyclotides have previously been reported to be encoded by dedicated genes or as a domain within a knottin-encoding PA1-albumin-like gene. Here we report the discovery of cyclotides and related non-cyclic peptides we called “acyclotides” from petunia of the agronomically important Solanaceae plant family. Transcripts for petunia cyclotides and acyclotides encode the shortest known cyclotide precursors. Despite having a different precursor structure, their sequences suggest that petunia cyclotides mature via the same biosynthetic route as other cyclotides. We assessed the spatial distribution of cyclotides within a petunia leaf section by MALDI imaging and observed that the major cyclotide component Phyb A was non-uniformly distributed. Dissected leaf midvein extracts contained significantly higher concentrations of this cyclotide compared with the lamina and outer margins of leaves. This is the third distinct type of cyclotide precursor, and Solanaceae is the fourth phylogenetically disparate plant family to produce these structurally conserved cyclopeptides, suggesting either convergent evolution upon the CCK structure or movement of cyclotide-encoding sequences within the plant kingdom.

Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase

Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of cyclotide biosynthesis and confers a measure of stability on the resultant cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the cyclotide-producing plant Oldenlandia affinis. We recombinantly express OaAEP1b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, OaAEP1b cannot cleave at the N-terminal site of O. affinis cyclotide precursors, implicating additional proteases in cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to cyclotides. We propose that recombinant OaAEP1b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired.

Cycloquest: Identification of cyclopeptides via database search of their mass spectra against genome databases

Hundreds of ribosomally synthesized cyclopeptides have been isolated from all domains of life, the vast majority having been reported in the last 15 years. Studies of cyclic peptides have highlighted their exceptional potential both as stable drug scaffolds and as biomedicines in their own right. Despite this, computational techniques for cyclopeptide identification are still in their infancy, with many such peptides remaining uncharacterized. Tandem mass spectrometry has occupied a niche role in cyclopeptide identification, taking over from traditional techniques such as nuclear magnetic resonance spectroscopy (NMR). MS/MS studies require only picogram quantities of peptide (compared to milligrams for NMR studies) and are applicable to complex samples, abolishing the requirement for time-consuming chromatographic purification. While database search tools such as Sequest and Mascot have become standard tools for the MS/MS identification of linear peptides, they are not applicable to cyclopeptides, due to the parent mass shift resulting from cyclization and different fragmentation patterns of cyclic peptides. In this paper, we describe the development of a novel database search methodology to aid in the identification of cyclopeptides by mass spectrometry and evaluate its utility in identifying two peptide rings from Helianthus annuus, a bacterial cannibalism factor from Bacillus subtilis, and a θ-defensin from Rhesus macaque.

The Evolution of Momordica Cyclic Peptides

Cyclic proteins have evolved for millions of years across all kingdoms of life to confer structural stability over their acyclic counterparts while maintaining intrinsic functional properties. Here, we show that cyclic miniproteins (or peptides) from Momordica (Cucurbitaceae) seeds evolved in species that diverged from an African ancestor around 19 Ma. The ability to achieve head-to-tail cyclization of Momordica cyclic peptides appears to have been acquired through a series of mutations in their acyclic precursor coding sequences following recent and independent gene expansion event(s). Evolutionary analysis of Momordica cyclic peptides reveals sites that are under selection, highlighting residues that are presumably constrained for maintaining their function as potent trypsin inhibitors. Molecular dynamics of Momordica cyclic peptides in complex with trypsin reveals site-specific residues involved in target binding. In a broader context, this study provides a basis for selecting Momordica species to further investigate the biosynthesis of the cyclic peptides and for constructing libraries that may be screened against evolutionarily related serine proteases implicated in human diseases.

The role of disulfide bonds in structure and activity of chlorotoxin

BACKGROUND Chlorotoxin is a small scorpion peptide that inhibits glioma cell migration. We investigated the importance of a major component of chlorotoxins chemical structure - four disulfide bonds - to its tertiary structure and biological function. RESULTS Five disulfide bond analogs of chlorotoxin were synthesized, with l-α-aminobutyric acid residues replacing each or all of the disulfide bonds. Chemical oxidation and circular dichroism experiments revealed that Cys III-VII and Cys V-VIII were essential for native structure formation. Cys I-IV and Cys II-VI were important for stability of enzymatic proteolysis but not for the inhibition of human umbilical vein endothelial cell migration. CONCLUSION The disulfide bonds of chlorotoxin are important for its structure and stability and have a minor role in its activity against cell migration.

Glycine-containing flaxseed orbitides.

Five new orbitides, cyclolinopeptides 21-25, were identified in flaxseed oil (Linum usitatissimum) extracts. Their HPLC-ESIMS quasimolecular ion peaks at m/z 1097.7 (21), 1115.6 (22), 1131.6 (23), 1018.6 (24), and 1034.6 (25) [M + H](+) corresponded to the molecular formulae C59H89N10O10, C58H87N10O10S, C58H87N10O11S, C53H80N9O9S, and C53H80N9O10S, respectively. Their structures were elucidated by extensive HPLC-ESIMS/MS analyses, and their presence was confirmed by precursor proteins identified in flax genomic DNA sequence data. The amino acid sequences of these orbitides were confirmed as [1-10-NαC]-GILVPPFFLI, [1-10-NαC]-GMLIPPFFVI, [1-10-NαC]-GOLIPPFFVI, [1-9-NαC]-GMLVFPLFI, and [1-9-NαC]-GOLVFPLFI for cyclolinopeptides 21-25, respectively. Previously reported orbitides, [1-9-NαC]-ILVPPFFLI (1), [1-9-NαC]-MLIPPFFVI (2), [1-9-NαC]-OLIPPFFVI (3), [1-8-NαC]-MLVFPLFI (7), and [1-8-NαC]-OLVFPLFI (8), were also present in flaxseed oil. The precursors of orbitides 21, 22, and 24 also produced orbitides 1, 2, and 7 by alternative cyclization. Cyclolinopeptides 3, 8, 23, and 25 contain MetO (O) and are not directly encoded, but are products of post-translational modification of the Met present in 2, 7, 22, and 24, respectively. Sufficient cyclolinopeptide 23 was isolated for characterization via 1D ((1)H and (13)C) and 2D (NOESY and HMBC) NMR spectroscopy. These compounds have been named as cyclolinopeptides U, V, W, X, and Y for 21, 22, 23, 24, and 25, respectively.

A comparative study of extraction methods reveals preferred solvents for cystine knot peptide isolation from Momordica cochinchinensis seeds

MCoTI-I and MCoTI-II (short for Momordica cochinchinensis Trypsin Inhibitor-I and -II, respectively) are attractive candidates for developing novel intracellular-targeting drugs because both are exceptionally stable and can internalize into cells. These seed-derived cystine knot peptides are examples of how natural product discovery efforts can lead to biomedical applications. However, discovery efforts are sometimes hampered by the limited availability of seed materials, highlighting the need for efficient extraction methods. In this study, we assessed five extraction methods using M. cochinchinensis seeds, a source of well-characterized cystine knot peptides. The most efficient extraction of nine known cystine knot peptides was achieved by a method based on acetonitrile/water/formic acid (25:24:1), followed by methods based on sodium acetate (20 mM, pH 5.0), ammonium bicarbonate (5 mM, pH 8.0), and boiling water. On average, the yields obtained by these four methods were more than 250-fold higher than that obtained using dichloromethane/methanol (1:1) extraction, a previously applied standard method. Extraction using acetonitrile/water/formic acid (25:24:1) yielded the highest number of reconstructed masses within the majority of plant-derived cystine knot peptide mass range but only accounted for around 50% of the total number of masses, indicating that any single method may result in under-sampling. Applying acetonitrile/water/formic acid (25:24:1), boiling water, and ammonium bicarbonate (5 mM, pH 8.0) extractions either successively or discretely significantly increased the sampling number. Overall, acetonitrile/water/formic acid (25:24:1) can facilitate efficient extraction of cystine-knot peptides from M. cochinchinensis seeds but for discovery purposes the use of a combination of extraction methods is recommended where practical.