An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.     

Conserved structural and sequence elements implicated in the processing of gene-encoded circular proteins

The cyclotides are the largest family of naturally occurring circular proteins. The mechanism by which the termini of these gene-encoded proteins are linked seamlessly with a peptide bond to form a circular backbone is unknown. Here we report cyclotide-encoding cDNA sequences from the plant Viola odorata and compare them with those from an evolutionarily distinct species, Oldenlandia affinis. Individual members of this multigene family encode one to three mature cyclotide domains. These domains are preceded by N-terminal repeat regions (NTRs) that are conserved within a plant species but not between species. We have structurally characterized peptides corresponding to these NTRs and show that, despite them having no sequence homology, they form a structurally conserved alpha-helical motif. This structural conservation suggests a vital role for the NTR in the in vivo folding, processing, or detoxification of cyclotide domains from the precursor protein.     

The role of conserved Glu residue on cyclotide stability and activity: a structural and functional study of kalata B12, a naturally occurring Glu to Asp mutant

Cyclotides are a family of plant defense proteins with a unique cyclic backbone and cystine knot. Their remarkable stability under harsh thermal, enzymatic, and chemical conditions, combined with their range of bioactivities, including anti-HIV activity, underpins their potential as protein drug scaffolds. The vast majority of cyclotides possess a conserved glutamate residue in loop 1 of the sequence that is involved in a structurally important network of hydrogen bonds to an adjacent loop (loop 3). A single native cyclotide sequence, kalata B12, has been discovered that has an aspartic acid in this otherwise conserved position. Previous studies have determined that methylation of the glutamate or substitution with alanine abolishes the membrane disrupting activity that is characteristic of the family. To further understand the role of this conserved structural feature, we studied the folding, structure, stability, and activity of the natural aspartic acid variant kalata B12 and compared it to the prototypical cyclotide kalata B1, along with its glutamate to alanine or aspartate mutants. We show that the overall fold of kalata B12 is similar to the structure of other cyclotides, confirming that the cyclotide framework is robust and tolerant to substitution, although the structure appears to be more flexible than other cyclotides. Modification of the glutamate in kalata B1 or replacing the aspartate in kalata B12 with a glutamate reduces the efficiency of oxidative folding relative to the native peptides. The bioactivity of all modified glutamate cyclotides is abolished, suggesting an important functional role of this conserved residue. Overall, this study shows that the presence of a glutamic acid in loop 1 of the cyclotides improves stability and is essential for the membrane disrupting activity of cyclotides.     

Conformation and mode of membrane interaction in cyclotides. Spatial structure of kalata B1 bound to a dodecylphosphocholine micelle

Cyclotides are a family of bioactive plant peptides that are characterized by a circular protein backbone and three conserved tightly packed disulfide bonds. The antimicrobial and hemolytic properties of cyclotides, along with the relative hydrophobicity of the peptides, point to the biological membrane as a target for cyclotides. To assess the membrane-induced conformation and orientation of cyclotides, the interaction of the M?bius cyclotide, kalata B1, from the African perennial plant Oldenlandia affinis, with dodecylphosphocholine micelles was studied using NMR spectroscopy. Under conditions where the cyclotide formed a well-defined complex with micelles, the spatial structure of kalata B1 was calculated from NOE and J couplings data, and the model for the peptide-micelle complex was built using 5- and 16-doxylstearate relaxation probes. The binding of divalent cations to the peptide-micelle complex was quantified by Mn2+ titration. The results show that the peptide binds to the micelle surface, with relatively high affinity, via two hydrophobic loops (loop 5, Trp19-Val21; and loop6, Leu27-Val29). The charged residues (Glu3 and Arg24), along with the cation-binding site (near Glu3) are segregated on the other side of the molecule and in contact with polar head groups of detergent. The spatial structure of kalata B1 is only slightly changed during incorporation into micelles and represents a distorted triple-stranded beta-sheet cross-linked by a cystine knot. Detailed structural analysis and comparison with other knottins revealed structural conservation of the two-disulfide motif in cyclic and acyclic peptides. The results thus obtained provide the first model for interaction of cyclotides with membranes and permit consideration of the cyclotides as membrane-active cationic antimicrobial peptides.     

Scale‐up of Oldenlandia affinis suspension cultures in photobioreactors for cyclotide production

Cyclotides are a family of backbone‐cyclized cystine‐knot‐containing macrocyclic peptides from plants that possess extremely interesting biological activities. Suspension cultures of Oldenlandia affinis, a model plant containing cyclotides, were scaled‐up from shake flask to photobioreactor operation in order to produce these plant peptides under controlled conditions. Cell growth was highly dependent on inoculation culture; cell density as well as culture age had an effect on the growth rates and thus affected the kalata B1 productivity of the bioprocess. In a 25 l scale bioreactor the maximum doubling time was about 1.12 days compared to 2.24 days in shake flasks. The accumulation of kalata B1 of 0.09 mg g^?1 DW and 0.07–0.10 mg g^?1 DW respectively, however, was on a similar level during the corresponding stationary growth phases in both bioreactor and flask processes. An adjustment of cell culture growth via culture preparation and inoculum density to high cyclotide accumulation results in an estimated output during the most productive retardation phase of about 21 mg kalata B1 per day in the 25 l system. This makes the biotechnological cyclotide synthesis under GMP conditions a competitive production tool compared to field cultivation, chemical, and recombinant synthesis in drug discovery for structure analysis and bioactivity assays.     

Subcellular targeting and biosynthesis of cyclotides in plant cells

? Premise of the study: The cyclotide kalata B1 is found in the leaves of Oldenlandia affinis and is a potent insecticidal and nematocidal molecule. This peptide is cleaved from a precursor protein, Oak1, and ligation of the N- and C-termini occurs to form a continuous peptide backbone. The subcellular location of the excision and cyclization reactions is unknown, and there is debate as to which enzyme catalyzes the event. To determine where in the plant cell Oak1 is processed, we prepared constructs encoding GFP (green fluorescent protein) linked to the cyclotide precursor Oak1. ? Methods: The GFP constructs were transiently expressed in the leaves of Nicotiana benthamiana, and GFP fluorescence was observed in living cells using confocal microscopy. A Fei Mao (FM) styryl dye was infiltrated into whole leaves that were still growing and expressing GFP constructs, enabling the plasma membrane and the tonoplast to be highlighted for visualization of the vacuole in living cells. ? Key results: The full length Oak1 precursor directed GFP to the vacuole, suggesting that excision and cyclization of the cyclotide domain occurs in the vacuole where the cyclotides are then stored. The N-terminal propeptide and N-terminal repeat of Oak1 were both sufficient to target GFP to the vacuole, although the C-terminal propeptide, which is essential for cyclization, was not a targeting signal. ? Conclusions: The vacuolar location of cyclotides supports our hypothesis that the vacuolar processing enzyme, asparaginyl endoproteinase, has a pivotal role in excision and cyclization from cyclotide precursors.     

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.     

Isolation, solution structure, and insecticidal activity of kalata B2, a circular protein with a twist: do Möbius strips exist in nature?

A large number of macrocyclic miniproteins with diverse biological activities have been isolated from the Rubiaceae, Violaceae, and Cucurbitaceae plant families in recent years. Here we report the three-dimensional structure determined using (1)H NMR spectroscopy and demonstrate potent insecticidal activity for one of these peptides, kalata B2. This peptide is one of the major components of an extract from the leaves of the plant Oldenlandia affinis. The structure consists of a distorted triple-stranded beta-sheet and a cystine knot arrangement of the disulfide bonds and is similar to those described for other members of the cyclotide family. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals that they can be separated into two subfamilies, one of which contains 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-peptidyl-proline bond and may conceptually be regarded as a molecular Mobius strip. Kalata B2 is the second putative member of the Mobius cyclotide family to be structurally characterized and has a cis-peptidyl-proline bond, thus validating the suggested name for this subfamily of cyclotides. The observation that kalata B2 inhibits the growth and development of Helicoverpa armigera larvae suggests a role for the cyclotides in plant defense. A comparison of the sequences and structures of kalata B1 and B2 provides insight into the biological activity of these peptides.     

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 B2, 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.     

Insights into processing and cyclization events associated with biosynthesis of the cyclic Peptide kalata b1

Plant cyclotides are the largest family of gene-encoded cyclic proteins. They act as host defense molecules to protect plants and are promising candidates as insecticidal and nematocidal agents in agriculture. For this promise to be realized a greater understanding of the post-translational processing of these proteins is needed. Cyclotides are cleaved from precursor proteins with subsequent ligation of the N and C termini to form a continuous peptide backbone. This cyclization step is inefficient in transgenic plants and our work aims to shed light on the specificity requirements at the excision sites for cyclic peptide production. Using the prototypic cyclotide kalata B1 (kB1) expressed from the Oak1 gene, MALDI-TOF mass spectrometry was used to examine the cyclization efficiency when mutants of the Oak1 gene were expressed in transgenic Nicotiana benthamiana. Cleavage at the N terminus of the cyclotide domain occurs rapidly with no strict specificity requirements for amino acids at the cleavage site. In contrast, the C-terminal region of the cyclotide domain in the P2, P1, P1', and P2' positions is highly conserved and only specific amino acids can occupy these positions. The cyclization reaction requires an Asn at position P1 followed by a small amino acid (Ala, Gly, Ser) at the P1' position. The P2' position must be filled by Leu or Ile; in their absence an unusual post-translational modification occurs. Substitution of the P2' Leu with Ala leads to hydroxylation of the neighboring proline. Through mutational analysis this novel proline hydroxylation motif was determined to be Gly-Ala-Pro-Ser.     

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.     

Capped acyclic permutants of the circular protein kalata B1

The cyclotides are a family of head-to-tail cyclized peptides that display exceptionally high stability and a range of biological activities. Acyclic permutants that contain a break in the circular backbone have been reported to be devoid of the haemolytic activity of the prototypic cyclotide kalata B1, but the potential role of the charges at the introduced termini in this loss of membraneolytic activity has not been fully determined. In this study, acyclic permutants of kalata B1 with capped N- and C-termini were synthesized and found to adopt a native fold. These variants were observed to cause no measurable lysis of erythrocytes, strengthening the connection between backbone cyclization and haemolytic activity.     

Linearization of a naturally occurring circular protein maintains structure but eliminates hemolytic activity

Cyclotides are a recently discovered family of disulfide rich proteins from plants that contain a circular protein backbone. They are exceptionally stable, as exemplified by their use in native medicine of the prototypic cyclotide kalata B1. The peptide retains uterotonic activity after the plant from which it is derived is boiled to make a medicinal tea. The circular backbone is thought to be in part responsible for the stability of the cyclotides, and to investigate its role in determining structure and biological activity, an acyclic derivative, des-(24-28)-kalata B1, was chemically synthesized and purified. This derivative has five residues removed from the 29-amino acid circular backbone of kalata B1 in a loop region corresponding to a processing site in the biosynthetic precursor protein. Two-dimensional NMR spectra of the peptide were recorded, assigned, and used to identify a series of distance, angle, and hydrogen bonding restraints. These were in turn used to determine a representative family of solution structures. Of particular interest was a determination of the structural similarities and differences between des-(24-28)-kalata B1 and native kalata B1. Although the overall three-dimensional fold remains very similar to that of the native circular protein, removal of residues 24-28 of kalata B1 causes disruption of some structural features that are important to the overall stability. Furthermore, loss of hemolytic activity is associated with backbone truncation and linearization.     

Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides

Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.     

A novel plant protein-disulfide isomerase involved in the oxidative folding of cystine knot defense proteins

We have isolated a protein-disulfide isomerase (PDI) from Oldenlandia affinis (OaPDI), a coffee family (Rubiaceae) plant that accumulates knotted circular proteins called cyclotides. The novel plant PDI appears to be involved in the biosynthesis of cyclotides, since it co-expresses and interacts with the cyclotide precursor protein Oak1. OaPDI exhibits similar isomerase activity but greater chaperone activity than human PDI. Since domain c of OaPDI is predicted to have a neutral pI, we conclude that this domain does not have to be acidic in nature for PDI to be a functional chaperone. Its redox potential of -157 +/- 4 mV supports a role as a functional oxidoreductase in the plant. The mechanism of enzyme-assisted folding of plant cyclotides was investigated by comparing the folding of kalata B1 derivatives in the presence and absence of OaPDI. OaPDI dramatically enhanced the correct oxidative folding of kalata B1 at physiological pH. A detailed investigation of folding intermediates suggested that disulfide isomerization is an important role of the new plant PDI and is an essential step in the production of insecticidal cyclotides. The nucleotide sequence(s) reported in this paper have been submitted to the GenBank/EBI Data Bank with accession number(s) 911777.     

An Efficient Approach for the Total Synthesis of Cyclotides by Microwave Assisted Fmoc-SPPS

Cyclotides are mini-proteins of approximately 30 amino acid residues that have a unique structure consisting of a head-to-tail cyclized backbone and a knotted arrangement of three disulfide bonds. This unique cyclotide structure provides exceptional stability to chemical, enzymatic and thermal treatments and has been implicated as an ideal drug scaffold for the development into agricultural and biotechnological agents. In the current work, we present the first method for microwave assisted Fmoc-SPPS of cyclotides. This protocol adopts a strategy that combines optimized microwave assisted chemical reactions for Fmoc-SPPS of the peptide backbone, the cleavage of the protected peptide and the introduction of a thioester at the C-terminal carboxylic acid to obtain the head-to-tail cyclized cyclotide backbone by native chemical ligation. To exemplify the utility of this protocol in the synthesis of a wide array of different cyclotide sequences we synthesized representative members from the three cyclotide subfamilies—the Möbius kalata B1, the bracelet cycloviolacin O2 and the trypsin inhibitory MCoTI-II. In addition, a “one pot” reaction promoting both cyclization and oxidative folding of crude peptide thioester was adapted for kalata B1 and MCoTI-II.     

Structural characterization of an unusually stable cyclic peptide, kalata B2 from Oldenlandia affinis

Kalata peptides are isolated from an African medicinal plant, Oldenlandia affinis, an aqueous decoction of which can be ingested to accelerate uterine contraction during childbirth. The closely packed disulfide core of kalata peptides confers unusual stability against thermal, chemical, and enzymatic degradation. The molecular arrangement may hamper NMR-assisted disulfide connectivity assignment. We have combined NMR with high-resolution mass spectrometry (MS) and MS/MS of native and chemically derivatized kalata B2 to determine its amino acid sequence and disulfide connectivity. Infrared multiphoton dissociation establishes the disulfide bond linkages in kalata B2 as I-IV, II-V and III-VI.     

Twists,knots, and rings in proteins. Structural definition of the cyclotide framework

In recent years an increasing number of miniproteins containing an amide-cyclized backbone have been discovered. The cyclotide family is the largest group of such proteins and is characterized by a circular protein backbone and six conserved cysteine residues linked by disulfide bonds in a tight core of the molecule. These form a cystine knot in which an embedded ring formed by two of the disulfide bonds and the connecting backbone segment is threaded by a third disulfide bond. In the current study we have undertaken high resolution structural analysis of two prototypic cyclotides, kalata B1 and cycloviolacin O1, to define the role of the conserved residues in the sequence. We provide the first comprehensive analysis of the topological features in this unique family of proteins, namely rings (a circular backbone), twists (a cis-peptide bond in the M?bius cyclotides) and knots (a knotted arrangement of the disulfide bonds).     

Cyclotides: natural, circular plant peptides that possess significant activity against gastrointestinal nematode parasites of sheep

The cyclotides are a novel family of backbone-cyclized cystine-knot containing peptides from plants that have been shown to possess insecticidal activity against Helicoverpa larvae, an important pest of corn and cotton. In the current study, we investigated the in vitro effects of the cyclotides on the viability of egg, larval, and adult life stages of two species of economically important gastrointestinal nematode parasites of livestock, Hemonchus contortus and Trichostrongylus colubriformis. The cyclotides showed significant activity in inhibiting development of nematode larvae and motility of adult worms. Activities were comparable to some currently used anthelmintic compounds in these in vitro assay systems. A series of alanine mutants of the prototypic cyclotide kalata B1 were assayed against larvae to determine regions of the peptide responsible for activity. It was observed that anthelmintic activity was dramatically reduced as a consequence of the mutation of a large number of residues that are found clustered on one surface. Activities toward larvae were equivalent in the naturally occurring L-isomer of kalata B1 and a synthetic all-D-isomer, indicating that there is no chiral requirement for anthelmintic activity. The clustering of important residues and the lack of chiral selectivity further support the proposed mode of action of the cyclotides, which involves a membrane-based interaction rather than an interaction at a specific receptor. The cyclotide-induced leakage of a fluorescent dye from vesicles used as a model membrane mimetic further confirms the membrane lytic ability of cyclotides. The relative potency of kalata B1 and kalata B2 in causing membrane leakage is consistent with the order of their anthelmintic activity. These results demonstrate that the cyclotides show potential for use in the control of gastrointestinal nematode parasites.