FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2143324852> ?p ?o ?g. }

• W2143324852 endingPage "10805" @default.
• W2143324852 startingPage "10797" @default.
• W2143324852 abstract "Cyclotides are a family of macrocyclic peptides that combine the unique features of a head-to-tail cyclic backbone and a cystine knot motif, the combination of which imparts them with extraordinary stability. The prototypic cyclotide kalata B1 is toxic against two economically important gastrointestinal nematode parasites of sheep, Haemonchus contortus and Trichostrongylus colubriformis. A lysine scan was conducted to examine the effect of the incorporation of positive charges into the kalata B1 cyclotide framework. Each of the non-cysteine residues in this 29-amino acid peptide was successively substituted with lysine, and the nematocidal and hemolytic activities of the suite of mutants were determined. Substitution of 11 residues within kalata B1 decreased the nematocidal activity dramatically. On the other hand, six other residues that are clustered on the surface of kalata B1 were tolerant to Lys substitution, and indeed the introduction of positively charged residues into this region increased nematocidal activity. This activity was increased further in double and triple lysine mutants, with a maximal increase (relative to the native kalata B1) of 13-fold obtained with a triple lysine mutant (mutated at positions Thr-20, Asn-29, and Gly-1). Hemolytic activity correlated with the nematocidal activity of all lysine mutants. Our data clearly highlight the residues crucial for nematocidal and hemolytic activity in cyclotides, and demonstrate that the nematocidal activity of cyclotides can be increased by incorporation of basic amino acids. Cyclotides are a family of macrocyclic peptides that combine the unique features of a head-to-tail cyclic backbone and a cystine knot motif, the combination of which imparts them with extraordinary stability. The prototypic cyclotide kalata B1 is toxic against two economically important gastrointestinal nematode parasites of sheep, Haemonchus contortus and Trichostrongylus colubriformis. A lysine scan was conducted to examine the effect of the incorporation of positive charges into the kalata B1 cyclotide framework. Each of the non-cysteine residues in this 29-amino acid peptide was successively substituted with lysine, and the nematocidal and hemolytic activities of the suite of mutants were determined. Substitution of 11 residues within kalata B1 decreased the nematocidal activity dramatically. On the other hand, six other residues that are clustered on the surface of kalata B1 were tolerant to Lys substitution, and indeed the introduction of positively charged residues into this region increased nematocidal activity. This activity was increased further in double and triple lysine mutants, with a maximal increase (relative to the native kalata B1) of 13-fold obtained with a triple lysine mutant (mutated at positions Thr-20, Asn-29, and Gly-1). Hemolytic activity correlated with the nematocidal activity of all lysine mutants. Our data clearly highlight the residues crucial for nematocidal and hemolytic activity in cyclotides, and demonstrate that the nematocidal activity of cyclotides can be increased by incorporation of basic amino acids. IntroductionGastrointestinal nematodes cause major losses to livestock industries worldwide. The control of these pests, until now, has been via synthetic anthelmintics, including benzimidazoles (e.g. thiabendazole), nicotinic acetylcholine receptor agonists (e.g. levamisole), and macrocyclic lactones (e.g. ivermectin and moxidectin). However, resistance of sheep and cattle nematodes to these broad-spectrum anthelmintics is a serious issue for livestock production globally (1.Besier B. Trends Parasitol. 2007; 23: 21-24Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 2.Kaplan R.M. Trends Parasitol. 2004; 20: 477-481Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 3.Wolstenholme A.J. Fairweather I. Prichard R. von Samson-Himmelstjerna G. Sangster N.C. Trends Parasitol. 2004; 20: 469-476Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar). Thus, the development of novel agents with potent anthelmintic activity is of great economic and agricultural importance.Cyclotides are circular miniproteins discovered originally in plants of the Rubiaceae, Violaceae, and Cucurbitaceae families (4.Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (631) Google Scholar, 5.Craik D.J. Daly N.L. Mulvenna J. Plan M.R. Trabi M. Curr. Protein Pept. Sci. 2004; 5: 297-315Crossref PubMed Scopus (161) Google Scholar). Kalata B1, the first cyclotide to be structurally characterized, is abundant in the tropical African plant Oldenlandia affinis (6.Craik D.J. Toxicon. 2001; 39: 1809-1813Crossref PubMed Scopus (101) Google Scholar, 7.Saether O. Craik D.J. Campbell I.D. Sletten K. Juul J. Norman D.G. Biochemistry. 1995; 34: 4147-4158Crossref PubMed Scopus (365) Google Scholar) but has also been reported in the European Sweet Violet (Viola odorata) (8.Ireland D.C. Colgrave M.L. Craik D.J. Biochem. J. 2006; 400: 1-12Crossref PubMed Scopus (140) Google Scholar) and several other Viola species (9.Claeson P. Göransson U. Johansson S. Luijendijk T. Bohlin L. J. Nat. Prod. 1998; 61: 77-81Crossref PubMed Scopus (139) Google Scholar, 10.Göransson U. Luijendijk T. Johansson S. Bohlin L. Claeson P. J. Nat. Prod. 1999; 62: 283-286Crossref PubMed Scopus (155) Google Scholar, 11.Hallock Y.F. Sowder 2nd, R.C. Pannell L.K. Hughes C.B. Johnson D.G. Gulakowski R. Cardellina 2nd, J.H. Boyd M.R. J. Org. Chem. 2000; 65: 124-128Crossref PubMed Scopus (104) Google Scholar, 12.Broussalis A.M. Göransson U. Coussio J.D. Ferraro G. Martino V. Claeson P. Phytochemistry. 2001; 58: 47-51Crossref PubMed Scopus (84) Google Scholar, 13.Trabi M. Mylne J.S. Sando L. Craik D.J. Org. Biomol. Chem. 2009; 7: 2378-2388Crossref PubMed Scopus (32) Google Scholar). Members of the cyclotide family possess a cyclic peptide backbone and a cystine knot motif, which is formed by three disulfide bonds at the core of their three-dimensional structure. The sequence and structure of kalata B1 is illustrated in Fig. 1, which highlights the six backbone loops between the six conserved cysteine residues that make up the cystine knot (14.Wang C.K. Hu S.H. Martin J.L. Sjögren T. Hajdu J. Bohlin L. Claeson P. Göransson U. Rosengren K.J. Tang J. Tan N.H. Craik D.J. J. Biol. Chem. 2009; 284: 10672-10683Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The cyclic cystine knot motif is thought to be responsible for the extraordinary enzymatic and chemical stability (15.Colgrave M.L. Craik D.J. Biochemistry. 2004; 43: 5965-5975Crossref PubMed Scopus (447) Google Scholar) of the cyclotides. Cyclotides have a wide range of bioactivities, including anti-HIV (16.Gustafson K.R. S.I.R. Henderson L.E. Parsons I.C. Kashman Y. Cardellina II, J.H. McMahon J.B. Buckheit Jr., R.W. Pannell L.K. Boyd M.R. J. Am. Chem. Soc. 1994; 116: 9337-9338Crossref Scopus (255) Google Scholar, 17.Daly N.L. Gustafson K.R. Craik D.J. FEBS Lett. 2004; 574: 69-72Crossref PubMed Scopus (103) Google Scholar, 18.Ireland D.C. Wang C.K. Wilson J.A. Gustafson K.R. Craik D.J. Biopolymers Peptide Science. 2008; 90: 51-60Crossref PubMed Scopus (117) Google Scholar, 19.Wang C.K. Colgrave M.L. Gustafson K.R. Ireland D.C. Goransson U. Craik D.J. J. Nat. Prod. 2008; 71: 47-52Crossref PubMed Scopus (143) Google Scholar), neurotensin antagonism (20.Witherup K.M. Bogusky M.J. Anderson P.S. Ramjit H. Ransom R.W. Wood T. Sardana M. J. Nat. Prod. 1994; 57: 1619-1625Crossref PubMed Scopus (226) Google Scholar), hemolytic (21.Barry D.G. Daly N.L. Clark R.J. Sando L. Craik D.J. Biochemistry. 2003; 42: 6688-6695Crossref PubMed Scopus (101) Google Scholar), antimicrobial (22.Tam J.P. Lu Y.A. Yang J.L. Chiu K.W. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 8913-8918Crossref PubMed Scopus (401) Google Scholar), anti-fouling (23.Göransson U. Sjögren M. Svangård E. Claeson P. Bohlin L. J. Nat. Prod. 2004; 67: 1287-1290Crossref PubMed Scopus (122) Google Scholar), and pesticidal activities (24.Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 10614-10619Crossref PubMed Scopus (398) Google Scholar, 25.Jennings C.V. Rosengren K.J. Daly N.L. Plan M. Stevens J. Scanlon M.J. Waine C. Norman D.G. Anderson M.A. Craik D.J. Biochemistry. 2005; 44: 851-860Crossref PubMed Scopus (193) Google Scholar, 26.Barbeta B.L. Marshall A.T. Gillon A.D. Craik D.J. Anderson M.A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1221-1225Crossref PubMed Scopus (161) Google Scholar, 27.Simonsen S.M. Sando L. Rosengren K.J. Wang C.K. Colgrave M.L. Daly N.L. Craik D.J. J. Biol. Chem. 2008; 283: 9805-9813Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 28.Colgrave M.L. Kotze A.C. Kopp S. McCarthy J.S. Coleman G.T. Craik D.J. Acta Trop. 2009; 109: 163-166Crossref PubMed Scopus (76) Google Scholar, 29.Gruber C.W. Cemazar M. Anderson M.A. Craik D.J. Toxicon. 2007; 49: 561-575Crossref PubMed Scopus (119) Google Scholar).In a recent study (30.Colgrave M.L. Kotze A.C. Huang Y.H. O'Grady J. Simonsen S.M. Craik D.J. Biochemistry. 2008; 47: 5581-5589Crossref PubMed Scopus (122) Google Scholar), the anthelmintic activity of a range of cyclotides was demonstrated. Kalata B1, B2, B6, and B7, cyclotides extracted from O. affinis, significantly inhibited the development of the larval life stages of two sheep gastrointestinal nematodes Haemonchus contortus (H. contortus) and Trichostrongylus colubriformis, as well as inhibiting motility of adult H. contortus. A subsequent study reported enhanced nematocidal activity of other natural cyclotides possessing multiple basic residues (31.Colgrave M.L. Kotze A.C. Ireland D.C. Wang C.K. Craik D.J. Chembiochem. 2008; 11: 1939-1945Crossref Scopus (98) Google Scholar). Cycloviolacin O2 (containing three basic residues) was found to be the most potent cyclotide tested against sheep parasites. The removal of charge through acetylation of lysine residues resulted in reduced anthelmintic activity, highlighting the importance of the positively charged residues in the anthelmintic activity of cyclotides.Cyclotides have been proven to be amenable to chemical synthesis (22.Tam J.P. Lu Y.A. Yang J.L. Chiu K.W. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 8913-8918Crossref PubMed Scopus (401) Google Scholar, 32.Tam J.P. Lu Y.A. Protein Sci. 1998; 7: 1583-1592Crossref PubMed Scopus (109) Google Scholar, 33.Daly N.L. Love S. Alewood P.F. Craik D.J. Biochemistry. 1999; 38: 10606-10614Crossref PubMed Scopus (190) Google Scholar), and as a result, mutated cyclotides can be readily synthesized, enabling detailed studies into their structure-activity relationships. In the current study, a “lysine scan” of the prototypic cyclotide kalata B1 was conducted to understand the structural and functional role of positive charge at different sites within cyclotides. Each of the non-cysteine residues in this 29-amino acid peptide was successively replaced with lysine using solid-phase peptide synthesis, and the suite of mutants was assayed against H. contortus and T. colubriformis. In addition, select double and triple lysine mutants were designed and assayed. Many of the substitutions resulted in a total loss of activity, thus helping to define residues implicated in bioactivity, but some substitutions resulted in increased anthelmintic activity, which was further increased in some double and triple mutants.Overall, this biochemical mutagenesis study has identified key residues in the prototypic cyclotide kalata B1 important for anthelmintic activity and demonstrated the ability to enhance activity through incorporation of positively charged residues into a localized region of the peptide. We refer to this localized region as the “amendable” face of cyclotides and describe a mechanistic model explaining its role in modulating cyclotide bioactivity.DISCUSSIONIn this study the structural and functional role of positive charge in the cyclotide framework was assessed by synthesis of a suite of lysine mutants, each with a single point mutation of the prototypic cyclotide kalata B1. The anthelmintic properties of the lysine mutants were evaluated by their ability to interfere with the development of eggs through to L3 larvae stage for two agriculturally important nematode species H. contortus and T. colubriformis. These nematodes are responsible for major stock and economic losses in the livestock industry. Eleven of 22 single point lysine mutants showed a dramatic decrease in anthelmintic activity, five showed equipotent or slightly decreased activity, and the remaining six mutants displayed an increased activity, which was ascribed to the incorporation of charge in a region amenable to amino acid substitution. Mutants with multiple lysine substitutions within this region further enhanced the anthelmintic activity. Overall, the results suggest that the anthelmintic potency of kalata B1 is decreased after residue substitution on one face of the molecule, maintained on substitution of another set of residues, and significantly increased by the incorporation of one or multiple basic residues in an “amendable” face. The term amendable is used here in its literal sense of “able to be modified to produce an improvement.”Recent studies (27.Simonsen S.M. Sando L. Rosengren K.J. Wang C.K. Colgrave M.L. Daly N.L. Craik D.J. J. Biol. Chem. 2008; 283: 9805-9813Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 41.Huang Y.H. Colgrave M.L. Daly N.L. Keleshian A. Martinac B. Craik D.J. J. Biol. Chem. 2009; 284: 20699-20707Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) identified two faces of the kalata B1 molecule that are associated with its mode of insecticidal action: a hydrophilic face that appears to be involved in cyclotide self-association and a hydrophobic face that interacts with membranes. It was hypothesized that insecticidal activity is due to pore formation in membranes that results from cyclotide self-association and membrane binding. The results from the current study support this hypothesis and suggest that it is a general mechanism for other cyclotide activities, including anthelmintic activity and hemolytic activity. The biochemical mutagenesis studies undertaken here have specifically identified regions of the surface of kalata B1 that are associated with bioactivity and membrane binding.The LDA data in the current study demonstrated that even at the highest concentration tested, the development of nematode larvae was not inhibited by 11 of the lysine mutants (G6K, E7K, T8K, V10K, G12K, N15K, T16K, W23K, V25K, P3K, and V4K). Except for G12K, 10 of the 11 residues that upon mutation profoundly reduced the anthelmintic activity of kalata B1 are co-localized on one face of this prototypic cyclotide (Fig. 3B, left). Thus, these results define a region of kalata B1 critical for anthelmintic activity. When combined with data from the recent Ala-scanning mutagenesis study that examined insecticidal activity (27.Simonsen S.M. Sando L. Rosengren K.J. Wang C.K. Colgrave M.L. Daly N.L. Craik D.J. J. Biol. Chem. 2008; 283: 9805-9813Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), it is apparent that this region is made up of two distinct subregions. The first is centered on Glu-7 and adjacent hydrophilic residues has been described previously as the “bioactive face” (circled in Fig. 3B, solid line) (27.Simonsen S.M. Sando L. Rosengren K.J. Wang C.K. Colgrave M.L. Daly N.L. Craik D.J. J. Biol. Chem. 2008; 283: 9805-9813Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The second subregion comprises a surface-exposed patch of hydrophobic residues (encircled by a dashed line in Fig. 3B). The Ala-scanning mutagenesis study (27.Simonsen S.M. Sando L. Rosengren K.J. Wang C.K. Colgrave M.L. Daly N.L. Craik D.J. J. Biol. Chem. 2008; 283: 9805-9813Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) suggested that the hydrophilic bioactive face is implicated in self-association and that the hydrophobic patch is implicated in membrane binding, and that both are essential for insecticidal activity.One fundamental difference between the Ala-scanning study and the current Lys-scanning study is that mutation of some of the residues in the hydrophobic patch to Ala did not reduce insecticidal activity much, but their mutagenesis to Lys reported here does dramatically reduce anthelmintic activity. However, rather than being a contradictory result, this difference is readily explicable on the basis of the nature of the mutational probe residues, Ala versus Lys. Alanine is a hydrophobic amino acid, and its substitution into an existing hydrophobic patch would not be expected to produce a functional perturbation. By contrast, inclusion of lysine, a highly hydrophilic residue, into a hydrophobic patch would be expected to disrupt it and lead to loss of function (provided that the hydrophobic patch has a functional role). Thus, the current results support the previously proposed dual-component (self-association and membrane binding) mechanism of action of kalata B1. However, the new mutagenesis data significantly extends the mechanistic interpretation by identifying a third precisely defined region of the surface that is amendable to enhance bioactivity.Substitution of Gly-18, Thr-20, Ser-22, Thr-27, Asn-29, or Gly-1 with Lys resulted in increased anthelmintic activity by up to 7-fold. These six residues form a patch on the opposite side to the face comprising the combined “bioactive” and membrane binding regions. Incorporation of charged residues into the newly identified amendable face of kalata B1 probably boosts its anthelmintic activity through electrostatic interactions between the positively charged side chains and the negatively charged phospholipid head groups of target membranes.In a recent study (30.Colgrave M.L. Kotze A.C. Huang Y.H. O'Grady J. Simonsen S.M. Craik D.J. Biochemistry. 2008; 47: 5581-5589Crossref PubMed Scopus (122) Google Scholar), the substitution of individual residues in kalata B1 with alanine resulted in significant decreases in inhibition observed in LDAs. The anthelmintic activity of some mutants dropped to <25% (G6A, E7A, T8A, V10A, and G12A), some decreased to between 25 and 40% (N15A, T16A, V25A, R28A, and P3A), and others to 65–90% (P17A, L2A and V4A). Similar trends were observed for decreasing lytic ability (41.Huang Y.H. Colgrave M.L. Daly N.L. Keleshian A. Martinac B. Craik D.J. J. Biol. Chem. 2009; 284: 20699-20707Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) of alanine mutants, as demonstrated by the leakage of a self-quenching dye from phospholipid vesicles. Although the anthelmintic activity of single point lysine mutants broadly correlates with those of alanine mutants (30.Colgrave M.L. Kotze A.C. Huang Y.H. O'Grady J. Simonsen S.M. Craik D.J. Biochemistry. 2008; 47: 5581-5589Crossref PubMed Scopus (122) Google Scholar, 41.Huang Y.H. Colgrave M.L. Daly N.L. Keleshian A. Martinac B. Craik D.J. J. Biol. Chem. 2009; 284: 20699-20707Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), there are two major differences between these data sets. First, a larger number of lysine mutants (11 of 22) showed decreased inhibition rates to <25% in LDA than the number of alanine mutants (5 of 21, as the anthelmintic activity of W23A was not determined). Second, incorporation of a lysine residue at a number of positions (Gly-18, Thr-20, Ser-22, Thr-27, Asn-29, and Gly-1) increased the anthelmintic activity, whereas none of the alanine mutants were observed to be more potent than the wild-type peptide.The current study has demonstrated that the anthelmintic activity of kalata B1 is lost upon replacement of any of the residues on the hydrophobic patch with a hydrophilic residue. Because the hydrophobic patch has been reported to be responsible for the binding of kalata B1 to the surface of dodecylphosphocholine (44.Shenkarev Z.O. Nadezhdin K.D. Sobol V.A. Sobol A.G. Skjeldal L. Arseniev A.S. FEBS J. 2006; 273: 2658-2672Crossref PubMed Scopus (108) Google Scholar), introduction of a different hydrophobic residue (alanine) at some positions in this region may not affect the binding significantly. However, placing a hydrophilic residue in this patch may have hindered its hydrophobic interactions with the membrane, thus resulting in a loss of activity.The LDA results obtained for the lysine mutants in this study further emphasize the functional role of the hydrophilic patch, because incorporation of a lysine (positively charged) residue in this area silences the anthelmintic activity of kalata B1. Glu-7 is a highly conserved residue in the cyclotides, which has been proven to have an important role in stabilizing the structure through hydrogen bonding interactions between loops 1 and 3, via the side chain oxygen atoms of the glutamic acid (45.Rosengren K.J. Daly N.L. Plan M.R. Waine C. Craik D.J. J. Biol. Chem. 2003; 278: 8606-8616Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Methylation of the side chain of the highly conserved glutamic acid in cyclotide cycloviolacin O2 resulted a 48-fold decrease in its cytotoxic activity (46.Herrmann A. Svångard E. Claeson P. Gullbo J. Bohlin L. Göransson U. Cell Mol. Life Sci. 2006; 63: 235-245Crossref PubMed Scopus (91) Google Scholar) and a 6-fold decrease in its anthelmintic activity against H. contortus (31.Colgrave M.L. Kotze A.C. Ireland D.C. Wang C.K. Craik D.J. Chembiochem. 2008; 11: 1939-1945Crossref Scopus (98) Google Scholar). These findings substantiate the important role of the glutamic acid residue in the cyclotide function. Thus, substituting the negatively charged Glu-7 or any adjacent residues with a positively charged residue resulted in a dramatic loss of activity. The R28K mutant (a relatively minor substitution) was the only lysine mutant present in the hydrophilic patch that retained 60–70% (relative activity 0.7 for H. contortus, 0.6 for T. colubriformis, Table 1) of nematocidal activity.The anthelmintic activity of natural variants extracted from Viola odorata was determined previously (31.Colgrave M.L. Kotze A.C. Ireland D.C. Wang C.K. Craik D.J. Chembiochem. 2008; 11: 1939-1945Crossref Scopus (98) Google Scholar) and cycloviolacin O2 was reported to be the most potent cyclotide in LDAs, with activity 18-fold greater than the prototypic cyclotide kalata B1. A substantial improvement in activity was observed in all peptides that contained three to four basic residues. To investigate the correlation between the enhanced anthelmintic activities and the number of charged residues within the peptide sequences, three double lysine mutants (T20K/S22K, T20K/N29K, and T20K/G1K) and one triple lysine mutant (T20K/N29K/G1K) were produced. As illustrated in Fig. 4, all four multiple lysine mutants showed higher activity than those with single point mutations at the same positions within the cyclotide structure. However, the observed lower nematocidal activity of T20K/S22K compared with T20K/N29K and T20K/G1K suggests the distribution of the positively charged residues on the amendable face is also a determinant in the relative potency of these novel anthelmintic analogues. Similar to the results of the LDAs, the double lysine mutants affected the motility of H. contortus adults to a greater degree than did the single mutants.The lysine mutants in the current study were also assayed for hemolytic activity to determine whether the increased potency observed in anthelmintic assays would correlate with increased efficacy of hemolysis. The trends observed for hemolytic activity correlated with those shown for anthelmintic activity of these lysine mutants. Overall, the collective results suggest that a common mechanism involving cyclotide-membrane interaction is responsible for the hemolytic, insecticidal, and anthelmintic activity of kalata B1.Fig. 6 shows a schematic representation of the proposed mechanism of action of kalata B1. The view shown in a highlights two important regions of the surface defined previously: the hydrophobic face and the bioactive face. Lysine incorporation into a third face, the amendable face, results in enhanced hemolytic and anthelmintic activities. The side view of kalata B1 in b clearly shows its three faces. The addition of a positive charge into the amendable face probably enhances activity by facilitating the binding of the peptide to the negatively charged head group region of the membrane, as illustrated in c. Subsequent interactions between the hydrophobic patch of kalata B1 and the membrane core would allow peptide insertion into the lipid bilayer (d). We speculate that self-association as indicated in e could then lead ultimately to pore formation. Overall, the activity of kalata B1 appears to be dependent on hydrophobic interactions with membranes, together with self-association involving residues clustered in the bioactive face, but it can be further modulated through the incorporation of a positive charge in the amendable face.FIGURE 6Schematic representation of functionally important regions of kalata B1 and their role in the proposed mechanism of action. a, representation of kalata B1 displaying the bioactive (dark gray) and hydrophobic face (black). b, rotation of kalata B1 by 90° reveals the amendable face of kalata B1, which is colored light gray. The incorporation of a lysine residue in the amendable face (indicated by the bold black arrow) probably facilitates electrostatic interactions between kalata B1 and the negatively charged membrane head group region as illustrated in c. In d, interactions between the hydrophobic patch on kalata B1 and the hydrophobic core of the membrane result in peptide insertion. We speculate that peptide self-association might occur, as shown in e, ultimately leading to pore formation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Although our focus here has been on structure-activity studies of the prototypic Möbius subfamily cyclotide kalata B1 with the aim of improving its agricultural potential, a recent report has demonstrated that a member of the trypsin inhibitor subfamily of cyclotides isolated from Momordica cochinchinensis (47.Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. Andrieu J.P. Heitz A. Trinh Hong T. Pham T.T. Le Nguyen D. Biochemistry. 2000; 39: 5722-5730Crossref PubMed Scopus (293) Google Scholar, 48.Felizmenio-Quimio M.E. Daly N.L. Craik D.J. J. Biol. Chem. 2001; 276: 22875-22882Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) also has exciting potential agricultural applications and can be re-engineered to inhibit a range of proteases other than trypsin (49.Thongyoo P. Roqué-Rosell N. Leatherbarrow R.J. Tate E.W. Org. Biomol. Chem. 2008; 6: 1462-1470Crossref PubMed Scopus (113) Google Scholar). Two synthetic MCoTI-II analogues were reported to exhibit significant activity against 3C protease, a cysteine protease involved in the formation of proteins required for the RNA replication of the foot-and-mouth-disease virus (FMDV) (50.Sweeney T.R. Roqué-Rosell N. Birtley J.R. Leatherbarrow R.J. Curry S. J. Virol. 2007; 81: 115-124Crossref PubMed Scopus (67) Google Scholar, 51.Curry S. Roqué-Rosell N. Sweeney T.R. Zunszain P.A. Leatherbarrow R.J. Biochem. Soc. Trans. 2007; 35: 594-598Crossref PubMed Scopus (19) Google Scholar). This report not only discovered the first peptide-based inhibitor of this agriculturally important viral enzyme, but also demonstrated that the enzyme specificity of this class of cyclotides can be redirected with simple point mutations.In conclusion, disruption of either the hydrophobic or bioactive patches of kalata B1 by incorporation of lysine into these regions abolished anthelmintic activity. This study has identified the presence of a third important face on the opposite side to the bioactive patch of kalata B1. Lysine substitution in this amendable face resulted in a dramatic improvement in anthelmintic activity. The structure-activity relationships described here pave the way for the further exploitation of cyclotides as valuable agents against agricultural pests. IntroductionGastrointestinal nematodes cause major losses to livestock industries worldwide. The control of these pests, until now, has been via synthetic anthelmintics, including benzimidazoles (e.g. thiabendazole), nicotinic acetylcholine receptor agonists (e.g. levamisole), and macrocyclic lactones (e.g. ivermectin and moxidectin). However, resistance of sheep and cattle nematodes to these broad-spectrum anthelmintics is a serious issue for livestock production globally (1.Besier B. Trends Parasitol. 2007; 23: 21-24Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 2.Kaplan R.M. Trends Parasitol. 2004; 20: 477-481Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 3.Wolstenholme A.J. Fairweather I. Prichard R. von Samson-Himmelstjerna G. Sangster N.C. Trends Parasitol. 2004; 20: 469-476Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar). Thus, the development of novel agents with potent anthelmintic activity is of great economic and agricultural importance.Cyclotides are circular miniproteins discovered originally in plants of the Rubiaceae, Violaceae, and Cucurbitaceae families (4.Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (631) Google Scholar, 5.Craik D.J. Daly N.L. Mulvenna J. Plan M.R. Trabi M. Curr. Protein Pept. Sci. 2004; 5: 297-315Crossref PubMed Scopus (161) Google Scholar). Kalata B1, the first cyclotide to be structurally characterized, is abundant in the tropical African plant Oldenlandia affinis (6.Craik D.J. Toxicon. 2001; 39: 1809-1813Crossref PubMed Scopus (101) Google Scholar, 7.Saether O. Craik D.J. Campbell I.D. Sletten K. Juul J. Norman D.G. Biochemistry. 1995; 34: 4147-4158Crossref PubMed Scopus (365) Google Scholar) but has also been reported in the European Sweet Violet (Viola odorata) (8.Ireland D.C. Colgrave M.L. Craik D.J. Biochem. J. 2006; 400: 1-12Crossref PubMed Scopus (140) Google Scholar) and several other Viola species (9.Claeson P. Göransson U. Johansson S. Luijendijk T. Bohlin L. J. Nat. Prod. 1998; 61: 77-81Crossref PubMed Scopus (139) Google Scholar, 10.Göransson U. Luijendijk T. Johansson S. Bohlin L. Claeson P. J. Nat. Prod. 1999; 62: 283-286Crossref PubMed Scopus (155) Google Scholar, 11.Hallock Y.F. Sowder 2nd, R.C. Pannell L.K. Hughes C.B. Johnson D.G. Gulakowski R. Cardellina 2nd, J.H. Boyd M.R. J. Org. Chem. 2000; 65: 124-128Crossref PubMed Scopus (104) Google Scholar, 12.Broussalis A.M. Göransson U. Coussio J.D. Ferraro G. Martino V. Claeson P. Phytochemistry. 2001; 58: 47-51Crossref PubMed Scopus (84) Google Scholar, 13.Trabi M. Mylne J.S. Sando L. Craik D.J. Org. Biomol. Chem. 2009; 7: 2378-2388Crossref PubMed Scopus (32) Google Scholar). Members of the cyclotide family possess a cyclic peptide backbone and a cystine knot motif, which is formed by three disulfide bonds at the core of their three-dimensional structure. The sequence and structure of kalata B1 is illustrated in Fig. 1, which highlights the six backbone loops between the six conserved cysteine residues that make up the cystine knot (14.Wang C.K. Hu S.H. Martin J.L. Sjögren T. Hajdu J. Bohlin L. Claeson P. Göransson U. Rosengren K.J. Tang J. Tan N.H. Craik D.J. J. Biol. Chem. 2009; 284: 10672-10683Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The cyclic cystine knot motif is thought to be responsible for the extraordinary enzymatic and chemical stability (15.Colgrave M.L. Craik D.J. Biochemistry. 2004; 43: 5965-5975Crossref PubMed Scopus (447) Google Scholar) of the cyclotides. Cyclotides have a wide range of bioactivities, including anti-HIV (16.Gustafson K.R. S.I.R. Henderson L.E. Parsons I.C. Kashman Y. Cardellina II, J.H. McMahon J.B. Buckheit Jr., R.W. Pannell L.K. Boyd M.R. J. Am. Chem. Soc. 1994; 116: 9337-9338Crossref Scopus (255) Google Scholar, 17.Daly N.L. Gustafson K.R. Craik D.J. FEBS Lett. 2004; 574: 69-72Crossref PubMed Scopus (103) Google Scholar, 18.Ireland D.C. Wang C.K. Wilson J.A. Gustafson K.R. Craik D.J. Biopolymers Peptide Science. 2008; 90: 51-60Crossref PubMed Scopus (117) Google Scholar, 19.Wang C.K. Colgrave M.L. Gustafson K.R. Ireland D.C. Goransson U. Craik D.J. J. Nat. Prod. 2008; 71: 47-52Crossref PubMed Scopus (143) Google Scholar), neurotensin antagonism (20.Witherup K.M. Bogusky M.J. Anderson P.S. Ramjit H. Ransom R.W. Wood T. Sardana M. J. Nat. Prod. 1994; 57: 1619-1625Crossref PubMed Scopus (226) Google Scholar), hemolytic (21.Barry D.G. Daly N.L. Clark R.J. Sando L. Craik D.J. Biochemistry. 2003; 42: 6688-6695Crossref PubMed Scopus (101) Google Scholar), antimicrobial (22.Tam J.P. Lu Y.A. Yang J.L. Chiu K.W. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 8913-8918Crossref PubMed Scopus (401) Google Scholar), anti-fouling (23.Göransson U. Sjögren M. Svangård E. Claeson P. Bohlin L. J. Nat. Prod. 2004; 67: 1287-1290Crossref PubMed Scopus (122) Google Scholar), and pesticidal activities (24.Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 10614-10619Crossref PubMed Scopus (398) Google Scholar, 25.Jennings C.V. Rosengren K.J. Daly N.L. Plan M. Stevens J. Scanlon M.J. Waine C. Norman D.G. Anderson M.A. Craik D.J. Biochemistry. 2005; 44: 851-860Crossref PubMed Scopus (193) Google Scholar, 26.Barbeta B.L. Marshall A.T. Gillon A.D. Craik D.J. Anderson M.A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1221-1225Crossref PubMed Scopus (161) Google Scholar, 27.Simonsen S.M. Sando L. Rosengren K.J. Wang C.K. Colgrave M.L. Daly N.L. Craik D.J. J. Biol. Chem. 2008; 283: 9805-9813Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 28.Colgrave M.L. Kotze A.C. Kopp S. McCarthy J.S. Coleman G.T. Craik D.J. Acta Trop. 2009; 109: 163-166Crossref PubMed Scopus (76) Google Scholar, 29.Gruber C.W. Cemazar M. Anderson M.A. Craik D.J. Toxicon. 2007; 49: 561-575Crossref PubMed Scopus (119) Google Scholar).In a recent study (30.Colgrave M.L. Kotze A.C. Huang Y.H. O'Grady J. Simonsen S.M. Craik D.J. Biochemistry. 2008; 47: 5581-5589Crossref PubMed Scopus (122) Google Scholar), the anthelmintic activity of a range of cyclotides was demonstrated. Kalata B1, B2, B6, and B7, cyclotides extracted from O. affinis, significantly inhibited the development of the larval life stages of two sheep gastrointestinal nematodes Haemonchus contortus (H. contortus) and Trichostrongylus colubriformis, as well as inhibiting motility of adult H. contortus. A subsequent study reported enhanced nematocidal activity of other natural cyclotides possessing multiple basic residues (31.Colgrave M.L. Kotze A.C. Ireland D.C. Wang C.K. Craik D.J. Chembiochem. 2008; 11: 1939-1945Crossref Scopus (98) Google Scholar). Cycloviolacin O2 (containing three basic residues) was found to be the most potent cyclotide tested against sheep parasites. The removal of charge through acetylation of lysine residues resulted in reduced anthelmintic activity, highlighting the importance of the positively charged residues in the anthelmintic activity of cyclotides.Cyclotides have been proven to be amenable to chemical synthesis (22.Tam J.P. Lu Y.A. Yang J.L. Chiu K.W. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 8913-8918Crossref PubMed Scopus (401) Google Scholar, 32.Tam J.P. Lu Y.A. Protein Sci. 1998; 7: 1583-1592Crossref PubMed Scopus (109) Google Scholar, 33.Daly N.L. Love S. Alewood P.F. Craik D.J. Biochemistry. 1999; 38: 10606-10614Crossref PubMed Scopus (190) Google Scholar), and as a result, mutated cyclotides can be readily synthesized, enabling detailed studies into their structure-activity relationships. In the current study, a “lysine scan” of the prototypic cyclotide kalata B1 was conducted to understand the structural and functional role of positive charge at different sites within cyclotides. Each of the non-cysteine residues in this 29-amino acid peptide was successively replaced with lysine using solid-phase peptide synthesis, and the suite of mutants was assayed against H. contortus and T. colubriformis. In addition, select double and triple lysine mutants were designed and assayed. Many of the substitutions resulted in a total loss of activity, thus helping to define residues implicated in bioactivity, but some substitutions resulted in increased anthelmintic activity, which was further increased in some double and triple mutants.Overall, this biochemical mutagenesis study has identified key residues in the prototypic cyclotide kalata B1 important for anthelmintic activity and demonstrated the ability to enhance activity through incorporation of positively charged residues into a localized region of the peptide. We refer to this localized region as the “amendable” face of cyclotides and describe a mechanistic model explaining its role in modulating cyclotide bioactivity." @default.
• W2143324852 created "2016-06-24" @default.
• W2143324852 creator A5021518732 @default.
• W2143324852 creator A5033914141 @default.
• W2143324852 creator A5040937753 @default.
• W2143324852 creator A5055209466 @default.
• W2143324852 creator A5079308758 @default.
• W2143324852 date "2010-04-01" @default.
• W2143324852 modified "2023-10-11" @default.
• W2143324852 title "Lysine-scanning Mutagenesis Reveals an Amendable Face of the Cyclotide Kalata B1 for the Optimization of Nematocidal Activity" @default.
• W2143324852 cites W1963677912 @default.
• W2143324852 cites W1968645374 @default.
• W2143324852 cites W1969126658 @default.
• W2143324852 cites W1969184513 @default.
• W2143324852 cites W1981873289 @default.
• W2143324852 cites W1982687124 @default.
• W2143324852 cites W1983851126 @default.
• W2143324852 cites W1984687223 @default.
• W2143324852 cites W1989706787 @default.
• W2143324852 cites W1992931410 @default.
• W2143324852 cites W1994048326 @default.
• W2143324852 cites W1995390287 @default.
• W2143324852 cites W2002132496 @default.
• W2143324852 cites W2003706352 @default.
• W2143324852 cites W2006767418 @default.
• W2143324852 cites W2009039916 @default.
• W2143324852 cites W2011464642 @default.
• W2143324852 cites W2024089776 @default.
• W2143324852 cites W2025227932 @default.
• W2143324852 cites W2028277934 @default.
• W2143324852 cites W2029780190 @default.
• W2143324852 cites W2031504189 @default.
• W2143324852 cites W2036465513 @default.
• W2143324852 cites W2037645437 @default.
• W2143324852 cites W2040091143 @default.
• W2143324852 cites W2041619855 @default.
• W2143324852 cites W2041997590 @default.
• W2143324852 cites W2042796433 @default.
• W2143324852 cites W2049202410 @default.
• W2143324852 cites W2052876027 @default.
• W2143324852 cites W2055870805 @default.
• W2143324852 cites W2056072225 @default.
• W2143324852 cites W2057918544 @default.
• W2143324852 cites W2059326736 @default.
• W2143324852 cites W2065109254 @default.
• W2143324852 cites W2071364873 @default.
• W2143324852 cites W2071570207 @default.
• W2143324852 cites W2073660388 @default.
• W2143324852 cites W2073764925 @default.
• W2143324852 cites W2082443443 @default.
• W2143324852 cites W2091528388 @default.
• W2143324852 cites W2092065212 @default.
• W2143324852 cites W2136544846 @default.
• W2143324852 cites W2146837416 @default.
• W2143324852 cites W2153726959 @default.
• W2143324852 cites W2162857162 @default.
• W2143324852 cites W2165055155 @default.
• W2143324852 cites W4213262935 @default.
• W2143324852 cites W4250921268 @default.
• W2143324852 doi "https://doi.org/10.1074/jbc.m109.089854" @default.
• W2143324852 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2856286" @default.
• W2143324852 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20103593" @default.
• W2143324852 hasPublicationYear "2010" @default.
• W2143324852 type Work @default.
• W2143324852 sameAs 2143324852 @default.
• W2143324852 citedByCount "98" @default.
• W2143324852 countsByYear W21433248522012 @default.
• W2143324852 countsByYear W21433248522013 @default.
• W2143324852 countsByYear W21433248522014 @default.
• W2143324852 countsByYear W21433248522015 @default.
• W2143324852 countsByYear W21433248522016 @default.
• W2143324852 countsByYear W21433248522017 @default.
• W2143324852 countsByYear W21433248522018 @default.
• W2143324852 countsByYear W21433248522019 @default.
• W2143324852 countsByYear W21433248522020 @default.
• W2143324852 countsByYear W21433248522021 @default.
• W2143324852 countsByYear W21433248522022 @default.
• W2143324852 countsByYear W21433248522023 @default.
• W2143324852 crossrefType "journal-article" @default.
• W2143324852 hasAuthorship W2143324852A5021518732 @default.
• W2143324852 hasAuthorship W2143324852A5033914141 @default.
• W2143324852 hasAuthorship W2143324852A5040937753 @default.
• W2143324852 hasAuthorship W2143324852A5055209466 @default.
• W2143324852 hasAuthorship W2143324852A5079308758 @default.
• W2143324852 hasBestOaLocation W21433248521 @default.
• W2143324852 hasConcept C104317684 @default.
• W2143324852 hasConcept C16318435 @default.
• W2143324852 hasConcept C185592680 @default.
• W2143324852 hasConcept C2776016237 @default.
• W2143324852 hasConcept C501734568 @default.
• W2143324852 hasConcept C515207424 @default.
• W2143324852 hasConcept C55493867 @default.
• W2143324852 hasConcept C70721500 @default.
• W2143324852 hasConcept C86803240 @default.
• W2143324852 hasConcept C98274493 @default.
• W2143324852 hasConceptScore W2143324852C104317684 @default.
• W2143324852 hasConceptScore W2143324852C16318435 @default.
• W2143324852 hasConceptScore W2143324852C185592680 @default.

• next