Gly6 of kalata B1 is critical for the selective binding to phosphatidylethanolamine membranes

Hall, Kristopher Norman; Lee, Tzong Hsien; Daly, Norelle L.; Craik, David J.; Aguilar, Marie Isabel

doi:10.1016/j.bbamem.2012.04.007

Cited by 17 publications

(13 citation statements)

References 39 publications

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“…showed weaker binding to immobilized lipid bilayers, 184,231,284 which was consistent with their diminished bioactivity. Interestingly, inserting an additional Trp residue into loop 5 or replacing Asn15 with Tyr (loop 3) led to improvements in both membrane binding and activity in hemolytic or cytotoxicity assays.…”

Section: Defining the Mode-of-action For Membrane-active Cyclotidesmentioning

confidence: 65%

Cyclotides: From Structure to Function

2019

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Early studies of cyclotides [7][8][9][13][14][15] were based on discovery and characterization efforts at the peptide level. The general approach for these studies involved either bioassay-guided or mass-guided fractionation of plant extracts, followed by peptide isolation and purification, amino acid analysis, Edman sequencing and chemical, NMR 9 or mass spectrometry-based 10 approaches to deduce disulfide connectivity. The first step in many of the early discovery programs was a pre-screening procedure to determine if a given plant tissue might contain cyclotides (Figure 3). Various protocols 13,15,57,69,116,123 have been reported for quickly screening if a plant might be cyclotide-positive, with the simplest based on extraction (using dichloromethane/methanol, aqueous acetonitrile, or aqueous buffer) and purification on a C18 reverse phase column followed by a main-screen. The latter typically involves (A) examination of a reversed phase HPLC-MS profile for late-eluting peaks that are (B) in the mass range 2800-4000 Da and (C) contain six Cys residues as judged by a mass shift of 348 Da after reduction and S-alkylation, as shown in Figure 3. 116 The rationale for the target HPLC elution range was that all of the cyclotides discovered in early studies were somewhat hydrophobic and eluted relatively late on HPLC. Similarly, many of the initial cyclotides were in the mass range 2500-4000 Da and it was assumed that new cyclotides would fall within a similar range. Finally, by definition, a cyclotide must contain six Cys residues so the mass shift test in step C of the screen provided a simple way of testing for that criterion. An example of the 348 Da mass shift after reduction and alkylation is illustrated for the identification of the cyclotide Cter A from Clitoria ternatea (butterfly pea) seeds in Figure 3. Chassalia chartacea chassatides 235 Chassalia curviflora chacur c Chassalia discolor CD-1 c Chassalia parvifolia circulins 7,22,406 Hedyotis biflora hedyotides 207,213,280,350 H. centranthoides hcf-1 c H. terminalis htf-1 c Oldenlandia affinis kalata peptides 1-5,9 Palicourea condensata palicoureins 25 P. rigida parigidin-br1 236,329,345 P. tetragona paltet 1 c Psychotria brachiata psybra 1 c Psychotria brachyceras Psyleio and psybry peptides c Psychotria leiocarpa Psyleio peptides 338 Psychotria leptothyrsa psyle peptides 157,158 Psychotria longipes cyclopsychotride A 8 Psychotria poeppigiana psypoe 1 Psychotria solitudinum psysol 2 305 Psychotria suterella PS-1 c Cucurbitaceae Momordica clarkeana TI peptides 315 These trypsin inhibitor cyclotides are also referred to as cyclic knottins; typically, these peptides are found only in seeds. M. cochinchinensis MCoTI peptides 24 M. denticulate TI peptides 315 M. diocia Modi peptides 424 M. gilgiana TI peptides 315 M. macrophylla TI peptides 315 M. subangulata TI peptides 315 Fabaceae Clitoria ternatea Cter peptides; 204 cliotides 206 These cyclotides are biosynthetically processed from 'hijacked' genes (i.e. from albumin genes in which an ancestral albumin subun...

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Section: Defining the Mode-of-action For Membrane-active Cyclotidesmentioning

confidence: 65%

Cyclotides: From Structure to Function

2019

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“…The previous study shows [G6A]kB1 and [G6K]kB1 lost their inhibition to insect and nematode larvae, respectively (55,65). Another study (66) suggests Gly6 in kB1 is very crucial for the PEheadgroup interaction, because [G6A]kB1 could not bind with PE-containing membrane. This could probably be because Gly-6 is at the edge of hydrophobic patch and plays a critical role on the effective surface topography.…”

Section: Discussionmentioning

confidence: 99%

Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb Hybanthus enneaspermus

Chan

Gilding

et al. 2020

Journal of Biological Chemistry

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Cyclotides are plant-derived peptides characterized by a ~30-amino-acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and one known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, M, and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide–membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.

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“…SPR experiments were carried out with a Biacore T100 analytical system on L1 sensor chip (Biacore, Uppsala, Sweden) as previously described 42 43 44 45 46 . A minimum of 5000RU was deposited which corresponds to 5 ng/mm 2 .…”

Section: Methodsmentioning

confidence: 99%

Real-time Measurement of Membrane Conformational States Induced by Antimicrobial Peptides: Balance Between Recovery and Lysis

Hall

Lee

Mechler

et al. 2014

Sci Rep

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The disruption of membranes by antimicrobial peptides is a multi-state process involving significant structural changes in the phospholipid bilayer. However, direct measurement of these membrane structural changes is lacking. We used a combination of dual polarisation interferometry (DPI), surface plasmon resonance spectroscopy (SPR) and atomic force microscopy (AFM) to measure the real-time changes in membrane structure through the measurement of birefringence during the binding of magainin 2 (Mag2) and a highly potent analogue in which Ser8, Gly13 and Gly18 has been replaced with alanine (Mag-A). We show that the membrane bilayer undergoes a series of structural changes upon peptide binding before a critical threshold concentration is reached which triggers a significant membrane disturbance. We also propose a detailed model for antimicrobial peptide action as a function of the degree of bilayer disruption to provide an unprecedented in-depth understanding of the membrane lysis in terms of the interconversion of different membrane conformational states in which there is a balance between recovery and lysis.

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Gly6 of kalata B1 is critical for the selective binding to phosphatidylethanolamine membranes

Cited by 17 publications

References 39 publications

Cyclotides: From Structure to Function

Cyclotides: From Structure to Function

Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb Hybanthus enneaspermus

Real-time Measurement of Membrane Conformational States Induced by Antimicrobial Peptides: Balance Between Recovery and Lysis

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