Lysine-rich Cyclotides: A New Subclass of Circular Knotted Proteins

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Lysine-rich cyclotides: a new subclass of circular knotted proteins from Violaceae Anjaneya S Ravipati, Sónia Troeira Henriques, Aaron G Poth, Quentin Kaas, Conan K Wang, Michelle L Colgrave, and David J Craik ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00454 • Publication Date (Web): 31 Aug 2015 Downloaded from http://pubs.acs.org on September 1, 2015

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Lysine-rich cyclotides: a new subclass of circular knotted proteins from Violaceae Anjaneya S. Ravipati1, Sónia Troeira Henriques1, Aaron G. Poth1, Quentin Kaas1, Conan K. Wang1, Michelle L. Colgrave2, and David J. Craik 1*

1

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, 4072,

Australia, 2Commonwealth Scientific and Industrial Research Organization, Division of Animal, Food and Health Sciences, St. Lucia, QLD, 4067, Australia

Keywords: cyclic peptides, cyclotides, cystine knot, phosphatidylethanolamine

*

To whom correspondence should be addressed

Institute for Molecular Bioscience The University of Queensland Brisbane 4072, Queensland, Australia Tel: +61-7-3346-2019 Fax: +61-7-334-2101 E-mail: [email protected]

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ABSTRACT

Cyclotides are macrocyclic proteins produced by plants for host defence. Although they occur sparsely in other plant families, cyclotides have been detected in every Violaceae plant species so far screened. Many of the Violaceae species examined until now have been from closely related geographical regions or habitats. To test the hypothesis that cyclotides are ubiquitous in this family, two geographically isolated (and critically endangered) species of Australasian Violaceae, namely Melicytus chathamicus and M. latifolius were examined. Surprisingly, we discovered a suite of cyclotides possessing novel sequence features, including a lysine-rich nature, distinguishing them from ‘conventional’ cyclotides and suggesting that they might have different physiological activities in plants to those reported to date. The newly discovered cyclotides were found to bind to lipid membranes and were cytotoxic against cancer cell lines but had low toxicity against red blood cells, which is advantageous for potential therapeutic applications. This suite of novel Lys-rich cyclotides emphasizes the broad diversity of cyclotides in Violaceae species.



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INTRODUCTION

Cyclotides1 are plant-derived disulfide-rich mini-proteins ranging from 28 to 37 amino acids in size, which feature a head-to-tail cyclic backbone as part of their characteristic cyclic cystine knot (CCK) structure.2 The CCK is formed by three disulfide bonds within a circular peptide backbone, whereby two disulfide bonds and their connecting backbone segments are penetrated by the third disulfide bond, forming a knot-like structure (Figure 1). This unique structural motif engenders cyclotides with exceptional resistance to thermal, chemical or enzymatic degradation.2,

3

Six

backbone loops, which vary widely in sequence, protrude from the CCK core of cyclotides and are considered to be combinatorial display elements that are responsible for the diverse bioactivities of cyclotides.4, 5

The first cyclotide was discovered during pharmacological investigation of “kalata-kalata”, a herbal decoction prepared by boiling leaves of Oldenlandia affinis, a plant belonging to the Rubiaceae (coffee) family, and used by Congolese women to accelerate childbirth.6 Chemical analysis of the decoction revealed the presence of heat-stable peptides, including the prototypic cyclotide kalata B1 (kB1) which was later discovered to be responsible for the uterotonic activity.7 The ethnomedicinal use of plant extracts containing cyclotides provided anecdotal evidence for their heat stable nature, bioavailability and exceptional stability against enzymatic degradation. The elucidation of the CCK structure of kalata B18 provided a molecular explanation for this exceptional stability.

Cyclotides are grouped into two major subfamilies, termed Möbius and bracelet, based on the presence or absence of a cis-Pro residue in loop 5 of the peptide backbone (Figure 1a). As the names suggest, the cis-Pro moiety induces a 180º twist in the backbone protein ribbon, similar to 3 ACS Paragon Plus Environment

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the topology of a Möbius strip, whereas the absence of this cis-Pro results in a cyclic backbone resembling an untwisted bracelet.2 A third subfamily of cyclotides referred to as the trypsin inhibitor cyclotides,9 exemplified by MCoTI-I and MCoTI-II, is found in the seeds of the tropical vine Momordica cochinchinensis, from the Cucurbitaceae family.10 Although members of this subfamily are not homologous to bracelet or Möbius cyclotides, their cystine knot pattern and biosynthetic cyclization mechanism justify their inclusion in the cyclotide family. Compared to cyclotides from the Möbius and bracelet subfamilies, MCoTI-I and II are more hydrophilic11 and have a greater number of positively charged residues. Of interest from a drug design perspective, a mutagenesis study on MCoTI-II suggested an important role for positively-charged residues in the cell penetrating properties of this cyclotide.11 Since their initial discovery in O. affinis (Rubiaceae), numerous cyclotides have been discovered in plants belonging to the Rubiaceae,12-14 Violaceae,15,

16

Cucurbitaceae,10 Fabaceae17,

18

and

Solanaceae19 families at both protein and DNA levels. Bioinformatics and expression analysis approaches have also shown the presence of cyclotide-like sequences in monocots from the Poaceae family,20 including rice, wheat and corn.21, 22 Although the Rubiaceae is one of the largest plant families, with 600 genera and 13,000 species,23 only a minority (10

0.31 ± 0.01

08.14 ± 0.69

1.18 ± 0.28

58.88 ± 13.51

>10

0.30± 0.02

12.28 ± 1.39

2.15 ± 0.42

0.30± 0.06

97.43 ± 40.19c

>10

0.32 ± 0.01

12.20± 1.14

1.51 ± 0.32

mela 6&7

0.24 ± 0.02

28.69 ± 3.25

1.53 ± 0.12

0.33 ± 0.00

13.37 ± 0.20

0.6 ± 0.09

mech 2&3

0.25 ± 0.01

33.62 ± 2.22

7.71 ± 0.28

0.35 ± 0.01

17.50 ± 0.72

8.93 ± 0.32

kB1

0.19 ± 0.01

16.35 ± 1.11

4.44 ± 0.21

0.26 ± 0.00

19.79 ± 0.43

a

>10

Membrane-binding dose-responses of cyclotides were studied by SPR (refer to Fig 3A) and the affinity compared with P/Lmax (the maximum binding response) and KD (concentration of peptide required to achieve half-maximum binding at equilibrium). KD and P/Lmax values were obtained by fitting dose-response curves with one-site specific binding with Hill slope. bConcentration required to induce leakage in 50% of the vesicles. Total lipid concentration is 5 µM. cDifficult to establish the maximum response as the curve did not reach the plateau in the concentration range tested.


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Table 4. Cytotoxicity of selected cyclotides against cultured cells and fresh red blood cells (RBCs)a Peptide

MM96L

HFF-1

HELA

RBCs

mela 1

2.09 ± 0.18

3.07 ± 0.15

9.83 ± 0.78

>64

mela 2

1.30 ± 0.44

5.86 ± 0.38

19.26 ± 1.37

>64

mela 3&4

2.04 ± 0.26

5.39 ± 0.33

18.73 ± 1.82

NDb

mela 6&7

1.58 ± 0.48

3.13 ± 0.16

11.42 ± 1.00

>64

mech 2&3

21.94 ± 1.90

12.45 ± 0.99

36.92 ± 3.25

>64

2.50 ± 0.50

7.43 ± 0.46

12.33 ± 0.72

27.58 ± 1.45

kB1 a

The concentration required to kill 50% of cells (CC50 ± SD) is given in µM. CC50 and SD were determined by fitting dose-response curves with nonlinear regression equation with Hill slope. Dose-responses were conducted in triplicates. bnot determined, as the amount of peptide was insufficient for the experiments.



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Figure 1


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Figure 2



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Figure 3


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Figure 4


Lysine-rich Cyclotides: A New Subclass of Circular Knotted Proteins

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