Biosynthesis and insecticidal properties of plant cyclotides: the cyclic knotted proteins from Oldenlandia affinis

Abstract

Several members of the Rubiaceae and Violaceae families produce a series of cyclotides or macrocyclic peptides of 29-31 amino acids with an embedded cystine knot. We aim to understand the mechanism of synthesis of cyclic peptides in plants and have isolated a cDNA clone that encodes the cyclotide kalata B1 as well as three other clones for related cyclotides from the African plant Oldenlandia affinis. The cDNA clones encode prepropeptides with a 20-aa signal sequence, an N-terminal prosequence of 46-68 amino acids and one, two, or three cyclotide domains separated by regions of about 25 aa. The corresponding cyclotides have been isolated from plant material, indicating that the cyclotide domains are excised and cyclized from all four predicted precursor proteins. The exact processing site is likely to lie on the N-terminal side of the strongly conserved GlyLeuPro or SerLeuPro sequence that flanks both sides of the cyclotide domain. Cyclotides have previously been assigned an antimicrobial function; here we describe a potent inhibitory effect on the growth and development of larvae from the Lepidopteran species Helicoverpa punctigera.

Figure 1

(A) Schematic representation of kalata B1 showing the cyclic cystine knot, the amino acid sequence in single letter code, and the regions used for oligonucleotide primer design (shaded). (B) The primers used in the PCR reactions. I represents inosine, Y represents C or T, and R represents A, C, T, or G. The introduced restriction enzyme sites are in italics. (C) Amino acid sequence of the protein encoded by the Oak1 clone. The sequence corresponding to the PCR product obtained with the Kal2 and oligo-dT primers is shaded.

Figure 2

(A) Block diagram of the precursor proteins predicted from the Oak1, 2, 3, and 4 clones showing the signal peptide (light shading), the regions corresponding to the mature kalata peptides (dark shading), the region of 22 aa on the N-terminal side of the kalata peptide sequence (N-T repeat, hatched). (B) Alignment of the amino acid sequence of the proteins encoded by the Oak1–Oak4 clones (labeled 1–4, respectively). The sequence begins with the first amino acid after the signal peptide. The N-T repeat sequences are boxed and hatched. The kalata sequences are shaded. Identical amino acids in the N-T repeat and kalata sequences are marked with an asterisk, and similar amino acids are marked with a dot. Gaps (−) were introduced to maximize the alignment. (C) The potential processing sites. The mature cyclic peptide retains one copy of the Gly-Leu-Pro sequence that could be derived entirely from one of the two flanking elements or partially from both depending on the initial cleavage sites for B1, B3, B6, and B7. The retention of Gly-Leu-Pro in the B2 cyclotide suggests cleavage before the Gly and Ser residues.

Figure 3

Gel blot analysis of RNA and genomic DNA from O. affinis. (A) Blot of genomic DNA digested with: (1) HindIII, (2) BamHI, (3) Nde1, and (4) EcoRV probed with radiolabeled Oak1. (Upper) (i) Blot of total RNA from 1 roots; 2, leaves; 3, shoots probed with radiolabeled Oak1. (Lower) Identical gel to (Upper) stained with ethidium bromide.

Figure 4

Effect of kalata B1 on growth and development of H. punctigera larvae. (A) Survival of larvae fed an artificial diet containing kalata B1 (■) and the control (●) diet. (B) Average mean weight of larvae fed on kalata B1 (■) and control (●) diet. (C) Size of larvae after 16 days on artificial diet containing kalata B1 (Left) or control diet (Right). (Bar = 1 cm.)