Identification of candidates for cyclotide biosynthesis and cyclisation by expressed sequence tag analysis of Oldenlandia affinis

Abstract

Background: Cyclotides are a family of circular peptides that exhibit a range of biological activities, including anti-bacterial, cytotoxic, anti-HIV activities, and are proposed to function in plant defence. Their high stability has motivated their development as scaffolds for the stabilisation of peptide drugs. Oldenlandia affinis is a member of the Rubiaceae (coffee) family from which 18 cyclotides have been sequenced to date, but the details of their processing from precursor proteins have only begun to be elucidated. To increase the speed at which genes involved in cyclotide biosynthesis and processing are being discovered, an expressed sequence tag (EST) project was initiated to survey the transcript profile of O. affinis and to propose some future directions of research on in vivo protein cyclisation.

Results: Using flow cytometry the holoploid genome size (1C-value) of O. affinis was estimated to be 4,210 - 4,284 Mbp, one of the largest genomes of the Rubiaceae family. High-quality ESTs were identified, 1,117 in total, from leaf cDNAs and assembled into 502 contigs, comprising 202 consensus sequences and 300 singletons. ESTs encoding the cyclotide precursors for kalata B1 (Oak1) and kalata B2 (Oak4) were among the 20 most abundant ESTs. In total, 31 ESTs encoded cyclotide precursors, representing a distinct commitment of 2.8% of the O. affinis transcriptome to cyclotide biosynthesis. The high expression levels of cyclotide precursor transcripts are consistent with the abundance of mature cyclic peptides in O. affinis. A new cyclotide precursor named Oak5 was isolated and represents the first cDNA for the bracelet class of cyclotides in O. affinis. Clones encoding enzymes potentially involved in processing cyclotides were also identified and include enzymes involved in oxidative folding and proteolytic processing.

Conclusion: The EST library generated in this study provides a valuable resource for the study of the cyclisation of plant peptides. Further analysis of the candidates for cyclotide processing discovered in this work will increase our understanding and aid in reconstructing cyclotide production using transgenic systems and will benefit their development in pharmaceutical applications and insect-resistant crop plants.

Figure 1

Fluorescence histogram of propidium iodide-stained nuclei isolated from fresh leaf tissues of Oldenlandia affinis seedlings. Glycine max 'Polanka' (2C = 2.50 pg) was used as an internal reference standard. Nuclei from the sample and the standard were simultaneously isolated, stained, and analysed. The actual ratio between both peaks in this histogram is 3.42. Each plant was re-estimated three times on different days.

Figure 2

The functional distribution of EST clones by modified MIPS based functional classification for O. affinis. BLASTX comparisons to predicted proteins from Arabidopsis were used to assign O. affinis ESTs based on functional annotation after MIPS FunCat schema. The cyclotide precursors are pointed out within "No match" group since Arabidopsis does not contain cyclotides.

Figure 3

Cyclotide precursor constructions and maturation in O. affinis. A) Cyclotide precursor constructions of O. affinis. (ER, endoplasmic reticulum; NTPD, N-terminal pro-domain; NTR, N-terminal repeat; "kB" is the abbreviation of kalatas). B) Alignment of cyclotide domains including their flanking sequences. Red letters under the sequences indicate conserved or relatively conserved amino acids. The six conserved cysteines are numbered using Roman numerals and their connectivity is shown. C) Proposed molecular model for in vivo formation of cyclotides in O. affinis. Formation of disulfide bond requires redox systems, which consists of the ferredoxin/thioredoxin system, the NADP/thioredoxin system and the glutathione/glutaredoxin system. Excision of the cyclotide domains from precursor proteins and their cyclisation are thought to be catalysed by endopeptidases [18,19]. Round black disks represent the cysteins and they are connected by disulfide bonds showing in the structures.

Figure 4

MALDI-TOF MS profile of crude peptide extracts from O. affinis leaves. The calculated (Calc) and observed (Obsv) monoisotopic masses for the cyclotides kalata B1, B2 and B3 are included and serve as internal controls. Peaks for kalata B6 (kB6) and kalata B7 (kB7) are marked without mass data. The calculated mass for kalata B19 (3092.34) is close to a mass in this profile (3091.47), but when its error (represented as ppm) is compared to the errors of other cyclotides in the same profile, the difference between calculated and observed is too great for this mass to be B19.