Understanding the Diversity and Distribution of Cyclotides from Plants of Varied Genetic Origin

Cyclotides are a large family of
naturally occurring plant-derived
macrocyclic cystine-knot peptides, with more than 400 having been
identified in species from the Violaceae, Rubiaceae, Cucurbitaceae,
Fabaceae, and Solanaceae families. Nevertheless, their specialized
distribution within the plant kingdom remains poorly understood. In
this study, the diversity of cyclotides was explored through the screening
of 197 plants belonging to 43 different families. In total, 28 cyclotides
were sequenced from 15 plant species, one of which belonged to the
Rubiaceae and 14 to the Violaceae. Every Violaceae species screened
contained cyclotides, but they were only sparsely represented in Rubiaceae
and nonexistent in other families. The study thus supports the hypothesis
that cyclotides are ubiquitous in the Violaceae, and it adds to the
list of plants found to express kalata S and cycloviolacin O12. Finally,
previous studies suggested the existence of cyclotide isoforms with
either an Asn or an Asp at the C-terminal processing site of the cyclotide
domain within the precursor proteins. Here we found that despite the
discovery of a few cyclotides genuinely containing an Asp in loop
6 as evidenced by gene sequencing, deamidation of Asn during enzymatic
digestion resulted in the artifactual presence of Asp isoforms. This
result is consistent with studies suggesting that peptides can undergo
deamidation after being subjected to external factors, including pH,
temperature, and enzymatic digestion.