Characterizing circular peptides in mixtures: sequence fragment assembly of cyclotides from a violet plant by MALDI-TOF/TOF mass spectrometry

Hossein Hashempour 0 1 2 Johannes Koehbach 0 1 2 Norelle L. Daly 0 1 2 Alireza Ghassempour 0 1 2 Christian W. Gruber 0 1 2 0 N. L. Daly School of Pharmacy and Molecular Sciences, Centre for Biodiscovery and Molecular Development of Therapeutics, Queensland Tropical Health Alliance, James Cook University , Cairns 4878, Australia 1 H. Hashempour A. Ghassempour Medicinal Plants and Drugs Research Institute, Shahid Beheshti University , G.C. Evin, Tehran, Iran 2 H. Hashempour J. Koehbach C. W. Gruber (&) Center for Physiology and Pharmacology, Medical University of Vienna , Schwarzspanierstrasse 17, 1090 Vienna, Austria Cyclotides are a very abundant class of plant peptides that display significant sequence variability around a conserved cystine-knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability. Their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals. However, laborious isolation and purification prior to de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development. Here we extend the knowledge about their sequence diversity by analysing the cyclotide content of a violet species native to Western Asia and the Caucasus region. Using an experimental approach, which was named H. Hashempour and J. Koehbach contributed equally to this study. - Cyclotides are a unique class of cysteine-rich macrocyclic mini-proteins of about 30 amino acids in size that are defined by a head-to-tail cyclized backbone and three disulfide bonds in a knotted arrangement referred to as cyclic cystine-knot (CCK) motif (Craik et al. 1999). Their knotted structure makes them exceptionally stable against thermal, chemical and enzymatic degradation (Colgrave and Craik 2004). Cyclotides have been discovered and isolated from plants of the violet (Violaceae), coffee (Rubiaceae), cucurbit (Cucurbitaceae) and legume family (Fabaceae) (Poth et al. 2010). Their distribution within the plant kingdom still remains unclear (Gruber 2010), but they are expected to be far more widespread and the number of different cyclotides may be around 50,000 (Gruber et al. 2008; Simonsen et al. 2005) making them one of the largest peptide classes within plants. In agreement with their anticipated number, recent studies report the presence of more than 70 different cyclotides within one single species (Seydel et al. 2007; Grundemann et al. 2012). The first cyclotide kalata B1 was discovered from kalata-kalata, a decoction from leaves of Oldenlandia affinis, which has been used as a remedy during childbirth in African ethnomedicine due to its uterotonic activity (Gran 1970; Gruber and OBrien 2011). In line with their reported antibacterial (Tam et al. 1999), antifouling (G oransson et al. 2004), anthelmintic (Colgrave et al. 2008) and insecticidal properties (Jennings et al. 2001; Gruber et al. 2007a; Barbeta et al. 2008) their native function seems to be part of the plant defence system. As a key feature, cyclotides are amenable to various amino acid changes by peptide engineering, which highlights the flexibility and plasticity of the cyclotide framework (Clark et al. 2006). Thus, their high sequence diversity is extensively under investigation for being utilized as scaffolds in the development of agrochemicals and pharmaceuticals (Henriques and Craik 2010). Besides these distinct differences in the sequences of the so-called intercysteine loops, cyclotides can be divided into two subfamilies, i.e., Mobius or bracelet type cyclotides based on the presence or absence of a cis-Pro residue in loop 5 (Fig. 1) (Craik et al. 1999). These differences have further implications regarding their physico-chemical properties. Whereas most Mobius cyclotides are slightly negatively charged or have an overall net-charge of zero, bracelet cyclotides are usually multiply positively charged. This ultimately influences their chemical behaviour and amenability to sequencing and oxidative folding, which are still challenges, in particular for bracelet cyclotides. Usually, amino acid sequencing of cyclotides is performed after enzymatic digestion of peptides that have been laboriously purified by reversed-phase high cycloviolacin O2 III loop 1 I II III IV V VI kalata B1 G LPVCGETCVGGTCNTP--GCTCSWPVCTR N cycloviolacin O2 G IP-CGESCVWIPCISSAIGCSCKSKVCYR N Fig. 1 Ribbon structures of the cyclotides kalata B1 (left panel), a representative of the Mobius subfamily and cycloviolacin O2 (right panel) belonging to the bracelet subfamily are shown as cartoons. The unique cyclic cystine-knot (CCK) motif with three conserved disulfide bonds (yellow) and the cyclized backbone (black dots and connecting line) as well as typical secondary structure elements of ahelices (blue) and b-sheets (red) and their respective sequences are shown (PDB code: 1NB1 and 2KNM, respectively). The disulfide connectivity CIIV, CIIV and CIIIVI has been indicated with black lines (color figure online) performance liquid chromatography (RP-HPLC) to produce single linearized peptides that are amenable to tandem mass spectrometry (MS) analysis. However, the complexity of cyclotide plant extracts, which comprise dozens of distinct peptides, limits their analysis and characterization by standard MS analysis. Using endoproteinase GluC (endo-GluC), cyclotides are mostly cleaved to yield a single (ring-opened) peptide fragment due to a conserved glutamic acid in loop 1, whereas the use of trypsin and chymotrypsin usually yields several fragments due to multiple cleavage sites. When applied to the analysis of cyclotide mixtures as they occur in plant extracts, mass spectra may be confusing and hard to evaluate caused by fragment ion overlays. Hence the application of combinations of digests to obtain peptide-specific fragments and the subsequent accurate assembly of sequence fragments may overcome this issue. Particularly for bracelet cyclotides this is of importance since until now the majority (*70 %) of more than 200 published cyclotide sequences accessible on CyBase (Wang et al. 2008b) belong to this subfamily. Besides the complexity of cyclotide sequence analysis, another issue associated with their great diversity is their chemical and biological synthesis. Previous studies have shown that different enzymes seem to be involved in backbone cyclization and disulfide bond formation (Gruber et al. 2007b; Saska et al. 2007) during biosynthesis of these gene-encoded peptides in planta. However, the community still lacks clarity about this process, in particular with respect to the sequence-folding relationship, i.e., how the inter-cysteine sequences of different cyclotides can influence the formation of the native CCK-motif and hence determine their folding yield. As a consequence, in vitro oxidative folding is still a major challenge in cyclotide engineering. Whereas high-yield chemi (...truncated)