New Flaxseed Orbitides: Detection, Sequencing and 15N Incorporation

Three new orbitides (cyclolinopeptides 17, 18 and 19) were identified in flaxseed (Linum
usitatissimum L.) extracts without any form of purification. Their structures were
elucidated by a combination of 15N-labeling experiments and extensive HPLC-ESIMS/
MS analyses. Putative linear peptide sequences of the new orbitides were used as the
query in TBLASTN searches of a flax genome database. These searches returned linear
sequences for the putative precursors of cyclolinopeptides 17 and 19 among others.
Cyclolinopeptide 18 contains MetO (O) and is not directly encoded, but is a product of
post-translation modification of the Met present in 17. The identification of precursor
proteins in flax mRNA transcripts and DNA sequences confirmed the occurrence and
amino acid sequences of these orbitides as [1-9-NαC]-MLKPFFFWI, [1-9-NαC]-
OLKPFFFWI and [1 9-NαC] GIPPFWLTL for cyclolinopeptides 17, 18 and 19,
respectively. (Authors abstract)

Plant cyclic peptides or cyclopeptides have been isolated from about 26 families of higher plants. They have been classified into eight types based on chemical structures. Type VI (Caryophyllaceae type homomonocyclopeptides) and Type VIII (Cyclotides) peptides are cyclic compounds that contain natural amino acids and are formed from the cyclization of linear precursors via fusion of the terminal amino and carboxy residues in a peptide bond. The peptide bonds can arise from 2 to 37 proteinaceous and/or nonproteinaceous amino acid residues.  Cyclopeptides exhibit a wide range of pharmacological activities and appear to have higher potency than linear peptides, a characteristic attributable to the stable configuration conferred by their cyclic structures.  Flax is an example of a higher plant that is rich in hydrophobic orbitides or cyclolinopeptides that occur in the seeds and roots.  Cyclolinopeptides are biologically active; induce peripheral to blood lymphocytes, suppress immune responses in human and mouse peripheral to blood lymphocytes, and possess antimalarial activity.   Flax orbitides were numbered in the order of their discovery. Isolation of the first orbitide, cyclolinopeptide A (1) was reported in 1959, whereas characterization of the second cyclolinopeptide (2) was reported almost 38 years later.  The isolation of cyclolinopetides C, D, and E (3, 6, and 8, respectively) were reported in 1999. Matsumoto et al. published the isolation of cyclolinopeptides F, G, H and I (12, 16, 14, 11), while Stefanowicz simultaneously reported cyclolinopeptides D’, E’, F and G (5, 7, 10, 13).   Matsumoto and coworkers also described two additional cyclolinopeptides, CLs J and K (9, 4), as the methionine S,S to dioxide derivatives of CLs B and E’ (2, 7), respectively.  Here the authors propose a naming system similar to that used by Olivia and coworkers19 and one that is based on current literature precedents, specifically IUPAC such that (i) the amino acid sequence reflects the gene sequences of their known precursor peptides, instead of being based on the order of fragmentation in a mass spectrometer; (ii) the numbering of the amino acid residues commence at the N terminal and proceed to higher numbers ending at the C to terminal where linkage is through the alpha amino group and (iii) a distinction is possible between linear and cyclic peptides possessing the same amino acid sequence. The proposed naming system enables the systematic representation of amino acid variants.

Orbitides occur in flaxseed at low concentrations, thus, isolation of individual peptides for complete spectroscopic characterization requires large quantities of starting material.  As a consequence, the discovery and characterization of new orbitides, has been limited. Development of sensitive analytical tools such as mass spectrometry (MS), however, allows the discovery of new compounds in complex mixtures, while using very small amounts of starting material. Following HPLC to MS analyses of fresh flaxseed extracts, we identified sixteen orbitide sequences, three of which were new. The orbitide structures were elucidated using detailed HPLC to MS/MS analyses and 15N labeling experiments. Additionally, we identified genes that encode for ribosomal biosynthesis of the linear precursors of these orbitides.

The current progress made in flax genome sequencing does provide some answers to address these uncertainties. For instance, a flaxseed expressed sequence tag (EST) library was screened with the linear peptide sequences MLLPFFWI, MLMPFFWV, and MLMPFFWI for 5, 10 and 13, respectively, resulting in the identification of several putative transcripts. Additionally, the genomic DNA sequence of a gene encoding for linear putative precursors of 1, 2, 7 has been identifed.  These sequences are consistent with the synthesis of these flax orbitides from precursor proteins that are genomically to encoded and ribosomally to derived. Thus, the availability of these genomic databases provides additional tools for unambiguous determination of the amino acid sequence of a given flaxseed orbitide.

Discovery of new flaxseed orbitides, like most valuable natural products, is limited by their occurrence in extremely low concentrations. However, we elucidated structures of new linseed orbitides in crude linseed extracts without any form of purification. This was achieved by a combination of HPLC to MS/MS analyses, labeling experiments, and identification of putative precursor genes of these orbitides. This work demonstrates the utility of this approach in the identification of novel natural products with potential pharmaceutical applications. Once identified, a target to compound purification approach can be employed to obtain sufficient quantities of such compounds to perform structure activity relationship studies.