Novel flax orbitide derived from genetic deletion

Abstract

BackgroundFlaxseed orbitides are homodetic plant cyclic peptides arising from ribosomal synthesis and post-translation modification (N to C cyclization), and lacking cysteine double bonds (Nat Prod Rep 30:108-160, 2013). Screening for orbitide composition was conducted on the flax core collection (FCC) grown at both Saskatoon, Saskatchewan and Morden, Manitoba over three growing seasons (2009-2011). Two flax (Linum usitatissimum L.) accessions ‘Hollandia’ (CN 98056) and ‘Z 11637’ (CN 98150) produce neither [1−9-NαC]-linusorb B2 (3) nor [1−9-NαC]-linusorb B3 (1). Mass spectrometry was used to identify novel compounds and elucidate their structure. NMR spectroscopy was used to corroborate structural information.ResultsExperimental findings indicated that these accessions produce a novel orbitide, identified in three oxidation states having quasimolecular ion peaks at m/z 1072.6 (18), 1088.6 (19), and 1104.6 (20) [M + H]+ corresponding to molecular formulae C57H86N9O9S, C57H86N9O10S, and C57H86N9O11S, respectively. The structure of 19 was confirmed unequivocally as [1−9-NαC]-OLIPPFFLI. PCR amplification and sequencing of the gene coding for 18, using primers developed for 3 and 1, identified the putative linear precursor protein of 18 as being comprised of the first three amino acid residues of 3 (MLI), four conserved amino acid residues of 3 and/or 1 (PPFF), and the last two residues of 1 (LI).ConclusionComparison of gene sequencing data revealed that a 117 base pair deletion had occurred that resulted in truncation of both 3 and 1 to produce a sequence encoding for the novel orbitide precursor of 18. This observation suggests that repeat units of flax orbitide genes are conserved and suggests a novel mechanism for evolution of orbitide gene diversity. Orbitides 19 and 20 contain MetO and MetO2, respectively, and are not directly encoded, but are products of post-translation modification of Met present in 18 ([1−9-NαC]-MLIPPFFLI).Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1303-8) contains supplementary material, which is available to authorized users.

Synthesis of [1‐8‐NαC]‐zanriorb A1, alanine‐containing analogues, and their cytotoxic and anti‐inflammatory activity

“…The cyclic[Ala 2 ]-zanriorb A1 (8) with Pro 2 substituted by Ala was more potent in the inhibition of NO production (IC 50 = 65.0 ± 3.5 μM) than the reference standard L-NMMA (IC 50 = 97.5 ± 3.2 μM). Cyclic [Ala 5,6,8 ]-zanriorb A1 (10) in which all achiral Gly residues were replaced with chiral Ala was even more active, with an IC 50 = 22.2 ± 0.2 μM. We probed this result further with an additional analogue, cyclic[Ala 1,6,8 , Glu 2 ]-zanriorb A1 (11), prepared by on-resin cyclization with the Wang resin using the strategy as described by us earlier.…”

“…In addition, we prepared several alanine containing analogues of the natural product 1, and these were also found to be not active against Jurkat and MCF-7 breast cancer cells as well as non-toxic against normal fibroblast NIH-3T3 cells. The cyclic[Ala 5,6,8 ]-zanriorb A1 (10) with all three Gly residues replaced with Ala was identified as a potent inhibitor of nitric oxide production in LPS activated J774.2 macrophages. The synthesized cyclic[Ala 5,6,8 ]-zanriorb A1 is noncytotoxic and possesses a great potential to be served as nitric oxide inhibitor, hence can act as a lead anti-inflammatory molecule.…”

“…Linear analogue 8a: (Phe 4 -Phe 3 -Ala 2 -Pro 1 -Gly 8 -Phe 7 -Gly 6 -Gly 5 for 781.3673, C 41 H 49 O 8 N 8 ). 1 H-NMR and 13 C-NMR (Table S3).…”

“…The homodetic orbitides have been isolated from at least nine plant families, and many have bioactivities such as antioxidative, antimalarial, anticancer and immunomodulation. [5][6][7] For example, cycloleonurinin exhibited a significant immunosuppressive activity comparable with the clinical drug cyclosporin A. 8 Phytochemical investigation of Zanthoxylum riedelianum Engl.…”

Plant derived cyclic peptides

“…Orbitides from other plant families have also been found recently, including a cyclic peptide from Pseudostellaria heterophylla [ 14 ] and a novel orbitide derived from a genetic deletion was detected from flaxseed [ 15 ]. Furthermore, a study that revisited the discovery of evolidine from the Rutaceae species M. xanthoxyloides also resulted in the discovery of six novel orbitides (xanthoxycyclin A–F) encoded by similar transcripts [ 3 ].…”