Poster Presentation 10th Australian Peptide Conference 2013

Hunting for Cystine Knot Peptides in Seeds (#198)

Tunjung Mahatmanto 1 , Aaron Poth 1 , David Craik 1
  1. Institute for Molecular Bioscience, The University of Queensland, Brisbane/St. Lucia, QLD, Australia

Plant-derived cystine knot peptides – unique peptides sharing a disulfide knotted core, which endows them high stability against enzymatic, chemical and thermal degradation – have promising use in medicine and agriculture. To fully exploit cystine knot peptides for these purposes, it is imperative to understand their diversity (inter-relationship between sequence, structure, function) and distribution (origin, localization, evolution); knowledge grounded on the discovery of cystine knot peptides. At times, discovery efforts are hampered by the availability of plant materials. However, current screening strategies have not taken into account the efficiency of extraction methods and availability of plant materials. Therefore, the development of an efficient screening strategy is vital. Here, we evaluate five methods commonly used to extract cystine knot peptides: acetonitrile/formic acid1, boiled water, dichloromethane/methanol2, ammonium bicarbonate3, and sodium acetate/acetone4, using small amounts of Momordica cochinchinensis (gac) seeds as the plant material. The relative abundances of nine known cystine knot peptides, i.e. MCoTI-I, II, IV, V, VI, VII, VIII, Mco-1 and Mco-2, inferred from their extracted ion chromatogram peak intensities, revealed large differences in extraction yield with the acetonitrile/formic acid method having an average of 42% higher yield than the second best (sodium acetate/acetone). Having established the best extraction method, the next challenge is sequencing the cyclic version of cystine knot peptides in crude extracts; given the complexity of fragments that could be generated by enzymatic digestion. Therefore, ongoing work attempts to develop a one pot reduction-alkylation-cleavage to produce full-length linearized cyclic cystine knot peptides in crude extracts for tandem MS sequencing. These data may then be interrogated against transcriptomic data, which would only require relatively small amounts of plant materials to generate, to confirm the peptide sequences and thus providing a new approach for the discovery of cystine knot peptides from rare samples.

  1. Poth, A. G.; Colgrave, M. L.; Philip, R.; Kerenga, B.; Daly, N. L.; Anderson, M. A.; Craik, D. J., Discovery of cyclotides in the Fabaceae plant family provides new insights into the cyclization, evolution, and distribution of circular proteins. ACS Chem. Biol. 2011, 6 (4), 345-355.
  2. Craik, D. J.; Daly, N. L.; Bond, T.; Waine, C., Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif. J. Mol. Biol. 1999, 294 (5), 1327-1336.
  3. Hamato, N.; Koshiba, T.; Pham, T. N.; Tatsumi, Y.; Nakamura, D.; Takano, R.; Hayashi, K.; Hong, Y. M.; Hara, S., Trypsin and elastase inhibitors from bitter gourd (Momordica charantia LINN.) seeds: Purification, amino acid sequences, and inhibitory activities of four new inhibitors. J. Biochem. 1995, 117 (2), 432-437.
  4. Chan, L. Y.; Wang, C. K. L.; Major, J. M.; Greenwood, K. P.; Lewis, R. J.; Craik, D. J.; Daly, N. L., Isolation and characterization of peptides from Momordica cochinchinensis seeds. J. Nat. Prod. 2009, 72 (8), 1453-1458.