Sunflower trypsin inhibitor‐1, proteolytic studies on a trypsin inhibitor peptide and its analogs

Abstract

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14 amino acid cyclic peptide from sunflower seeds, which possesses exceptionally potent trypsin-inhibitory activity, and has promise as a stable peptide-based drug template. Within its compact structure, SFTI-1 combines a head-to-tail cyclized backbone and a disulfide bond. In this study, we synthesized a range of acyclic and disulfide-deficient analogs of SFTI-1 to investigate enzyme-assisted cyclization of the peptide backbone and proteolytic degradation that occurs as a result of incubation with trypsin. Electrospray and matrix-assisted laser desorption ionization mass spectrometry allowed the characterization of a range of novel degradation products and elucidation of the time-course for cyclization and/or proteolysis. Trypsin displayed the ability to resynthesize the scissile bond(s) and hence cyclize two of the linear permutants, whereas irreversible degradation was observed for another two permutants. An interesting ring contraction mediated by trypsin was observed, supporting a role for protease catalyzed splicing as a way of increasing the combinatorial diversity of cyclic peptides in nature. Disulfide-deficient mutants were degraded within minutes, emphasizing the critical role of the cysteine bridge in maintaining proteolytic stability of SFTI-1. Overall, the study provides additional support for the proposal that naturally occurring cyclic peptides like SFTI-1 are biosynthesized by proteolytic enzymes effectively catalyzing the reverse of their normal reaction to make, rather than break peptide bonds.