Defense proteins from seed of Cassia fistula include a lipid transfer protein homologue and a protease inhibitory plant defensin

A novel trypsin inhibitor was extracted from the seeds of Cassia fistula by a process successively involving soaking seeds in water, extraction of the seeds in methanol, and extraction of the cell wall material at high ionic strength. The protease inhibitor (PI) was subsequently purified by chromato graphy on carboxymethylcellulose, gel filtration and reversed phase HPLC (R P-HPLC). Electrospray ionization mass spectrometry (ESMS) of the oxidized f rom of the PI yielded an average molecular mass of 5458.6 +/- 0.8 Da. Edman sequencing of the PI yielded a full-length 50 amino acid sequence inferred to contain eight cysteines and with a calculated average molecular mass (f ully oxidized form) of 5459.3 Da, in agreement with the observed mass. The C. fistula seed PI is homologous to the family of plant defensins (gamma -t hionins), which have four disulfide linkages at highly conserved locations. The C. fistula PI inhibits trypsin (IC50 2 muM), and is the first known ex ample of a plant defensin with protease inhibitory activity, suggesting a p ossible additional function for some members of this class of plant defensi ve proteins. C. fistula seeds also contain a 9378 Da lipid transfer protein (LTP) homologue, other LTPs, a 7117 Da protein copurifying with PI activit y and a 5144 Da defensin which does not inhibit trypsin. The complete seque nce of the 5144 Da defensin was determined by Edman sequencing, yielding a calculated average molecular mass (oxidized form) of 5144.1 Da, in agreemen t with the mass observed by ESMS. The likely trypsin inhibitory residue on the 5459 Da defensin is Lysine-25, the corresponding amino acid being Tyros ine-25 in the homologous 5144 Da defensin that is not a trypsin inhibitor. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.