Purification and mass spectrometry-assisted sequencing of basic antifungal proteins from seeds of pumpkin ( Cucurbita maxima)

Abstract

A number of different basic polypeptides with in vitro antifungal activity were isolated from the soluble and cell wall-derived fractions of seeds of pumpkin ( Cucurbita maxima) using batchwise chromatography on carboxymethyl-cellulose (CM52) and reverse-phase HPLC. Polypeptide masses were determined by electrospray ionisation mass spectrometry (ESMS). Reverse-phase HPLC, on a C8 column subjected to a gradient of increasing CH 3CN concentration in 0.1% TFA, yielded successive peaks 1–4 containing a multiplicity of components with the following masses (most abundant components underlined): peak 1 and peak 2 ( 4650 , 4779, 4850, 4890, 4961, 5097 and 5253 Da), peak 3 (5253, 7084, 7194 , 7308 and 7350 Da) and peak 4 (11 696 Da). An additional 2249 Da component was isolated from the cell-wall enriched fraction, but was not present in the soluble fraction. Fractions containing the major 2249, 4650 and 11 696 Da components exhibit antifungal activity against a variety of fungi. The 11 696 Da component is a napin-like protein composed of a 3700 Da small chain (CS1) and an 8004 Da large chain (CL1) that are joined by two of the four disulphide linkages in the oxidised complex. The complete amino acid sequences of the napin-like protein small and large chains, the 2249 Da cell-wall protein and the 4650 Da protein (and its associated homologues) were determined from Edman sequencing, previously published cDNA sequence data and corroborative ESMS measurements of the masses of tryptic fragments and/or intact proteins. Within experimental uncertainty, the calculated masses for the napin-like protein major small and large chains and for the corresponding oxidised complex are in exact agreement with their observed masses. Unlike many other basic antifungal proteins, the 11 696 Da napin-like protein is neither a substrate for Ca 2+-dependent protein kinase (CDPK) nor a calmodulin (CaM) antagonist. CS1 and CL1 do not have the putative `two-sided' α-helical structural element found in all the related napin and napin-like protein small and large chains shown to be CaM antagonists.