Download ALTERNATIVE SPLICING AND BIOCHEMICAL FUNCTIONS OF

ALTERNATIVE SPLICING AND BIOCHEMICAL FUNCTIONS OF COCOA
CYSTATINS
Pirovani, CP1,2, Santiago, A.S 1, Alvim, FC, Santos, LS, Gesteira, AS1, Margis, R2 ;
Pereira, GAG3, Cascardo, JCM1
1
DCB/UESC, Bahia, Brasil;2CBIOT/UFRGS, 3 IB/UNICAMP, São Paulo, Brasil.
Phytocystatins are cysteine-proteinase inhibitors from plants implicated in the
endogenous regulation of protein turnover, programmed cell death, and defense
mechanisms against pathogens. We identified four cystatins ORFs named
TcCYS1, TcCYS2, TcCYS3 and TcCYS4. The cDNA encode 209, 127, 124 and
205 amino acid residues. Analyses of genes structures suggest that the TcCYS3
is an alternative splicing form of the TcCYS1 and represent family 2 cystatins . The
four ORFs were sub-cloned, and His-Tag fused proteins expressed in E.coli.
Polyclonal antibodies against the recombinant TcCYS1 and TcCYS4 were raised,
allowing the immunodetection of the endogenous proteins in the young plant
tissues. Recombinant proteins showed papain inhibition in gelatin/SDS-PAGE and
colorimetric method with BApNA substrate. We examined the biochemical and
structural properties of TcCYS4 under heat-stress conditions. The enzyme
inhibitory reactivity was reduced by heating in a temperature-dependent manner
and reached a minimal level by heating at 65 °C for 10 min. Size -exclusion
chromatography showed that TcCYS4 forms a homodimer at 65 oC and that the
reduction of inhibitory reactivity is due to the conversion of the monomeric to a
dimeric form. The recombinant proteins revealed a toxic effect on the mycelium
growth of Moniliophthora perniciosa. Similar toxic effect was observed against
tobacco suspension cultures cells. The TcCYS3 and TcCYS4-sepharose-CNBr
immobilized recruited different proteases isoforms in cacao and M. perniciosa
extracts.
Keywords: Theobroma cacao, plant-pathogen interaction, cisteine-protease
Inhibitor, Moniliophthora perniciosa .