Soyacystatin, a novel cysteine proteinase inhibitor in soybean, is distinct in protein structure and gene organization from other cystatins of animal and plant origin

Cystatins, cysteine proteinase inhibitors, deserve note because of their regulatory and protective functions in plant tissues. We isolated both genomic DNA and cDNA clones from soybean that encode a cystatin consisting of 245 amino acid residues (soyacystatin). It is, while basically similar in sequence to known cystatins that are generally in the range of 12 - 15 kDa, characterized by having extremely large extension sequences in both its amino and carboxyl termini. The genomic DNA encoding soyacystatin is also unique in that it consists of four exons with three introns in its coding regions. The mRNA for soyacystatin is distinctly expressed in soybean seeds 2 weeks after flowering. Soyacystatin purified from mature soybean seeds had a molecular mass of about 26 kDa on SDS/PAGE which suggests that it contains the extension sequences. Papain-inhibition experiments demonstrate that this endogenous soyacystatin has almost the same inhibitory activity as that of its deletion mutant (102 amino acid residues) recombinantly produced by truncation of the amino and carboxyl terminal extensions, indicating that the occurrence of the extensions does not affect the cystatin activity. Immunohistochemical experiments reveal that soyacystatin is expressed nearly uniformly in the cotyledons. These results also suggest the possible occurrence of a cysteine proteinase as the target enzyme of soyacystatin.