Molecular analysis of actinidin, the cysteine proteinase of Actinidia chinensis

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Abstract
We have isolated and sequenced two very similar cDNA clones of 1145 and 809 bp length, from a fruit-specific library of Actinidia chinensis, the larger encoding all 220 amino acids of actinidin, showing 91% homology to the published amino acid sequence. Both cDNAs code for an additional 25 amino acids following the mature carboxy terminus of actinidin. The larger clone has coding potential for 57 residues of an amino-terminal extension with considerable homology to amino-terminal sequences of other cysteine proteinases. From size determination of both mRNA (1.4 kb) and immunoprecipitated in vitro translation product (39 kDa) it was estimated that actinidin is synthesised as a precursor approximately 15 kDa larger than the mature protein. Both proteolytic cleavage sites are located on the surface of the molecule as illustrated by the hydropathy profile of the deduced amino acid sequence. Features of the prosegment primary sequence are considered with regard to a possible mechanism of inactivation of the proteinase, by analogy with other proteolytic zymogens. The presence of three potential glycosylation sites, one within the carboxy-terminal and two in the amino-terminal extension, are consistent with subcellular location of the enzyme within membrane-bound organelles. Results from a Southern blot suggest that actinidin is encoded by a multigene family of up to ten members. Actinidin gene expression, both at the level of mRNA and protein, is largely restricted to the fruit of the plant, where the level of actinidin mRNA accumulates early during development.