Biochemical and functional characterization of cysteine proteases from ginger rhizome (Zingiber officinale)

Abstract

The ginger rhizome contains thiol-dependent proteases [1], which are related to other plant cysteine proteases. There has been some biochemical characterization of two of the enzymes at protein level, including the protein sequences [2] and the 3D-structure of one of these [3].Papain is the best characterized plant cysteine protease and has found commercial application, for example, as a meat tenderizer. Ginger has been used as a food additive and herbal medicine for centuries. The primary incentive for the present study was to determine whether ginger proteases may have properties which better suit it for these applications.Two closely related cDNAs (designated gp2 and gp3) were cloned from the ginger rhizome, encoding two distinct cysteine proteases, each of which is represented by two isoforms, (gp2a and gp2b, and gp3a and gp3b). The mature enzyme encoded by gp2a cDNA is identical, except for 3 residues, to the amino acid sequence of the enzymatically characterized ginger protease II (GPU) reported by Choi and Laursen [2]. The deduced amino acid sequence of gp3 indicates that it encodes a novel protease which shares only 86 % sequence identity with GPU and 73 % identity with the published GPI. The most striking difference between gp2 and gp3 clones is that the gp3 cDNAs encode a long C-terminal 113 residue propeptide, compared with a 19 residue propeptide of gp2. Southern blot analysis confirms that these sequences constitute a small subfamily of cysteine proteases that may contain as few as three members.The gp2 and gp3 genes exhibit different patterns of temporal regulation during rhizome development. Whereas gp2 expression varies 350-fold over the growing season, gp3 exhibits a much higher basal level of gene expression throughout the year with an only 4-fold induction during rhizome maturation. The results indicate that ginger proteases are differentially expressed during the development of the rhizome, consistent with the measured changes of the proteolytic activity of ginger extracts over the same period. In addition, the expression of both transcripts was found to be rhizome-specific.Three ginger proteases were purified by ion exchange chromatography. The enzymes correspond to three of the four cloned cDNAs: GP2B, corresponding to gp2b cDNA, was found in both mature and young rhizome. GP3A was found in mature rhizome and GP2A in young rhizome. The enzyme corresponding to gp3b cDNA was not found.The changes of enzyme activity reflected the expression pattern of transcripts during rhizome development over the growing season of the rhizome. However, a time lag between transcription and enzyme activity was observed. A similar pattern was observed in Arabidopsis RD21. It has been suggested that the proenzymes may be stored up and processed slowly to produce active proteases, which degrade cellular proteins during Arabidopsis leaf senescence. Possible functions for the ginger proteases, based on their structures and patterns of expression are discussed.n n n