Molecular and cellular studies of a cysteine proteinase from germinated Vicia faba L.

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Yu, Wen-Jin
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University of Guelph

A cDNA library of germinated V. faba seeds was successfully constructed. From the three largest and overlapping positive clones a cDNA encoding a full length cysteine pre-pro-proteinase was constructed (GenBank access number: U59465). The cDNA contains a full coding region, a 15-base pair 5\sp′-end untranslated region, 334-base 3\sp′-untranslated region and a 19-base poly(A) tail. To my knowledge this the first cDNA coding for a cysteine proteinase constructed from the mRNA of V. faba L. The deduced full length cysteine pre-pro-proteinase was designated as VFCYSPRO. As in other reported cysteine proteinase zymogens, VFCYSPRO contains a N-terminal signal peptide sequence (18 amino acids), a pro-protein region (112 amino acids) and a mature proteinase portion (232 amino acids). The mature protein belongs to the well-characterized papain superfamily although it shares only 37% identity with papain. A 3-D model of the mature enzyme of VFCYSPRO was generated by aligning VFCYSPRO with known protein structures and confirmed this classification. The pro-protein and partial mature enzyme or VFCYSPRO were successfully synthesized in bacterial host E. coli and purified. Antibodies were raised to the fusion proteins and purified to be specific to VFCYSPRO using a simple, inexpensive and reliable method. VFCYSPRO expression is under temporal or developmental control, rather than tissue specific regulation. It is expressed not only in germinated seeds but also in developing leaves, shoots, roots and pod walls. It is not expressed in developing seeds. That both the mRNA and the protein are expressed in the same pattern indicates that the protein content and activity are controlled at the transcriptional level. The phytohormone ethylene significantly promotes the expression of the mRNA for VFCYSPRO, while neither ABA nor GA has a stimulating role. Based on the fact that both VFCYSPRO mRNA and protein are expressed in seeds at post-germinated stages (when most storage proteins, globulins, have been mobilized), in senescing pods, and in the vascular tissues, it is speculated that besides involvement in degradation of storage proteins in seeds, VFCYSPRO plays a role for cell content turnover during programmed cell death.

molecular, cellular, cDNA library, cysteine proteinase, germinated, Vicia faba L.