Abstract
In plant cells, many cysteine proteinases (CPs) are synthesized as precursors in the endoplasmic reticulum, and then are subject to post-translational modifications to form the active mature proteinases. They participate in various cellular and physiological functions. Here, AcCP2, a CP from pineapple fruit (Ananas comosus L.) belonging to the C1A subfamily is analyzed based on the molecular modeling and homology alignment. Transcripts of AcCP2 can be detected in the different parts of fruits (particularly outer sarcocarps), and gradually increased during fruit development until maturity. To analyze the substrate specificity of AcCP2, the recombinant protein was overexpressed and purified from Pichia pastoris. The precursor of purified AcCP2 can be processed to a 25 kDa active form after acid treatment (pH 4.3). Its optimum proteolytic activity to Bz-Phe-Val-Arg-NH-Mec is at neutral pH. In addition, the overexpression of AcCP2 gene in Arabidopsis thaliana can improve the resistance to fungal pathogen of Botrytis cinerea. These data indicate that AcCP2 is a multifunctional proteinase, and its expression could cause fruit developmental characteristics of pineapple and resistance responses in transgenic Arabidopsis plants.
Highlights
Cysteine proteinases (EC 3.4.22, cysteine proteinases (CPs)), known as thiol proteinases, are widely distributed among living organisms
For isolating the genes related to fruit development and ripening, cDNA libraries of pineapple fruits were constructed into pGEM-T easy vector using mRNAs of different developmental stages
The predicted AcCP2 protein contains several of characteristic elements of papain structure and active sites (Figure 1A,B), which are essential for catalytic activity and maintaining the tertiary structure [26]
Summary
Cysteine proteinases (EC 3.4.22, CPs), known as thiol proteinases, are widely distributed among living organisms. Mature papain-like proteinases share similar catalytic residues in the order of Cys....His....Asn/Asp and the three-dimensional structure [10,11] They are characterized by the presence of multiple disulfide bridges, and mainly accumulate in the vacuole, apoplast or specific vesicles [12,13]. An important source of plant proteinases used in the traditional medicine and industry is bromelain, which is a mixture of different CPs with similar amino acid sequences [15,16,17]. These enzymes display different proteolytic activities and molecular masses ranging from 20 to 38 kDa [18]. Transgenic Arabidopsis plants overexpressing the AcCP2 gene demonstrate the improved resistance to fungal pathogens
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