Biochemical characterization and immunolocalization studies of a Capsicum chinense Jacq. protein fraction containing DING proteins and anti-microbial activity

The DING protein family consists of proteins of great biological importance due to their ability to inhibit carcinogenic cell growth. A DING peptide with Mr similar to 7.57 kDa and pI similar to 5.06 was detected in G10P1.7.57, a protein fraction from Capsicum chinense Jacq. seeds. Amino acid sequencing of the peptide produced three smaller peptides showing identity to the DING protein family. G10P1.7.57 displayed a phosphatase activity capable of dephosphorylating different phosphorylated substrates and inhibited the growth of Saccharomyces cerevisiae cells. Western immunoblotting with a custom-made polyclonal antibody raised against a sequence (ITYMSPDYAAPTLAGLDDATK), derived from the similar to 7.57 kDa polypeptide, immunodetected an similar to 39 kDa polypeptide in G10P1.7.57. Purification by electroelution followed by amino acid sequencing of the similar to 39 kDa polypeptide yielded seven new peptide sequences and an additional one identical to that of the initially identified peptide. Western immunoblotting of soluble proteins from C. chinense seeds and leaves revealed the presence of the similar to 39 kDa polypeptide at all developmental stages, with increased accumulation when the organs reached maturity. Immunolocalization using Dabsyl chloride- or Alexa fluor 488-conjugated antibodies revealed a specific fluorescent signal in the cell cytoplasm at all developmental stages, giving support to the idea that the similar to 39 kDa polypeptide is a soluble DING protein. Thus, we have identified and characterized a protein fraction with a DING protein from C. chinense. (C) 2016 Elsevier Masson SAS. All rights reserved.