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Molecular analysis of the interaction between cardosin A and phospholipase Dα
Author(s) -
Simões Isaura,
Mueller EvaChristina,
Otto Albrecht,
Bur Daniel,
Cheung Alice Y.,
Faro Carlos,
Pires Euclides
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04967.x
Subject(s) - phospholipase , biochemistry , homology modeling , biology , phospholipase a2 , chemistry , microbiology and biotechnology , enzyme
Cardosin A is an RGD‐containing aspartic proteinase from the stigmatic papillae of Cynara cardunculus L. A putative cardosin A‐binding protein has previously been isolated from pollen suggesting its potential involvement in pollen–pistil interaction [Faro C, Ramalho‐Santos M, Vieira M, Mendes A, Simões I, Andrade R, Verissimo P, Lin X, Tang J & Pires E (1999) J Biol Chem 274 , 28724–28729]. Here we report the identification of phospholipase Dα as a cardosin A‐binding protein. The interaction was confirmed by coimmunoprecipitation studies and pull‐down assays. To investigate the structural and molecular determinants involved in the interaction, pull‐down assays with cardosin A and various glutathione S ‐transferase‐fused phospholipase Dα constructs were performed. Results revealed that the C2 domain of phospholipase Dα contains the cardosin A‐binding activity. Further assays with mutated recombinant forms of cardosin A showed that the RGD motif as well as the unprecedented KGE motif, which is structurally and charge‐wise very similar to RGD, are indispensable for the interaction. Taken together our results indicate that the C2 domain of plant phospholipase Dα can act as a cardosin A‐binding domain and suggest that plant C2 domains may have an additional role as RGD/KGE‐recognition domains.