Molecular analysis of the interaction between cardosin A and phospholipase Dα

Identification of RGD/KGE sequences as binding motifs for C2 domains

Authors

  • Isaura Simões,

    1. Departamento de Biologia Molecular e Biotecnologia, Centro de Neurociências e Biologia Celular, Universidade de Coimbra and Departamento de Bioquímica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Portugal
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  • Eva-Christina Mueller,

    1. Max Delbrueck Center for Molecular Medicine, Berlin, Germany
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  • Albrecht Otto,

    1. Max Delbrueck Center for Molecular Medicine, Berlin, Germany
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  • Daniel Bur,

    1. Actelion Pharmaceuticals Ltd, Allschwil, Switzerland
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  • Alice Y. Cheung,

    1. Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA, USA
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  • Carlos Faro,

    1. Departamento de Biologia Molecular e Biotecnologia, Centro de Neurociências e Biologia Celular, Universidade de Coimbra and Departamento de Bioquímica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Portugal
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  • Euclides Pires

    1. Departamento de Biologia Molecular e Biotecnologia, Centro de Neurociências e Biologia Celular, Universidade de Coimbra and Departamento de Bioquímica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Portugal
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  • Note
    The nucleotide sequence of PLDα from C. cardunculus L has been submitted to the EBI Data Bank with the accession number AJ583515

C. Faro, Departamento de Bioquímica, Universidade de Coimbra, Apt. 3126, 3000 Coimbra, Portugal
Fax: +351 239 480208
Tel: +351 239 480210
E-mail: cfaro@imagem.ibili.uc.pt

Abstract

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 Chem274, 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.

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