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Journal of Biochemistry Advance Access originally published online on February 10, 2008
Journal of Biochemistry 2008 143(5):633-639; doi:10.1093/jb/mvn014
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© 2008 The Japanese Biochemical Society.

Characterization of the Molecular Interaction between Tropoelastin and DANCE/Fibulin-5

Hiroshi Wachi1,*, Risa Nonaka1, Fumiaki Sato1, Kayoko Shibata-Sato1, Marie Ishida1, Saori Iketani1, Iori Maeda2, Koji Okamoto2, Zsolt Urban3,4, Satoshi Onoue5 and Yoshiyuki Seyama1

1Department of Clinical Chemistry, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501; 2Department of Biochemical Engineering and Science, Kyushu Institute of Technology, 680-4 kawazu, Iizuka-shi, Fukuoka 804-8550, Japan; 3Department of Pediatrics and; 4Department of Genetics, Washington University, 660 S. Euclid Ave., St Louis, Missouri 63110, USA; and 5Materials Research Department, Advanced Cosmetic Research Laboratories, KOSÉ Corporation, 1-8-4 Azusawa, Itabashi-ku, Tokyo 174-0051, Japan

*To whom correspondence should be addressed. Tel: +81-3-5498-5243, Fax: +81-3-5498-5243, E-mail: wchrs_1107{at}hoshi.ac.jp

Received November 1, 2007; Accepted January 27, 2008


  Abstract

Fibulin-5 is believed to play an important role in the elastic fiber formation. The present experiments were carried out to characterize the molecular interaction between fibulin-5 and tropoelastin. Our data showed that the divalent cations of Ca2+, Ba2+ and Mg2+ significantly enhanced the binding of fibulin-5 to tropoelastin. In addition, N-linked glycosylation of fibulin-5 does not require for the binding to tropoelastin. To address the fibulin-5 binding site on tropoelastin constructs containing, exons 2–15 and exons 16–36, of tropoelastin were used. Fibulin-5 binding was significantly reduced to either fragment and also to a mixture of the two fragments. These results suggested that the whole molecule of tropoelastin was required for the interaction with fibulin-5. In co-immunoprecipitation experiments, tropoelastin binding to fibulin-5 was enhanced by an increase of temperature and sodium chloride concentration, conditions that enhance the coacervation of tropoelastin. The binding of tropoelastin fragments to fibulin-5 was directly proportional to their propensity to coacervate. Furthermore, the addition of fibulin-5 to tropoelastin facilitated coacervation. Taken together, the present study shows that fibulin-5 enhances elastic fiber formation in part by improving the self-association properties of tropoelastin.

Key Words: coacervation, DANCE/fibulin-5, extracellular matrix, molecular interaction, tropoelastin

Abbreviations: BH, C-terminal half-fragment of bovine tropoelastin encoded by exons 16–36; BTE, bovine tropoelastin; cbEGF, calcium-binding epidermal growth factor; CHO-K1, Chinese hamster Ovary-K1 cells; DANCE, developmental arteries and neural crest epidermal growth factor-like; DTT, dithiothreitol; EMIKIN, elastin microfibril interface located protein; FH, N-terminal half-fragment of bovine tropoelastin encoded by exons 2–15; MAGP, microfibril-associated glycoprotein; PBS, phosphate buffer saline; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; TMB, 3,3',5,5'-tetramethylbenzidine; T-TBS, Tris buffered saline supplemented with 0.05% Tween 20


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