ABSTRACT

Human fibrillin-1 is a 350-kDa glycoprotein found in 10-nm connective tissue microfibrils. Mutations in the gene encoding this protein cause the Marfan syndrome, a disease characterized by cardiovascular, ocular, and skeletal abnormalities. Fibrillin-1 has a modular structure that includes 47 epidermal growth factor-like (EGF-like) domains, 43 of which contain a consensus sequence associated with calcium binding. A mutation causing an Asn-2144 Ser amino acid change in one of the potential calcium binding residues has been described in a patient with the Marfan syndrome. We have chemically synthesized a wild-type EGF-like domain (residues 2126-2165 of human fibrillin-1) and a mutant EGF-like domain containing the Asn-2144 Ser amino acid change and measured calcium binding to each using H-NMR spectroscopy. The wild-type domain binds calcium with a similar affinity to isolated EGF-like domains from coagulation factors IX and X; however, the mutant domain exhibits >5-fold reduction in affinity. Rotary shadowing of fibrillin-containing microfibrils, isolated from dermal fibroblast cultures obtained from the Marfan patient, shows that the mutation does not prevent assembly of fibrillin into microfibrils but does alter the appearance of the interbead region. We have modeled a region of fibrillin-1 (residues 2126-2331) encompassing five calcium binding EGF-like domains, using data derived from the recently determined crystal structure of a calcium binding EGF-like domain from human factor IX. Our model suggests that these fibrillin-1 EGF-like domains adopt a helical arrangement stabilized by calcium and that defective calcium binding to a single EGF-like domain results in distortion of the helix. We propose a mechanism for the interaction of contiguous arrays of calcium binding EGF-like domains within the microfibril.

FOOTNOTES

*

This paper is a contribution from the Oxford Center for Molecular Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Members of the Oxford Center for Molecular Sciences.

¶

Supported by a Royal Society University Research Fellowship. To whom correspondence should be addressed. Tel.: 865-275583, Fax: 865-275556.

**

An MRC Senior Research Fellow.

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The abbreviations used are: EGF, epidermal growth factor; HPLC, high pressure liquid chromatography; FCS, fetal calf serum; PBS, phosphate-buffered saline.

()

Z. Rao, P. A. Handford, M. Mayhew, V. Knott, G. G. Brownlee, and D. Stuart, submitted for publication.

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