Regulation of fibrillin carboxy-terminal furin processing

Fibrillins are large cysteine-rich glycoproteins (~350 kDa) which form the molecular scaffold of a class of beaded microfibrils that are key structural elements of dynamic connective tissues (Kielty and Shuttleworth, 1995). These microfibrils are extensible polymers which act as a structural lattice for elastin deposition during elastic fibre formation (Mecham and Heusar, 1991). Linkage of the fibrillin-1 and fibrillin-2 genes to the heritable connective tissue disorders Marfan syndrome and congenital contractural arachnodactyly, respectively, highlights their critical contribution to connective tissue integrity (Ramirez, 1996). Fibrillin molecules have a cysteine-rich multidomain organisation dominated by calcium-binding epidermal growth factor-like domains (cbEGF-like domains) interspersed with eight-cysteine-containing motifs (TB modules; Pereira et al., 1993; Zhang et al., 1994). The contiguous arrays of cbEGFlike domains form rod-like structures in the presence of calcium (Downing et al., 1996; Reinhardt et al., 1997). Each isoform contains a unique hydrophobic sequence towards the amino terminus which may act as a potential molecular hinge; in fibrillin-1 this sequence is proline-rich sequence, and in fibrillin-2 it is glycine-rich. Aminoand carboxy-terminal fibrillin sequences contain furin/PACE proprotein convertase tetrabasic consensus sequences, and processing at these sites may be important regulatory steps in fibrillin assembly (Raghunath et al., 1999; Ritty et al., 1999). The molecular pathway of fibrillin assembly remains poorly understood. Intermediates have proved difficult to identify due to the large size of fibrillin molecules and their propensity to form disulphide-bonded aggregates (Sakai, 1990). The possibility that dimers may occur has been suggested by SDS-PAGE analysis of metabolically labelled fibrillin immunoprecipitated from cell culture medium (Kielty and Shuttleworth, 1993), and recent recombinant studies (Trask et al., 1999; Ashworth et al., 1999). Efforts have concentrated on resolving the precise arrangement of fibrillin monomers within microfibrils, in order to understand their structural properties and to gain insights into how they form. A parallel head-to-tail alignment model of unstaggered fibrillin monomers with aminoand carboxy-termini at, or close to, the beads was suggested on the basis of antibody epitope mapping and measured molecular dimensions (Reinhardt et al., 1996). Alternative staggered arrangements based on extrapolation of molecular length from cbEGF-like domain dimensions (Downing et al., 1996), or on alignment of transglutaminase cross-link sites (Qian and Glanville, 1998), have also been proposed. In this study, we have investigated human fibrillin-1 4163 Journal of Cell Science 112, 4163-4171 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS0811