Biogenesis of extracellular microfibrils: Multimerization of the fibrillin-1 C terminus into bead-like structures enables self-assembly

Abstract

Microfibrils are essential elements in elastic and nonelastic tissues contributing to homeostasis and growth factor regulation. Fibrillins form the core of these multicomponent assemblies. Various human genetic disorders, the fibrillinopathies, arise from mutations in fibrillins and are frequently associated with aberrant microfibril assembly. These disorders include Marfan syndrome, Weill-Marchesani syndrome, Beals syndrome, and others. Although homotypic and heterotypic fibrillin self-interactions are considered to provide critical initial steps, the detailed mechanisms for microfibril assembly are unknown. We show here that the C-terminal recombinant half of fibrillin-1 assembles into disulfidebonded multimeric globular structures with peripheral arms and a dense core. These globules are similar to the beaded structures observed in microfibrils isolated from tissues. Only these C-terminal fibrillin-1 multimers interacted strongly with the fibrillin-1 N terminus, whereas the monomers showed very little self-interaction activity. The multimers strongly inhibited microfibril formation in cell culture, providing evidence that these recombinant assemblies can also interact with endogenous fibrillin-1. The Cterminal self-interaction site was fine-mapped to the last three calcium-binding EGF domains in fibrillin-1. These results suggest a new mechanism for microfibril formation where fibrillin-1 first oligomerizes via its C terminus before the partially or fully assembled bead-like structures can further interact with other beads via the fibrillin-1 N termini. extracellular matrix ͉ fibrillinopathies ͉ Marfan syndrome ͉ protein assembly

Fibrillin Assembly Requires Fibronectin

“…Heparin did not inhibit this interaction in concentrations up to 200 g/ml (ϳ13 M), indicating different binding epitopes (data not shown). Recently, we demonstrated that C-terminal fibrillin-1 fragments have the propensity to multimerize into structures with similarity to the beads found in the bead-on-a-string-structured, extracted microfibrils (Hubmacher et al, 2008). It was further shown that this multimerization significantly increases the avidity for the self-interaction of the fibrillin-1 C-terminus with the N-terminus.…”

“…The multimerization of the fibrillin-2 (rFBN2-C) and fibrillin-3 (rFBN3-C) recombinant C-terminal halves were analyzed by Superose 6 gel filtration chromatography (100-ml column on a high-precision Ä ktaPurifier 10; GE Healthcare) as described in detail for the fibrillin-1 C-terminal construct rFBN1-C (Hubmacher et al, 2008). Recombinant protein, 2.7 mg, previously purified by metal-chelating chromatography were loaded onto the column equilibrated with TBS, including 2 mM CaCl 2 at a flow rate of 0.5 ml/min.…”

“…In this process, cell-associated multimerization of the fibrillin-1 C-terminus appears to be an important determinant in generating high-affinity binding sites for the fibrillin-1 N-terminus (Hubmacher et al, 2008). In addition to linear N-C interactions, lateral homotypic interactions in different regions of the fibrillin-1 molecule may play a role in stabilizing initial multimers or the lateral associa-tions of individual microfibrils (Trask et al, 1999;Ashworth et al, 1999;Marson et al, 2005).…”

“…One of the early mechanisms in the multistep, cell-associated fibrillin assembly process involves N-to-C-terminal (N-C) self-interactions, whereby the interaction sites for fibrillin-1 have been mapped to the N-and C-terminal ends of the molecule (Lin et al, 2002;Marson et al, 2005;Hubmacher et al, 2008). In this process, cell-associated multimerization of the fibrillin-1 C-terminus appears to be an important determinant in generating high-affinity binding sites for the fibrillin-1 N-terminus (Hubmacher et al, 2008).…”

“…Although many publications over recent years have addressed the spatial organization of fibrillins as it relates to the bead-on-a-string microfibrillar structure Reinhardt et al, 1996;Downing et al, 1996;Liu et al, 1996;Qian and Glanville, 1997;Baldock et al, 2001Baldock et al, , 2006Lee et al, 2004;Kuo et al, 2007), little information is available about the dynamic processes and components involved in microfibril formation. The pathogenetic relevance of this is highlighted by the fact that microfibril assembly is frequently disturbed in patients with fibrillinopathies (Tiedemann et al, 2004).One of the early mechanisms in the multistep, cell-associated fibrillin assembly process involves N-to-C-terminal (N-C) self-interactions, whereby the interaction sites for fibrillin-1 have been mapped to the N-and C-terminal ends of the molecule (Lin et al, 2002;Marson et al, 2005;Hubmacher et al, 2008). In this process, cell-associated multimerization of the fibrillin-1 C-terminus appears to be an important determinant in generating high-affinity binding sites for the fibrillin-1 N-terminus (Hubmacher et al, 2008).…”