Fibrillin-1 Interactions with Fibulins Depend on the First Hybrid Domain and Provide an Adaptor Function to Tropoelastin

Abstract

Fibrillin-containing microfibrils in elastic and nonelastic extracellular matrices play important structural and functional roles in various tissues, including blood vessels, lung, skin, and bone. Microfibrils are supramolecular aggregates of several protein and nonprotein components. Recently, a large region in the N-terminal portion of fibrillin-1 was characterized as a multifunctional protein interaction site, including binding sites for fibulin-2 and -5 among others. Using a panel of recombinant fibrillin-1 swapped domain and deletion fragments, we demonstrate here that the conserved first hybrid domain in fibrillin-1 is essential for binding to fibulin-2, -4, and -5. Fibulin-3 and various isoforms of fibulin-1 did not interact with fibrillin-1. Although the first hybrid domain in fibrillin-1 is located in close vicinity to the self-assembly epitope, binding of fibulin-2, -4, and -5 did not interfere with self-assembly. However, these fibulins can associate with microfibrils at various levels of maturity. Formation of ternary complexes between fibrillin-1, fibulins, and tropoelastin demonstrated that fibulin-2 and -5 but much less fibulin-4, are able to act as molecular adaptors between fibrillin-1 and tropoelastin.The microfibril/elastic fiber system provides tissues, such as lung, blood vessels, and skin, with elastic properties. Microfibrils with a diameter of 10 -12 nm are typically located on the outer surface of elastic fibers and are thought to play an essential role in elastogenesis (1). Whereas elastic fibers are always associated with microfibrils, microfibrils themselves can occur in the absence of elastin in certain tissues such as ocular ciliary zonules, the kidney, or in close proximity to various basement membranes. The microfibril/elastic fiber system is a multicomponent assembly in the extracellular matrix, and for both, the microfibrils and the elastic fibers, a number of constituents have been described (for a review, see Ref 2). For most of the associated molecules, the exact relationship in terms of physical interaction with microfibrils and/or elastic fibers, and their functional relevance is not clear.The best described components of the microfibrils are a family of proteins consisting of three highly homologous members, fibrillin-1, -2, and -3 (3-9). Fibrillins, like many other extracellular glycoproteins, are characterized by a number of tandemly arranged domains. The most prominent domain is an epidermal growth factor-like domain (EGF), 2 which occurs 46 -47 times in fibrillins. These domains are stabilized by three intramolecular disulfide bonds, and the majority (42-43 domains) contain a consensus sequence for calcium binding (cbEGF) (10 -12). The tandemly arranged EGF and cbEGF domains are interspersed by two other types of domains, the transforming growth factor ␤-binding protein (TB) or 8-Cys domains and the hybrid domains. The seven TB/8-Cys domains are characterized by four intramolecular disulfide bonds, and a similar arrangement is predicted for the two hybrid domains, alth...

Fibulin-2 Is Dispensable for Mouse Development and Elastic Fiber Formation

“…While fibulin-2 and -4 are present at the interface between the central elastin core and its surrounding fibrillin microfibrils (8,18), fibulin-1 is located within the elastin core and fibulin-5 is associated with fibrillin microfibrils (8,19). Consistent with these observations, in vitro protein binding studies have shown that all fibulins are capable of binding to tropoelastin, albeit with different affinities (8,20,27), and that fibulin-2, -4, and -5 interact with the N-terminal region of fibrillin-1 (5,6,18).…”

New insights into elastic fiber assembly

“…Unlike fibulin-1, fibulin-2 binds directly to fibrillin-1 and has been shown to localize to the interface between the amorphous elastin core and microfibrils in skin . Fibulin-3 does not bind to fibrillin-1 (El-Hallous et al, 2007) and has been shown to only weakly interact with tropoelastin (Kobayashi et al, 2007). Fibulin-3 is expressed in capillaries, but not large blood vessels (Giltay et al, 1999) and most likely does not have a role in elas-tic fiber assembly.…”

“…Fibulin-3 is expressed in capillaries, but not large blood vessels (Giltay et al, 1999) and most likely does not have a role in elas-tic fiber assembly. Fibulin-4 binds fibrillin-1 (El-Hallous et al, 2007) and shows moderate affinity for tropoelastin. Fibulin-4 has been localized to microfibrils (Kobayashi et al, 2007).…”

“…The microfibrils bind to the cell surface through integrins. Fibulin-4 and -5 both bind fibrillin-1 (El-Hallous et al, 2007;Yanagisawa et al, 2002) and may help transfer the elastin aggregates to the microfibril. The elastin aggregates on the microfibril coalesce into larger structures and are further crosslinked by LOX and/or LOXL to form the complete and functional elastic fiber.…”

Fibrillin Assembly Requires Fibronectin

“…After subcloning the 1416-base pair PCR product in the pCRII-TOPO vector (Invitrogen, Carlsbad, CA), an 874-base pair SphI ϫ NotI fragment from the resulting plasmid was ligated with the 5677-base pair SphI ϫ NotI fragment from pBS-rFBN3C-1, resulting in pBS-rFBN3-C. This plasmid was digested with NotI ϫ NheI, and the 4127-base pair fragment was ligated with the vector backbone of the pDNSP-rF1F plasmid (El-Hallous et al, 2007). The final expression plasmid pDNSP-rFBN3-C encodes the BM40 signal peptide for secretion, generating an artificial APLA sequence, followed by the human fibrillin-3 amino acid sequence and a heptahistidine tag (APLAD 1321 -R 2684 HHHHHHH).…”