Self-assembly and Supramolecular Organization of EMILIN

Abstract

The primary structure of human Elastin microfibril interface-located protein (EMILIN), an elastic fiber-associated glycoprotein, consists of a globular C1q domain (gC1q) at the C terminus, a short collagenous stalk, a long region with a high potential for forming coiled-coil ␣ helices, and a cysteine-rich N-terminal sequence. It is not known whether the EMILIN gC1q domain is involved in the assembly process and in the supramolecular organization as shown for the similar domain of collagen X. By employing the yeast two-hybrid system the EMILIN gC1q domains interacted with themselves, proving for the first time that this interaction occurs in vivo. The gC1q domain formed oligomers running as trimers in native gels that were less stable than the comparable trimers of the collagen X gC1q domain since they did not withstand heating. The collagenous domain was trypsin-resistant and migrated at a size corresponding to a triple helix under native conditions. In reducing agarose gels, EMILIN also migrated as a trimer, whereas under non-reducing conditions it formed polymers of many millions of daltons. A truncated fragment lacking gC1q and collagenous domains assembled to a much lesser extent, thus deducing that the C-terminal domain(s) are essential for the formation of trimers that finally assemble into large EMILIN multimers.Elastin microfibril interface-located protein (EMILIN) belongs to the C1q/TNF 1 superfamily of proteins (1) and is a considerable component of the elastic fiber system. Elastic fibers confer the properties of resiliency and elastic recoil to connective tissues and are secreted in the extracellular matrix (ECM) of many tissues in various forms. In the elastic ligaments they are deposited as solid branching and unbranching fine and thick rod-like fibers, in the blood vessels as concentric sheets of lamellae, in the elastic cartilages as a three-dimensional meshwork of fine fibrils, and in skin and lung as a combination of these (2). As previously studied in the chick model, the major characteristics of EMILIN, first extracted by means of denaturating and reducing agents, are as follows. It is preferentially extracted from tissues using buffers containing guanidine HCl and reducing agents; it forms a fibrillar network in the ECM of in vitro grown smooth muscle cells and fibroblasts and in the ECM of several tissues including blood vessels, skin, heart, lung, kidney, and cornea; it codistributes with elastin in most sites (3-7); it is a component of elastic fibers being mainly localized at the interface between amorphous elastin and the surrounding microfibrils (8); finally and more important for the functional significance of EMILIN, the process of in vitro elastin deposition was perturbed by the addition of anti-EMILIN antibodies to the culture medium suggesting that this protein plays a leading role during the elastic fiber formation process (8).Recently, the primary structure of the human EMILIN has been elucidated (1). The mature form of this multimodular protein consists of 996 amino acids and d...

The Solution Structure of EMILIN1 Globular C1q Domain Reveals a Disordered Insertion Necessary for Interaction with the α4β1 Integrin

“…Based on the homotrimeric model of ACRP30 gC1q crystal structure (10), it was supposed that the EMILIN1 gC1q domain could also trimerize via several hydrophobic residues. This was confirmed, first by biochemical (3,7) and later by preliminary structural evidence (14).…”

“…Thus, EMILIN1 favorably located at the subendothelium of blood vessels is a new specific antagonist of tumor growth factor-␤, and the function of this constituent of elastic tissues is linked to the pathogenesis of hypertension. The C-terminal gC1q domain is involved in the oligomerization of EMILIN1 (7), in cell adhesion and migration via interaction with the ␣4␤1 integrin (8), and in trophoblast invasion (9).…”

Molecular Cloning and Characterization of EndoGlyx-1, an EMILIN-like Multisubunit Glycoprotein of Vascular Endothelium

“…Exploiting the yeast two-hybrid system with EMI-LIN-1 indicates that trimerization is initiated by the C1q-like domain followed by a subsequent quarternary assembly mediated by intermolecular disulfide bridges, which was further supported by the fact that a deletion mutant of EMILIN-1, lacking the C1q-like domain, was incapable in multimer formation (21). Our analysis shows that the C-terminal portion of EndoGlyx-1 p125/140 consists of an C1q-like domain homologous to EMILIN-1 and multimerin, with 26 and 32% amino acid sequence identity, respectively, which is in accordance with the oligomerization of the different EndoGlyx-1 subunits mediated by intermolecular disulfide bridges.…”

“…Additionally, a significant level of conservation was found for the C-terminal C1q-like domain, between amino acids 818 and 949, with 26% sequence identity with the respective region in the human EMILIN-1 precursor protein and 32% sequence identity with the human multimerin precursor protein (Fig. 4B), indicating a shared functional feature provided by this segment, such as the formation of homomultimers, which was demonstrated for several members of the TNF/C1q superfamily (21). Additional sequence analysis details can be viewed at mendel.imp.univie.ac.at.SEQUENCES/endoglyx/.…”

α-Helical Coiled-coil Oligomerization Domains Are Almost Ubiquitous in the Collagen Superfamily

“…Early studies on the procollagen precursors of the fibrillar collagens (types I-III, V, and XI) (6) showed that the C-propeptide regions are necessary to direct correct chain association, which is followed by zipper-like folding of the triple helix in the C-to N-terminal direction (7). This concept was subsequently extended to basement membrane collagen type IV (8 -10), microfibril-forming collagen VI (11,12), members of the C1q family (13) including collagens VIII and X (14 -17) and the emilins (18), and the FACITs 1 (19 -21).…”