Self-assembly and Supramolecular Organization of EMILIN

Maurizio Mongiat,Gabriella Mungiguerra,Simonetta Bot,Maria Teresa Mucignat,Emiliana Giacomello,Roberto Doliana,Alfonso Colombatti

doi:10.1074/jbc.m001426200

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 alpha 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.

Highlights

Elastin microfibril interface-located protein (EMILIN) belongs to the C1q/TNF1 superfamily of proteins [1] and is a considerable component of the elastic fiber system
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 extracellular matrix (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 [3,4,5,6,7]; it is a component of elastic fibers being mainly localized at the interface between amorphous elastin and the surrounding microfibrils [8]; 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]
It is not clear if the leucine zipper domain enhances the interaction; for the first time we demonstrate that the globular C1q domain (gC1q) domain, which in these experiments is the essential element for the binding, is able to self-assemble in vivo

Summary

EXPERIMENTAL PROCEDURES

Construction of Plasmids for the Two-hybrid System—To study the EMILIN intermolecular interactions, several constructs encoding LexA fusion proteins containing the C-terminal domains (see below) were generated by polymerase chain reaction (PCR) employing 1 ng of EMILIN-pGEM-T as a template [1], 10 pM each primer (see below), 2.5 units of Taq polymerase (Promega Corp.), and 0.2 mM each of the four deoxynucleotide triphosphates (Pharmacia Ultrapure, Amersham Pharmacia Biotech). Production of Recombinant Prokaryotic Domains of Human EMILIN—The sequence corresponding to the C-terminal domain of EMILIN (gC1q construct pB) was amplified from the EMILIN-pGEM-T template (see above) with the same primers used for generating yB, in which the 5Ј-ends were replaced with the BamHI and KpnI recognition sites for sense and antisense primers, respectively It was ligated in frame in the His6-tagged pQE-30 expression vector (Qiagen GmbH) and transformed in M15 cells. Puromycin-resistant cells were selected and assayed for recombinant protein expression by precipitating the spent (10 min at 13,000 rpm) serum-free culture medium with 50% (w/v) trichloroacetic acid and and 1% Triton X-100 and analyzing the precipitate by SDS-PAGE. The spectra were analyzed using the Menendez-Arias program [23]

RESULTS

DISCUSSION

Methods