Biological Activity of Ectodysplasin A Is Conditioned by Its Collagen and Heparan Sulfate Proteoglycan-binding Domains

Lee Kim Swee,Karine Ingold-Salamin,Aubry Tardivel,Laure Willen,Olivier Gaide,Manuel Favre,Stéphane Demotz,Marja Mikkola,Pascal Schneider

doi:10.1074/jbc.m109.042259

Lee Kim Swee, Karine Ingold-Salamin + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m109.042259

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Abstract

Mutations in the TNF family ligand EDA1 cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition characterized by defective development of skin appendages. The EDA1 protein displays a proteolytic processing site responsible for its conversion to a soluble form, a collagen domain, and a trimeric TNF homology domain (THD) that binds the receptor EDAR. In-frame deletions in the collagen domain reduced the thermal stability of EDA1. Removal of the collagen domain decreased its activity about 100-fold, as measured with natural and engineered EDA1-responsive cell lines. The collagen domain could be functionally replaced by multimerization domains or by cross-linking antibodies, suggesting that it functions as an oligomerization unit. Surprisingly, mature soluble EDA1 containing the collagen domain was poorly active when administered in newborn, EDA-deficient (Tabby) mice. This was due to a short stretch of basic amino acids located at the N terminus of the collagen domain that confers EDA1 with proteoglycan binding ability. In contrast to wild-type EDA1, EDA1 with mutations in this basic sequence was a potent inducer of tail hair development in vivo. Thus, the collagen domain activates EDA1 by multimerization, whereas the proteoglycan-binding domain may restrict the distribution of endogeneous EDA1 in vivo.

Highlights

Vaudois (CHUV), 1011 Lausanne, Switzerland. 3 Present address: Department of Dermatology, University of Geneva, ically active
Ectodysplasin A (EDA) is a Tumor necrosis factor (TNF) family ligand involved in the development of various structures derived from the ectoderm, such as hair, teeth, and sweat glands, and EDA loss of function in mouse and human is associated with X-linked hypohidrotic ectodermal dysplasia (OMIM 305100)
We have used a fusion protein, in which the receptor-binding portion of EDA1 was fused to the Fc portion of human immunoglobulin G1 (FcEDA1) (12), as a probe to detect surface expression of EDA1 receptor (EDAR) in HaCat cells (Fig. 2A)

Summary

MATERIALS AND METHODS

Plasmid with 50 ␮l of 2.5 M CaCl2. Sterile water was added to 500 ␮l. 293T fresh DMEM (no serum) was added that contained the ligand of cells were transiently transfected with pMSCVpuro-EDAR:Fas interest either in a purified form (between 0.05 and 1 ␮g/ml) or and co-transfected with the pHIT60 and VSV-G plasmids, con- as Opti-MEM supernatants. For heparin-Sepharose pull-downs (GE Healthcare), 10 ␮l of beads freshly rehydrated in PBS were mixed with 750 ␮l of Opti-MEM supernatant in PBS (1:1 v/v), incubated for 1 h at 4 °C on a rotating wheel, transferred into a mini column, washed with PBS, and eluted with 15 ␮l of PBS supplemented with 0.8 M NaCl. Cell Lysis—HaCat cells were harvested at defined time points. Various Flag-EDA and Flag-FasL constructs in Opti-MEM supernatants were analyzed In this case, 2, 5, 10, or 50 ␮l of the 500 ␮l fractions were used to detect the ligands by ELISA, essentially as described previously (3, 11).

RESULTS

The HaCat assay provided qualitative rather than quantitative

Estimated number of subunitsc ml kDa

DISCUSSION