Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia

X-linked hypohidrotic ectodermal dysplasia (XLHED) is a heritable disorder of the ED-1 gene disrupting the morphogenesis of ectodermal structures. The ED-1 gene product, ectodysplasin-A (EDA), is a tumor necrosis factor (TNF) family member and is synthesized as a membrane-anchored precursor protein with the TN F core motif located in the C-terminal domain. The stalk region of EDA contains the sequence -Arg-Val-Arg-Arg(156)-Asn-Lys-Arg(159)-, repr esenting overlapping consensus cleavage sites (Arg-X-Lys/Arg-Arg (down arro w)) for the proprotein convertase furin. Missense mutations in four of the five basic residues within this sequence account for approximate to 20% of all known XLHED cases, with mutations occurring most frequently at Arg(156) which is shared by the two consensus furin sites. These analyses suggest t hat cleavage at the furin site(s) in the stalk region is required for the E DA-mediated cell-to-cell signaling that regulates the morphogenesis of ecto dermal appendages. Here we show that the 50-kDa EDA parent molecule is clea ved at -Arg(156)Asn-Lys-Arg(159) (down arrow) - to release the soluble C-te rminal fragment containing the TNF core domain. This cleavage appears to be catalyzed by furin, as release of the TNF domain was blocked either by exp ression of the furin inhibitor alpha (1)-PDX or by expression of EDA in fur in-deficient LoVo cells. These results demonstrate that mutation of a funct ional furin cleavage site in a developmental signaling molecule is a basis for human disease (XLHED) and raise the possibility that furin cleavage may regulate the ability of EDA to act as a juxtacrine or paracrine factor.