Generation and Characterization of Function-blocking Anti-ectodysplasin A (EDA) Monoclonal Antibodies That Induce Ectodermal Dysplasia

Christine Kowalczyk-Quintas,Laure Willen,Anh Thu Dang,Heidi Sarrasin,Aubry Tardivel,Katharina Hermes,Holm Schneider,Olivier Gaide,Olivier Donzé,Neil Kirby,Denis J Headon,Pascal Schneider

doi:10.1074/jbc.m113.535740

Abstract

Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated.

Highlights

The TNF family ligand EDA1 is required for development of hair, teeth, and many glands
An ELISA assay was performed in which the antigen Fc-EDA1 was either coated directly to the plate, a process that partially denatures proteins, or else was captured via its Fc portion to maintain its native structure
EctoD1, EctoD2, and EctoD3 recognized both coated and captured Fc-EDA1 with similar signal intensities, suggesting that they recognize surface-exposed epitopes in EDA1 (Fig. 2C). These results suggest that Renzo2 may recognize a linear, partially buried epitope, whereas EctoD1, EctoD2, and EctoD3 preferentially bind to surface epitopes that may not be linear in the primary sequence

Summary

Background

The TNF family ligand EDA1 is required for development of hair, teeth, and many glands. The action of the TNF family ligand EDA2 on its receptor EDAR, a target gene of Wnt signaling, is required shortly thereafter at least for sweat glands and some hair types, to stimulate the transcription factor NF-␬B, itself necessary for the formation of morphologically distinct cellular condensates called placodes [3, 4]. Some placodes, such as those of guard hair developing at embryonic day (E) 14.5, are fully dependent on EDA-EDAR interactions and NF-␬B for their formation, whereas others, such as those of undercoat hair developing at around birth, can form in the absence of EDA but produce morphologically abnormal hair [4]. These antibodies will be useful for immunodetection of EDA protein and to explore the developmental and homeostatic roles of EDA signaling in birds and mammals

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION