The autoantibodies in the blistering disease pemphigus foliaceus (PF) have been shown to bind to a transmembrane desmosomal glycoprotein called desmoglein I (DGI). It has been suggested that these antibodies might directly inhibit cell adhesion by binding to the extracellular domain of DGI, thereby causing acantholysis. Although desmosomes are one of the most prominent types of adhesive junctions in epithelia, until recently very little was known regarding the nature of the molecules responsible for desmosome-mediated adhesion. Recently, sequence analysis has demonstrated that DGI is related to the calcium-dependent class of cell adhesion molecules known as cadherins. In spite of this, DGI has not yet been demonstrated experimentally to be an adhesion molecule. In this proposal a combined cell biological and molecular genetic approach will be taken to study the function of the PF antigen, desmoglein I, and its relationship to the disease process.
The first aim will be to investigate whether DGI is sufficient for mediating adhesion in cultured fibroblasts that are normally non- adherent. Constructs encoding full length DGI will be transfected into non-adherent fibroblasts, and the ability of these cells to adhere in the presence of calcium will be assessed. If DGI alone is not sufficient, the possibility that additional cytoplasmic proteins are required will be tested by co-expression experiments. As an alternative approach, in aim two antisense mRNA experiments will be carried out to test whether DGI is necessary for adhesion or plaque integrity in human keratinocytes.
In aim three, the ability of latex beads coated with extracellular domains of desmosomal cadherins to bind to desmosome-containing cells and induce signals necessary for induction of plaque assembly will be investigated. In the fourth aim, deletion mutants and chimeric molecules combining specific portions of DGI with portions of E-cadherin will be expressed in L cells and/or junction bearing cells, and the fat of expressed protein, and effect on endogenous junctions and filament systems will be assessed. Finally, the relationship of DGI autoantibodies to acantholysis in PF will be determined. the goal of this final aim will be to a) generate fusion proteins encoding specific regions in the extracellular domain of DGI, b) affinity purify individual IgG that bind these epitopes from PF patient sera, and c) assess the ability of these antibodies to cause acantholysis in vivo and in vitro. This work should provide insights into the function of the PF antigen and may provide a groundwork for the development of a plasmapheresis-based therapy for PF.
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