Wspólnotowy Serwis Informacyjny Badan i Rozwoju - CORDIS

Identification of molecular mechanisms of proteolytic processing (shedding) of BP180 (collagen XVII)

The major autoantigen of bullous pemphigoid, BP180 or collagen XVII, has two forms, a transmembrane and a short, soluble form, which results from shedding of the ectodomain from the keratinocyte surface. The major objective here was to identify the molecular mechanisms of shedding. Using a broad panel of proteinase inhibitors, recombinant enzymes and substrates, and enzyme-deficient cells from knockout mice we demonstrated that collagen XVII is shed by ADAMs (a disintegrin and metalloproteinase) proteinases. The prototype sheddase TACE (TNF-alpha converting enzyme, or ADAM-17), ADAM-10 and ADAM-9 contribute to shedding of collagen XVII. This was the first description of shedding of a transmembrane collagen by ADAMs, which possibly release also other transmembrane proteins from the keratinocyte surface. In order to define the cleavage region and to assess its structure and requirements for shedding, deletion mutants of the NC16A domain were expressed in COS-7 cells. Analysis of the deletions pointed to the stretch of amino acids 528-547 as important for sheddase recognition and cleavage. Secondary protein structure predictions showed that deletion of this stretch resulted in conformational changes in the collagen XVII homotrimer, indicating that the conformation of the NC16A domain and steric availability of the cleavage site influence shedding and are important for folding of collagen XVII. Monoclonal antibodies recognizing the proteolytically released ectodomain of collagen XVII were also produced and characterized. As a development of studies on the regulation of BP180 shedding, we investigated the role of plasma membrane microenvironment in BP180 shedding. ADAMs-mediated BP180 shedding was induced by disintegration of lipid rafts and by low-cholesterol membrane levels, and confocal microscopy analysis indicated that BP180 is incorporated in lipid rafts. These data represent the first evidence of the role of plasma membrane lipid organization in the regulation of BP180 shedding and therefore of keratinocyte migration and differentiation.New highly relevant knowledge has been obtained about basic aspects of keratinocyte biology, such as cell adhesion and migration. Besides, the data on shedding are important for understanding the generation of autoantigen epitopes in bullous pemphigoid and for design of biologically valid novel therapeutic strategies. Finally, the monoclonal antibodies to the shedded ectodomain of collagen XVII can contribute both to the research reagent repertoire for study of hemidesmosomes and to the development of clinical diagnostic tests (a patent application has been submitted by the involved participant, University of Freiburg).

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