The anchoring complex is an ultrastructural feature of the dermal-epidermal basement membrane that includes the hemidesmosomes, anchoring filaments and anchoring fibrils. These structures are believed to work as a unit to stabilize epithelial attachment to the underlying basement membrane. Failure of components of the anchoring complex results in spontaneous blistering characteristic of the inherited bullous diseases. We have recently identified two laminin variants that are components of the anchoring filaments. We have termed these kalinin and K-laminin. We know that kalinin is absent from the basement membrane zone of patients with lethal epidermolysis bullosa. Kalinin is a cell adhesion macromolecule specific for certain types of epithelial cells including keratinocytes. For these cells, kalinin substitutes for laminin as the favored growth substrate. Monoclonal antibodies made to kalinin cause cell detachment and de-epithelialization of whole skin in vitro. This application is focused upon continued characterization of the structure and function of kalinin and K-laminin. We propose to isolate kalinin and K-laminin for protein sequence determinations and for preparing protein fragments; to complete the deduced amino acid sequences of these chains and to express selected subdomains in vitro; and to generate domain specific monoclonal antibodies. These reagents will be used to evaluate the binding function of kalinin and K-laminin subdomains. We will interfere with these functions in an in vitro skin equivalent model. These probes will also be used to evaluate mutations in the kalinin and K-laminin chains relative to the pathogenesis of junctional epidermolysis bullosa.
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