Type VII collagen is a recently described collagen from human amnion. The biochemical and structural features of this molecule suggest that it is a component of anchoring fibrils. Anchoring fibrils are collagenase-sensitive, fibrous structures that are thought to anchor certain epithelial cell layers onto the underlying connective tissue. Anchoring fibrils are altered in many disease states, such as in recessive epidermoly sis bullosa dystrophica, a heritable skin disorder. This disorder is characterized by degeneration of the dermis and disruption of anchoring fibrils, resulting in severe blistering of the skin. Different regions of the dermal-epidermal junction are affected in patients with other forms of epidermolysis bullosa. The objectives of this research are (1) to define new proteins of the dermal-epidermal junction, (2) to identify the molecular components of the anchoring fibrils and (3) to determine the nature of the interaction between anchoring fibrils, the basement membrane and the underlying stroma.
Specific aims are (1) to isolate an anchoring fibril-basement membrane (AF-BM) complex from skin, using non-degradative, mechanical methods, (2) to solubilize, purify and characterize the various components of this complex, using standard techniques of self fractionation, column chromatography, HPLC and peptide mapping (3) to define the interactive sites on the molecules by performing binding studies using whole molecules and derived peptides and (4) to reconstitute anchoring fibrils from the purified components, monitoring fiber formation by electron microscopy. Human skin, fetal calf skin and skin from lathyritic chicken embryos will be used. Preliminary studies from this laboratory indicate that there is a collagenous protein in the AF-BM complex that resembles Type VII collagen. This protein will be compared to Type VII collagen from amnion to establish whether or not they are identical. In addition, there is a large, non-collagenous protein that differs from other previously described proteins of the epidermal-dermal junction. This protein will be characterized to define its chemical nature, precise location and ability to interact with other components of the AF-BM complex. Several monoclonal antibodies are available to aid in the localization of these proteins. These studies will help define the role of anchoring fibrils in the body and will provide a foundation for the study of connective tissue disorders that involve anchoring fibrils.
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