Experiments will be performed to investigate the structure and degradation of the extracellular matrix assembled by human gingival fibroblasts in cell culture. It will initially be shown that gingival fibroblasts can assemble the three major fibrillar systems found in most connective tissues: namely, the interstitial collagen fibrils assembled from type I, III and V collagens (and possibly type XII and/or type XIV collagen), the elastin (beaded, oxytalan) fibers and the microfibrils of type VI collagen. Monoclonal or polyclonal antibodies will be used to demonstrate the assembly of these fibrils and, where necessary, additional monoclonal antibodies will be prepared (e.g. to human type V collagen). Biochemical analyses will be performed to determine the relative proportions of type I, III and V collagens present in the matrix. Experiments will be performed in which the cells, cell membranes and soluble proteins of the matrix will be extracted by mild detergent (Triton X-100) to leave a fibrillar matrix whose structure will be investigated by i) transmission electron microscopy, ii) electron microscopic immunolocalization using a second antibody coupled to colloidal gold, iii) rotary shadowing of a homogenate of the matrix. We will determine that the structure of the three fibril systems in the extracellular matrix are unaffected by detergent extraction. We will also explore the effects of removal of glycosaminoglycans from the matrix by digestion with chondroitinase ABC or hyaluronidase. Once these experiments are completed, gingival fibroblasts will be grown and the matrix radioactively-labeled with [3H]glycine. The matrix preparation will be cut into small squares and used as a substrate for the three metalloproteinases thought to be required for successful matrix degradation: namely, collagenase, stromelysin and 72 kD gelatinase. We presently have available recombinant collagenase and stromelysin and recombinant 72 kD gelatinase will be obtained from Dr. Jeffrey Engler's laboratory, UAB. The release of peptide fragments from the matrix will be examined initially by fluorography and large-scale preparations will be made of any significant peptides that are released. Initial identification of peptides will be based on N-terminal amino acid sequencing after blotting onto Immobilon membrane. Cultures will be examined by light and electron microscopy and, additionally by rotary shadowing, to determine the effects of each enzyme either separately or in combination on the structure of the three fibril systems. These experiments will explore the enzymatic requirements for the degradation of a matrix which closely resembles the matrix as it exists in the gingiva. Nothing is known of the requirements for the degradation either of the type VI fibrils or the elastin (oxytalan) fibers and yet both are key fibrils of the matrix. The results will provide important new insights into the degradation of naturally occurring fibril systems in a manner which previously has not been achieved.
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