The high molecular weight, disulfide-bonded aggregate is an atypical collagenous macromolecule which possesses unique structural and chemical features which clearly differentiate it from other pepsin-solubilized, collagen molecules. This proposal has been formulated to address key issues relevant to the aggregate's precursor structure, its function and location in extracellular matrices, and its cellular biosynthesis and origins. Proposed studies will be concerned with completing the biochemical purification of collagenous and noncollagenous constituents of the previously described pepsin-solubilized aggregate by high performance liquid chromatography coupled with molecular sieve and ion-exchange chromatography and amino acid analysis to characterize the primary structural features of these polypeptides. In addition, monospecific antibodies prepared by hybridoma technique from mice against purified components of the aggregate will be used for localization of the protein in extracellular sites of placental tissues by immunoelectron microscopy. Monospecific antibodies to the aggregate will also be used to determine by indirect immunofluorescent light microscopy the relative degree of synthesis of this macromolecule by different cells in culture. Subsequently, attempts will be made to radiolabel the precursor macromolecule by biosynthesis in cell culture and isolate it by extraction in native form by conventional solvent systems including selection by immunoprecipitation or as denatured components using denaturing solvents or detergents in the presence of thiol reducing agents. Finally, extractions of the selected placental tissues under denaturing conditions coupled with thiol reducing agents will be attempted to isolate and characterize aggregate constituents and corroborate this data with that of in vitro cell culture. The results of the proposed studies will advance current knowledge about the biochemistry and biology of the high molecular weight aggregate as it presently stands to other collagen types. Valuable information will be obtained as to the involvement of the aggregate as a potential structural entity of transitional regions, such as those interphases between stroma and basement membranes as well as the functional significance of a diverse structural protein comprised of noncollagenous and collagenous sequences in its protein backbone.
| Furuto, D K; Gay, R E; Stewart, T E et al. (1991) Immunolocalization of types V and XI collagen in cartilage using monoclonal antibodies. Matrix 11:144-9 |
