We have isolated and biochemically characterized several chondroitin sulfate proteoglycans of brain identified with monoclonal antibodies, and will now extend this work using other available antibodies. We have also begun to determine the primary structure of a developmentally regulated chondroitin sulfate proteoglycan (designated 3F8), using amino acid sequence data for the synthesis of a cDNA probe by the PCR. The major objectives of our cloning studies include: a.) elucidating the structural relationship of 3F8 to other chondroitin sulfate proteoglycans of brain; b.) using cloned probes for in situ hybridization studies of its cellular sites of synthesis and tissue-distribution of message; c.) identification of core protein domains of potential functional importance, such as the growth factor-like and lectin-like sequences which we have found in another proteoglycan; and d.) eventual use of determined cDNA sequences of brain proteoglycans to better understand their biological functions by examining the effects of antisense constructs in transfected cells. We also plan to obtain information on the molecular interactions and specific biological roles of chondroitin sulfate proteoglycans in nervous tissue. In preliminary studies we have demonstrated that very low concentrations of the core proteins of characterized chondroitin sulfate proteoglycans of brain are capable of inhibiting the homophilic binding of the neuron-glia cell adhesion molecule, Ng-CAM. Using other model systems and cell adhesion molecules, we will now examine the possibility that extracellular chondroitin sulfate proteoglycans act as repulsive molecules which provide a mechanism for diminishing adhesive forces, thereby permitting cell rounding, division, differentiation, and cell movement in developing brain. Related studies will attempt to define the oligosaccharide binding specificity of the lectin-like domain present in the 1D1 (and possibly other) chondroitin sulfate proteoglycans of brain which we have isolated and characterized, and the roles of chondroitin sulfate proteoglycans in adhesive or repulsive processes will be studied by evaluating the effects of identified protein domains (expressed as fusion proteins) in cloned proteoglycans on cell-cell and cell-matrix interactions. Finally, we will determine the primary structure and cellular localization of a major heparan sulfate proteoglycan of brain which we have previously isolated and biochemically characterized. Amino acid sequence data derived from the 55 kDa core glycoprotein will be used for its cloning by our established PCR methodology. Cloning this putative membrane-intercalated cell surface proteoglycan will also enable us to employ synthetic peptides or fusion proteins as immunogens for its immunocytochemical localization in developing brain at the light and electron microscopic levels, and will provide the foundation for transfection studies aimed at understanding its possible roles in such processes as cell adhesion and interactions with growth factors.
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