This proposal is designed to study the relationship between the structure of aggrecan and its functional role in the matrix of articular cartilage. Aggrecan is a proteoglycan whose core protein bears over 100 chondroitin sulfate chains, and which assembles into large aggregates that create a strong swelling pressure in the tissue and help it to serve as a cushioning material within articulating joints. This application is based on the recent observation that the human aggrecan core protein exhibits a unique degree of polymorphism, which produces core proteins of different lengths in different individuals, and results in aggrecan molecules whose potential number of chondroitin sulfate attachment sites may vary by up to 30%. Such structural differences could potentially influence the function of the molecule, and this scenario is made more intriguing by the observation that one of the polymorphic aggrecan alleles is preferentially associated with bilateral osteoarthritis of the hand. To study the relationship between the variant polymorphic chondroitin sulfate-attachment regions and cartilage function, an immortalized chondrocyte cell line will be stably transfected with mini-gene constructs in which the polymorphic region is flanked by aggrecan G1 and G3 domains. Different cells lines exhibiting high and low levels of recombinant expression will be analyzed to study variations that may occur in the size, type of sulfation and position of substitution of the chondroitin sulfate chains. The nature of the matrix produced by the recombinant-expressing cell lines will also be studied in an agarose culture system. The structure of the resulting matrix will be assessed by microscopy techniques, and its function by biomechanical testing. The resulting data will be used to test the hypothesis that the different aggrecan alleles can affect the functional properties of the cartilage, and will establish a basis for future study of their role in the development of osteoarthritis.
