The major components of cartilage extracellular matrix consist of collagen, fibrils, proteoglycans and water. The chondroitin sulfate proteoglycan (aggrecan) is comprised of proteoglycan core protein substituted with multiple chains of chrondroitin sulfate and keratan sulfate. The proteoglycan complex is, in turn, non-covalently bound to hyaluronate, interactions which are stabilized by a third component, the link protein. Link protein and the aggregating proteoglycan are related macromolecules, showing significant primary sequence homology in regions with similar function. Hyaluronate binding domains in the link protein and proteoglycan are also related to domains found in the lymphocyte homing receptor (CD44). Detailed structural information about the molecular interactions between these components of the proteoglycan aggregate of cartilage upon which cartilage structure and function depend, will enhance our understanding of cartilage biochemistry in normal and disease states such as arthritis and structural information concerning functional domains of the link protein molecule will related to the homologous functionally similar domains in the aggrecan monomer. Furthermore, these studies may help to explain the functional significance of homologous proteins on lymphocyte cell surfaces. The atomic resolution structure of link protein will be studied using a combination of molecular biological, biochemical and x-ray crystallographic techniques. Over the three years of the proposed grant period we will obtain crystals suitable for three dimensional atomic resolution studies of the intact link protein and isolated structural domains produced using recombinant DNA techniques. Once appropriate crystals of link protein are available structure analysis by single crystal x-ray diffraction methods will be undertaken. Finally the resultant structures will be examined in light of their known functional roles. This development and feasibility study will provide the basis for ongoing collaborative studies between the principal investigators. The information obtained will be used to initiate more comprehensive investigations into cartilage extracellular matrix macromolecular structure, as well as to influence ongoing studies to determine sites of functional interactions between components of the proteoglycan aggregate. Related studies already in progress involve cloning, site directed mutagenesis and functional determinations of link protein and aggrecan domains. Three dimensional atomic resolution structural information once obtained will enable the rational design of mutant forms of link protein that can be produced by recombinant DNA technology, studied using crystallographic techniques and assessed for function by well established biochemical methods.
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