THROMBOSPONDIN 4

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The thrombospondins are a family of extracellular, calcium-binding proteins with adhesive and counter-adhesive activity. Thrombospondin-1 is the most extensively characterized member of the family. It has been shown to modulate cell adhesion, migration and growth. The objective of this proposal is to characterize two new members of the thrombospondin gene family. Thrombospondin-4 is upregulated in muscle after denervation and supports neuronal attachment and neurite outgrowth. Cartilage oligomeric matrix protein (COMP or thrombospondin-5) has been shown to contain mutations in bone dysplasias. The proposed study is designed to correlate in vitro and in vivo data that is obtained from protein biochemical, cell biological and genetic approaches. Specific focus will be directed toward the following areas:(1) Functional properties of thrombospondin-4 and COMP. A specific aim of the proposed study is to specifically map the sites for glycoasminoglycan binding, calcium binding, and cell binding within these proteins through the production of (1) proteolytic fragments and fusion proteins that retain functional activity, (2) polyclonal and monoclonal antibodies that block function, (3) site-directed mutagenesis, and (4) synthetic peptides that mimic or inhibit the activity of the intact proteins. These approaches will also be used to map active sites within thrombospondin-4 that support neurite outgrowth. The normal and mutant forms of thrombospondin-4 and its domains will be assayed for the ability to correct abnormalities in thrombospondin-4-deficient mice and cell lines. (2) Deletion of the thrombospondin-4 gene. A goal of the proposed study is to delete the thrombospondin-4 gene from the mouse genome by homologous recombination in embryonic stem cells. Selected embryonic stem cells will be used to produce chimeric mice that can be bred to establish a strain of mice that are heterozygous or homozygous for thrombospondin-4 deficiency in two genetic backgrounds. If the homozygotes are not viable, we will determine the developmental stage and cause of the embryonic lethality. If the homozygous animals are viable, the morphology of the major organ systems will be analyzed, with particular focus on brain, heart and skeletal muscle. The possibility of compensation by other members of the thrombospondin gene family will be investigated by quantitating mRNA levels and by crossing two strains of mice that are deficient in other thrombospondins. (3) COMP mutations in chondrodysplasias. A specific aim of the proposed study is to determine how these mutations and others cause PSACH and multiple epiphyseal dysplasia (MED). We hypothesize that these chondrodysplasias are a result of either decreased chondrocyte viability and premature cessation of growth or a defect in the interaction of chondrocytes with the extracellular matrix. To determine the role of COMP mutations in PSACH and MED, we plan to examine the effect of the mutations on the structure, biosynthesis and function of COMP. Mutant proteins will be made by site directed mutagenesis and baculovirus expression. The mutant proteins will be compared to normal COMP in terms of their (1) calcium-dependent folding, (2) interaction with chaperones, (3) ability to bind glycosaminoglycans, and (4) their interaction with cell surfaces.