Clusterin is the major protein secreted by cultured Sertoli cells. Clusterin is heavily glycosylated protein consisting of disulfide linked heteromonomers and is expressed by many cells but primarily by those of epithelial origin. It is secreted into fluid-tissue interfaces and the basal level of secretion is greatly increased under conditions of cellular stress such as heat shock or anoxia. In addition, it accumulates in apoptotic germ cells in the testis but it is synthesized primarily by the Sertoli cells in response to the impeding death of the nearby cells. The function of clusterin is unknown but we present a hypothesis supported by data from the literature and our own work that clusterin is necessary for solubilizing insoluble macromolecular complexes and presenting them for endocytosis by the surviving cells. This function would make clusterin important for keeping fluid-tissue interfaces free from cellular debris and would have implications for stressed or damaged or aged tissue. The evidence supports the notion that the biological function of clusterin is a result of its ability to bind tightly to hydrophobic macromolecules and molecular complexes. In this proposal we present experiments to directly test our proposed function for clusterin. In the first specific aim we will thoroughly analyze the impact of a knock-out of the clusterin gene on male reproductive function under normal and toxic conditions. We will examine the basis for the binding of clusterin to a variety of target proteins. In the second specific aim we will analyze a number of structural properties of clusterin and attempt to relate structure to function. We will make deletion and site-specific mutations and analyze the binding of modified clusterin to a defined substrate. We will analyze structure by limited proteolysis, by CD spectroscopy and by fluorescence and cross-linking. Since clusterin is clearly implicated as a marker for cellular damage and pathology, the elucidation of its function is very important.
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