Purification and characterization of a cell-aggregating factor (clusterin), the major glycoprotein in ram rete testis fluid.

Abstract

Clusterin has been purified from ram rete testis fluid by conventional techniques and by immunoaffinity chromatography. The molecule is characterized as a glycoprotein having a molecular mass of approximately 80,000 Da and an isoelectric point of 3.6. The purified protein retains the capacity to elicit clustering of cells in an in vitro assay. Under reducing conditions in the presence of sodium dodecyl sulfate, clusterin dissociates into subunits of about 40,000 Da. Heterogeneities in apparent molecular mass were eliminated after treatment of clusterin with neuraminidase. Gel filtration chromatography revealed that clusterin exists in dimeric and tetrameric forms under conditions of neutral pH and low salt concentrations. In the presence of 6 M urea, only the monomeric form is evident, with an apparent molecular mass of approximately 85,000 Da. Clusterin, which was found to contain 4.5% glucosamine, binds to concanavalin A-Sepharose and also to wheat germ agglutinin Sepharose. The amino acid composition of clusterin is reported. The possible cellular source of clusterin in rete testis fluid is discussed. It is shown that Sertoli cells in the seminiferous tubule are one potential source, since primary cultures of rat Sertoli cells secrete a protein having the same immunochemical and physical properties as clusterin isolated from ram rete testis fluid. Possible functions of clusterin are discussed.