Biochemical and ultrastructural studies of proteoheparan sulfates synthesized by PYS-2, a basement membrane-producing cell line.

Abstract

The mouse teratocarcinoma-derived cell line, PYS-2, has been shown to produce laminin, a basement membrane-specific glycoprotein. In these studies we demonstrate that PYS-2 cells synthesize and secrete into the culture medium a proteoglycan which contains only heparan sulfate as its sulfated polysaccharide side chains, as well as type IV procollagen and laminin. The apparent molecular weights of the proteoglycan and its heparan sulfate side chain were estimated to be 400,000 and 25,000, respectively, by gel chromatography. A proteoheparan sulfate with properties closely similar, if not identical, to those of the proteoglycan in the medium, together with two heparan sulfate single chains of different molecular size, were extracted from the cell layer with 2% SDS in the presence of protease inhibitors. Ultrastructurally, a fine fibrillar intercellular matrix was recognized which contained discrete 100-200 A diameter ruthenium red-positive granules interspersed throughout the filamentous meshwork. The PYS-2 cultures were shown by immunofluorescence to react with antibodies against the heparan sulfate-containing proteoglycan isolated from the mouse EHS sarcoma (Hassell, J. R., P. G. Robey, H. J. Barrach, J. Wilczek, S. I. Rennard, and G. R. Martin. 1980. Proc. Natl. Acad. Sci. U. S. A. 77:4494-4498). Immunoelectron microscopic examination, using the same antibodies, revealed that the proteoheparan sulfate was located not only at the edges but also within the interstices of the matrix. These findings indicate that PYS-2 cells synthesize and secrete a proteoglycan with properties similar to those of basement membrane proteoglycan. These cells may therefore serve as a useful model system for the study of the biosynthesis and structure of basement membranes.