Biosynthesis of Sulfated Proteoglycanin vitroby Cells Derived from Human Osteochondrophytic Spurs of the Femoral Head

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Abstract
Cells derived from organ-explant culture of the cartilaginous component of osteochondrophytic spurs of human femoral heads were incubated with [35S]-sulfate to study sulfated proteoglycan biosynthesis in vitro. Secondary monolayer cultures incorporated [35S]-sulfate into macromolecules which were recovered in the bottom fraction (dA1) of a CsCl gradient after ultracentrifugation in associative buffer (0.5 M guanidine .cntdot. HCl). The incorporated [35S]-sulfate in fraction dA1 from the culture medium eluted in 2 with average partition coefficients (Kav) of 0.14 and 0.45, respectively, on Sepharose CL-2B eluted with dissociative buffer (4 M guanidine .cntdot. HCl). A signficant percentage of incorporated [35S]-sulfate was found in the medium dA4 fraction (44%). The Kav of this fraction on Sepharose CL-2B was 0.66 with a shoulder of incorporated [35S]-sulfate at Kav, 0.22. In contrast to the culture medium, cellular CsCl gradient fractions dA1-dA3 showed Kav''s on Sepharose CL-2B ranging from 0.63-0.75. Cellular fraction dA4 was even more polydisperse. A dD1 fraction (proteoglycan monomer) prepared by CsCl ultracentrifugation in dissociative buffer of [35S]-sulfate labeled culture medium eluted with a Kav of 0.25 on Sepharose CL-2B identical to the Kav of bovine nasal cartilage A1D1 and human tissue osteophyte A1D1 chromatographed under identical conditions. Glycosaminoglycan analysis demonstrated significant amounts of chondroitin 6- and 4-sulfate in unfractionated culture medium and in those proteoglycan fractions generated from culture medium (dA1, dA2 and dD1). Cellular fractions dA1-dA3 and medium fraction dA4 were enriched in dermatan sulfate. The size of the [35S]-sulfated glycosaminoglycan chains analyzed by Sepharose CL-6B chromatography showed considerable polydispersity (Kav range, 0.29-0.52). Cells derived from the cartilaginous component of human osteophyte apparently synthesized several distinct populations of sulfated proteoglycans. These results may reflect the heterogeneity of cells which grow out from osteophyte organ explants and become established in monolayer culture.