Biosynthesis of Sulfated Proteoglycan in vitro by Cells Derived from Human Osteochondrophytic Spurs of the Femoral Head

Abstract

Cells derived from organ-explant culture of the cartilaginous component of osteochondrophytic spurs of human femoral heads were incubated with [³⁵S]-sulfate in order 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 · HcI). The incorporated [³⁵S]-sulfate in fraction dA1 from the culture medium eluted in two peaks with average partition coefficients (K_av) of 0.14 and 0.45 respectively, on Sepharose CL-2B eluted with dissociative buffer (4 M guanidine. HCl). A significant percentage of incorporated [³⁵S]-sulfate was found in the medium dA4 fraction (44%). The K_av of this fraction on Sepharose CL-2B was 0.66 with a shoulder of incorporated [³⁵S]-sulfate at K_av 0.22. In contrast to the culture medium, cellular CsCl gradient fractions dA1-dA3 showed K_av'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 labelled culture medium eluted with a K_av of 0.25 on Sepharose CL-2B identical to the K_av 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). In contrast, 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 (K_av range, 0.29–0.52). The results of this study indicated that cells derived from the cartilaginous component of human osteophyte 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.