Phosphorylation of proteoglycans from human articular cartilage.
Previous studies have shown that sulfated proteoglycans from human articular and epiphyseal cartilage were phosphorylated. These macromolecules contribute to the stiffness and resiliency of this tissue. We demonstrate here that the phosphate moieties are an integral part of proteoglycan subunits. Specifically, evidence is presented which indicates that proteoglycan monomers contain 3 to 4 phosphate moieties per core protein and that these appear to exist as phosphoserine residues. Furthermore, the data illustrate that human articular cartilage also contains more than 20 different phosphoproteins, some of which are closely associated with proteoglycan aggregates. Proteoglycan subunits were purified from extracts of articular cartilage or from media fractions which had been used to label tissue specimens with 32P-orthophosphate. Chemical and radiographic analyses revealed that the phosphate concentration with respect to sulfate and uronic acid content remained constant when purified proteoglycan monomers were subjected to equilibrium ultracentrifugation and size-exclusion chromatography. That the phosphate moieties were bound to proteoglycan monomers via monoester linkages was indicated by the release of 32P-orthophosphate from proteoglycan subunits incubated under mild alkaline conditions or reacted with acid or alkaline phosphatases. Identification of serine residues in the core protein as the sites of phosphorylation was made by autoradiography of thin layer plates on which hydrolyzed samples of purified 32P-proteoglycan subunits had been subjected to 2-dimensional electrophoresis/chromatography. Quantification of 3 to 4 phosphate moieties per core protein of 200,000 daltons was made by chemical analysis of inorganic phosphate released from proteoglycans by acid hydrolysis.
D000328:Adult; D000368:Aged; D002358:Cartilage, Articular / Q000378:metabolism*; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D010750:Phosphoproteins / Q000502:physiology; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011509:Proteoglycans / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011509:Proteoglycans; D013462:Sulfur Radioisotopes