Characterization of Proteoglycans Synthesized by Cultured Arterial Smooth Muscle Cells of the Rat

Abstract

Arterial smooth muscle cells cultured from rat aorta were labeled with Na [35S]-sulfate in combination with either [3H]glucosamine or [3H]mannose. The newly synthesized hyaluronate and sulfated proteoglycans obtained from the growth medium (M-PG) and extracted from the cell layer (C-PG) with 4M guanidinium chloride in the presence of proteinase inhibitors were purified by sequential fractionation on Sepharose 4B CL, equilibrium density gradient centrifugation and ion exchange chromatography under dissociative conditions. Gel filtration of M-PG resulted in the separation of free hyaluronate and 2 size classes of [35S]-proteoglycan populations eluted at Kav 0.15 (fraction M-A) and 0.48 (fraction M-B). On further fractionation M-A dissociated into hyaluronate (Mr 1.6 .times. 106) and a proteoglycan monomer (M-PG, A, MW 180,000), which contained chondroitin 4-sulfate (MW 21,000) as the main glycosaminoglycan moiety. The proteoglycan isolated from M-B (M-PG B) was identified as a proteoglycan monomer (MW 200,000) containing mainly chondroitin sulfate/dermatan sulfate hybrid side chains (MW 34,000). [3H]Mannose labelling and binding to ConA [concanavalin A] Sepharose of both M-PG A and B indicated the presence of oligosaccharides of the glycoprotein type. An analogous fractionation of proteoglycans associated with the cell layer yielded 2 hyaluronate-proteoglycan complexes (C-PreA and C-A). The proteoglycan monomers of these complexes (C-PG PreA and C-PG A) had MW values of 420,000 and 130,000. A non-complexed proteoglycan monomer C-PG B (MW 90,000) was also found. All cell layer bound proteoglycans had glycosaminoglycan side chains with MW 36,000 but the predominant glycosaminoglycan component was either heparan sulfate, chondroitin sulfate or dermatan sulfate. All cell layer bound proteoglycans contained [3H]mannose radioactivity, about 15% of which was bound to ConA Sepharose.