Isolation and characterization of proteoglycans synthesized by cultured mesangial cells

• Published in: The Journal of biological chemistry Vol. 265; no. 1; pp. 522 - 531
• Main Authors: YAOITA, E, OGURI, K, OKAYAMA, E, KAWASAKI, K, KOBAYASHI, S, KIHARA, I, OKAYAMA, M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.01.1990
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cells, Cultured; Centrifugation, Density Gradient; Chondroitin Lyases; Chondroitin Sulfate Proteoglycans - biosynthesis; Chondroitin Sulfate Proteoglycans - isolation & purification; Chromatography; Clusterin; Female; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Glomerular Mesangium - metabolism; Glucosamine - metabolism; Glycoproteins; Glycoproteins - biosynthesis; Glycosaminoglycans - analysis; Heparan Sulfate Proteoglycans; Heparitin Sulfate - biosynthesis; Hyaluronic Acid - analysis; kidney; Molecular Chaperones; Molecular Weight; Proteins; Proteoglycans - biosynthesis; Proteoglycans - isolation & purification; Rats; Rats, Inbred F344; Sulfates - metabolism; Proteoglycan; Cell culture; Radiolabelling; Rat; Mesangial cell; Rodentia; Characterization; Vertebrata; Mammalia; Isolation; Density gradient; Immunofluorescence; Column chromatography
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)40262-7

Rat mesangial cells selected by long-term culture of glomeruli exhibited a hill and valley appearance in the confluent state and were stained with antibodies against vimentin and desmin, suggesting that they are smooth muscle-like mesangial cells. The glycoconjugates produced by the cells were metabolically labeled with [35S]sulfate and [3H]glucosamine and extracted with 4 M guanidine HCl containing 0.5% Triton X-100. The radiolabeled glycoconjugates were separated on DEAE-Sephacel and compared with those synthesized by glomeruli labeled in the same conditions. Of the three major sulfated glycoconjugates, sulfated glycoprotein (17% of the total 35S-labeled macromolecules), heparan sulfate proteoglycan (35%), and chondroitin sulfate proteoglycan (30%) synthesized by glomeruli, the cultured mesangial cells synthesized mainly chondroitin sulfate proteoglycan (more than 90%). After purification by CsCl density-gradient centrifugation, the chondroitin sulfate proteoglycan from the cell layer was separated on Bio-Gel A-5m into three molecular species with estimated Mr values of 230,000, 150,000, and 40,000-10,000, whereas that released into the medium consisted of a single species with an Mr of 135,000. In the beta-elimination reaction, the former two larger proteoglycans released chondroitin sulfate chains with Mr of an apparent 30,000 and the latter from the medium released the glycosaminoglycan chains with an Mr of 36,000. The Mr of the smallest proteoglycan from the cell layer was not significantly changed after beta-elimination, indicating that this species had only a small peptide, if any. Analysis with chondroitinase AC-II and ABC demonstrated that all the chondroitin sulfates were copolymers consisting of glucuronosyl-N-acetylgalactosamine (65-74%) having sulfate groups at position 4 (53-57%) or positions 4 and 6 (10-14%) of hexosamine moieties and iduronosyl-N-acetylgalactosamine (21-26%) having sulfate groups at position 4 (17-23%) or positions 4 and 6 (about 3%) of hexosamine moieties; namely chondroitin sulfate H type. These characteristics of the chondroitin sulfate H proteoglycans synthesized by the cultured mesangial cells were very similar to those of the proteoglycans synthesized by glomeruli. Thus, we conclude that most, if not all, of the glomerular chondroitin sulfate proteoglycans are synthesized by mesangial cells. The cultured mesangial cells were also found to synthesize hyaluronic acid at a similar level to chondroitin sulfate proteoglycan. Based on the characteristics of this glycosaminoglycan, we discuss the possible role of hyaluronic acid produced by mesangial cells.