Purification and characterization of decorin from the culture media of MRC-5 cells.
Myofibroblasts play an important role in fibrogenesis. Myofibroblasts secrete several components of the extracellular matrix, including decorin. To clarify the properties of decorin synthesized by myofibroblasts, we have purified and characterized decorin secreted into culture medium by the myofibroblast cell line MRC-5. Decorin was purified by successive chromatography steps using Hitrap Q and Superdex 200. Purified decorin showed a broad band on SDS-polyacrylamide gel electrophoresis, which was resolved into two smaller molecular weight bands after digestion with chondroitinase ABC. Further digestion with N-glycanase resolved these two bands into a single band, indicating that the N-glycation pattern of decorin is heterogeneous. The N-terminal amino acid sequence analysis of the purified protein and its reactivity towards an antibody raised against a C-terminal peptide of decorin indicate that MRC-5 cells secrete full-length decorin into the culture medium. To characterize the glycosaminoglycan chains attached to decorin, glycosaminoglycans from the purified protein were treated with chondroitinase ACI, chondroitinase ACII, chondroitinase ABC and chondroitinase B. The resulting disaccharides were analyzed by chromatography, which indicated that decorin secreted by MRC-5 cells is a dermatan sulfate proteoglycan. In conclusion, the decorin secreted by MRC-5 cells has similar characteristics to the decorin expressed in several tissues. Thus, culturing MRC-5 cells may be highly useful for studying the role of decorin and myofibroblasts in fibrosis.
Honda
Eiko
E
Life Science Institute, School of Medicine, Kinki University, 377-2 Ohno-Higashi, Osaka-Sayama 589-8511, Japan.
Munakata
Hiroshi
H
D002460:Cell Line; D002810:Chondroitinases and Chondroitin Lyases / Q000378:metabolism; D003470:Culture Media; D058575:Decorin; D003871:Dermatan Sulfate; D004187:Disaccharides / Q000032:analysis; D016326:Extracellular Matrix Proteins; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D011509:Proteoglycans / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011658:Pulmonary Fibrosis / Q000209:etiology
D003470:Culture Media; C546878:DCN protein, human; D058575:Decorin; D004187:Disaccharides; D016326:Extracellular Matrix Proteins; D011509:Proteoglycans; D003871:Dermatan Sulfate; D002810:Chondroitinases and Chondroitin Lyases