title: Characterization of chondroitin/dermatan sulfate proteoglycans synthesized by bovine retinal pericytes in culture.
abstract: Pericytes associate with the outside of endothelial cells in microvessels. Previous studies have shown that these cells synthesize glycosaminoglycans (GAGs) but the nature of the core proteins to which these GAGs are attached is unknown. In the present study, cultured bovine retinal pericytes were metabolically labeled with [(3)H]glucosamine, [(35)S]sodium sulfate or (35)S-labeled amino acids and the proteoglycans synthesized by these cells were purified by DEAE-Sephacel ion exchange and molecular sieve Sepharose CL-4B chromatography. Separated proteoglycans were digested with papain, heparitinase or chondroitin ABC lyase and the GAGs characterized by Sepharose CL-6B chromatography. Proteoglycans were also assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after digestion with chondroitin ABC lyase. Pericytes predominantly synthesize and secrete chondroitin or dermatan sulfate proteoglycans (CS/DS PGs) rather than heparan sulfate proteoglycans (HSPGs). Two subclasses of CS/DS PGs are synthesized by pericytes; one is a high M(r) subclass with high charge density. This subclass eluted at the void volume of a Sepharose CL-4B molecular sieve column, was susceptible to chondroitin ABC lyase, and contained core proteins of ca. 550 and 450 kD which were recognized by antibody to versican. The other major subclass eluted at a K(av) ca. 0.45 on a Sepharose CL-4B molecular sieve column, was susceptible to chondroitin ABC lyase, and contained core proteins recognized by antibodies to either biglycan or decorin that separated as a broad band of ca. 50 kDa in SDS-PAGE. A small amount of HSPG was also synthesized by these cells and could be separated from the CS/DS PGs by DEAE-Sephacel chromatography using a linear gradient of 0.1-0.7 M NaCl. Release of GAG chains by protease digestion indicated that the length of GAG chains was approximately M(r) 45000 in biglycan and decorin, approximately M(r) 48000 in the small amount of HSPGs and approximately M(r) 66000 in versican. These proteoglycans resemble those synthesized by vascular smooth muscle cells but differ markedly from those synthesized by vascular endothelial cells.
authors.affiliation: Department of Environmental Health, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Japan. t-kaji@hokuriku-u.ac.jp
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Kaji
authors.forename: Shigeru
authors.initials: S
authors.lastname: Sakurai
authors.forename: Chika
authors.initials: C
authors.lastname: Yamamoto
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Fujiwara
authors.forename: Sho-ichi
authors.initials: S
authors.lastname: Yamagishi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Michael G
authors.initials: MG
authors.lastname: Kinsella
authors.forename: Thomas N
authors.initials: TN
authors.lastname: Wight
mesh_terms: D000818:Animals; D058578:Biglycan; D002417:Cattle; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis; D002809:Chondroitin Sulfates / Q000096:biosynthesis / Q000737:chemistry*; D002847:Chromatography, Agarose; D058575:Decorin; D003871:Dermatan Sulfate / Q000096:biosynthesis / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D020286:Pericytes / Q000378:metabolism*; D011509:Proteoglycans / Q000096:biosynthesis / Q000737:chemistry*; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D053675:Versicans
chemical_list: D058578:Biglycan; D011508:Chondroitin Sulfate Proteoglycans; D058575:Decorin; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D053675:Versicans; D003871:Dermatan Sulfate; D002809:Chondroitin Sulfates