title: Isolation and characterization of dermatan sulfate proteoglycans synthesized by cultured bovine aortic endothelial cells.
abstract: Three glucuronic acid-rich dermatan sulfate proteoglycans (DS-PGs) have been isolated by chromatographic and electrophoretic techniques from cultures of bovine aortic endothelial cells and characterized structurally. The smallest of the DS-PGs (DS-II) has an apparent Mr of approximately 100,000 and glycosaminoglycan chains of Mr approximately 29,000. Core glycoprotein samples prepared by chondroitin ABC lyase digestion run as doublets of Mr = 45,000 and 48,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A decrease in core size is apparent after N-glycanase digestion, or when DS-PG is isolated from tunicamycin-treated cultures, providing evidence that the core protein is N-glycosylated. Isolated DS-II shows evidence of self-association when subjected to liquid chromatography under conditions of reduced ionic strength, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, DS-II, but not other endothelial cell DS-PG subclasses, is bound by an antibody against human skin fibroblast DS-PG, indicating that this DS-PG belongs to a family of widely distributed small DS-PGs, previously isolated from various connective tissues. A slightly larger (Mr approximately 220,000) DS-PG (DS-I) can be separated from DS-II by preparative electrophoresis. Despite similarities in core size and extent of N-glycosylation between DS-I and DS-II, DS-I shows only limited ability to self-associate, and does not interact with the anti-fibroblast DS-PG antibody. DS-I glycosaminoglycan chains are also smaller (Mr approximately 18,000) than those from DS-II, similar in size to the chains borne by the DS-PG subclass of largest size (high molecular weight (HMW)-DS). HMW-DS, which predominated in cell layer extracts, runs with a Kav of 0.45 on Sepharose CL-2B and is estimated to have an Mr greater than 700,000. Reduction and alkylation of HMW-DS indicates that it forms disulfide-bonded aggregates with other matrical proteins within the cell layer. HMW-DS displayed multiple protein cores (Mr greater than 200,000) upon chondroitin ABC lyase treatment. Despite some similarity in size to the family of large, aggregating chondroitin sulfate proteoglycans and DS-PGs, immunological evidence suggests that it lacks a hyaluronic acid binding region.
authors.affiliation: Department of Pathology, School of Medicine, University of Washington, Seattle 98195.
authors.forename: M G
authors.initials: MG
authors.lastname: Kinsella
authors.forename: T N
authors.initials: TN
authors.lastname: Wight
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002807:Chondroitin / Q000031:analogs & derivatives*; D011508:Chondroitin Sulfate Proteoglycans / Q000302:isolation & purification*; D002850:Chromatography, Gel; D003871:Dermatan Sulfate / Q000302:isolation & purification*; D004730:Endothelium, Vascular / Q000378:metabolism*; D005470:Fluorometry; D007150:Immunohistochemistry; D008715:Methionine / Q000378:metabolism; D011509:Proteoglycans / Q000302:isolation & purification*
chemical_list: D011508:Chondroitin Sulfate Proteoglycans; D011509:Proteoglycans; C032649:dermatan sulfate proteoglycan; D003871:Dermatan Sulfate; D002807:Chondroitin; D008715:Methionine