title: Effect of disulfide bonding and multimerization on proteoglycan 4's cartilage boundary lubricating ability and adsorption.
abstract: OBJECTIVE
  The objectives of this study were to assess the cartilage boundary lubricating ability of (1) nonreduced (NR) disulfide-bonded proteoglycan 4 (PRG4) multimers versus PRG4 monomers and (2) NR versus reduced and alkylated (R/A) PRG4 monomers and to assess (3) the ability of NR PRG4 multimers versus monomers to adsorb to an articular cartilage surface.
  
  
  METHODS
  PRG4 was separated into two preparations, PRG4 multimer enriched (PRG4Multi+) and PRG4 multimer deficient (PRG4Multi-), using size exclusion chromatography (SEC) and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cartilage boundary lubricating ability of PRG4Multi+ and PRG4Multi- was compared at a physiological concentration (450 μg/mL) and assessed over a range of concentrations (45, 150, and 450 μg/mL). R/A and NR PRG4Multi- were evaluated at 450 μg/mL. Immunohistochemistry with anti-PRG4 antibody 4D6 was performed to visualize the adsorption of PRG4 preparations to the surface of articular cartilage explants.
  
  
  RESULTS
  Separation into enriched populations of PRG4Multi+ and PRG4Multi- was achieved using SEC and was confirmed by SDS-PAGE. PRG4Multi+ and PRG4Multi- both functioned as effective friction-reducing cartilage boundary lubricants at 450 μg/mL, with PRG4Multi+ being more effective than PRG4Multi-. PRG4Multi+ lubricated in a dose-dependent manner, however, PRG4Multi- did not. R/A PRG4Multi- lubricated similar to NR PRG4Multi-. PRG4-containing solutions showed 4D6 immunoreactivity at the articular surface; the immunoreactive intensity of PRG4Multi+ appeared to be similar to SF, whereas PRG4Multi- appeared to have less intensity.
  
  
  CONCLUSIONS
  These results demonstrate that the intermolecular disulfide-bonded multimeric structure of PRG4 is important for its ability to adsorb to a cartilage surface and function as a boundary lubricant. These findings contribute to a greater understanding of the molecular basis of cartilage boundary lubrication of PRG4. Elucidating the PRG4 structure-lubrication function relationship will further contribute to the understanding of PRG4's role in diarthrodial joint homeostasis and disease.
authors.affiliation: a Biomedical Engineering Graduate Program, University of Calgary , Calgary , AB , Canada.|b McCaig Institute of Bone and Joint Health , University of Calgary , Calgary , AB , Canada.
authors.forename: Saleem
authors.initials: S
authors.lastname: Abubacker
authors.affiliation: b McCaig Institute of Bone and Joint Health , University of Calgary , Calgary , AB , Canada.|c Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine , University of Calgary , Calgary , AB , Canada.
authors.forename: Dragana
authors.initials: D
authors.lastname: Ponjevic
authors.affiliation: d Biomedical Engineering Department , Northwestern University , Evanston , IL , USA.
authors.forename: Hyun O
authors.initials: HO
authors.lastname: Ham
authors.affiliation: d Biomedical Engineering Department , Northwestern University , Evanston , IL , USA.|e Departments of Bioengineering and Materials Science and Engineering Department , University of California , Berkeley , CA , USA.
authors.forename: Phillip B
authors.initials: PB
authors.lastname: Messersmith
authors.affiliation: b McCaig Institute of Bone and Joint Health , University of Calgary , Calgary , AB , Canada.|c Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine , University of Calgary , Calgary , AB , Canada.
authors.forename: John R
authors.initials: JR
authors.lastname: Matyas
authors.affiliation: a Biomedical Engineering Graduate Program, University of Calgary , Calgary , AB , Canada.|b McCaig Institute of Bone and Joint Health , University of Calgary , Calgary , AB , Canada.|f Faculty of Kinesiology , University of Calgary , Calgary , AB , Canada.
authors.forename: Tannin A
authors.initials: TA
authors.lastname: Schmidt
mesh_terms: D000327:Adsorption; D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism*; D002417:Cattle; D002850:Chromatography, Gel; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D017276:Friction; D007150:Immunohistochemistry; D007700:Kinetics; D008153:Lubrication; D055503:Protein Multimerization; D011509:Proteoglycans / Q000737:chemistry* / Q000378:metabolism*; D000099158:Proteoglycan 4
chemical_list: D004220:Disulfides; D011509:Proteoglycans; C498836:PRG4 protein, Bos taurus; D000099158:Proteoglycan 4
keywords: Boundary lubrication; PRG4 disulfide-bonded structure; cartilage adsorption; proteoglycan 4 (PRG4)