Journal of Bone and Mineral Metabolism

, Volume 29, Issue 1, pp 23–30

A functional role of the glycosylated N-terminal domain of chondromodulin-I

Authors

  • Jun Kondo
    • Advanced Medical Research Laboratory, Research DivisionMitsubishi Tanabe Pharma Corporation
  • Hiroyuki Shibata
    • Department of Cellular Differentiation, Institute for Frontier Medical SciencesKyoto University
  • Shigenori Miura
    • Department of Cellular Differentiation, Institute for Frontier Medical SciencesKyoto University
  • Akira Yamakawa
    • Department of Life Science, Graduate School of Life ScienceUniversity of Hyogo
  • Koji Sato
    • Department of Cellular Differentiation, Institute for Frontier Medical SciencesKyoto University
  • Yoshiki Higuchi
    • Department of Life Science, Graduate School of Life ScienceUniversity of Hyogo
  • Chisa Shukunami
    • Department of Cellular Differentiation, Institute for Frontier Medical SciencesKyoto University
    • Department of Cellular Differentiation, Institute for Frontier Medical SciencesKyoto University
Original Article

DOI: 10.1007/s00774-010-0193-0

Cite this article as:
Kondo, J., Shibata, H., Miura, S. et al. J Bone Miner Metab (2011) 29: 23. doi:10.1007/s00774-010-0193-0

Abstract

Chondromodulin-I (ChM-I) is a 25-kDa glycoprotein that specifically localizes in the extracellular matrix of cartilage and negatively regulates angiogenesis. ChM-I comprises two domains: an N-terminal hydrophilic domain (domain 1) containing an N-linked glycosylation site and a C-terminal hydrophobic domain (domain 2) with all four disulfide bonds that are present in this protein. We generated a nonglycosylated recombinant human ChM-I (NG-hChM-I) and compared its bioactivity with that of the glycosylated form of human ChM-I (G-hChM-I) expressed in Chinese hamster ovary cells in vitro. NG-hChM-I exhibited the growth factor/inhibitor activity in the cultures of chondrocytes and vascular endothelial cells but required markedly higher doses. Although domain 1 is predicted to be hydrophilic per se on the basis of its amino acid sequence, NG-hChM-I remains insoluble in aqueous solution as much as ΔN-hChM-I that lacks the N-terminal 37 amino acids containing an N-glycosylation site. Circular dichroism measurements revealed that the content of α-helix was calculated to be 34% in G-hChM-I, whereas the content of the characteristic secondary structures in NG-hChM-I was distinctly lower than those in G-hChM-I. These results indicate that glycosylation in domain 1 is critical for the structural integrity for biological functions of ChM-I in vitro.

Keywords

Chondromodulin-I Glycosylation Angiogenesis inhibitor Vascular endothelial cells Chondrocytes

Supplementary material

774_2010_193_MOESM1_ESM.pdf (192 kb)
Supplemental Fig. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (15%) of NG-hChM-I under reduced conditions during purification. Proteins were stained with Coomassie Brilliant Blue R250. Lane 1, total cells; lane 2, extracts with urea and DTT; lane 3, Q Sepharose eluate; lane 4, SP Sepharose eluate; lane 5, Butyl-Toyopearl eluate; lane 6, molecular weight markers. (PDF 192 kb)

Copyright information

© The Japanese Society for Bone and Mineral Research and Springer 2010