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

  • Jun Kondo
  • Hiroyuki Shibata
  • Shigenori Miura
  • Akira Yamakawa
  • Koji Sato
  • Yoshiki Higuchi
  • Chisa Shukunami
  • Yuji Hiraki
Original Article

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

Authors and Affiliations

  • Jun Kondo
    • 1
  • Hiroyuki Shibata
    • 2
  • Shigenori Miura
    • 2
  • Akira Yamakawa
    • 3
  • Koji Sato
    • 2
  • Yoshiki Higuchi
    • 3
  • Chisa Shukunami
    • 2
  • Yuji Hiraki
    • 2
  1. 1.Advanced Medical Research Laboratory, Research DivisionMitsubishi Tanabe Pharma CorporationYokohamaJapan
  2. 2.Department of Cellular Differentiation, Institute for Frontier Medical SciencesKyoto UniversityKyotoJapan
  3. 3.Department of Life Science, Graduate School of Life ScienceUniversity of HyogoAko-gunJapan

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