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

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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.

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Acknowledgments

We thank Dr. Y. Sato (Institute for Development, Aging and Cancer, Tohoku University) for providing us with MSS31 endothelial cells. The measurement of CD spectra was carried out by using a spectrophotometer installed at Institute for Protein Research, Osaka University. We are indebted to Emeritus Prof. Hideo Akutsu at Osaka University and Dr. Yuki Takayama at National Institute of Health for their helpful discussions for the analysis of CD data. This work was supported in part by the Grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (no. 21510224).

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Authors and Affiliations

  1. Advanced Medical Research Laboratory, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, 227-0033, Japan

    Jun Kondo

  2. Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan

    Hiroyuki Shibata, Shigenori Miura, Koji Sato, Chisa Shukunami & Yuji Hiraki

  3. Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo, 678-1297, Japan

    Akira Yamakawa & Yoshiki Higuchi

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  1. Jun Kondo
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  2. Hiroyuki Shibata
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  3. Shigenori Miura
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  4. Akira Yamakawa
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  5. Koji Sato
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  6. Yoshiki Higuchi
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  7. Chisa Shukunami
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  8. Yuji Hiraki
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Correspondence to Yuji Hiraki.

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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)

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Kondo, J., Shibata, H., Miura, S. et al. A functional role of the glycosylated N-terminal domain of chondromodulin-I. J Bone Miner Metab 29, 23–30 (2011). https://doi.org/10.1007/s00774-010-0193-0

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  • DOI: https://doi.org/10.1007/s00774-010-0193-0

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