A functional role of the glycosylated N-terminal domain of chondromodulin-I.
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.
Kondo
Jun
J
Advanced Medical Research Laboratory, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Japan.
Shibata
Hiroyuki
H
Miura
Shigenori
S
Yamakawa
Akira
A
Sato
Koji
K
Higuchi
Yoshiki
Y
Shukunami
Chisa
C
Hiraki
Yuji
Y
D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism; D002942:Circular Dichroism; D006224:Cricetinae; D003412:Cricetulus; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data
D020533:Angiogenesis Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C068675:CNMD protein, human