The N-terminal cleavage of chondromodulin-I in growth-plate cartilage at the hypertrophic and calcified zones during bone development.
Chondromodulin-I (ChM-I) is a 20-25 kDa anti-angiogenic glycoprotein in cartilage matrix. In the present study, we identified a novel 14-kDa species of ChM-I by immunoblotting, and purified it by immunoprecipitation with a newly raised monoclonal antibody against ChM-I. The N-terminal amino acid sequencing indicated that it was an N-terminal truncated form of ChM-I generated by the proteolytic cleavage at Asp37-Asp38. This 14-kDa ChM-I was shown by the modified Boyden chamber assay to have very little inhibitory activity on the VEGF-A-induced migration of vascular endothelial cells in contrast to the intact 20-25 kDa form of ChM-I (ID50 = 8 nM). Immunohistochemistry suggested that 20-25 kDa ChM-I was exclusively localized in the avascular zones, i.e. the resting, proliferating, and prehypertrophic zones, of the cartilaginous molds of developing long bone, whereas the 14-kDa form of ChM-I was found in hypertrophic and calcified zones. Immunoblotting demonstrated that mature growth-plate chondrocytes isolated from rat costal cartilage actively secrete ChM-I almost exclusively as the intact 20-25 kDa form into the medium in primary culture. Taken together, our results suggest that intact 20-25 kDa ChM-I is stored as a component of extracellular matrix in the avascular cartilage zones, but it is inactivated by a single N-terminal proteolytic cleavage in the hypertrophic zone of growth-plate cartilage.
Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Shigenori
S
Miura
Research and Development Division, Science and Technology Research Center Inc., Mitsubishi Chemical Group, Kanagawa, Japan.
Jun
J
Kondo
Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Aki
A
Takimoto
Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Hiroko
H
Sano-Takai
Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Long
L
Guo
Department of Dental and Medical Biochemistry, Basic Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan.
Chisa
C
Shukunami
Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Hideyuki
H
Tanaka
Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Yuji
Y
Hiraki
D000595:Amino Acid Sequence; D000818:Animals; D001846:Bone Development; D002113:Calcification, Physiologic; D002356:Cartilage / Q000378:metabolism*; D019902:Chondrocytes / Q000378:metabolism; D006132:Growth Plate / Q000166:cytology / Q000378:metabolism* / Q000473:pathology / Q000502:physiology; D006801:Humans; D006984:Hypertrophy; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D059748:Proteolysis; D051381:Rats
D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; C068675:CNMD protein, human