Module-specific antibodies against human connective tissue growth factor: utility for structural and functional analysis of the factor as related to chondrocytes.
Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24/CCN2) shows diverse functions in the process of endochondral ossification. It promotes not only the proliferation and differentiation of chondrocytes and osteoblasts in vitro, but also angiogenesis in vivo. The ctgf gene is a member of the gene family called CCN, and it encodes the characteristic 4-module structure of this family, with the protein containing IGFBP, VWC, TSP and CT modules. We raised several monoclonal antibodies and polyclonal antisera against CTGF, and located the epitopes in the modules by Western blotting. For mapping the epitopes, Brevibacillus-produced independent modules were utilized. As a result, at least 1 antibody or antiserum was prepared for the detection of each module in CTGF. Western blotting with these antibodies is expected to be useful for the analysis of CTGF fragmentation. Moreover, we examined the effects of these monoclonal antibodies on the biological functions of CTGF. One out of 3 humanized monoclonal antibodies was found to neutralize efficiently the stimulatory effect of CTGF on chondrocytic cell proliferation. This particular antibody bound to the CT module. In contrast, surprisingly, all of the 3 antibodies recognizing IGFBP, VWC and CT modules stimulated proteoglycan synthesis in chondrocytic cells. Together with previous findings, these results provide insight into the structural-functional relationships of CTGF in executing multiple functions.
Minato
Masanao
M
Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8525.
Kubota
Satoshi
S
Kawaki
Harumi
H
Nishida
Takashi
T
Miyauchi
Akira
A
Hanagata
Hiroshi
H
Nakanishi
Tohru
T
Takano-Yamamoto
Teruko
T
Takigawa
Masaharu
M
D020816:Amino Acid Motifs; D000906:Antibodies / Q000276:immunology* / Q000494:pharmacology; D000918:Antibody Specificity; D001951:Brevibacterium / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism; D003198:Computer Simulation; D055513:Connective Tissue Growth Factor; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D007106:Immune Sera / Q000276:immunology / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D011509:Proteoglycans / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013329:Structure-Activity Relationship
D000906:Antibodies; C526317:CCN2 protein, human; D000939:Epitopes; D017874:Immediate-Early Proteins; D007106:Immune Sera; D036341:Intercellular Signaling Peptides and Proteins; D011509:Proteoglycans; D011994:Recombinant Proteins; D055513:Connective Tissue Growth Factor