Monoclonal antibody Rip specifically recognizes 2',3'-cyclic nucleotide 3'-phosphodiesterase in oligodendrocytes.
The antigen recognized with monoclonal antibody (mAb) Rip (Rip-antigen) has been long used as a marker of oligodendrocytes and myelin sheaths. However, the identity of Rip-antigen has yet to be elucidated. We herein identified the Rip-antigen. No signal recognized by mAb-Rip was detected by immunoblot analyses in the rat brain, cultured rat oligodendrocytes, or the oligodendrocyte cell line CG-4. As this antibody worked very well on immunocytochemistry and immunohistochemistry, Rip-antigen was immunopurified with mAb-Rip from the differentiated CG-4 cells. Eight strong-intensity bands thus appeared on 5-20% SDS-PAGE with SYPRO ruby fluorescence staining. To identify these molecules, each band extracted from the gel was analyzed by MALDI-QIT/TOF mass spectrometry. We found an interesting molecule in the oligodendrocytes from an approximately 44-kDa band as 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). To test whether CNP was recognized by mAb-Rip, double-immunofluorescence staining was performed by using Alexa Fluor 488-conjugated mAb-Rip and Alexa Fluor 568-conjugated mAb-CNP in the rat cerebellum, mouse cerebellum, cultured rat oligodendrocytes, and CG-4 cells. The Rip-antigen was colocalized with CNP in these cells and tissues. To provide direct evidence that CNP was recognized by mAb-Rip, rat Cnp1-transfected HEK293T cells were used for double-immunofluorescence staining with mAb-Rip and mAb-CNP. The Rip-antigen was colocalized with CNP in rat Cnp1-transfected HEK293T cells, but the antigen was not detected by mAb-Rip and mAb-CNP in mock-transfected HEK293T cells. Overall, we have demonstrated that the antigen labeled with mAb-Rip is CNP in the oligodendrocytes.
Watanabe
Masatomo
M
Laboratory of Molecular Pharmacology, Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Sanuki, Kagawa, Japan.
Sakurai
Yoko
Y
Ichinose
Tatsuya
T
Aikawa
Yoshikatsu
Y
Kotani
Masaharu
M
Itoh
Kouichi
K
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology* / Q000302:isolation & purification; D001921:Brain / Q000166:cytology / Q000201:enzymology / Q000276:immunology; D002460:Cell Line; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique / Q000379:methods; D005456:Fluorescent Dyes; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008813:Mice, Inbred ICR; D009474:Neurons / Q000166:cytology / Q000201:enzymology; D009836:Oligodendroglia / Q000166:cytology / Q000201:enzymology* / Q000276:immunology*; D051381:Rats; D017208:Rats, Wistar; D014162:Transfection
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D005456:Fluorescent Dyes; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases