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The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin‐like protease(s) in a subpopulation of microglia in neonatal rat brain

To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross‐reaction with rat peritoneal macrophages (Mφ). W...

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Autores principales: Kawahara, Kohichi, Hirata, Hiroshi, Ohbuchi, Kengo, Nishi, Kentaro, Maeda, Akira, Kuniyasu, Akihiko, Yamada, Daisuke, Maeda, Takehiko, Tsuji, Akihiko, Sawada, Makoto, Nakayama, Hitoshi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129557/
https://www.ncbi.nlm.nih.gov/pubmed/27464357
http://dx.doi.org/10.1002/glia.23034
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author Kawahara, Kohichi
Hirata, Hiroshi
Ohbuchi, Kengo
Nishi, Kentaro
Maeda, Akira
Kuniyasu, Akihiko
Yamada, Daisuke
Maeda, Takehiko
Tsuji, Akihiko
Sawada, Makoto
Nakayama, Hitoshi
author_facet Kawahara, Kohichi
Hirata, Hiroshi
Ohbuchi, Kengo
Nishi, Kentaro
Maeda, Akira
Kuniyasu, Akihiko
Yamada, Daisuke
Maeda, Takehiko
Tsuji, Akihiko
Sawada, Makoto
Nakayama, Hitoshi
author_sort Kawahara, Kohichi
collection PubMed
description To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross‐reaction with rat peritoneal macrophages (Mφ). We identified the antigen molecule for 9F5: the 50‐ to 70‐kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)/osteoactivin, which started at Lys(170). In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin‐like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and Mφ did not, although all these cells expressed mRNA and the full‐length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and Mφ, may have furin‐like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double‐immunοstaining with 9F5 antibody and anti‐Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist in vivo. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG/Mφ and to reveal the role of the GPNMB fragments during developing brain. GLIA 2016;64:1938–1961
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spelling pubmed-51295572016-11-30 The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin‐like protease(s) in a subpopulation of microglia in neonatal rat brain Kawahara, Kohichi Hirata, Hiroshi Ohbuchi, Kengo Nishi, Kentaro Maeda, Akira Kuniyasu, Akihiko Yamada, Daisuke Maeda, Takehiko Tsuji, Akihiko Sawada, Makoto Nakayama, Hitoshi Glia Research Articles To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross‐reaction with rat peritoneal macrophages (Mφ). We identified the antigen molecule for 9F5: the 50‐ to 70‐kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)/osteoactivin, which started at Lys(170). In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin‐like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and Mφ did not, although all these cells expressed mRNA and the full‐length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and Mφ, may have furin‐like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double‐immunοstaining with 9F5 antibody and anti‐Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist in vivo. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG/Mφ and to reveal the role of the GPNMB fragments during developing brain. GLIA 2016;64:1938–1961 John Wiley and Sons Inc. 2016-07-27 2016-11 /pmc/articles/PMC5129557/ /pubmed/27464357 http://dx.doi.org/10.1002/glia.23034 Text en © 2016 The Authors. Glia Published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs (http://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
spellingShingle Research Articles
Kawahara, Kohichi
Hirata, Hiroshi
Ohbuchi, Kengo
Nishi, Kentaro
Maeda, Akira
Kuniyasu, Akihiko
Yamada, Daisuke
Maeda, Takehiko
Tsuji, Akihiko
Sawada, Makoto
Nakayama, Hitoshi
The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin‐like protease(s) in a subpopulation of microglia in neonatal rat brain
title The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin‐like protease(s) in a subpopulation of microglia in neonatal rat brain
title_full The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin‐like protease(s) in a subpopulation of microglia in neonatal rat brain
title_fullStr The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin‐like protease(s) in a subpopulation of microglia in neonatal rat brain
title_full_unstemmed The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin‐like protease(s) in a subpopulation of microglia in neonatal rat brain
title_short The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin‐like protease(s) in a subpopulation of microglia in neonatal rat brain
title_sort novel monoclonal antibody 9f5 reveals expression of a fragment of gpnmb/osteoactivin processed by furin‐like protease(s) in a subpopulation of microglia in neonatal rat brain
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129557/
https://www.ncbi.nlm.nih.gov/pubmed/27464357
http://dx.doi.org/10.1002/glia.23034
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