title: Mass spectrometric identification of citrullination sites and immunohistochemical detection of citrullinated glial fibrillary acidic protein in Alzheimer's disease brains.
abstract: Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that convert protein arginine to citrulline residues in a calcium ion-dependent manner. Previously, we reported the abnormal accumulation of citrullinated proteins and the increase in the amount of PAD2 in hippocampi from Alzheimer's disease (AD) patients. Moreover, glial fibrillary acidic protein (GFAP), an astrocyte-specific marker protein, and vimentin were identified as citrullinated proteins by using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. To clarify the substrate specificity of PADs against GFAP, we prepared recombinant human (rh)PAD1, rhPAD2, rhPAD3, rhPAD4, and rhGFAP. After incubation of rhGFAP with rhPAD1, rhPAD2, rhPAD3, and rhPAD4, citrullinated (cit-)rhGFAP was detected by Western blotting. The citrullination of rhGFAP by rhPAD2 was unique, specific, and time dependent; additionally, rhPAD1 slightly citrullinated rhGFAP. We then generated eight anti-cit-rhGFAP monoclonal antibodies, CTGF-125, -128, -129, -1212, -1213, -1221, -122R, and -1224R, which reacted specifically with cit-rhGFAP. Two of those eight monoclonal antibodies, CTGF-122R and -1224R, reacted with both cit-rhGFAP and rhGFAP in Western blots. By using the CTGF-1221 antibody and a tandem mass spectrometer, we identified the two independent citrullination sites (R270Cit and R416Cit) of cit-rhGFAP. Immunohistochemical analysis with CTGF-1221 antibody revealed cit-GFAP staining in the hippocampus of AD brain, and the cit-GFAP-positive cells appeared to be astrocyte-like cells. These collective results strongly suggest that PAD2 is responsible for the citrullination of GFAP in the progression of AD and that the monoclonal antibody CTGF-1221, reacting with cit-GFAP at R270Cit and R416Cit, is useful for immunohistochemical investigation of AD brains.
authors.affiliation: Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.
authors.forename: Akihito
authors.initials: A
authors.lastname: Ishigami
authors.affiliation: Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Masutomi
authors.affiliation: Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.
authors.forename: Setsuko
authors.initials: S
authors.lastname: Handa
authors.affiliation: Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Nakamura
authors.affiliation: Global Applications Development Center, Shimadzu Corp., Kyoto, Japan.
authors.forename: Shuuichi
authors.initials: S
authors.lastname: Nakaya
authors.affiliation: Research & Development Division, Fujirebio Inc., Tokyo, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Uchida
authors.affiliation: Department of Neuropathology, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Saito
authors.affiliation: Department of Neuropathology, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo, Japan.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Murayama
authors.affiliation: Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Korea.
authors.forename: Byungki
authors.initials: B
authors.lastname: Jang
authors.affiliation: Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Korea.
authors.forename: Yong-Chul
authors.initials: YC
authors.lastname: Jeon
authors.affiliation: Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Korea.
authors.forename: Eun-Kyoung
authors.initials: EK
authors.lastname: Choi
authors.affiliation: Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Korea.
authors.forename: Yong-Sun
authors.initials: YS
authors.lastname: Kim
authors.affiliation: Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.|Research & Development Division, Fujirebio Inc., Tokyo, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kasahara
authors.affiliation: Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Maruyama
authors.affiliation: Advanced Medical Research Center, Yokohama City University, Yokohama, Japan.
authors.forename: Tosifusa
authors.initials: T
authors.lastname: Toda
mesh_terms: D000544:Alzheimer Disease / Q000473:pathology*; D015153:Blotting, Western; D001921:Brain / Q000473:pathology*; D002956:Citrulline / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D007150:Immunohistochemistry; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005904:Glial Fibrillary Acidic Protein; D011994:Recombinant Proteins; D002956:Citrulline; D006867:Hydrolases; C000620352:PADI2 protein, human; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases
keywords: Alzheimer's disease; citrullinated glial fibrillary acidic protein; mass spectrometric analysis; neurodegenerative disorder; peptidylarginine deiminase