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.