C-terminal region-dependent change of antibody-binding to the Eighth Reelin repeat reflects the signaling activity of Reelin.
Reelin is a secreted glycoprotein that plays pivotal roles in the development and function of the brain, but how it activates downstream intracellular signaling is not fully understood. We have recently reported that the highly conserved C-terminal region (CTR) of Reelin is required for its full signaling activity, although the underlying mechanism remains unknown. During biochemical study of Reelin, we serendipitously found that one commercially available anti-Reelin antibody G20 can bind to CTR-lacking mutant Reelin proteins, but not wild-type Reelin, on Western blotting. The G20 epitope resides in the last 19 residues of Reelin-repeat 8 (RR8), and neither posttranslational modification nor proteolysis can explain this effect. Furthermore, when an unrelated sequence, such as FLAG-tag, is inserted between RR8 and CTR, the reactivity of the corresponding antibody greatly decreases. These results suggest that RR8 and CTR form a tight structure that makes the surrounding sequence inaccessible to an antibody. Taking advantage of this phenomenon, we show the existence of CTR-lacking Reelin isoform in vivo for the first time and estimate its contribution to the total amount of secreted Reelin. Importantly, the extent to which Reelin mutants react with G20 is inversely correlated with their signaling activity, indicating that the CTR-induced structural change of RR8 is a prerequisite for downstream signaling activation, presumably via binding to a certain neuronal membrane molecule(s).
Kohno
Takao
T
Department of Biomedical Science, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan.
Nakano
Yoshimi
Y
Kitoh
Noriko
N
Yagi
Hirokazu
H
Kato
Koichi
K
Baba
Atsushi
A
Hattori
Mitsuharu
M
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D019556:COS Cells; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000091042:Reelin Protein; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D000906:Antibodies; D015816:Cell Adhesion Molecules, Neuronal; D016326:Extracellular Matrix Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D000091042:Reelin Protein; C000717127:RELN protein, human; C000717128:Reln protein, mouse; D012697:Serine Endopeptidases