Journal of Biochemistry Advance Access originally published online on November 26, 2007
Journal of Biochemistry 2008 143(3):325-332; doi:10.1093/jb/mvm229
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© 2007 The Japanese Biochemical Society.
Anti-Peptide Antibodies for Examining the Conformation, Molecular Assembly and Localization of an Intracellular Protein, Ribosomal Protein S6, In vivo
1Division of Biosignaling, Research Institute for Biological Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan; 2Faculty of Human Development, Kobe University, Tsurukabuto 3-11, Nada, Kobe, Hyogo 657-8501, Japan; and 3Laboratory of Dynamic Cell Biology, Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
*To whom correspondence should be addressed. Tel: +81-4-7121-4082, Fax: +81-4-7121-4089, E-mail: tazuma{at}rs.noda.tus.ac.jp
Received November 4, 2007; Accepted November 9, 2007
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Abstract |
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Ribosomal protein S6 (rpS6) is known to relate to cell proliferation. Our recent proteome analysis of human metaphase chromosomes revealed the enrichment of rpS6 during mitosis. Here, structure, localization and molecular assembly in vitro and in vivo of a human rpS6, were examined using antibodies (Abs) prepared by immunizing rabbits with synthetic peptides. Five peptides, Ser6-Asp20 (S6-1), Ile52-Gly66 (S6-2), Asp103-Gly117 (S6-3), Asn146-Lys160 (S6-4) and Arg178-Ile192 (S6-5) were chosen as epitopes of human rpS6. These peptides except for S6-3 induced strong Ab production, and with an enzyme-linked immunosorbent assay, anti-S6-2, anti-S6-4 and anti-S6-5, showed high reactivity to recombinant rpS6 (r-rpS6), while anti-S6-1 did not, suggesting that S6-2, S6-4 and S6-5 were exposed on the r-rpS6 surface, while S6-1 was less exposed or possessed a different conformation. The immunostaining of HeLa cells as well as isolated chromosomes suggested that rpS6 occurs in endoplasmic reticulum (ER) but less possible on chromosomes since no Abs showed localization of rpS6 to chromosomes. In addition, the immunostaining suggested that only S6-4 is exposed on the protein surface, while S6-2 and S6-5 are buried by the interaction with other macromolecules in HeLa cells. Present our result shows new possibility of antibodies as tools for structure-oriented cell biology.
Key Words: anti-peptide antibody, conformation, peptides, ribosomal protein S6, epitope
Abbreviations: Ab, antibody; Ag, antigen; HPLC, high-pressure liquid chromatography; H. sapiens, Homo sapiens; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; rpS6, ribosomal protein S6; r-rpS6, recombinant ribosomal protein S6; S. cerevisiae, Saccharomyces cerevisiae; S6K, ribosomal protein S6 kinase; T. thermophilus, Thermus thermophilus