Anti-peptide antibodies for examining the conformation, molecular assembly and localization of an intracellular protein, ribosomal protein S6, in vivo.
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.
Nakagawa
Masatoshi
M
Division of Biosignaling, Research Institute for Biological Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba, Japan.
Ohmido
Nobuko
N
Ishikawa
Katsumi
K
Uchiyama
Susumu
S
Fukui
Kiichi
K
Azuma
Takachika
T
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000941:Antigens / Q000276:immunology; D002877:Chromosomes, Human / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D000939:Epitopes; D006367:HeLa Cells; D006801:Humans; D007114:Immunization; D042541:Intracellular Space / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D038601:Ribosomal Protein S6 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D000941:Antigens; D000939:Epitopes; D010455:Peptides; D011994:Recombinant Proteins; D038601:Ribosomal Protein S6