Specific photoaffinity-labeling of Tyr-50 on heavy chain and of Tyr-32 on the light chain in the steroid combining site of a mouse monoclonal anti-estradiol antibody using C3-, C6-, and C7-linked 5-azido-2-nitrobenzoylamidoestradiol photoreagents

Autor: Elisabeth Mappus, Dessalces G, Coulon S, Cuilleron Cy, C. Grenot, Delolme F, Rolland de Ravel M, Blachère T, Daniel Baty
Přispěvatelé: Pathologie Hormonale Moléculaire, IFR62-Institut National de la Santé et de la Recherche Médicale (INSERM), INSERM U 329, Pathologie Hormonale Moléculaire, Institut National de la Santé et de la Recherche Médicale (INSERM), Service Central d'Analyse (SCA), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Bussy, Agnès
Jazyk: angličtina
Rok vydání: 2001
Předmět:
Zdroj: Biochemistry
Biochemistry, 2001, 40 (49), pp.14907-14920
Biochemistry, American Chemical Society, 2001, 40 (49), pp.14907-14920
ISSN: 7860-7868
0006-2960
1520-4995
Popis: International audience; A mouse monoclonal anti-7-(O carboxymethyl)oximinoestradiol antibody 9D3, raised against the same immunogen as that employed for generating the reported anti-estradiol antibody 15H11 [Rousselot, P., et al. (1997) Biochemistry 36, 7860-7868], was found to exhibit an opposite specificity profile with a much stronger recognition of the D-ring than of the A-ring extremity of the steroid, but a similar lack of specificity for both 6- and 7-positions of the B-ring. This antibody was photoaffinity-labeled with five (5-azido-2-nitrobenzoyl)amido (ANBA) derivatives of [17 alpha-H-3]estradiol, synthesized from 3-aminoethyloxy, 3-(aniinoethylamido)carboxymethyloxy, 6 alpha- and 6 beta -amino, and 7-[O-(aminoethylamido)carboxymethyl]oximino precursors. After tryptic digestion, the radioactive peptides on L and H chains were immunopurified with the immobilized antibody 9D3, separated by reversed-phase liquid chromatography, sequenced, and characterized by mass spectrometry, including post-source decay-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The long 3-(ANBA-ethylamido)carboxymethyl ether photoreagent was found to label TyrL-32 (on CDR L1), whereas no labeling was observed with the shorter 3-derivative, a result in agreement with a binding pocket large enough to explain the high crossreactivity with estradiol 3-conjugates. The two 6 alpha- and 6 beta -ANBA-estradiol isomers, as well as the 7-[O-(ANBA-ethylamido)carboxymethyl]oximinoestradioI photoreagent derived from the steroid hapten, labeled the same TyrL-32 residue. The 6 beta -ANBA epimer also labeled TyrH-50 (at the basis of CDR H2). These experiments indicate that TyrL-32 is freely accessible from the three C3, C6, and C7 positions, all presumed to be exposed to solvent, while TyrH-50 is probably located on the beta -face of estradiol. These results, obtained in solution, provide experimental data useful for molecular modeling of the steroid-antibody complex.
Databáze: OpenAIRE
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