Specific photoaffinity-labeling of Tyr-50 on the 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.
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 [17alpha-(3)H]estradiol, synthesized from 3-aminoethyloxy, 3-(aminoethylamido)carboxymethyloxy, 6alpha- and 6beta-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 cross-reactivity with estradiol 3-conjugates. The two 6alpha- and 6beta-ANBA-estradiol isomers, as well as the 7-[O-(ANBA-ethylamido)carboxymethyl]oximinoestradiol photoreagent derived from the steroid hapten, labeled the same TyrL-32 residue. The 6beta-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.
Rolland de Ravel
M
M
Institut National de la Santé et de la Recherche Médicale, Unité INSERM U 329, Pathologie Hormonale Moléculaire, Hôpital Debrousse, 69322 Lyon, France.
Blachère
T
T
Delolme
F
F
Dessalces
G
G
Coulon
S
S
Baty
D
D
Grenot
C
C
Mappus
E
E
Cuilleron
C Y
CY
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D001483:Base Sequence; D001665:Binding Sites; D004958:Estradiol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019920:Photoaffinity Labels / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D016415:Sequence Alignment; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism
C106726:6-(5-azido-2-nitrobenzoyl)amidoestradiol; D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019920:Photoaffinity Labels; D014443:Tyrosine; D004958:Estradiol