Specific Photoaffinity Labeling of Tyr-49 on the Light Chain in the Steroid-Combining Site of a Mouse Monoclonal Anti-Estradiol Antibody Using Two Epimeric 6α- and 6β-(5-Azido-2-nitrobenzoyl)amidoestradiol Photoreagents^,

Abstract

A mouse monoclonal anti-7-(O-carboxymethyl)oximinoestradiol antibody was photoaffinity labeled with two cross-reactive 6alpha- and 6beta-(5-azido-2-nitrobenzoyl)amido[17alpha-3H]estradiol photoreagents (6alpha- and 6beta-ANBA-[17alpha-3H]estradiol). Covalently bound radioactivity was found exclusively on the light chain. The maximal level of specific incorporation was 0.18 mol of label per mole of antibody for both photoreagents. In both cases, tryptic digestion of the photolabeled light chain, immunopurification with the immobilized antibody, reverse-phase liquid chromatography, and Edman degradation showed the presence of radioactive peptide GLM-([3H]X)-HGNTLEDGIPSR derived from peptide 46-61 of the light chain sequence (determined from cDNA) in which the unidentified amino acid corresponding to X is a Tyr residue. Two other radioactive peptides were also isolated, one corresponding probably to the methionine sulfoxide derivative of the peptide 46-61 photolabeled with the 6beta-reagent and the other to the N-terminal tetrapeptide 46-49 of the peptide 46-61 photolabeled with the 6alpha-reagent. In all cases, the main peak of radioactivity was released at the fourth Edman cycle, thus suggesting that the same Tyr-49 residue on the light chain was photolabeled. This residue is contiguous to the N-terminal amino acid of the second hypervariable complementary determining region 50-56 of light chain. Covalent labeling was confirmed by mass spectrometry of photolabeled peptides which showed molecular ion values corresponding to the addition of the photoactive 6alpha- or 6beta-ANBA-estradiol nitrene derivatives to the peptide.

Functional characterization of an anti‐estradiol antibody by site‐directed mutagenesis and molecular modelling: modulation of binding properties and prominent role of the VL domain in estradiol recognition

“…The amino acid sequences of the variable domains of H and L chains of anti-estradiol antibody 9D3 were aligned with those reported for two other anti-estradiol antibodies 15H11 (Rousselot et al, 1997) and 57-2 (Lamminmäki et al, 1997), the anti-cortisol antibody 5A4 (Le Calvez et al, 1995) and the anti-progesterone antibody DB3 [Arevalo et al, 1993; Fig. 1(a)].…”

“…To construct the 9D3 scFv, V H and V L genes were amplified by PCR from k light chain and g heavy chain and inserted in the plasmid pBlueScript SK (Stratagene; Rousselot et al, 1997). The 3' primer used to amplify V H contained the first part of the coding sequence for the flexible linker GSTSGSGKPGSGEGSTKG (Whitlow et al, 1993).…”

“…3 H]-estradiol, according to protocols similar to those reported for the anti-estradiol antibody 15H11 (Rousselot et al, 1997). Photoaffinity labelled tryptic peptides isolated by immunopurification and HPLC were analysed by Edman sequencing and mass spectrometry.…”

“…To further assess the role of V H and V L domains, the two single V H and V L antibody fragments were constructed from mAb 9D3 and from mAb 15H11, a previously reported antiestradiol antibody, with very different binding properties (Rousselot et al, 1997). In addition, two chimeric scFvs corresponding to the V H 15H11/V L 9D3 and to the V H 9D3/ V L 15H11 hybrids were also constructed.…”

Stereoselective Interactions of a Specialized Antibody with Cholesterol and Epicholesterol Monolayers

“…Stereoselective recognition has been demonstrated in monoclonal antibodies raised against steroid haptens, [8] enzyme inhibitors, [9,10] and drugs. [11] Antibodies that catalyze chemical transformations with stereochemical control have been generated.…”