Effect of heavy chain signal peptide mutations and NH2-terminal chain length on binding of anti-digoxin antibodies.

Ping, Jiang; Schildbach, Joel F.; Shaw, Shyh-Yu; Quertermous, Thomas; Novotný, Jiří; Bruccoleri, Robert E.; Margolies, Michael N.

doi:10.1016/s0021-9258(19)49417-9

Cited by 14 publications

(4 citation statements)

References 47 publications

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“…The amino acid sequence of the leader might, for example, have altered the site at which the leader is trimmed from the mature protein. Such an effect has been described in several proteins (43)(44)(45)(46)(47), including an antidigoxin antibody in which single substitutions in the heavy chain leader peptide changed the length of the mature chain by up to 10 residues (48). Some of these length changes were sufficient to change the affinity of the antibody.…”

Section: Resultsmentioning

confidence: 89%

“…One study comparing Ig crystal structures concluded that the conformation and position of CDR2 of the Ig heavy chain is largely shaped by the nature of the side chain on framework residue 71 (2). In another study, a spontaneous variant of an antidigoxin antibody with 580-fold less affinity for digoxin than wild type was found to have a substitution from S to R at position 94 in its heavy chain, a residue predicted to lie in the framework at the base of CDR3 (48,(51)(52)(53). Computer modeling suggested that the substitution increased hydrogen bonding between CDR3 of the heavy chain and CDR2 of the light chain so that the digoxin binding surface of the Ig was altered.…”

Section: Discussionmentioning

confidence: 99%

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Influence of the NH2-terminal Amino Acid of the T Cell Receptor α Chain on Major Histocompatibility Complex (MHC) Class II + Peptide Recognition

Seibel

Wilson

Kozono

et al. 1997

The Journal of Experimental Medicine

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The α/β T cell receptor (TCR) recognizes peptide fragments bound in the groove of major histocompatibility complex (MHC) molecules. We modified the TCR α chain from a mouse T cell hybridoma and tested its ability to reconstitute TCR expression and function in an α chain–deficient variant of the hybridoma. The modified α chain differed from wild type only in its leader peptide and mature NH2-terminal amino acid. Reconstituted cell surface TCR complexes reacted normally with anti-TCR and anti-CD3 antibodies. Although cross-linking of this TCR with an antibody to the TCR idiotype elicited vigorous T cell hybridoma activation, stimulation with its natural MHC + peptide ligand did not. We demonstrated that this phenotype could be reproduced simply by substituting the glutamic acid (E) at the mature NH2 terminus of the wild type TCR α chain with aspartic acid (D). The substitution also dramatically reduced the affinity of soluble α/β-TCR heterodimers for soluble MHC + peptide molecules in a cell-free system, suggesting that it did not exert its effect simply by disrupting TCR interactions with accessory molecules on the hybridoma. These results demonstrate for the first time that amino acids which are not in the canonical TCR complementarity determining regions can be critical in determining how the TCR engages MHC + peptide.

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Section: Resultsmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Influence of the NH2-terminal Amino Acid of the T Cell Receptor α Chain on Major Histocompatibility Complex (MHC) Class II + Peptide Recognition

Seibel

Wilson

Kozono

et al. 1997

The Journal of Experimental Medicine

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“…The primers used to construct the template for reversion mutagenesis were located distally with respect to the variable gene coding sequences to accurately recreate all features of the unmutated (and mutated) variable genes, including the leader sequences. This was done because mutations in leader sequences can affect antibody binding (Ping et al, 1993). The reverted constructs were transfected in the context of an IgG2b genomic expression vector into SP2/0 cells to produce antibody, which was affinity encoded by somatically mutated V region genes, carrying from 2 to 24 aa replacements (Table I).…”

Section: A Large Autoreactive Lineage Derived From a Nonautoreactive B Cellmentioning

confidence: 99%

Somatic hypermutation as a generator of antinuclear antibodies in a murine model of systemic autoimmunity

Guo

Smith

Aviszus

et al. 2010

Journal of Experimental Medicine

102

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Systemic lupus erythematosus (SLE) is characterized by high-avidity IgG antinuclear antibodies (ANAs) that are almost certainly products of T cell–dependent immune responses. Whether critical amino acids in the third complementarity-determining region (CDR3) of the ANA originate from V(D)J recombination or somatic hypermutation (SHM) is not known. We studied a mouse model of SLE in which all somatic mutations within ANA V regions, including those in CDR3, could be unequivocally identified. Mutation reversion analyses revealed that ANA arose predominantly from nonautoreactive B cells that diversified immunoglobulin genes via SHM. The resolution afforded by this model allowed us to demonstrate that one ANA clone was generated by SHM after a VH gene replacement event. Mutations producing arginine substitutions were frequent and arose largely (66%) from base changes in just two codons: AGC and AGT. These codons are abundant in the repertoires of mouse and human V genes. Our findings reveal the predominant role of SHM in the development of ANA and underscore the importance of self-tolerance checkpoints at the postmutational stage of B cell differentiation.

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“…Recent progress in antibody engineering methodologies has stimulated new promising approaches aimed at modifying the binding properties of anti-steroid antibodies, as illustrated by several reports on anti-testosterone mAbs (17), antidigoxin mAbs (18,19), anti-cortisol mAbs (20), and antiestradiol mAbs (1,2,13,21).…”

mentioning

confidence: 99%

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

et al. 2001

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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.

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Effect of heavy chain signal peptide mutations and NH2-terminal chain length on binding of anti-digoxin antibodies.

Cited by 14 publications

References 47 publications

Influence of the NH2-terminal Amino Acid of the T Cell Receptor α Chain on Major Histocompatibility Complex (MHC) Class II + Peptide Recognition

Influence of the NH2-terminal Amino Acid of the T Cell Receptor α Chain on Major Histocompatibility Complex (MHC) Class II + Peptide Recognition

Somatic hypermutation as a generator of antinuclear antibodies in a murine model of systemic autoimmunity

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

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