Heavy and light chain contributions to antigen binding in an anti-digoxin chain recombinant antibody produced by transfection of cloned anti-digoxin antibody genes (original) (raw)
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Journal of Immunology, 1991
Five murine A/J strain anti-digoxin mAb (35-20, 1) Digoxin is unusually large for a hapten, and probably 40-40. 40-120, 40-140, and 40-160) have highly hooccupies most of the antibody combining site (1, 2); 2) xmologous H and L chain V regions, only differing by ray crystallographic studies of the structure of digoxin Somatic mutation, yet differ in affinity and Specificand several analogs are known and show that the cardiac itye The Of the VH and vL genomic glycoside steroid ring is conformationally rigid, regardless from One 40-1409 has now of functional group substitutions (3); 3) the availability lowed studies in vitro mutagenesis and of more than 60 structural analogs of digoxin is useful among these five hybridomas. for the investigation of fine specificity of Ag binding (4); To determine the relative contributions of the mutations found in either VH or VL to the overall and 4) monoclonal anti-digoxin antibodies generated binding properties of these antibodies, we recornfrom immunized A / J strain mice have unusually high bined the 4 0-4 4 0~~ with the VL of each hybridoma. affinities (more than 10' "I). which suggests that mul-The 4 0-1 4 0~~ gene was transfected into hybridoma tiple antibody-Ag Contacts exist within the antibody comvariants that produce only VL. The recombinant bining site (5). antibodies show that the mutations present in VH, Sets of mAb that have homologous H and L chains are rather than in VL, affect the fine specificity prop-useful in structure-function studies because the small erties of these antibodies, whereas, the mutations mutational sequence differences are responsible for among both V H and VL chains are important in binding alterations. This allows the assignment of cerdetermining antigen affinity. From mutations Prestain residues to certain contributions to Ag binding. Such ent in VH that affect fine Specificity properties, the sets of mAb often are a large part of certain immune comparison Of the antibody and from the responses, comprising a major idiotypic family whose dicted and tested selected VH mutations for their gene segments (6-8). Among anti-digoxin mAb, we had
Contribution of a single heavy chain residue to specificity of an anti-digoxin monoclonal antibody
Protein Science, 2008
Two distinct spontaneous variants of the murine anti-digoxin hybridoma 26-10 were isolated by fluorescenceactivated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26-10. Replacement of Asn with Gln reduced affinity for digoxin 10-fold relative to the wild-type antibody, but maintained wild-type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90-fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16-acetylated haptens and haptens with a saturated lactone.
Journal of Biological Chemistry
Antibody produced by a variant of the murine antidigoxin hybridoma 26-10 has reduced affinity for digoxin but enhanced recognition of the digoxin 12-hydroxyl due to a Tyr to His substitution at heavy chain position 50 (Schildbach, J. F., Panka, D. J., Parks, D. R., Jager, G. C., Novotny, J., Herzenberg, L. A., Mudgett-Hunter, M., Bruccoleri, R. E., Haber, E., and Margolies, M. N. (1991) J. Biol. Chem. 266, 4640-4647). Consistent with these data, the 26-10 Fab-digoxin x-ray crystal structure (Jeffrey, P. D., Strong, R. K., Sieker, L. C., Chang, C. Y., Campbell, R. L., Petsko, G. A., Haber, E., Margolies, M. N., and Sheriff, S. (1993) Proc. Natl. Acad. Sci. U. S. A., in press) reveals that Tyr-50 contacts a region of digoxin that includes the hapten-12 carbon. To determine the effects of other heavy chain position 50 substitutions, mutant antibodies were engineered, and their affinities for digoxin and digoxin analogues were measured. The affinity of the mutant antibodies for digoxin ...
Journal of Biological Chemistry, 1993
In certain instances, antibody variable region mutations outside of the antigen-combining site influence antigen binding. We reported previously that a heavy chain mutation (Ser-94 + Arg) decreased binding of the anti-digoxin antibody 40-150, whereas an additional signal peptide mutation at the-2 position (Gln + Pro) causing NHz-terminal 2-residue truncation partially restored binding. To assess the combined effects on binding of two seemingly distant mutations, we constructed signal peptide mutations and NHz-terminal deletions in the presence of Ser-94 and Arg-94. Deletions of one to three amino acids had little effect on binding for Ser-94 mutants, whereas 2-residue truncations produced directly or by signal peptide mutation increased affinity approximately 40-fold for Arg-94 mutants. These observations are consistent with the reported computer-generated model of anti-* This work was supported by National Institutes of Health grant R01 HL47415-01A1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession numbed4 L20974.
Contribution of the Vλ Light Chain to the Development of the Primary Antibody Repertoirea
Annals of The New York Academy of Sciences, 2008
The development of the primary antibody repertoire is influenced by mechanistic processes that generate germline diversity and endogenous selection of immunoglobulin (Ig) receptors expressed on B lymphocytes. Most mammalian Ig molecules bear either K or A light chains,' and their usage varies with different species, for example, horse (5% K , 95% A), man (60% K , 40% A), and mouse (95% K , 5% A).
FEBS Journal
The antigen-binding site of immunoglobulins is formed by six regions, three from the light and three from the heavy chain variable domains, which, on association of the two chains, form the conventional antigenbinding site of the antibody. The mode of interaction between the heavy and light chain variable domains affects the relative position of the antigen-binding loops and therefore has an effect on the overall conformation of the binding site. In this article, we analyze the structure of the interface between the heavy and light chain variable domains and show that there are essentially two different modes for their interaction that can be identified by the presence of key amino acids in specific positions of the antibody sequences. We also show that the different packing modes are related to the type of recognized antigen.
Contribution of the VK4 light chain to antibody specificity for lysozyme and β(1,6)d-galactan
Molecular Immunology, 1986
The V, amino acid sequence of an anti-lysozyme hybridoma protein, HyHEL-5, was determined. HyHEL-5 expresses a V region of the V,4 family and J, 1. The V,4 family also includes light chains from galactan binding antibodies, although sequence comparisons suggest that a different member of this family is used to encode HyHEL-5. The HyHEL-5 light chain has a deletion of residue 96, such that L3 is one residue shorter than the majority of murine L3. Chain recombination experiments, employing H and L chains from different anti-gaiactan and anti-lysozyme binding antibodies, were performed to examine the contrjbution of the H and L chain in dictating specificity for either galactan or the tysozyme epitope recognized by HyHEL-5. The results indicate that, although the ability to bind galactan vs lysozyme is absolutely heavy-chain dependent, having the appropriate heavy chain is not sufficient for specific high affinity binding. Both the L chains from HyHEL-5 and 5539 (a gaiactan-binding myeloma protein) were capable of supporting binding to galactan in combination with the 5539 H chain, but affinity for galactan is less with the HyHEL-5 L chain. Only V,4 L chains supported binding of the HyHEL-5 heavy chain to the HyHEL-5 epitope. although binding with the 5539 L chain was low affinity and relatively nonspecific, INTRODUCTlON Recently, in a survey of V,-Vu expression by BALB/c antibodies to a protein antigen, lysozyme, we reported that many V, and V, isotypes were expressed 920 SANDRA J. SMITH-GILL et al.
Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies
Proceedings of The National Academy of Sciences, 2006
Clefts on protein surfaces are avoided by antigen-combining sites of conventional antibodies, in contrast to heavy-chain antibodies (HCAbs) of camelids that seem to be attracted by enzymes' substrate pockets. The explanation for this pronounced preference of HCAbs was investigated. Eight single domain antigen-binding fragments of HCAbs (VHH) with nanomolar affinities for lysozyme were isolated from three immunized dromedaries. Six of eight VHHs compete with small lysozyme inhibitors. This ratio of active site binders is also found within the VHH pool derived from polyclonal HCAbs purified from the serum of the immunized dromedary. The crystal structures of six VHHs in complex with lysozyme and their interaction surfaces were compared to those of conventional antibodies with the same antigen. The interface sizes of VHH and conventional antibodies to lysozyme are very similar as well as the number and chemical nature of the contacts. The main difference comes from the compact prolate shape of VHH that presents a large convex paratope, predominantly formed by the H3 loop and interacting, although with different structures, into the concave lysozyme substrate-binding pocket. Therefore, a single domain antigen-combining site has a clear structural advantage over a conventional dimeric format for targeting clefts on antigenic surfaces.
Immunology, 1991
Human-mouse hybridoma cell lines producing human monoclonal antibodies against the cardiac glycoside digoxin were established after in vitro immunization or direct immortalization of human peripheral blood lymphocytes with digoxin. Three antibodies, designated MO6, LH92 and LH1114, displayed different patterns of fine specificity against digoxin and several digoxin analogues, as elucidated by inhibition ELISA. All three monoclonal antibodies had mu heavy chains, two of them (MO6 and LH114) had kappa light chains and one (LH92) lambda light chains. DNA encoding the variable regions of both heavy and light chains of the three antibodies were amplified from cDNA using the polymerase chain reaction (PCR). The nucleotide sequences of the amplified DNA were determined after subcloning of PCR fragments in M13 vectors. The deduced amino acid sequences revealed considerable sequence differences in the complementarity determining regions between the three antibodies.