Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.17 (lysozyme)
 21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

For elucidation of the role of salt bridge formation in the antigen-antibody complex, the interaction between hen egg white lysozyme (HEL) and its monoclonal antibody HyHEL10, the structure of which has been well characterized and forms one salt bridge (Lys97 of HEL and Asp32 of HyHEL10 heavy chain variable region (VH)), was investigated. Asp32 of VH was substituted with Ala, Asn, or Glu by site-directed mutagenesis, and the interaction between HEL and the mutant fragments of the variable region of light chain was investigated by inhibition of the enzymatic activity of HEL and isothermal titration calorimetry. Inhibition assay indicated that these mutations lowered the inhibition only slightly. Thermodynamic study indicated that the negative enthalpic change in the interaction between each of the mutant variable regions of light chain and HEL was significantly increased, although the association constant was slightly decreased, suggesting that these mutations increased the entropy change upon antigen-antibody binding. These results indicate that the role of salt bridge formation in the HyHEL10-HEL interaction is to lower the entropic loss due to binding. In the mutant proteins, the numbers of residues that were perturbed structurally on binding increased, suggesting that the salt bridge suppresses excess structural movement of the antibody upon binding.
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PMID:Role of salt bridge formation in antigen-antibody interaction. Entropic contribution to the complex between hen egg white lysozyme and its monoclonal antibody HyHEL10. 895 89

An important aspect of the study of antibody structure-function relationships involves analysis of natural or synthetic mutations of antigen-combining sites. The anti-hen egg lysozyme monoclonal antibody HyHEL-10 has been a focus for antibody structure-function studies. We have displayed on bacteriophage of a hybrid single chain Fv, containing the light chain variable region of HyHEL-10 and the heavy chain variable region of a structurally related but functionally distinct antibody, AS32. By using a combination of site-directed mutagenesis, complementary determining region grafting and molecular modeling, we have identified a number of contact and non-contact residues that are important in the affinity of HyHEL-10 for lysozyme. In particular, the heavy chain variable region framework residue at position 94 was shown to be an important determinant of high-affinity binding. The phage display approach eliminates the need for purification of antibodies and, when used in combination with polymerase chain reaction for variable region sequence mutagenesis, facilitates the rapid generation and characterization of mutant antibodies.
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PMID:Study of antibody-antigen interaction through site-directed mutagenesis of the VH region of a hybrid phage-antibody fragment. 901 Sep 35

Although the heavy and light chain domains of some antibody variable region fragments (Fvs) readily dissociate under physiological conditions, the Fvs are stable in the presence of antigen. This 'antigen-driven Fv stabilization mechanism' was applied to the selection of clones with specificity toward target antigens. The results can be summarized as follows. (i) Some of the residues in the heavy chain complementarity determining region 2 (HCDR2) of anti-hen egg white lysozyme (HEL) monoclonal antibody HyHEL10 heavy chain variable region (VH) were randomized. (ii) The randomized VH fragments of HyHEL10 were displayed on a filamentous bacteriophage and mixed with the target antigen, before being applied to a light chain variable region (VL) which was immobilized on microtiter plates and subjected to selection by panning. (iii) After four rounds of panning, four clones that showed significant binding to human lysozyme (hL), which HyHEL10 recognized poorly, were selected from the HCDR2 library. (iv) The soluble Fv fragments selected were expressed in Escherichia coli, purified, and subjected to an inhibition assay of lysozyme enzymatic activities and an isothermal titration calorimetry. These Fv fragments had increased affinity toward hL, and thermodynamic analysis suggested that the reduced entropy loss due to binding by the replacement of residues in HCDR2 resulted in the higher hL binding activity.
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PMID:Novel selection method for engineered antibodies using the mechanism of Fv fragment stabilization in the presence of antigen. 951 20

Using phage-display, an anti-turkey egg-white lysozyme single-chain Fv fragment was selected from a naive light chain variable region repertoire in combination with a heavy chain variable region 'mini library' of anti-hen egg-white lysozyme single-domain binders (Ward et al., 1989). Whereas the selected VH domain alone binds somewhat better hen egg-white lysozyme than turkey egg-white lysozyme, but both with comparatively low affinity, the specificity of VH is converted by addition of the VL domain. Thus, the single-chain Fv fragment is more specific for turkey egg-white lysozyme, with markedly increased affinities towards both lysozymes. The complex of single-chain Fv with turkey lysozyme has been crystallized and characterized by preliminary X-ray analysis.
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PMID:A phage library-derived single-chain Fv fragment in complex with turkey egg-white lysozyme: characterization, crystallization and preliminary X-ray analysis. 969 19

We describe an optimized noncompetitive and homogeneous immunoassay based on the antigen-dependent reassociation of antibody variable domains and beta-galactosidase (beta-gal) complementation (open sandwich enzymatic complementation immunoassay, OS-ECIA). The reassociation of two fusion proteins, an antibody heavy chain variable region fragment tethered to an N-terminal deletion mutant of beta-gal, V(H)Deltaalpha, and the light chain variable region fragment tethered to a C-terminal deletion mutant of beta-gal, V(L)Deltaomega, was monitored by the enzymatic complementation between the two. With the use of anti-hen egg lysozyme (HEL) antibody HyHEL10, an antigen-dependent enhancement in the enzymatic activity was clearly observed. To optimize the assay, the lengths of the linkers connecting the two domains of each fusion protein were varied, and the optimal pair V(H)(G(4)S)(2)Deltaalpha/V(L)(G(4)S)Deltaomega showed much improved antigen-responsive beta-gal activity. After various optimizations, almost 1000-fold improvement in sensitivity compared with that of our corresponding homogeneous open sandwich (OS) assays based on the energy transfer was observed, possibly due to lower V(H)/V(L) concentration and background heterodimer association.
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PMID:An optimized homogeneous noncompetitive immunoassay based on the antigen-driven enzymatic complementation. 1296 62

While many antibodies with strong antigen-binding affinity have stable variable regions with a strong antibody heavy chain variable region fragment (V(H))/antibody light chain variable region fragment (V(L)) interaction, the anti-lysozyme IgG HyHEL-10 has a fairly strong affinity, yet a very weak V(H)/V(L) interaction strength, in the absence of antigen. To investigate the possible relationship between antigen-binding affinity and V(H)/V(L) interaction strength, a novel phage display system that can switch two display modes was employed. We focused on the two framework region 2 regions of the HyHEL-10 V(H) and V(L), facing each other at the domain interface, and a combinatorial library was made in which each framework region 2 residue was mixed with that of D1.3, which has a far stronger V(H)/V(L) interaction. The phagemid library, encoding V(H) gene 7 and V(L) amber codon gene 9, was used to transform TG-1 (sup+), and the phages displaying functional variable regions were selected. The selected phages were then used to infect a nonsuppressing strain, and the culture supernatant containing V(H)-displaying phages and soluble V(L) fragment was used to evaluate the V(H)/V(L) interaction strength. The results clearly showed the existence of a key framework region 2 residue (H39) that strongly affects V(H)/V(L) interaction strength, and a marked positive correlation between the antigen-binding affinity and the V(H)/V(L) interaction, especially in the presence of a set of particular V(L) residues. The effect of the H39 mutation on the wild-type variable region was also confirmed by a SPR biosensor as a several-fold increase in antigen-binding affinity owing to an increased association rate, while a slight decrease was observed for the single-chain variable region.
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PMID:The role of interface framework residues in determining antibody V(H)/V(L) interaction strength and antigen-binding affinity. 1664 95