Heavy Chain Constant Domains Research Articles

Roles of immunoglobulin valency and the heavy-chain constant domain in antibody-mediated downregulation of Sindbis virus replication in persistently infected neurons

Clearance of infectious Sindbis virus from neurons is mediated by antibody to the E2 glycoprotein. Properties of the antibody important for downregulation of Sindbis virus replication are unknown. Immunoglobulin isotypes and valency determine many biological properties of antibodies. An immunoglobulin G1 (IgG1) isotype switch mutant and F(ab')2 and Fab fragments of IgG3 monoclonal antibody 209 were prepared and tested for clearance of infectious virus from persistently infected rat dorsal root ganglion neurons in vitro. IgG1, IgG3, and IgG3-derived F(ab')2 fragments were similarly efficacious, while IgG3-derived Fab fragments had no effect on virus replication. Cross-linking of Fab with secondary antibodies restored antiviral activity. Therefore, we found no evidence that IgG subclass plays a role in control of intracellular Sindbis virus replication. However, bivalency appears to be crucial for the ability of E2-specific IgG molecules to mediate clearance of infectious virus from neuron cells, suggesting that cross-linking of E2 molecules is essential.

Antibody constant region: Potential to bind metal and nucleic acid

Environmental challenges appear to elicit similar patterns of cellular responses such as positive autoregulation and autoamplification whether one considers the generation of antibodies with identical antigen specificity or the accumulation of host-protective transcription factors. Therefore, I analyzed the structure of immunoglobulins (Ig) for motifs commonly found in transcription factors. Specifically, the well-known abundance and periodic location of cysteine residues in immunoglobulin chains prompted me to check antibody constant regions for the presence of putative metal-binding domains and zinc finger-like sequences. The constant regions of Ig light and heavy chains were found to harbor one or several copies, respectively, of a short cysteine and histidine-containing sequence. Moreover, all four IgG subclasses were detected to comprise zinc finger-like motifs in their heavy chain constant and hinge domains. Yet another finding is the occurrence of several sequences of the form serine-proline-X-X and/or threonine-proline-X-X in the hinge sections of IgA and IgG3. These results suggest that antibody constant regions, as a fragment and/or embedded in a full-length immunoglobulin chain, may complex metal, thus acquiring conformations conducive to dimerization and nucleic acid binding. As such, my study provides a putative structural basis for the known requirement of divalent metal cations, particularly of zinc ions, for a normal immune response, and warrants further investigations, both theoretical and experimental, into the potential of antibody constant regions for metal binding and gene regulation. Moreover, future testing of the proposed zinc finger peptides from Ig constant domains should yield information relevant to zinc finger design with potentially wide applications in research and clinical medicine. Finally, the structural evidence described here allows the prediction that immunoglobulins and transcription factors may have diverged from a common ancestor molecule.

Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment.

Europium chelates provide a non-radioactive alternative for sensitive labelling of antibodies for diagnostic immunoassays. Lysine residues at antibody surfaces are ready targets for labelling by an isothiocyanate derivative of the europium chelate (Eu3+). Here the labelling efficiency of a recombinant anti-human alpha-fetoprotein (hAFP) Fab fragment has been improved by increasing its lysine content by protein engineering. Molecular modelling was used to identify three light chain constant domain surface arginine residues, R154, R187 and R210, which were mutated to lysine residues. The mutations did not influence the affinity of the lysine-enriched Fab fragment and its labelling efficiency was found to be approximately 40% higher than that of the wild-type Fab fragment. With low degree of labelling, the affinities of the two Fab fragments were identical and comparable with that of the original monoclonal anti-hAFP IgG. With a higher degree of labelling the affinities of both Fab fragments decreased more than that of the intact IgG since more lysine residues are available for labelling in the additional heavy chain constant domains of the larger molecule. Electrostatic adsorption and covalent immobilization of the Fab fragments were characterized by BIAcore and the lysine-enriched Fab fragment was found to be more efficiently immobilized to an activated carboxymethyl surface.

Variable domain-identical antibodies exhibit IgG subclass-related differences in affinity and kinetic constants as determined by surface plasmon resonance

We have analysed the binding of variable domain-identical mouse monoclonal antibodies (mAb) of the IgG3, IgG1 and IgG2b subclasses, as well as F(ab') 2 fragments derived from the IgG3 and IgG1 mAb, to a multivalent glycoprotein target. Using a biosensor device (BIAcore TM, Pharmacia Biosensor) that measures the mass of the antibody (or other receptor molecule) deposited on a sensor chip displaying the relevant epitopes, we found that the IgG3 mAb binds more effectively than the other antibody species at a high but not a low epitope density. The greater functional affinity associated with the IgG3 mAb, at high epitope density, was correlated with both slower dissociation rate constants and faster association rate constants in comparison with the IgG1 and IgG2b mAb and the F(ab') 2 fragments derived from the IgG3 and IgG1 mAb. Evidence for slower dissociation kinetics for the IgG3 mAb versus the IgG1 and IgG2b mAb was also obtained by ELISA and flow cytometry. These results demonstrate that: (1) differences in heavy chain constant (C H) domains can significantly influence apparent functional affinity for multivalent antigen, as determined without the use of covalently modified primary or secondary antibodies; (2) differences in C H domains can alter both association and dissociation rate constants for interactions between IgG antibodies and multivalent antigen; and (3) these effects of C H domains depend on epitope density. The effect of constant region differences on the apparent association rate constants suggests new approaches for achieving better binding or functional effectiveness through antibody engineering.

Immunoglobulin V H regions in Atlantic cod ( Gadus morhua L.): Their diversity and relationship to V H families from other species

Variable regions (V H) of Atlantic cod cDNA clones have been isolated by the polymerase chain reaction (PCR) technique, using primers for the first constant domain of heavy chain (C H1) and λgt11. Based upon sequence analysis and comparisons these clones have been divided into three different V H families. The approximate number of members in each family was estimated by genomic Southern blot. Comparisons of complementarity determining regions (CDRs) and frameworks (FRs) show that FR2 and the last part of FR3 are the most conserved regions. The CDR3 is very heterogeneous and gives a major contribution to V H diversity. Possible relationships between V H sequences from 17 species are shown graphically. Different V H families are often more conserved between species than within any one species. Two genomic V H clones have been isolated and partially sequenced. The V H genes have an octamer and TATA motif in the 5′ region, followed by an 18-amino-acid-long hydrophobic leader, and the mature V H coding region. The characteristic heptamer-nonamer recombination signals for V H to D joining are present 3′ of the V H segment.

A genetically engineered human IgG with limited flexibility fully initiates cytolysis via complement

A causal link between antigen induced conformational change and functional activity has been previously suggested for the triggering of immunoglobulin effector functions. To test this hypothesis an immunoglobulin with greatly restricted Fab flexibility has been created by site-directed mutagenesis. Serine 119, a residue at the elbow in the first heavy chain constant domain of a mouse-human chimeric immunoglobulin, was replaced by cysteine 119. The resulting mutant immunoglobulin had an additional intramolecular disulfide bond connecting the two heavy chains. This newly introduced covalent bond between antigen binding arms gave rise to a compact ‘tethered’ conformation which displayed lowered segmental flexibility as determined by nanosecond fluorescence depolarization anisotropy. However, the ability of this tethered immunoglobulin to initiate lysis of target cells via the complement cascade was unimpaired. Therefore, it is likely that allosteric or distortive mechanisms of conformational change induced complement activation which require unhindered ‘twist’ and/or ‘waggle’ motions of Fab are untenable.

Role of heavy chain constant domains in antibody-antigen interaction. Apparent specificity differences among streptococcal IgG antibodies expressing identical variable domains.

In this report, we examine the influence of CH domains on antibody specificity, in the context of variable epitope density on bacteria and synthetic glycoconjugates. Hybridomas secreting IgG1 and IgG2b mAb, specific for the N-acetyl-glucosamine (GlcNAc) residues of streptococcal group A carbohydrate, were previously generated from a hybridoma secreting a mouse IgG3 mAb. We show that these three mAb have identical H and L chain V domains, as determined by 1) cDNA sequencing, 2) binding to soluble Ag, and 3) binding to nine monoclonal anti-idiotopes. Nevertheless, the IgG3 mAb binds more effectively than the V region-identical IgG1 or IgG2b mAb to each of three strains of group A streptococci that display different amounts of terminal GlcNAc residues on their cell walls. The magnitude of the subclass-associated differential in binding varies with the target strain, and, whereas the IgG3 mAb binds best to the strain expressing an intermediate amount of GlcNAc, the IgG1 and IgG2b mAb and IgG3-derived F(ab')2 fragments bind best to the strain expressing the highest amount of GlcNAc. The IgG3 mAb also binds better than the IgG1 and IgG2b mAb to solid-phase GlcNAc50-BSA, but the IgG2b mAb binds best to otherwise identical conjugates with lower ratios of GlcNAc to BSA (20:1, 10:1, 5:1, and 1:1). These results suggest that epitope density can significantly influence the magnitude of IgG subclass-associated binding differences, and that structural differences in the CH regions, particularly the CH2 and CH3 domains, can influence the apparent specificities of IgG molecules for multivalent Ag.

Crystal structure of a streptococcal protein G domain bound to an Fab fragment.

Protein G is a cell-surface protein from Streptococcus which binds to IgG molecules from a wide range of species with an affinity comparable to that of antigen. The high affinity of protein G for the Fab portion of IgG poses a particular challenge in molecular recognition, given the variability of heavy chain subclass, light chain type and complementarity-determining regions. Here we report the crystal structure of a complex between a protein G domain and an immunoglobulin Fab fragment. An outer beta-strand in the protein G domain forms an antiparallel interaction with the last beta-strand in the constant heavy chain domain of the immunoglobulin, thus extending the beta-sheet into the protein G. The interaction between secondary structural elements in Fab and protein G provides an ingenious solution to the problem of maintaining a high affinity for many different IgG molecules. The structure also contrasts with Fab-antigen complexes, in which all contacts with antigen are mediated by the variable regions of the antibody, and to our knowledge provides the first details of interaction of the constant regions of Fab with another protein.

Mapping and comparison of the interaction sites on the Fc region of IgG responsible for triggering antibody dependent cellular cytotoxicity (ADCC) through different types of human Fcγ receptor

In the present study 3-iodo-4-hydroxy-5-nitrophenacetyl (NIP)-specific antibodies were compared for induction of antibody dependent lysis of NIP-derivatised red blood cells effected by pre-stimulated U937 or HL-60 cells and by K cells. The chimaeric antibodies have heavy chains corresponding to human IgG subclasses 1–4, and include site-directed mutants of IgG3 as well as the aglycosylated form of IgG3; a mouse IgG2b antibody and a site-directed mutant IgG2b were also examined. rIFN stimulated U937 or HL-60 cells express increased levels of Fcγ R1 compared to unstimulated cells; PMA stimulated HL-60 and U937 cells express an increased level of Fcγ R11 compared to unstimulated cells; K cells express Fcγ R111. Using these effector cell populations and the target cells mentioned above, we have compared anti-NIP antibodies with different heavy chain constant domains for their ability to induce ADCC through human Fcγ R1, Fcγ R11 and Fcγ R111. The results suggest that all three human Fcγ receptors appear to recognise a binding site on IgG within the lower hinge (residues 234–237) and trigger ADCC via this site, but that each receptor sees this common site in a different way. The possibility that other amino acid residues also participate in the binding/triggering site(s) cannot be excluded.

Anti-viral antibodies in HIV (HTLV-III) infection possess auto-antibody activity against a CH1 domain determinant in human IgG: possible immunological consequences

Two regions of the envelope glycoprotein gp120 of the human immunodeficiency virus were shown to have a significant degree of homology to human immunoglobulin-γ heavy-chain constant domains. We have now synthesized three short linear peptides, the first representing a sequence within the CH1 domain, the second an analogue of it, and the third representative of a region within the viral gp120. Polyclonal antibodies against these peptides were raised in rabbits and used to demonstrate that they all reacted well with human native IgG. Vice-versa, we observed the reaction of these antisera to the virus in an ELISA system. The proportion of sera reacting with the human γ-chain peptide was significantly higher in HIV-positive individuals than in HIV-negative individuals, suggesting production of anti-viral antibodies in AIDS patients with auto-antibody activity against a CH1 domain determinant in human IgG.

The molecular organization of the protein HC-IgA complex (HC-IgA).

Complexes of protein HC and monoclonal IgA1 or IgA2 or polyclonal IgA were isolated from human blood plasma. Dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that all complexes contain three types of chains: two light immunoglobulin chains, one regular IgA alpha-chain, and one chain with Mr = 90,000 carrying both alpha-chain and protein HC epitopes. The complexes were split into Fab alpha and Fc alpha fragments by bacterial IgA proteases. The protein HC epitopes were linked to the Fc fragments. Complexes of protein HC and an alpha-chain devoid of the variable region and the first heavy chain constant domain could also be demonstrated to be present in the blood plasma of a patient with alpha-heavy chain disease. Pepsin digestion of HC-IgA released a fragment containing all the protein HC epitopes and the C-terminal nonapeptide of the IgA alpha-chain. The light immunoglobulin chains, the regular alpha-chain, and the 90,000-Da chain from monoclonal HC-IgA1 were isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis and by repeated gel filtration in dodecyl sulfate-containing buffer. The N-terminal amino acid sequence of the alpha-chain was identical with that of a regular human heavy immunoglobulin chain of subgroup III. Subtractive degradations of the 90,000-Da chain displayed 2 amino acid residues in each position in a pattern suggesting simultaneous degradations of a chain identical with the regular alpha-chain of HC-IgA and of uncomplexed, low molecular weight, protein HC. All the results are compatible with a model for HC-IgA in which a single low molecular weight protein HC polypeptide chain is covalently linked, side by side, to the C-terminal nonapeptide of one of the two alpha-chains of a regular monomeric IgA unit.

IgE class-restricted tolerance induced by neonatal administration of soluble or cell-bound IgE. Cellular mechanisms.

Certain aspects of the phenomenon of IgE class-restricted tolerance induced in mice by neonatal treatment with monoclonal IgE, either in soluble form or coupled to syngeneic spleen cells, were examined. The present studies document that this tolerance results from exposure to IgE molecules, irrespective of their antigen specificity, and the resulting effects are polyclonal in nature since IgE responses directed against antigenic determinants unrelated to the tolerance-inducing IgE molecules are affected. Moreover, such findings indicate that the molecular subregion(s) responsible for inducing IgE class-restricted tolerance resides in the epsilon heavy chain constant region domain(s) of IgE. When soluble IgE is employed, tolerance induction results from neonatal treatment with doses as low as 2.5 micrograms per injection per mouse; cell-bound IgE is considerably more potent, in terms of total dose required, since tolerance results from treatment with as few as 1 X 10(6) cells per injection (per mouse), equivalent to an absolute quantity of 0.2 ng of IgE per injection. This long-term class-specific tolerance appears to be a unique feature of the IgE antibody system, since treatment of mice with monoclonal antibodies of the IgA, IgG1, or IgG2b isotypes, either in soluble or cell-bound form, does not perturb antibody responses of their corresponding isotypes or in the IgE class. By analyzing the lymphoid cells of IgE-tolerant mice after they reached adulthood, the following observations were made: (a) lymphoid cells from such tolerant mice fail to develop FcR epsilon + cells upon in vitro stimulation with IgE, as is characteristically observed with lymphoid cells from nontolerant mice; and (b) mice rendered tolerant by neonatal treatment with soluble IgE possess IgE class-restricted suppressor T cells, demonstrable in adoptive transfer experiments, whereas no such suppressor cells are evident in mice in which cell-bound IgE was used for neonatal treatment. The latter observations could mean that two different mechanisms underlie the IgE class-restricted tolerance, or both mechanisms operate coordinately to varying degrees depending upon which regimen is used for tolerance induction, as discussed herein.

Complete amino acid sequence of a mouse mu chain: homology among heavy chain constant region domains.

The complete amino acid sequence of the mouse mu chain secreted by the MOPC 104E myeloma tumor has been determined. There are four constant region domains in the mu chain and a 20-residue COOH-terminal segment that plays a role in the polymerization of pentameric immunoglobulin M molecules. There are six sites of carbohydrate attachment in the MOPC 104E mu chain. Three complex-type and two high-mannose oligosaccharides are located in the mu chain constant region. The general type and location of carbohydrate moieties in the mu chain constant region are completely conserved between mouse and human mu chains. Homology in the location of carbohydrate structures on different classes of heavy chains is discussed.

The amino acid sequences of the three heavy chain constant region domains of a human IgG2 myeloma protein.

The amino acid sequences of most of the CH1, CH2 and CH3 domains of IgG Zie, a myeloma protein belonging to the IgG2 subclass, are presented. These data make possible a comparison of the sequences of residues 253-446 of all four subclasses of immunoglobulins: these residues make up almost the entire Fc regions. A comparison can also be made of the CH1 domain of IgG1 Eu and the CH1 domain of IgG2 Zie. Earlier sequence analyses of the Fc regions of subclass 1 and 3 proteins, and parts of the Fc regions of subclass 2 and 4 proteins showed that about 95% of these sequences were identical. The extended comparisons made possible by the data presented here show that this very high degree of identity is maintained throughout the four subclasses. Similarly, the CH1 domains of gamma 1 and gamma 2 chains were found to have about 93% sequence identity. It is unlikely that the few single amino acid changes within the constant region domains can account for the marked differences between subclasses observed in the region domains can account for the marked differences between subclasses observed in the biological effector functions of immunoglobulin Fc regions, especially since most of the changes are highly conservative. Rather, it seems probable that these functional differences are caused by conformational differences between the subgroups, which result from sequence differences in the hinge regions.