Immunodominant Residues Research Articles

Structural modeling and biochemical studies reveal insights into the molecular basis of the recognition of β‐2‐microglobulin by antibody BBM.1

Human beta-2-microglobulin (beta2m) is the light chain of human leucocyte antigen-I (HLA-I). It can disassociate from HLA-I and accumulate to cause serious dialysis-related amyloidosis (DRA) in long-term hemodialysis patients. Monoclonal antibody (mAb) BBM.1 can recognize both free-form and HLA-I associated beta2m. It can be used for specific elimination of beta2m from serum and can induce apoptosis of several types of tumor cells, and thus has great therapeutic potential. In this study, we constructed structural models of the BBM.1 Fv (fragment of the variable domain) and the BBM.1 Fv-beta2m complex, followed by biochemical evaluation. Analysis of the optimal complex model reveals that the previously identified immunodominant residues Glu(44) and Arg(45) of beta2m have direct interactions with BBM.1, while Asp(38) exerts its function mainly via stabilization of Arg(45). In addition, Arg(81) of beta2m is a newly identified immunodominant residue to have direct interaction with BBM.1. Further modeling study shows no steric conflict between the antibody and the HLA-I heavy chain. These results provide insights into the molecular basis of the recognition of beta2m by BBM.1 and explain why BBM.1 can bind both free-form and HLA-1 associated beta2m. This information could be exploited in the engineering and improvement of BBM.1 and the development of other beta2m-targeting mAbs for therapeutic purposes.

Recombinant staphylokinase variants with altered immunoreactivity. III: Species variability of antibody binding patterns.

The "charged cluster-to-alanine" substitution variants SakSTAR(K35A,E38A,K74A,E75A,R77A) and SakSTAR(K74A,E75A,R77A,E80A,D82A), previously identified as SakSTAR.M38 and SakSTAR.M89, respectively, induce less antibody formation in patients than wild-type recombinant staphylokinase (SakSTAR), but their specific activities are reduced by 50%. Therefore, the effect of the reversal of one or more of these substituted amino acids on the ratio of activity to antigenicity was studied. Fourteen mutants with one to four "alanine-to-wild-type" reversals were expressed in Escherichia coli and highly purified (> 95%). In rabbits immunized with wild-type SakSTAR, the combined K35,E38,K74,E75,R77 or K74,E75,R77,E80,E82 epitope accounted for only 30% of antibody absorption from plasma, and no clear immunodominant residue could be identified. In baboons immunized with SakSTAR, the K35,E38 and K74,E75,R77 epitopes or the K74,E75,R77 and E80,D82 epitopes contributed equally to account for 50% of total antibody binding, but no immunodominant residues were apparent. In pooled plasma from patients with peripheral arterial occlusion treated with wild-type SakSTAR, about 40% of the antibodies depended on K74 of epitope K74,E75,R77 for binding, whereas epitopes K35,E38 and E80,D82 had a negligible contribution toward antibody recognition. The recognition pattern by SakSTAR variants of antibodies induced with wild-type SakSTAR differs markedly among species. This implies that a systematic evaluation of reduced antigen recognition and antibody induction in humans will require the development of human or humanized systems. Surprisingly, SakSTAR(K74), with a single substitution of Lys74 with Ala, had an intact specific activity but did not absorb 40% of the antibodies induced in patients by treatment with wild-type SakSTAR.

IgM rheumatoid factors react with human class I HLA molecules.

Half of 30 human polyclonal IgM rheumatoid factors (RF) showed positive ELISA reactions with affinity-isolated human class I molecules (A2 and B7). Positive RF reactivity with isolated class I heavy chains indicated that anti-class I RF interaction did not merely reflect RF anti-beta 2m specificity. Cross-reactions between antigenic determinants on human IgG, class I molecules, and beta 2m reacting with RF could be demonstrated by ELISA inhibition. When gene products of A2 alpha 2 exons 2, 3, and 4 were synthesized as overlapping heptamers on polypropylene pins, six RF-reactive epitopes within solvent accessible class I HLA regions were identified: EPRAPWI, QEGPEYW, QTHRVDL (second A2 exon), EQLRAYL, GTCVEWL, and WLRRYLE (third A2 exon). Glycine-alanine substitution for each residue within these RF-reactive sites identified R48, W51, E55, Y107, R108, W147, Q155, and E172 as immunodominant residues for RF reactivity. Many of these RF-reactive class I regions also showed positive ELISA reactions with monoclonal human IgM RFs derived from rheumatoid arthritis synovial B cells. Whole serum from patients with rheumatoid arthritis showed a spectrum of IgM, IgA, and IgG Abs that adsorbed to and could be eluted from monomeric IgG or HLA A2 affinity columns. Normal serum similarly analyzed showed only trace reactions with IgG, A2, or beta 2m. Flow cytometry using RF incubated with A2 cell lines showed definite immunofluorescence reactivity with cell membranes not observed with normal non-RF IgM. Reactivity of human IgM RF with determinants on class I molecules may reflect antigenic overlap within several members of Ig gene superfamily products.

IgG Anti-F(ab′) 2 Antibodies from SLE Patients React with Immunodominant Residues in κ CDRs, but Show Reduced Cκ Region Reactivity

We studied reactivity of affinity-purified human IgG anti-F(ab′) 2 antibodies for antigenic determinants on κ light chains. Variable (VL) and constant (CL) regions of a human κI light chain (EU) were studied for enzyme-linked immunosorbent assay reactivity with monoclonal anti-κ antibodies and human IgG anti-F(ab′) 2 using overlapping heptamers of primary sequence synthesized as peptides on polypropylene pins. Polyclonal IgG anti-F(ab′) 2 antibodies isolated by affinity chromatography from the serum of patients with systemic lupus erythematosus (SLE) were found to react primarily with CDR1- and CDR3-related peptides. The most reactive residues included IL29, TRY32, LEU33, ALA34, GLU90, TYR91, and ASN 92. No striking difference in overall V-region anti-F(ab)2 reactivity profiles was noted when 10 IgG anti-F(ab′) 2 preparations from normal subjects and SLE patients were compared. In contrast, however, when tested against overlapping Cκ heptamer sequences, the reactivity in SLE-associated anti-F(ab′) 2 antibodies was markedly decreased. We report that this difference may reflect a defect in anti-idiotypic control mechanisms in SLE.

Isolation, purification and partial analysis of the lipopolysaccharide antigenic determinant recognized by a monoclonal antibody to legionella pneumophila serogroup 1

Monoclonal antibody II-6-18 recognizes a serogroup-1-specific Legionella pneumophila antigenic determinant which has been shown to be virulence-associated. We previously reported the physicochemical characterization by means of a quantitative fluorometric assay of monoclonal antibody II-6-18 binding to L. pneumophila, and its implications concerning the nature of the antigen. We describe here the isolation and the purification of the antigen by chemical and immunological methods, followed by its partial chemical analysis. The results demonstrate that the epitope - an immunodominant carbohydrate which includes a fucosamine-like residue — is part of the cell wall lipopolysaccharide (LPS). It is localized in the polysaccharide moiety of the LPS which contains KDO, rhamnose, mannose, glucosamine and an unidentified aminodideoxyhexose X1, but no heptose. The aminodideoxyhexose X1 could be fucosamine and is probably the immunodominant residue in the epitope, localized, at least partially, at the end of the polysaccharide chain.

The Epitope Specificity and Idiotypy of Monoclonal Rheumatoid Factors

Monoclonal rheumatoid factors (MRF) of the Wa idiotypic family have been studied for Fc gamma epitope specificity and the expression of idiotopes recognized by murine monoclonal antibodies. The majority of MRF recognized the Ga antigen or a closely similar iso-allotypic epitope expressed on IgG1, 2, 4 and IgG3.G3m(s.t.) proteins. MRF having the iso-allotypic specificity expressed a similar profile of V-region sub-group and idiotypic serological markers. One protein, Kas, whilst recognizing a distinct Fc gamma epitope also expressed these V-region markers and was highly homologous, from primary sequence analysis, with a MRF of Ga specificity. We conclude that these MRF recognize overlapping non-identical epitopes possibly including the same immuno-dominant residue and consequently demonstrate restricted V-gene usage.

A monoclonal antibody with reactivity to asialo GM 1 and murine natural killer cells

A monoclonal antibody (MAb) was prepared by the fusion of murine SP 2-O myeloma cells with BALB/cByJ spleen cells that were immunized with the glycolipid asialo GM 1 adsorbed to naked Salmonella. The specificity of the IgM antibody obtained was denned using various glycolipids, cell extracts and saccharides in ELISA assays and thin-layer chromatography (TLC) immunoblots. The non-reducing terminal galactose is the immunodominant residue for this antibody; however, there is undetectable reactivity to free galactose, galactosylceramide or compounds with an α-linked galactose. The SH-34 antibody specifically lyses asialo GM 1-expressing macrophages in the presence of complement and removes NK cells in vitro from spleen cell populations. When the specificity of the MAb was compared to that of a commercially available rabbit antiserum to asialo GM 1, it was found that both cross-reacted with GM 1 and asialo GM 3 at high antibody concns; however, the MAb did not bind asialo GM 2 while the rabbit antiserum showed substantial reactivity to this glycolipid. It is anticipated that this MAb will be useful for the study of murine and rat natural killer cells.

15] Preparation of site-specific anti-cytochrome c antibodies and their application

Publisher Summary This chapter discusses the preparation of site-specific anticytochrome c antibodies and their application. Antibodies bind to distinct the determinants on the surface of a protein antigen. It is found that when they are separated into individual populations that recognize single sites, they become useful reagents for the analysis of the changes in conformation and of the distribution of the functions on the molecular surface of the antigen. The fractionation of antibodies elicited in rabbits against cytochrome c into site-specific populations, using a series of immunoadsorption columns, is also discussed in the chapter. Antigen-binding fragments (Fab') of the antibodies have been used to detect the conformational changes in the chemically modified cytochrome c, and to study the interaction between the cytochrome c and its respiratory chain electron exchange partners. The cross-reactivities between homologous antigens and a given antibody population appear to be controlled by bulk, charge, and polarity of the immunodominant residues. It is found that in every case observed for cytochrome c antigens, additional amino acid side chain bulk at the immunodominant residue position prevents antibody binding.

The anti-I activity of the Sophora Japonica lectin

The erythroagglutinating activity of the purified lectin from Sophora japonica seeds appears to be related to the presence of the I antigen. It was found that 10–40 times greater concentrations of lectin was needed to agglutinate A or B adult or cord red blood cells which are relatively deficient in the I antigen than required to agglutinate type A or B adult human erythrocytes possessing the I determinant. The relationship of structures other than the A and B immunodominant residues of blood group substance to the ability of the lectin to react with the substance is demonstrated by the similar and relatively strong interaction of untreated and Smith degraded blood group substances with the lectin. The concept that reactivity of blood group antigens with the S. japonica agglutinin depends upon the presence of either the A or B as well as I determinants is supported by the findings that O erythrocytes are not agglutinated by the lectin and that α-galactosidase degradation of blood group substance causes a decrease in reactivity with the lectin. It is proposed that the S. japonica hemagglutinin preferentially reacts with AI and BI cells due to a higher concentration of reactive groupings (sum of non-reducing terminal 2-acetamido-2-deoxy-α- D-galactosyl and β- D-galactosyl or α- D-galactosyl residues) on I cells.