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R Jefferis

Publications and source records attributed to R Jefferis.

At least 19 recordsLinked to original sources
PubMed1997

Glycosylation of polyclonal and paraprotein IgG in multiple myeloma.

It has previously been shown that in multiple myeloma (MM) each IgG paraprotein exhibits a unique oligosaccharide profile. It has been assumed that this results from a clone specific glycosylation machinery. However, the abnormal physiological environment of the bone marrow in this disease may also affect normal plasma cells producing polyclonal IgG. We present data to show that this is so and that, in two cases, the oligosaccharide profile of the polyclonal IgG reflected that of the paraprotein from the same patient rather than that of normal polyclonal IgG.

Chromatography, DEAE-Cellulose↗
PubMed1997

Structure of human IgM rheumatoid factor Fab bound to its autoantigen IgG Fc reveals a novel topology of antibody-antigen interaction.

Rheumatoid factors are the characteristic autoantibodies of rheumatoid arthritis, which bind to the Fc regions of IgG molecules. Here we report the crystal structure of the Fab fragment of a patient-derived IgM rheumatoid factor (RF-AN) complexed with human IgG4 Fc, at 3.2 A resolution. This is the first structure of an autoantibody-autoantigen complex. The epitope recognised in IgG Fc includes the C gamma 2/C gamma 3 cleft region, and overlaps the binding sites of bacterial Fc-binding proteins. The antibody residues involved in autorecognition are all located at the edge of the conventional combining site surface, leaving much of the latter available, potentially, for recognition of a different antigen. Since an important contact residue is somatic mutation, the structure implicates antigen-driven selection, following somatic mutation of germline genes, in the production of pathogenic rheumatoid factors.

Antigen-Antibody Reactions↗
PubMed1996

Multiple interactions of IgG with its core oligosaccharide can modulate recognition by complement and human Fc gamma receptor I and influence the synthesis of its oligosaccharide chains.

Glycosylation at Asn297 within the CH2 domains of IgG is important for recognition by the effector ligands Fc gammaR and C. Protein engineering has been used to replace amino acid residues within the extensive oligosaccharide interaction site that contact the core hexasaccharide (GlcNAc2Man3GlcNAc). Replacement of residues Phe241, Val264, or Asp265, in particular, results in reduced recognition of human chimeric anti-nitroiodophenacetyl IgG3 produced in Chinese hamster ovary cells, by guinea pig C and human C1q. Replacement of residues Val264 or Asp265, in particular, results in reduced superoxide production triggered through human Fc gammaRI expressed on U937 cells. These results suggest that noncovalent interactions of multiple amino acid residues of IgG with oligosaccharide residues that include the primary and secondary GlcNAc are necessary for optimal recognition of IgG by human Fc gammaRI and C1q. Replacement of residues 241, 243, 264, 265, or 301 with alanine in each case resulted in increased galactosylation and sialylation relative to the wild-type oligosaccharide chains. In particular, for the mutant FA243 there was much increased sialylation of its oligosaccharide chains (73%) relative to the wild-type (4%). Thus, even single residue replacements within the oligosaccharide interaction site of the C region can influence galactosylation and sialylation of its oligosaccharide chains. These data suggest a protein engineering route to the production of more homogeneously glycosylated IgG molecules with or without compromised biologic activities.

Animals↗
PubMed1996

Evaluation of monoclonal antibodies with specificity for human IgA, IgA subclasses and allotypes and secretory component. Results of an IUIS/WHO collaborative study.

51 monoclonal antibodies (McAb) with putative specificity for human IgA, the IgA subclasses, Am allotypes or secretory component (SC) were evaluated for immunoreactivity and specificity by nine laboratories employing immunodiffusion, agglutination, immunohistological assays, immunoblotting and direct binding and competitive inhibition enzyme immunoassays. McAbs specific for IgA PAN (n = 24), IgA1 (n = 7), IgA2 (n = 3), IgA2m(2) (n = 2), non-IgA2m(2) (n = 4) and SC or secretory IgA (n = 5) were identified that were immunoreactive and specific in the assays employed. The McAbs identified as IgA- or SC-reactive were shown to be non-reactive to human IgG, IgM, IgD, IgE, kappa and lambda by direct binding and competitive inhibition immunoassays. Interestingly, no McAbs with restricted specificity for IgA2m(1) were identified. Some McAbs displayed higher affinity and/or better performance in one or several of the assay groups. The IgA- and SC-specific McAbs identified in this international collaborative study have potential as immunochemical reference reagents to identify and quantitate monomeric and polymeric IgA in human serum and secretions.

Animals↗
PubMed1996

Crystallization of a complex between the Fab fragment of a human immunoglobulin M (IgM) rheumatoid factor (RF-AN) and the Fc fragment of human IgG4.

Rheumatoid factors (RF) are the characteristic autoantibodies found in patients with rheumatoid arthritis. They recognize epitopes in the Fc region of immunoglobulin G (IgG) and are often of the IgM isotype. In order to analyse the nature of RF-Fc interactions, we have crystallized a complex between the Fab fragment of a human monoclonal IgM rheumatoid factor (RF-AN) and the Fc fragment of human IgG4. The stoichiometry of the complex within the crystals was found to be 2:1 Fab:Fc. The crystals diffracted X-rays to 0.3 nm resolution, and the space group was C2, with cell dimensions a = 16.03 nm, b = 8.19 nm, c = 6.42 nm, beta = 98.3 degrees. We have also determined the sequence of the variable region of the RF-AN light chain, not hitherto reported. This belongs to the V lambda III-a subgroup and is closely related to the germline gene Humlv318, from which it differs in three amino acid residues. This is the first reported crystallized complex between a human autoantibody and its autoantigen.

Amino Acid Sequence↗
PubMed1995

Anti-CD40 plus interleukin-4-activated human naive B cell lines express unmutated immunoglobulin genes with intraclonal heavy chain isotype variability.

Combination of anti-CD40 antibody and interleukin-4 (IL-4) induces B cell clonal expansion reminiscent of the T-dependent proliferation following antigenic challenge in vivo. We have analyzed the usage of CH genes and the presence or absence of somatic mutations within the progeny of a single human naive B cell activated with anti-CD40 + IL-4. To address this issue, single-cell cultures of naive (sIgD+) tonsillar B lymphocytes expressing the VH1-restricted G8 idiotype were set up. After culture and RNA extraction, VH1+ Ig mRNA were reverse-transcribed, amplified by polymerase chain reaction and sequenced. A single sIgD+ B cell could generate clones expressing mu, gamma 1, gamma 3, or epsilon, illustrating that the progeny of a single cell can express different isotypes in response to the same stimulus in vitro. The rate of somatic mutations affecting the immunoglobulin variable heavy chain gene was indistinguishable from the background of errors introduced by Taq polymerase.

Antibodies, Anti-Idiotypic↗
PubMed1995

Recognition sites on human IgG for Fc gamma receptors: the role of glycosylation.

Human IgG subclass proteins exhibit more than 95% primary amino acid sequence homology in their Fc regions, but each has a unique profile for recognition by the 3 human Fc gamma receptors. The Fc gamma Rs are themselves highly homologous members of the immunoglobulin supergene family. Consistent with these data we have proposed that Fc gamma RI, Fc gamma RII and Fc gamma RIII recognise overlapping non-identical interaction sites in the lower hinge region of the CH2 domain of the IgG molecule. Evidence in support was provided by protein engineering effecting single amino acid replacements in the proposed site. Alternatively, we have demonstrated that the primary amino acid sequence alone is not sufficient for IgG molecules to fold with the generation of Fc gamma R interaction sites and that glycosylation of Asn 297 of the CH2 domain is essential. We have further defined a 'core' oligosaccharide structure that provides for the generation of Fc gamma R interaction sites which suggests that the addition of outer-arm sugar residues does not affect this primary activity; although in vivo it could influence other essential biological activities. These findings have opened up a new approach to engineering antibody function--by protein engineering of amino acid residues that form contacts with the oligosaccharide moiety. In the present report we demonstrate that replacement of contact residues for galactose on the alpha(1-6) arm does not affect Fc gamma RI and Fc gamma RII recognition while replacement of Asp 265, a contact for a 'core' N-acetylglucosamine residue, results in a loss of Fc gamma RI and Fc gamma RII recognition.

Binding Sites, Antibody↗
PubMed1995

Rheumatoid factors, B cells and immunoglobulin genes.

The paradigm of self, non-self discrimination in the immune system is under review as autoreactive B or T cells are increasingly delineated within normal individuals. The products of autoreactive B cells are, mostly, polyspecific IgM antibodies of low affinity. These 'natural' antibodies include rheumatoid factors (RF) encoded by unmutated germline immunoglobulin genes. In rheumatoid arthritis (RA) the RF may be of the IgM, IgG or IgA isotype, show evidence of somatic mutation and have increased affinity; consistent with maturation of an antigen driven immune response. This response could be initiated or driven by an auto-immunogenic form of IgG or an exogenous cross-reactive antigen. Changes in galactosylation of IgG have been reported to be a valuable diagnostic and prognostic indicator in RA. Speculation that these changes may precipitate some of the disease processes is critically reviewed.

Autoantibodies↗
PubMed1995

Oligosaccharide-protein interactions in IgG can modulate recognition by Fc gamma receptors.

X-ray crystal structures of IgG-Fc provide evidence of extensive noncovalent interactions between the protein and carbohydrate moieties, and glycosylation, at Asn-297 within the Fc, has been shown to be important for effector functions mediated through Fc gamma receptors expressed on leukocytes. We have applied protein engineering in an attempt to define protein/carbohydrate interactions essential to wild-type biological activity. We demonstrate that replacement of Lys-246, Asp-249, and Glu-258, which make contacts with GlcNac and Gal on the outer alpha[1-->6] arm, do not affect recognition of human chimeric IgG3 by human Fc gamma RI and Fc gamma RII. However, replacement of Asp-265, which make contacts with the primary GlcNac sugar residue and is covalently attached to Asn-297, resulted in loss of recognition of both Fc gamma RI and Fc gamma RII. Similarly, replacement of Asp-265 in mouse IgG2b resulted in loss of recognition by mouse Fc gamma RII. These results suggest that noncovalent contacts of Asp-265 with the primary GlcNac residue are important for maintenance of recognition of IgG by Fc gamma receptors whereas contacts with GlcNac and Gal on the alpha [1-->6] arm do not have a measurable effect. This conclusion was supported by experiments in which galactose-deficient and fully galactosylated forms of a human IgG4-Fc fragment were shown to be equivalent in their ability to inhibit superoxide generation by IgG4 stimulated U937 cells.

Animals↗
PubMed1995

Activation of effector functions by immune complexes of mouse IgG2a with isotype-specific autoantibodies.

Analysis of five monoclonal autoantibodies, rheumatoid factors produced by hybridomas generated from spleen cells of BALB/c mice repeatedly infected with A/PR/8/34 human influenza A virus, revealed that they recognized distinct but spatially related epitopes. The differing isoallotypic specificity of the IgM and IgA monoclonal antibodies correlated with the presence of Ile258 and Ala305, respectively. Although these data suggest that the epitopes recognized are within the CH2 domain, all antibodies failed to inhibit IgG antigen reactivity with Staphylococcus aureus protein A (SpA), C1q, mouse C3, human Fc gamma RI or mouse Fc gamma RII, activities known to be predominantly determined by CH2 domain structures. Reactivity of the IgA antibody, Z34, with IgG2b allowed further specificity studies using a panel of 26 mutant IgG2b proteins, each having single amino acid replacements over the surface of the CH2 domain. The only substitution that affected Z34 reactivity was Asn/Ala297, which destroyed the glycosylation sequon, resulting in secretion of an aglycosylated IgG molecule. The epitope recognized by Z34 therefore seems to be located outside of the Fc gamma R and C1q binding sites, but to be dependent on the presence of carbohydrate for expression. In contrast to the binding studies, complement activation by aggregated IgG2a, through classical or alternative pathways, was inhibited by the presence of autoantibodies. The functional significance of isotype-specific autoantibody in immune regulation is discussed.

Animals↗
PubMed1994

A novel 13C isotopic labelling strategy for probing the structure and dynamics of glycan chains in situ on glycoproteins.

A protocol is described for uniform 13C labelling of terminal galactose residues of the glycan chains of glycoproteins, using an enzymatic method which does not perturb the protein. The technique is illustrated by application to the biantennary N-linked glycan chains attached at Asn 297 of immunoglobulin G (IgG). Isotope-edited NMR experiments on this glycoprotein yield data which suggest that the galactose residues on the glycan exist in two discrete environments, with the galactose in one environment having greater mobility than that in the other. These data are qualitatively consistent with crystallographic data on an Fc fragment, which suggest that one arm of the glycan is in contact with the protein, while the other projects into the space between the C gamma 2 domains. Quantitatively, however, these data cannot be rationalized with the crystallographic data, which implies subtle differences in oligosaccharide structure and dynamics between the solution and crystal states of Fc.

Carbohydrate Sequence↗
PubMed1994

Characterisation of putative monoclonal anti-G3m(u) and anti-G3m(g) reagents and their antigenic determinants.

Monoclonal antibodies (MAbs) against IgG3 allotypic markers were evaluated in haemagglutination inhibition and IgG3 capture ELISA. MAbs PNF69C and 200D1 exhibited G3m(u) and G3m(g) specificity respectively. In HAI target epitopes detected by MAbs were remarkably stable to physiochemical degradation. Western blotting revealed that MAb 200D1, bound to intact IgG3 heavy chain disease protein and not its pFc' fragment; a result consistent with the CH2 domain location of the G3m(g) allotope. The G3m(u) allotope is also located within this domain. Surprisingly anti-G3m(u) MAb PNF69C bound to the pFc' of IgG3-related protein, HW, and to the pFc' of IgG1-related protein, PR, in Western blot.

Antibodies, Monoclonal↗