Maninder K. Sohi

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 lgG4 Fc, at 3.2 Å resolution. This is the first structure of an autoantibody–autoantigen complex. The epitope recognised in IgG Fc includes the Cγ2/Cγ3 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 a somatic mutation, the structure implicates antigen-driven selection, following somatic mutation of germline genes, in the production of pathogenic rheumatoid factors.

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, β = 98.3°. We have also determined the sequence of the variable region of the RF–AN light chain, not hitherto reported. This belongs to the VλIII‐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.

Regulation of erythroid cell differentiation by haemin.

The differentiation of immature erythroblasts, isolated from anaemic rabbit bone marrow by density centrifugation to bovine serum albumin gradients, is accelerated by the addition of 10(-5)-10(-4) M haemin to the culture medium. Both the proportion of benzidine-positive cells and the synthesis of haemoglobin relative to the total protein were increased, whereas cell growth and DNA synthesis were decreased. Some of these changes were detected within 4 h and were maximal after 18-40 h. It is suggested that haem may have a physiological role in regulating in vivo erythropoiesis during haemolysis by accelerating terminal erythroid cell differentiation.

The role of cAMP and calcium in the stimulation of proliferation of immature erythroblasts by erythropoietin.

The hypothesis that cAMP or calcium are the second messengers of erythropoietin (Epo) was tested on fractionated, Epo-responsive immature erythroblasts from anemic rabbit bone marrow by examining whether the proliferative effects of the hormone could be mimicked by agents that increase the intracellular concentration of cAMP or Ca2+. None of the compounds tested (including 10(-6)-10(-4) M db-cAMP, forskolin, isoprenaline or 10(-7)-10(-6) M of the calcium ionophore A23187) alone or in combination could either initiate or potentiate the mitogenic action of the hormone. Furthermore, addition of 0.2 U/ml erythropoietin produced no permanent or transient increase in the uptake of 45Ca2+ by erythroblasts at 37 degrees C. However, cells cultured with imidazole or cordycepin (which reduce the level of intracellular cAMP), or with the calcium chelator EGTA, or the drugs verapamil or TMB-8 (which interfere with the utilization of extracellular or intracellular calcium) showed a decreased stimulation of DNA synthesis by Epo. Finally, the tumour promoter phorbol ester TPA could partially mimic the action of Epo when added to cultures containing more immature progenitor cells. We conclude then that an artificial increase in the cytoplasmic concentration of either cAMP or Ca2+ is not sufficient to elicit the proliferation of Epo-responsive cells.

The Structure of a Human Rheumatoid Factor Bound to IgG Fc

This is the first crystal structure analysis of a complex between an autoantibody and its autoantigen, and it reveals a mode of interaction never before seen in an antibody-antigen complex. Not only are there relatively few antibody contact residues, contributing perhaps to its very low affinity, but these residues are to be found on only one side of the potential combining site surface. Indeed, so many CDR residues are not involved in Fc binding, including those in the central region of the combining site, that it is easy to envisage that this RF may have another, entirely different, specificity. The antibody may therefore have originated in response to another, as yet unidentified, antigen, and the reactivity with IgG Fc may be an unfortunate cross-reactivity. Certainly some of the CDR residues which do interact with IgG Fc are germline encoded, but significantly one of only two residues in the light chain, Pro56, which makes many contacts with Fc, is a somatic mutation. Since this mutation would appear to make a significant contribution to the binding affinity, it is therefore evidence for an antigen driven response to the IgG Fc in the generation of this autoantibody. The Fc epitope recognised by RF-AN is strikingly similar to the binding sites for the bacterial binding proteins A and G, but the significance of this is not clear. What is clear however is that the epitope does not include any part of the Fc carbohydrate residues, although the structure of the complex does reveal that there is an alteration in the carbohydrate conformation when the galactose residues are absent. Loss of the interaction between the terminal galactose residue on the alpha (1-6) linked branch and the C gamma 2 domain appears to allow the carbohydrate chains to become mobile, at the same time exposing a predominantly hydrophobic patch on the C gamma 2 surface. Accessibility to either the agalactosyl carbohydrate chains or the newly exposed residues may account for the enhanced reactivity for G0-IgG that has been reported for certain RFs, and such an epitope need not be very different to that recognised by RF-AN. In order to understand more completely the effect of the presence or absence of the terminal galactose residue, the fully galactosylated glycoform of Fc must be studied for comparison; this work is underway. It is also important now to study a RF which is known to sense this difference in oligosaccharide composition, and also to study RFs of higher affinity, of the IgG class, and from the synovium. RF-AN was the first RF to be immortalised as a cell line, and in many ways it is a typical RF (in terms of specificity, relationship to germline sequence and affinity), but we must now establish whether the novel structural features revealed in this analysis are indeed typical of other RFs. Only when comparisons can be made between RFs of different origin and with contrasting functional properties will we begin to understand what constitutes a pathogenic RF, and the mechanism by which such auto-reactive antibodies are generated.