Michael J. Taussig

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

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.

Production of human antibody repertoires in transgenic mice

Transgenic mice have been created that carry human immunoglobulin heavy and light chain genes in germline configuration and that have the corresponding endogenous genes silenced. The transgenes are either minigene constructs or large, almost authentic, transloci on yeast artificial chromosomes and undergo B-cell-specific DNA rearrangement and hypermutation in the mouse lymphoid tissue. Monoclonal antibodies with good affinities for human antigens have been obtained after immunisation. These mice may be a future source of human antibodies for therapy.

Protein microarrays: high-throughput tools for proteomics.

Protein microarrays are versatile tools for parallel, miniaturized screening of binding events involving large numbers of immobilized proteins in a time- and cost-effective manner. They are increasingly applied for high-throughput protein analyses in many research areas, such as protein interactions, expression profiling and target discovery. While conventionally made by the spotting of purified proteins, recent advances in technology have made it possible to produce protein microarrays through in situ cell-free synthesis directly from corresponding DNA arrays. This article reviews recent developments in the generation of protein microarrays and their applications in proteomics and diagnostics.

Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins

BACKGROUND Peptostreptococcus magnus protein L (PpL) is a multidomain, bacterial surface protein whose presence correlates with virulence. It consists of up to five homologous immunoglobulin binding domains that interact with the variable (VL) regions of kappa light chains found on two thirds of mammalian antibodies. RESULTS We refined the crystal structure of the complex between a human antibody Fab fragment (2A2) and a single PpL domain (61 residues) to 2.7 A. The asymmetric unit contains two Fab molecules sandwiching a single PpL domain, which contacts similar VL framework regions of two light chains via independent interfaces. The residues contacted on VL are remote from the hypervariable loops. One PpL-Vkappa interface agrees with previous biochemical data, while the second is novel. Site-directed mutagenesis and analytical-centrifugation studies suggest that the two PpL binding sites have markedly different affinities for VL. The PpL residues in both interactions are well conserved among different Peptostreptococcus magnus strains. The Fab contact positions identified in the complex explain the high specificity of PpL for antibodies with kappa rather than lambda chains. CONCLUSIONS The PpL-Fab complex shows the first interaction of a bacterial virulence factor with a Fab light chain outside the conventional combining site. Structural comparison with two other bacterial proteins interacting with the Fab heavy chain shows that PpL, structurally homologous to streptococcal SpG domains, shares with the latter a similar binding mode. These two bacterial surface proteins interact with their respective immunoglobulin regions through a similar beta zipper interaction.

In situ synthesis of protein arrays

In situ or on-chip protein array methods use cell free expression systems to produce proteins directly onto an immobilising surface from co-distributed or pre-arrayed DNA or RNA, enabling protein arrays to be created on demand. These methods address three issues in protein array technology: (i) efficient protein expression and availability, (ii) functional protein immobilisation and purification in a single step and (iii) protein on-chip stability over time. By simultaneously expressing and immobilising many proteins in parallel on the chip surface, the laborious and often costly processes of DNA cloning, expression and separate protein purification are avoided. Recently employed methods reviewed are PISA (protein in situ array) and NAPPA (nucleic acid programmable protein array) from DNA and puromycin-mediated immobilisation from mRNA.

The structure and origin of rheumatoid factors

Abstract The recently determined X-ray crystal structure of a human rheumatoid factor Fab bound to IgG Fc provides the basis of a new hypothesis for the origin of these autoantibodies in rheumatoid arthritis. The observation that Fc is bound outside the conventional antigen combining site suggests a novel form of crossreactivity with simultaneous binding of another antigen, potentiated by somatic mutation. This article discusses the implications for the induction of these autoantibodies.

Generating recombinant antibodies to the complete human proteome

In vitro antibody generation technologies have now been available for two decades. Research reagents prepared via phage display are becoming available and several recent studies have demonstrated that these technologies are now sufficiently advanced to facilitate generation of a comprehensive renewable resource of antibodies for any protein encoded by the approximately 22,500 human protein-coding genes. Antibody selection in vitro offers properties not available in animal-based antibody generation methods. By adjusting the biochemical milieu during selection, it is possible to control the antigen conformation recognized, the antibody affinity or unwanted cross-reactivity. For larger-scale antibody generation projects, the handling, transport and storage logistics and bacterial production offer cost benefits. Because the DNA sequence encoding the antibody is available, modifications, such as site-specific in vivo biotinylation and multimerization, are only a cloning step away. This opinion article summarizes opportunities for the generation of antibodies for proteome research using in vitro technologies.

Up-regulation of microsphere transport across the follicle-associated epithelium of Peyer’s patch by exposure to Streptococcus pneumoniae R36a

Transport of antigens through the follicle‐associated epithelium (FAE) of Peyers patch (PP) is the critical first step in the induction of mucosal immune responses. We have previously described that short‐term exposure to Streptococcus pneumoniae R36a induced dramatic morphological alterations of the FAE in rabbit PP. These results prompted us to investigate whether the pneumococci‐induced modifications were accompanied by enhanced ability of the FAE to transport antigens. We addressed this problem by evaluating the ability of the FAE to bind, internalize, and transport fluorescent polystyrene microparticles, highly specific to rabbit M cells, after exposure to S. pneumoniae. Quantitative study revealed a marked increase in the number of microspheres in PP tissues exposed to S. pneumoniae compared to tissues exposed to either phosphate‐buffered saline or Escherichia coli DH5a as controls. No sign of bacterially induced damage to the epithelial barrier was observed. Further confocal microscopy analysis of the FAE surface showed that a significant increase in the number of cells that showed both morphological and functional features of M cells took place within pneumococci‐treated PP tissues. These data provide the first direct evidence that the FAE‐specific antigen sampling function may be manipulated to improve antigen and drug delivery to the intestinal immune system.—Meynell, H. M., Thomas, N. W., James, P. S., Holland, J., Taussig, M. J., Nicoletti, C. Up‐regulation of microspheres transport across the follicle‐associated epithelium of Peyers patch by exposure to Streptococcus pneumoniae R36a. FASEB J. 13, 611–619 (1999)

A sense of closeness: protein detection by proximity ligation.

Highly specific and sensitive procedures will be required to evaluate proteomes. Proximity ligation is a recently introduced mechanism for protein analysis. In this technique, the convergence of sets of protein-binding reagents on individual target molecules juxtaposes attached nucleic acid sequences. Through a ligation reaction a DNA reporter sequence is created, which can be amplified. The procedure thus encodes detected proteins as specific nucleic acid sequences in what may be viewed as a reverse translation reaction.