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Brian J Sutton

Publications and source records attributed to Brian J Sutton.

16 recordsLinked to original sources
PubMed2006

Structural insights into autoreactive determinants in thyroid peroxidase composed of discontinuous and multiple key contact amino acid residues contributing to epitopes recognized by patients' autoantibodies.

Thyroid peroxidase (TPO) is a major autoantigen of thyroid autoimmune disease, and the autoantibodies that are produced recognize two immunodominant regions (IDR) of the molecule, termed IDR-A and -B. Based upon our structural model of the TPO ectodomain, we recently identified R225 and K627 as key residues in IDR-A and -B, respectively. We report here on rational mutagenic investigations to identify additional residues surrounding R225 and K627 that affect the binding of recombinant human Fabs (rhFabs) specific for each IDR. Two residues R646 and D707 were identified from the model as promising surface-exposed amino acids adjacent to R225. Similarly, residues E604, D620, D624, and D630 were identified in the vicinity of K627. These residues were substituted in different combinations of single, double, and multiple mutations, and stably expressed in Chinese hamster ovary cells. By fluorescence-activated cell sorting and capture ELISA, we found that R225A, R646A, and D707N specifically led to the loss of binding of IDR-A rhFabs, whereas E604A, D620R, K627G, and D630N specifically abrogated the binding of IDR-B rhFabs. Further supportive evidence of the importance of these residues for the IDR epitopes was obtained with patients' sera. We conclude that R646 and D707 together with R225 constitute a functional epitope within IDR-A, and that residues E604, D620, and D630, together with K627, constitute a functional epitope within IDR-B. This identification of key residues within the autoreactive epitopes will help in understanding the structural basis for the breakdown of immune tolerance to TPO in thyroid autoimmune disease.

Animals↗
PubMed2005

An attempt to define allergen-specific molecular surface features: a bioinformatic approach.

Allergens are proteins that elicit T helper lymphocyte type 2 (Th2) responses culminating in IgE antibody production and allergic disease. However, we have no answer to the fundamental question of why certain proteins are allergens, while others are not. We hypothesized that analysis of the surface of diverse allergens may reveal common structural features which might enable them to be recognized as Th2-inducing antigens by cells of the innate immune system. We have therefore used the ConSurf server to search for allergen-specific motifs. This has enabled us to identify residue conservation patterns in the homologues of Ara t 8 (plant profilin), Act c 1 (actinidin), Bet v 1 (plant pathogenesis-related protein) and Ves v 5 (venom allergen). The results demonstrate the presence of allergen-specific patches consisting of an unusually high proportion of surface-exposed hydrophobic residues. The patches that have been identified may represent molecular patterns recognizable by cells of the innate immune system.

Algorithms↗
PubMed2005

The structure of human CD23 and its interactions with IgE and CD21.

The low-affinity immunoglobulin E (IgE) receptor, CD23 (FcepsilonRII), binds both IgE and CD21 and, through these interactions, regulates the synthesis of IgE, the antibody isotype that mediates the allergic response. We have determined the three-dimensional structure of the C-type lectin domain of CD23 in solution by nuclear magnetic resonance spectroscopy. An analysis of concentration-dependent chemical shift perturbations have allowed us to identify the residues engaged in self-association to the trimeric state, whereas ligand-induced changes have defined the binding sites for IgE and CD21. The results further reveal that CD23 can bind both ligands simultaneously. Despite the C-type lectin domain structure, none of the interactions require calcium. We also find that IgE and CD23 can interact to form high molecular mass multimeric complexes. The interactions that we have described provide a solution to the paradox that CD23 is involved in both up- and down-regulation of IgE and provide a structural basis for the development of inhibitors of allergic disease.

Binding Sites↗
PubMed2005

Homology modelling of transferrin-binding protein A from Neisseria meningitidis.

Neisseria meningitidis, a causative agent of bacterial meningitis, obtains transferrin-bound iron by expressing two outer membrane located transferrin-binding proteins, TbpA and TbpB. TbpA is thought to be an integral outer membrane pore that facilitates iron uptake. Evidence suggests that TbpA is a useful antigen for inclusion in a vaccine effective against meningococcal disease, hence the identification of regions involved in ligand binding is of paramount importance to design strategies to block uptake of iron. The protein shares sequence and functional similarities to the Escherichia coli siderophore receptors FepA and FhuA, whose structures have been determined. These receptors are composed of two domains, a 22-stranded beta-barrel and an N-terminal plug region that sits within the barrel and occludes the transmembrane pore. A three-dimensional TbpA model was constructed using FepA and FhuA structural templates, hydrophobicity analysis and homology modelling. TbpA was found to possess a similar architecture to the siderophore receptors. In addition to providing insights into the highly immunogenic nature of TbpA and allowing the prediction of potentially important ligand-binding epitopes, the model also reveals a narrow channel through its entire length. The relevance of this channel and the spatial arrangement of external loops, to the mechanism of iron translocation employed by TbpA is discussed.

Amino Acid Sequence↗
PubMed2005

Effect of pH on the active site of an Arg121Cys mutant of the metallo-beta-lactamase from Bacillus cereus: implications for the enzyme mechanism.

The zinc-dependent metallo-beta-lactamases are a group of bacterial enzymes that pose a threat to the future efficacy of present-day antibiotics. Their mechanism is poorly understood, and there are no clinically useful inhibitors. While most members of the group contain two tightly bound zinc ions in their active sites, the Bacillus cereus enzyme has a much lower affinity for its second zinc (Zn2), thought to be due to the presence of Arg121 immediately beneath the floor of the active site (cf. Cys/Ser/His121 in the bizinc enzymes). Crystal structures of the Arg121Cys mutant of the B. cereus 569/H/9 enzyme were solved at pH 7.0, 5.0, and 4.5, each in the presence of either 20 microM or 20 mM Zn(2+) to generate the mono- and bizinc forms, respectively. Surprisingly, the structure of the active site was unaffected by the mutation; a network of ordered water molecules replaced the interactions made by the arginine side chain, and the occupancy of Zn2 appeared minimally changed. As the pH was lowered, Zn2 moved away from one of its ligands, Asp120, but was "tracked" by two others, Cys221 and His263. Furthermore, the hydroxide ion (and proposed nucleophile for beta-lactam hydrolysis) was bound to Zn1 at pH 5 and above but absent at pH 4.5. This provides experimental evidence for an earlier proposed mechanism in which protonation of Asp120 and the Zn1-bound hydroxide are the two events that lead to the loss of activity at low pH.

Amino Acid Substitution↗
PubMed2005

Disulfide linkage controls the affinity and stoichiometry of IgE Fcepsilon3-4 binding to FcepsilonRI.

IgE antibodies cause long-term sensitization of tissue mast cells and blood basophils toward allergen-induced cross-linking and triggering of allergic inflammation. This persistence of IgE binding is due to its uniquely high affinity for the receptor FcepsilonRI and in particular its slow rate of dissociation once bound. The binding interface consists of two subsites, one contributed by each Cepsilon3 domain of IgE Fc in a 1:1 complex. We have investigated the contributions of Cepsilon3 disulfide linkage and glycosylation to the kinetics and affinity of binding of an Fc subfragment (Fcepsilon3-4) to a soluble receptor fragment (sFcepsilonRIalpha). In contrast to IgG Fc where deglycosylation abrogates receptor binding activity, the removal of the N-linked carbohydrate at Asn-394 in Fcepsilon3-4 only reduces binding affinity by a factor of 4, principally because of a faster off-rate. Removal of the inter-heavy chain disulfide bond unexpectedly resulted in a fragment with a much faster off-rate and the potential to form a complex with a 2:1 stoichiometry (sFcepsilonRIalpha:Fcepsilon3-4). This permitted the determination of the affinity of a single, natively folded Cepsilon3 domain for the first time. The low affinity Ka approximately 10(5)-10(6) m-1, similar to that determined previously for an isolated and partially folded Cepsilon3 domain, demonstrates that substantial reduction in affinity can be achieved by preventing the engagement of one of the two Cepsilon3 domains. Recent structural data indicate that conformational change in IgE is required to allow both Cepsilon3 domains to bind, and thus an allosteric inhibitor that prevents access to the second Cepsilon3 has the potential to reduce the ability of IgE to sensitize allergic effector cells.

Allosteric Site↗
PubMed2005

Biased use of VH5 IgE-positive B cells in the nasal mucosa in allergic rhinitis.

BACKGROUND: IgE antibody-producing B cells are enriched in the nasal mucosa in patients with allergic rhinitis because of local class switching to IgE. The expressed IgE VH genes also undergo somatic hypermutation in situ to generate clonal families. The antigenic driving force behind these events is unknown. OBJECTIVE: To examine the possible involvement of a superantigen in allergic rhinitis, we compared the variable (VH) gene use and patterns of somatic mutation in the expressed IgE heavy-chain genes in nasal biopsy specimens and blood from allergic patients and the IgA VH use in the same biopsy specimens and also those from nonallergic controls. METHODS: We extracted mRNA from the nasal biopsy specimens of 13 patients and 4 nonallergic control subjects and PBMCs from 7 allergic patients. IgE and IgA VH regions were RT-PCR amplified, and the DNA sequences were compared with those of control subjects. We constructed a molecular model of VH5 to locate amino acids of interest. RESULTS: We observed a significantly increased frequency of IgE and IgA VH5 transcripts in the nasal mucosa of the allergic patients compared with the normal PBMC repertoire. Within IgE and IgA VH5 sequences in the nasal mucosa, the distribution of replacement amino acids was skewed toward the immunoglobulin framework regions. Three of 4 nonintrinsic hotspots of mutation identified in the VH5 sequences were in framework region 1. The hotspots and a conserved VH5-specific framework residue form a tight cluster on the surface of VH5. CONCLUSION: Our results provide evidence for the activity of a superantigen in the nasal mucosa in patients with allergic rhinitis.

Adolescent↗
PubMed2004

Key residues contributing to dominant conformational autoantigenic epitopes on thyroid peroxidase identified by mutagenesis.

Thyroid peroxidase (TPO) is a major autoantigen in thyroid autoimmune disease where pathogenic autoantibodies recognise conformational epitopes restricted to two overlapping immunodominant regions (IDR) termed IDR-A and -B. Based upon our structural model of TPO, we report on the localisation of the IDRs to specific amino acids important for autoantibody binding. Using a panel of recombinant human Fabs (rhFabs) from autoimmune patients, specific for the IDR-A or -B epitopes, in combination with eukaryotic expression of 14 single amino acid mutants of TPO, we identify R225 and K627 as key components of IDR-A and -B, respectively. Moreover, each mutant specifically led to the loss of binding of three different IDR-A- or -B-specific rhFabs, without affecting the binding of autoantibodies to the other determinant. Further supportive evidence for the role of amino acids R225 and K627 was obtained with murine monoclonal antibodies that first defined the IDRs. The identification of amino acids R225 and K627 as key residues for the IDR epitopes on TPO will advance our understanding of the molecular basis of autoreactivity and facilitate the design of novel therapeutic agents.

Amino Acids↗
PubMed2002

Evidence for involvement of a hydrophobic patch in framework region 1 of human V4-34-encoded Igs in recognition of the red blood cell I antigen.

The monoclonal IgM cold agglutinins that bind to the I/i carbohydrate Ags on the surface of RBCs all have Ig H chains encoded by the V4-34 gene segment. This mandatory use indicates that distinctive amino acid sequences may be involved in recognition. Critical amino acids exist in framework region 1 (FR1) of V4-34-encoded Ig, and these generate a specific Id determinant which apparently lies close to the I binding site. However, I binding by Id-expressing Ig can be modulated by sequences in complementarity-determining region (CDR)(H)3. Examination of the crystal structure of an anti-I cold agglutinin has revealed a hydrophobic patch in FR1 involving residue W7 on beta-strand A and the AVY motif (residues 23-25) on beta-strand B. In this study we used mutagenesis to show that each of the strand components of the hydrophobic patch is required for binding the I carbohydrate Ag. In addition, the crystal structure reveals that amino acids in the carboxyl-terminal region of CDR(H)3 form a surface region adjacent to the hydrophobic patch. We propose that the I carbohydrate Ag interacts simultaneously with the entire hydrophobic patch in FR1 and with the outside surface of CDR(H)3. This interaction could leave most of the conventional binding site available for binding other Ags.

Agglutinins↗
PubMed2002

Scaffolds for protein crystallisation.

In the absence of a method to ensure that crystals can be obtained for any given protein, the possibility of developing scaffolds for protein crystallisation becomes attractive. Among several approaches that could yield scaffolds, two are particularly promising: the first is based on immunoglobulin Fab fragments and immunoglobulin binding proteins while the second is based on fusion proteins. In the Fab based scaffold, the protein of interest is the antigen recognised by the antibody. In the second case, it is a protein fused to one of the scaffold components. The operational difference between the two methods is the existence of a flexible covalent tether compared to a highly specific interaction. The relative merits and disadvantages of each approach are discussed here. We also describe a lattice obtained through a combinatorial approach which appears to have the required properties to be considered a scaffold. The system makes use of an Fab derived from a rheumatoid factor and an Fc-fusion protein. The Fc-fusion system is ideal for enhanced expression of glycoproteins in mammalian cells and provides a useful tag for their purification. The molecular replacement shows a mode of binding for this rheumatoid factor that is not competitive with bacterial Fc-binding proteins. Hence it may be possible to generalize the method to include bacterial expression of fusion proteins with either protein A or protein G as the fusion partner.

Amino Acid Sequence↗
PubMed2002

Evidence for plasticity and structural mimicry at the immunoglobulin light chain-protein L interface.

The multidomain bacterial surface protein L (PpL) is a virulence factor expressed by only 10% of Peptostreptococcus magnus strains, and its expression is correlated with bacterial vaginosis. The molecular basis for its ability to recognize 60% of mammalian immunoglobulin light chain variable regions (V(L)) has been described recently by x-ray crystallography, which suggested the presence of two V(L) binding sites on each protein L domain (Graille, M., Stura, E. A., Housden, N. G., Beckingham, J. A., Bottomley, S. P., Beale, D., Taussig, M. J., Sutton, B. J., Gore, M. G., and Charbonnier, J. (2001) Structure 9, 679-687). Here, we report the crystal structure at 2.1 A resolution of a protein L mutant complexed to an Fab' fragment with only 50% of the V(L) residues interacting with PpL site 1 conserved. Comparison of the site 1 interface from both structures shows how protein L is able to accommodate these sequence differences and therefore bind to a large repertoire of Ig. The x-ray structure and NMR results confirm the existence of two V(L) binding sites on a single protein L domain. These sites exhibit a remarkable structural mimicry of growth factors binding to their receptors. This could explain the protein L superantigenic activity on human B lymphocytes.

Amino Acid Sequence↗
PubMed2002

The crystal structure of IgE Fc reveals an asymmetrically bent conformation.

The distinguishing structural feature of immunoglobulin E (IgE), the antibody responsible for allergic hypersensitivity, is the C epsilon 2 domain pair that replaces the hinge region of IgG. The crystal structure of the IgE Fc (constant fragment) at a 2.6-A resolution has revealed these domains. They display a distinctive, disulfide-linked Ig domain interface and are folded back asymmetrically onto the C epsilon 3 and C epsilon 4 domains, which causes an acute bend in the IgE molecule. The structure implies that a substantial conformational change involving C epsilon 2 must accompany binding to the mast cell receptor Fc epsilon RI. This may be the basis of the exceptionally slow dissociation rate of the IgE-Fc epsilon RI complex and, thus, of the ability of IgE to cause persistent allergic sensitization of mast cells.

Carbohydrate Conformation↗
PubMed2002

Mutagenesis within human FcepsilonRIalpha differentially affects human and murine IgE binding.

Soluble fragments of the alpha-chain of FcepsilonRI, the high-affinity receptor for IgE, compete with membrane-bound receptors for IgE and may thus provide a means to combat allergic responses. Mutagenesis within FcepsilonRIalpha is used in this study, in conjunction with the crystal structure of the FcepsilonRIalpha/IgE complex, to define the relative importance of specific residues within human FcepsilonRIalpha for IgE binding. We have also compared the effects of these mutants on binding to both human and mouse IgE, with a view to evaluating the mouse as an appropriate model for the analysis of future agents designed to mimic the human FcepsilonRIalpha and attenuate allergic disease. Three residues within the C-C' region of the FcepsilonRIalpha2 domain and two residues within the alpha2 proximal loops of the alpha1 domain were selected for mutagenesis and tested in binding assays with human and mouse IgE. All three alpha2 mutations (K117D, W130A, and Y131A) reduced the affinity of human IgE binding to different extents, but K117D had a far more pronounced effect on mouse IgE binding, and although Y131A had little effect, W130A modestly enhanced binding to mouse IgE. The mutations in alpha1 (R15A and F17A) diminished binding to both human and mouse IgE, with these effects most likely caused by disruption of the alpha1/alpha2 interface. Our results demonstrate that the effects of mutations in human FcepsilonRIalpha on mouse IgE binding, and hence the inhibitory properties of human receptor-based peptides assayed in rodent models of allergy, may not necessarily reflect their activity in a human IgE-based system.

Animals↗
PubMed2002

A novel immunoglobulin superfamily receptor (19A) related to CD2 is expressed on activated lymphocytes and promotes homotypic B-cell adhesion.

A novel lymphocyte-specific immunoglobulin superfamily protein (19A) has been cloned. The predicted 335-amino-acid sequence of 19A represents a Type 1 membrane protein with homology with the CD2 family of receptors. A molecular model of the two predicted extracellular immunoglobulin-like domains of 19A has been generated using the crystal structure of CD2 as a template. In isolated lymphocytes, expression of 19A is induced by various activation stimuli, and enforced expression of the 19A gene promotes homotypic cell adhesion in a B-cell-line model. Collectively these data imply that the 19A protein plays a role in regulation of lymphocyte adhesion.

Amino Acid Sequence↗
PubMed2001

The biology of IGE and the basis of allergic disease.

Allergic individuals exposed to minute quantities of allergen experience an immediate response. Immediate hypersensitivity reflects the permanent sensitization of mucosal mast cells by allergen-specific IgE antibodies bound to their high-affinity receptors (FcepsilonRI). A combination of factors contributes to such long-lasting sensitization of the mast cells. They include the homing of mast cells to mucosal tissues, the local synthesis of IgE, the induction of FcepsilonRI expression on mast cells by IgE, the consequent downregulation of FcgammaR (through an insufficiency of the common gamma-chains), and the exceptionally slow dissociation of IgE from FcepsilonRI. To understand the mechanism of the immediate hypersensitivity phenomenon, we need explanations of why IgE antibodies are synthesized in preference to IgG in mucosal tissues and why the IgE is so tenaciously retained on mast cell-surface receptors. There is now compelling evidence that the microenvironment of mucosal tissues of allergic disease favors class switching to IgE; and the exceptionally high affinity of IgE for FcepsilonRI can now be interpreted in terms of the recently determined crystal structures of IgE-FcepsilonRI and IgG-FcgammaR complexes. The rate of local IgE synthesis can easily compensate for the rate of the antibody dissociation from its receptors on mucosal mast cells. Effective mechanisms ensure that allergic reactions are confined to mucosal tissues, thereby minimizing the risk of systemic anaphylaxis.

Allergens↗