Sandra J. Smith-Gill

Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice

Immunological tolerance has been demonstrated in double-transgenic mice expressing the genes for a neo-self antigen, hen egg lysozyme, and a high affinity anti-lysozyme antibody. The majority of anti-lysozyme B-cells did not undergo clonal deletion, but were no longer able to secrete anti-lysozyme antibody and displayed markedly reduced levels of surface IgM while continuing to express high levels of surface IgD. These findings indicate that self tolerance may result from mechanisms other than clonal deletion, and are consistent with the hypothesis that IgD may have a unique role in B-cell tolerance.

Epitopes on protein antigens: misconceptions and realities.

Afin de determiner les structures des epitopes a la surface de proteines, des complexes entre anticorps monoclonal Fab et antigene sont formes et analyses par cristallisation et diffraction des rayons-X. De tels complexes Fab-lysozyme et Fab neuraminidase ont ete etudies dans cet article

Antibody recognition imaging by force microscopy

We have developed a method that combines dynamic force microscopy with the simultaneous molecular recognition of an antigen by an antibody, during imaging. A magnetically oscillated atomic force microscopy tip carrying a tethered antibody was scanned over a surface to which lysozyme was bound. By oscillating the probe at an amplitude of only a few nanometers, the antibody was kept in close proximity to the surface, allowing fast and efficient antigen recognition and gentle interaction between tip and sample. Antigenic sites were evident from reduction of the oscillation amplitude, as a result of antibody–antigen recognition during the lateral scan. Lysozyme molecules bound to the surface were recognized by the antibody on the scanning tip with a few nanometers lateral resolution. In principle, any ligand can be tethered to the tip; thus, this technique could potentially be used for nanometer-scale epitope mapping of biomolecules and localizing receptor sites during biological processes.

X-ray snapshots of the maturation of an antibody response to a protein antigen

The process whereby the immune system generates antibodies of higher affinities during a response to antigen (affinity maturation) is a prototypical example of molecular evolution. Earlier studies have been confined to antibodies specific for small molecules (haptens) rather than for proteins. We compare the structures of four antibodies bound to the same site on hen egg white lysozyme (HEL) at different stages of affinity maturation. These X-ray snapshots reveal that binding is enhanced, not through the formation of additional hydrogen bonds or van der Waals contacts or by an increase in total buried surface, but by burial of increasing amounts of apolar surface at the expense of polar surface, accompanied by improved shape complementarity. The increase in hydrophobic interactions results from highly correlated rearrangements in antibody residues at the interface periphery, adjacent to the central energetic hot spot. This first visualization of the maturation of antibodies to protein provides insights into the evolution of high affinity in other protein–protein interfaces.

Organ reactive autoantibodies from non-immunized adult BALB/c mice are polyreactive and express non-biased VH gene usage

To examine the naturally activated autoreactive B cell repertoire, we analyzed a panel of hybridomas from unmanipulated adult BALB/c spleen cells for reactivity patterns and VH gene usage. We found a pattern of VH usage that was diverse and appeared to reflect the germline repertoire. Although all but one natural antibody hybridoma (NAb) were initially selected for organ rather than antigen binding, the majority of organ reactive IgM NAbs were polyreactive, expressing a broad range of reactivity patterns for both self and foreign antigens, that were unique for each NAb and were not indiscriminate. Our results are consistent with the hypothesis that many naturally activated adult B cells are highly polyreactive and that autoreactivity is a consequence of polyreactivity. We suggest that the population of NAbs exhibiting organ reactivity overlaps the populations of other IgM autoantibodies that have been described previously, and that these all derive from a pool of polyreactive IgM antibodies which are polyclonally activated in the early immune response. These polyreactive natural antibodies may represent a first line of defense and offer protection for the host against a variety of foreign agents.

Dissection of binding interactions in the complex between the anti-lysozyme antibody HyHEL-63 and its antigen

Alanine-scanning mutagenesis, X-ray crystallography, and double mutant cycles were used to characterize the interface between the anti-hen egg white lysozyme (HEL) antibody HyHEL-63 and HEL. Eleven HEL residues in contact with HyHEL-63 in the crystal structure of the antigen-antibody complex, and 10 HyHEL-63 residues in contact with HEL, were individually truncated to alanine in order to determine their relative contributions to complex stabilization. The residues of HEL (Tyr20, Lys96, and Lys97) most important for binding HyHEL-63 (Delta G(mutant) - Delta G(wild type) > 3.0 kcal/mol) form a contiguous patch at the center of the surface contacted by the antibody. Hot spot residues of the antibody (Delta Delta G > 2.0 kcal/mol) are organized in two clusters that juxtapose hot spot residues of HEL, resulting in energetic complementarity across the interface. All energetically critical residues are centrally located, shielded from solvent by peripheral residues that contribute significantly less to the binding free energy. Although HEL hot spot residues Lys96 and Lys97 make similar interactions with antibody in the HyHEL-63/HEL complex, alanine substitution of Lys96 results in a nearly 100-fold greater reduction in affinity than the corresponding mutation in Lys97. To understand the basis for this marked difference, we determined the crystal structures of the HyHEL-63/HEL Lys96Ala and HyHEL-63/HEL Lys97Ala complexes to 1.80 and 1.85 A resolution, respectively. Whereas conformational changes in the proteins and differences in the solvent networks at the mutation sites appear too small to explain the observed affinity difference, superposition of free HEL in different crystal forms onto bound HEL in the wild type and mutant HyHEL-63/HEL complexes reveals that the side-chain conformation of Lys96 is very similar in the various structures, but that the Lys97 side chain displays considerable flexibility. Accordingly, a greater entropic penalty may be associated with quenching the mobility of the Lys97 than the Lys96 side chain upon complex formation, reducing binding. To further dissect the energetics of specific interactions in the HyHEL-63/HEL interface, double mutant cycles were constructed to measure the coupling of 13 amino acid pairs, 11 of which are in direct contact in the crystal structure. A large coupling energy, 3.0 kcal/mol, was found between HEL residue Lys97 and HyHEL-63 residue V(H)Asp32, which form a buried salt bridge surrounded by polar residues of the antigen. Thus, in contrast to protein folding where buried salt bridges are generally destabilizing, salt bridges in protein-protein interfaces, whose residual composition is more hydrophilic than that of protein interiors, may contribute significantly to complex stabilization.

A substantial proportion of the adult BALB/c available B cell repertoire consists of multireactive B cells

A variety of studies have documented multireactive antibodies in both the preimmune and naturally activated repertoire, but the relationship of these primarily IgM multireactive antibodies to antigen-specific primary and secondary response antibodies is currently not defined. In order to characterize the BALB/c preimmunization specificity repertoire and the baseline of naturally activated antibodies from which the immune response to a specific antigen (hen egg-white lysozyme, HEL) develops, panels of polyclonally activated blast-derived hybridomas (BlAbs) and natural antibody hybridomas (NAbs) from the spleens of unimmunized mice were screened for binding to a panel of nine complex antigens. Over half of the IgM-secreting BlAbs produced antibodies that were antigen-reactive; of these, over half were multireactive, i.e. capable of binding more than one complex antigen. There was no bias towards self vs foreign or thymus-dependent vs thymus-independent antigens. The frequency of antigen-reactive NAbs was about half the frequency of antigen-reactive antibodies found among the BlAbs. However, over half of the antigen-reactive NAbs were also multireactive, and the reactivity profile within the antigen-reactive subset of NAbs was similar to that within the antigen-reactive subset of BlAbs. These results suggest that the available repertoire of adult spleen cells contains a high proportion of multireactive antibodies, and that a subset of the available repertoire is randomly activated, yielding a small proportion of natural antibodies which closely reflect a random sampling of the available repertoire. Although monospecific precursor cells are rare, monospecific IgM BlAbs were found for all antigens in the panel except staphylococcal nuclease and mouse IgG. Monospecific as well as multireactive HEL-binding BlAbs were found at frequencies comparable to other protein antigens in the panel, and HEL-reactive NAbs were also present. On the other hand, it has previously been shown that HEL-reactive IgM antibodies (including multireactive antibodies whose specificities include HEL) are rare or absent in both the primary and secondary response to HEL. This cannot be attributed to an absence of available precursor B cells, and most likely reflects an early recruitment of HEL-reactive clones into the peripheral B cell pool. The possibility that polyreactive B cells may serve as precursors for some HEL-specific IgG antibodies is discussed.

Protein Structure to Function Via Dynamics

Protein folding, binding, catalytic activity and molecular recognition all involve molecular movements, with varying extents. The molecular movements are brought upon via flexible regions. Stemming from sequence, a fine tuning of electrostatic and hydrophobic properties of the protein fold determine flexible and rigid regions. Studies show flexible regions usually lack electrostatic interactions, such as salt-bridges and hydrogen-bonds, while the rigid regions often have larger number of such electrostatic interactions. Protein flexible regions are not simply an outcome of looser packing or instability, rather they are evolutionally selected. In this review article we highlight the significance of protein flexibilities in folding, binding and function, and their structural and thermodynamic determinants. Our electrostatic calculations and molecular dynamic simulations on an antibody-antigen complex further illustrate the importance of protein flexibilities in binding and function.

Refined structures of bobwhite quail lysozyme uncomplexed and complexed with the HyHEL-5 Fab fragment.

The HyHEL‐5 antibody has more than a thousandfold lower affinity for bobwhite quail lysozyme (BWQL) than for hen egg‐white lysozyme (HEL). Four sequence differences exist between BWQL and HEL, of which only one is involved in the interface with the Fab. The structure of bobwhite quail lysozyme has been determined in the uncomplexed state in two different crystal forms and in the complexed state with HyHEL‐5, an anti‐hen egg‐white lysozyme Fab. Similar backbone conformations are observed in the three molecules of the two crystal forms of uncomplexed BWQL, although they show considerable variability in side‐chain conformation. A relatively mobile segment in uncomplexed BWQL is observed to be part of the HyHEL‐5 epitope. No major backbone conformational differences are observed in the lysozyme upon complex formation, but side‐chain conformational differences are seen in surface residues that are involved in the interface with the antibody. The hydrogen bonding in the interface between BWQL and HyHEL‐5 is similar to that in previously determined lysozyme‐HyHEL‐5 complexes.

Association Energetics of Cross-Reactive and Specific Antibodies†

HyHEL-8, HyHEL-10, and HyHEL-26 (HH8, HH10, and HH26, respectively) are murine monoclonal IgG(1) antibodies which share over 90% variable-region amino acid sequence identity and recognize identical structurally characterized epitopes on hen egg white lysozyme (HEL). Previous immunochemical and surface plasmon resonance-based studies have shown that these antibodies differ widely in their tolerance of mutations in the epitope. While HH8 is the most cross-reactive, HH26 is rigidified by a more extensive network of intramolecular salt links and is highly specific, with both association and dissociation rates strongly affected by epitope mutations. HH10 is of intermediate specificity, and epitope mutations produce changes primarily in the dissociation rate. Calorimetric characterization of the association energetics of these three antibodies with the native antigen HEL and with Japanese quail egg white lysozyme (JQL), a naturally occurring avian variant, shows that the energetics of interaction correlate with cross-reactivity and specificity. These results suggest that the greater cross-reactivity of HH8 may be mediated by a combination of conformational flexibility and less specific intermolecular interactions. Thermodynamic calculations suggest that upon association HH8 incurs the largest configurational entropic penalty and also the smallest loss of enthalpic driving force with variant antigen. Much smaller structural perturbations are expected in the formation of the less flexible HH26 complex, and the large loss of enthalpic driving force observed with variant antigen reflects its specificity. The observed thermodynamic parameters correlate well with the observed functional behavior of the antibodies and illustrate fundamental differences in thermodynamic characteristics between cross-reactive and specific molecular recognition.