Impact of antibody subclass and disulfide isoform differences on the biological activity of CD200R and ßklotho agonist antibodies.

Agonism of cell surface receptors by monoclonal antibodies is dependent not only on its ability to bind the target, but also to deliver a biological signal through receptors to the cell. Immunoglobulin G2 antibodies (IgG2s) are made up of a mixture of distinct isoforms (IgG2-A, -B and A/B), which differ by the disulfide connectivity at the hinge region. When evaluating panels of agonistic antibodies against CD200 receptor (CD200R) or ßklotho receptor (ßklotho), we noticed striking activity differences of IgG1 or IgG2 antibodies with the same variable domains. For the CD200R antibody, the IgG2 antibody demonstrated higher activity than the IgG1 or IgG4 antibody. More significantly, for ßklotho, agonist antibodies with higher biological activity as either IgG2 or IgG1 were identified. In both cases, ion exchange chromatography was able to isolate the bioactivity to the IgG2-B isoform from the IgG2 parental mixture. The subclass-related increase in agonist activity was not correlated with antibody aggregation or binding affinity, but was driven by enhanced avidity for the CD200R antibody. These results add to the growing body of evidence that show that conformational differences in the antibody hinge region can have a dramatic impact on the antibody activity and must be considered when screening and engineering therapeutic antibody candidates. The results also demonstrate that the IgG1 (IgG2-A like) or the IgG2-B form may provide the most active form of agonist antibodies for different antibodies and targets.

Structural characterization of apical membrane antigen 1 (AMA1) from Toxoplasma gondii.

Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Apicomplexan parasites to invade host cells. We report the 2.0 A resolution x-ray crystal structure of the full ectodomain (domains I, II, and III) of AMA1 from the pervasive protozoan parasite Toxoplasma gondii. The structure of T. gondii AMA1 (TgAMA1) is the most complete of any AMA1 structure to date, with more than 97.5% of the ectodomain unambiguously modeled. Comparative sequence analysis reveals discrete segments of divergence in TgAMA1 that map to areas of established functional importance in AMA1 from Plasmodium vivax (PvAMA1) and Plasmodium falciparum (PfAMA1). Inspection of the TgAMA1 structure reveals a network of apical surface loops, reorganized in both size and chemistry relative to PvAMA1/PfAMA1, that appear to serve as structural filters restricting access to a central hydrophobic groove. The terminal portion of this groove is formed by an extended loop from DII that is 14 residues shorter in TgAMA1. A pair of tryptophan residues (Trp(353) and Trp(354)) anchor the DII loop in the hydrophobic groove and frame a conserved tyrosine (Tyr(230)), forming a contiguous surface that may be critical for moving junction assembly. The minimalist DIII structure folds into a cystine knot that probably stabilizes and orients the bulk of the ectodmain without providing excess surface area to which invasion-inhibitory antibodies can be generated. The detailed structural characterization of TgAMA1 provides valuable insight into the mechanism of host cell invasion by T. gondii.

Structural and functional characterization of SporoSAG: a SAG2-related surface antigen from Toxoplasma gondii.

Toxoplasma gondii, the etiological agent of toxoplasmosis, utilizes stage-specific expression of antigenically distinct glycosylphosphatidylinositol-tethered surface coat proteins to promote and establish chronic infection. Of the three infective stages of T. gondii, sporozoites are encapsulated in highly infectious oocysts that have been linked to large scale outbreaks of toxoplasmosis. SporoSAG (surface antigen glycoprotein) is the dominant surface coat protein expressed on the surface of sporozoites. Using a bioinformatic approach, we show that SporoSAG clusters with the SAG2 subfamily of the SAG1-related superfamily (SRS) and is non-polymorphic among the 11 haplogroups of T. gondii strains. In contrast to the immunodominant SAG1 protein expressed on tachyzoites, SporoSAG is non-immunogenic during natural infection. We report the 1.60 A resolution crystal structure of SporoSAG solved using cadmium single anomalous dispersion. SporoSAG crystallized as a monomer and displays unique features of the SRS beta-sandwich fold relative to SAG1 and BSR4. Intriguingly, the structural diversity is localized to the upper sheets of the beta-sandwich fold and may have important implications for multimerization and host cell ligand recognition. The structure of SporoSAG also reveals an unexpectedly acidic surface that contrasts with the previously determined SAG1 and BSR4 structures where a basic surface is predicted to play a role in binding negatively charged glycosaminoglycans. Our structural and functional characterization of SporoSAG provides a rationale for the evolutionary divergence of this key SRS family member.

Ral GTPase interacts with the N-terminal in addition to the C-terminal region of PLC-delta1.

Previously, we have shown that RalA, a calmodulin (CaM)-binding protein, binds to the C2 region in the C-terminal of PLC-delta1, and increases its enzymatic activity. Since PLC-delta1 contains a CaM-like region in its N-terminus, we have investigated if RalA can also bind to the N-terminus of PLC-delta1. Therefore, we created a GST-PLC-delta1 construct consisting of the first 294 amino acids of PLC-delta1 (GST-PLC-delta1(1-294)). In vitro binding experiments confirmed that PLC-delta1(1-294) was capable of binding directly to RalA. W-7 coupled to polyacrylamide beads bound pure PLC-delta1, demonstrating that PLC-delta1 contains a CaM-like region. Competition assays with W-7, peptides representing RalA and the newly identified RalB CaM-binding regions, or the IQ peptide from PLC-delta1 were able to inhibit RalA binding to PLC-delta1(1-294). This study demonstrates that there are two binding sites for RalA in PLC-delta1 and provides further insight into the role of Ral GTPase in the regulation of PLC-delta1 function.

Insect-cell expression, crystallization and X-ray data collection of the bradyzoite-specific antigen BSR4 from Toxoplasma gondii.

Toxoplasma gondii is an important global pathogen that infects nearly one third of the world's adult population. A family of developmentally expressed structurally related surface-glycoprotein adhesins (SRSs) mediate attachment to and are utilized for entry into host cells. The latent bradyzoite form of T. gondii persists for the life of the host and expresses a distinct family of SRS proteins, of which the bradyzoite-specific antigen BSR4 is a prototypical member. Structural studies of BSR4 were initiated by first recombinantly expressing BSR4 in insect cells, which was followed by crystallization and preliminary X-ray data collection to 1.95 A resolution. Data processing showed that BSR4 crystallized with one molecule in the asymmetric unit of the P4(1)2(1)2 or P4(3)2(1)2 space group, with a solvent content of 60% and a corresponding Matthews coefficient of 2.98 A(3) Da(-1).

Characterization of the molecular pharmacology of AMD3100: a specific antagonist of the G-protein coupled chemokine receptor, CXCR4.

The chemokine receptor CXCR4 is widely expressed on different cell types, is involved in leukocyte chemotaxis, and is a co-receptor for HIV. AMD3100 has been shown to be a CXCR4 receptor antagonist, and to block HIV infection of T-tropic, X4-using, virus in vitro and in vivo. AMD3100 is an effective mobilizer of hematopoietic stem cells and is being investigated in clinical trials in multiple myeloma and non-Hodgkins lymphoma patients. Using the CCRF-CEM T-cell line that constitutively expresses CXCR4 we confirmed that AMD3100 was an antagonist of SDF-1/CXCL12 ligand binding (IC50=651+/-37 nM). We have also shown that AMD3100 inhibits SDF-1 mediated GTP-binding (IC50=27+/-2.2 nM), SDF-1 mediated calcium flux (IC50=572+/-190 nM), and SDF-1 stimulated chemotaxis (IC50=51+/-17 nM). AMD3100 did not inhibit calcium flux against cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7 when stimulated with their cognate ligands, nor did it inhibit receptor binding of LTB4. AMD3100 did not, on its own, induce a calcium flux in the CCRF-CEM cells, which express multiple GPCRs including CXCR4, CCR4 and CCR7. Furthermore, AMD3100 neither stimulated GTP-binding, an assay for GPCR activation, in CEM cell membranes; nor chemotaxis of CCRF-CEM cells. These data therefore demonstrate that AMD3100 is a specific antagonist of CXCR4, is not cross-reactive with other chemokine receptors, and is not an agonist of CXCR4.

Structure of the micronemal protein 2 A/I domain from Toxoplasma gondii.

Toxoplasma gondii is a widespread zoonotic pathogen capable of causing serious disease in humans and animals. As an obligate intracellular parasite, T. gondii relies on the orchestrated secretion of proteins from its apical complex organelles including the multimodular, transmembrane micronemal protein 2 (MIC2) that couples recognition of the host cell with cytoskeletal reorganization of the parasite to drive invasion. To probe the basis by which the von Willebrand Factor A (vWA)-Integrin like module of TgMIC2 engages the host cell, we solved the crystal structure of a truncated form of TgMIC2A/I (TgMIC2A/Ic) phased by iodide SIRAS and refined to a resolution of 2.05 Å. The TgMIC2A/Ic core is organized into a central twisted beta sheet flanked by α-helices consistent with a canonical vWA fold. A restricted basic patch serves as the putative heparin binding site, but no heparin binding was detected in native gel shift assays. Furthermore, no metal was observed in the metal ion dependent adhesion site (MIDAS). Structural overlays with homologous A/I domains reveal a divergent organization of the MIDAS ß4-α4 loop in TgMIC2A/Ic, which is stabilized through the burial of Phe195 into a deep pocket formed by Gly185. Intriguingly, Gly185 appears to be unique among A/I domains to TgMIC2A/I suggesting that the divergent loop conformation may also be unique to TgMIC2A/I. Although lacking the C-terminal extension, the TgMIC2A/Ic structure reported here is the first of an A/I domain from an apicomplexan parasite and provides valuable insight into defining the molecular recognition of host cells by these widespread pathogens.

Structural characterization of the bradyzoite surface antigen (BSR4) from Toxoplasma gondii, a unique addition to the surface antigen glycoprotein 1-related superfamily.

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects nearly one-third of the human population. The success of T. gondii is based on its complex life cycle; a lytic tachyzoite form disseminates infection, whereas an encysted bradyzoite form establishes a latent, chronic infection. Persistence and transmissibility is central to the survival of the parasite and is, in part, mediated by a family of antigenically distinct surface antigen glycoprotein (SAG)-related sequences (SRS) adhesins that play a dual role in host cell attachment and host immune evasion. More than 160 members of the SRS family have been identified with only the tachyzoite-expressed SAG1 structurally characterized. Here we report the first structural description of the bradyzoite adhesin BSR4 using x-ray crystallography and small angle x-ray scattering. The 1.90-A crystal structure of BSR4 reveals an architecture comprised of tandem beta sandwich domains organized in a head to tail fashion with the N-terminal domain responsible for dimer formation. A restructured topology in BSR4 results in a ligand-binding site that is significantly reorganized in both structure and chemistry relative to SAG1, consistent with BSR4 binding a distinct physiological ligand. The small angle x-ray scattering solution structure of BSR4 highlights a potentially important structural role for the interdomain polymorphic linker that imparts significant flexibility that may promote structural adaptation during ligand binding. This study reveals an unexpected level of structural diversity within the SRS superfamily and provides important insight into the role of these virulence factors.

Ral-GTPase interacts with the beta1 subunit of Na+/K+-ATPase and is activated upon inhibition of the Na+/K+ pump.

Na+/K+-ATPase functions as both an ion pump and a signal transducer. Cardiac glycosides partially inhibit Na+/K+-ATPase, causing activation of multiple interrelated growth pathways via the Na+/K+-ATPase/c-Src/epidermal growth factor receptor complex. Such pathways include Ras/MEK/ERK and Ral/RalGDS cascades, which can lead to cardiac hypertrophy. In search of novel Ral-GTPase binding proteins, we used RalB as the bait to screen a human testes cDNA expression library using the yeast 2-hybrid system. The results demonstrated that 1 of the RalB interacting clones represented the C-terminal region of the beta1 subunit of Na+/K+-ATPase. Further analysis using the yeast 2-hybrid system and full-length beta1 subunit of Na+/K+-ATPase confirmed the interaction with RalA and RalB. In vitro binding and pull-down assays demonstrated that the beta1 subunit of Na+/K+-ATPase interacts directly with RalA and RalB. Ral-GTP pull-down assays demonstrated that short-term ouabain treatment of A7r5 cells, a rat aorta smooth muscle cell line, caused activation of Ral GTPase. Maximal activation was observed 10 min after ouabain treatment. Ouabain-mediated Ral activation was inhibited upon the stimulation of Na+/K+-ATPase activity by Ang II. We propose that Ral GTPase is involved in the signal transducing function of Na+/K+-ATPase and provides a possible molecular mechanism connecting Ral to cardiac hypertrophy during diseased conditions.

Calmodulin binding to the small GTPase Ral requires isoprenylated Ral.

Ral, a member of the Ras-p21 superfamily of small GTPases, has been shown to require the calcium-signaling protein calmodulin (CaM) for activation. In the present work, we investigated the properties of the Ral-CaM interaction. Using CaM affinity binding assay with lysates from mammalian cells overexpressing various Ral mutants, we found that RalB(V23, DeltaCAAX) lacking the C-terminal isoprenylation region bound significantly less efficiently to CaM. Binding of other mutants containing critical amino acid changes in the nucleotide or substrate binding regions (residues 23, 28, and 49) was not affected. In addition, all mutants bound significantly better in the presence of calcium versus the calcium chelator EGTA. Using in vitro transcription-translation in the presence of geranylgeranyl pyrophosphate, we demonstrate enhanced Ral binding to CaM. Inhibition of isoprenylation in cells in culture with lovastatin resulted in decreased binding of CaM to Ral. The present results show that post-translational isoprenylation of Ral is important in Ral-CaM interaction.