Isolation and cloning of a Drosophila homolog to the mammalian RACK1 gene, implicated in PKC-mediated signalling

AbstractThe mammalian RACK1 protein binds activated protein kinase C, acting as an intracellular receptor to anchor the activated PKC to the cytoskeleton. Genes encoding RACK1-like proteins have been isolated from a wide range of eucaryotic organisms; we report the isolation of a Drosophila member of this family. This Drosophila RACK1-like protein shows 76% identity to the mammalian RACK1 proteins, but only about 60% identity to related proteins from plants and fungi. The Drosophila rack1 gene has a dynamic pattern of expression during early embryogenesis with the highest expression in the mesodermal and endodermal lineages

B-cell epitopes in GroEL of Francisella tularensis.

The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other's binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange-mass spectrometry (DXMS) and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL

Accurate identification of paraprotein antigen targets by epitope reconstruction

We describe the first successful clinical application of a new discovery technology, epitope-mediated antigen prediction (E-MAP), to the investigation of multiple myeloma. Until now, there has been no reliable, systematic method to identify the cognate antigens of paraproteins. E-MAP is a variation of previous efforts to reconstruct the epitopes of paraproteins, with the significant difference that it provides enough epitope sequence data so as to enable successful protein database searches. We first reconstruct the paraprotein's epitope by analyzing the peptides that strongly bind. Then, we compile the data and interrogate the nonredundant protein database, searching for a close match. As a clinical proof-of-concept, we apply this technology to uncovering the protein targets of para-proteins in multiple myeloma (MM). E-MAP analysis of 2 MM paraproteins identified human cytomegalovirus (HCMV) as a target in both. E-MAP sequence analysis determined that one para-protein binds to the AD-2S1 epitope of HCMV glycoprotein B. The other binds to the amino terminus of the HCMV UL-48 gene product. We confirmed these predictions using immunoassays and immunoblot analyses. E-MAP represents a new investigative tool for analyzing the role of chronic antigenic stimulation in B-lymphoproliferative disorders

DXMS-epitopes of FtGroEL mAbs on the molecular surface of Ft GroEL-GroES homology models.

<p>(<b>A</b>) FtGroEL monomers. (<b>B</b>) FtGroEL tetradecamers complexed with GroES. The epitopes of the five mAbs and the binding-site of GroES on GroEL are color-coded as indicated, and are divided into two images to separate the overlapping epitopes. FtGroEL residue Y476 is indicated in black. Note that the N30 epitope is exposed on both the GroES-bound and non-GroES-bound monomers but is blocked from view by the apical domain in the head-on view of the former. The proximity of the N3 and N30 epitopes in the tetradecamer, especially in the non-GroES-bound heptamer, reflects the juxtaposition of epitopes from neighboring monomers.</p

Homology model of FtGroEL.

<p>(<b>A–C</b>) Ribbon diagrams of GroEL tetradecamer complexed with GroES and of the non-GroES-bound and GroES-bound monomers. The positions of ADP (space-filling model in purple), of FtGroEL residues K344 and Y476 (space-filling models), and of the amino (N) and carboxyl (C) termini of the monomers are indicated. The GroES-bound monomer in C top is also shown after a 180° rotation about the z axis in C bottom right, to facilitate comparison with the non-GroES-bound monomer in C bottom left. (<b>D</b>) Linear amino acid sequence (in one-letter code) and secondary structure representation of FtGroEL. α-helices, β-strands, and loops are represented as boxes, thick arrows, and lines, respectively, blue for the equatorial domain, green for the intermediate region, and red for the apical domain. Helix letters and strand numbers are indicated. The FtGroEL amino acid residues involved in the interaction with GroES are highlighted in magenta.</p

X-ray crystallographic structures of the antigen-binding sites of Ab53 and Ab64.

<p>(<b>A</b>) Head-on views of the molecular surfaces, colored gray for the H chain and purple for the L chain, with the CDR loops colored and indicated as H1, H2, H3 for the H chain and L1, L2, L3 for the L chain. (<b>B</b>) Ribbon diagrams of the binding-sites, colored gray for VH and purple for VL, and clipped/depth-cued for clarity. Selected side chains are shown in stick and labeled in black for VH residues and purple for VL residues. Solvent molecules present in the structures (sulfate, chloride, tris) are also shown. Hydrogen bonds indicated in the text are shown as dotted lines. (<b>C</b>) Ribbon diagrams of the binding-sites with the docked FtGroEL model, shown in red for Ab53 with the side-chain of R362 and in green for Ab64 with the side-chain of K344. The positions of some of the residues in the docked antibody structures are somewhat shifted compared with the crystal structures in B due to the energy minimization steps used during the docking protocol. Figure made with Maestro (version 9.3.5, Schrödinger, Inc., New York, NY).</p

Ab64 and N200 reduce SchuS4 burden in a mouse model of respiratory tularemia.

<p>(<b>A</b>) BALB/cJ female mice (n = 15) were inoculated i.n. with 91–164 CFU of SchuS4, injected i.p. with 50 µg of the indicated mAbs 2 hours post inoculation, and bled then euthanized 3 days post inoculation for blood CFU determination. Data were pooled from 3 experiments with 5 mice per group for each mAb and compared for statistical significance with PBS only for groups that were tested at the same time (same panel). (<b>B</b>) BALB/cJ female mice (n = 4) were inoculated i.n. with 93 (n = 2) or 114 (n = 2) CFU of SchuS4, injected i.p. with 50 µg of the indicated mAbs 2 hours post inoculation, and bled then euthanized 3 days post inoculation for blood, lung and spleen CFU determination. Percent CFU reduction compared with PBS was calculated from the median CFU numbers and the P value (indicated below the percent CFU reduction by *p≤0.05, **p≤0.01, or ***p≤0.001) was determined using the two-tailed Mann-Whitney test. All mAbs are IgG2a except for Ab12 and TIB-114, which are IgG3 (indicated as G3). The Ab52 mAb (anti-Ft O-antigen) was used as standard. The TIB-114 (anti-sheep red blood cells) and CO17-1A (anti-human tumor-associated antigen EpCam) mAbs were used as isotype controls. All other mAbs are anti-Ft GroEL.</p

DXMS-mapping of mAb epitopes in FtGroEL.

<p>(<b>A</b>) Difference heat-maps for FtGroEL complexed with Fab (left) or IgG (right) of the indicated mAbs in the indicated molar ratio of mAb to FtGroEL monomer. The times of FtGroEL-Ab interaction (10 seconds, 100 seconds, and 1000 seconds) are specified. As indicated in the color bar, blue shades suggest more buried regions (which exchange slower upon antibody-binding), and red shades suggest more exposed regions (which exchange faster upon antibody-binding). The darkest blue region(s) in each difference heat-map were taken as the DXMS-epitopes and their spans are indicated. Note that the heat map for FtGroEL in complex with Ab53 Fab (top left, the first one to be performed) is in a slightly different format than the other heat-maps. (<b>B</b>) Linear representation of the DXMS-epitopes on the sequence/secondary structure template of FtGroEL (see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099847#pone-0099847-g007" target="_blank">Figure 7D</a> for description, except partial view). When both Fab and IgG data were available, the results were combined. Epitopes for the indicated mAbs are represented as labeled colored boxes below the FtGroEL sequence.</p

Data collection and refinement statistics for Ab53 and Ab64 Fabs.

<p>Data collection and refinement statistics for Ab53 and Ab64 Fabs.</p