Glutathione S-transferase Fusion Protein Research Articles

The Dystonia-associated Protein TorsinA Modulates Synaptic Vesicle Recycling

The loss of a glutamic acid residue in the AAA-ATPase (ATPases associated with diverse cellular activities) torsinA is responsible for most cases of early onset autosomal dominant primary dystonia. In this study, we found that snapin, which binds SNAP-25 (synaptosome-associated protein of 25,000 Da) and enhances the association of the SNARE complex with synaptotagmin, is an interacting partner for both wild type and mutant torsinA. Snapin co-localized with endogenous torsinA on dense core granules in PC12 cells and was recruited to perinuclear inclusions containing mutant DeltaE-torsinA in neuroblastoma SH-SY5Y cells. In view of these observations, synaptic vesicle recycling was analyzed using the lipophilic dye FM1-43 and an antibody directed against an intravesicular epitope of synaptotagmin I. We found that overexpression of wild type torsinA negatively affects synaptic vesicle endocytosis. Conversely, overexpression of DeltaE-torsinA in neuroblastoma cells increases FM1-43 uptake. Knockdown of snapin and/or torsinA using small interfering RNAs had a similar inhibitory effect on the exo-endocytic process. In addition, down-regulation of torsinA causes the persistence of synaptotagmin I on the plasma membrane, which closely resembles the effect observed by the overexpression of the DeltaE-torsinA mutant. Altogether, these findings suggest that torsinA plays a role together with snapin in regulated exocytosis and that DeltaE-torsinA exerts its pathological effects through a loss of function mechanism. This may affect neuronal uptake of neurotransmitters, such as dopamine, playing a role in the development of dystonic movements.

Corynebacterium glutamicum σ E is involved in responses to cell surface stresses and its activity is controlled by the anti-σ factor CseE

In this study, we demonstrate that sigma(E), an alternative sigma factor of Corynebacterium glutamicum, is involved in cell surface stresses. Cells in which the sigE gene was deleted evidenced increased sensitivity to magnesium deficiency, as well as to SDS, lysozymes, EDTA and heat. We utilized physiological analyses to show that the downstream gene, designated cseE, encodes an anti-sigma factor. The retarded growth of the cseE mutant cells under ordinary growth conditions could be recovered by an additional deletion of sigE encoding sigma(E). Under stress conditions, the phenotype of the cseE-overexpressing cells mimicked that of the sigE mutant. The sigE and cseE genes were transcribed into a single transcript, and gene transcription was stimulated by heat. The SigE and CseE proteins interacted physically in vitro, in the form of glutathione S-transferase (GST) and maltose binding protein (MBP) fusion proteins, respectively. 2D-PAGE analysis of the wild-type and mutant crude extracts showed that the sigE mutant failed to synthesize a 34 kDa polypeptide that was normally induced in wild-type cells grown under heat (or SDS)-stressed conditions. The protein turned out to be expressed from ORF NCgl1070 and showed similarity to methyltransferases which may confer resistance to antibiotics. Accordingly, the sigE mutant evidenced extreme sensitivity to antibiotics, including nalidixic acid, penicillin and vancomycin. Finally, we present a discussion of the possible role of the sigE and cseE genes in the acclimation of C. glutamicum to cell surface stress conditions.

Investigation of GST fusion proteins for screening protein interactions

Proteins of interest are commonly expressed as fusion protein conjugates with Glutathione S‐Transferase (GST) due to the relative ease with which GST can be purified. Expression vectors may have proteolytic cleavage sites engineered into the proteins sequence so that the protein of interest may be separated from GST and isolated for further study; however, these enzymes can be expensive and cleavage may be incomplete, reducing the usable yield. We investigated fluorescence anisotropy as a method for examining the interaction of GST fusion proteins. Fluorescence studies often use ligands specific for the protein of interest; however some proteins may not have natural probes suitable for fluorescence studies. We investigated the possibility of measuring the interaction of GST fusion proteins via glutathione (Glu‐Cys‐Gly, or GSH) labeled with a fluorecein‐maleimide. Titration with GST demonstrated an increase in fluorescence anisotropy, indicating GSH‐Fl ligand binding. Results suggest that fluorescence anisotropy of GST‐fusion protein interaction could be a preliminary or complementary screening method for examining protein interactions, especially those whose natural ligands are not spectroscopically observable.

Mapping and Characterization of the Binding Site for Specific Oxidized Phospholipids and Oxidized Low Density Lipoprotein of Scavenger Receptor CD36

Recent studies have identified a novel family of oxidized phosphatidylcholines (oxPC(CD36)) that serve as highly specific ligands for scavenger receptor CD36. oxPC(CD36) accumulate in vivo and mediate macrophage foam cell formation as well as promote platelet hyper-reactivity in hyperlipidemia via CD36. The structural basis of oxPC(CD36) binding to CD36 has not been elucidated. We used liquid-phase binding to glutathione S-transferase fusion proteins containing various regions of CD36 to initially identify the region spanning CD36 amino acids 157-171 to contain a major binding site for oxPC(CD36). A bell-shaped pH profile and salt concentration dependence suggest an electrostatic mechanism of the binding. Two conserved, positively charged amino acids in the region 157-171 (lysines at positions 164 and 166) were identified as critical for oxPC(CD36) and oxidized low density lipoprotein (oxLDL) binding to CD36. Lysine neutralization with chemical modifier or site-directed mutagenesis of lysine 164/166 to alanine or glutamate, but not to arginine, abolished binding. Cells expressing full-length CD36 with mutated lysines (164 and 166) failed to recognize oxPC(CD36) and oxLDL. Synthetic peptides mimicking the CD36 binding site, but not mutated or scrambled peptides, effectively prevented: (i) oxLDL binding to CD36, (ii) macrophage foam cell formation induced by oxLDL, and (iii) platelet activation by oxPC(CD36). These data indicate that CD36 (160-168) represents the core of the oxPC(CD36) binding site with lysines 164/166 being indispensable for the binding.

Potential artifacts in using a glutathione S-transferase fusion protein system and spin labeling electron paramagnetic resonance methods to study protein–protein interactions

Site-directed spin labeling electron paramagnetic resonance methods have been an important tool in studying protein–protein interactions. Labels are often attached to a cysteine residue, and spectra are acquired with and without binding partner(s) to provide information on the binding. This requires a knowledge of the label location which is simplified if the label remains faithfully attached to the designated residue in the complex. We report a system where this is not the case because the label was extracted by dialysis-resistant glutathione molecules. Once this artifact is identified, spectral subtraction provides a solution for meaningful data interpretation.

Solitary and Repetitive Binding Motifs for the AP2 Complex α-Appendage in Amphiphysin and Other Accessory Proteins

Adaptor protein (AP) complexes bind to transmembrane proteins destined for internalization and to membrane lipids, so linking cargo to the accessory internalization machinery. This machinery interacts with the appendage domains of APs, which have platform and beta-sandwich subdomains, forming the binding surfaces for interacting proteins. Proteins that interact with the subdomains do so via short motifs, usually found in regions of low structural complexity of the interacting proteins. So far, up to four motifs have been identified that bind to and partially compete for at least two sites on each of the appendage domains of the AP2 complex. Motifs in individual accessory proteins, their sequential arrangement into motif domains, and partial competition for binding sites on the appendage domains coordinate the formation of endocytic complexes in a temporal and spatial manner. In this work, we examine the dominant interaction sequence in amphiphysin, a synapse-enriched accessory protein, which generates membrane curvature and recruits the scission protein dynamin to the necks of coated pits, for the platform subdomain of the alpha-appendage. The motif domain of amphiphysin1 contains one copy of each of a DX(F/W) and FXDXF motif. We find that the FXDXF motif is the main determinant for the high affinity interaction with the alpha-adaptin appendage. We describe the optimal sequence of the FXDXF motif using thermodynamic and structural data and show how sequence variation controls the affinities of these motifs for the alpha-appendage.

Acetylation-dependent ADP-ribosylation by Trypanosoma brucei Sir2

Sirtuins are a highly conserved family of proteins implicated in diverse cellular processes such as gene silencing, aging, and metabolic regulation. Although many sirtuins catalyze a well characterized protein/histone deacetylation reaction, there are a number of reports that suggest protein ADP-ribosyltransferase activity. Here we explored the mechanisms of ADP-ribosylation using the Trypanosoma brucei Sir2 homologue TbSIR2rp1 as a model for sirtuins that reportedly display both activities. Steady-state kinetic analysis revealed a highly active histone deacetylase (k cat = 0.1 s(-1), with Km values of 42 microm and for NAD+ and 65 microm for acetylated substrate). A series of biochemical assays revealed that TbSIR2rp1 ADP-ribosylation of protein/histone requires an acetylated substrate. The data are consistent with two distinct ADP-ribosylation pathways that involve an acetylated substrate, NAD+ and TbSIR2rp1 as follows: 1) a noncatalytic reaction between the deacetylation product O-acetyl-ADP-ribose (or its hydrolysis product ADP-ribose) and histones, and 2) a more efficient mechanism involving interception of an ADP-ribose-acetylpeptide-enzyme intermediate by a side-chain nucleophile from bound histone. However, the sum of both ADP-ribosylation reactions was approximately 5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed.

ECRG2 Disruption Leads to Centrosome Amplification and Spindle Checkpoint Defects Contributing Chromosome Instability

Cancer cells contain an abnormal number of chromosomes (aneuploidy), which is a prevalent form of genetic instability in human cancers. Abnormal amplification of centrosomes and defects of spindle assembly checkpoint are the major causes of chromosome instability in cancer cells. Here we present biochemical evidence to suggest a role of ECRG2, a novel tumor suppressor gene, in maintaining chromosome stability. ECRG2 localized to centrosomes during interphase and kinetochores during mitosis. Further analysis revealed that ECRG2 participates in centrosome amplification in a p53-dependent manner. Depletion of ECRG2 not only destabilized p53, down-regulated p21, and increased the cyclin E/CDK2 activity, thus initiating centrosome amplification, but also abolished the ability of p53 localize to centrosomes. Overexpression of ECRG2 restored the p53-dependent suppression of centrosome duplication. Furthermore, ECRG2-depleted cells show severely disrupted spindle phenotype but fail to maintain the mitotic arrest due to minimal BUBR1 protein levels. Taken together, our results indicate that ECRG2 is important for ensuring centrosome duplication, spindle assembly checkpoint, and accurate chromosome segregation, and its depletion may contribute to chromosome instability and aneuploidy in human cancers.

General Transcription Factor IIA-γ Increases Osteoblast-specific Osteocalcin Gene Expression via Activating Transcription Factor 4 and Runt-related Transcription Factor 2

ATF4 (activating transcription factor 4) is an osteoblast-enriched transcription factor that regulates terminal osteoblast differentiation and bone formation. ATF4 knock-out mice have reduced bone mass (severe osteoporosis) throughout life. Runx2 (runt-related transcription factor 2) is a runt domain-containing transcription factor that is essential for bone formation during embryogenesis and postnatal life. In this study, we identified general transcription factor IIA gamma (TFIIA gamma) as a Runx2-interacting factor in a yeast two-hybrid screen. Immunoprecipitation assays confirmed that TFIIA gamma interacts with Runx2 in osteoblasts and when coexpressed in COS-7 cells or using purified glutathione S-transferase fusion proteins. Chromatin immunoprecipitation assay of MC3T3-E1 (clone MC-4) preosteoblast cells showed that in intact cells TFIIA gamma is recruited to the region of the osteocalcin promoter previously shown to bind Runx2 and ATF4. A small region of Runx2 (amino acids 258-286) was found to be required for TFIIA gamma binding. Although TFIIA gamma interacts with Runx2, it does not activate Runx2. Instead, TFIIA gamma binds to and activates ATF4. Furthermore, TFIIA gamma together with ATF4 and Runx2 stimulates osteocalcin promoter activity and endogenous mRNA expression. Small interfering RNA silencing of TFIIA gamma markedly reduces levels of endogenous ATF4 protein and Ocn mRNA in osteoblastic cells. Overexpression of TFIIA gamma increases levels of ATF4 protein. Finally, TFIIA gamma significantly prevents ATF4 degradation. This study shows that a general transcription factor, TFIIA gamma, facilitates osteoblast-specific gene expression through interactions with two important bone transcription factors ATF4 and Runx2.

Effect of a fungal immunomodulatory protein from Ganoderma tsugae on cell cycle and interferon-gamma production through phosphatidylinositol 3-kinase signal pathway

A fungal immunomodulatory protein (FIP-gts) was purified from Ganoderma tsugae. Recombinant FIP-gts was expressed as glutathione S-transferase fusion protein in E. coli. In this study, recombinant FIP-gts promoted cell cycle progression from G0/G1 to S phase and signaling pathways were investigated in human peripheral mononuclear cells (HPBMCs) by PI staining and flow cytometry. FIP-gts significantly induced cytokine interferon-gamma (IFN-γ) expression. Pre-treatment of cells with phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002 abolished FIP-gts, induced increase in S phase, and inhibited IFN-γ secretion. Akt, a downstream effector of PI 3-kinase was phosphorylated and activated by FIP-gts. Results show that FIP-gts is a potent activator in PBMC and its effects are mediated via cytokine regulation of PI 3-kinase.

Regulation of Platelet Dense Granule Secretion by the Ral GTPase-Exocyst Pathway

Non-hydrolyzable GTP analogues, such as guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp), induce granule secretion from permeabilized platelets in the absence of increased intracellular Ca(2+). Here, we show that the GppNHp-induced dense granule secretion from permeabilized platelets occurred concomitantly with the activation of small GTPase Ral. This secretion was inhibited by the addition of GTP-Ral-binding domain (RBD) of Sec5, which is a component of the exocyst complex known to function as a tethering factor at the plasma membrane for vesicles. We generated an antibody against Sec5-RBD, which abolished the interaction between GTP-Ral and the exocyst complex in vitro. The addition of this antibody inhibited the GppNHp-induced secretion. These data indicate that Ral mediates the GppNHp-induced dense granule secretion from permeabilized platelets through interaction with its effector, the exocyst complex. Furthermore, GppNHp enhanced the Ca(2+) sensitivity of dense granule secretion from permeabilized platelets, and this enhancement was inhibited by Sec5-RBD. In intact platelets, the association between Ral and the exocyst complex was induced by thrombin stimulation with a time course similar to that of dense granule secretion and Ral activation. Taken together, our results suggest that the Ral-exocyst pathway participates in the regulation of platelet dense granule secretion by enhancing the Ca(2+) sensitivity of the secretion.

Cyclophilin A Is Required for CXCR4-mediated Nuclear Export of Heterogeneous Nuclear Ribonucleoprotein A2, Activation and Nuclear Translocation of ERK1/2, and Chemotactic Cell Migration

The chemokine receptor CXCR4-mediated signaling cascades play an important role in cell proliferation and migration, but the underlying mechanisms by which the receptor signaling is regulated remain incompletely understood. Here, we demonstrate that CXCR4 was co-immunoprecipitated with cyclophilin A (CyPA) from the lysate of HEK293 cells stably expressing CXCR4. Although both the glutathione S-transferase-CXCR4 N- and C-terminal fusion proteins were associated with the purified CyPA, truncation of the C-terminal domain of CXCR4 robustly inhibited the receptor co-immunoprecipitation with CyPA in intact cells, thereby suggesting a critical role of the receptor C terminus in this interaction. Ligand stimulation of CXCR4 induced CyPA phosphorylation and nuclear translocation, both of which were inhibited by truncation of the C-terminal domain of CXCR4. CyPA was associated with transportin 1, and knockdown of transportin 1 by RNA interference (RNAi) blocked CXCL12-induced nuclear translocation of CyPA, thereby suggesting a transportin 1-mediated nuclear import of CyPA. CyPA formed a complex with heterogeneous nuclear ribonucleoprotein (hnRNP) A2, which underwent nuclear export in response to activation of CXCR4. Interestingly, the CXCR4-mediated nuclear export of hnRNP A2 was blocked by RNAi of CyPA. Moreover, CXCR4-evoked activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was attenuated by CyPA RNAi, by overexpression of a PPIase-deficient mutant of CyPA (CyPA-R55A), and by pretreatment of the immunosuppressive drugs, cyclosporine A and sanglifehrin A. Finally, CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 or Jurkat T cells was inhibited by CyPA RNAi or CsA treatment.

SOX9 is expressed in normal prostate basal cells and regulates androgen receptor expression in prostate cancer cells: Wang H, McKnight NC, Zhang T, Lu ML, Balk SP, Yuan X, Cancer Biology Program, Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Urology Research Laboratory, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA

SOX9 is a member of the SOX [Sry-related high-mobility group (HMG) box] family of HMG DNA-binding domain transcription factors and is required for the development and differentiation of multiple cell lineages. This report shows that basal epithelial cells express SOX9 in normal prostate, with no detectable expression in luminal epithelial cells. In contrast, SOX9 is expressed in primary prostate cancers in vivo, at a higher frequency in recurrent prostate cancer and in prostate cancer cell lines (LNCaP, CWR22, PC3, and DU145). SOX9 message and protein levels in prostate cancer cells were increased by treatment with glycogen synthase kinase 3β inhibitor ( SB415286 ), and SOX9 was reduced when β-catenin was down-regulated by small interfering RNA (siRNA), indicating that SOX9 expression in prostate cancer is regulated by Wnt/β-catenin signaling. SOX9 bound specifically to androgen receptor (AR) DNA-binding domain glutathione S-transferase fusion proteins, and this interaction was dependent on a short peptide immediately COOH-terminal to the DNA-binding domain (the C-terminal extension), which is required for interactions between steroid hormone receptors and the architectural HMG proteins. Exogenous SOX9 expressed at high nonphysiologic levels decreased AR expression and activity; however, at lower levels, SOX9 increased AR protein expression. Significantly, down-regulation of SOX9 by siRNA in prostate cancer cells reduced endogenous AR protein levels and cell growth, indicating that SOX9 contributes to AR regulation and decreased cellular proliferation. These results indicate that SOX9 in prostate basal cells supports the development and maintenance of the luminal epithelium and that a subset of prostate cancer cells may escape basal cell requirements through SOX9 expression.

Significance of Zinc in a Regulatory Protein, CCM1, Which Regulates the Carbon-Concentrating Mechanism in Chlamydomonas reinhardtii

In conditions with the poor availability of inorganic carbon (CO(2) and HCO(3) (-): Ci) for photosynthesis, aquatic photosynthetic organisms induce active Ci uptake systems that allow accumulation of Ci within the cell, the so-called carbon-concentrating mechanism (CCM). In a unicellular green alga, Chlamydomonas reinhardtii, a regulatory factor CCM1 is indispensable for the regulation of the CCM by sensing CO(2) availability. CCM1 has two putative zinc-binding domains with several conserved cysteine and histidine residues in its N-terminal region. To determine whether the domains actually bind zinc atoms, the N-terminal parts of CCM1 were expressed as glutathione S-transferase fusion proteins and subjected to atomic absorption spectrometry. It was found that 1 mol of zinc is bound to 1 mol of amino acid regions 1-71 and 72-101 of CCM1, respectively. In the case of the site-directed mutant proteins, H54Y, C77V and C80V, the zinc-binding ability was lost. Physiological analyses of the transgenic Chlamydomonas cells harboring a mutated Ccm1 gene revealed that amino acid residues such as C36, C41, H54, C77, C80, H90 and C93 were indispensable for induction of the CCM in response to Ci-limiting stress conditions. Size exclusion chromatography followed by immunoblot analyses indicated that CCM1 is present as a protein complex of approximately 290-580 kDa independent of Ci availability.

Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope

Human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein gp41 is targeted by broadly-reactive neutralizing antibodies 2F5 and 4E10, making it an attractive target for vaccine development. To better assess immunogenic properties of gp41, we generated five soluble glutathione S-transferase fusion proteins encompassing C-terminal 30, 64, 100, 142, or 172 (full-length) amino acids of gp41 ectodomain from M group consensus envelope sequence. Antibody responses in HIV-1-infected patients were evaluated using these proteins and overlapping peptides. We found (i) antibody responses against different regions of gp41 varied tremendously among individual patients, (ii) patients with stronger antibody responses against membrane-proximal external region exhibit broader and more potent neutralizing activity, and (iii) several patients mounted antibodies against epitopes that are near, or overlap with, those targeted by 2F5 or 4E10. These soluble gp41 fusion proteins could be an important source of antigens for future vaccine development efforts.

Preparation and Properties of Recombinant Rat and Human Procholecystokinin57 –95

Recombinant human progastrin(6-80) binds two ferric ions with an apparent dissociation constant of 2.2 +/- 0.1 microM [Baldwin (2004) Protein J 23:65-70]. The aims of the present study were to express fragments of recombinant procholecystokinin and to determine whether or not they bound ferric ions. Recombinant rat and human procholecystokinin(57-95) were expressed as glutathione S-transferase fusion proteins in E. coli. The fusion proteins were bound to glutathione-agarose, cleaved with thrombin, and purified by reverse phase HPLC. Recombinant procholecystokinin(57-95) did not bind to either the CCK1 or CCK2 receptor with high affinity. No change in absorption spectrum was observed on addition of ferric ions, and analysis of the quenching of tryptophan fluorescence observed in the presence of ferric ions indicated that binding to procholecystokinin(57-95) was at least 40-fold weaker than the binding of ferric ions to progastrin(6-80).

Hst3 Is Regulated by Mec1-dependent Proteolysis and Controls the S Phase Checkpoint and Sister Chromatid Cohesion by Deacetylating Histone H3 at Lysine 56

The SIR2 homologues HST3 and HST4 have been implicated in maintenance of genome integrity in the yeast Saccharomyces cerevisiae. We find that Hst3 has NAD-dependent histone deacetylase activity in vitro and that it functions during S phase to deacetylate the core domain of histone H3 at lysine 56 (H3K56). In response to genotoxic stress, Hst3 undergoes rapid Mec1-dependent phosphorylation and is targeted for ubiquitin-mediated proteolysis, thus providing a mechanism for the previously observed checkpoint-dependent accumulation of Ac-H3K56 at sites of DNA damage. Loss of Hst3-mediated regulation of H3K56 acetylation results in a defect in the S phase DNA damage checkpoint. The pathway that regulates H3K56 acetylation acts in parallel with the Rad9 pathway to transmit a DNA damage signal from Mec1 to Rad53. We also observe that loss of Hst3 function impairs sister chromatid cohesion (SCC). Both S phase checkpoint and SCC defects are phenocopied by H3K56 point mutants. Our findings demonstrate that Hst3-regulated H3K56 acetylation safeguards genome stability by controlling the S phase DNA damage response and promoting SCC.

Evidence for nifU and nifS Participation in the Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

The nifU and nifS genes encode the components of a cellular machinery dedicated to the assembly of [2Fe-2S] and [4Fe-4S] clusters required for growth under nitrogen-fixing conditions. The NifU and NifS proteins are involved in the production of active forms of the nitrogenase component proteins, NifH and NifDK. Although NifH contains a [4Fe-4S] cluster, the NifDK component carries two complex metalloclusters, the iron-molybdenum cofactor (FeMo-co) and the [8Fe-7S] P-cluster. FeMo-co, located at the active site of NifDK, is composed of 7 iron, 9 sulfur, 1 molybdenum, 1 homocitrate, and 1 unidentified light atom. To investigate whether NifUS are required for FeMo-co biosynthesis and to understand at what level(s) they might participate in this process, we analyzed the effect of nifU and nifS mutations on the formation of active NifB protein and on the accumulation of NifB-co, an isolatable intermediate of the FeMo-co biosynthetic pathway synthesized by the product of the nifB gene. The nifU and nifS genes were required to accumulate NifB-co in a nifN mutant background. This result clearly demonstrates the participation of NifUS in NifB-co synthesis and suggests a specific role of NifUS as the major provider of [Fe-S] clusters that serve as metabolic substrates for the biosynthesis of FeMo-co. Surprisingly, although nifB expression was attenuated in nifUS mutants, the assembly of the [Fe-S] clusters of NifB was compensated by other non-nif machinery for the assembly of [Fe-S] clusters, indicating that NifUS are not essential to synthesize active NifB.

A NOVEL GENE FROM BRUGIA SP. THAT ENCODES A CYTOTOXIC FATTY ACID BINDING PROTEIN ALLERGEN RECOGNIZED BY CANINE MONOCLONAL IgE AND SERUM IgE FROM INFECTED DOGS

Brugia pahangi infection of dogs is a well characterized model of human lymphatic filariasis in which sera consistently show IgG or IgE reactivity to a 35-kDa antigen. Using dog lymph node B cells, we previously established a heterohybridoma cell line producing canine monoclonal IgE (cmAb 2.39) that activates and degranulates canine mast cells, and specifically recognizes a 35-kDa B. pahangi antigen. By affinity purification and sequencing of the native protein from B. pahangi adults, a 19-amino acid sequence was obtained; the derived nucleotide sequence showed homology to a Brugia malayi and 2 related Onchocerca volvulus expressed sequence tag (EST) clones from the Filarial Genome Project database. Consensus primers amplified a 244-bp product from adult and infective larval stage cDNA libraries of B. malayi, O. volvulus, and Wuchereria bancrofti, but not from those of nonfilarial nematodes. The B. malayi EST clone only showed nucleotide sequence homology to O. volvulus EST sequences. A 684-bp region from the open reading frame was expressed as a glutathione S-transferase fusion protein designated BmAl-1. CmAb 2.39, as well as serum IgE from dogs infected with B. pahangi and canine filarial heartworm, Dirofilaria immitis, recognized BmAl-1 on enzyme-linked immunosorbent assay and Western blots. BmAl-1 showed high binding affinity for a fatty acid; however, a search for sequence homology with known fatty acid binding proteins indicated that BmAl-1 is a unique fatty acid binding protein. This 35-kDa protein seems to be highly conserved in different stages and species of filarids, and it represents a previously unknown allergen that is possibly involved in the pathogenesis of filarial disease.

Characterization of the Human Herpesvirus 6 U69 Gene Product and Identification of Its Nuclear Localization Signal

To elucidate the function of the U69 protein kinase of human herpesvirus 6 (HHV-6) in vivo, we first analyzed its subcellular localization in HHV-6-infected Molt 3 cells by using polyclonal antibodies against the U69 protein. Immunofluorescence studies showed that the U69 signal localized to the nucleus in a mesh-like pattern in both HHV-6-infected and HHV6-transfected cells. A computer program predicted two overlapping classic nuclear localization signals (NLSs) in the N-terminal region of the protein; this NLS motif is highly conserved in the N-terminal region of most of the herpesvirus protein kinases examined to date. An N-terminal deletion mutant form of the protein failed to enter the nucleus, whereas a fusion protein of green fluorescent protein (GFP) and/or glutathione S-transferase (GST) and the U69 N-terminal region was transported into the nucleus, demonstrating that the predicted N-terminal NLSs of the protein actually function as NLSs. The nuclear transport of the GST-GFP fusion protein containing the N-terminal NLS of U69 was inhibited by wheat germ agglutinin and by the Q69L Ran-GTP mutant, indicating that the U69 protein is transported into the nucleus from the cytoplasm via classic nuclear transport machinery. A cell-free import assay showed that the nuclear transport of the U69 protein was mediated by importin alpha/beta in conjunction with the small GTPase Ran. When the import assay was performed with a low concentration of each importin-alpha subtype, NPI2/importin-alpha7 elicited more efficient transport activity than did Rch1/importin-alpha1 or Qip1/importin-alpha3. These results suggest a relationship between the localization of NPI2/importin-alpha7 and the cell tropism of HHV-6.