Acid activation of Helicobacter pylori vacuolating cytotoxin (VacA) results in toxin internalization by eukaryotic cells.

Helicobacter pylori VacA is a secreted toxin that induces multiple structural and functional alterations in eukaryotic cells. Exposure of VacA to either acidic or alkaline pH ('activation') results in structural changes in the protein and a marked enhancement of its cell-vacuolating activity. However, the mechanism by which activation leads to increased cytotoxicity is not well understood. In this study, we analysed the binding and internalization of [125I]-VacA by HeLa cells. We detected no difference in the binding of untreated and activated [125I]-VacA to cells. Binding of acid-activated [125I]-VacA to cells at 4 degrees C was not saturable, and was only partially inhibited by excess unlabelled toxin. These results suggest that VacA binds either non-specifically or to an abundant, low-affinity receptor on HeLa cells. To study internalization of VacA, we used a protease protection assay. Analysis by SDS-PAGE and autoradiography indicated that the intact 87 kDa toxin was internalized in a time-dependent process at 37 degrees C but not at 4 degrees C. Furthermore, internalization of the intact toxin was detected only if VacA was acid or alkaline activated before being added to cells. The internalization of activated [125I]-VacA was not substantially inhibited by the presence of excess unlabelled toxin, but was blocked if cells were depleted of cellular ATP by the addition of sodium azide and 2-deoxy-D-glucose. These results indicate that acid or alkaline pH-induced structural changes in VacA are required for VacA entry into cells, and that internalization of the intact 87 kDa toxin is required for VacA cytotoxicity.

A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation.

Most Helicobacter pylori strains secrete a toxin (VacA) that causes structural and functional alterations in epithelial cells and is thought to play an important role in the pathogenesis of H. pylori-associated gastroduodenal diseases. The amino acid sequence, ultrastructural morphology, and cellular effects of VacA are unrelated to those of any other known bacterial protein toxin, and the VacA mechanism of action remains poorly understood. To analyze the functional role of a unique strongly hydrophobic region near the VacA amino terminus, we constructed an H. pylori strain that produced a mutant VacA protein (VacA-(Delta6-27)) in which this hydrophobic segment was deleted. VacA-(Delta6-27) was secreted by H. pylori, oligomerized properly, and formed two-dimensional lipid-bound crystals with structural features that were indistinguishable from those of wild-type VacA. However, VacA-(Delta6-27) formed ion-conductive channels in planar lipid bilayers significantly more slowly than did wild-type VacA, and the mutant channels were less anion-selective. Mixtures of wild-type VacA and VacA-(Delta6-27) formed membrane channels with properties intermediate between those formed by either isolated species. VacA-(Delta6-27) did not exhibit any detectable defects in binding or uptake by HeLa cells, but this mutant toxin failed to induce cell vacuolation. Moreover, when an equimolar mixture of purified VacA-(Delta6-27) and purified wild-type VacA were added simultaneously to HeLa cells, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. A dominant negative effect also was observed when HeLa cells were co-transfected with plasmids encoding wild-type and mutant toxins. We propose a model in which the dominant negative effects of VacA-(Delta6-27) result from protein-protein interactions between the mutant and wild-type VacA proteins, thereby resulting in the formation of mixed oligomers with defective functional activity.

Kinetics and mechanisms of extracellular protein release by Helicobacter pylori.

To investigate the kinetics and mechanisms of extracellular protein release by Helicobacter pylori, we analyzed the entry of metabolically radiolabeled bacterial proteins into broth culture supernatant. At early time points, vacuolating cytotoxin (VacA) constituted a major extracellular protein. Subsequently, culture supernatants accumulated many proteins that were components of intact bacterial cells. This nonselective release of proteins was associated with a decreasing turbidity of cultures and loss of bacterial viability, indicative of an autolytic process. The rates of VacA secretion and autolysis were each influenced by medium composition, and therefore these may be regulated phenomena. Extracellular release of proteins by H. pylori may be an important adaptation that facilitates the persistence of H. pylori in the human gastric mucus layer. Moreover, entry of proinflammatory proteins into the gastric mucosa may contribute to the induction of a mucosal inflammatory response.

Ligand-induced desensitization of the human CXC chemokine receptor-2 is modulated by multiple serine residues in the carboxyl-terminal domain of the receptor.

We have characterized the ligand-enhanced phosphorylation of the CXC chemokine receptor-2 (CXCR2) in a series of clonal 3ASubE cell lines expressing receptors truncated or mutated in the carboxyl-terminal domain. Truncation of CXCR2 by substitution of a stop codon for Ser-342 (342T) or Ser-331 (331T) results in total loss of melanoma growth stimulatory activity/growth-related protein (MGSA/GRO)-enhanced receptor phosphorylation, which cannot be explained based upon altered ligand binding affinity or receptor number. 3ASubE cells expressing 342T or CXCR2 with mutation of Ser-342, -346, -347, and -348 to alanine (4A) exhibit strong mobilization of Ca2+ in response to ligand (interleukin-8 or MGSA/GRO), with a recovery phase significantly slower than that of cells expressing wild type (WT) CXCR2. In contrast to the WT CXCR2, which is 93% desensitized by 20 nM ligand, the 331T, 342T, and 4A CXCR2 mutants do not undergo significant ligand-induced desensitization, and respond to a second ligand challenge by mobilizing Ca2+. The 3ASubE cells expressing CXCR2 with mutation of Ser-346, -347, and -348 to alanine, or with mutation of only one serine in this domain, continue to be phosphorylated in response to ligand and are 60-70% desensitized following the initial ligand challenge. WT CXCR2 phosphorylation and desensitization occur in <1 min, while receptor sequestration is a much later event (30-60 min). However, mutant receptors that are neither phosphorylated nor desensitized in response to ligand are <10% sequestered 60 min following ligand challenge. These data demonstrate for the first time that ligand binding to CXCR2 results in receptor phosphorylation, desensitization, and sequestration and that serine residues 342 and 346-348 participate in the desensitization and sequestration processes.

Interruption of G protein-coupling in CXCR2 does not alter ligand binding, but eliminates ligand-activation of GTPgamma35S binding, calcium mobilization, and chemotaxis.

CXCR2 is a seven-transmembrane receptor that transduces intracellular signals in response to the chemokines IL-8, MGSA/GRO, and other ELR motif-containing CXC chemokines by coupling to heterotrimeric GTP-binding proteins. In this study, we have mutated two putative G protein-coupling regions of CXCR2 and characterized the effects of these mutations on ligand-activated signal transductions: aspartic acid 89 in the second transmembrane domain and the HRAMR sequence (BBXXB motif, found in the third intracellular loop where B indicates a basic amino acid and X represents any amino acid). The Asp89 was replaced by either asparagine (D89N) or glutamic acid (D89E). For the BBXXB motif, the first two basic amino acids were mutated to two neutral isoleucines (HR-II), or alternatively, two isoleucines were inserted between alanine and methionine (II-insert). When expressed in human embryonic kidney 293 cells, the D89E mutant was localized intracellularly with no detectable cell surface expression. In contrast, D89N, HR-II, and II-insert mutants displayed cell surface expression, with Kd values and expression levels similar to that of the wild-type transfectant. The ability of the mutants to transduce signal was assessed by ligand-stimulated GTPgamma35S binding, mobilization of intracellular free Ca2+, and chemotaxis assays. Both D89N and HR-II mutants signaled similarly to a wild-type receptor in all three assays. However, the II-insert mutant exhibited a loss of ligand-stimulated GTPgamma35S binding, calcium mobilization, and chemotaxis. Unexpectedly, this receptor underwent ligand-induced sequestration comparable to wild-type CXCR2. These data indicate that Asp89 and the basic amino acids in the third intracellular domain do not play essential roles in ligand-induced signal transduction through CXCR2. However, proper secondary structure and orientation of the third intracellular loop of CXCR2 are essential for ligand-mediated signal transduction but not for receptor sequestration.

Melanoma growth stimulatory activity signaling through the class II interleukin-8 receptor enhances the tyrosine phosphorylation of Crk-associated substrate, p130, and a 70-kilodalton protein.

Binding of the CXC chemokine, melanoma growth stimulatory activity (MGSA), to the class II IL-8 receptor on cells which overexpress this G-protein coupled receptor results in enhanced phosphorylation on serine residues. In experiments described herein, it is demonstrated that MGSA also enhances the tyrosine phosphorylation of two endogenously tyrosine phosphorylated proteins approximately 130 and 70 kDa in size. MGSA treatment (5 nM) of the clonally selected, stably transfected placental cell line, 3ASubE P-3, which overexpresses the class II IL-8 receptor, results in the maximal tyrosine phosphorylation of the 130 kDa protein before 2 min. This enhanced phosphorylation of the 130 kDa protein returns to basal level after a 5 min treatment. Based upon cell fractionation studies, the 130 kDa protein is concentrated in the membrane fraction of the cells. The 70 kDa protein which also shows tyrosine phosphorylation is predominantly cytosolic. The identity of the 130 kDa tyrosine phosphorylated protein was determined by immunoprecipitation and Western blot analyses. In these experiments, the 130 kDa tyrosine phosphorylated protein was shown to immunoprecipitate with antibody to the cas antigen (crk-associated substrate) and with antibody to the p130 tyrosine phosphorylated protein described as undergoing tyrosine phosphorylation in src transformed cells. The data suggest that MGSA binding to the class II IL-8 receptor is associated with tyrosine phosphorylation of p130/cas. The data also suggest that p130 and the cas antigen are the same protein.

Activation of protein kinase C enhances the phosphorylation of the type B interleukin-8 receptor and stimulates its degradation in non-hematopoietic cells.

We have previously characterized the stably transfected, clonally selected human placental cell line, 3ASubE P-3, which overexpresses the type B interleukin-8 receptor (IL-8RB) and responds to the chemokine melanoma growth stimulatory activity (MGSA) with enhanced phosphorylation of this receptor. In work described here, we demonstrate that the MGSA-enhanced phosphorylation of this receptor is mediated via a process involving pertussis toxin-sensitive G proteins. Furthermore, treatment of the 3ASubE P-3 cells with either 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol (diC8), two different activators of protein kinase C (PKC), results in a concentration-dependent increase in the phosphorylation of the IL-8RB. Inhibition of PKC, by treatment with staurosporin (50 nM for 2 h), or down-regulation of PKC, by prolonged treatment with TPA (400 nM for 40 h) suppresses the TPA-enhanced receptor phosphorylation, but has no effect on the MGSA-enhanced receptor phosphorylation. These data suggest that the isoforms of PKC that are sensitive to these manipulations may not play a role in mediating the MGSA-enhanced phosphorylation of the IL-8RB. TPA treatment also results in a time-dependent decrease in 125I-MGSA binding to the 3ASubE P-3 cells. A 30-min treatment with 400 nM TPA results in approximately a 50% decrease in binding, whereas a 2-h treatment essentially eliminates specific binding of 125I-MGSA to these cells. The TPA-induced decrease in 125I-MGSA binding is accompanied by enhanced degradation of the IL-8RB, as indicated by Western blot analysis and pulse-chase experiments, suggesting a potential role for PKC as a negative regulator of the IL-8RB. MGSA treatment (50 nM for 2 h) also stimulates receptor degradation in the 3ASubE P-3 cells, indicating that this receptor is down-regulated in response to prolonged exposure to its ligand. In similar studies conducted on the promonocytic cell line, U937, MGSA treatment of the U937 cells resulted in receptor phosphorylation, whereas PKC activation failed to significantly modulate the phosphorylation state of the IL-8RB. Treatment of the U937 cells with MGSA, TPA, or diC8 resulted in a loss of receptor protein present in these cell types. These data imply that MGSA signaling through the IL-8RB is similar in both the non-hematopoietic and hematopoietic cell types, whereas activation of PKC by TPA or diC8 elicits different responses in these two distinct cell types.

Melanoma growth stimulatory activity enhances the phosphorylation of the class II interleukin-8 receptor in non-hematopoietic cells.

The class II IL-8 receptor (IL-8R) binds both melanoma growth stimulatory activity (MGSA) and IL-8 with high affinity. Reverse transcriptase polymerase chain reaction demonstrates that the class II IL-8R mRNA, which has previously been detected only in cells of hematopoietic lineage, is also expressed in non-hematopoietic cell types shown to respond to MGSA or IL-8. To study the signaling mechanism by MGSA through the class II IL-8R in non-hematopoietic cells, this receptor was overexpressed in the 3ASubE human placental and the 293 human kidney cell lines. Membrane preparations of the class II IL-8R expressing 3ASubE transfectants exhibited a 2.3 +/- 0.2-fold increase in GTP gamma 35S binding, which was sensitive to pertussis toxin, in response to MGSA treatment (0.2 microM). This MGSA response was not observed in cells transfected with the parental expression vector. In vivo phosphorylation studies demonstrated that the class II IL-8R was basally phosphorylated in the untreated transfectants, and MGSA (5 nM) treatment markedly enhanced the phosphorylation of this receptor. The MGSA-induced receptor phosphorylation was both time and concentration dependent and could be mimicked by treatment with the calcium ionophore A23187. Phosphoamino acid analysis indicated that the MGSA-induced receptor phosphorylation was on serine residue(s), suggesting that a serine kinase is activated in response to MGSA binding to the class II IL-8R in non-hematopoietic cells.

Inhibition and labeling of sodium, potassium ATPase by the dialdehyde derivative of ATP.

Canine renal Na,K-ATPase was treated with ATP dialdehyde, "oxATP" (20 microM), as described by G. Ponzio, B. Rossi, and M. Lazdunski (1983, J. Biol. Chem. 258, 8201-8205). In this system, a by-product, formaldehyde, was the inactivator. We modified the system to minimize such inhibition and to speed up the reaction. oxATP itself inactivated the enzyme at a rate that was slow at first and later speeded up. We fitted a precursor-product model to the data. Labeling with [3H]oxATP indicated about three sites per alpha beta protomer at complete inactivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled enzyme showed radioactivity in many components, in the alpha and beta subunits and in small molecules at the tracker dye region. ATP (20 mM) prevented all labeling and inactivation. Ponzio et al. concluded that oxATP labels covalently an ATP binding site. Our experiments did not support this conclusion. Ouabain did not affect labeling. Sodium stimulated both inhibition and labeling more than potassium did, indicating a high-affinity ATP binding site, if any. But nucleotide specificity for preventing or producing inhibition did not correspond to nucleotide specificity for binding of ATP to the native enzyme. Blocking the ATP binding center with fluorescein isothiocyanate or fluorosulfonyl benzoyl adenosine had no effect on [3H]oxATP labeling. ATP also prevented [3H]oxATP labeling of bovine serum albumin or of integral-membrane proteins.

Reconstitution of the D-glucose transporter of bovine thymocyte plasma membrane: partial purification of transport activity by chromatography on agarose lentil lectin and agarose ethanethiol.

The D-glucose transporter of bovine-thymocyte plasma membrane was partially purified using several procedures in sequence. Dimethylmaleic anhydride extraction removed extrinsic membrane proteins (approximately 50% of the total membrane protein) after which sodium cholate solubilized 40% of the residual protein. Reconstitution of solubilized proteins into phospholipid liposomes indicated a 2.5-fold increase in sugar transport specific activity relative to membrane solubilized without dimethylmaleic anhydride extraction. Detergent removal by gel filtration on G-50 Sephadex resulted in reaggregation of intrinsic membrane proteins. Ultracentrifugation of the reaggregated proteins generated a particulate fraction (pellet 1) which contained about 50% of the total D-glucose transport activity of the preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pellet 1 demonstrated removal of a major band at 68,000 daltons and two minor bands not removed by dimethylmaleic anhydride. The 68,000-dalton protein was not removed by any other method tested. Chromatography of resolubilized pellet 1 on a tandem-bed column of agarose ethanethiol and agarose lentil lectin resulted in a 6-fold increase in transport specific activity of nonabsorbed proteins relative to pellet 1. Approximately 15% of the protein (80-90% of the transport activity) applied to the tandem-bed column was recovered in the nonabsorbed fraction. Sodium dodecyl sulfate-gel electrophoresis of proteins in the nonabsorbed fraction showed apparent enrichment of a diffuse zone at 52,000-45,000 daltons. The overall increase in specific activity of the partially purified preparation was about 12-fold relative to unpurified solubilized proteins.

Effects of thiol reagents on glucose transport in thymocytes.

Rat thymocytes were incubated with 3-O-[14C]methyl D-glucose for 1 h and diluted 100X and the efflux was followed for 1 h. In control cells, about half of the methyl glucose efflux was rapid (t 1/2 approximately 3 min) and about half was slow (t 1/2 congruent to 40 min). The fast and slow compartments represent active and quiescent cells, respectively. A physiological mixture of amino acids present during the loading period dramatically increased the amount of methyl glucose exiting rapidly at the expense of that exiting slowly. Further studies revealed that cysteine was entirely responsible for the action. Cysteine (0.06 mM), glutathione (0.5 mM) and dithiothreitol (0.02 mM) added after completion of fast-phase exit, stimulated subsequent exit about 3-4-fold with no detectable delay. This action was inhibited by catalase and mimicked by 0.04 mM H2O2 and by 0.03 mM N-ethylmaleimide. It did not require extracellular or intracellular Ca2+. These effects are analogous to those seen in adipocytes, implicating sulfhydryl groups in glucose transport regulation [12]. Sulfhydryl oxidation may be a late event in the chain of events leading to glucose transport stimulation by physiological agents.