Judy M. Callaghan

EEA1 links PI(3)K function to Rab5 regulation of endosome fusion.

GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidyl-inositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5 (ref. 8). Excess Rab5–GTP complex restores endosome fusion when PI(3)K is inhibited,. Here we identify the early-endosomal autoantigen EEA1 (refs 10–12) which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5–GTP and PI(3)K activity, and excess Rab5–GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.

The endosome fusion regulator early-endosomal autoantigen 1 (EEA1) is a dimer

EEA1, an early-endosomal protein originally identified as an autoantigen, is essential for endocytic membrane fusion. It interacts with early endosomes via binding to the membrane lipid phosphatidylinositol 3-phosphate (PtdIns3P) and the active form of the small GTPase Rab5. Most of the EEA1 sequence contains heptad repeats characteristic of proteins involved in coiled-coil protein-protein interactions. Here we have investigated the ability of EEA1 to self-interact. Crosslinking of cytosolic and recombinant EEA1 resulted in the disappearance of the 180-kDa monomer in SDS/PAGE and the strong appearance of a approximately 350-kDa crosslinked product. Glycerol gradient centrifugation experiments indicated that native EEA1 had the same hydrodynamic properties as the approximately 350-kDa crosslinked complex. Two-hybrid analysis indicated that N- and C-terminal fragments of EEA1 can interact with themselves, but not with each other, suggesting that EEA1 forms parallel coiled-coil dimers. The ability of the C-terminus of EEA1 to dimerize correlates with its ability to bind to Rab5 and early endosomes, whereas its binding to PtdIns3P is independent of dimerization. These data enable us to propose a model for the quaternary structure of EEA1.

The parietal cell autoantibodies recognized in neonatal thymectomy-induced murine gastritis are the α and β subunits of the gastric proton pump

Murine autoimmune gastritis, induced by neonatal thymectomy, bears a striking similarity in pathology to the human autoimmune disease, pernicious anemia. Autoantibodies to parietal cells are found in both murine and human diseases. Monoclonal immunoglobulin G autoantibodies, obtained from neonatally thymectomized mice, have previously been shown to recognize two groups of gastric parietal cell antigens. In the present study, it is shown that two of these monoclonal autoantibodies, designated 1H9 and 2B6, are directed against the alpha subunit and beta subunit, respectively, of the gastric hydrogen-potassium-stimulated adenosine triphosphatase (H+,K(+)-ATPase; proton pump). Monoclonal antibody 1H9 showed reactivity by immunoblotting with a 95-kilodalton component of dog gastric tubulovesicular membranes and with a fusion protein containing the hydrophilic domain of the alpha subunit of the H+,K(+)-ATPase. Monoclonal antibody 2B6 reacted by immunoblotting with the 60-90-kilodalton glycoprotein (beta subunit) of the tomato lectin-purified dog H+,K(+)-ATPase and with the 60-90-kilodalton autoantigen purified with human parietal cell autoantibodies. Monoclonal antibody 2B6 also reacted with the deglycosylated 35-kilodalton core protein of the tomato lectin-purified 60-90-kilodalton beta subunit and of the purified 60-90-kilodalton autoantigen. Parietal cell autoantibody-positive sera from 20 mice with experimentally induced gastritis showed reactivity predominantly with the alpha and/or beta subunit of the gastric H+,K(+)-ATPase. Therefore, it is concluded that the major molecules targeted by parietal cell autoantibodies from mice with neonatal thymectomy-induced murine autoimmune gastritis and from humans with pernicious anemia are identical.

Evidence that intracellular β1-2 mannan is a virulence factor in Leishmania parasites

The protozoan parasite Leishmania mexicana proliferates within macrophage phagolysosomes in the mammalian host. In this study we provide evidence that a novel class of intracellular β1-2 mannan oligosaccharides is important for parasite survival in host macrophages. Mannan (degree of polymerization 4-40) is expressed at low levels in non-pathogenic promastigote stages but constitutes 80 and 90% of the cellular carbohydrate in the two developmental stages that infect macrophages, non-dividing promastigotes, and lesion-derived amastigotes, respectively. Mannan is catabolized when parasites are starved of glucose, suggesting a reserve function, and developmental stages having low mannan levels or L. mexicana GDPMP mutants lacking all mannose molecules are highly sensitive to glucose starvation. Environmental stresses, such as mild heat shock or the heat shock protein-90 inhibitor, geldanamycin, that trigger the differentiation of promastigotes to amastigotes, result in a 10-25-fold increase in mannan levels. Developmental stages with low mannan levels or L. mexicana mutants lacking mannan do not survive heat shock and are unable to differentiate to amastigotes or infect macrophages in vitro. In contrast, a L. mexicana mutant deficient only in components of the mannose-rich surface glycocalyx differentiates normally and infects macrophages in vitro. Collectively, these data provide strong evidence that mannan accumulation is important for parasite differentiation and survival in macrophages.

Serum amyloid P colocalizes with apolipoproteins in human atheroma: functional implications.

Serum amyloid P (SAP) is a common component of human amyloid deposits and has been identified in atherosclerotic lesions. We investigated the extent of the colocalization of SAP with apolipoprotein A-I (apoA-I), apoB, apoC-II, and apoE in human coronary arteries and explored potential roles for SAP in these regions, specifically the effect of SAP on the rate of formation and macrophage recognition of amyloid fibrils composed of apoC-II. Analysis of 42 human arterial sections by immunohistochemistry and double label fluorescence microscopy demonstrated that SAP and apoA-I, apoB, apoC-II, and apoE were increased significantly in atherosclerotic lesions compared with nonatherosclerotic segments. SAP colocalized with all four apolipoproteins to a similar extent, whereas plaque macrophages were found to correlate most strongly with apoC-II and apoB. In vitro studies showed that SAP accelerated the formation of amyloid fibrils by purified apoC-II. Furthermore, SAP strongly inhibited the phagocytosis of apoC-II amyloid fibrils by primary macrophages and macrophage cell lines and blocked the resultant production of reactive oxygen species. The ability of SAP to accelerate apoC-II amyloid fibril formation and inhibit macrophage recognition of apoC-II fibrils suggests that SAP may modulate the inflammatory response to amyloid fibrils in atherosclerosis.

α and β Subunits of the Gastric H/K -ATPase Are Concordantly Targeted by Parietal Cell Autoantibodies Associated with Autoimmune Gastritis

We have previously shown that parietal cell autoantibodies predominantly react with a 60-90 kDa gastric autoantigen1,2, subsequently identified as the (3 subunit of the gastric H+/K+-ATPase (EC 3.6.1.3) (proton pump)3-5 whereas Karlsson et al6 showed that these autoantibodies primarily target the 95 kDa α subunit of the pump. In view of these discordant results, we have reassessed the reactivity of parietal cell autoantibodies with the two subunits of the gastric H+/K+-ATPase. We show here that all 26 parietal cell autoantibody-positive sera immunoblot both subunits under appropriate, but mutually exclusive, conditions. Thus, reactivity of anti- parietal cell autoantibodies with the 95 kDa α subunit is optimal when the SDS-PAGE is carried out with samples which are reduced but not boiled. Whereas reactivity with the 60-90 kDa β subunit is optimal with samples which are boiled but not reduced. Autoantibody reactivity with the β subunit is critically dependent on the presence of a full complement of N-linke...

Nontypeable Haemophilus influenzae Induces Sustained Lung Oxidative Stress and Protease Expression

Nontypeable Haemophilus influenzae (NTHi) is a prevalent bacterium found in a variety of chronic respiratory diseases. The role of this bacterium in the pathogenesis of lung inflammation is not well defined. In this study we examined the effect of NTHi on two important lung inflammatory processes 1), oxidative stress and 2), protease expression. Bronchoalveolar macrophages were obtained from 121 human subjects, blood neutrophils from 15 subjects, and human-lung fibroblast and epithelial cell lines from 16 subjects. Cells were stimulated with NTHi to measure the effect on reactive oxygen species (ROS) production and extracellular trap formation. We also measured the production of the oxidant, 3-nitrotyrosine (3-NT) in the lungs of mice infected with this bacterium. NTHi induced widespread production of 3-NT in mouse lungs. This bacterium induced significantly increased ROS production in human fibroblasts, epithelial cells, macrophages and neutrophils; with the highest levels in the phagocytic cells. In human macrophages NTHi caused a sustained, extracellular production of ROS that increased over time. The production of ROS was associated with the formation of macrophage extracellular trap-like structures which co-expressed the protease metalloproteinase-12. The formation of the macrophage extracellular trap-like structures was markedly inhibited by the addition of DNase. In this study we have demonstrated that NTHi induces lung oxidative stress with macrophage extracellular trap formation and associated protease expression. DNase inhibited the formation of extracellular traps.

PROTON AND POTASSIUM TRANSPORT BY H+/K+‐ATPases

1. H+/K+‐ATPases are members of the P‐type ATPase multigene family. The prototypical H+/K+‐ATPase is the protein that acidifies gastric luminal contents. The physiological and pharmacological significance of this pump has led to a detailed investigation of its biochemistry and molecular and cell biology.

Diagnostic Elisa for Parietal Cell Autoantibody Using Tomato Lectin-Purified Gastric H+/K+-Atpase (Proton Pump)

Circulating parietal cell autoantibodies, a useful diagnostic marker for autoimmune gastritis and pernicious anaemia, are currently routinely tested by serum immunofluorescence reactivity with frozen sections of rodent stomach. The major molecular targets of these parietal cell autoantibodies have recently been demonstrated to be the alpha- and the beta-subunits of the gastric H+/K(+)-ATPase (proton pump). We have demonstrated that tomato lectin binds specifically to the beta-subunit of the proton pump and concomittantly co-purifies the alpha-subunit. In the present study, we have exploited the latter observation for the development of a diagnostic ELISA for the detection of parietal cell autoantibodies and compared the performance of this assay with an ELISA using crude gastric membranes. The ELISAs were tested on 72 parietal cell autoantibody-positive sera, 72 parietal cell autoantibody-negative sera and 72 disease-control sera. The ELISA using lectin-purified canine proton pump was superior to that using crude canine gastric membranes in that it was about two-fold more sensitive (82% vs. 43%). With an assay sensitivity of 82% and a specificity of 90%, we propose that the ELISA using the lectin-purified proton pump is a rapid, simple, sensitive and specific diagnostic immunoassay for parietal cell autoantibodies.

Stress-induced Synthesis of Phosphatidylinositol 3-Phosphate in Mycobacteria

Phosphoinositides play key roles in regulating membrane dynamics and intracellular signaling in eukaryotic cells. However, comparable lipid-based signaling pathways have not been identified in bacteria. Here we show that Mycobacterium smegmatis and other Actinomycetes bacteria can synthesize the phosphoinositide, phosphatidylinositol 3-phosphate (PI3P). This lipid was transiently labeled with [3H]inositol. Sensitivity of the purified lipid to alkaline phosphatase, headgroup analysis by high-pressure liquid chromatography, and mass spectrometry demonstrated that it had the structure 1,2-[tuberculostearoyl, octadecenoyl]-sn-glycero 3-phosphoinositol 3-phosphate. Synthesis of PI3P was elevated by salt stress but not by exposure to high concentrations of non-ionic solutes. Synthesis of PI3P in a cell-free system was stimulated by the synthesis of CDP-diacylglycerol, a lipid substrate for phosphatidylinositol (PI) biosynthesis, suggesting that efficient cell-free PI3P synthesis is dependent on de novo PI synthesis. In vitro experiments further indicated that the rapid turnover of this lipid was mediated, at least in part, by a vanadate-sensitive phosphatase. This is the first example of de novo synthesis of PI3P in bacteria, and the transient synthesis in response to environmental stimuli suggests that some bacteria may have evolved similar lipid-mediated signaling pathways to those observed in eukaryotic cells.