The gastric H+/K+-transporting adenosine triphosphatase ( H + / K + ATPase) (proton pump) consist... more The gastric H+/K+-transporting adenosine triphosphatase ( H + / K + ATPase) (proton pump) consists of a catalytic a-subunit and a recently proposed 60 -90-kDa glycoprotein p-subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60 -90-kDa glycoprotein.
Early endosomes are cellular compartments receiving endocytosed material and sorting them for ves... more Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved ...
... 39, 520-524. Lipids 14, 505-510. 3 57-358. 253, 6281-6288. Purification and Partial Amino Aci... more ... 39, 520-524. Lipids 14, 505-510. 3 57-358. 253, 6281-6288. Purification and Partial Amino Acid Sequence of Papain-Solubilized Class I1 Transplantation Antigens? Klas Wiman,*Lena Claesson, Lars Rask, Lena Traghrdh, and Per A. Peterson ...
EEA1, an early-endosomal protein originally identified as an autoantigen, is essential for endocy... more 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 " 350-Abbreviations used : PI 3-kinase, phosphatidylinositol 3-kinase ; EEA1, early-endsomal autoantigen 1 ; PtdIns3P, phosphatidylinositol 3-phosphate ;
Serum amyloid P (SAP) is a common component of human amyloid deposits and has been identified in ... more 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. Serum amyloid P colocalizes with apolipoproteins in human atheroma: functional implications. J. Lipid Res.
The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia ar... more The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia are largely uncharacterized. We have identified a new family of small myristoylated proteins (SMPs) that are present in Leishmania spp and related trypanosomatid parasites. One of these proteins, termed SMP-1, is targeted to the Leishmania flagellum. SMP-1 is myristoylated and palmitoylated in vivo, and mutation of Gly-2 and Cys-3 residues showed that both fatty acids are required for flagellar localization. SMP-1 is associated with detergent-resistant membranes based on its recovery in the buoyant fraction after Triton X-100 extraction and sucrose density centrifugation and coextraction with the major surface glycolipids in Triton X-114. However, the flagellar localization of SMP-1 was not affected when sterol biosynthesis and the properties of detergent-resistant membranes were perturbed with ketoconazole. Remarkably, treatment of Leishmania with ketoconazole and myriocin (an inhibitor of sphingolipid biosynthesis) also had no affect on SMP-1 localization, despite causing the massive distension of the flagellum membrane and the partial or complete loss of internal axoneme and paraflagellar rod structures, respectively. These data suggest that flagellar membrane targeting of SMP-1 is not dependent on axonemal structures and that alterations in flagellar membrane lipid composition disrupt axoneme extension.
Purification and partial amino acid sequence of plantaricin 1.25a a a and 1.25b b b, two bacterio... more Purification and partial amino acid sequence of plantaricin 1.25a a a and 1.25b b b, two bacteriocins produced by Lactobacillus plantarum TMW1.25 A . R EM I GE R, V .G .H . EI JS I NK , M . A. EH R MA NN , K. SL E TT EN , I. F. N ES AN D R. F. V OG EL . 1999.
FYVE domain proteins play key roles in regulating membrane traffic in eukaryotic cells. The FYVE ... more FYVE domain proteins play key roles in regulating membrane traffic in eukaryotic cells. The FYVE domain displays a remarkable specificity for the head group of the target lipid, phosphatidylinositol 3-phosphate (PtdIns[3]P). We have identified five putative FYVE domain proteins in the genome of the protozoan parasite Leishmania major, three of which are predicted to contain a functional PtdIns(3)Pbinding site. The FYVE domain of one of these proteins, LmFYVE-1, bound PtdIns(3)P in liposomebinding assays and targeted GFP to acidified late endosomes/lysosomes in mammalian cells. The highresolution solution structure of its N-terminal FYVE domain (LmFYVE-1[1-79]) was solved by nuclear magnetic resonance. Functionally significant clusters of residues of the LmFYVE-1 domain involved in PtdIns(3)P binding and dependence on low pH for tight binding were identified. This structure is the first trypanosomatid membrane trafficking protein to be determined and has been refined to high precision and accuracy using residual dipolar couplings.
Phosphoinositides play key roles in regulating membrane dynamics and intracellular signaling in e... more 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 [(3)H]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.
Circulating parietal cell autoantibodies, a useful diagnostic marker for autoimmune gastritis and... more 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.
The gastric mucosal parietal cells and cells of the renal collecting duct both possess H(+)-K(+)-... more The gastric mucosal parietal cells and cells of the renal collecting duct both possess H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) activities. In the stomach, the H(+)-K(+)-ATPase (EC 3.6.1.3) is responsible for acidification of luminal contents. The kidney H(+)-K(+)-ATPase protein(s) contribute to potassium reabsorption and secretion of hydrogen ions to maintain potassium and acid-base homeostasis. The stomach H(+)-K(+)-ATPase is well defined and consists of an alpha-catalytic subunit of apparent molecular mass of 95 kDa and a highly glycosylated beta-subunit of 60-90 kDa. The molecular identity of the protein that mediates the H(+)-K(+)-ATPase activity in the kidney has been addressed in this paper. A combination of RNA hybridizations, polymerase chain reaction analysis of kidney RNA, and sequence analysis of cDNAs indicated that gastric H(+)-K(+)-ATPase beta-subunit mRNA is present in kidney. Immunoblotting with antibodies specific for the gastric H(+)-K(+)-ATPase beta-subunit detected proteins, which, after deglycosylation, had the same molecular mass as the gastric beta-subunit in membrane protein preparations from rabbit, pig, rat, and mouse kidneys. Furthermore, we have used transgenic mice to demonstrate that the gastric H(+)-K(+)-ATPase beta-subunit gene contains cis-acting regulatory sequences that are active in both gastric parietal cells and the renal collecting ducts. Overall, these data indicate that the gastric H(+)-K(+)-ATPase beta-subunit is found in the kidney and probably associates with the gastric H(+)-K(+)-ATPase alpha-subunit and/or other P-type ATPase alpha-subunits, thus contributing to acid-base and potassium homeostasis.
Page 1. Clinical and Experimental Pharmacology and Physiology (1995) 22,952-960 BRIEF REVIEW: PRO... more Page 1. Clinical and Experimental Pharmacology and Physiology (1995) 22,952-960 BRIEF REVIEW: PROCEEDINGS OF 'MOLECULAR BIOLOGY IN THE SERVICE OF PHYSIOLOGY, PHARMACOLOGY AND ENDOCRINOLOGY' ...
We have previously shown that parietal cell autoantibodies predominantly react with a 60-90 kDa g... more We have previously shown that parietal cell autoantibodies predominantly react with a 60-90 kDa gastric autoantigen, subsequently identified as the beta subunit of the gastric H+/K(+)-ATPase (EC 3.6.1.3) (proton pump) whereas Karlsson et al showed that these autoantibodies primarily target the 95 kDa alpha 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 alpha 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 beta subunit is optimal with samples which are boiled but not reduced. Autoantibody reactivity with the beta subunit is critically dependent on the presence of a full complement of N-linked glycans since partially deglycosylated protein, and recombinant beta subunit expressed in COS cells, bearing high mannose N-glycans, failed to bind to the autoantibody. These studies also suggest that B cell auto-epitopes are located on the lumenal domain of the beta subunit. Reactivity of parietal cell autoantibodies with a bacterial fusion protein incorporating the catalytic cytoplasmic domain of the alpha subunit suggests the presence of auto-epitopes in this region of the molecule.
We have previously shown that tomato lectin binds specifically to the 60-90 kDa membrane glycopro... more We have previously shown that tomato lectin binds specifically to the 60-90 kDa membrane glycoprotein of parietal cell tubulovesicles, the fl-subunit of the gastric H+/K+-ATPase (proton pump) ) J. Cell Sci. 95, 563-(1990 Proc. Natl. Acad. Sci. U.S.A. 87, 6418-6422]. Here we have exploited this interaction for the development of a rapid single-step chromatography procedure for the purification of an active pig gastric proton pump complex. Initially, H+/K+-ATPase-enriched membranes, prepared from pig gastric microsomes by density-gradient centrifugation, were extracted in 1 % Triton X-100 and passed through a 1 ml tomato lectin-Sepharose 4B column. The bound material, eluted with 20 mM-chitotriose, showed a major band with an apparent molecular mass of 95 kDa, and a faint broad band of 60-90 kDa, by SDS/PAGE. N-Glycanase treatment of the bound material resulted in the appearance of a 35 kDa band, the size of the protein core of the 60-90 kDa glycoprotein f-subunit. The two components were identified as the 95 kDa a-subunit and the 60-90 kDa fl-subunit of the gastric H+/K+-ATPase, by immunoreactivity with monospecific antibodies, and by tryptic peptide sequences of the tomato-lectin-bound material. The fl-subunit was present in approximately equimolar amounts to the catalytic a-subunit.
The gastric H,K-ATPase (EC 3.6.1.3) is responsible for acid secretion into the stomach and is com... more The gastric H,K-ATPase (EC 3.6.1.3) is responsible for acid secretion into the stomach and is composed of two subunits, alpha and beta. The structure of the gene encoding the mouse beta subunit and the expression of both subunits during ontogeny are reported. The gene spans approximately 12 kilobase pairs and contains 7 exons. The positions at which introns interrupt the coding regions of the mouse H,K-ATPase beta subunit and mouse Na,K-ATPase (EC 3.6.1.37) beta 2 subunit genes are identical. The alternative beta subunit isoform of the Na,K-ATPase, beta 1, has a similar but not identical gene structure. Primer extension and S1 nuclease analysis of RNA isolated from mouse stomachs aged between 2 and 25 days indicated that major transcription-initiation sites are between 22 and 25 base pairs 5' of the translation initiation site at all ages. The expression of the H,K-ATPase alpha and beta subunit genes during ontogeny (day 1-40) was found to be co-ordinated. Protein levels of both the ATPase alpha and beta subunits were very low until day 15 and then increased to adult levels by day 30. In any mucosal cell throughout ontogeny, expression of the beta subunit gene invariably coincided with the expression of the alpha subunit gene. Cells detected by anti-H,K-ATPase beta subunit antibodies in sections from 10- and 30-day-old mice all had typical morphology of parietal cells and were arranged in glandular structures. Co-ordinate expression of the two subunit genes suggest that the regulatory mechanisms will be similar and that the beta subunit may be required for localization and function of the catalytic alpha subunit.
The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylino... more The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein-and hemagglutinin-tagged forms of dolicholphosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras are found in the same subcompartment of the endoplasmic reticulum (ER) as endogenous DPMS but are degraded as logarithmically growing promastigotes reach stationary phase, coincident with the down-regulation of endogenous DPMS activity and GPI biosynthesis in these cells. We provide evidence that these chimeras are constitutively transported to and degraded in a novel multivesicular tubule (MVT) lysosome. This organelle is a terminal lysosome, which is labeled with the endocytic marker FM 4-64, contains lysosomal cysteine and serine proteases and is disrupted by lysomorphotropic agents. Electron microscopy and subcellular fractionation studies suggest that the DPMS chimeras are transported from the ER to the lumen of the MVT via the Golgi apparatus and a population of 200-nm multivesicular bodies. In contrast, soluble ER proteins are not detectably transported to the MVT lysosome in either log or stationary phase promastigotes. Finally, the increased degradation of the DPMS chimeras in stationary phase promastigotes coincides with an increase in the lytic capacity of the MVT lysosome and changes in the morphology of this organelle. We conclude that lysosomal degradation of DPMS may be important in regulating the cellular levels of this enzyme and the stage-dependent biosynthesis of the major surface glycolipids of these parasites. ‡ These authors contributed equally to this work. § Corresponding
The 60-to 90-kDa parietal cell autoantigen associated with autoimmune gastritis is a ,0 subunit o... more The 60-to 90-kDa parietal cell autoantigen associated with autoimmune gastritis is a ,0 subunit of the gastric ABSTRACT Autoantibodies in the sera of patients with pernicious anemia recognize, in addition to the a subunit of the gastric H+/K+-ATPase, an abundant gastric microsomal glycoprotein of apparent Mr 60,000-90,000. Herein we have colocalized the glycoprotein and the a subunit of the gastric H+/K+-ATPase to the tubulovesicular membranes of the parietal cell by immunogold electron microscopy. Moreover, the glycoprotein and the a subunit were coimmunoprecipitated, and copurified by immunoaffinity chromatography, with an anti-glycoprotein monoclonal antibody. The pig glycoprotein was purified by chromatography on tomato lectin-Sepharose, and five tryptic peptides from the purified glycoprotein were partially sequenced. The complete amino acid sequence, deduced from the nucleotide sequence of overlapping cDNA clones, showed 33% similarity to the sequence of the (B subunit of the pig kidney Na+/K+-ATPase. We therefore propose that the 60to 90-kDa glycoprotein autoantigen is the (8 subunit of the gastric H+/K+-ATPase and that the a and 13 subunits of the proton pump are major targets for autoimmunization in autoimmune gastritis.
The gastric H+/K+-transporting adenosine triphosphatase ( H + / K + ATPase) (proton pump) consist... more The gastric H+/K+-transporting adenosine triphosphatase ( H + / K + ATPase) (proton pump) consists of a catalytic a-subunit and a recently proposed 60 -90-kDa glycoprotein p-subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60 -90-kDa glycoprotein.
Early endosomes are cellular compartments receiving endocytosed material and sorting them for ves... more Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved ...
... 39, 520-524. Lipids 14, 505-510. 3 57-358. 253, 6281-6288. Purification and Partial Amino Aci... more ... 39, 520-524. Lipids 14, 505-510. 3 57-358. 253, 6281-6288. Purification and Partial Amino Acid Sequence of Papain-Solubilized Class I1 Transplantation Antigens? Klas Wiman,*Lena Claesson, Lars Rask, Lena Traghrdh, and Per A. Peterson ...
EEA1, an early-endosomal protein originally identified as an autoantigen, is essential for endocy... more 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 " 350-Abbreviations used : PI 3-kinase, phosphatidylinositol 3-kinase ; EEA1, early-endsomal autoantigen 1 ; PtdIns3P, phosphatidylinositol 3-phosphate ;
Serum amyloid P (SAP) is a common component of human amyloid deposits and has been identified in ... more 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. Serum amyloid P colocalizes with apolipoproteins in human atheroma: functional implications. J. Lipid Res.
The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia ar... more The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia are largely uncharacterized. We have identified a new family of small myristoylated proteins (SMPs) that are present in Leishmania spp and related trypanosomatid parasites. One of these proteins, termed SMP-1, is targeted to the Leishmania flagellum. SMP-1 is myristoylated and palmitoylated in vivo, and mutation of Gly-2 and Cys-3 residues showed that both fatty acids are required for flagellar localization. SMP-1 is associated with detergent-resistant membranes based on its recovery in the buoyant fraction after Triton X-100 extraction and sucrose density centrifugation and coextraction with the major surface glycolipids in Triton X-114. However, the flagellar localization of SMP-1 was not affected when sterol biosynthesis and the properties of detergent-resistant membranes were perturbed with ketoconazole. Remarkably, treatment of Leishmania with ketoconazole and myriocin (an inhibitor of sphingolipid biosynthesis) also had no affect on SMP-1 localization, despite causing the massive distension of the flagellum membrane and the partial or complete loss of internal axoneme and paraflagellar rod structures, respectively. These data suggest that flagellar membrane targeting of SMP-1 is not dependent on axonemal structures and that alterations in flagellar membrane lipid composition disrupt axoneme extension.
Purification and partial amino acid sequence of plantaricin 1.25a a a and 1.25b b b, two bacterio... more Purification and partial amino acid sequence of plantaricin 1.25a a a and 1.25b b b, two bacteriocins produced by Lactobacillus plantarum TMW1.25 A . R EM I GE R, V .G .H . EI JS I NK , M . A. EH R MA NN , K. SL E TT EN , I. F. N ES AN D R. F. V OG EL . 1999.
FYVE domain proteins play key roles in regulating membrane traffic in eukaryotic cells. The FYVE ... more FYVE domain proteins play key roles in regulating membrane traffic in eukaryotic cells. The FYVE domain displays a remarkable specificity for the head group of the target lipid, phosphatidylinositol 3-phosphate (PtdIns[3]P). We have identified five putative FYVE domain proteins in the genome of the protozoan parasite Leishmania major, three of which are predicted to contain a functional PtdIns(3)Pbinding site. The FYVE domain of one of these proteins, LmFYVE-1, bound PtdIns(3)P in liposomebinding assays and targeted GFP to acidified late endosomes/lysosomes in mammalian cells. The highresolution solution structure of its N-terminal FYVE domain (LmFYVE-1[1-79]) was solved by nuclear magnetic resonance. Functionally significant clusters of residues of the LmFYVE-1 domain involved in PtdIns(3)P binding and dependence on low pH for tight binding were identified. This structure is the first trypanosomatid membrane trafficking protein to be determined and has been refined to high precision and accuracy using residual dipolar couplings.
Phosphoinositides play key roles in regulating membrane dynamics and intracellular signaling in e... more 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 [(3)H]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.
Circulating parietal cell autoantibodies, a useful diagnostic marker for autoimmune gastritis and... more 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.
The gastric mucosal parietal cells and cells of the renal collecting duct both possess H(+)-K(+)-... more The gastric mucosal parietal cells and cells of the renal collecting duct both possess H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) activities. In the stomach, the H(+)-K(+)-ATPase (EC 3.6.1.3) is responsible for acidification of luminal contents. The kidney H(+)-K(+)-ATPase protein(s) contribute to potassium reabsorption and secretion of hydrogen ions to maintain potassium and acid-base homeostasis. The stomach H(+)-K(+)-ATPase is well defined and consists of an alpha-catalytic subunit of apparent molecular mass of 95 kDa and a highly glycosylated beta-subunit of 60-90 kDa. The molecular identity of the protein that mediates the H(+)-K(+)-ATPase activity in the kidney has been addressed in this paper. A combination of RNA hybridizations, polymerase chain reaction analysis of kidney RNA, and sequence analysis of cDNAs indicated that gastric H(+)-K(+)-ATPase beta-subunit mRNA is present in kidney. Immunoblotting with antibodies specific for the gastric H(+)-K(+)-ATPase beta-subunit detected proteins, which, after deglycosylation, had the same molecular mass as the gastric beta-subunit in membrane protein preparations from rabbit, pig, rat, and mouse kidneys. Furthermore, we have used transgenic mice to demonstrate that the gastric H(+)-K(+)-ATPase beta-subunit gene contains cis-acting regulatory sequences that are active in both gastric parietal cells and the renal collecting ducts. Overall, these data indicate that the gastric H(+)-K(+)-ATPase beta-subunit is found in the kidney and probably associates with the gastric H(+)-K(+)-ATPase alpha-subunit and/or other P-type ATPase alpha-subunits, thus contributing to acid-base and potassium homeostasis.
Page 1. Clinical and Experimental Pharmacology and Physiology (1995) 22,952-960 BRIEF REVIEW: PRO... more Page 1. Clinical and Experimental Pharmacology and Physiology (1995) 22,952-960 BRIEF REVIEW: PROCEEDINGS OF 'MOLECULAR BIOLOGY IN THE SERVICE OF PHYSIOLOGY, PHARMACOLOGY AND ENDOCRINOLOGY' ...
We have previously shown that parietal cell autoantibodies predominantly react with a 60-90 kDa g... more We have previously shown that parietal cell autoantibodies predominantly react with a 60-90 kDa gastric autoantigen, subsequently identified as the beta subunit of the gastric H+/K(+)-ATPase (EC 3.6.1.3) (proton pump) whereas Karlsson et al showed that these autoantibodies primarily target the 95 kDa alpha 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 alpha 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 beta subunit is optimal with samples which are boiled but not reduced. Autoantibody reactivity with the beta subunit is critically dependent on the presence of a full complement of N-linked glycans since partially deglycosylated protein, and recombinant beta subunit expressed in COS cells, bearing high mannose N-glycans, failed to bind to the autoantibody. These studies also suggest that B cell auto-epitopes are located on the lumenal domain of the beta subunit. Reactivity of parietal cell autoantibodies with a bacterial fusion protein incorporating the catalytic cytoplasmic domain of the alpha subunit suggests the presence of auto-epitopes in this region of the molecule.
We have previously shown that tomato lectin binds specifically to the 60-90 kDa membrane glycopro... more We have previously shown that tomato lectin binds specifically to the 60-90 kDa membrane glycoprotein of parietal cell tubulovesicles, the fl-subunit of the gastric H+/K+-ATPase (proton pump) ) J. Cell Sci. 95, 563-(1990 Proc. Natl. Acad. Sci. U.S.A. 87, 6418-6422]. Here we have exploited this interaction for the development of a rapid single-step chromatography procedure for the purification of an active pig gastric proton pump complex. Initially, H+/K+-ATPase-enriched membranes, prepared from pig gastric microsomes by density-gradient centrifugation, were extracted in 1 % Triton X-100 and passed through a 1 ml tomato lectin-Sepharose 4B column. The bound material, eluted with 20 mM-chitotriose, showed a major band with an apparent molecular mass of 95 kDa, and a faint broad band of 60-90 kDa, by SDS/PAGE. N-Glycanase treatment of the bound material resulted in the appearance of a 35 kDa band, the size of the protein core of the 60-90 kDa glycoprotein f-subunit. The two components were identified as the 95 kDa a-subunit and the 60-90 kDa fl-subunit of the gastric H+/K+-ATPase, by immunoreactivity with monospecific antibodies, and by tryptic peptide sequences of the tomato-lectin-bound material. The fl-subunit was present in approximately equimolar amounts to the catalytic a-subunit.
The gastric H,K-ATPase (EC 3.6.1.3) is responsible for acid secretion into the stomach and is com... more The gastric H,K-ATPase (EC 3.6.1.3) is responsible for acid secretion into the stomach and is composed of two subunits, alpha and beta. The structure of the gene encoding the mouse beta subunit and the expression of both subunits during ontogeny are reported. The gene spans approximately 12 kilobase pairs and contains 7 exons. The positions at which introns interrupt the coding regions of the mouse H,K-ATPase beta subunit and mouse Na,K-ATPase (EC 3.6.1.37) beta 2 subunit genes are identical. The alternative beta subunit isoform of the Na,K-ATPase, beta 1, has a similar but not identical gene structure. Primer extension and S1 nuclease analysis of RNA isolated from mouse stomachs aged between 2 and 25 days indicated that major transcription-initiation sites are between 22 and 25 base pairs 5' of the translation initiation site at all ages. The expression of the H,K-ATPase alpha and beta subunit genes during ontogeny (day 1-40) was found to be co-ordinated. Protein levels of both the ATPase alpha and beta subunits were very low until day 15 and then increased to adult levels by day 30. In any mucosal cell throughout ontogeny, expression of the beta subunit gene invariably coincided with the expression of the alpha subunit gene. Cells detected by anti-H,K-ATPase beta subunit antibodies in sections from 10- and 30-day-old mice all had typical morphology of parietal cells and were arranged in glandular structures. Co-ordinate expression of the two subunit genes suggest that the regulatory mechanisms will be similar and that the beta subunit may be required for localization and function of the catalytic alpha subunit.
The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylino... more The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein-and hemagglutinin-tagged forms of dolicholphosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras are found in the same subcompartment of the endoplasmic reticulum (ER) as endogenous DPMS but are degraded as logarithmically growing promastigotes reach stationary phase, coincident with the down-regulation of endogenous DPMS activity and GPI biosynthesis in these cells. We provide evidence that these chimeras are constitutively transported to and degraded in a novel multivesicular tubule (MVT) lysosome. This organelle is a terminal lysosome, which is labeled with the endocytic marker FM 4-64, contains lysosomal cysteine and serine proteases and is disrupted by lysomorphotropic agents. Electron microscopy and subcellular fractionation studies suggest that the DPMS chimeras are transported from the ER to the lumen of the MVT via the Golgi apparatus and a population of 200-nm multivesicular bodies. In contrast, soluble ER proteins are not detectably transported to the MVT lysosome in either log or stationary phase promastigotes. Finally, the increased degradation of the DPMS chimeras in stationary phase promastigotes coincides with an increase in the lytic capacity of the MVT lysosome and changes in the morphology of this organelle. We conclude that lysosomal degradation of DPMS may be important in regulating the cellular levels of this enzyme and the stage-dependent biosynthesis of the major surface glycolipids of these parasites. ‡ These authors contributed equally to this work. § Corresponding
The 60-to 90-kDa parietal cell autoantigen associated with autoimmune gastritis is a ,0 subunit o... more The 60-to 90-kDa parietal cell autoantigen associated with autoimmune gastritis is a ,0 subunit of the gastric ABSTRACT Autoantibodies in the sera of patients with pernicious anemia recognize, in addition to the a subunit of the gastric H+/K+-ATPase, an abundant gastric microsomal glycoprotein of apparent Mr 60,000-90,000. Herein we have colocalized the glycoprotein and the a subunit of the gastric H+/K+-ATPase to the tubulovesicular membranes of the parietal cell by immunogold electron microscopy. Moreover, the glycoprotein and the a subunit were coimmunoprecipitated, and copurified by immunoaffinity chromatography, with an anti-glycoprotein monoclonal antibody. The pig glycoprotein was purified by chromatography on tomato lectin-Sepharose, and five tryptic peptides from the purified glycoprotein were partially sequenced. The complete amino acid sequence, deduced from the nucleotide sequence of overlapping cDNA clones, showed 33% similarity to the sequence of the (B subunit of the pig kidney Na+/K+-ATPase. We therefore propose that the 60to 90-kDa glycoprotein autoantigen is the (8 subunit of the gastric H+/K+-ATPase and that the a and 13 subunits of the proton pump are major targets for autoimmunization in autoimmune gastritis.
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Papers by Judy callaghan