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Nigel M. Hooper - One of the best experts on this subject based on the ideXlab platform.

  • Association of a GPI-anchored protein with detergent-resistant Membranes facilitates its trafficking through the early secretory pathway
    Experimental cell research, 2008
    Co-Authors: Zeynep Hein, Nigel M. Hooper, Hassan Y. Naim
    Abstract:

    Membrane microdomains are implicated in the trafficking and sorting of several Membrane proteins. In particular GPI-anchored proteins cluster into Triton X-100 resistant, cholesterol- and sphingolipid-rich Membrane microdomains and are sorted to the apical Membrane. A growing body of evidence has pointed to the existence of other types of microdomains that are insoluble in detergents, such as Lubrol WX and Tween-20. Here, we report on the role of detergent-resistant Membranes formed at early stages in the biosynthesis of Membrane Dipeptidase (MDP), a GPI-anchored protein, on its trafficking and sorting. Pulse-chase experiments revealed a retarded maturation rate of the GPI-anchor deficient mutant (MDPDeltaGPI) as compared to the wild type protein (wtMDP). However, Golgi to cell surface delivery rate did not show a significant difference between the two variants. On the other hand, early biosynthetic forms of wtMDP were partially insoluble in Tween-20, while MDPDeltaGPI was completely soluble. The lack of association of MDPDeltaGPI with detergent-resistant Membranes prior to maturation in the Golgi and the reduction in its trafficking rate strongly suggest the existence of an early trafficking control mechanisms for Membrane proteins operating at a level between the endoplasmic reticulum and the cis-Golgi.

  • Sphingomyelin chain length influences the distribution of GPI-anchored proteins in rafts in supported lipid bilayers.
    Molecular membrane biology, 2007
    Co-Authors: Ashley E. Garner, D. Alastair Smith, Nigel M. Hooper
    Abstract:

    Glycosyl-phosphatidylinositol (GPI)-anchored proteins are enriched in cholesterol- and sphingolipid-rich lipid rafts within the Membrane. Rafts are known to have roles in cellular organization and function, but little is understood about the factors controlling the distribution of proteins in rafts. We have used atomic force microscopy to directly visualize proteins in supported lipid bilayers composed of equimolar sphingomyelin, dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The transMembrane anchored angiotensin converting enzyme (TM-ACE) was excluded from the liquid ordered raft domains. Replacement of the transMembrane and cytoplasmic domains of TM-ACE with a GPI anchor (GPI-ACE) promoted the association of the protein with rafts in the bilayers formed with brain sphingomyelin (mainly C18:0). Association with the rafts did not occur if the shorter chain egg sphingomyelin (mainly C16:0) was used. The distribution of GPI-anchored proteins in supported lipid bilayers was investigated further using Membrane Dipeptidase (MDP) whose GPI anchor contains distearoyl phosphatidylinositol. MDP was also excluded from rafts when egg sphingomyelin was used but associated with raft domains formed using brain sphingomyelin. The effect of sphingomyelin chain length on the distribution of GPI-anchored proteins in rafts was verified using synthetic palmitoyl or stearoyl sphingomyelin. Both GPI-ACE and MDP only associated with the longer chain stearoyl sphingomyelin rafts. These data obtained using supported lipid bilayers provide the first direct evidence that the nature of the Membrane-anchoring domain influences the association of a protein with lipid rafts and that acyl chain length hydrophobic mismatch influences the distribution of GPI-anchored proteins in rafts.

  • Isolation and characterization of glycosylphosphatidylinositol-anchored peptides by hydrophilic interaction chromatography and MALDI tandem mass spectrometry.
    Analytical chemistry, 2006
    Co-Authors: Miren Josu Omaetxebarria, Nigel M. Hooper, Felix Elortza, Jesus M. Arizmendi, Per Hägglund, Ole Nørregaard Jensen
    Abstract:

    Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are posttranslationally processed proteins that become tethered to the extracellular leaflet of the plasma Membrane via a C-terminal glycan-like moiety. Since the first GPI-AP was described in the 1970s, more than 500 GPI-APs have been reported in a range of species, including plants, microbes, and mammals. GPI-APs are probably involved in cell signaling, cell recognition, and cell remodeling processes, and they may potentially serve as cell surface antigens or vaccine targets in pathogenic microorganisms or transformed mammalian cells. Due to the structural complexity and physicochemical properties of GPI-APs, their identification and structural characterization is a demanding analytical task. Here, we report a simple, fast and sensitive method for isolation and structural analysis of GPI-anchors using a combination of hydrophilic interaction liquid chromatography and matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight tandem mass spectrometry. This method allowed analysis of GPI peptides derived from low picomole levels of the porcine kidney Membrane Dipeptidase. Furthermore, it allowed unambiguous assignment of the omega site via amino acid sequencing of the modified peptides. GPI-anchor-specific diagnostic ions were observed by MALDI-MS/MS at m/z 162, 286, 422, and 447, corresponding to glucosamine, mannose ethanolamine phosphate, glucosamine inositol phosphate, and mannose ethanolamine phosphate glucosamine, respectively. Thus, the methodology described herein may enable sensitive and specific detection of GPI-anchored peptides in large-scale proteomic studies of plasma Membrane proteins.

  • N-glycans, not the GPI anchor, mediate the apical targeting of a naturally glycosylated, GPI-anchored protein in polarised epithelial cells.
    Journal of cell science, 2004
    Co-Authors: Susan Pang, Paula Urquhart, Nigel M. Hooper
    Abstract:

    The glycosyl-phosphatidylinositol (GPI) anchor mediates the apical sorting of proteins in polarised epithelial cells through its interaction with lipid rafts. Here we investigated the signals required for the apical targeting of the naturally N-glycosylated and GPI-anchored Membrane Dipeptidase by selective point mutation to remove the GPI anchor addition signal or the sites for N-linked glycosylation, or both. Activity assays, immunoblotting and immunofluorescence microscopy revealed that the constructs lacking the GPI anchor were secreted from Madin-Darby canine kidney (MDCK) cells, whereas those retaining the GPI anchor were attached at the cell surface, irrespective of the glycosylation status. Wild-type Membrane Dipeptidase was expressed preferentially on the apical surface of both MDCK and CaCo-2 cells. By contrast, the GPI-anchored construct lacking the N-glycans was targeted preferentially to the basolateral surface of both cell types. In constructs lacking the GPI anchor, the N-glycans also targeted the protein to the apical surface. Both the apically targeted, glycosylated and the basolaterally targeted, unglycosylated GPI-anchored forms of the protein were located in detergent-insoluble lipid rafts. These data indicate that it is the N-glycans, not the association of the GPI anchor with lipid rafts, which determine apical targeting of an endogenously N-glycosylated, GPI-anchored protein in polarised epithelial cells.

  • Glycosylation efficiency of Asn-Xaa-Thr sequons is independent of distance from the C-terminus in Membrane Dipeptidase.
    Glycobiology, 2003
    Co-Authors: Adrian R. Walmsley, Nigel M. Hooper
    Abstract:

    In vitro transcription/translation studies with model proteins have shown that glycosylation of Asn-Xaa-Thr sequons is reduced when the sequon is within 60 residues of the C-terminus of the protein. We have previously shown that in living cells N-glycosylation of the prion protein (PrP) is also abolished when its Asn-Ile-Thr and Asn-Phe-Thr sequons are less than 60 residues from the C-terminus (Walmsley and Hooper [2003] Biochemical Journal, 370, 351-355). To investigate whether sequon distance to the C-terminus is a general determinant of N-glycosylation in living cells, Asn-Ile/Phe-Thr sequons were introduced into another glycosylphosphatidylinositol (GPI) anchored protein, Membrane Dipeptidase (MDP), at similar distances from the C-terminus as those in PrP. When expressed in the human neuroblastoma SH-SY5Y cell line, the introduced sequons were fully N-glycosylated even when they were less than 60 residues from the C-terminus in both GPI-anchored and secreted forms of MDP. These data demonstrate that the utilization of sequons in some proteins is independent of their distance from the C-terminus.

Bernd Rüstow - One of the best experts on this subject based on the ideXlab platform.

  • Vitamin E as an antioxidant of the lung: mechanisms of vitamin E delivery to alveolar type II cells.
    American Journal of Respiratory and Critical Care Medicine, 2002
    Co-Authors: Ingrid Kolleck, Pranav Sinha, Bernd Rüstow
    Abstract:

    Oxidants play an important role in the development of acute and chronic lung injuries. Alveolar surfactant is the first target of air-borne oxidants. Surfactant contains, besides dipalmitoyl phosphatidylcholine, cholesterol and polyunsaturated phospholipids that play an important functional role. Therefore, vitamin E could be important for protecting surfactant lipids against oxidation and subsequent lung injury. Alveolar type II cells play a central role in synthesis and secretion of surfactant lipids and also supplement the surfactant with vitamin E during intracellular assembly. High density lipoprotein (HDL) is the primary source of vitamin E for type II cells. The uptake of vitamin E by specific lipid transfer is mediated by at least three HDL-specific receptors (scavenger receptor BI, Membrane Dipeptidase, and HDL-binding protein-2). In addition, cubilin and megalin mediate in a cooperative manner HDL-holoparticle uptake by alveolar type II cells. A temporary vitamin E deficiency induces a reversibl...

  • Vitamin E as an antioxidant of the lung: mechanisms of vitamin E delivery to alveolar type II cells.
    American journal of respiratory and critical care medicine, 2002
    Co-Authors: Ingrid Kolleck, Pranav Sinha, Bernd Rüstow
    Abstract:

    Oxidants play an important role in the development of acute and chronic lung injuries. Alveolar surfactant is the first target of air-borne oxidants. Surfactant contains, besides dipalmitoyl phosphatidylcholine, cholesterol and polyunsaturated phospholipids that play an important functional role. Therefore, vitamin E could be important for protecting surfactant lipids against oxidation and subsequent lung injury. Alveolar type II cells play a central role in synthesis and secretion of surfactant lipids and also supplement the surfactant with vitamin E during intracellular assembly. High density lipoprotein (HDL) is the primary source of vitamin E for type II cells. The uptake of vitamin E by specific lipid transfer is mediated by at least three HDL-specific receptors (scavenger receptor BI, Membrane Dipeptidase, and HDL-binding protein-2). In addition, cubilin and megalin mediate in a cooperative manner HDL-holoparticle uptake by alveolar type II cells. A temporary vitamin E deficiency induces a reversible change of the expression of pro- and antiinflammatory markers and of markers defining apoptosis, and reduces surfactant lipid synthesis in alveolar type II cells. These metabolic changes of type II cells may prime the lung to develop clinically manifest injury in response to an additional insult, e.g., hyperoxia.

  • identification of high density lipoprotein binding proteins including a glycosyl phosphatidylinositol anchored Membrane Dipeptidase in rat lung and type ii pneumocytes
    American Journal of Respiratory Cell and Molecular Biology, 2000
    Co-Authors: Wolfgang Witt, Ingrid Kolleck, Bernd Rüstow
    Abstract:

    Numerous communications have indicated that specific binding proteins for high density lipoprotein (HDL) exist in addition to the well characterized candidate HDL receptor SR-BI, but structural information was presented only in a few cases, and most of the work was aimed at the liver and steroidogenic glands. In this study, we purified two HDL-binding proteins by standard procedures from rat lung tissue. One of these Membrane glycoproteins was identified by N-terminal sequencing and with specific antibodies as HB2, a previously described HDL-binding protein, whereas the other one was identified as a glycosyl phosphatidylinositol–anchored Membrane Dipeptidase (MDP). The apparent dissociation constant of the HDL binding was determined by solid phase assay to be 2.1 μ g/ml (HB2) and 25 μ g/ml (MDP). MDP also exerts affinity to low density lipoprotein (LDL) on ligand blots, and competition between HDL and LDL was observed, but analysis by solid phase assay showed that very high concentrations of LDL are requi...

  • Identification of High Density Lipoprotein-Binding Proteins, Including a Glycosyl Phosphatidylinositol–Anchored Membrane Dipeptidase, in Rat Lung and Type II Pneumocytes
    American journal of respiratory cell and molecular biology, 2000
    Co-Authors: Wolfgang Witt, Ingrid Kolleck, Bernd Rüstow
    Abstract:

    Numerous communications have indicated that specific binding proteins for high density lipoprotein (HDL) exist in addition to the well characterized candidate HDL receptor SR-BI, but structural information was presented only in a few cases, and most of the work was aimed at the liver and steroidogenic glands. In this study, we purified two HDL-binding proteins by standard procedures from rat lung tissue. One of these Membrane glycoproteins was identified by N-terminal sequencing and with specific antibodies as HB2, a previously described HDL-binding protein, whereas the other one was identified as a glycosyl phosphatidylinositol–anchored Membrane Dipeptidase (MDP). The apparent dissociation constant of the HDL binding was determined by solid phase assay to be 2.1 μ g/ml (HB2) and 25 μ g/ml (MDP). MDP also exerts affinity to low density lipoprotein (LDL) on ligand blots, and competition between HDL and LDL was observed, but analysis by solid phase assay showed that very high concentrations of LDL are requi...

Anthony J. Turner - One of the best experts on this subject based on the ideXlab platform.

  • Differential effects of glycosphingolipids on the detergent-insolubility of the glycosylphosphatidylinositol-anchored Membrane Dipeptidase.
    Biochemical Journal, 2001
    Co-Authors: Edward T. Parkin, Anthony J. Turner, Nigel M. Hooper
    Abstract:

    The insolubility of glycosylphosphatidylinositol (GPI)-anchored proteins in certain detergents appears to be an intrinsic property of their association with sphingolipids and cholesterol in lipid rafts. We show that the GPI-anchored protein Membrane Dipeptidase is localized in detergent-insoluble lipid rafts isolated from porcine kidney microvillar Membranes, and that these rafts, which lack caveolin, are enriched not only in sphingomyelin and cholesterol, but also in the glycosphingolipid lactosylceramide (LacCer). Dipeptidase purified from porcine kidney was reconstituted into artificial liposomes in order to investigate the relationship between glycosphingolipids and GPI-anchored protein detergent-insolubility. Dipeptidase was insoluble in liposomes containing extremely low concentrations of LacCer. In contrast, identical concentrations of glucosylceramide or galactosylceramide failed to promote significant detergent-insolubility. Cholesterol was shown to enhance the detergent-insoluble effect of LacCer. GC–MS analysis revealed dramatic differences between the fatty acyl compositions of LacCer and those of the other glycosphingolipids. However, despite these differences, we show that the unusually marked effect of LacCer to promote the detergent-insolubility of Dipeptidase cannot be singularly attributed to the fatty acyl composition of this glycosphingolipid molecule. Instead, we suggest that the ability of LacCer to confer detergent-insolubility on this GPI-anchored protein is dependent on the structure of the lipid molecule in its entirety, and that this glycosphingolipid may have an important role to play in the stabilization of lipid rafts, particularly the caveolin-free glycosphingolipid signalling domains.

  • Stable and temperature-sensitive transformation of baby rat kidney cells by SV40 suppresses expression of Membrane Dipeptidase
    Oncogene, 1997
    Co-Authors: Shoshana Keynan, Nigel M. Hooper, Anthony J. Turner, Aruna Asipu, G Eric Blair
    Abstract:

    Membrane Dipeptidase (MDP) is a zinc metalloenzyme located in the lungs and on the brush border Membranes of the kidney and intestine. The gene for MDP (also termed DPEP1) is both frequently lost in Wilm's tumours and is located on human chromosome 16q24.3, a region of the genome known to contain a tumour suppressor gene(s). We now report on the regulation of MDP gene expression in normal and transformed cells. MDP enzyme activity and mRNA was detected in primary baby rat kidney (BRK) cells maintained in culture for up to 4 weeks. In contrast all stable transformed cell lines that were tested, derived either by transformation with the DNA tumour viruses SV40 or adenovirus, or in human tumour cell lines, contained very low levels of or no detectable MDP mRNA or enzyme activity. In BRK cells transformed by the temperature-sensitive tsA58 mutant of SV40 T antigen, MDP activity was not detectable, in cell lines grown at the permissive temperature (33°C) but after 5 – 14 days of incubation at the non-permissive temperature (39.5°C), MDP protein and enzyme activity could be readily detected. Taken together, these results indicate that MDP expression is characteristic of differentiated kidney epithelial cells and is down-regulated in proliferating, transformed cells.

  • Identification by site-directed mutagenesis of three essential histidine residues in Membrane Dipeptidase, a novel mammalian zinc peptidase.
    Biochemical Journal, 1997
    Co-Authors: Shoshana Keynan, Nigel M. Hooper, Anthony J. Turner
    Abstract:

    Membrane Dipeptidase (EC 3.4.13.19) is a plasma Membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D 4 . The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig Membrane Dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and K m for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney Membrane Dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin–Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane Dipeptidase shares some similarity with a Dipeptidase recently cloned from Acinetobacter calcoaceticus . In particular, His-20 and His-198 of Membrane Dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.

  • SITE-DIRECTED MUTAGENESIS OF CONSERVED CYSTEINE RESIDUES IN PORCINE Membrane Dipeptidase. CYS 361 ALONE IS INVOLVED IN DISULFIDE-LINKED DIMERIZATION
    Biochemistry, 1996
    Co-Authors: Shoshana Keynan, Nigel M. Hooper, Nicolette T. Habgood, Anthony J. Turner
    Abstract:

    Membrane Dipeptidase (EC 3.4.13.19) is a glycosylphosphatidylinositol-anchored glycoprotein of the renal brush border which exists as a disulfide-linked homodimer. Porcine Membrane Dipeptidase has a subunit M(r) of 47 kDa, and the mature protein contains seven cysteine residues per subunit, six of which are conserved in the human enzyme. Chemical modification established that cysteine residues are not involved in enzyme activity. In order to determine which of the cysteine residues are involved in the interchain disulfide bond, we have used a site-directed mutagenesis approach. Each of the conserved cysteine residues was replaced by glycine or alanine. The single mutants (C71G, C93A, C154G, C226A, C258G, and C361G) were expressed in COS-1 cells and their enzymatic activity and oligomeric structure determined. Only the C361G mutant migrated as a polypeptide of 47 kDa when subjected to denaturing polyacrylamide gel electrophoresis under nonreducing conditions. Thus, cysteine 361 is the only residue involved in disulfide linkage between the subunits. This places the disulfide bond close to the site of GPI anchor addition (Ser 368 in the porcine enzyme) and to the Membrane surface. Titration of the human and porcine proteins with 2-nitro-5-thiosulfabenzoate indicates that Membrane Dipeptidase additionally possesses two intrachain disulfide bonds. On native polyacrylamide gel electrophoresis, the C361G mutant migrates in a manner identical to that of the wild type, indicating that the protein remains associated as a noncovalent homodimer. The expressed C361G mutant, unlike the wild type, is released from COS-1 cell Membranes by trypsin and by an endogenous serine protease.

  • Structures of the Glycosyl-phosphatidylinositol Anchors of Porcine and Human Renal Membrane Dipeptidase COMPREHENSIVE STRUCTURAL STUDIES ON THE PORCINE ANCHOR AND INTERSPECIES COMPARISON OF THE GLYCAN CORE STRUCTURES
    The Journal of biological chemistry, 1995
    Co-Authors: Ian Andrew Brewis, Anthony J. Turner, Michael A. J. Ferguson, Angela Mehlert, Nigel M. Hooper
    Abstract:

    The glycan core structures of the glycosyl-phosphatidylinositol (GPI) anchors on porcine and human renal Membrane Dipeptidase (EC 3.4.13.19) were determined following deamination and reduction by a combination of liquid chromatography, exoglycosidase digestions, and methylation analysis. The glycan core was found to exhibit microheterogeneity with three structures observed for the porcine GPI anchor: Manα1-2Manα1-6Manα1-4GlcN (29% of the total population), Manα1-2Manα1-6(GalNAcβ1-4)Manα1-4GlcN (33%), and Manα1-2Manα1-6(Galβ1-3GalNAcβ1-4)Manα1-4GlcN (38%). The same glycan core structures were also found in the human anchor but in slightly different proportions (25, 52, and 17%, respectively). Additionally, a small amount (6%) of the second structure with an extra mannose α(1-2)-linked to the non-reducing terminal mannose was also observed in the human Membrane Dipeptidase GPI anchor. A small proportion (maximally 9%) of the porcine GPI anchor structures was found to contain sialic acid, probably linked to the GalNAc residue. The porcine GPI anchor was found to contain 2.5 mol of ethanolamine/mol of anchor. Negative-ion electrospray-mass spectrometry revealed the presence of exclusively diacyl-phosphatidylinositol (predominantly distearoyl-phosphatidylinositol with a minor amount of stearoyl-palmitoyl-phosphatidylinositol) in the porcine Membrane Dipeptidase anchor. Porcine Membrane Dipeptidase was digested with trypsin and the C-terminal peptide attached to the GPI anchor isolated by removal of the other tryptic peptides on anhydrotrypsin-Sepharose. The sequence of this peptide was determined as Thr-Asn-Tyr-Gly-Tyr-Ser, thereby identifying the site of attachment of the GPI anchor as Ser368. This work represents a comprehensive study of the GPI anchor structure of porcine Membrane Dipeptidase and the first interspecies comparison of mammalian GPI anchor structures on the same protein.

Ingrid Kolleck - One of the best experts on this subject based on the ideXlab platform.

  • Vitamin E as an antioxidant of the lung: mechanisms of vitamin E delivery to alveolar type II cells.
    American Journal of Respiratory and Critical Care Medicine, 2002
    Co-Authors: Ingrid Kolleck, Pranav Sinha, Bernd Rüstow
    Abstract:

    Oxidants play an important role in the development of acute and chronic lung injuries. Alveolar surfactant is the first target of air-borne oxidants. Surfactant contains, besides dipalmitoyl phosphatidylcholine, cholesterol and polyunsaturated phospholipids that play an important functional role. Therefore, vitamin E could be important for protecting surfactant lipids against oxidation and subsequent lung injury. Alveolar type II cells play a central role in synthesis and secretion of surfactant lipids and also supplement the surfactant with vitamin E during intracellular assembly. High density lipoprotein (HDL) is the primary source of vitamin E for type II cells. The uptake of vitamin E by specific lipid transfer is mediated by at least three HDL-specific receptors (scavenger receptor BI, Membrane Dipeptidase, and HDL-binding protein-2). In addition, cubilin and megalin mediate in a cooperative manner HDL-holoparticle uptake by alveolar type II cells. A temporary vitamin E deficiency induces a reversibl...

  • Vitamin E as an antioxidant of the lung: mechanisms of vitamin E delivery to alveolar type II cells.
    American journal of respiratory and critical care medicine, 2002
    Co-Authors: Ingrid Kolleck, Pranav Sinha, Bernd Rüstow
    Abstract:

    Oxidants play an important role in the development of acute and chronic lung injuries. Alveolar surfactant is the first target of air-borne oxidants. Surfactant contains, besides dipalmitoyl phosphatidylcholine, cholesterol and polyunsaturated phospholipids that play an important functional role. Therefore, vitamin E could be important for protecting surfactant lipids against oxidation and subsequent lung injury. Alveolar type II cells play a central role in synthesis and secretion of surfactant lipids and also supplement the surfactant with vitamin E during intracellular assembly. High density lipoprotein (HDL) is the primary source of vitamin E for type II cells. The uptake of vitamin E by specific lipid transfer is mediated by at least three HDL-specific receptors (scavenger receptor BI, Membrane Dipeptidase, and HDL-binding protein-2). In addition, cubilin and megalin mediate in a cooperative manner HDL-holoparticle uptake by alveolar type II cells. A temporary vitamin E deficiency induces a reversible change of the expression of pro- and antiinflammatory markers and of markers defining apoptosis, and reduces surfactant lipid synthesis in alveolar type II cells. These metabolic changes of type II cells may prime the lung to develop clinically manifest injury in response to an additional insult, e.g., hyperoxia.

  • identification of high density lipoprotein binding proteins including a glycosyl phosphatidylinositol anchored Membrane Dipeptidase in rat lung and type ii pneumocytes
    American Journal of Respiratory Cell and Molecular Biology, 2000
    Co-Authors: Wolfgang Witt, Ingrid Kolleck, Bernd Rüstow
    Abstract:

    Numerous communications have indicated that specific binding proteins for high density lipoprotein (HDL) exist in addition to the well characterized candidate HDL receptor SR-BI, but structural information was presented only in a few cases, and most of the work was aimed at the liver and steroidogenic glands. In this study, we purified two HDL-binding proteins by standard procedures from rat lung tissue. One of these Membrane glycoproteins was identified by N-terminal sequencing and with specific antibodies as HB2, a previously described HDL-binding protein, whereas the other one was identified as a glycosyl phosphatidylinositol–anchored Membrane Dipeptidase (MDP). The apparent dissociation constant of the HDL binding was determined by solid phase assay to be 2.1 μ g/ml (HB2) and 25 μ g/ml (MDP). MDP also exerts affinity to low density lipoprotein (LDL) on ligand blots, and competition between HDL and LDL was observed, but analysis by solid phase assay showed that very high concentrations of LDL are requi...

  • Identification of High Density Lipoprotein-Binding Proteins, Including a Glycosyl Phosphatidylinositol–Anchored Membrane Dipeptidase, in Rat Lung and Type II Pneumocytes
    American journal of respiratory cell and molecular biology, 2000
    Co-Authors: Wolfgang Witt, Ingrid Kolleck, Bernd Rüstow
    Abstract:

    Numerous communications have indicated that specific binding proteins for high density lipoprotein (HDL) exist in addition to the well characterized candidate HDL receptor SR-BI, but structural information was presented only in a few cases, and most of the work was aimed at the liver and steroidogenic glands. In this study, we purified two HDL-binding proteins by standard procedures from rat lung tissue. One of these Membrane glycoproteins was identified by N-terminal sequencing and with specific antibodies as HB2, a previously described HDL-binding protein, whereas the other one was identified as a glycosyl phosphatidylinositol–anchored Membrane Dipeptidase (MDP). The apparent dissociation constant of the HDL binding was determined by solid phase assay to be 2.1 μ g/ml (HB2) and 25 μ g/ml (MDP). MDP also exerts affinity to low density lipoprotein (LDL) on ligand blots, and competition between HDL and LDL was observed, but analysis by solid phase assay showed that very high concentrations of LDL are requi...

Shoshana Keynan - One of the best experts on this subject based on the ideXlab platform.

  • Stable and temperature-sensitive transformation of baby rat kidney cells by SV40 suppresses expression of Membrane Dipeptidase
    Oncogene, 1997
    Co-Authors: Shoshana Keynan, Nigel M. Hooper, Anthony J. Turner, Aruna Asipu, G Eric Blair
    Abstract:

    Membrane Dipeptidase (MDP) is a zinc metalloenzyme located in the lungs and on the brush border Membranes of the kidney and intestine. The gene for MDP (also termed DPEP1) is both frequently lost in Wilm's tumours and is located on human chromosome 16q24.3, a region of the genome known to contain a tumour suppressor gene(s). We now report on the regulation of MDP gene expression in normal and transformed cells. MDP enzyme activity and mRNA was detected in primary baby rat kidney (BRK) cells maintained in culture for up to 4 weeks. In contrast all stable transformed cell lines that were tested, derived either by transformation with the DNA tumour viruses SV40 or adenovirus, or in human tumour cell lines, contained very low levels of or no detectable MDP mRNA or enzyme activity. In BRK cells transformed by the temperature-sensitive tsA58 mutant of SV40 T antigen, MDP activity was not detectable, in cell lines grown at the permissive temperature (33°C) but after 5 – 14 days of incubation at the non-permissive temperature (39.5°C), MDP protein and enzyme activity could be readily detected. Taken together, these results indicate that MDP expression is characteristic of differentiated kidney epithelial cells and is down-regulated in proliferating, transformed cells.

  • Identification by site-directed mutagenesis of three essential histidine residues in Membrane Dipeptidase, a novel mammalian zinc peptidase.
    Biochemical Journal, 1997
    Co-Authors: Shoshana Keynan, Nigel M. Hooper, Anthony J. Turner
    Abstract:

    Membrane Dipeptidase (EC 3.4.13.19) is a plasma Membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D 4 . The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig Membrane Dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and K m for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney Membrane Dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin–Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane Dipeptidase shares some similarity with a Dipeptidase recently cloned from Acinetobacter calcoaceticus . In particular, His-20 and His-198 of Membrane Dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.

  • SITE-DIRECTED MUTAGENESIS OF CONSERVED CYSTEINE RESIDUES IN PORCINE Membrane Dipeptidase. CYS 361 ALONE IS INVOLVED IN DISULFIDE-LINKED DIMERIZATION
    Biochemistry, 1996
    Co-Authors: Shoshana Keynan, Nigel M. Hooper, Nicolette T. Habgood, Anthony J. Turner
    Abstract:

    Membrane Dipeptidase (EC 3.4.13.19) is a glycosylphosphatidylinositol-anchored glycoprotein of the renal brush border which exists as a disulfide-linked homodimer. Porcine Membrane Dipeptidase has a subunit M(r) of 47 kDa, and the mature protein contains seven cysteine residues per subunit, six of which are conserved in the human enzyme. Chemical modification established that cysteine residues are not involved in enzyme activity. In order to determine which of the cysteine residues are involved in the interchain disulfide bond, we have used a site-directed mutagenesis approach. Each of the conserved cysteine residues was replaced by glycine or alanine. The single mutants (C71G, C93A, C154G, C226A, C258G, and C361G) were expressed in COS-1 cells and their enzymatic activity and oligomeric structure determined. Only the C361G mutant migrated as a polypeptide of 47 kDa when subjected to denaturing polyacrylamide gel electrophoresis under nonreducing conditions. Thus, cysteine 361 is the only residue involved in disulfide linkage between the subunits. This places the disulfide bond close to the site of GPI anchor addition (Ser 368 in the porcine enzyme) and to the Membrane surface. Titration of the human and porcine proteins with 2-nitro-5-thiosulfabenzoate indicates that Membrane Dipeptidase additionally possesses two intrachain disulfide bonds. On native polyacrylamide gel electrophoresis, the C361G mutant migrates in a manner identical to that of the wild type, indicating that the protein remains associated as a noncovalent homodimer. The expressed C361G mutant, unlike the wild type, is released from COS-1 cell Membranes by trypsin and by an endogenous serine protease.

  • The renal Membrane Dipeptidase (dehydropeptidase I) inhibitor, cilastatin, inhibits the bacterial metallo-beta-lactamase enzyme CphA.
    Antimicrobial agents and chemotherapy, 1995
    Co-Authors: Shoshana Keynan, Nigel M. Hooper, Antonio Felici, Gianfranco Amicosante, Andanthony J. Turner
    Abstract:

    The Aeromonas hydrophila AE036 chromosome contains a cphA gene encoding a metallo-beta-lactamase which is highly active against carbapenem antibiotics such as imipenem. Here we show that the cphA gene product shares inhibitory similarities with a mammalian zinc peptidase, Membrane Dipeptidase (MDP; dehydropeptidase I). Both enzymes are able to hydrolyze imipenem and are inhibited by cilastatin. The active site similarities of these enzymes are not reflected in any significant primary sequence similarity.

  • Directed mutagenesis of pig renal Membrane Dipeptidase. His219 is critical but the DHXXH motif is not essential for zinc binding or catalytic activity.
    FEBS letters, 1994
    Co-Authors: Shoshana Keynan, Nigel M. Hooper, Anthony J. Turner
    Abstract:

    Pig renal Membrane Dipeptidase cDNA has been expressed in COS-1 cells. Directed mutagenesis was used to investigate the roles of some conserved histidyl and aspartyl residues. Mutation of His219 to Arg, Lys or Leu results in complete abolition of enzyme activity, although the mutants are expressed at the cell-surface. Residues in a proposed motif (DHXDH; residues 269−273) for zinc binding have been mutated individually. Each retained activity comparable to that of the wild-type, excluding an essential role for components of this motif. The zinc-binding ligands in Membrane Dipeptidase therefore represent a novel domain for a metallopeptidase with His219 being one candidate.