Chandra Dodia

Phospholipid Hydroperoxides Are Substrates for Non-selenium Glutathione Peroxidase

This study investigated phospholipid hydroperoxides as substrates for non-selenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin. Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clone (HA0683) showed GSH peroxidase activity withsn-2-linolenoyl- orsn-2-arachidonoyl-phosphatidylcholine hydroperoxides as substrate; NADPH or thioredoxin could not substitute for GSH. Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM−1min−1) were k 1 = 1–3 × 105 and k 2 = 4–11 × 104. In the presence of 0.36 mm GSH, apparentK m was 120–130 μm and apparentV max was 1.5–1.6 μmol/min/mg of protein. Assays with H2O2 and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroperoxides. Maximal enzymatic activity was at pH 7–8. Activity with phospholipid hydroperoxide substrate was inhibited noncompetitively by mercaptosuccinate with K i 4 μm. The enzyme had no GSH S-transferase activity. Bovine cDNA encoding NSGPx, isolated from a lung expression library using a polymerase chain reaction probe, showed >95% similarity to previously published human, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases. The molecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da. These results identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides. Thus, this enzyme may be an important component of cellular antioxidant defense systems.

Identification of a human cDNA clone for lysosomal type Ca2+-independent phospholipase A2 and properties of the expressed protein.

A Ca2+-independent phospholipase A2 (PLA2) maximally active at pH 4 and specifically inhibited by the transition-state analogue 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) was isolated from rat lungs. The sequence for three internal peptides (35 amino acids) was used to identify a 1653-base pair cDNA clone (HA0683) from a human myeloblast cell line. The deduced protein sequence of 224 amino acids contained a putative motif (GXSXG) for the catalytic site of a serine hydrolase, but showed no significant homology to known phospholipases. Translation of mRNA produced from this clone in both a wheat germ system and Xenopus oocytes showed expression of PLA2 activity with properties similar to the rat lung enzyme. Apparent kinetic constants for PLA2 with dipalmitoylphosphatidylcholine as substrate were Km = 0.25 mM and Vmax = 1.89 nmol/h. Activity with alkyl ether phosphatidylcholine as substrate was decreased significantly compared with diacylphosphatidylcholine. Significant lysophospholipase, phospholipase A1, or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase activity was not observed. Enzyme activity was insensitive to p-bromophenacyl bromide, bromoenol lactone, trifluoromethylarachidonoyl ketone, mercaptoethanol, and ATP, but was inhibited by MJ33 and diethyl p-nitrophenyl phosphate, a serine protease inhibitor. SDS-polyacrylamide gel electrophoresis with autoradiography of the translated [35S]methionine-labeled protein confirmed a molecular mass of 25.8 kDa, in good agreement with the enzyme isolated from rat lung. By Northern blot analysis, mRNA corresponding to this clone was present in both rat lung and isolated rat granular pneumocytes. These results represent the first molecular cloning of a cDNA for the lysosomal type Ca2+-independent phospholipase A2 group of enzymes.

ABCA3 Is Critical for Lamellar Body Biogenesis in Vivo

Mutations in ATP-binding cassette transporter A3 (human ABCA3) protein are associated with fatal respiratory distress syndrome in newborns. We therefore characterized mice with targeted disruption of the ABCA3 gene. Homozygous Abca3–/– knock-out mice died soon after birth, whereas most of the wild type, Abca3+/+, and heterozygous, Abca3+/–, neonates survived. The lungs from E18.5 and E19.5 Abca3–/– mice were less mature than wild type. Alveolar type 2 cells from Abca3–/– embryos contained no lamellar bodies, and expression of mature SP-B protein was disrupted when compared with the normal lung surfactant system of wild type embryos. Small structural and functional differences in the surfactant system were seen in adult Abca3+/– compared with Abca3+/+ mice. The heterozygotes had fewer lamellar bodies, and the incorporation of radiolabeled substrates into newly synthesized disaturated phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine in both lamellar bodies and surfactant was lower than in Abca3+/+ mouse lungs. In addition, since the fraction of near term Abca3–/– embryos was significantly lower than expected from Mendelian inheritance ABCA3 probably plays roles in development unrelated to surfactant. Collectively, these findings strongly suggest that ABCA3 is necessary for lamellar body biogenesis, surfactant protein-B processing, and lung development late in gestation.

Peroxiredoxin 6 phosphorylation and subsequent phospholipase A2 activity are required for agonist-mediated activation of NADPH oxidase in mouse pulmonary microvascular endothelium and alveolar macrophages.

Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA2) activities, participates in the activation of NADPH oxidase 2 (NOX2) in neutrophils, but the mechanism for this effect is not known. We now demonstrate that Prdx6 is required for agonist-induced NOX2 activation in pulmonary microvascular endothelial cells (PMVEC) and that the effect requires the PLA2 activity of Prdx6. Generation of reactive oxygen species (ROS) in response to angiotensin II (Ang II) or phorbol 12-myristate 13-acetate was markedly reduced in perfused lungs and isolated PMVEC from Prdx6 null mice. Rac1 and p47phox, cytosolic components of NOX2, translocated to the endothelial cell membrane after Ang II treatment in wild-type but not Prdx6 null PMVEC. MJ33, an inhibitor of Prdx6 PLA2 activity, blocked agonist-induced PLA2 activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA2s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA2 active site (S32A, H26A, and D140A), “rescued” Ang II-induced PLA2 activity and ROS generation. Ang II treatment of wild-type cells resulted in phosphorylation of Prdx6 and its subsequent translocation from the cytosol to the cell membrane. Phosphorylation as well as PLA2 activity and ROS generation were markedly reduced by the MAPK inhibitor, U0126. Thus, agonist-induced MAPK activation leads to Prdx6 phosphorylation and translocation to the cell membrane, where its PLA2 activity facilitates assembly of the NOX2 complex and activation of the oxidase.

Cloning and expression of rat lung acidic Ca2+-independent PLA2 and its organ distribution

A clone for a rat acidic Ca2+-independent phospholipase A2(aiPLA2) was isolated from a cDNA library prepared from rat granular pneumocytes with a probe based on the human aiPLA2 sequence (T. S. Kim, C. S. Sundaresh, S. I. Feinstein, C. Dodia, W. R. Skach, M. K. Jain, T. Nagase, N. Seki, K. Ishikawa, N. Nomura, and A. B. Fisher. J. Biol. Chem. 272: 2542-2550, 1997). In addition, a consensus sequence for mouse aiPLA2 was constructed from several mouse cDNA clones in the GenBank and dbEST databases. Each sequence codes for a 224-amino acid protein with 88% identity of the amino acids among the three species and conservation of a putative lipase motif (GDSWG). Translation of mRNA produced from the rat clone in a wheat germ system resulted in expression of PLA2 activity with properties similar to those of the human enzyme, i.e., acidic pH optimum and Ca2+ independence. The localization of aiPLA2 in rat tissues was studied with the human cDNA probe, polyclonal and monoclonal antibodies, and aiPLA2activity. aiPLA2 is present in the lung as evidenced by high levels of mRNA and protein expression and by enzymatic activity that is inhibited by anti-PLA2 antibody and by the transition state analog 1-hexadecyl-3-trifluoroethylglycero- sn-2-phosphomethanol (MJ33). Immunocytochemistry showed the presence of aiPLA2 in alveolar type II cells, alveolar macrophages, and bronchiolar epithelium. In the brain, heart, liver, kidney, spleen, and intestine, aiPLA2 mRNA content was <50% of that in the lung, immunoreactive protein was not detectable, and enzymatic activity was not inhibited by MJ33 or aiPLA2 antibody. These results show marked enrichment of aiPLA2in the lung compared with the other organs and suggest translational control of aiPLA2 expression.

Perfusate Composition and Edema Formation in Isolated Rat Lungs

The effect of perfusate composition on duration of lung perfusion until development of alveolar edema was evaluated in isolated ventilated rat lungs perfused at a rate of 25 ml/min in a recirculating system. When the perfusate was Krebs-Ringer bicarbonate solution (KRB) alone, alveolar edema developed in 40-85 min. Addition of glucose (5 mM) slightly prolonged the time to edema while addition of 3% fatty acid-poor bovine serum albumin (BSA) extended mean survival to 3.5 hr. With KRB containing both glucose and BSA, mean survival was greater than 4 hr and three of eight lungs had not become edematous when the experiments were terminated at 5 hr. Similar results were obtained when a synthetic plasma-stimulating solution (SPSS) that is essentially free of protein or other colloid was used as a perfusate. Perfusion at reduced flow rates (12 ml/min) with KRB plus glucose and BSA or with SPSS gave 5-hr survival rates of 100%. These results indicate that prolonged lung perfusion is possible with a colloid-free artificial medium and suggest that both mechanical and metabolic factors are important in maintaining isolated perfused rat lungs free of alveolar edema.

Interaction of Surfactant Protein A with Peroxiredoxin 6 Regulates Phospholipase A2 Activity

Peroxiredoxin 6 (Prdx6) is a “moonlighting” protein with both GSH peroxidase and phospholipase A2 (PLA2) activities. This protein is responsible for degradation of internalized dipalmitoylphosphatidylcholine, the major phospholipid component of lung surfactant. The PLA2 activity is inhibited by surfactant protein A (SP-A). We postulate that SP-A regulates the PLA2 activity of Prdx6 through direct protein-protein interaction. Recombinant human Prdx6 and SP-A isolated from human alveolar proteinosis fluid were studied. Measurement of kinetic constants at pH 4.0 (maximal PLA2 activity) showed Km0.35 mm and Vmax 138 nmol/min/mg of protein. SP-A inhibited PLA2 activity non-competitively with Ki 10 μg/ml and was Ca2+ -independent. Activity at pH 7.4 was ∼50% less, and inhibition by SP-A was partially dependent on Ca2+. Interaction of SP-A and Prdx6 at pH 7.4 was shown by Prdx6-mediated inhibition of SP-A binding to agarose beads, a pull-down assay using His-tagged Prdx6 and Ni2 -chelating beads, co-immunoprecipitation from lung epithelial cells and from a binary mixture of the two proteins, binding after treatment with a trifunctional cross-linker, and size-exclusion chromatography. Analysis by static light scattering and surface plasmon resonance showed calcium-independent SP-A binding to Prdx6 at pH 4.0 and partial Ca2+ dependence of binding at pH 7.4. These results indicate a direct interaction between SP-A and Prdx6, which provides a mechanism for regulation of the PLA2 activity of Prdx6 by SP-A.

Mitogen-activated protein kinase-mediated phosphorylation of peroxiredoxin 6 regulates its phospholipase A2 activity

Prdx6 (peroxiredoxin 6), a bifunctional protein with both GSH peroxidase and PLA(2) (phospholipase A(2)) [aiPLA(2) (acidic calcium-independent PLA(2))] activities, is responsible for the metabolism of lung surfactant phospholipids. We propose that the aiPLA(2) activity of the enzyme is regulated through phosphorylation. Incubation of isolated rat alveolar type II cells (AECII) with PMA, a PKC (protein kinase C) agonist, had no effect on Prdx6 expression but led to approximately 75% increase in aiPLA(2) activity that was abolished by pretreatment of cells with the MAPK (mitogen-activated protein kinase) inhibitors, SB202190 or PD98059. Prdx6 phosphorylation after incubation of AECII with PMA was demonstrated by autoradiography after immunoprecipitation with either anti-phosphothreonine o-phosphoserine antibodies. in vitro, several active isoforms of ERK (extracellular-signal-regulated kinase) and p38 phosphorylated Prdx6, resulting in an 11-fold increase in aiPLA(2) activity. The increased activity was calcium-independent and was abolished by the aiPLA(2) inhibitors, surfactant protein A and hexadecyl-3-trifluorethylglycero-sn-2-phospho-methanol (MJ33). The peroxidase activity of Prdx6 was unaffected by phosphorylation. Mass spectroscopic analysis of in vitro phosphorylated Prdx6 showed a unique phosphorylation site at Thr-177 and mutation of this residue abolished protein phosphorylation and the increase in MAPK-mediated activity. These results show that the MAPKs can mediate phosphorylation of Prdx6 at Thr-177 with a consequent marked increase in its aiPLA(2) activity.

Characterization of acidic Ca2+-independent phospholipase A2 of bovine lung

An acidic Ca(2+)-independent phospholipase A2 (aiPLA2) has been isolated previously from rat lung and a human cDNA has been described. This study applied the method to larger scale isolation of the native protein from the bovine lung. A polyclonal antibody was generated to a 15 amino acid synthetic peptide based on a conserved rat/human sequence. This antibody recognized a single protein band with an estimated molecular mass of approximately 29 kDa in a soluble fraction obtained from bovine lung homogenate. A 29 kDa protein that reacted with the aiPLA2 antipeptide antibody was detected in fractions containing aiPLA2 activity on sequential column chromatographies. The partially purified enzyme showed 176-fold increase over the homogenate in Ca(2+)-independent PLA2 activity at pH 4. Activity was maximal with phosphatidylcholine substrate and was significantly less with phosphatidylethanolamine and anionic phospholipids. The enzyme had no acyl group preference in phosphatidylcholine and showed no preference for oxidized substrate, but activity was less with 1-O-alkyl phosphatidylcholine. aiPLA2 activity was inhibited by a transition state phospholipid analog (MJ33, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol), serine protease inhibitors, and the anti-peptide antibody but was insensitive to arachidonoyl trifluoromethyl ketone, bromoenol lactone, p-bromophenacyl bromide, and ATP. Analysis of N-terminal amino acid sequence for the 29 kDa protein demonstrated its high homology to human 26 kDa aiPLA2. These was no significant change in molecular mass of the protein following treatment with endoglycosidase F. Western blot of subcellular fractions from rat lung indicated aiPLA2 immunoreactivity with lamellar body, lysosomal, and cytosolic fractions. These results indicate isolation from bovine lung of a 29 kDa acidic Ca(2+)-independent phospholipase A2 homologue of the rat and human enzyme and provide evidence for specificity in the metabolism of lung surfactant phosphatidylcholine.

Isolation of lamellar bodies from rat granular pneumocytes in primary culture

A lamellar body fraction was isolated from rat alveolar granular pneumocytes in primary culture by upward flotation on a discontinuous sucrose gradient and compared with a similar fraction isolated from lung homogenates. Lamellar bodies from granular pneumocytes were free of detectable contamination with either succinate dehydrogenase or NADPH-cytochrome c reductase. There was an enrichment of acid phosphatase activity, which, based on distribution of enzyme activity on the gradient, did not appear to be a contamination from other fractions. The lamellar body fraction of granular pneumocytes yielded approx. 1 microgram protein/10(6) cells with a phospholipid-to-protein ratio (mg/mg) of 9.6 +/- 0.4 (n = 7). Composition with respect to total phospholipids was 71.0% phosphatidylcholine (disaturated phosphatidylcholine, 45.2%), 8.4% phosphatidylglycerol and 12.8% phosphatidylethanolamine. Palmitic acid comprised 66% of the fatty acids in phosphatidylcholine and 34% of those in phosphatidylglycerol. The lamellar body fraction from granular pneumocytes was similar to that from whole lung with respect to phospholipid-to-protein ratio and phospholipid composition and showed only minor differences in fatty acid composition. Ultrastructurally, lamellar bodies showed generally intact limiting membranes and lamellated structure. Lamellar bodies from granular pneumocytes showed occasional multinucleated whorls which were not seen in those isolated from lung homogenates. This study describes a method for preparing a homogeneous fraction of intact lamellar bodies from small amounts of material (6 X 10(7) granular pneumocytes). The yield on a per cell basis was higher when compared with a similar preparation from whole lung, although overall yield is small, due to loss of cells during the cell isolation procedure. This preparation may be useful to evaluate the role of lamellar bodies in the synthesis and secretion of lung surfactant by isolated granular pneumocytes.