120-kD surface glycoprotein of Pneumocystis carinii is a ligand for surfactant protein A.

Pneumocystis carinii is the most common cause of life-threatening pneumonia in immunocompromised patients. In the current study, surfactant protein A (SP-A), the major nonserum protein constituent of pulmonary surfactant, is demonstrated to bind P. carinii in a specific and saturable manner. SP-A is surface bound and does not appear to be internalized or degraded by the P. carinii organism. Furthermore, SP-A binding to P. carinii is time- and calcium-dependent and is competitively inhibited by mannosyl albumin. In the absence of calcium or the presence of excess mannosyl albumin, SP-A binding to P. carinii is reduced by 95 and 71%, respectively. SP-A avidly binds P. carinii with a Kd of 8 x 10(-9) M and an estimated 8.4 x 10(6) SP-A binding sites per P. carinii organism, as determined from Scatchard plots. SP-A is shown to bind P. carinii in vivo, and a putative binding site for SP-A on P. carinii is demonstrated to be the mannoserich surface membrane glycoprotein gp120. These findings suggest that P. carinii can interact with the phospholipid-rich material in the alveolar spaces by specifically binding a major protein constituent of pulmonary surfactant.

Generation of lyso-phospholipids from surfactant in acute lung injury is mediated by type-II phospholipase A2 and inhibited by a direct surfactant protein A-phospholipase A2 protein interaction.

Lyso-phospholipids exert a major injurious effect on lung cell membranes during Acute Respiratory Distress Syndrome (ARDS), but the mechanisms leading to their in vivo generation are still unknown. Intratracheal administration of LPS to guinea pigs induced the secretion of type II secretory phospholipase A2 (sPLA2-II) accompanied by a marked increase in fatty acid and lyso-phosphatidylcholine (lyso-PC) levels in the bronchoalveolar lavage fluid (BALF). Administration of LY311727, a specific sPLA2-II inhibitor, reduced by 60% the mass of free fatty acid and lyso-PC content in BALF. Gas chromatography/mass spectrometry analysis revealed that palmitic acid and palmitoyl-2-lyso-PC were the predominant lipid derivatives released in BALF. A similar pattern was observed after the intratracheal administration of recombinant guinea pig (r-GP) sPLA2-II and was accompanied by a 50-60% loss of surfactant phospholipid content, suggesting that surfactant is a major lung target of sPLA2-II. In confirmation, r-GP sPLA2-II was able to hydrolyze surfactant phospholipids in vitro. This hydrolysis was inhibited by surfactant protein A (SP-A) through a direct and selective protein-protein interaction between SP-A and sPLA2-II. Hence, our study reports an in vivo direct causal relationship between sPLA2-II and early surfactant degradation and a new process of regulation for sPLA2-II activity. Anti-sPLA2-II strategy may represent a novel therapeutic approach in lung injury, such as ARDS.

Phosphatidylserine decarboxylase.

Phosphatidylserine decarboxylase (PSD) is an important enzyme in the synthesis of phosphatidylethanolamine in both prokaryotes and eukaryotes. The cloned bacterial gene encodes an integral membrane protein that is first made as a proenzyme, and subsequently proteolyzed to an alpha subunit, containing a pyruvoyl prosthetic group, and a beta subunit. Two types of decarboxylases are found in yeast, PSD1 and PSD2, that localize to the inner mitochondrial membrane and the Golgi/vacuole membrane, respectively. The mammalian enzyme is also found in the inner mitochondrial membrane. The yeast genes and mammalian cDNA have been cloned and sequenced. The yeast genes contain 5' sequences associated with regulation of expression by inositol and choline. The yeast PSD1 and the mammalian PSD both contain an LGST amino acid motif that identifies the site of proteolysis and pyruvoyl prosthetic group attachment in the bacterial enzyme. The yeast PSD1 and mammalian PSD also have mitochondrial targeting and inner membrane sorting sequences. Processing intermediates have been defined in the mammalian enzyme that correspond to the sequential removal of the mitochondrial targeting and inner membrane sorting sequence, followed by formation of the alpha and beta subunits. In contrast, the PSD2 enzyme contains a putative Golgi localization/retention sequence and a C2 homology domain, in addition to predicted alpha and beta subunits. The transport requirements for substrate access to the PSD enzymes have provided important information about lipid trafficking, and the availability of yeast mutants is likely to provide important new genetic selections in the future.

Interorganelle transport of aminoglycerophospholipids.

The aminoglycerophospholipids of eukaryotic cells, phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho), can be synthesized by multiple pathways. The PtdSer pathway encompasses the synthesis of PtdSer, its decarboxylation to PtdEtn and subsequent methylation reactions to form PtdCho. The Kennedy pathways consist of the synthesis of PtdEtn and PtdCho from Etn and Cho precursors via CDP-Etn and CDP-Cho intermediates. The reactions along the PtdSer pathway are spatially segregated with PtdSer synthesis occurring in the endoplasmic reticulum or mitochondria-associated membrane (MAM), PtdEtn formation occurring in the mitochondria and Golgi/vacuole compartments and PtdCho formation occurring in the endoplasmic reticulum or MAM. The organelle-specific metabolism of the different lipids in the PtdSer pathway has provided a convenient biochemical means for defining events in the interorganelle transport of the aminoglycerophospholipids in intact cells, isolated organelles and permeabilized cells. Studies with both mammalian cells and yeast demonstrate many significant similarities in lipid transport processes between the two systems. Genetic experiments in yeast now provide the tools to create new strains with mutations along the PtdSer pathway that can be conditionally rescued by the Kennedy pathway reactions. The genetic studies in yeast indicate that it is now possible to begin to define genes that participate in the interorganelle transport of the aminoglycerophospholipids.

Regulatory mechanisms of surfactant secretion.

Surfactant secretion is a critical regulated process in the metabolism of pulmonary surfactant. Presumably, because this process is vital to the survival of the organism, there are several independent pathways for stimulating secretion which work through different cell surface receptors and signaling mechanisms. In addition, there is apparent homeostatic regulation in that two components of surfactant, namely SP-A and dipalmitoylphosphatidylcholine, can inhibit secretion. Although secretion of surfactant has been studied for over two decades, there remains some important issues to be resolved. In vivo secretion can be stimulated by hyperventilation or even a single large breath. However, we do not know the biochemical mechanism for this physiologically important form of stimulation. In vitro, we know many of the proximal events in signaling, but we do not know how the lamellar bodies move within a cell or the docking mechanism at the plasma membrane. Many investigators have demonstrated that SP-A will inhibit secretion in vitro, but the mechanism is not known. Finally, there is a route of secretion of SP-A independent of lamellar bodies, but we do not know details of this pathway nor its regulation.

Tetradecanoylphorbol acetate and terbutaline stimulate surfactant secretion in alveolar type II cells without changing the membrane potential.

Alveolar type II cells were isolated from adult rat lungs after tissue dissociation with elastase. The effect of known secretagogues on transmembrane potential was examined in freshly isolated cells (day 0 cells) and in cells after one day of primary culture (day 1 cells). Freshly isolated type II cells were incubated with 3,3'-dipentyloxacarbocyanine (di-O-C5(3)) or 3,3'-dipropylthiadicarbocyanine (di-S-C3(5)), dyes whose intracellular fluorescence intensity is a direct function of the cellular transmembrane potential. Fluorescence was continuously recorded by fluorescence spectrophotometry. Type II cells rapidly incorporated the dyes, and the addition of gramicidin (1 microgram/ml) depolarized the cells as indicated by a change in fluorescence. Neither 12-O-tetradecanoylphorbol 13-acetate (TPA) nor terbutaline plus 3-isobutyl-1-methylxanthine (IBMX), which stimulate surfactant secretion from isolated alveolar type II cells, changed the transmembrane potential. The lipophilic cation triphenylmethylphosphonium (TPMP+) was used to quantitate the transmembrane potential of type II cells cultured for one day. Addition of TPA or terbutaline plus IBMX induced surfactant secretion but did not alter the transmembrane potential. To study further the relationship of secretion to the transmembrane potential, secretion was also determined in the presence of high extracellular potassium which depolarizes the cells and in the presence of choline in place of sodium. High potassium enhanced the basal secretion of phosphatidylcholine from 1.8% to 3.4% (P less than 0.01, n = 7). Substitution of sodium chloride by choline chloride had no effect on basal secretion but enhanced TPA-induced secretion (P less than 0.01). We conclude that high extracellular potassium induces membrane depolarization and stimulates surfactant secretion, but TPA or terbutaline plus IBMX stimulates secretion without detectable membrane depolarization and stimulation of secretion by TPA does not require extracellular sodium.

Pulmonary surfactant phosphatidylcholine transport bypasses the brefeldin A sensitive compartment of alveolar type II cells.

Brefeldin A (BFA) causes disassembly of the Golgi apparatus and blocks protein transport to this organelle from the endoplasmic reticulum. However, there still remains considerable ambiguity regarding the involvement of the Golgi apparatus in glycerolipid transport pathways. We examined the effects of BFA upon the intracellular translocation of phosphatidylcholine in alveolar type II cells, that synthesize, transport, store and secrete large amounts of phospholipid for regulated exocytosis. BFA at concentrations as high as 10 microg/ml failed to alter the assembly of phosphatidylcholine into lamellar bodies, the specialized storage organelles for pulmonary surfactant. The same concentration of BFA was also ineffective at altering the secretion of newly synthesized phosphatidylcholine from alveolar type II cells. In contrast, concentrations of the drug of 2.5 microg/ml completely arrested newly synthesized lysozyme secretion from the same cells, indicating that BFA readily blocked protein transport processes in alveolar type II cells. The disassembly of the Golgi apparatus in alveolar type II cells following BFA treatment was also demonstrated by showing the redistribution of the resident Golgi protein MG-160 to the endoplasmic reticulum. These results indicate that intracellular transport of phosphatidylcholine along the secretory pathway in alveolar type II cells proceeds via a BFA insensitive route and does not require a functional Golgi apparatus.

The Na+/H+ antiporter in rat alveolar type II cells and its role in stimulated surfactant secretion.

We used the pH-sensitive fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to identify Na+/H+ exchange in freshly isolated rat alveolar type II cells and alveolar type II cells in primary culture. The intracellular pH (pHi) of freshly isolated alveolar type II cells was 7.36 +/- 0.05 (n = 3). When freshly isolated alveolar type II cells were acid loaded with nigericin in sodium-free buffer, the pHi dropped to 6.59 +/- 0.04 and remained low in sodium-free buffer. When acid-loaded cells were subsequently incubated with NaCl, pHi increased in a dose-dependent manner. Amiloride (0.1 mM) inhibited the sodium-induced increase in pHi. When the acid-loaded cells were resuspended in an unbuffered choline chloride solution, the cells secreted H+ in a sodium-dependent and amiloride-inhibitable manner. Alveolar type II cell monolayers, which were cultured for 22 h on glass coverslips and then loaded with BCECF, had a resting pHi of 7.48 +/- 0.05 (n = 4). Nigericin acidified these cultured cells in the absence of sodium and NaCl increased the pHi of these acid loaded cells as observed in freshly isolated cells. Secretagogues of pulmonary surfactant, 12-O-tetradecanoylphorbol 13-acetate (TPA) and terbutaline, did not change pHi. Inhibition of the Na+/H+ antiporter by the addition of amiloride to a Na+ containing medium or the substitution of choline for Na+ did not inhibit stimulated phosphatidylcholine secretion. We conclude that pHi regulation in rat alveolar type II cells is in part mediated by an amiloride-sensitive Na+/H+ antiporter, but this system appears not to be involved in TPA- or terbutaline-induced pulmonary surfactant secretion in primary culture.

A yeast strain defective in oleic acid utilization has a mutation in the RML2 gene.

The molecular mechanisms of cellular long-chain fatty acid assimilation and its regulation remain unclear. In an attempt to identify essential mediators of these processes, we have isolated mutant strains of the yeast Saccharomyces cerevisiae unable to utilize oleic acid as sole carbon source, while retaining the ability to utilize acetate. These strains are then subjected to several secondary screening assays to identify mutants of interest. Here we describe a mutant (denoted fat21) that, despite a temperature-sensitive inability to utilize oleic acid as sole carbon source, displays no general defect in oleic acid uptake or incorporation of oleic acid into glycerolipids. Oxidation of acetate after growth in acetate medium is increased similarly in the mutant and parent strains. Oleic acid beta-oxidation in acetate grown cells is also comparable between strains. Induction of oleic acid oxidation following exposure to oleic acid is, however, defective in the fat21 mutant. The fat21 mutant allele displays conditional synthetic lethality in combination with a null allele of the OLE1 gene, which encodes Delta9-desaturase and is required for proper mitochondrial segregation. Clones capable of complementing the fat21 defect contained the RML2 gene, encoding a yeast mitochondria ribosomal protein. Segregation analysis and gene replacement experiments demonstrate that RML2 is the gene defective in the fat21 mutant. These observations of a defect in a mitochondrial protein differentially affecting the adaptation to oleic acid and acetate as carbon sources suggest that the phenotype of fat21 is associated with a novel pathway of mitochondrial-nuclear-peroxisomal communication.

A genetic screen for ethanolamine auxotrophs in Saccharomyces cerevisiae identifies a novel mutation in Mcd4p, a protein implicated in glycosylphosphatidylinositol anchor synthesis.

A genetic screen for ethanolamine auxotrophs has identified a novel mutant allele of the morphogenesis checkpoint dependent (MCD)-4 gene, designated mcd4-P301L. In the presence of a null allele for the phosphatidylserine (PtdSer) decarboxylase 1 gene (psd1 Delta), the mcd4-P301L mutation confers temperature sensitivity for growth on minimal medium. This growth defect is reversed by either ethanolamine or choline supplementation. Incubation of mutant cells with [(3)H]serine followed by analysis of the aminoglycerophospholipids demonstrated a 60% decrease in phosphatidylethanolamine (PtdEtn) formation compared to parental cells. Chemical analysis of phospholipid content after culture under non-permissive conditions also demonstrated a 60% decrease in the PtdEtn pool compared to the parental strain. Although the morphogenesis checkpoint dependent (MCD)-4 gene and its homologues have been shown to play a role in glycosylphosphatidylinositol (GPI) anchor synthesis, the mcd4-P301L strain displayed normal incorporation of [(3)H]inositol into both proteins and lipids. Thus, a defect in GPI anchor synthesis does not explain either the ethanolamine auxotrophy or biochemical phenotype of this mutant. We also examined the growth characteristics and PtdSer metabolism of a previously described mcd4-174 mutant strain, with defects in GPI anchor synthesis, protein modification and cell wall maintenance. The mcd4-174, psd1 Delta strain is a temperature sensitive ethanolamine auxotroph that requires osmotic support for growth, and displays normal PtdEtn formation compared to parental cells. These results reveal important genetic interactions between PSD1 and MCD4 genes, and provide evidence that Mcd4p can modulate aminoglycerophospholipid metabolism, in a way independent of its role in GPI anchor synthesis.

Properties and purification of an arachidonoyl-hydrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7.

The lipid mediators, platelet activating factor (PAF) and the eicosanoids, can be coordinately produced from the common phospholipid precursor, 1-O-alkyl-2-arachidonoylglycerophosphocholine (1-O-alkyl-2-arachidonoyl-GPC), through the initial action of a phospholipase A2 that cleaves arachidonic acid from the sn-2 position. The mouse macrophage cell line RAW 264.7, which was used as a model macrophage system to study the arachidonoyl-hydrolyzing phospholipase A2 enzyme(s), could be induced to release arachidonic acid in response to inflammatory stimuli. A phospholipase A2 that hydrolyzed 1-O-hexadecyl-2-[3H]arachidonoyl-GPC was identified in the cytosolic fraction of these macrophages. This phospholipase activity was optimal at pH 8 and dependent on calcium. Enzyme activity could be stimulated 3-fold by heparin, suggesting the presence of phospholipase inhibitory proteins in the macrophage cytosol. Compared to 1-alkyl-2-arachidonoyl-GPC, the enzyme hydrolyzed 1-acyl-2-arachidonoylglycerophosphoethanolamine (1-acyl-2-arachidonoyl-GPE) with similar activity but showed slightly greater activity against 1-acyl-2-arachidonoyl-GPC, suggesting no specificity for the sn-1 linkage or the phospholipid base group. Although comparable activity against 1-acyl-2-arachidonoylglycerophosphoinositol (1-acyl-2-arachidonoyl-GPI) could be achieved, the enzyme exhibited much lower affinity for the inositol-containing substrate. The enzyme did, however, show apparent specificity for arachidonic acid at the sn-2 position, since much lower activity was observed against choline-containing substrates with either linoleic or oleic acids at the sn-2 position. The cytosolic phospholipase A2 was purified by first precipitating the enzyme with ammonium sulfate followed by chromatography over Sephadex G150, where the phospholipase A2 eluted between molecular weight markers of 67,000 and 150,000. The active peak was then chromatographed over DEAE-cellulose, phenyl-Sepharose, Q-Sepharose, Sephadex G150 and finally hydroxylapatite. The purification scheme has resulted in over a 1000-fold increase in specific activity (2 mumol/min per mg protein). Under non-reducing conditions, a major band on SDS-polyacrylamide gels at 70 kDa was observed, which shifted to a lower molecular weight, 60,000, under reducing conditions. The properties of the purified enzyme including the specificity for sn-2-arachidonoyl-containing phospholipids was similar to that observed for the crude enzyme. The results demonstrate the presence of a phospholipase A2 in the macrophage cell line. RAW 264.7, that preferentially hydrolyzes arachidonoyl-containing phospholipid substrates.

Expression and characterization of rat surfactant protein A synthesized in Chinese hamster ovary cells.

Rat surfactant protein A (SP-A) was expressed in a Chinese hamster ovary (CHO-K1) cell line and characterized for biologic activity using assays for receptor binding and modulation of phospholipid secretion from isolated type II cells. The CHO-K1 cell line was cotransfected with separate plasmids encoding for the rat SP-A, dihydrofolate reductase and neomycin phosphotransferase, respectively. Antibiotic (Geneticin-G418)-resistant transformants were screened by ELISA for the secretion of recombinant SP-A into the media. Northern analysis of the transfected cell lines demonstrated the expression of both 1.6 kb and 0.9 kb mRNA species for SP-A, consistent with the proposed differential polyadenylation of the primary transcript. Amplification with methotrexate resulted in a dose-dependent increase in mRNA for SP-A and a 20-fold increase in the production of recombinant SP-A relative to untreated cells. Maximum production of SP-A was 370 micrograms of SP-A/l of media in a 4-day incubation. Recombinant SP-A was purified from the serum-free media of large scale cultures of transfected, amplified CHO cells by affinity chromatography on mannose-Sepharose. The recombinant SP-A migrated similarly to native SP-A by NaDodSO4-PAGE analysis under reducing and nonreducing conditions and under reducing conditions after digestion with N-glycanase. Recombinant SP-A effectively competed with 125I-native SP-A for binding to the high affinity receptor for SP-A on isolated plasma membranes from rat alveolar type II cells. The recombinant SP-A was as effective as native SP-A in the inhibition of secretion of phospholipid from isolated type II cells. We conclude that recombinant rat SP-A produced in Chinese hamster ovary cells is physically and functionally similar to native rat SP-A.

Site-directed mutagenesis of surfactant protein A reveals dissociation of lipid aggregation and lipid uptake by alveolar type II cells.

Surfactant protein A (SP-A) binds to dipalmitoylphosphatidylcholine (DPPC) and induces phospholipid vesicle aggregation. It also regulates the uptake and secretion of surfactant lipids by alveolar type II cells. We introduced the single mutations Glu195-->Gln (rE195Q), Lys201-->Ala (rK201A) and Lys203-->Ala (rK203A) for rat SP-A, Arg199-->Ala (hR199A) and Lys201-->Ala (hK201A) for human SP-A, and the triple mutations Arg197, Lys201 and Lys203-->Ala (rR197A/K201A/K203A) for rat SP-A, into cDNAs for SP-A, and expressed the recombinant proteins using baculovirus vectors. All recombinant proteins avidly bound to DPPC liposomes. rE195Q, rK201A, rK203A, hR199A and hK201A function with activity comparable to wild type SP-A. Although rR197A/K201A/K203A was a potent inducer of phospholipid vesicle aggregation, it failed to stimulate lipid uptake. rR197A/K201A/K203A was a weak inhibitor for lipid secretion and did not competed with rat [125I]SP-A for receptor occupancy. From these results, we conclude that Lys201 and Lys203 of rat SP-A, and Arg199 and Lys201 of human SP-A are not individually critical for the interaction with lipids and type II cells, and that Glu195 of rat SP-A can be replaced with Gln without loss of SP-A functions. This study also demonstrates that the SP-A-mediated lipid uptake is not directly correlated with phospholipid vesicle aggregation, and that specific interactions of SP-A with type II cells are involved in the lipid uptake process.

Internalization of surfactant protein A (SP-A) into lamellar bodies of rat alveolar type II cells in vitro.

Pulmonary surfactant is thought to be internalized and processed for reuse by alveolar Type II cells. In the present study we followed the internalization and intracellular trafficking of purified surfactant protein A (SP-A) by primary cultures of alveolar Type II cells. Internalization of native rat SP-A was compared with that of recombinant rat and human SP-A isolated from a patient with alveolar proteinosis. All SP-A species were conjugated with colloidal gold for visualization by electron microscopy. The gold conjugates were biologically active, as demonstrated by inhibition of phospholipid secretion from alveolar Type II cells. The SP-A-gold conjugates were internalized to lamellar bodies (LB) via the endosomal system, which included both electron-lucent and -dense multivesicular bodies. Labeling of LB was time dependent, and after 7 hr 30-40% of these organelles were labeled. Alkylation of SP-A greatly reduced internalization, as did an excess of non-conjugated SP-A. No qualitative differences in uptake were observed with the three forms of SP-A. The percent of labeled LB was similar (30-40%) after 7 hr of internalization with the three species of SP-A. The recombinant SP-A produced using a baculovirus vector lacked hydroxyproline and had an altered oligosaccharide, but these features did not affect its internalization or the rate of LB labeling. Internalization of the gold-conjugated SP-A and endocytosis of the fluid-phase marker Lucifer Yellow were related to the shape of Type II cells. Both uptake of SP-A, which is receptor mediated, and fluid-phase endocytosis were found to be less active in the flattened than in the rounded cells. Therefore, cell shape and hence cytoskeletal organization may play an important role in SP-A recycling. However, it is possible that both morphology and decreased endocytosis are independent manifestations related to the loss of differentiated function of cultured Type II cells.

Macromolecular assemblies regulate nonvesicular phosphatidylserine traffic in yeast.

PtdSer (phosphatidylserine) is synthesized in the endoplasmic reticulum and the related MAM (mitochondria-associated membrane), and transported to the PtdSer decarboxylases, Pds1p in the mitochondria, and Psd2p in the Golgi. Genetic and biochemical analyses of PtdSer transport are now revealing the role of specific protein and lipid assemblies on different organelles that regulate non-vesicular PtdSer transport. The transport of PtdSer from MAM to mitochondria is regulated by at least three genes: MET30 (encoding a ubiquitin ligase), MET4 (encoding a transcription factor), and one or more unknown genes whose transcription is regulated by MET4. MET30-dependent ubiquitination is required for the MAM to function as a competent donor membrane and for the mitochondria to function as a competent acceptor membrane. Non-vesicular transport of PtdSer to the locus of Psd2p is under the control of at least three genes, STT4 [encoding Stt4p (phosphatidylinositol 4-kinase)], PSTB2 (encoding the lipid-binding protein PstB2p) and PSD2 (encoding Psd2p). Stt4p is proposed to produce a pool of PtdIns4P that is necessary for lipid transport. PstB2p and Psd2p must be present on the acceptor membrane for PtdSer transport to occur. Psd2p contains a C2 (Ca(2+) and phospholipid binding sequence) domain that is required for lipid transport. Reconstitution studies with chemically defined donor membranes demonstrate that membrane domains rich in the anionic lipids, PtdSer, PtdIns4P and phosphatidic acid function as the most efficient donors of PtdSer to Psd2p. The emerging view is that macromolecular complexes dependent on protein-protein and protein-lipid interactions form between donor and acceptor membranes and serve to dock the compartments and facilitate phospholipid transport.