SP-D-dependent regulation of NO metabolism in lipopolysaccharide-stimulated peritoneal macrophages.

This work was designed to study the role of surfactant protein D in the regulation of NO synthesis by "non-alveolar" microphages. We evaluated whether the effects of surfactant protein D depend on the phenotype of macrophages. In the absence of surfactant protein D, the LPS-induced iNOS response was shown to decrease in macrophages of native and proinflammatory phenotypes by 30%, and in macrophages of the antiinflammatory phenotype (by 63%). Under the influence of lipopolysaccharide in high doses (500 ng/ml), NO(2)*- production by mouse macrophages without surfactant protein D was reduced in native cells (by 25%), but increased in proinflammatory (by 40%) and antiinflammatory phenotypes (by 12% compared to mouse macrophages with surfactant protein D). Our results suggest that surfactant protein D is involved in the immune response in the whole organism, but not only in the lungs. The effect of surfactant protein D depends on the phenotype of macrophages.

Nitric oxide production and perivascular nitration in brain after carbon monoxide poisoning in the rat.

Nitric oxide is a short-lived free radical and physiological mediator which has the potential to cause cytotoxicity. Studies were conducted to investigate whether nitric oxide, and the potent oxidant peroxynitrite, were generated in brain during experimental carbon monoxide (CO) poisoning in the rat. Nitric oxide production was documented by electron paramagnetic resonance spectroscopy, and found to be increased by ninefold immediately after CO poisoning. Evidence that peroxynitrite was generated was sought by looking for nitrotyrosine in the brains of CO-poisoned rats. Nitrotyrosine was found deposited in vascular walls, and also diffusely throughout the parenchyma in inummocytochemical studies. The affinity and specificity of an anti-nitrotyrosine antibody was investigated and a solid phase immunoradiochemical assay was developed to quantity nitrotyrosine in brain homogenates. A 10-fold increase in nitrotyrosine was found in the brains of CO-poisoned rats. Platelets were involved with production of nitrotyrosine in the early phase of exposure to CO. However, nitrotyrosine formation and leukocyte sequestration were not decreased in thrombocytopenic rats poisoned with CO according to the standard model. When rats were pre-treated with the nitric oxide synthase inhibitor, L-nitroarginine methyl ester, formation of both nitric oxide and nitrotyrosine in response to CO poisoning were abolished, as well as leukocyte sequestration in the microvasculature, endothelial xanthine dehydrogenase conversion to xanthine oxidase, and brain lipid peroxidation. We conclude that perivascular reactions mediated by peroxynitrite are important in the cascade of events which lead to brain oxidative stress in CO poisoning.

Susceptibility to ozone-induced airway inflammation is associated with decreased levels of surfactant protein D.

BACKGROUND: Ozone (O3), a common air pollutant, induces exacerbation of asthma and chronic obstructive pulmonary disease. Pulmonary surfactant protein (SP)-D modulates immune and inflammatory responses in the lung. We have shown previously that SP-D plays a protective role in a mouse model of allergic airway inflammation. Here we studied the role and regulation of SP-D in O3-induced inflammatory changes in the lung. METHODS: To evaluate the effects of O3 exposure in mouse strains with genetically different expression levels of SP-D we exposed Balb/c, C57BL/6 and SP-D knockout mice to O3 or air. BAL cellular and cytokine content and SP-D levels were evaluated and compared between the different strains. The kinetics of SP-D production and inflammatory parameters were studied at 0, 2, 6, 12, 24, 48, and 72 hrs after O3 exposure. The effect of IL-6, an O3-inducible cytokine, on the expression of SP-D was investigated in vitro using a primary alveolar type II cell culture. RESULTS: Ozone-exposed Balb/c mice demonstrated significantly enhanced acute inflammatory changes including recruitment of inflammatory cells and release of KC and IL-12p70 when compared with age- and sex-matched C57BL/6 mice. On the other hand, C57BL/6 mice had significantly higher levels of SP-D and released more IL-10 and IL-6. Increase in SP-D production coincided with the resolution of inflammatory changes. Mice deficient in SP-D had significantly higher numbers of inflammatory cells when compared to controls supporting the notion that SP-D has an anti-inflammatory function in our model of O3 exposure. IL-6, which was highly up-regulated in O3 exposed mice, was capable of inducing the expression of SP-D in vitro in a dose dependent manner. CONCLUSION: Our data suggest that IL-6 contributes to the up-regulation of SP-D after acute O3 exposure and elevation of SP-D in the lung is associated with the resolution of inflammation. Absence or low levels of SP-D predispose to enhanced inflammatory changes following acute oxidative stress.

Inhibition of Trimeresurus flavoviridis phospholipase A2 by lung surfactant protein A (SP-A).

A marked sequence homology has been noted between lung surfactant protein A (SP-A) and an inhibitor of phospholipase A2 (PLA2) isolated from the serum of Trimeresurus flavoviridis (Habu snake). This study evaluated the effect of SP-A on PLA2 activity from several sources. SP-A was isolated from bovine or rat lung surfactant by extraction with 1-butanol and octyl beta-D-glucopyranoside. The addition of SP-A produced a concentration-dependent inhibition of T. flavoviridis PLA2 that indicated non-competitive kinetics with Ki 5 micrograms/ml. Inhibition was reversed by heat inactivation, disulfide bond reduction or alkylation of SP-A, or by the presence of anti-SP-A antibody. Treatment of SP-A with endoglycosidase F or the presence of variation monosaccharides or lectins did not alter SP-A inhibition. Binding of PLA2 to SP-A was shown by ultrafiltration and was abolished by SP-A alkylation or the presence of SDS. The SP-A/PLA2 complex recovered from the ultrafilter had essentially no enzymatic activity, but activity was restored by treatment with mercaptoethanol. SP-A had no effect on activity of PLA2 from Naja naja, Crotalus atrox, or bovine pancreas. These results indicate that surfactant protein A selectively inhibits Trimeresurus phospholipase A2 activity and suggest that binding to the enzyme is the mechanism for inhibition.

Partial deficiency of surfactant protein B in an infant with chronic lung disease.

OBJECTIVE: To evaluate components of pulmonary surfactant and identify mutations in the surfactant protein B gene (SP-B) of a term infant with severe respiratory distress and chronic lung disease. PATIENT AND TESTING: Respiratory distress developed in an infant delivered at term, and he required extracorporeal bypass support for 2 weeks. Until his unexpected death at 9.5 months, he was ventilator and oxygen dependent and required continual dexamethasone therapy. Tracheobronchial lavage samples were analyzed for content of surfactant proteins (SPs), and DNA from blood samples were sequenced and analyzed by polymerase chain reaction restriction analysis for the presence of SP-B gene mutations. Surfactant lipid composition and function, the contents of SPs and their messenger RNAs (mRNAs), and the immunostaining pattern for SPs were determined in postmortem lung tissue. RESULTS: The lavage sample contained SP-A but not SP-B, and DNA restriction analysis indicated that the patient and his mother were heterozygous for the previously described 121ins2 mutation of SP-B. Postmortem lung tissue contained normal levels of SP-A and its mRNA, a low but detectable level of SP-B, and near normal content of SP-B mRNA. SP-C was abundant on staining, and some 6-kd precursor was present in tissue. A surfactant fraction was deficient in phosphatidylglycerol and was not surface active. On DNA sequencing, a point mutation was found in exon 7 of the patient's SP-B gene allele without the 121ins2 mutation, resulting in a cysteine for arginine substitution, and the father was a carrier for the same mutation. CONCLUSIONS: We describe a patient who is a compound heterozygote with a new mutation and only a partial deficiency of SP-B. Some forms of inherited SP-B deficiency may have low expression of immunoreactive and possibly functional SP-B with milder lung disease and longer survival. These infants may benefit from glucocorticoid therapy and may not develop antibodies to SP-B after either lung transplant or gene therapy.

Recurrent pneumothorax in AIDS patients with Pneumocystis pneumonia. A clinicopathologic report of three cases and review of the literature.

Spontaneous pneumothorax associated with Pneumocystis carinii pneumonia (PCP) in AIDS patients has been reported with increasing frequency; however, little is known about the causative histopathology. In the past year, we treated three patients with documented PCP subsequently complicated by multiple spontaneous pneumothoraces. All patients underwent open surgical repair. In contrast to traditional pathologic findings of PCP in AIDS, histologic sections of lung from each patient consistently demonstrated an extensive interstitial inflammatory process with destruction of lung tissue primarily involving the periphery of the lung. Subpleural necrosis with bleb formation as well as bullous changes persisted even in the absence of an alveolar filling process. We conclude that the mechanism for pneumothorax in PCP is spontaneous rupture of necrotic lung tissue occurring in a subgroup of AIDS patients in which the interstitial inflammatory response to Pneumocystis has been accelerated.

Inhibition of cellular processing of surfactant protein C by drugs affecting intracellular pH gradients.

Surfactant protein C (SP-C) is a hydrophobic protein synthesized and secreted exclusively by alveolar type II cells through proteolysis of a 21-kDa propeptide (SP-C21) to produce the 3.7-kDa surface active form. Previous studies from this laboratory have demonstrated that early processing of proSP-C involves extensive intracellular proteolysis of the COOH terminus of proSP-C21 in subcellular compartments, which include the acidic type II cell-specific subcellular organelle, the lamellar body. (Beers, M. F., Kim, C. Y., Dodia, C., and Fisher, A. B.(1994) J. Biol. Chem. 269, 20318-20328). The role of intracellular pH gradients in SP-C processing was studied in freshly isolated rat type II cells. Using vital fluorescence microscopy, the pH indicator acridine orange (AO) identified intense fluorescence staining of acidic cytoplasmic vesicles within fresh type II cells. The AO vesicular staining pattern was similar in cells labeled with the lamellar body marker phosphine 3R and the phospholipid dye nile red. AO fluorescence was quenched by the addition of a membrane-permeable weak base, methylamine. Immunoprecipitation of cell lysates with anti-proSP-C antisera following pulse-chase labeling (0-2 h) with 35S-Translabel demonstrated rapid synthesis of 35S-proSP-C21 with a time-dependent appearance of 16- and 6-kDa intermediates (SP-C16 and SP-C6). Tricine polyacrylamide gel electrophoresis analysis of organic extracts of cell lysates showed time-dependent appearance of mature SP-C3.7. The addition of 5 mM methylamine significantly blocked the post-translational processing of proSP-C resulting in disruption of normal precursor-product relationships and inhibition of SP-C3.7 formation. Methylamine-treated cells exhibited slow accumulation of SP-C16 and SP-C6, a persistence of SP-C21, and an absence of SP-C3.7 for the duration of the chase period. The lysosomotropic agent chloroquine, the proton ionophore monensin, and bafilomycin A1, a specific vacuolar H+-ATPase inhibitor, each caused inhibition of proSP-C processing in a similar manner. These results demonstrate that normal post-translational proteolysis of proSP-C occurs in acidic intracellular compartments, which include the lamellar body, and that complete processing to SP-C3.7 is dependent upon maintenance of transmembrane pH gradients by a vacuolar H+-ATPase.

Surfactant protein C: a review of its unique properties and metabolism.

Traditionally, our thinking about surfactant proteins has centered around their effects on the biophysical properties of surfactant phospholipids. It is now apparent that the three major surfactant proteins (SP-A, SP-B, and SP-C) are a biochemically and functionally diverse group of mammalian peptides. Accumulated data suggest that they have roles beyond modulation of alveolar surface tension. SP-C is a 33-35 amino acid peptide found in organic extracts of pulmonary surfactant. In part, because of its extreme hydrophobicity, a full understanding of SP-C is presently incomplete. Progress to date has included evaluation of the biophysical properties and investigations of the SP-C gene, including studies of the SP-C promoter. This review describes the unique structural and functional properties of the SP-C molecule and summarizes available data on its molecular biology and metabolism. Studies from literature show that SP-C represents a physiologically important peptide with novel structural properties; namely, extreme hydrophobicity, an alpha-helical membrane spanning region, and a unique posttranslational modification: palmitoylation. From data on similarly modified proteins, we propose that the properties of SP-C, including the covalent addition of palmitic acid, render it capable of being targeted to and interacting with specific cell membranes. A complete understanding of SP-C, especially with regard to its metabolism and function, may require a reorientation of our thinking to consider SP-C as a membrane peptide and not just as a "surfactant protein."

Synthesis and processing of hydrophobic surfactant protein C by isolated rat type II cells.

Surfactant protein C (SP-C) is a 3.7-kDa hydrophobic peptide isolated from organic extracts of pulmonary surfactant which is secreted by alveolar type II cells after synthesis and posttranslational processing of a 21-kDa proSP-C peptide (SP-C21). Previously characterized epitope-specific proSP-C antisera were used to study early proteolytic steps of proSP-C processing by adult rat type II cells. Western blotting and immunocytochemistry using anti-NPROSP-C (epitope = Met10-Glu23) each demonstrated marked attenuation of proSP-C protein expression by culture on plastic. Processing was therefore studied by metabolic labeling of freshly isolated type II cells maintained in suspension in serum-free media. With the use of anti-NPROSP-C, immunoprecipitation of cell lysates continuously labeled for 4 h with [35S]methionine demonstrated radiolabeled bands of M(r) 21, 16, and 10-6,000 while anti-CTERMSP-C (epitope = Ser149-Ser166) failed to detect 35S-bands of M(r) < 16,000. Pulse-chase studies demonstrated synthesis of 35S-proSP-C21 with a time-dependent dependent appearance of 16-kDa and 10- to 6-kDa forms which was blocked by addition of brefeldin A. SP-C precursors were not detected in the media. Quantitative analysis of the major bands by direct beta-counting indicated a precursor-product relationship between SP-C21 and SP-C16. These results demonstrate the utility of freshly isolated type II cells for characterization of SP-C synthetic pathways and show that early proSP-C processing events include synthesis of a 21-kDa primary translation product followed by extensive intracellular proteolysis of the proSP-C COOH-terminal in subcellular compartments of type II cells which are distal to the trans-Golgi network.

Binding and uptake of surfactant protein B by alveolar type II cells.

Surfactant protein B (SP-B, mol wt 9,000, reduced) is a low-molecular-weight hydrophobic protein found in organic extracts of lung surfactant. The interaction of iodinated bovine SP-B (125I-SP-B) and isolated rat alveolar type II cells was examined. The association of SP-B with the lung cells was time and temperature dependent; type II cells exhibited time-dependent binding (at 4 degrees C) and uptake (at 37 degrees C) of SP-B. Binding of phospholipid-poor 125I-SP-B was linearly related to the external SP-B concentration from 0.25 to 60 microgram/ml and was not inhibited by a 60-fold excess of unlabeled SP-B. However, the binding of 125I-SP-B reconstituted with bovine surfactant or with phospholipid-containing liposomes occurred through a high-affinity, saturable process and could be inhibited with unlabeled SP-B. By Scatchard analysis, half-maximum binding in the presence of surfactant occurred at 3.1 +/- 0.7 micrograms SP-B/ml. Saturable binding of SP-B reconstituted with surfactant also occurred with other cell types. The results indicate that SP-B was bound and internalized by type II cells. The apparent lack of specificity in the absence of phospholipid may have been due to the self-association of SP-B. The reconstitution of SP-B with phospholipid altered the binding of phospholipid-poor SP-B from a nonspecific process to a high-affinity process consistent with a cell surface binding site.

Synthesis and post-translational processing of surfactant protein C.

Traditional thinking about surfactant proteins has centered around their effects on the biophysical properties of surfactant phospholipids. Accumulated data now suggests that the four major surfactant proteins (SPs) are a biochemically and functionally diverse group of mammalian peptides that have function beyond modification of alveolar surface tension. Alveolar SP-C (SP-C3.7, Mr 21,000) is 35 amino acid peptide isolated from lung surfactant that is synthesized and processed from a 191-197 amino acid precursor (proSP-C21). Although its solubility in organic solvents and avidity for lipid membranes impart properties important for its biophysical activity, SP-C represents a structurally and functionally challenging protein for the alveolar type II cell that must synthesize and traffic the peptide through the regulated secretory pathway. Despite technical and analytical difficulties imposed by its unique structure, our current understanding of SP-C biosynthesis has evolved over the past 10 years. Recent data now require us to consider proSP-C21 as a hybrid molecule incorporating structural and functional features both of bitopic integral membrane proteins as well us more classically recognized propeptide hormones. Our article highlights major developments related to characterization of molecular and cellular mechanisms underlying expression, post-translational processing, and targeting of proSP-C21 that result in production of secreted SP-C3.7.

Differential extraction for the rapid purification of bovine surfactant protein B.

Surfactant protein B (SP-B), a peptide found in organic solvent extracts of mammalian surfactant, has been isolated from surfactant previously by column chromatography and/or preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE). We have developed a method for isolation of SP-B from bovine surfactant utilizing differential organic extraction. Dried surfactant, isolated from lavage of excised cow lungs, was delipidated by extraction with diisopropyl ether-butanol (3:2). The aqueous layer, containing surfactant proteins, was dried and then was sequentially extracted with diethyl ether-ethanol (3:1) and CHCl3:MeOH:HCl (3:2:0.005 N). SP-B partitioned into chloroform-methanol, which was evaporated under N2. Purified SP-B, quantitated by Coomassie dye binding, represented 1% (wt/wt) of the original surfactant with a final phospholipid-to-protein ratio less than 1. Silver-stained SDS/PAGE of the SP-B extract revealed a single band at 9 kDa (reduced) and 18 kDa (nonreduced), which by immunoblotting reacted strongly with monospecific anti-SP-B antibody. Amino acid sequence analysis confirmed the presence of NH2 and N-1 terminal sequences of bovine SP-B. This procedure offers a rapid, reliable method for isolation of purified SP-B from whole surfactant.

Synthetic processing of surfactant protein C by alevolar epithelial cells. The COOH terminus of proSP-C is required for post-translational targeting and proteolysis.

Surfactant protein C (SP-C) is synthesized by alveolar type II cells as a 21-kDa propeptide (proSP-C21) which is proteolytically processed in subcellular compartments distal to the trans-Golgi network to yield a 35-residue mature form. Initial synthetic processing events for SP-C include post-translational cleavages of the COOH terminus of proSP-C21 yielding two intermediates (16 and 6 kDa). To test the role of specific COOH-terminal domains in intracellular targeting and proteolysis of proSP-C21, synthesis and processing of SP-C was evaluated using a lung epithelial cell line (A549) transfected with a eukaryotic expression vector containing either the full-length cDNA for rat SP-C (SP-Cwt) or one of six polymerase chain reaction (PCR)-generated COOH terminally truncated forms (SP-C1-185, SP-C1-175, SP-C1-147, SP-C1-120, SP-C1-72, and SP-C1-59). Using in vitro transcription/translation, each of the seven constructs produced a 35S-labeled product of appropriate length which could be immunoprecipitated by epitope specific proSP-C antisera. Immunoprecipitation of 35S-labeled A549 cell lysates from SP-Cwt transfectants demonstrated rapid synthesis of [35S]proSP-C21 with processing to SP-C16 and SP-C6 intermediates via cleavages of the COOH-terminal propeptide. Both the intermediates as well as the kinetics of processing in A549 cells were similar to that observed in rat type II cells. In contrast, constructs SP-C1-185, SP-C1-175, SP-C1-147, SP-C1-120, SP-C1-72, and SP-C1-59 were each translated but degraded without evidence of proteolytic processing. Fluorescence immunocytochemistry identified proSP-Cwt in cytoplasmic vesicles of A549 cells while all COOH-terminal deletional mutants were restricted to an endoplasmic reticulum/Golgi compartment identified by co-localization with fluorescein isothiocyanate-concanavalin A. We conclude that SP-Cwt expressed in A549 cells is directed to cytoplasmic vesicles where it is proteolytically processed in a manner similar to native type II cells and that amino acids Cys186-Ile194 located at the COOH terminus of proSP-C21 are necessary for correct intracellular targeting and subsequent cleavage events.

H+ ATPase of chromaffin granules. Kinetics, regulation, and stoichiometry.

The chromaffin granule ATPase mediates an inwardly directed transport of H+ against concentration gradients, thereby forming and maintaining an electrochemical transmembrane H+ gradient. The kinetics of this ATPase, its activity modulation by changes in electrochemical H+ gradients, and the stoichiometry between H+ transport and ATP hydrolysis were studied in intact bovine chromaffin granules, resealed chromaffin granule ghosts, and highly purified fragmented chromaffin granule membranes. In fragmented membranes the H+ ATPase has a KM for ATP of 69 microM, a maximum of activity at pH 7.3, and a Vmax of 111 nmol/min/mg of protein at 20 degrees C. Trimethyl tin inhibits the ATPase at much lower concentrations than dicyclohexylcarbodiimide, whereas oligomycin, reserpine, and other inhibitors were without effect. In intact chromaffin granules, the ATPase activity was stimulated up to 300% by collapsing the H+ transmembrane gradients. H+/ATP stoichiometry was measured in resealed chromaffin ghosts devoid of ATP and catecholamines under conditions where no net pH changes occur upon ATP hydrolysis. After addition of ATP, the rates of H+ accumulation in the ghosts and ATP hydrolysis were both linear for about 60-100 s, and the ratio of H+ to ATP was 1.71. These data indicate that the H+ ATPase of chromaffin granules has both kinetic similarities and dissimilarities with other known H+ ATPases. The regulation by changes in H+ gradients and the fixed H+/ATP ratio of this ATPase is further evidence of its primary role in establishing electrogenic H+ translocation and H+ gradients in chromaffin granules.

Evidence for an ascorbate shuttle for the transfer of reducing equivalents across chromaffin granule membranes.

Adrenal chromaffin granules must shuttle reducing equivalents from the cytosol inward to reduce ascorbic acid oxidized during norepinephrine biosynthesis by intragranular dopamine-beta-hydroxylase. A transmembrane electron shuttle between the external (cytosolic) and intragranular ascorbate pools was demonstrated in vitro in intact bovine chromaffin granules undergoing tyramine- or dopamine-stimulated dopamine-beta-hydroxylase turnover. Incubation of intact chromaffin granules with tyramine results in a time-dependent decrease in reduced intragranular ascorbate and production of octopamine. The rate of ascorbate oxidation is a function of the extragranular concentrations of tyramine over the range 50 microM to 2 mM and is 95% inhibited by addition of the dopamine-beta-hydroxylase inhibitor disulfiram. The stoichiometry of octopamine synthesized/ascorbate oxidized closely approximates unity. The presence of extragranular dopamine also induces oxidation of intragranular ascorbate which is inhibited by blocking dopamine transport with reserpine. On the other hand, incubation with octopamine, which is also transported by the granules, causes no net decrease in reduced intragranular ascorbate. The presence of 400 microM extragranular ascorbate abolishes the observed tyramine-induced intragranular ascorbate oxidation. The addition of ascorbate extragranularly 30 min after addition of tyramine reverses the oxidation of intragranular ascorbate. The measurement of [14C]ascorbate distribution ratios in granule pellets and supernatants indicates that there is no transmembrane transport of ascorbate. Extravesicular NADH had no significant effect on matrix ascorbate levels during beta-hydroxylation. These data provide new in vitro evidence that chromaffin granules shuttle reducing equivalents inwardly from an extra- to an intravesicular ascorbate pool and that cytosolic ascorbate is the source of the intragranular reducing equivalents required during norepinephrine biosynthesis.

Synthesis of type II cell lamellar body lysozyme-15 kD protein (lbl-15) by perfused rat lung.

Isolated alveolar type II pneumocytes of the rat have been shown to secrete a 14 to 15 kD protein that has some sequence homology and immunoreactivity with lysozyme. Using immunochemical analyses of rat lung subcellular fractions and 35S metabolic labeling of isolated perfused lung preparations, we studied the subcellular distribution and synthetic pathway for this protein. SDS-PAGE and Western blotting of lamellar bodies (LB) using a polyclonal anti-human lysozyme (anti-HLZ) demonstrated a single band at 15 kD that was significantly enriched over rat lung homogenates, isolated lysosomes, and type II cell lysates. This 15 kD protein isolated from LB by immunoprecipitation with anti-HLZ also demonstrated functional lysozyme activity and was termed lamellar body lysozyme (lbl-15). Analysis of LB and surfactant (SF) isolated from 12 separate perfused lung preparations labeled for 6 h with 35S-labeled amino acids demonstrated that lbl-15 represented a significant portion of the radiolabeled LB proteins (5.9% of total LB radioactivity). Lamellar bodies and an extracellular fraction (surfactant) obtained from rat lungs perfused with 35S-methionine-cysteine for varying times both showed time-dependent appearance of lbl-15. At all time points, the specific activity of lbl-15 was greater in LB than in SF. The kinetics for appearance of lbl-15 in LB and SF was similar to that for surfactant protein A. These results indicate that in the rat lung, type II cells synthesize a 15 kD protein (lbl-15) that is secreted into the alveolar space via an organellar pathway involving LB.(ABSTRACT TRUNCATED AT 250 WORDS)

Pneumocystis carinii glycoprotein A inhibits surfactant phospholipid secretion by rat alveolar type II cells.

Pneumocystis carinii pneumonia (PCP) remains a major cause of morbidity in AIDS. The pathogenesis of PCP is poorly understood, but evidence of surfactant abnormalities is mounting. The role of the major surface glycoprotein of P. carinii, gpA, in producing surfactant abnormalities was investigated. Rat type II pneumocytes were incubated with [3H]choline, purified gpA, and modulators. Lipid was extracted, and [3H]dipalmitoyl phosphatidylcholine (DPPC) secretion was calculated. Contaminating endotoxin had no effect on DPPC secretion. gpA inhibited basal and ATP-stimulated DPPC secretion in a dose- and time-dependent manner. An anti-gpA monoclonal antibody attenuated inhibition of DPPC secretion. Unglycosylated recombinant gpA inhibited secretion, suggesting that functional activity resides in the protein moiety of gpA. These results suggest that gpA is a specific trigger for abnormalities of surfactant lipids in PCP. This is a unique role for a microbial product in disease pathogenesis and a potentially exploitable therapeutic target.

Surfactant protein B processing in human fetal lung.

Surfactant protein B (SP-B8), an 8-kDa hydrophobic protein essential for surfactant and normal lung function, is produced from the intracellular processing of preproSP-B. To characterize SP-B processing in human type 2 cells, we used human fetal lung in explant culture and polyclonal antibodies to human SP-B8 (Phe201-Met279) and to specific epitopes within the NH2- and COOH-terminal propeptide domains (Ser145-Leu160, Gln186-Gln200, and Gly284-Ser304). Western blot analysis revealed a novel intermediate at approximately 9 kDa, representing mature SP-B8, with a residual NH2-terminal peptide of approximately 10 amino acids. Pulse-chase studies showed a precursor-product relationship between the 9- and 8-kDa forms. During differentiation of type 2 cells in explant culture, the rate of proSP-B conversion to 25-kDa intermediate remained constant, whereas the rate of 25-kDa intermediate conversion to SP-B8 increased, resulting in a net increase in tissue SP-B8. Dexamethasone did not affect the rate of proSP-B processing but markedly enhanced the rate of SP-B8 accumulation. We conclude that NH2-terminal propeptide cleavage of proSP-B is a multistep process and that more distal processing events are rate limiting and both developmentally and hormonally regulated.

Biosynthesis of surfactant protein C: characterization of aggresome formation by EGFP chimeras containing propeptide mutants lacking conserved cysteine residues.

Surfactant protein C (SP-C) is a lung-specific secreted protein, which is synthesized as a 21-kDa propeptide (SP-C(21)) and then proteolytically processed as a bitopic transmembrane protein in subcellular compartments distal to the medial Golgi to produce a 3.7 kDa mature form. We have shown that initial processing of SP-C(21) involves two endoproteolytic cleavages of the C terminus and that truncation of nine amino acids from the C-flanking peptide resulted in retention of mutant protein in proximal compartments. Because these truncations involved removal of a conserved cysteine residue (Cys(186)), we hypothesized that intralumenal disulfide-mediated folding of the C terminus of SP-C(21) is required for intracellular trafficking. To test this, cDNA constructs encoding heterologous fusion proteins consisting of enhanced green fluorescent protein (EGFP) attached to the N terminus of wild-type rat proSP-C (EGFP/SP-C(1-194)), C-terminally deleted proSP-C (EGFP/SP-C(1-185); EGFP/SP-C(1-191)) or point mutations of conserved cysteine residues (EGFP/SP-C(C122G); EGFP/SP-C(C186G); or EGFP/SP-C(C122/186G)) were transfected into A549 cells. Fluorescence microscopy revealed that transfected EGFP/SP-C(1-194) and EGFP/SP-C(1-191 )were expressed in a punctate pattern within CD-63 positive, EEA-1 negative cytoplasmic vesicles. In contrast, EGFP/SP-C(1-185), EGFP/SP-C(C122G), EGFP/SP-C(C186G) and EGFP/SP-C(C122/186G) were expressed but retained in a juxtanuclear compartment that stained for ubiquitin and that contained (&ggr;)-tubulin and vimentin, consistent with expression in aggresomes. Treatment of cells transfected with mutant proSP-C with the proteasome inhibitor lactacysteine enhanced aggresome formation, which could be blocked by coincubation with nocodazole. Western blots using a GFP antibody detected a single form in lysates of cells transfected with EGFP/SP-C cysteine mutants, without evidence of smaller degradation fragments. We conclude that residues Cys(122) and Cys(186) of proSP-C are required for proper post-translational trafficking. Mutation or deletion of one or both of these residues results in misfolding with mistargeting of unprocessed mutant protein, leading to formation of stable aggregates within aggresomes.