Antisense inhibition of surfactant protein A decreases tubular myelin formation in human fetal lung in vitro

Surfactant protein A (SP-A) is the most abundant of the surfactant-associated proteins. SP-A is involved in the formation of tubular myelin, the modulation of the surface tension-reducing properties of surfactant phospholipids, the metabolism of surfactant phospholipids, and local pulmonary host defense. We hypothesized that elimination of SP-A would alter the regulation of SP-B gene expression and the formation of tubular myelin. Midtrimester human fetal lung explants were cultured for 3-5 days in the presence or absence of an antisense 18-mer phosphorothioate oligonucleotide (ON) complementary to SP-A mRNA. After 3 days in culture, SP-A mRNA was undetectable in antisense ON-treated explants. After 5 days in culture, levels of SP-A protein were also decreased by antisense treatment. SP-B mRNA levels were not affected by the antisense SP-A ON treatment. However, there was decreased tubular myelin formation in the antisense SP-A ON-treated tissue. We conclude that selective elimination of SP-A mRNA and protein results in a decrease in tubular myelin formation in human fetal lung without affecting SP-B mRNA. We speculate that SP-A is critical to the formation of tubular myelin during human lung development and that the regulation of SP-B gene expression is independent of SP-A gene expression.     

Isolation and characterization of the cDNA for pulmonary surfactant-associated protein-B (SP-B) in the rabbit

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.     

Distribution and surfactant association of carcinoembryonic cell adhesion molecule 6 in human lung

Carcinoembryonic cell adhesion molecule 6 (CEACAM6) is a glycosylated, glycophosphatidylinositol-anchored protein expressed in epithelial cells of various primate tissues. It binds gram-negative bacteria and is overexpressed in human cancers. CEACAM6 is associated with lamellar bodies of cultured type II cells of human fetal lung and protects surfactant function in vitro. In this study, we characterized CEACAM6 expression in vivo in human lung. CEACAM6 was present in lung lavage of premature infants at birth and increased progressively in intubated infants with lung disease. Of surfactant-associated CEACAM6, ～80% was the fully glycosylated, 90-kDa form that contains the glycophosphatidylinositol anchor, and the concentration (3.9% of phospholipid for adult lung) was comparable to that for surfactant proteins (SP)-A/B/C. We examined the affinity of CEACAM6 by purification of surfactant on density gradient centrifugation; concentrations of CEACAM6 and SP-B per phospholipid were unchanged, whereas levels of total protein and SP-A decreased by 60%. CEACAM6 mRNA content decreased progressively from upper trachea to peripheral fetal lung, whereas protein levels were similar in all regions of adult lung, suggesting proximal-to-distal developmental expression in lung epithelium. In adult lung, most type I cells and ～50% of type II cells were immunopositive. We conclude that CEACAM6 is expressed by alveolar and airway epithelial cells of human lung and is secreted into lung-lining fluid, where fully glycosylated protein binds to surfactant. Production appears to be upregulated during neonatal lung disease, perhaps related to roles of CEACAM6 in surfactant function, cell proliferation, and innate immune defense.     

Synthesis of surfactant components by cultured type II cells from human lung

We examined the effect of monolayer culture on surfactant phospholipids and proteins of type II cells isolated from human adult and fetal lung. Type II cells were prepared from cultured explants of fetal lung (16-24 weeks gestation) and from adult surgical specimens. Cells were maintained for up to 6 days on plastic tissue culture dishes. Although incorporation of [methyl-3H]choline into phosphatidylcholine (PC) by fetal cells was similar on day 1 and day 5 of culture, saturation of PC fell from 35 to 26%. In addition, there was decreased distribution of labeled acetate into PC, whereas distribution into other phospholipids increased or did not change. The decrease in saturation of newly synthesized PC was not altered by triiodothyronine (T3) and dexamethasone treatment or by culture as mixed type II cell/fibroblast monolayers. The content of surfactant protein SP-A (28-36 kDa) in fetal cells, as measured by ELISA and immunofluorescence microscopy, rose during the first day and then fell to undetectable levels by the fifth. Synthesis of SP-A, as measured by [35S]methionine labeling and immunoprecipitation, was detectable on day 1 but not thereafter. Levels of mRNAs for SP-A and for the two lipophilic surfactant proteins SP-B (18 kDa) and SP-C (5 kDa) fell with half-times of maximally 24 h. In contrast, total protein synthesis measured by [35S]methionine incorporation increased and then plateaued. In adult cells, the content of SP-A and its mRNA decreased during culture, with time-courses similar to those for fetal cells. We conclude that in monolayer culture on plastic culture dishes, human type II cells lose their ability to synthesize both phospholipids and proteins of surfactant. The control of type II cell differentiation under these conditions appears to be at a pretranslational level.     

Synthesis of surfactant-associated protein, 35,000 daltons, in fetal lung

The major human pulmonary surfactant-associated protein of 35,000 daltons (Da) (SAP-35), consists of a group of related proteins of 27,000-36,000 Da, with isoelectric points ranging from pH 4.6 to 5.2. SAP-35 precursors were identified by immunoprecipitation of protein products of in vitro translation of normal adult human poly(A)+ mRNA with human SAP-35 antiserum. The translation products nearly comigrated with the most basic components of alveolar SAP-35 (mol mass = 24,500-27,000 Da). Processing of the primary translation products by canine pancreatic microsomal membranes increased their apparent molecular weight to 29,000-30,000-Da forms, which were sensitive to endoglycosidase F, suggesting the addition of asparagine-linked oligosaccharides to the molecules. A smaller protein of 24,500 Da was generated during treatment with canine microsomal membranes likely representing cleavage of a signal peptide. SAP-35 was not detected in explants of [35S]methionine-labeled fetal lung (20-24 wk gestation) after 1 day of culture or immunoprecipitates of in vitro translated poly(A)+ mRNA isolated from fetal human lung. However, after 3-5 days of organ culture, synthesis of SAP-35 was readily detected by immunoprecipitation of [35S] methionine-labeled tissue. Fully sialylated (neuraminidase-sensitive forms) comigrated with fully glycosylated SAP-35 isolated from human surfactant. High mannose (endoglycosidase H-sensitive precursors) were also synthesized by the organ cultures and were distinct from the secreted form in surfactant. Synthesis of surfactant-associated SAP-35 and its precursors was induced in association with morphological maturation of the type II epithelial cell during organ culture of human fetal lung.     

Ontogeny of surfactant apoprotein D, SP-D, in the rat lung

Surfactant protein D (SP-D) is a collagenous surfactant-associated glycoprotein synthesized by alveolar type II cells. Antiserum against rat SP-D was raised in rabbits and an enzyme-linked immunosorbant assay (ELISA) has been developed using anti-rat SP-D IgG. In the present study we examined the developmental profile of SP-D in the rat lung compared with that of surfactant protein A (SP-A). SP-A content in the lungs increased during late gestation and reached its maximum on day 1 of neonate, and then gradually decreased until at least day 5. SP-D content during early gestation was less than 10 ng/mg protein until day 18, but on day 19 there was a 4-fold increase in SP-D (compared to that on day 18). It increased twice between day 21 and the day of birth, when it reached the adult level of 250 ng/mg protein, which is about one fourth that of the adult level of SP-A. Unlike SP-A there seemed to be no decrease in SP-D content after birth. These results demonstrate that SP-D is regulated developmentally as are the other components of surfactant, but the inconsistency in the developmental profiles of SP-A and SP-D suggests that these proteins may play different roles in lung maturation.     

Differential effects of glucocorticoid on expression of surfactant proteins in a human lung adenocarcinoma cell line

Synthesis of pulmonary surfactant-associated glycoproteins of Mr 28,000-36,000 (SP-A) and Mr 42,000-46,000 (proSP-B) has been identified in a continuous cell line derived from a human lung adenocarcinoma. SP-A was detected by immunoblot analysis, ELISA assay and by [35S]methionine labelling of the cells. SP-A was secreted into the media as an endoglycosidase F sensitive glycoprotein which co-migrated with the isoforms of SP-A identified in human lavage fluid by 2D-IEF-SDS-PAGE. Hybridization of cellular RNA with SP-A-specific cDNA identified an abundant 2.2 kb mRNA species, identical to that observed in human lung. SP-A RNA and protein content were markedly inhibited by dexamethasone in a dose-dependent fashion. Under identical culture conditions, synthesis of a distinct surfactant protein, SP-B, was markedly stimulated by the glucocorticoid. The SP-B precursor was secreted into the media as heterogeneous Mr 42,000-46,000 protein, pI 4.6-5.1, and was sensitive to endoglycosidase F. Synthesis of proSP-B was enhanced by the glucocorticoid in a dose-dependent fashion and was associated with increased SP-B mRNA of 2.0 kb detected by Northern blot analysis. The cell line secreted proSP-B as Mr 42,000-46,000 glycosylated protein and did not process the precursor to the Mr 7000-8000 surfactant peptide. In summary, a human adenocarcinoma cell line has been identified which synthesizes and secretes two surfactant-associated proteins, SP-A and proSP-B. Glucocorticoid enhanced SP-B but inhibited SP-A expression in this cell line. The identification of a continuous cell line secreting surfactant proteins may be useful in the study of synthesis and secretion of these important proteins and for production of the proteins for clinical uses.     

Regulation of human pulmonary surfactant protein gene expression by 1alpha,25-dihydroxyvitamin D3

1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] has been reported to stimulate lung maturity, alveolar type II cell differentiation, and pulmonary surfactant synthesis in rat lung. We hypothesized that 1,25(OH)(2)D(3) stimulates expression of surfactant protein-A (SP-A), SP-B, and SP-C in human fetal lung and type II cells. We found that immunoreactive vitamin D receptor was detectable in fetal lung tissue and type II cells only when incubated with 1,25(OH)(2)D(3). 1,25(OH)(2)D(3) significantly decreased SP-A mRNA in human fetal lung tissue but did not significantly decrease SP-A protein in the tissue. In type II cells, 1,25(OH)(2)D(3) alone had no significant effect on SP-A mRNA or protein levels but reduced SP-A mRNA and protein in a dose-dependent manner when the cells were incubated with cAMP. SP-A mRNA levels in NCI-H441 cells, a nonciliated bronchiolar epithelial (Clara) cell line, were decreased in a dose-dependent manner in the absence or presence of cAMP. 1,25(OH)(2)D(3) had no significant effect on SP-B mRNA levels in lung tissue but increased SP-B mRNA and protein levels in type II cells incubated in the absence or presence of cAMP. Expression of SP-C mRNA was unaffected by 1,25(OH)(2)D(3) in lung tissue incubated +/- cAMP. These results suggest that regulation of surfactant protein gene expression in human lung and type II cells by 1,25(OH)(2)D(3) is not coordinated; 1,25(OH)(2)D(3) decreases SP-A mRNA and protein levels in both fetal lung tissue and type II cells, increases SP-B mRNA and protein levels only in type II cells, and has no effect on SP-C mRNA levels.     

Synthesis of surfactant-associated glycoprotein A by rat type II epithelial cells. Primary translation products and post-translational modification

Surfactant-associated glycoproteins A, 38 (A3), 32 (A2) and 26 (A1) kDa, pI (4.2-4.8), were identified as related proteins present in surfactant isolated from rat lung lavage fluid. Differences in size and charge among surfactant-associated glycoproteins A were related to differences in glycosylation as determined by reduction of the larger forms (38 and 32 kDa) to 26 kDa by endoglycosidase F and by increased isoelectric points of the glycosylated forms after treatment with neuraminidase. Synthesis and secretion of surfactant-associated glycoproteins A and precursors were demonstrated in purified rat Type II epithelial cells by immunoprecipitation of [35S]methionine-labelled proteins with anti-surfactant-associated glycoprotein A antisera. In pulse-chase experiments, labelled proteins 26-34 kDa, appeared within 10 min and smaller forms co-migrated with surfactant-associated glycoprotein A from alveolar lavage. The relative abundance of the larger molecular mass forms (30-34 kDa, pI 4.8) increased at later times up to 3 h. More acidic mature forms, which co-migrated with surfactant-associated glycoproteins A2 and A3 in surfactant (38 and 32 kDa), were readily detectable in the media, but were not abundant forms in lysates of labelled Type II cells after 1-3 h of incubation. Primary translation products of surfactant-associated glycoprotein A were immunoprecipitated with monospecific anti-surfactant-associated glycoprotein A antiserum after in vitro translation of poly(A)+ mRNA isolated from adult rat lung. The immunoprecipitated translation product migrated at 26 kDa, pI 4.8, and migrated slightly faster than surfactant-associated glycoprotein A1 from surfactant. Treatment of surfactant-associated glycoprotein A with bacterial collagenase resulted in proteolytic fragments 23-20 kDa, pI 4.2-4.8, which no longer underwent sulfhydryl-dependent cross-linking, suggesting that the collagen-like domain was required for the sulfhydryl-dependent oligomerization. Surfactant-associated glycoproteins A are synthesized by rat Type II epithelial cells as pre-proteins, 26-34 kDa. Larger forms result primarily from N-linked glycosylation of the 26 kDa primary translation product. Mature, more acidic forms result from further addition of sialic acid.     

肺表面活性物质相关蛋白A与肺部免疫防御研究进展

肺表面活性物质相关蛋白A（SP—A）是一种高度保守的亲水性糖蛋白,属于C-型凝素家族成员,相对分子质量为29～36kDa。SP—A是肺部重要的天然免疫防御分子,在肺的局部防御和天然免疫反应中起着十分重要的作用。它不仅可调节局部免疫和炎症反应、调理吞噬作用,还可凝集病原微生物、影响趋化作用及促进杀菌作用等。     

Proteomic analysis of human bronchoalveolar lavage fluid: expression profiling of surfactant-associated protein A isomers derived from human pulmonary alveolar proteinosis using immunoaffinity detection

Human bronchoalveolar lavage fluid (BALF) proteins from pulmonary alveolar proteinosis (PAP) obtained by washing the epithelial lining of the lung with phosphate-buffered saline, were separated using high resolution two-dimensional gel electrophoresis (2-DE) under denaturing and reducing conditions. By Western blotting, the proteins were transferred from polyacrylamide gel onto a chemical resilient membrane. The surfactant-associated protein A (SP-A) isomers were then identified with enhanced chemiluminescence detection (ECL) using antibody-antigen reaction. Some of the gels were treated with silver staining after 2-DE. The molecular masses of SP-A isomers in BALF from PAP ranged from 20.5 to 26, 26 to 32, and 32 to 42 kDa, respectively; and isoelectric points (pI) were in pH range of 4.5-5.4 under denaturing and reducing conditions. In the mass range of 20.5-26 kDa and pI of 4.5-5.4, there were five isomers, and in mass range of 26-32 kDa and pI of 4.5 to 5.4, there were at least eight isomers on the ECL detection film. However, in the mass range of 32-42 kDa and pI of 4.5-5.4, there were three isomers separated one from another but there was also a cluster of overlapping spots on the ECL detection film. Thus, this communication describes a characteristic 2-DE pattern of SP-A isomers in BALF from PAP as follows. (1) The five isomers of mass 20.5-26 kDa and pI of 4.5-5.4; (2) the eight isomers of mass 26-32 kDa and pI of 4.5-5.4; and (3) the three isomers of mass 32-42 kDa and pI of 4.5-5.4.     

Primary structure differences of human surfactant-associated proteins isolated from normal and proteinosis lung.

The primary structures of human pulmonary surfactant-associated proteins SP-A, SP-B and SP-C isolated from lung lavage of patients with alveolar proteinosis exhibit significant differences from lung surfactant proteins isolated from lungs of healthy individuals. In contrast to SP-A from normal lungs, proteinosis SP-A was shown by SDS gel electrophoresis to contain large amounts of unreducibly cross-linked beta chains. Specific primary structure modifications of SP-C and SP-B proteins were established by direct molecular weight and structural analysis, using [252Cf]plasma desorption mass spectrometry (PD/MS) as the principal method. In comparison to normal lung surfactant SP-B, proteinosis SP-B showed a significantly increased molecular weight by approx. 500 Da for the unreduced protein dimer. SP-C proteins from normal lungs were identified to possess a bis-cysteinyl-5,6-(thioester)palmitoylated structure, and to contain a frayed N-terminus resulting in two sequences of 34 and 35 amino acid residues. In contrast, SP-C from proteinosis patients was modified by (i) partial or even complete removal of palmitate residues and (ii) additional N-terminal proteolytic degradation. These results indicate the presence of pathophysiological structure modifications, which are likely to occur in the alveolar space, and may lead to a reduced surfactant function.     

Structural relationships of the major glycoproteins from human alveolar proteinosis surfactant

Alveolar proteinosis is a disease characterized by accumulation of proteinaceous material in the alveolar space of the lung. Two major collagenase-sensitive polypeptides, alveolar proteinosis peptides of 34 kDa kilodaltons (APP-34) and of 62 kDa (APP-62), were isolated from bronchioalveolar lavage of patients with alveolar proteinosis. These proteins co-purified during fast-performance liquid chromatography (FPLC) chromatofocusing and were separated from each other by electroelution following SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of these proteins demonstrated that both shared antigenic sites with the normal human surfactant-associated protein of Mr 34,000 (SAP-34) using both polyclonal and monoclonal antibodies generated against SAP-34. Removal of asparagine-linked oligosaccharides from the 34 kDa and 62 kDa alveolar proteinosis proteins with endoglycosidase F resulted in polypeptides of 28 kDa from APP-34 and 56 kDa from APP-62. Amino acid analysis and tryptic peptide maps of the electroeluted APP-34 and APP-62 proteins were essentially identical and similar to that previously reported for human SAP-34, supporting the likely relationship of APP-34 and APP-62 as monomer and dimer of the normal SAP-34. APP-34 and APP-62 were both sensitive to bacterial collagenase, yielding collagenase-resistant fragments of 21 kDa, similar in migration and amino acid composition to the fragment generated by collagenase digestion of normal human SAP-34. High molecular weight aggregates of APP-34 and APP-62 were the result of sulfhydryl-dependent and non-sulfhydryl-dependent cross-linking. A domain in the C-terminal non-collagenous portion of the molecules which forms sulfhydryl-dependent oligomers was identified. The two major polypeptides accumulating in the airway of patients with alveolar proteinosis are monomeric (34 kDa) and dimeric (62 kDa) forms of the major surfactant-associated glycoprotein, SAP-34.