Induction of surfactant protein in fetal lung. Effects of cAMP and dexamethasone on SAP-35 RNA and synthesis

Synthesis of surfactant-associated glycoprotein of Mr = 30,000-35,000 (SAP-35) was induced in explant culture of human fetal lung obtained from 8 to 24 weeks of gestation. SAP-35 synthesis and content increased markedly during 1-5 days in organ culture in association with the morphologic maturation of Type II epithelial cells and the appearance of lamellar bodies. [35S] Methionine labeling of the explants and subsequent immunoprecipitation of SAP-35 demonstrated distinct high-mannose precursors and sialylated SAP-35 forms as early in culture as SAP-35 synthesis was detectable. The increase in SAP-35 synthesis was associated with increased SAP-35 RNA of 2.1 kilobases as assessed by hybridization assay with [32P]cDNA specific for human SAP-35. Specific SAP-35 RNA increased during organ culture and both SAP-35 content and SAP-35 RNA increased in the absence of exogenous hormones in 2% carbon-stripped fetal calf serum. SAP-35 content and synthesis was stimulated by 8-Br-cAMP. Addition of 100 microM 8-Br-cAMP, enhanced both the concentration of SAP-35 protein and the SAP-35 RNA as assessed by hybridization assay. In contrast, treatment of the explants with dexamethasone was associated with decreased SAP-35 protein synthesis, SAP-35 content, and decreased SAP-35 RNA levels compared to untreated explants. Inhibition by dexamethasone occurred at all gestational ages tested, was dose-dependent, and detectable within 24-48 h during organ culture. Dexamethasone significantly inhibited both basal and cAMP-induced SAP-35 synthesis. Induction of pulmonary surfactant protein (SAP-35) synthesis during organ culture of human fetal lung was associated with increased SAP-35 RNA. SAP-35 synthesis and SAP-35 RNA were inhibited by dexamethasone and enhanced cAMP.     

Identification of canine pulmonary surfactant-associated glycoprotein A precursors

Surfactant-associated glycoproteins A were identified by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude surfactant from canine alveolar lavage: an unglycosylated form (protein A1), 27,000-28,000 daltons; glycoprotein A2, 32,000-34,000 daltons; and glycoprotein A3, 37,000-38,000 daltons; pH at isoelectric point (pI) 4.5-5.0. Glycoproteins A2 and A3 were electroeluted and used to prepare a monospecific antiserum that identified proteins A1, A2, and A3 in immunoblots of crude surfactant obtained from dog lung lavage. This antiserum precipitated several proteins from in vitro translated canine lung poly(A)+ mRNA; proteins of 27,000 daltons, pI 5.0, and 28,000 daltons, pI 4.8-5.0, which precisely comigrated with proteins A1 from canine surfactant. Cotranslational processing of the primary translation products by canine pancreatic microsomal membranes resulted in larger proteins of 31,000-34,000 daltons, pI 4.8-5.0. Treatment of these processed forms of glycoprotein A with endoglycosidase F, to remove N-linked carbohydrate, resulted in proteins of 27,000-28,000 daltons which precisely comigrated with surfactant protein A1. These observations demonstrate that the polypeptide precursors to the glycoproteins A complex are extensively modified by addition of asparagine N-linked complex carbohydrate and are subsequently secreted as glycoproteins A2 and A3.     

Glycosylation and secretion of surfactant-associated glycoprotein A

Synthesis of glycoprotein A, the major surfactant-associated protein, was demonstrated in Type II epithelial cells isolated from rat lung. Predominant, secreted forms migrated as glycoproteins with asparagine-linked, complex-type oligosaccharides (32,000-36,000 daltons, pI 4.2-4.8). Primary in vitro translation products of the glycoprotein migrated as five distinct proteins of approximately 26,000 daltons which were processed by pancreatic microsomal membranes in vitro to 30,000-34,000-dalton, endoglycosidase F-sensitive forms. These in vitro processed forms of glycoprotein A co-migrated with intracellular forms immunoprecipitated from [35S]methionine-labeled, Type II cells. Pulse-chase experiments with [35S]methionine-labeled cells demonstrated rapid synthesis of endoglycosidase H-sensitive precursors of 34,000 daltons, pI 4.7-4.8, which were neither secreted from Type II cells nor detected in surfactant from alveolar lavage. These high-mannose forms were slowly processed to more acidic, endoglycosidase H-resistant, neuraminidase-sensitive forms. At between 10 and 180 min, fully sialylated or other endoglycosidase H-resistant forms were a minor fraction of intracellular glycoprotein A. After 16 h, intracellular glycoproteins A were primarily present as endoglycosidase H-resistant forms. Secretion of mature, sialylated, glycoprotein A was first detected 1 h after labeling, and was also readily detected after 16-24 h chase period. Tunicamycin, which blocks N-linked protein glycosylation, resulted in synthesis of three major 26,000-dalton proteins which co-migrated with the nonglycosylated, surfactant-associated proteins A1 present in surfactant from alveolar lavage and with the major in vitro translation products of rat lung poly(A+) mRNA. Tunicamycin inhibited secretion of glycoprotein A. Swainsonine, which inhibits Golgi alpha-mannosidase II, completely inhibited synthesis of the fully sialylated molecule. Swainsonine produced forms of glycoprotein A which were both neuraminidase- and endoglycosidase H-sensitive and were readily secreted. Monensin, an ionophore that alters protein transport, markedly inhibited intracellular sialylation and secretion. These studies demonstrate that pulmonary Type II cells rapidly synthesize and process surfactant-associated glycoprotein A precursors to endoglycosidase H-sensitive forms, which are slowly sialylated prior to secretion.     

Glucocorticoid enhances surfactant proteolipid Phe and pVal synthesis and RNA in fetal lung

Two newly described surfactant proteolipids (SPL), Phe and pVal, are produced by proteolytic processing of distinct precursors of Mr = 40,000 and 22,000, respectively. These proteins are structurally related and intimately associated with surfactant phospholipids. We now demonstrate the expression of both SPL(Phe) and SPL(pVal) in explants of human fetal lung from 16-24 weeks of gestation. Content, synthesis, and mRNA for the proteolipids were low prior to organ culture of fetal lung. Induction of synthesis of the proteolipids occurred rapidly in explant culture in the absence of exogenous hormones and was enhanced by addition of dexamethasone. Increased synthesis of the proteolipids was detected by enzyme-linked immunosorbent assay and by [35S]methionine incorporation into the glycosylated Mr = 40,000-43,000 SPL (Phe) precursor. The response to dexamethasone occurred rapidly and contrasted with effects of dexamethasone on the expression of surfactant-associated protein- (SAP) 35, a distinct surfactant glycoprotein. 8-Br-cAMP did not significantly increase proteolipid content but markedly increased synthesis of SAP-35 in identical cultures. Increased proteolipid content was associated with increased mRNA for each protein as determined by the Northern blot analysis. Proteolipid RNA was also increased by 8-Br-cAMP, however, not to the extent observed with the glucocorticoid. Immunohistochemical analysis of fetal lung with anti-proteolipid antiserum confirmed that the dexamethasone-enhanced synthesis of the proteins by Type II epithelial cells. The time and hormone dependence of the regulation of expression of both SPL(Phe) and SPL(pVal) precursors were distinct from that of SAP-35. Expression of the surfactant proteolipids increased during explant culture of human fetal lung and was further enhanced by glucocorticoid. Developmental and hormonal regulation of the surfactant proteolipids may be important factors in surfactant function at birth.     

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.     

Differential effects of epidermal growth factor and transforming growth factor-beta on synthesis of Mr = 35,000 surfactant-associated protein in fetal lung

Differentiation of pulmonary Type II epithelial cells in late gestation is associated with the synthesis of pulmonary surfactant required for adaptation to air breathing at birth. In the present work, induction of synthesis of a Type II epithelial cell protein, surfactant-associated glycoprotein of Mr = 35,000 (SAP-35) was studied in human fetal lung tissue obtained at 15-24 weeks of gestation. SAP-35 content increased during organ culture in the absence of exogenous hormones. Epidermal growth factor or triiodothyronine stimulated the induction of SAP-35 synthesis during culture. Stimulation by epidermal growth factor (EGF) was detected as early as 2 days and persisted for up to 5 days in culture. Response to EGF was dose-dependent (0.01-10 ng/ml) and was associated with enhanced incorporation of [35S]methionine into immunoprecipitable SAP-35. Increased SAP-35 synthesis was associated with increased SAP-35 RNA as assessed by Northern blot and hybridization assays with human SAP-35 cDNA. Effects of EGF were comparable to the induction of SAP-35 synthesis by 8-bromo-cAMP. In contrast to the stimulatory effect of EGF and triiodothyronine, SAP-35 content was inhibited by transforming growth factor-beta. Both the stimulatory and inhibitory effects of these agents on SAP-35 content were associated with concomitant changes in SAP-35 synthesis. These findings demonstrate multihormonal control of SAP-35 expression and strongly implicate both EGF and transforming growth factor-beta in the regulation of surfactant apoprotein synthesis.     

Primary translation products, biosynthesis, and tissue specificity of the major surfactant protein in rat

Rat lung tissue was labeled with [35S]methionine and the major surfactant-associated proteins immunoprecipitated using a specific antiserum. The protein pattern obtained was very similar to that seen in rat bronchoalveolar lavage. Rat lung mRNA was subsequently translated in an in vitro rabbit reticulocyte system, and surfactant-associated protein-related polypeptides were immunoprecipitated. A 26-kDa polypeptide was identified and characterized as follows. (a) Unlabeled surfactant proteins added to the immunoprecipitation mixture completely inhibited its immunoprecipitation. (b) Two-dimensional gel electrophoresis of the 26-kDa protein resolved it into 3 isoforms. (c) Inclusion of dog pancreatic microsomes in the translation mixture resulted in the formation of two distinct higher molecular weight groups of isoforms, suggesting that the 26-kDa protein is destined to become a glycoprotein. Immunoprecipitation of [35S]methionine-labeled rat lung tissue proteins after tunicamycin treatment resulted in 3 isoforms, identical to the ones seen in the primary translation products. In addition, expression of the surfactant proteins appears specific to the lung.     

In vitro sulfation of pulmonary surfactant-associated protein-35

Surfactant-associated protein-35 consists of a group of phospholipid-associated proteins of 26-36 kDa isolated from pulmonary alveolar surfactant. In the rat, surfactant-associated protein-35 is synthesized from 26-kDa primary translation products which are cotranslationally acetylated and glycosylated to heterogeneous 30 and 34 kDa forms. High-mannose oligosaccharide-containing precursors of surfactant-associated protein-35 are processed in the rough endoplasmic reticulum and Golgi to complex-type oligosaccharides, resulting in a mature glycoprotein which exhibits extensive charge heterogeneity in two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis. Much of this charge heterogeneity is related to terminal sialylation of the two asparagine-linked oligosaccharides. In the present study, we report that surfactant-associated protein-35 is also sulfated. Sulfation of the 30 and 34 kDa forms of surfactant-associated protein-35 was clearly detected in primary cultures of rat Type II epithelial cells. These sulfated isoforms were sensitive to endoglycosidase F digestion, but resistant to neuraminidase, suggesting that sulfation occurred at oligosaccharide residues other than sialic acid. The lack of sulfation of the 26 kDa forms of surfactant-associated protein-35 and the resistance of the sulfated isoforms to endoglycosidase H digestion are consistent with Golgi-associated sulfation of the complex type oligosaccharides of surfactant-associated protein-35. Thus, sulfation is another component of the complex post-translational processing of surfactant-associated protein-35, which includes acetylation, hydroxylation, glycosylation, sialylation, sulfhydryl-dependent oligomerization and sulfation.     

Surfactant-associated protein inhibits phospholipid secretion from type II cells

Secretion of [3H]phosphatidylcholine ([3H]PC) from isolated rat pulmonary type II epithelial cells was inhibited by the surfactant-associated protein of Mr = 35,000 (SAP-35) purified from canine lung surfactant. SAP-35 inhibited [3H]PC secretion in a dose-dependent manner and significantly inhibited basal, phorbol ester, beta-adrenergic, and P2-purinergic agonist-induced [3H]PC secretion. SAP-35 significantly inhibited [3H]PC secretion from 1 to 3 h after treatment. The IC50 for inhibition of [3H]PC secretion by canine SAP-35 was 1-5 X 10(-6) g/ml and was similar for inhibition of both basal and secretagogue-stimulated release. Heat denaturation of SAP-35, addition of monoclonal anti-SAP-35 antibody, reduction and alkylation of SAP-35, or association of SAP-35 with phospholipid vesicles reversed the inhibitory effect on secretagogue-induced secretion. Inhibitory effects of SAP-35 were observed 3 h after cells were washed with buffer that did not contain SAP-35. Although SAP-35 enhanced reassociation of surfactant phospholipid with isolated type II cells, its inhibitory effect on secretion of [3H]PC did not result from stimulation of reuptake of secreted [3H]PC by type II cells. The inhibition of phospholipid secretion by SAP-35 was also not due to inhibition of PC or disaturated PC synthesis by SAP-35. SAP-35, the major phospholipid-associated protein in pulmonary surfactant, is a potent inhibitor of surfactant secretion from type II cells in vitro and may play an important role in homeostasis of surfactant in the alveolar space.     

In vitro acetylation of rat pulmonary surfactant-associated glycoprotein(s) A primary translation products

The primary translation products of pulmonary surfactant-associated glycoprotein(s) A, the major apolipoprotein in mammalian surfactants, exhibit extensive charge heterogeneity. After in vitro translation of poly(A)⁺ mRNa from rat lung, the primary translation products of glycoprotein(s) A were identified as a charge train of five proteins of 26 kDa (pI 4.6–5.0), the predominant forms being the more acidic members (pI < 4.8). Inhibition of acetylation during in vitro translationof rat lung poly(A)⁻ mRNA resulted in a predominance of the more basic isoforms (pI ≥ 4.8). Intracellular forms of glycoprotein(s) A were immunoprecipitated from rat Type II epithelial cells after treatment with tunicamycin or after deglycosylation with endoglycosidase H. Five intracellular precursors consisting primarily of acidic members of the charge train were identified, this being consistent with the intracellular acetylation of the protein. In contrast, canine glycoprotein(s) A translation products consisted of only three proteins of 26 kDa (pI 4.8–5.0), in which most of the radiolabel was concentrated in the more basic components. Acetylation may account for some, but not all, of the charge heterogeneity in the primary translation products and processed forms of surfactant-associated glycoprotein(s) A in the rat.     

Structural differences between insulin and somatomedin-C/insulin-like growth factor-1 receptors revealed by autoantibodies to the insulin receptor

Polyadenylated RNA prepared from first trimester human placenta was translated in a membrane-free cell-free system derived from wheat germ. Analysis of the [³⁵S]methionine-labeled products by SDS-polyacrylamide electrophoresis demonstrated two proteins with apparent M_rs of 14,500 and 16,000 that were specifically immunoprecipitated by antiserum to reduced and carboxylated bovine LH_α, and two different proteins with apparent M_rs of 18,500 and 21,000 that were specifically immunoprecipitated by antiserum to hCG_β. None of these products was sensitive to cleavage by endoglycosidase H, whereas the M_r 21,000 product precipitated by antisera to bovine LH_α and to hCG_α from translations supplemented by canine pancreatic microsomes was processed to a product with M_r 13,000 by endoglycosidase H. We suggest that the two forms of the α and β subunit precursors could arise from the translation of two distinct mRNAs encoding each subunit.     

Biophysical activity of synthetic phospholipids combined with purified lung surfactant 6000 dalton apoprotein

This research studies the biophysical surface activity of synthetic phospholipids combined in vitro with purified lung surfactant apoprotein, having an Mr of 6000. Hydrophobic surfactant-associated protein (SAP-6) was delipidated and purified from both bovine and canine lung lavage, and was combined in vitro with a synthetic phospholipid mixture (SM) of similar composition to natural lung surfactant phospholipids. SM phospholipids were also combined and studied biophysically with another purified surfactant-associated protein, SAP-35. The biophysical activity of synthetic phospholipid-apoprotein combinants was assessed by measurements of adsorption facility and dynamic surface tension lowering ability at 37 degrees C. The SM-SAP-6 combinants had adsorption facility equivalent to natural lung surfactant, and to the surfactant extract preparations CLSE and surfactant-TA used in exogenous surfactant replacement therapy for the neonatal Respiratory Distress Syndrome (RDS). The synthetic phospholipid-SAP-6 combinants also lowered surface tension to less than 1 dyne/cm under dynamic compression in an oscillating bubble apparatus at concentrations as low as 0.5 mg phospholipid/ml. A striking finding was that this excellent dynamic surface activity was preserved as SAP-6 composition was reduced to values as low as 5 micrograms/5 mg SM phospholipid (0.1% SAP-6 protein), an order of magnitude less than the 1% protein content of CLSE and surfactant-TA. Mixtures of SM phospholipids plus SAP-35, the major surfactant glycoprotein, had significantly lower biophysical activity, which did not approach that of a functional lung surfactant. These results suggest that synthetic exogenous surfactants of potential utility for replacement therapy in RDS can be formulated by combining synthetic phospholipids in vitro with specifically purified, hydrophobic surfactant-associated protein, SAP-6.     

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.     

Assembly of the sarcoplasmic reticulum. Cell-free synthesis of te Ca2+ + Mg2+-adenosine triphosphatase and calsequestrin

Polyadenylated RNA prepared from neonatal rat muscle was translated in a rabbit reticulocyte cell-free system. Two sarcoplasmic reticulum proteins, the Ca2+ + Mg2+-dependent adenosine triphosphatase (ATPase) and calsequestrin, were isolated from the translation mixture by immunoprecipitation, followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The [35S]methionine-labeled translation products were characterized by molecular weight, peptide mapping, and NH2-terminal sequence analysis. The ATPase synthesized in the cell-free system was found to have the same molecular weight (Mr = 100,000) and [35S]-methionine-labeled peptide map as the mature ATPase. The methionine residue present at the NH2 terminus of the mature ATPase was donated by initiator methionyl-tRNArMet and it became acetylated during translation. These results suggest that the ATPase was synthesized without an NH2-terminal signal sequence. Calsequestrin (Mr - 63,000) was synthesized as a higher molecular weight precursor (Mr = 66,000) that contained an additional [35S]methionine-labeled peptide when compared to mature calsequestrin. The NH2-terminal sequence of the precursor was different from the mature protein. The precursor was processed to a polypeptide with a molecular weight identical with mature calsequestrin when microsomal membranes prepared from canine pancreas were included during translation. These results show that calsequestrin is synthesized with an NH2-terminal signal sequence that is removed during translation. These data add to the evidence that the ATPase and calsequestrin follow distinctly different biosynthetic pathways, even though, ultimately, they are both located in the same membrane.     

Histochemical characterization of an antigen specific for the great alveolar cell in the mouse lung

Summary Previous papers reported on a specific antigenic marker for the great alveolar (type-II) cell of the mouse lung and described its recognition by a specific rabbit antiadult mouse lung serum. In the present study light- and electron-microscopical immunohistochemistry on fixed mouse lung sections showed the presence of the marker on the alveolar surface. The antigenic determinants recognized by the antibody were further characterized by immunoblotting and immunoprecipitation studies after in vitro translation of mouse lung messenger RNA.Immunoblots of a surfactant-enriched pellet of a bronchoalveolar lavage fraction of mouse lung showed that the antibody reacted with surfactant-associated proteins having apparent molecular weights of about 27,000, 32,000, and 38,000 daltons in SDS gels. Immunoblots of mouse-lung homogenate revealed the presence of 27,000, 30,000, 39,000, and 41,000 dalton proteins, presumably also surfactant-associated proteins. Immunoprecipitation after in vitro translation of mouse-lung mRNA showed specific reactivity only with a 12,000 dalton polypeptide, a component of the cell marker we were unable to relate to surfactant. Our findings indicate that the 12,000 dalton component of the antigenic marker for the great alveolar cell is a polypeptide whose synthesis is a lung-specific process and that the immunoreaction of the larger and surfactant-associated components is due to post-translational modifications.     

Histochemical characterization of an antigen specific for the great alveolar cell in the mouse lung

Previous papers reported on a specific antigenic marker for the great alveolar (type-II) cell of the mouse lung and described its recognition by a specific rabbit anti-adult mouse lung serum. In the present study light- and electron-microscopical immunohistochemistry on fixed mouse lung sections showed the presence of the marker on the alveolar surface. The antigenic determinants recognized by the antibody were further characterized by immunoblotting and immunoprecipitation studies after in vitro translation of mouse lung messenger RNA. Immunoblots of a surfactant-enriched pellet of a bronchoalveolar lavage fraction of mouse lung showed that the antibody reacted with surfactant-associated proteins having apparent molecular weights of about 27,000, 32,000, and 38,000 daltons in SDS gels. Immunoblots of mouse-lung homogenate revealed the presence of 27,000, 30,000, 39,000, and 41,000 daltons proteins, presumably also surfactant-associated proteins. Immunoprecipitation after in vitro translation of mouse-lung mRNA showed specific reactivity only with a 12,000 dalton polypeptide, a component of the cell marker we were unable to relate to surfactant. Our findings indicate that the 12,000 dalton component of the antigenic marker for the great alveolar cell is a polypeptide whose synthesis is a lung-specific process and that the immunoreaction of the larger and surfactant-associated components is due to post-translational modifications.     

In vitro translation, post-translational processing and secretion of pulmonary surfactant protein B precursors

Surfactant proteolipid SP-B is a hydrophobic protein of Mr = 8000 identified in organic solvent extracts of pulmonary surfactant. Analysis of the human SP-B RNA predicts that the active surfactant peptide is derived by proteolysis of an Mr = 40,000 precursor. In the present work, characteristics of synthesis, secretion and processing of SP-B were demonstrated in a pulmonary adenocarcinoma cell line by immunoprecipitation of radiolabelled precursors. Treatment of cells with tunicamycin resulted in synthesis and secretion of unglycosylated proSP-B of Mr = 39,000. Immunoprecipitation of protein produced by in vitro translation of human lung poly(A)+ RNA detected an Mr = 40,000 protein; the size discrepancy is likely related to cleavage of a leader signal sequence. Endoglycosidase-H-sensitive precursors of Mr = 41,000-43,000, pI = 5.1-5.4 were the first isoforms detected within the cells and were processed to endoglycosidase-H-resistant isoforms and secreted. Neuraminidase and endoglycosidase-F-sensitive forms of proSP-B were first detected in the media at 60 min as Mr = 42-46,000 isoforms with pI = 4.6-5.1. Proteolytically processed isoforms of proSP-B were detected primarily in the media and were generated by cleavage of an amino-terminal Mr = 16,000 peptide resulting in Mr = 27,000-33,000 isoforms (pH = 5.6-6.8). The Mr = 27,000-33,000 isoforms were sensitive to neuraminidase, resulting in isoforms with pH = 6.0-6.8. Digestion of the Mr = 27,000-33,000 peptide with endoglycosidase-F resulted in isoforms of Mr = 23,000, pH = 6.0-6.8. The endoglycosidase-F-resistant peptide of Mr = 16,000, pI = 4.2-4.4 was identified with an antiserum generated against synthetic peptides derived from the amino-terminal domain, as deduced from the SP-B DNA sequence. Further proteolytic processing of the Mr = 27,000-33,000 isoforms to the Mr = 8000 peptide detected in surfactant was not observed in this cell line. Thus, in the H441-4 cells (a cell line with morphologic features of Clara cells), SP-B is synthesized as a preproprotein which undergoes cleavage of a signal sequence and addition of asparagine-linked carbohydrate; proSP-B is secreted by processes which are independent of glycosylation. SP-B peptides of Mr = 27,000-33,000 and Mr = 16,000, representing carboxy and amino-terminal domains, accumulate in the media.     

Expression of the major surfactant-associated protein, SP-A, in type II cells of human lung before 20 weeks of gestation

In a previous paper (Otto-Verberne et al., Anat. Embryol. 178, 29-39 (1988) we reported that the type II alveolar epithelial cell can be identified in fetal human lung on the basis of morphological and immunological characteristics from 10 to 12 weeks after conception (a.c.) onward. For immunological recognition we used a lung-specific antibody, called SALS-Hu (specific anti-lavage serum, rabbit antihuman). The present immunoblotting experiments, after one-and two-dimensional electrophoresis, showed that SALS-Hu-reactive proteins in lavage fractions obtained from alveolar proteinosis patients exhibited molecular masses of mainly 29, 31 to 36, and 62 to 66 kDa. All SALS-Hu-reactive proteins migrated in the same acidic isoelectric point range (pI 4.4-5.1) and were almost undetectable when we used SALS-Hu preabsorbed with recombinant surfactant-associated protein A. We concluded that SALS-Hu recognizes exclusively isoforms of the major surfactant-associated protein, SP-A. In vitro translation assays in which we used mRNA isolated from adult human lung confirmed that SALS-Hu recognized the 29 to 31 kDa SP-A precursor proteins. These SALS-Hu-immunoreactive precursors for SP-A were already detectable (though in much lower amounts) in human fetuses aged 17 to 18 weeks, indicating that mRNA coding for SP-A is present at that time. We concluded that the cytoplasmic staining of fetal (from 10-12 weeks a.c. onward) and adult human type II cells by SALS-Hu is due to the presence of SP-A.     

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.