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.     

Combined and independent action of proteins SP-B and SP-C in the surface behavior and mechanical stability of pulmonary surfactant films

The hydrophobic proteins SP-B and SP-C are essential for pulmonary surfactant function, even though they are a relatively minor component (<2% of surfactant dry mass). Despite countless studies, their specific differential action and their possible concerted role to optimize the surface properties of surfactant films have not been completely elucidated. Under conditions kept as physiologically relevant as possible, we tested the surface activity and mechanical stability of several surfactant films of varying protein composition in vitro using a captive bubble surfactometer and a novel (to our knowledge) stability test. We found that in the naturally derived surfactant lipid mixtures, surfactant protein SP-B promoted film formation and reextension to lower surface tensions than SP-C, and in particular played a vital role in sustaining film stability at the most compressed states, whereas SP-C produced no stabilization. Preparations containing both proteins together revealed a slight combined effect in enhancing film formation. These results provide a qualitative and quantitative framework for the development of future synthetic therapeutic surfactants, and illustrate the crucial need to include SP-B or an efficient SP-B analog for optimal function.     

Tissue-specific regulation of glucocorticoid receptor mRNA by dexamethasone

The effect of glucocorticoids on tissue-specific regulation of glucocorticoid receptor mRNA was studied in intact and adrenalectomized rats. Glucocorticoid receptor mRNA was examined by Northern blot hybridization and quantitated by slot blot hybridization using a glucocorticoid cRNA probe. Glucocorticoid receptor mRNA was greatest in the lung with the relative levels in other tissues as follows: spleen, 70%; brain, 55%; liver, 50%; kidney, 43%; heart, 35%; adrenal, 13%; and testis only 8%. A tissue-specific difference in glucocorticoid receptor mRNA accumulation was found after adrenalectomy. There was little change in glucocorticoid receptor mRNA levels in liver and lung, but the brain and kidney demonstrated a 40 and 80% increase in mRNA, respectively. In contrast, dexamethasone treatment resulted in a consistent decrease of 40-60% in the accumulation of glucocorticoid receptor mRNA in all tissues studied. These results provide in vivo evidence for the autoregulation of the glucocorticoid receptor by its homologous ligand and demonstrate the existence of tissue-specific regulation of the glucocorticoid receptor mRNA levels in states of glucocorticoid excess and depletion.     

Sequences of a hairpin structure in the 3'-untranslated region mediate regulation of human pulmonary surfactant protein B mRNA stability

The ability of pulmonary surfactant to reduce alveolar surface tension requires adequate expression of surfactant protein B (SP-B). Dexamethasone (DEX, 10(-7) M) increases human SP-B mRNA stability by a mechanism that requires a 126-nt-long segment (the 7.6S region) of the 3'-untranslated region (3'-UTR). The objective of this study was to identify sequences in the 7.6S region that mediate regulation of SP-B mRNA stability. The 7.6S region was found to be sufficient for DEX-mediated stabilization of mRNA. Sequential substitution mutagenesis of the 7.6S region indicates that a 90-nt region is required for DEX-mediated stabilization and maintenance of intrinsic stability. In this region, one 30-nt-long element (002), predicted to form a stem-loop structure, is sufficient for DEX-mediated stabilization of mRNA and intrinsic mRNA stability. Cytosolic proteins specifically bind element 002, and binding activity is unaffected whether proteins are isolated from cells incubated in the absence or presence of DEX. While loop sequences of element 002 have no role in regulation of SP-B mRNA stability, the proximal stem sequences are required for DEX-mediated stabilization and specific binding of proteins. Mutation of the sequences that comprise the proximal or distal arm of the stem negates the destabilizing activity of element 002 on intrinsic SP-B mRNA stability. These results indicate that cytosolic proteins bind a single hairpin structure that mediates intrinsic and hormonal regulation of SP-B mRNA stability via mechanisms that involve sequences of the stems of the hairpin structure.     

Expression of mature pulmonary surfactant-associated protein B (SP-B) in Escherichia coli using truncated human SP-B cDNAs

The present communication documents attempts to produce the mature form of human surfactant-associated protein B (SP-B) by modification of the 5' and 3' regions of the cDNA and expression of the truncated cDNAs after insertion into the vector pKK223-3. The 5' end of a cDNA for human SP-B (1407 base pairs) was reconstructed through the ligation of synthetic oligonucleotides to an internal PstI site in the 5' region. This construction coded for the initiation of protein synthesis at a Met codon adjacent to a codon for the N-terminal Phe of the mature polypeptide. Variable amounts of the 3' end of the human SP-B cDNA were deleted with mung bean nuclease and exonuclease III. The resulting blunt-ended 3' fragments were then ligated to a synthetic oligonucleotide linker designed to create a stop codon. The modified 5' and 3' ends were ligated to a short PstI-BamHI fragment isolated from the SP-B cDNA and inserted into the expression vector pKK223-3. In vitro translation of sense mRNAs derived from the truncated SP-B cDNAs yielded oligopeptides of appropriate molecular weights, as indicated by urea - sodium dodecyl sulphate - polyacrylamide gel electrophoresis of either intact or immunoprecipitated reaction mixtures. Expression of SP-B in Escherichia coli was confirmed by Northern blot analysis for the mRNAs corresponding to the truncated cDNAs in appropriately transformed bacteria induced with the galactose analog isopropyl-beta-thiogalactoside. Western blot analysis using rabbit antisera prepared against bovine SP-B confirmed the presence of mature SP-B in lipid extracts of transformed E. coli, but the amounts were very small.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional tests for the characterization of surfactant protein B (SP-B) and a fluorescent SP-B analog

Surfactant protein B (SP-B) enhances lipid insertion into the alveolar air/liquid interface upon inhalation. The aim of this study was (i) to apply a palette of tests for a detailed biochemical and biophysical characterization of SP-B and (ii) to use these tests to compare native SP-B with a fluorescent (Bodipy) SP-B analog. The method of labeling was fast and resulted in a covalent fluorophore-protein bond. The ability of both proteins to spread a surfactant film on top of a buffer surface was determined in a spreading tray using the Wilhelmy plate technique to allow detection of alterations in surface tension and calculation of spreading velocities. In a captive bubble surfactometer surface tensions of spread films were measured. Similar biophysical properties were found for both native and Bodipy-labeled SP-B. It is concluded that the combination of tests used allows detection of small differences in structure and activity between the two proteins.     

Raman spectroscopic studies of model human pulmonary surfactant systems: phospholipid interactions with peptide paradigms for the surfactant protein SP-B

The temperature dependence of dipalmitoylphosphatidylcholine (DPPC)/phosphatidylglycerol (PG) multilayers, reconstituted with various synthetic peptides for modeling human lung surfactant, was monitored by vibrational Raman spectroscopy. The synthetic peptides consisted, respectively, of residues 59-81 of the human surfactant protein SP-B and 21 amino acid residue peptides containing repeating units of arginine separated by either four or eight leucines (RL4 or RL8). Each peptide demonstrated the ability to reduce significantly the surface tension of analogues of the phospholipid mixture used in the Raman studies. Raman spectroscopic integrated band intensities and relative peak height intensity ratios, two spectral parameters used to determine bilayer disorder, provided sensitive probes for characterizing multilayer perturbations in the reconstituted liposomes. Temperature profiles derived from the various Raman intensity parameters for the 3100-2800-cm-1 carbon-hydrogen (C-H) stretching mode region, a spectral interval representative of acyl chain vibrations, reflected lipid reorganizations due to the bilayer interactions of these peptides. For the three reconstituted multilamellar surfactant systems, the gel-to-liquid-crystalline phase-transition temperatures Tm, defined by acyl chain C-H stretching mode order/disorder parameters, increased from 35 degrees C in the peptide free system to 37-38 degrees C, indicating increased lipid headgroup constraints for the model liposomes. Although the values of Tm were similar for the three recombinant lipid/peptide assemblies, individual phase-transition cooperativities varied significantly between systems and between spectroscopically derived order/disorder parameters.     

NMR structure of lung surfactant peptide SP-B(11-25)

Surfactant protein B (SP-B) is a 79-residue essential component of lung surfactant, the film of lipid and protein lining the alveoli, and is the subject of great interest for its role in lung surfactant replacement therapies. Here we report circular dichroism results and the solution NMR structure of SP-B(11-25) (CRALIKRIQAMIPKG) dissolved in CD(3)OH at 5 degrees C. This is the first report of NMR data related to the protein SP-B, whose structure promises to help elucidate the mechanism of its function. Sequence-specific resonance assignments were made for all observable (1)H NMR signals on the basis of standard 2D NMR methods. Structures were determined by the simulated annealing method using restraints derived from 2D NOESY data. The calculations yielded 17 energy-minimized structures, three of which were subjected to 0.95 ns of restrained dynamics to assess the relevance of the static structures to more realistic dynamic behavior. Our CD and NMR data confirm that this segment is an amphiphilic alpha helix from approximately residue L14 through M21. The backbone heavy-atom RMSD for residues L14 through M21 is 0.09 +/- 0.12 A, and the backbone heavy-atom RMSD for the whole peptide is 0.96 +/- 2.45 A, the difference reflecting fraying at the termini. Aside from the disordered termini, the minimized structures represent dynamic structures well. Structural similarity to the homologous regions of related saposin-like proteins and the importance of the distribution of polar residues about the helix axis are discussed.     

Activation of mitogen activated protein (MAP) kinases by vanadate is independent of insulin receptor autophosphorylation

Treatment of Chinese hamster ovary (CHO) cells over-expressing the human insulin receptor (CHO-HIRc) with the insulin mimetic agent, vanadate, resulted in a dose- and time-dependent tyrosine phosphorylation of two proteins with apparent molecular sizes of 42 kDa (p42) and 44 kDa (p44). However, vanadate was unable to stimulate the tyrosyi phosphorylation of theβ-subunit of the insulin receptor. By using myelin basic protein (MBP) as the substrate to measure mitogen-activated protein (MAP) kinase activity in whole cell lysates, vanadate-stimulated tyrosyl phosphorylation of p42 and p44 was associated with a dose- and time-dependent activation of MAP kinase activity. Furthermore, affinity purification of cell lysates on anti-phosphotyrosine agarose column followed by immunoblotting with a specific antibody to MAP kinases demonstrated that vanadate treatment increased the tyrosyl phosphorylation of both p44^mapk and p42^mapk by several folds, as compared to controls, in concert with MAP kinase activation. In addition, retardation in gel mobility further confirmed that vanadate treatment increased the phosphorylation of p44^mapk and p42^mapk in CHO-HIRc. A similar effect of vanadate on MAP kinase tyrosyl phosphorylation and activation was also observed in CHO cells over-expressing a protein tyrosine kinase-deficient insulin receptor (CHO-1018). These results demonstrate that the protein tyrosine kinase activity of the insulin receptor may not be required in the signaling pathways leading to the vanadate-mediated tyrosyl phosphorylation and activation of MAP kinases.     

Lymphokine regulation of activated (G1) lymphocytes. II. Glucocorticoid and anti-Tac-induced inhibition of human T lymphocyte proliferation

The regulation of the first cell cycle of human, activated (G1) PBL was analyzed by flow cytometry and [3H]thymidine incorporation. Endogenous IL 2 production was blocked in situ by pharmacologic concentration of DEX (100 to 1000 nM), resulting in an 80 to 90% reduction of thymidine uptake. Although T lymphocyte activation (G0-G1a transition) by PHA was unaltered, cells remained in the G1a phase of the cell cycle due to insufficient RNA synthesis for proliferation. The addition of IL 2-containing supernatants reversed this inhibitory effect of DEX by allowing the cells to synthesize more RNA (G1a-G1b transition). Such cells could enter the S phase and proliferate. Similar studies were performed on cells treated with a monoclonal antibody (anti-Tac) against the IL 2 receptor. In these studies, IL 2-induced RNA synthesis, and subsequent proliferation of DEX-treated and PHA-stimulated cells was inhibited by anti-Tac. Anti-Tac did not, however, inhibit the effect of endogenous IL 2 (PHA-stimulated PBL without DEX treatment), although it did bind equally well to such cells. Thus, IL 2 directly or indirectly regulates human T cell proliferation at the level of RNA synthesis. Furthermore, anti-Tac can inhibit the mitogenic signal given by endogenous IL 2, but not by in situ produced IL 2, an observation of importance to further investigations of the mechanisms by which IL 2 interacts with specific receptors to elicit proliferation.     

Palmitoylation of pulmonary surfactant protein SP-C is critical for its functional cooperation with SP-B to sustain compression/expansion dynamics in cholesterol-containing surfactant films

Recent data suggest that a functional cooperation between surfactant proteins SP-B and SP-C may be required to sustain a proper compression-expansion dynamics in the presence of physiological proportions of cholesterol. SP-C is a dually palmitoylated polypeptide of 4.2 kDa, but the role of acylation in SP-C activity is not completely understood. In this work we have compared the behavior of native palmitoylated SP-C and recombinant nonpalmitoylated versions of SP-C produced in bacteria to get a detailed insight into the importance of the palmitic chains to optimize interfacial performance of cholesterol-containing surfactant films. We found that palmitoylation of SP-C is not essential for the protein to promote rapid interfacial adsorption of phospholipids to equilibrium surface tensions (∼22 mN/m), in the presence or absence of cholesterol. However, palmitoylation of SP-C is critical for cholesterol-containing films to reach surface tensions ≤1 mN/m at the highest compression rates assessed in a captive bubble surfactometer, in the presence of SP-B. Interestingly, the ability of SP-C to facilitate reinsertion of phospholipids during expansion was not impaired to the same extent in the absence of palmitoylation, suggesting the existence of palmitoylation-dependent and -independent functions of the protein. We conclude that palmitoylation is key for the functional cooperation of SP-C with SP-B that enables cholesterol-containing surfactant films to reach very low tensions under compression, which could be particularly important in the design of clinical surfactants destined to replacement therapies in pathologies such as acute respiratory distress syndrome.     

AUUUA motifs in the 3'UTR of human glucocorticoid receptor alpha and beta mRNA destabilize mRNA and decrease receptor protein expression

An association between a gene polymorphism of the human glucocorticoid receptor (hGR) gene and rheumatoid arthritis has recently been suggested. This polymorphism contains an A to G mutation in the 3'UTR of exon 9beta, which encodes the 3'UTR of the mRNA of the hGRbeta isoform. The hGRbeta isoform can act as a dominant negative inhibitor of hGRalpha, and therefore may contribute to glucocorticoid resistance. The A to G mutation is located in an AUUUA motif, which is known to destabilize mRNA. In the present study, the importance of the mutation in this AUUUA motif was further characterized and mutations in other AUUUA motifs in the 3'UTR of hGRbeta and hGRalpha mRNA were studied. hGRbeta and hGRalpha expression vectors, carrying mutations in one AUUUA motif or all AUUUA motifs were transiently transfected into COS-1 cells. Each transfected vector was analyzed for the mRNA expression level, the mRNA turnover rate and the protein expression level. The naturally occurring mutation in the 3'UTR of hGRbeta mRNA increased mRNA stability and protein expression. Mutation of two other AUUUA motifs in the 3'UTR of hGRbeta, or mutation of all four AUUUA motifs resulted in a similar effect. Mutation of the most 5' AUUUA motif did not alter hGRbeta mRNA expression or mRNA stability. Mutation of all 10 AUUUA motifs in the 3'UTR of hGRalpha mRNA increased hGRalpha mRNA expression and mRNA stability as well as expression of the receptor protein level. Thus, the naturally occurring mutation in an AUUUA motif in the 3'UTR of hGRbeta mRNA results not only in increased mRNA stability, but also in increased receptor protein expression, which may contribute to glucocorticoid resistance. A similar role is suggested for two other AUUUA motifs in the 3'UTR of hGRbeta mRNA and for the 10 AUUUA motifs that are present in the 3'UTR of hGRalpha.