Mature Surfactant Protein B Research Articles

Conformational Stability of the NH2-Terminal Propeptide of the Precursor of Pulmonary Surfactant Protein SP-B.

Assembly of pulmonary surfactant lipid-protein complexes depends on conformational changes coupled with proteolytic maturation of proSP-B, the precursor of pulmonary surfactant protein B (SP-B), along the surfactant biogenesis pathway in pneumocytes. Conformational destabilization of the N-terminal propeptide of proSP-B (SP-BN) triggers exposure of the mature SP-B domain for insertion into surfactant lipids. We have studied the conformational stability during GdmCl- or urea-promoted unfolding of SP-BN with trp fluorescence and circular dichroism spectroscopies. Binding of the intermediate states to bis-ANS suggests their molten globule-like character. ΔG0H2O was ~ 12.7 kJ·mol-1 either with urea or GdmCl. None of the thermal transitions of SP-BN detected by CD correspond to protein unfolding. Differential scanning calorimetry of SP-BN evidenced two endothermic peaks involved in oligomer dissociation as confirmed with 2 M urea. Ionic strength was relevant since at 150 mM NaCl, the process originating the endotherm at the highest temperature was irreversible (Tm2 = 108.5°C) with an activation energy of 703.8 kJ·mol-1. At 500 mM NaCl the process became reversible (Tm2 = 114.4°C) and data were fitted to the Non-two States model with two subpeaks. No free thiols in the propeptide could be titrated by DTNB with or without 5.7 M GdmCl, indicating disulfide bonds establishment.

Synergistic Effect of Caffeine and Glucocorticoids on Expression of Surfactant Protein B (SP-B) mRNA

Administration of glucocorticoids and caffeine is a common therapeutic intervention in the neonatal period, but possible interactions between these substances are still unclear. The present study investigated the effect of caffeine and different glucocorticoids on expression of surfactant protein (SP)-B, crucial for the physiological function of pulmonary surfactant. We measured expression levels of SP-B, various SP-B transcription factors including erythroblastic leukemia viral oncogene homolog 4 (ErbB4) and thyroid transcription factor-1 (TTF-1), as well as the glucocorticoid receptor (GR) after administering different doses of glucocorticoids, caffeine, cAMP, or the phosphodiesterase-4 inhibitor rolipram in the human airway epithelial cell line NCI-H441. Administration of dexamethasone (1 µM) or caffeine (5 mM) stimulated SP-B mRNA expression with a maximal of 38.8±11.1-fold and 5.2±1.4-fold increase, respectively. Synergistic induction was achieved after co-administration of dexamethasone (1 mM) in combination with caffeine (10 mM) (206±59.7-fold increase, p<0.0001) or cAMP (1 mM) (213±111-fold increase, p = 0.0108). SP-B mRNA was synergistically induced also by administration of caffeine with hydrocortisone (87.9±39.0), prednisolone (154±66.8), and betamethasone (123±6.4). Rolipram also induced SP-B mRNA (64.9±21.0-fold increase). We detected a higher expression of ErbB4 and GR mRNA (7.0- and 1.7-fold increase, respectively), whereas TTF-1, Jun B, c-Jun, SP1, SP3, and HNF-3α mRNA expression was predominantly unchanged. In accordance with mRNA data, mature SP-B was induced significantly by dexamethasone with caffeine (13.8±9.0-fold increase, p = 0.0134). We found a synergistic upregulation of SP-B mRNA expression induced by co-administration of various glucocorticoids and caffeine, achieved by accumulation of intracellular cAMP. This effect was mediated by a caffeine-dependent phosphodiesterase inhibition and by upregulation of both ErbB4 and the GR. These results suggested that caffeine is able to induce the expression of SP-transcription factors and affects the signaling pathways of glucocorticoids, amplifying their effects. Co-administration of caffeine and corticosteroids may therefore be of benefit in surfactant homeostasis.

Pepsinogen C Proteolytic Processing of Surfactant Protein B

Surfactant protein B (SP-B) is essential to the function of pulmonary surfactant and to lamellar body genesis in alveolar epithelial type 2 cells. The bioactive, mature SP-B is derived from multistep post-translational proteolysis of a larger proprotein. The identity of the proteases involved in carboxyl-terminal cleavage of proSP-B remains uncertain. This cleavage event distinguishes SP-B production in type 2 cells from less complete processing in bronchiolar Clara cells. We previously identified pepsinogen C as an alveolar type 2 cell-specific protease that was developmentally regulated in the human fetal lung. We report that pepsinogen C cleaved recombinant proSP-B at Met(302) in addition to an amino-terminal cleavage at Ser(197). Using a well described model of type 2 cell differentiation, small interfering RNA knockdown of pepsinogen C inhibited production of mature SP-B, whereas overexpression of pepsinogen C increased SP-B production. Inhibition of SP-B production recapitulated the SP-B-deficient phenotype evident by aberrant lamellar body genesis. Together, these data support a primary role for pepsinogen C in SP-B proteolytic processing in alveolar type 2 cells.

Alteration of the Pulmonary Surfactant System in Full-Term Infants with Hereditary ABCA3 Deficiency

ABCA3 mutations are known to cause fatal surfactant deficiency. We studied ABCA3 protein expression in full-term newborns with unexplained respiratory distress syndrome (URDS) as well as the relevance of ABCA3 mutations for surfactant homeostasis. Lung tissue of infants with URDS was analyzed for the expression of ABCA3 in type II pneumocytes. Coding exons of the ABCA3 gene were sequenced. Surfactant protein expression was studied by immunohistochemistry, immunoelectron microscopy, and Western blotting. ABCA3 protein expression was found to be greatly reduced or absent in 10 of 14 infants with URDS. Direct sequencing revealed distinct ABCA3 mutations clustering within vulnerable domains of the ABCA3 protein. A strong expression of precursors of surfactant protein B (pro-SP-B) but only low levels and aggregates of mature surfactant protein B (SP-B) within electron-dense bodies in type II pneumocytes were found. Within the matrix of electron-dense bodies, we detected precursors of SP-C (pro-SP-C) and cathepsin D. SP-A was localized in small intracellular vesicles, but not in electron-dense bodies. SP-A and pro-SP-B were shown to accumulate in the intraalveolar space, whereas mature SP-B and SP-C were reduced or absent, respectively. Our data provide evidence that ABCA3 mutations are associated not only with a deficiency of ABCA3 but also with an abnormal processing and routing of SP-B and SP-C, leading to severe alterations of surfactant homeostasis and respiratory distress syndrome. To identify infants with hereditary ABCA3 deficiency, we suggest a combined diagnostic approach including immunohistochemical, ultrastructural, and mutation analysis.

Processing of Pulmonary Surfactant Protein B by Napsin and Cathepsin H

Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. SP-B is synthesized in alveolar type II cells as a preproprotein and processed to the mature peptide by the cleavage of NH2- and COOH-terminal peptides. An aspartyl protease has been suggested to cleave the NH2-terminal propeptide resulting in a 25-kDa intermediate. Napsin, an aspartyl protease expressed in alveolar type II cells, was detected in fetal lung homogenates as early as day 16 of gestation, 1 day before the onset of SP-B expression and processing. Napsin was localized to multivesicular bodies, the site of SP-B proprotein processing in type II cells. Incubation of SP-B proprotein from type II cells with a crude membrane extract from napsin-transfected cells resulted in enhanced levels of a 25-kDa intermediate. Purified napsin cleaved a recombinant SP-B/EGFP fusion protein within the NH2-terminal propeptide between Leu178 and Pro179, 22 amino acids upstream of the NH2 terminus of mature SP-B. Cathepsin H, a cysteine protease also implicated in pro-SP-B processing, cleaved SP-B/EGFP fusion protein 13 amino acids upstream of the NH2 terminus of mature SP-B. Napsin did not cleave the COOH-terminal peptide, whereas cathepsin H cleaved the boundary between mature SP-B and the COOH-terminal peptide and at several other sites within the COOH-terminal peptide. Knockdown of napsin by small interfering RNA resulted in decreased levels of mature SP-B and mature SP-C in type II cells. These results suggest that napsin, cathepsin H, and at least one other enzyme are involved in maturation of the biologically active SP-B peptide.

Surfactant protein B in type II pneumocytes and intra-alveolar surfactant forms of human lungs.

Surfactant protein B (SP-B) is synthesized by type II pneumocytes as a proprotein (proSP-B) that is proteolytically processed to an 8-kD protein. In human type II pneumocytes, we identified not only proSP-B, processing intermediates of proSP-B, and mature SP-B, but also fragments of the N-terminal propeptide. By means of immunoelectron microscopy, proSP-B and processing intermediates were localized in the endoplasmic reticulum, Golgi vesicles, and few multivesicular bodies in type II pneumocytes in human lungs. A colocalization of fragments of the N-terminal propeptide and mature SP-B was found in multivesicular, composite, and some lamellar bodies. Mature SP-B was localized over the projection core of lamellar bodies and core-like structures in tubular myelin figures. In line with immunoelectron microscopy and Western blot analysis of human type II pneumocytes, a fragment of the N-terminal propeptide was also detected in isolated rat lamellar bodies. In conclusion, our data indicate that the processing of proSP-B occurs between the Golgi complex and multivesicular bodies and provide evidence that a fragment of the N-terminal propeptide and mature SP-B are transported together to the lamellar bodies. In human lungs, mature SP-B is involved in the structural organization of lamellar bodies and tubular myelin by the formation of core particles.

Differences in N-linked glycosylation between human surfactant protein-B variants of the C or T allele at the single-nucleotide polymorphism at position 1580: implications for disease.

Human surfactant protein-B (SP-B), a hydrophobic protein, is essential for normal lung function. SP-B is expressed and secreted by specific lung cell types, i.e. alveolar type II and Clara cells, of the respiratory epithelium. The SP-B precursor (42 kDa) undergoes post-translational processing to generate an 8 kDa mature SP-B. A single-nucleotide polymorphism (SNP) at nucleotide 1580 (C/T) in exon 4 of SP-B that changes amino acid 131 from threonine to isoleucine (Thr131-->Ile) is associated with several pulmonary diseases. The Thr131-->Ile substitution can eliminate a potential N-linked glycosylation site, Asn129-Gln-Thr131, which is present in the SP-B variant of the C allele (ACT/Thr) but not in that of the T allele (ATT/Ile). To determine whether the C allele SP-B variant is indeed glycosylated at Asn(129)-Gln-Thr131, we first generated stably transfected Chinese hamster ovary cell lines that expressed each version of SP-B, and developed specific SP-B polyclonal anti-peptide antibodies. Using both the stably transfected cell lines and fetal lung explants, we observed that the C allele variant is indeed glycosylated at the Asn129-Gln-Thr131 site, whereas the T allele variant, which served as a control, is not. In addition, we also confirmed that both SP-B variants contain another N-linked glycosylation site, Asn311-Ser-Ser313. Given its association with several pulmonary diseases, this finding provides useful information for future studies in disease systems associated with this SNP. Further, we speculate that, given the fact that this SNP is found frequently in the general population, N-linked glycosylation at residue Asn129 interferes with SP-B processing, secretion and folding under certain disease conditions.

Allelic heterogeneity in hereditary surfactant protein B (SP-B) deficiency.

Inability to produce surfactant protein B (SP-B) causes fatal neonatal respiratory disease. A frame-shift mutation (121ins2) is the predominant but not exclusive cause of disease. To determine the range of mechanisms responsible for SP-B deficiency, both alleles from 32 affected infants were characterized. Sixteen infants were homozygous for the 121ins2 mutation, 10 infants were heterozygous for the 121ins2 and another mutation, and six infants were homozygous for other mutations. Thirteen novel SP-B gene mutations were identified, which were not found in a control population. One novel mutation was found in two unrelated families. Surfactant protein expression was evaluated by immunohistochemistry and/or protein blotting. Absence of proSP-B and mature SP-B was associated with nonsense and frame-shift mutations. In contrast, proSP-B expression was associated with missense mutations, or mutations causing in-frame deletions or insertions, and low levels of mature SP-B expression were associated with four mutations. Extracellular staining for proSP-C and/or aberrantly processed SP-C was observed in lungs of all infants with SP-B gene mutations. Hereditary SP-B deficiency is caused by a variety of distinct mutations in the SP-B gene and may be associated with reduced, as well as absent, levels of mature SP-B, likely caused by impaired processing of proSP-B.

Intracellular Localization of Processing Events in Human Surfactant Protein B Biosynthesis

Surfactant protein B (SP-B) is essential to the function of pulmonary surfactant and to alveolar type 2 cell phenotype. Human SP-B is the 79-amino acid product of extensive post-translational processing of a 381-amino acid preproprotein. Processing involves modification of the primary translation product from 39 to 42 kDa and at least 3 subsequent proteolytic cleavages to produce the mature 8-kDa SP-B. To examine the intracellular sites of SP-B processing, we carried out immunofluorescence cytochemistry and inhibitor studies on human fetal lung in explant culture and isolated type 2 cells in monolayer culture using polyclonal antibodies to human SP-B(8) (Phe(201)-Met(279)) and specific epitopes within the N- (NFProx, Ser(145)-Leu(160); NFlank Gln(186)-Gln(200)) and C-terminal (CFlank, Gly(284)-Ser(304)) propeptides of pro-SP-B. Fluorescence immunocytochemistry using epitope-specific antisera showed colocalization of pro-SP-B with the endoplasmic reticulum resident protein BiP. The 25-kDa intermediate was partially endo H-sensitive, colocalized with the medial Golgi resident protein MG160, and shifted into the endoplasmic reticulum in the presence of brefeldin A, which interferes with anterograde transport from endoplasmic reticulum to Golgi. The 9-kDa intermediate colocalized in part with MG160 but not with Lamp-1, a transmembrane protein resident in late endosomes and lamellar bodies. Brefeldin A induced a loss of colocalization between MG160 and NFlank, shifting NFlank immunostaining to a juxtanuclear tubular array. In pulse-chase studies, brefeldin A blocked all processing of 42-kDa pro-SP-B whereas similar studies using monensin blocked the final N-terminal processing event of 9 to 8 kDa SP-B. We conclude that: 1) the first enzymatic cleavage of pro-SP-B to the 25-kDa intermediate is in the brefeldin A-sensitive, medial Golgi; 2) cleavage of the 25-kDa intermediate to a 9-kDa form is a trans-Golgi event that is slowed but not blocked by monensin; 3) the final cleavage of 9 to 8 kDa SP-B is a monensin-sensitive, post-Golgi event occurring prior to transfer of SP-B to lamellar bodies.

Surfactant Protein B (SP-B) −/− Mice Are Rescued by Restoration of SP-B Expression in Alveolar Type II Cells but Not Clara Cells

Surfactant protein B (SP-B) mRNA and protein are restricted to alveolar Type II and Clara cells in the respiratory epithelium. In order to investigate the function of SP-B in these distinct cell types, transgenic mice were generated in which SP-B expression was selectively restored in Type II cells or Clara cells of SP-B -/- mice. The 4.8-kilobase murine SP-C promoter was used to generate 3 transgenic lines which expressed human SP-B in Type II cells (mSP-C/hSP-B). Likewise, the 2.3-kilobase murine CCSP promoter was used to generate two transgenic lines which expressed human SP-B in Clara cells (mCCSP/hSP-B). mSP-C/hSP-B and mCCSP/hSP-B transgenic mice were subsequently bred to SP-B +/- mice in order to selectively express SP-B in Type II cells or Clara cells of SP-B -/- mice. Selective restoration of SP-B expression in Type II cells completely rescued the neonatal lethal phenotype in SP-B -/- mice. Expression of SP-B in some, but not all Type II cells of SP-B -/- mice, allowed postnatal survival, but resulted in significantly altered lung architecture and function. Selective restoration of SP-B expression in Clara cells of SP-B -/- mice resulted in respiratory dysfunction and invariable neonatal death, related to the complete absence of mature SP-B peptide in these mice. These results indicate that expression and processing of the SP-B proprotein to the mature peptide in Type II cells is absolutely required for lung function in vivo and that SP-B expression in Clara cells cannot substitute for this function.

Rescue of SP-B Knockout Mice with a Truncated SP-B Proprotein: FUNCTION OF THE C-TERMINAL PROPEPTIDE

The function of the 102-amino acid C-terminal propeptide of surfactant protein B (SP-B) was analyzed by characterizing the phenotype associated with loss of expression of this peptide domain in transgenic mice. A construct encoding the signal peptide, N-terminal propeptide, and mature peptide of human SP-B (hSP-BDeltac) was cloned under the control of the 3.7-kilobase human SP-C promoter and injected into fertilized eggs of the FVB/N mouse strain. Founder mice expressing the hSP-BDeltac transgene were bred with heterozygous SP-B knockout mice (SP-B +/-). Offspring containing the transgene and one allele of mouse SP-B were identified and subsequently crossed to generate a transgenic line that expressed SP-BDeltac in a null background (SP-B(-/-)/hSP-BDeltac(+/+)). Expression of hSP-BDeltac in SP-B(-/-) mice was restricted to type II cells and resulted in a 2-fold increase in mature SP-B relative to wild type littermates. These mice survived without any evidence of respiratory problems and had normal lung function, normal alveolar surfactant phospholipid pool sizes, and typical tubular myelin indicating that the 102-residue C-terminal propeptide of SP-B is not required for normal structure and function of extracellular surfactant. However, proteolytic processing of the SP-C proprotein was perturbed resulting in the accumulation of a processing intermediate, Mr = 11,000, similar to the phenotype detected in SP-B(-/-) mice; furthermore, lamellar bodies in type II cells of SP-B(-/-)/hSP-BDeltac(+/+) mice were much larger than in the wild type animal and saturated phosphatidylcholine content in lung tissue was significantly increased although the incorporation of choline into saturated phosphatidylcholine was normal. Collectively, these results demonstrate a role for the C-terminal propeptide of SP-B in SP-C proprotein processing and the maintenance of lamellar body size. The C-terminal propeptide may be an important determinant of intracellular surfactant pool size.

Thyroid transcription factor-1, hepatocyte nuclear factor-3beta, surfactant protein B, C, and Clara cell secretory protein in developing mouse lung.

We used immunohistochemical analysis to localize thyroid transcription factor-1 (TTF-1), hepatocyte nuclear factor-3beta (HNF-3beta), prosurfactant proteins B and C (pro-SP-B, pro-SP-C), surfactant protein B (SP-B), and Clara cell secretory protein (CCSP) in developing mouse lung. TTF-1 and HNF-3beta were expressed at the onset of lung morphogenesis (gestational Day 10) and throughout fetal lung development, being detected in the nuclei of airway epithelial cells. TTF-1 was most prominent in distal airway epithelial cells in embryonic lung and HNF-3beta in proximal bronchial and bronchiolar epithelial cells. Pro-SP-B and pro-SP-C were first detected on gestational Day 11, being localized to the cytoplasm of airway epithelial cells. Expression of both pro-proteins was confined to distal airway epithelial cells from gestational Day 12 to Day 16. From gestational Day 17 and thereafter, pro- SP-B was detectable in Type II cells and bronchiolar epithelial cells, whereas pro-SP-C was restricted to Type II cells. SP-B peptide was first detected on gestational Day 17 in the cytoplasm of Type II cells and within the lumen of distal airways. SP-B peptide was detectable only in the cytoplasm of Type II cells in adult lung. CCSP was first detected on gestational Day 17, being localized to the cytoplasm of columnar epithelial cells lining the conducting airways. Pro-SP-B, SF-B, pro-SP-C, and CCSP staining increased before birth. The early expression of TTF-1 and HNF-3beta, preceding and overlapping that of pro-SP-B, mature SP-B, pro-SP-C, and CCSP, supports a regulatory role for TTF-1 and HNF-3beta in lung-specific gene expression.

Structural Requirements for Targeting of Surfactant Protein B (SP-B) to Secretory Granules in Vitro and in Vivo

Human surfactant protein B (SP-B) is synthesized by type II cells as a 381-residue preproprotein which is proteolytically processed to a 79-residue mature peptide and targeted to lamellar bodies for secretion. To identify secretory granule targeting determinants, constructs encoding the SP-B preproprotein (SP-B), COOH-terminally deleted SP-B (SP-BDeltaC), the NH2-terminal propeptide (SP-BN), and a chimeric molecule consisting of albumin and the mature peptide (ALB/SP-BM) were transfected into AtT-20 and PC12 cells. Pulse-chase studies demonstrated that 10-30% of SP-B and SP-BDeltaC remained in cells in an endoglycosidase H-resistant form. Secretion of stored SP-B was stimulated by forskolin/12-O-tetradecanoylphorbol-13-acetate and intracellular SP-B was localized to secretory granules by immunoelectron microscopy. In contrast, SP-BN and ALB/SP-BM were constitutively secreted and not detected in secretory granules. Specific processing of SP-B was not detected in either AtT-20 or PC12 cells. Expression of SP-BDeltaC in transgenic mice resulted in secretion of fully processed mature SP-B, indicating correct processing and targeting of this construct in vivo. We conclude that 1) SP-B processing occurs in a cell-specific manner, 2) the proprotein contains secretory granule targeting determinants that are not cell-specific, 3) the NH2-terminal propeptide and the mature peptide are required for targeting SP-B to lamellar body, and 4) the COOH-terminal propeptide is not required for processing or sorting of SP-B.

Structural requirements for intracellular transport of pulmonary surfactant protein B (SP-B).

Human SP-B is synthesized by the alveolar Type II epithelial cell as a 381 amino acid preproprotein. The 79 residue mature SP-B peptide is extremely hydrophobic and flanked by propeptides of 200 and 102 amino acids at its NH2- and COOH-termini, respectively. The purpose of this study was to identify peptide domains of the SP-B proprotein necessary for trafficking of the mature peptide in the secretory pathway. To this end several constructs were generated, by subcloning the full length human SP-B (SP-B), COOH-terminally truncated SP-B (SP-B delta C, in which residues 201-381 were deleted), NH2-terminally deleted SP-B (SP-B delta N, in which residues 28-200 were deleted), NH2-terminal propeptide (SP-BN), mature SP-B (SP-BM) and COOH-terminal propeptide (SP-BC), into the mammalian expression vector pcDNA3. The resulting expression constructs were characterized by DNA sequencing and in vitro transcription/translation and subsequently transfected into Chinese hamster ovary cells. 48 h after transfection, cells were labeled with [35S]-met/cys and analyzed by immunoprecipitation, SDS-PAGE and autoradiography. Proteins encoded by SP-B, SP-B delta C, SP-BN and SP-BC constructs were secreted into media; in contrast, SP-B constructs lacking the NH2-terminal propeptide (SP-B delta N) remained in the endoplasmic reticulum (as assessed by endoglycosidase H sensitivity) and were rapidly degraded. We conclude that (1) 27 amino acids at the NH2-terminus of SP-B contain a functional signal peptide and (2) the NH2-terminal propeptide of the SP-B precursor is necessary and sufficient for intracellular trafficking of the mature peptide.

AMNIOTIC FLUID (AF) FETAL LUNG PROTEINS IN CONGENITAL DIAPHRAGMATIC HERNIA(CDH). † 1373

We have recently reported that the AF concentrations of surfactant protein A (SP-A) and saturated phosphatidylcholine (Sat PC) are decreased in fetuses with CDH (Am J Ob Gyn 173:1401, 1995). Nonetheless, it is still unclear if these abnormalities correlate with outcome and whether other fetal lung proteins are also decreased in AF in the presence of CDH. In this study we measured SP-A by ELISA, Sat PC by thin layer chromatography after exposure to osmium tetroxide, as well as surfactant protein B (SP-B) and clara cell protein (CCP) using Western blotting and RIA, respectively, in AF samples obtained by amniocentesis from a group of fetuses with CDH diagnosed by prenatal ultrasound (n=27, GA 16-41 wks), and compared them with measurements from a control population of normal fetuses (n=95, GA 14-41 wks). In the control group there were progressive increases in SP-A (R=0.53, p<0.00001), Sat PC (R=0.59, p<0.00001) and CCP (R=0.43, p<0.00001) with advancing gestation. Comparisons between both groups were done by ANCOVA using gestational age as covariate. The AF concentrations of SP-A (F=9.55, p=0.002), Sat PC (F=14.44, p=0.0002) and CCP (F=13.77, p=0.0003) were significantly lower in fetuses with CDH than in controls. However, the AF concentration of total SP-B of controls (n=35, GA 30-41 wks) was quite variable and was not different from fetuses with CDH (n=20, F=2.33, p=0.13). Also, no differences were found in the AF concentration of mature SP-B (8 KD) or its 26 KD and 42 KD precursors. Using discriminant analysis or multiple logistic regression to determine the outcome of fetuses with CDH, CCP in AF correctly predicted survival vs death or need for ECMO in 80% of cases (sens. 56%, spec. 94%) compared with 76% of cases for SP-A and Sat PC (sens. 56%, spec. 88%). Using a combination of these measurements plus GA did not improve their predictive ability. These findings suggest that the AF concentration of several fetal lung products are markedly decreased in AF from pregnancies with fetuses with CDH, and that these abnormalities exhibit some correlation with outcome in this population.

Surfactant protein B in human fetal lung: developmental and glucocorticoid regulation.

Pulmonary surfactant protein B (SP-B) enhances phospholipid film formation in vitro and is essential for normal surfactant function in vivo. We examined human fetal lung before and during explant culture for content and cellular localization of SP-B mRNA and protein. SP-B mRNA was low in preculture specimens (18-20 wk) but hybridization signal increased over epithelial cells during culture and was enhanced by dexamethasone treatment (10 nM). SP-B immunofluorescence was very low in preculture specimens, increased during culture, and was uniformly intense in epithelial cells of dexamethasone-treated tissue. With a newly developed immunoassay, SP-B protein was undetectable in preculture lung (< 2% of adult), appeared during culture (26% of adult), and was further increased approximately 3-fold by dexamethasone treatment (86% of adult); lung tissue of two newborn infants contained 7-9-fold more SP-B than is found in the adult. Using Western blot with enhanced chemiluminescence, mature SP-B was undetectable in 16-wk specimens but was present in 19-24-wk preculture tissue at 0.2-2.9% of the adult level. By comparison, SP-B mRNA content is 14 and 50% of adult level in 19- and 24-wk lung tissue, respectively; levels increase 3-fold during culture and a further 3-fold with dexamethasone. Based on these observed differences between mRNA and protein content, we conclude that basal SP-B gene expression in epithelial cells of human fetal lung is regulated primarily at the level of translation or protein stability, whereas glucocorticoids act transcriptionally. We speculate that SP-B protein accumulates only as type II cells differentiate and acquire lamellar bodies for processing and storage of SP-B.

Intracellular processing of pulmonary surfactant protein B in an endosomal/lysosomal compartment.

The hydrophobic surfactant protein B (SP-B) is synthesized in alveolar type II cells as a 40-kDa precursor protein that is processed via 39- and 23-kDa intermediates to the mature 8-kDa size. To determine the site of SP-B processing, the subcellular distribution of the precursor and mature forms of SP-B was investigated on ultrathin cryosections of human lung using two polyclonal antibodies that discriminate between precursor forms and the mature form of SP-B. An antibody against the human lysosomal membrane glycoprotein CD63 was used to identify organelles in the endosomal/lysosomal pathway. Precursor SP-B was present in the endoplasmic reticulum, the Golgi complex, and multivesicular bodies but was absent from lamellar bodies and plasma membrane. Mature SP-B was present in multivesicular bodies and lamellar bodies but almost completely absent in the endoplasmic reticulum and the Golgi complex. Semiquantitative evaluation of the immunogold labeling by counting the gold particles representing precursor or mature SP-B in the different compartments showed that the mature-to-precursor ratio was low in the endoplasmic reticulum and the Golgi complex (0.13 and 0.11, respectively), increased in the multivesicular bodies to 3.4, and was very high (65) in lamellar bodies. Multivesicular bodies and lamellar bodies contain the lysosomal membrane marker CD63 and are therefore part of the lysosomal pathway. These data strongly suggest that precursor SP-B is proteolytically processed to its mature 8-kDa form intracellularly in an endosomal/lysosomal compartment, most probably in multivesicular bodies.

Processing of surfactant protein B proprotein by a cathepsin D-like protease.

Surfactant protein B (SP-B) is a hydrophobic peptide of relative molecular weight (M(r)) = 8,000 that is associated with pulmonary surfactant phospholipids. SP-B is synthesized by the alveolar type II epithelial cell as a proprotein of M(r) = 42,000 which requires at least two proteolytic cleavages to generate the 79 residue mature SP-B peptide. We have previously reported that cleavage of the NH2-terminal propeptide, to generate a processing intermediate of M(r) = 25,000, occurs in close temporal approximation to secretion. In the present study we demonstrate that SP-B proprotein, isolated from stably transfected Chinese hamster ovary cells, is processed to M(r) = 25,000 by a crude type II cell membrane fraction but not by intact type II cells or type II cell conditioned media. In vitro processing of the proprotein by the type II cell membrane preparation resulted in release of a single peptide of M(r) = 16,000-17,000, which was detected by antiserum directed against antigenic epitopes in propeptide of the precursor. SP-B processing activity was extracted by Na2CO3 lysis of the type II cell membrane preparation, had a pH optimum of 5.0-6.0, and was inhibited by 10(-7) M pepstatin A, suggesting that the NH2-terminal peptide of the precursor is cleaved by an aspartyl protease. Consistent with this hypothesis, processing of SP-B by a crude type II cell membrane preparation was blocked by antiserum directed against the aspartyl protease cathepsin D; further, purified cathepsin D efficiently processed the SP-B precursor to M(r) = 25,000. Collectively these results suggest that cleavage of the NH2-terminal propeptide of the SP-B precursor is mediated by cathepsin D or a cathepsin D-like protease localized within the secretory pathway of the type II epithelial cell.

Processing of hydrophobic pulmonary surfactant protein B in rat type II cells

The amino acid sequence of surfactant protein B (SP-B), derived from human genomic and cDNA sequences, indicates that the active polypeptide is contained within the sequence of a preproprotein of 381 residues. Synthesis of mature SP-B, which requires proteolytic processing at both the NH2- and COOH-termini of the proprotein, was studied in primary cultures of rat alveolar type II epithelial cells. Type II cells were pulse labeled with [35S]methionine-cysteine for 15-30 min and chased for 0-18 h. SP-B proprotein (Mr = 42,000) accumulated in the medium at early time points but declined at later time points suggesting extracellular proteolysis of the proprotein. In contrast, surfactant protein A (SP-A), another surfactant protein, continued to accumulate extracellularly during this time period. A proteolytic fragment of SP-B (Mr = 25,000) accumulated in the medium with a slightly slower time course, consistent with extracellular proteolysis of the proprotein. Intracellular processing of SP-B was also detected. SP-B polypeptides of Mr 8,000 and 12,000 were detected intracellularly and in the medium at late time points. These forms of SP-B (Mr = 8,000 and 12,000) comigrated in two-dimensional isoelectric-focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mature SP-B isolated from rat alveolar lavage fluid. The mature form of SP-B (Mr = 8,000), but not the Mr 42,000 and 25,000 SP-B precursors, was also detected in lamellar bodies isolated from rat lung homogenates. These experiments demonstrate complete proteolytic processing of prepro-SP-B to the alveolar Mr 8,000 form by type II epithelial cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)