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.     

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.     

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.     

Production in Escherichia coli of a recombinant C-terminal truncated precursor of surfactant protein B (rproSP-B Delta C). Structure and interaction with lipid interfaces

SP-B, a protein absolutely required to maintain the lungs open after birth, is synthesized in the pneumocytes as a precursor containing C-terminal and N-terminal domains flanking the mature sequence. These flanking-domains are cleaved to produce mature SP-B, coupled with its assembly into pulmonary surfactant lipid-protein complexes. In the present work we have optimized over-expression in Escherichia coli and purification of rproSP-B(DeltaC), a recombinant form of human proSP-B lacking the C-terminal flanking peptide, which is still competent to restore SP-B function in vivo. rProSP-B(DeltaC) has been solubilized, purified and refolded from bacterial inclusion bodies in amounts of about 4 mg per L of culture. Electrophoretic mobility, immunoreactivity, N-terminal sequencing and peptide fingerprinting all confirmed that the purified protein had the expected mass and sequence. Once refolded, the protein was soluble in aqueous buffers. Circular dichroism and fluorescence emission spectra of bacterial rproSP-B(DeltaC) indicated that the protein is properly folded, showing around 32% alpha-helix and a mainly hydrophobic environment of its tryptophan residues. Presence of zwitterionic or anionic phospholipids vesicles caused changes in the fluorescence emission properties of rproSP-B(DeltaC) that were indicative of lipid-protein interaction. The association of this SP-B precursor with membranes suggests an intrinsic amphipathic character of the protein, which spontaneously adsorbs at air-liquid interfaces either in the absence or in the presence of phospholipids. The analysis of the structure and properties of recombinant proSP-B(DeltaC) in surfactant-relevant environments will open new perspectives on the investigation of the mechanisms of lipid and protein assembly in surfactant complexes.     

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.     

Proteolytic activity of human cytomegalovirus UL80 protease cleavage site mutants.

The human cytomegalovirus UL80 open reading frame encodes protease and assembly protein from its N- and C-terminal regions, respectively. We reported previously that a 30-kDa protease is derived by autoproteolytic processing of a polyprotein which is the translation product of the entire UL80 open reading frame (E. Z. Baum, G. A. Bebernitz, J. D. Hulmes, V. P. Muzithras, T. R. Jones, and Y. Gluzman, J. Virol. 67:497-506, 1993). Three autoproteolytic cleavage sites within the UL80 polyprotein were characterized; site 143 is within the protease domain and inactivates the protease. In this article, we report (i) expression analyses of UL80 in infected cells, including the processing kinetics of the UL80 polyprotein; (ii) the existence of an additional cleavage site (site 209) within the protease domain of the UL80 polyprotein; and (iii) the effect of mutagenesis at each of the cleavage sites upon proteolytic activity and steady-state levels of the UL80 processing products. During the course of infection, UL80 polyprotein processing begins at cleavage site 643 and follows at sites 256 and 143. Cleavage at site 643 and/or 256 within the polyprotein is not a prerequisite for efficient protease activity, since all three proteases (85-, 80-, and 30-kDa proteins) were equally active in cleaving the assembly protein precursor to its mature form. Inhibition of cleavage at site 143 resulted in a three- to sixfold increase in the steady-state level of the 30-kDa protease, supporting the hypothesis that cleavage at this site may represent a mechanism by which cytomegalovirus regulates the level of active protease.     

Dissecting a cyanobacterial proteolytic system: efficiency in inducing degradation of the D1 protein of photosystem II in cyanobacteria and plants

A chromatography fraction, prepared from isolated thylakoids of a fatty acid desaturation mutant (Fad6/desA Colon, two colons Km(r)) of the cyanobacterium Synechocystis 6803, could induce an initial cleavage of the D1 protein in Photosystem II (PSII) particles of Synechocystis 6803 mutant and Synechococcus 7002 wild type as well as in supercomplexes of PSII-light harvesting complex II of spinach. Proteolysis was demonstrated both in darkness and in light as a reduction in the amount of full-length D1 protein or as a production of C-terminal initial degradation fragments. In the Synechocystis mutant, the main degradation fragment was a 10-kDa C-terminal one, indicating an initial cleavage occurring in the cytoplasmic DE-loop of the D1 protein. A protein component of 70-90 kDa isolated from the chromatographic fraction was found to be involved in the production of this 10-kDa fragment. In spinach, only traces of the corresponding fragment were detected, whereas a 24-kDa C-terminal fragment accumulated, indicating an initial cleavage in the lumenal AB-loop of the D1 protein. Also in Synechocystis the 24-kDa fragment was detected as a faint band. An antibody raised against the Arabidopsis DegP2 protease recognized a 35-kDa band in the proteolytically active chromatographic fraction, suggesting the existence of a lumenal protease that may be the homologue DegP of Synechocystis. The identity of the other protease cleaving the D1 protein in the DE-loop exposed on the stromal (cytoplasmic) side of the membrane is discussed.     

Autoprocessing of the HIV-1 protease using purified wild-type and mutated fusion proteins expressed at high levels in Escherichia coli

Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli. The full-length fusion proteins did not exhibit self-processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease. Rapid purification involving two column steps gave an HIV-1 protease preparations of greater than 95% purity (specific activity approximately 8500 pmol.min-1.micrograms protease-1) with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV-1 fusion proteins containing mutations at either the N- or the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site. Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity (approximately 5 pmol.min-1.micrograms protein-1).     

The elevation of serum napsin A in idiopathic pulmonary fibrosis, compared with KL-6, surfactant protein-A and surfactant protein-D

ABSTRACT: BACKGROUND: Napsin A, an aspartic protease, is mainly expressed in alveolar type-II cells and renal proximal tubules and is a putative immunohistochemical marker for pulmonary adenocarcinomas. This study sought to determine whether napsin A could be measured in the serum to evaluate its relationship to idiopathic pulmonary fibrosis (IPF) and determine whether renal dysfunction might affect serum napsin A levels. METHODS: Serum levels of napsin A were measured in 20 patients with IPF, 34 patients with lung primary adenocarcinoma, 12 patients with kidney diseases, and 20 healthy volunteers. Surfactant protein (SP)-A, SP-D, and Krebs von den Lungen-6 (KL-6) levels in serum and pulmonary function tests were also evaluated in IPF patients. RESULTS: Circulating levels of napsin A were increased in patients with IPF, as compared with healthy controls, and they correlated with the severity of disease. Moreover, the serum napsin A levels were not elevated in patients with pulmonary adenocarcinoma or renal dysfunction. The distinguishing point between IPF and the controls was that the area under the receiver operating characteristic curve (ROC) of napsin A was larger than that of KL-6, SP-A, or SP-D. CONCLUSION: These findings suggest that serum napsin A may be a candidate biomarker for IPF.     

Production in Escherichia coli of a recombinant C-terminal truncated precursor of surfactant protein B (rproSP-BΔc). Structure and interaction with lipid interfaces

SP-B, a protein absolutely required to maintain the lungs open after birth, is synthesized in the pneumocytes as a precursor containing C-terminal and N-terminal domains flanking the mature sequence. These flanking-domains are cleaved to produce mature SP-B, coupled with its assembly into pulmonary surfactant lipid-protein complexes. In the present work we have optimized over-expression in Escherichia coli and purification of rproSP-B_ΔC, a recombinant form of human proSP-B lacking the C-terminal flanking peptide, which is still competent to restore SP-B function in vivo. rProSP-B_ΔC has been solubilized, purified and refolded from bacterial inclusion bodies in amounts of about 4 mg per L of culture. Electrophoretic mobility, immunoreactivity, N-terminal sequencing and peptide fingerprinting all confirmed that the purified protein had the expected mass and sequence. Once refolded, the protein was soluble in aqueous buffers. Circular dichroism and fluorescence emission spectra of bacterial rproSP-B_ΔC indicated that the protein is properly folded, showing around 32% α-helix and a mainly hydrophobic environment of its tryptophan residues. Presence of zwitterionic or anionic phospholipids vesicles caused changes in the fluorescence emission properties of rproSP-B_ΔC that were indicative of lipid-protein interaction. The association of this SP-B precursor with membranes suggests an intrinsic amphipathic character of the protein, which spontaneously adsorbs at air-liquid interfaces either in the absence or in the presence of phospholipids. The analysis of the structure and properties of recombinant proSP-B_ΔC in surfactant-relevant environments will open new perspectives on the investigation of the mechanisms of lipid and protein assembly in surfactant complexes.     

Isolation of lamellar bodies from rat granular pneumocytes in primary culture

A lamellar body fraction was isolated from rat alveolar granular pneumocytes in primary culture by upward flotation on a discontinuous sucrose gradient and compared with a similar fraction isolated from lung homogenates. Lamellar bodies from granular pneumocytes were free of detectable contamination with either succinate dehydrogenase or NADPH-cytochrome c reductase. There was an enrichment of acid phosphatase activity, which, based on distribution of enzyme activity on the gradient, did not appear to be a contamination from other fractions. The lamellar body fraction of granular pneumocytes yielded approx. 1 microgram protein/10(6) cells with a phospholipid-to-protein ratio (mg/mg) of 9.6 +/- 0.4 (n = 7). Composition with respect to total phospholipids was 71.0% phosphatidylcholine (disaturated phosphatidylcholine, 45.2%), 8.4% phosphatidylglycerol and 12.8% phosphatidylethanolamine. Palmitic acid comprised 66% of the fatty acids in phosphatidylcholine and 34% of those in phosphatidylglycerol. The lamellar body fraction from granular pneumocytes was similar to that from whole lung with respect to phospholipid-to-protein ratio and phospholipid composition and showed only minor differences in fatty acid composition. Ultrastructurally, lamellar bodies showed generally intact limiting membranes and lamellated structure. Lamellar bodies from granular pneumocytes showed occasional multinucleated whorls which were not seen in those isolated from lung homogenates. This study describes a method for preparing a homogeneous fraction of intact lamellar bodies from small amounts of material (6 X 10(7) granular pneumocytes). The yield on a per cell basis was higher when compared with a similar preparation from whole lung, although overall yield is small, due to loss of cells during the cell isolation procedure. This preparation may be useful to evaluate the role of lamellar bodies in the synthesis and secretion of lung surfactant by isolated granular pneumocytes.     

Identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus Avian infectious bronchitis virus and characterization of the cleavage products

The coronavirus Avian infectious bronchitis virus (IBV) employs polyprotein processing as a strategy to express its gene products. Previously we identified the first cleavage event as proteolysis at the Gly(673)-Gly(674) dipeptide bond mediated by the first papain-like proteinase domain (PLPD-1) to release an 87-kDa mature protein. In this report, we demonstrate a novel cleavage activity of PLPD-1. Expression, deletion, and mutagenesis studies showed that the product encoded between nucleotides 2548 and 8865 was further cleaved by PLPD-1 at the Gly(2265)-Gly(2266) dipeptide bond to release an N-terminal 195-kDa and a C-terminal 41-kDa cleavage product. Characterization of the cleavage activity revealed that the proteinase is active on this scissile bond when expressed in vitro in rabbit reticulocyte lysates and can act on the same substrate in trans when expressed in intact cells. Both the N- and C-terminal cleavage products were detected in virus-infected cells and were found to be physically associated. Glycosidase digestion and site-directed mutagenesis studies of the 41-kDa protein demonstrated that it is modified by N-linked glycosylation at the Asn(2313) residue encoded by nucleotides 7465 to 7467. By using a region-specific antiserum raised against the IBV sequence encoded by nucleotides 8865 to 9786, we also demonstrated that a 33-kDa protein, representing the 3C-like proteinase (3CLP), was specifically immunoprecipitated from the virus-infected cells. Site-directed mutagenesis and expression studies showed that a previously predicted cleavage site (Q(2583)-G(2584)) located within the 41-kDa protein-encoding region was not utilized by 3CLP, supporting the conclusion that the 41-kDa protein is a mature viral product.     

Identification of a segment in the precursor of pulmonary surfactant protein SP-B, potentially involved in pH-dependent membrane assembly of the protein

In the present work, the hydrophobic properties of proSP-B, the precursor of pulmonary surfactant protein SP-B, have been analyzed under different pH conditions, and the sequence segment at position 111-135 of the N-terminal domain of the precursor has been detected as potentially possessing pH-dependent hydrophobic properties. We have studied the structure and lipid-protein interactions of the synthetic peptides BpH, with sequence corresponding to the segment 111-135 of proSP-B, and BpH-W, bearing the conservative substitution F127W to use the tryptophan as an intrinsic fluorescent probe. Peptide BpH-W interacts with both zwitterionic and anionic phospholipid vesicles at neutral pH, as monitored by the blue-shifted maximum emission of its tryptophan reporter. Insertion of tryptophan into the membranes is further improved at pH 5.0, especially in negatively-charged membranes. Peptides BpH and BpH-W also showed pH-dependent properties to insert into phospholipid monolayers. We have also found that the single sequence variation F120K decreases substantially the interaction of this segment with phospholipid surfaces as well as its pH-dependent insertion into deeper regions of the membranes. We hypothesize that this region could be involved in pH-triggered conformational changes occurring in proSP-B along the exocytic pathway of surfactant in type II cells, leading to the exposure of the appropriate segments for processing and assembly of SP-B within surfactant lipids.     

Characterization of the Niemann-Pick C pathway in alveolar type II cells and lamellar bodies of the lung

The Niemann-Pick C (NPC) pathway plays an essential role in the intracellular trafficking of cholesterol by facilitating the release of lipoprotein-derived sterol from the lumen of lysosomes. Regulation of cellular cholesterol homeostasis is of particular importance to lung alveolar type II cells because of the need for production of surfactant with an appropriate lipid composition. We performed microscopic and biochemical analysis of NPC proteins in isolated rat type II pneumocytes. NPC1 and NPC2 proteins were present in the lung, isolated type II cells in culture, and alveolar macrophages. The glycosylated and nonglycosylated forms of NPC1 were prominent in the lung and the lamellar body organelles. Immunocytochemical analysis of isolated type II pneumocytes showed localization of NPC1 to the limiting membrane of lamellar bodies. NPC2 and lysosomal acid lipase were found within these organelles, as confirmed by z-stack analysis of confocal images. All three proteins also were identified in small, lysosome-like vesicles. In the presence of serum, pharmacological inhibition of the NPC pathway with compound U18666A resulted in doubling of the cholesterol content of the type II cells. Filipin staining revealed a striking accumulation of cholesterol within lamellar bodies. Thus the NPC pathway functions to control cholesterol accumulation in lamellar bodies of type II pneumocytes and, thereby, may play a role in the regulation of surfactant cholesterol content.     

Identification of a segment in the precursor of pulmonary surfactant protein SP-B, potentially involved in pH-dependent membrane assembly of the protein

In the present work, the hydrophobic properties of proSP-B, the precursor of pulmonary surfactant protein SP-B, have been analyzed under different pH conditions, and the sequence segment at position 111-135 of the N-terminal domain of the precursor has been detected as potentially possessing pH-dependent hydrophobic properties. We have studied the structure and lipid-protein interactions of the synthetic peptides BpH, with sequence corresponding to the segment 111-135 of proSP-B, and BpH-W, bearing the conservative substitution F127W to use the tryptophan as an intrinsic fluorescent probe. Peptide BpH-W interacts with both zwitterionic and anionic phospholipid vesicles at neutral pH, as monitored by the blue-shifted maximum emission of its tryptophan reporter. Insertion of tryptophan into the membranes is further improved at pH 5.0, especially in negatively-charged membranes. Peptides BpH and BpH-W also showed pH-dependent properties to insert into phospholipid monolayers. We have also found that the single sequence variation F120K decreases substantially the interaction of this segment with phospholipid surfaces as well as its pH-dependent insertion into deeper regions of the membranes. We hypothesize that this region could be involved in pH-triggered conformational changes occurring in proSP-B along the exocytic pathway of surfactant in type II cells, leading to the exposure of the appropriate segments for processing and assembly of SP-B within surfactant lipids.     

Distribution of surfactant proteins in type II pneumocytes of newborn, 14-day old,and adult rats: an immunoelectron microscopic and stereological study

Surfactant proteins (SP) have an important impact on the function of the pulmonary surfactant. In contrast to humans, rat lungs are immature at birth. Alveolarization starts on postnatal day 4. Little is known about the distribution of SP during postnatal alveolarization. By immunoelectron microscopy, we studied the distribution of SP-A, SP-D, SP-B, and precursors of SP-C in type II pneumocytes before, near the end and after alveolarization and in mature lungs. We determined the subcellular volume fractions and the relative labeling index to obtain information about preferential labeling of compartments and non-randomness of labeling. Independently of alveolarization, the overall cellular distribution of SP was non-random. A preferential labeling for SP-A and SP-D was found in small vesicles and multivesicular bodies (mvb). SP-B and precursors of SP-C were localized in mvb and lamellar bodies (lb). There are no postnatal changes in labeling for all three SP in these compartments. Labeling intensity for SP-B in lb increased in close correlation with a significant increase in the volume fractions of lb during alveolarization. Our results support the concept that postnatal alveolarization in rat lungs is associated with significant increases in the SP-B content in lb and volume fraction of lb in type II pneumocytes. The postnatal compartment-specific distribution of SP-A, precursors of SP-C and SP-D does not change. Both the authors Andreas Schmiedl and Matthias Ochs contributed equally to this work.     

Definition of a C-reactive protein binding determinant on histones

C-reactive protein (CRP) is an acute phase inflammatory protein in man which binds to phosphocholine, chromatin, histones, and the 70-kDa protein of the U1 small nuclear ribonucleoprotein particle in a calcium-dependent, phosphocholine-inhibitable manner. CRP also binds to other proteins including fibronectin. The determinants involved in CRP binding to these diverse proteins have not been identified. The binding of CRP to histones was examined as these proteins are available in large quantity at high purity and subject to protease digestion with well characterized products. Histone H1 was digested with thrombin and trypsin to produce three distinct fragments, N-terminal, central globular, and C-terminal. CRP was shown only to bind to the C-terminal fragment. Binding to histone H2A was also examined. CRP binding was not diminished by cleavage of the C-terminal fragment but was greatly decreased when the central globular region of H2A was tested. Peptides were prepared to be identical to the N- and C-terminal fragments of H2A. The N-terminal (15 amino acid) fragment of H2A blocked CRP-induced precipitation of phosphocholine-coupled bovine serum albumin and histone H2A, whereas the C-terminal fragment showed no inhibition. Thus we have defined the first reported CRP binding determinant on a protein.     

The role of charged amphipathic helices in the structure and function of surfactant protein B.

Surfactant protein B (SP-B) is essential for normal lung surfactant function. Theoretical models predict that the disulfide cross-linked, N- and C-terminal domains of SP-B fold as charged amphipathic helices, and suggest that these adjacent helices participate in critical surfactant activities. This hypothesis is tested using a disulfide-linked construct (Mini-B) based on the primary sequences of the N- and C-terminal domains. Consistent with theoretical predictions of the full-length protein, both isotope-enhanced Fourier transform infrared (FTIR) spectroscopy and molecular modeling confirm the presence of charged amphipathic alpha-helices in Mini-B. Similar to that observed with native SP-B, Mini-B in model surfactant lipid mixtures exhibits marked in vitro activity, with spread films showing near-zero minimum surface tensions during cycling using captive bubble surfactometry. In vivo, Mini-B shows oxygenation and dynamic compliance that compare favorably with that of full-length SP-B. Mini-B variants (i.e. reduced disulfides or cationic residues replaced by uncharged residues) or Mini-B fragments (i.e. unlinked N- and C-terminal domains) produced greatly attenuated in vivo and in vitro surfactant properties. Hence, the combination of structure and charge for the amphipathic alpha-helical N- and C-terminal domains are key to SP-B function.     

Fo portion of Escherichia coli ATP synthase. Further resolution of trypsin-generated fragments from subunit b

F1-stripped everted membrane vesicles of the ATP synthase-overproducing Escherichia coli strain KY 7485 were treated with trypsin for different lengths of time. Subsequently, the Fo complex was isolated and analyzed by sodium dodecyl sulfate-gel electrophoresis, as well as immunoblotting using antibodies raised against subunit b. By these techniques 3 degradation products with apparent molecular masses of about 16 kDa could be detected in accordance with previous findings (Perlin, D.S., and Senior, A.E. (1985) Arch. Biochem. Biophys. 236, 603-611). Labeling of isolated trypsin-treated Fo fractions with the thiol-specific reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide, which has been demonstrated recently to specifically modify subunit b (Schneider, E., and Altendorf, K. (1985) Eur. J. Biochem. 153, 105-109) revealed that the 16-kDa digestion products were degraded into two stable fragments of 12 and 8.3 kDa. These polypeptides do not react with the anti-b antibodies. Treatment of purified liposome-integrated Fo with trypsin resulted in a similar cleavage pattern. In both cases protease digestion inhibited F1 binding while proton-translocating activity remained unaffected. However, liposomes reconstituted with Fo isolated from trypsin-treated membranes were impaired in both binding of F1 and proton translocation. These activities could be restored when reconstitution was carried out in the presence of native subunit b. From this we conclude that the C-terminal region of subunit b is necessary for proper reconstitution of Fo into liposomes.