ABSTRACT
The molecular basis involving adsorption of pulmonary surfactant at the respiratory air-liquid interface and the specific roles of the surfactant proteins SP-B and SP-C in this process have not been completely resolved. The reasons might be found in the largely unknown structural assembly in which surfactant lipids and proteins are released from alveolar type II cells, and the difficulties to sample, manipulate and visualize the adsorption of these micron-sized particles at an air-liquid interface under appropriate physiological conditions. Here, we introduce several approaches to overcome these problems. First, by immunofluorescence we could demonstrate the presence of SP-B and SP-C on the surface of exocytosed surfactant particles. Second, by sampling the released particles and probing their adsorptive capacity we could demonstrate a remarkably high rate of interfacial adsorption, whose rate and extent was dramatically affected by treatment with antibodies against SP-B and SP-C. The effect of both antibodies was additive and specific. Third, direct microscopy of an inverted air-liquid interface revealed that the blocking effect is due to a stabilization of the released particles when contacting the air-liquid interface, precluding their transformation and the formation of surface films. We conclude that SP-B and SP-C are acting as essential, preformed molecular keys in the initial stages of surfactant unpacking and surface film formation. We further propose that surfactant activation might be transduced by a conformational change of the surfactant proteins upon contact with surface forces acting on the air-liquid interface.
Subject(s)
Alveolar Epithelial Cells/metabolism , Pulmonary Surfactant-Associated Protein B/physiology , Pulmonary Surfactant-Associated Protein C/physiology , Adsorption , Alveolar Epithelial Cells/drug effects , Alveolar Epithelial Cells/ultrastructure , Animals , Boron Compounds , Cells, Cultured , Exocytosis , Fluorescent Dyes , Heterocyclic Compounds, 3-Ring , Hydrophobic and Hydrophilic Interactions , Microscopy, Confocal , Organelles/drug effects , Organelles/metabolism , Pulmonary Surfactant-Associated Protein B/antagonists & inhibitors , Pulmonary Surfactant-Associated Protein B/pharmacology , Pulmonary Surfactant-Associated Protein C/antagonists & inhibitors , Pulmonary Surfactant-Associated Protein C/pharmacology , Pulmonary Surfactants/chemistry , Rats , Rats, Sprague-Dawley , Surface Properties , Surface TensionABSTRACT
Amyloid fibrils are found in approximately 25 different diseases, including Alzheimer's disease. Lung surfactant protein C (SP-C) forms fibrils in association with pulmonary disease. It was recently found that the C-terminal domain of proSP-C (CTC), which is localized to the endoplasmic reticulum (ER) lumen, protects the transmembrane (TM) part of (pro)SP-C from aggregation into amyloid until it has a folded into an alpha-helix. CTC appears to have a more general anti-amyloid effect by also acting on TM regions of other proteins. Here we investigate interactions of CTC with the amyloid beta-peptide (Abeta) associated with Alzheimer's disease and medin, a peptide that forms fibrils in the most common form of human amyloid. CTC prevents fibril formation in Abeta and medin and forms a complex with Abeta oligomers, as judged by size-exclusion chromatography and electrospray ionization mass spectrometry. These data suggest that CTC functions as a chaperone that acts preferentially against unfolded TM segments and structural motifs found during amyloid fibril formation, a mechanism that may be exploited in forming a basis for future anti-amyloid therapy.
Subject(s)
Amyloid beta-Peptides/antagonists & inhibitors , Milk Proteins/antagonists & inhibitors , Peptide Fragments/antagonists & inhibitors , Protein Precursors/physiology , Pulmonary Surfactant-Associated Protein C/physiology , Amino Acid Sequence , Amyloid beta-Peptides/ultrastructure , Antigens, Surface/ultrastructure , Humans , Milk Proteins/ultrastructure , Molecular Chaperones/chemistry , Molecular Chaperones/physiology , Molecular Chaperones/ultrastructure , Molecular Sequence Data , Peptide Fragments/ultrastructure , Protein Folding , Protein Precursors/ultrastructure , Protein Structure, Tertiary/physiology , Pulmonary Surfactant-Associated Protein C/chemistry , Pulmonary Surfactant-Associated Protein C/ultrastructureABSTRACT
The newly synthesized proSP-C (surfactant protein C precursor) is an integral ER (endoplasmic reticulum) membrane protein with a single metastable polyvaline alpha-helical transmembrane domain that comprises two-thirds of the mature peptide. More than 20 mutations in the ER-lumenal CTC (C-terminal domain of proSP-C), are associated with ILD (interstitial lung disease), and some of the mutations cause intracellular accumulation of cytotoxic protein aggregates and a corresponding decrease in mature SP-C. In the present study, we showed that: (i) human embryonic kidney cells expressing the ILD-associated mutants proSP-C(L188Q) and proSP-C(DeltaExon4) accumulate Congo Red-positive amyloid-like inclusions, whereas cells transfected with the mutant proSP-C(I73T) do not; (ii) transfection of CTC into cells expressing proSP-C(L188Q) results in a stable CTC-proSP-C(L188Q) complex, increased proSP-C(L188Q) half-life and reduced formation of Congo Red-positive deposits; (iii) replacement of the metastable polyvaline transmembrane segment with a stable polyleucine transmembrane segment likewise prevents formation of amyloid-like proSP-C(L188Q) aggregates; and (iv) binding of recombinant CTC to non-helical SP-C blocks SP-C amyloid fibril formation. These results suggest that CTC can prevent the polyvaline segment of proSP-C from promoting formation of amyloid-like deposits during biosynthesis, by binding to non-helical conformations. Mutations in the Brichos domain of proSP-C may lead to ILD via loss of CTC chaperone function.
Subject(s)
Amyloid/physiology , Lung Diseases, Interstitial/genetics , Mutation , Pulmonary Surfactant-Associated Protein C/physiology , Amino Acid Sequence , Amino Acid Substitution , Amyloid/antagonists & inhibitors , Cell Aggregation/physiology , Cell Line , Exons , Humans , Kidney , Molecular Sequence Data , Pulmonary Surfactant-Associated Protein C/chemistry , Pulmonary Surfactant-Associated Protein C/genetics , Pulmonary Surfactant-Associated Protein C/ultrastructure , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Recent evidence suggests that dysfunctional type II alveolar epithelial cells (AECs) contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Based on the hypothesis that disease-causing mutations in surfactant protein C (SFTPC) provide an important paradigm for studying IPF, we investigated a potential mechanism of AEC dysfunction suggested to result from mutant SFTPC expression: induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). We evaluated biopsies from 23 IPF patients (including 3 family members with L188Q SFTPC mutations, 10 individuals with familial interstitial pneumonia without SFTPC mutations, and 10 individuals with sporadic IPF) and sections from 10 control lungs. After demonstrating UPR activation in cultured A549 cells expressing mutant SFTPC, we identified prominent expression of UPR markers in AECs in the lungs of patients with SFTPC mutation-associated fibrosis. In individuals with familial interstitial pneumonia without SFTPC mutations and patients with sporadic IPF, we also found UPR activation selectively in AECs lining areas of fibrotic remodeling. Because herpesviruses are found frequently in IPF lungs and can induce ER stress, we investigated expression of viral proteins in lung biopsies. Herpesvirus protein expression was found in AECs from 15/23 IPF patients and colocalized with UPR markers in AECs from these patients. ER stress and UPR activation are found in the alveolar epithelium in patients with IPF and could contribute to disease progression. Activation of these pathways may result from altered surfactant protein processing or chronic herpesvirus infection.
Subject(s)
Endoplasmic Reticulum/physiology , Herpesviridae Infections/physiopathology , Pulmonary Alveoli/ultrastructure , Pulmonary Fibrosis/physiopathology , Pulmonary Surfactant-Associated Protein C/physiology , Stress, Physiological/physiopathology , Antigens, Viral/biosynthesis , Cells, Cultured , DNA-Binding Proteins/biosynthesis , Endoplasmic Reticulum Chaperone BiP , Glycoproteins/biosynthesis , Heat-Shock Proteins/biosynthesis , Herpesviridae Infections/complications , Humans , Immunohistochemistry , Molecular Chaperones/biosynthesis , Nuclear Proteins/biosynthesis , Protein Folding , Pulmonary Fibrosis/complications , Pulmonary Surfactant-Associated Protein C/genetics , Regulatory Factor X Transcription Factors , Transcription Factors , alpha-Mannosidase/biosynthesisABSTRACT
Modified natural surfactant preparations, used for treatment of respiratory distress syndrome in premature infants, contain phospholipids and the hydrophobic surfactant protein (SP)-B and SP-C. Herein, the individual and combined effects of SP-B and SP-C were evaluated in premature rabbit fetuses treated with airway instillation of surfactant and ventilated without positive end-expiratory pressure. Artificial surfactant preparations composed of synthetic phospholipids mixed with either 2% (wt/wt) of porcine SP-B, SP-C, or a synthetic poly-Leu analog of SP-C (SP-C33) did not stabilize the alveoli at the end of expiration, as measured by low lung gas volumes of approximately 5 ml/kg after 30 min of ventilation. However, treatment with phospholipids containing both SP-B and SP-C/SP-C33 approximately doubled lung gas volumes. Doubling the SP-C33 content did not affect lung gas volumes. The tidal volumes were similar in all groups receiving surfactant. This shows that SP-B and SP-C exert different physiological effects, since both proteins are needed to establish alveolar stability at end expiration in this animal model of respiratory distress syndrome, and that an optimal synthetic surfactant probably requires the presence of mimics of both SP-B and SP-C.
Subject(s)
Animals, Newborn/physiology , Pulmonary Alveoli/physiology , Pulmonary Surfactant-Associated Protein B/physiology , Pulmonary Surfactant-Associated Protein C/physiology , Respiratory Distress Syndrome, Newborn/physiopathology , 1,2-Dipalmitoylphosphatidylcholine/pharmacology , Amino Acid Sequence , Animals , Female , Humans , Infant, Newborn , Lung Volume Measurements , Molecular Sequence Data , Phospholipids/metabolism , Positive-Pressure Respiration , Pregnancy , Pulmonary Surfactants/pharmacology , Rabbits , Respiratory Mechanics/physiology , Tidal Volume/physiologyABSTRACT
Lung surfactant is a complex mixture of phospholipids and four surfactant-associated proteins (SP-A, SP-B, SP-C and SP-D). Its major function in the lung alveolus is to reduce surface tension at the air-water interface in the terminal airways by the formation of a surface-active film enriched in surfactant lipids, hence preventing cellular collapse during respiration. Surfactant therapy using bovine or porcine lung surfactant extracts, which contain only polar lipids and native SP-B and SP-C, has dramatically improved the therapeutic outcomes of preterm infants with respiratory distress syndrome (RDS). One important goal of surfactant researchers is to replace animal-derived therapies with fully synthetic preparations based on SP-B and SP-C, produced by recombinant technology or peptide synthesis, and reconstituted with selected synthetic lipids. Here, we review recent research developments with peptide analogues of SP-B and SP-C, designed using either the known primary sequence and three-dimensional (3D) structure of the native proteins or, alternatively, the known 3D structures of closely homologous proteins. Such SP-B and SP-C mimics offer the possibility of studying the mechanisms of action of the respective native proteins, and may allow the design of optimized surfactant formulations for specific pulmonary diseases (e.g., acute lung injury (ALI) or acute respiratory distress syndrome (ARDS)). These synthetic surfactant preparations may also be a cost-saving therapeutic approach, with better quality control than may be obtained with animal-based treatments.
Subject(s)
Lung/physiology , Pulmonary Surfactant-Associated Proteins/chemistry , Pulmonary Surfactant-Associated Proteins/physiology , Pulmonary Surfactants/chemistry , Amino Acid Sequence , Disulfides/analysis , Humans , Models, Molecular , Molecular Sequence Data , Protein Conformation , Pulmonary Surfactant-Associated Protein A/chemistry , Pulmonary Surfactant-Associated Protein A/physiology , Pulmonary Surfactant-Associated Protein B/chemistry , Pulmonary Surfactant-Associated Protein B/physiology , Pulmonary Surfactant-Associated Protein C/chemistry , Pulmonary Surfactant-Associated Protein C/physiology , Pulmonary Surfactant-Associated Protein D/physiology , Respiratory Mechanics , Sequence AlignmentABSTRACT
Surfactant proteins A and D (SP-A and SP-D) are members of the collectin family of host defense proteins that are expressed in epithelial cells lining the lung. SP-A and SP-D interact with surfactant lipids in complex ways to determine the structure of tubular myelin, and the ratio of surfactant subfractions that, in turn, influences surfactant uptake and metabolism, respectively. SP-A and SP-D play critical roles in host defense of the lung against diverse viral, fungal, and bacterial pathogens. The collectins bind to the surfaces of microbes via carbohydrate-dependent interaction, aggregating, opsonizing, and enhancing clearance of the organisms by alveolar macrophages in the lung. Pulmonary surfactant proteins A and D play dual roles in pulmonary homeostasis, determining the structure of alveolar lipids and mediating the innate host defense system of the lung.
Subject(s)
Lung Diseases/prevention & control , Pulmonary Surfactants , Animals , Bacterial Infections/prevention & control , Humans , Lung Diseases/microbiology , Lung Diseases/virology , Lung Diseases, Fungal/prevention & control , Pulmonary Surfactant-Associated Protein A/physiology , Pulmonary Surfactant-Associated Protein C/physiology , Pulmonary Surfactant-Associated Protein D/physiology , Virus Diseases/prevention & controlABSTRACT
Recent reports have linked mutations in the surfactant protein C gene (SFTPC) to familial forms of pulmonary fibrosis, but it is uncertain whether deficiency of mature SP-C contributes to disease pathogenesis. In this study, we evaluated bleomycin-induced lung fibrosis in mice with genetic deletion of SFTPC. Compared with wild-type (SFTPC+/+) controls, mice lacking surfactant protein C (SFTPC-/-) had greater lung neutrophil influx at 1 week after intratracheal bleomycin, greater weight loss during the first 2 weeks, and increased mortality. At 3 and 6 weeks after bleomycin, lungs from SFTPC-/- mice had increased fibroblast numbers, augmented collagen accumulation, and greater parenchymal distortion. Furthermore, resolution of fibrosis was delayed. Although remodeling was near complete in SFTPC+/+ mice by 6 weeks, SFTPC-/- mice did not return to baseline until 9 weeks after bleomycin. By terminal dUTP nick-end labeling staining, widespread cell injury was observed in SFTPC-/- and SFTPC+/+ mice 1 week after bleomycin; however, ongoing apoptosis of epithelial and interstitial cells occurred in lungs of SFTPC-/- mice, but not SFTPC+/+ mice, 6 weeks after bleomycin. Thus, SP-C functions to limit lung inflammation, inhibit collagen accumulation, and restore normal lung structure after bleomycin.
Subject(s)
Pulmonary Fibrosis/pathology , Pulmonary Surfactant-Associated Protein C/physiology , Animals , Apoptosis , Bleomycin/toxicity , Cells/pathology , Collagen/analysis , Disease Models, Animal , Fibroblasts , Hydroxyproline/analysis , In Situ Nick-End Labeling , Leukocyte Count , Lung/pathology , Mice , Mice, Knockout , Neutrophils , Peroxidase/analysis , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/genetics , Pulmonary Surfactant-Associated Protein C/genetics , Weight LossABSTRACT
Surfactant protein C (SP-C) is a small lipopeptide of which the main part consists of a typical valyl-rich transmembrane domain. The protein is expressed as a propeptide (proSP-C) which is processed and sorted via the regulated secretory pathway to the lamellar body, where mature SP-C is stored before secretion into the alveolar space. In this study we investigated the identity of the compartment to which proSP-C is sorted in cells that do not have a regulated secretory pathway, such as CHO cells. By electron microscopy we determined that proSP-C was localized in an uncommon membrane compartment with very regular morphology, which was not present in control cells. This membrane compartment is not influenced by the palmitoylation of proSP-C and is probably derived from the endoplasmic reticulum. However, proSP-C chimeras with artificial transmembrane domains induced a membrane compartment with a different morphology. Therefore we propose that the typical amino acid sequence of the transmembrane domain of proSP-C plays a role in membrane formation and morphology, which may be relevant under physiological conditions.
Subject(s)
CHO Cells/physiology , Intracellular Membranes/metabolism , Peptides/physiology , Pulmonary Surfactant-Associated Protein C/physiology , Amino Acid Sequence , Animals , CHO Cells/cytology , CHO Cells/ultrastructure , Cricetinae , Culture Media/chemistry , Culture Media/pharmacology , Gene Expression Regulation/drug effects , Intracellular Membranes/ultrastructure , Microscopy, Immunoelectron , Molecular Sequence Data , Peptides/genetics , Peptides/metabolism , Protein Processing, Post-Translational , Pulmonary Surfactant-Associated Protein C/genetics , Pulmonary Surfactant-Associated Protein C/metabolism , Pulmonary Surfactants/metabolism , Sequence Homology, Amino AcidABSTRACT
Incorporation of pulmonary surfactant into fibrin inhibits its plasmic degradation. In the present study we investigated the influence of surfactant proteins (SP)-A, SP-B, and SP-C on the fibrinolysis-inhibitory capacity of surfactant phospholipids. Plasmin-induced fibrinolysis was quantified by means of a (125)I-fibrin plate assay, and surfactant incorporation into polymerizing fibrin was analyzed by measuring the incorporation of (3)H-labeled L-alpha-dipalmitoylphosphatidylcholine into the insoluble clot material. Incorporation of a calf lung surfactant extract (Alveofact) and an organic extract of natural rabbit large surfactant aggregates (LSA) into a fibrin clot revealed a stronger inhibitory effect on plasmic cleavage of this clot than a synthetic phospholipid mixture (PLX) and unprocessed LSA. Reconstitution of PLX with SP-B and SP-C increased, whereas reconstitution with SP-A decreased, the fibrinolysis-inhibitory capacity of the phospholipids. The SP-B effect was paralleled by an increased incorporation of phospholipids into fibrin. We conclude that the inhibitory effect of surfactant incorporation into polymerizing fibrin on its susceptibility to plasmic cleavage is enhanced by SP-B and SP-C but reduced by SP-A. In the case of SP-B, increased phospholipid incorporation may underlie this finding.