PERSPECTIVES
Abnormal Expression of Surfactant Protein C and Lung Disease

Lawrence M. Nogee

Division of Neonatology, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland

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Pulmonary surfactant is the mixture of lipids and proteins necessary to reduce alveolar surface tension and prevent end expiratory atelectasis. Deficiency of surfactant due to immaturity is the principal cause of the respiratory distress syndrome (RDS) observed in prematurely born infants (1). Exogenous surfactant preparations derived from animals are highly effective in treating RDS, and show promise for other lung diseases (2, 3). Although surfactant phospholipids are the principal components that allow surface tension reduction, two low molecular weight, hydrophobic proteins, surfactant protein (SP)-B and SP-C, are also important for surfactant function (4, 5). Addition of either SP-B or SP-C to surfactant phospholipids results in surfactant preparations that can rapidly lower surface tension in vitro and are effective in treating RDS in experimental animals, as is a modified recombinant SP-C-based surfactant (6). Both SP-B and SP-C are found in varying amounts in animal-derived exogenous surfactant preparations used clinically (9). However, the relative roles of SP-B and SP-C in surfactant function are unknown.

Both SP-B and SP-C are highly expressed in lung tissue, and their expression increases with advancing gestation, although changes in expression were not precisely coregulated during development, lung injury, and in vitro (10- 13). Both proteins are derived from proteolytical processing of larger precursor proteins (proSP-B and proSP-C) (14). proSP-B is expressed in both alveolar type II cells and Clara cells, although only type II cells fully process proSP-B to mature SP-B. SP-C expression is confined to alveolar type II cells, and its cell specificity has made the SP-C promoter an extremely useful reagent to drive the expression of a wide number of proteins in lungs of transgenic animals (15, 16).

Human proSP-C is a 197-amino acid protein encoded by a single, relatively small gene on the short arm of chromosome 8 that spans ~ 3,500 bases, and contains 6 exons, the last of which is untranslated (Figure 1) (17, 18). Alternative splicing at the beginning of exon 5 also results in a transcript encoding a 191-amino acid proprotein (17, 19). The relative degree to which each splice variant is translated and the roles of these SP-C splice variants are unknown. proSP-C does not contain a traditional signal peptide and is an integral membrane protein in which the region corresponding to mature SP-C acts as the membrane-anchoring domain (14). Most data are consistent with a type II orientation of proSP-C in the endoplasmic reticulum membrane with the amino terminus oriented toward the cytoplasm (14, 20). Proteolytic processing of proSP-C involves first, removal of the carboxy-terminus in multiple steps, and, subsequently, the amino-terminus, with the latter steps taking place in a post-golgi compartment, likely multivesicular bodies or lamellar bodies. Recent studies in both transgenic animals and in cell culture support the hypothesis that the amino-terminal domain of proSP-C is responsible for appropriate intracellular routing (21, 22). Mature SP-C is stored in the lamellar body and secreted by exocytosis along with surfactant phospholipids and SP-B (14).

View larger version (26K): [in this window] [in a new window]   Figure 1.   SP-C gene, mRNA, and protein. The SP-C gene is shown at the top, with exons represented by boxes and introns by lines. The locations of two single nucleotide polymorphisms located in exons 4 and 5 that result in amino acid substitutions (codon 138 Threonine or Asparagine, and codon 186 Serine or Asparagine) are shown, as is the site of alternative splicing at the beginning of exon 5 (arrow, hatched area in mRNA). Mature SP-C is encoded in exon 2 (gray). The normal SP-C mRNA, its translation, and post-translational processing of proSP-C are shown on the left; the mature SP-C domain is shown in gray. The exact structure and folding of the proprotein domains are not known. The dashed line indicates the presumed block in proSP-C processing associated with hereditary SP-B deficiency. On the right are shown the postulated effects of a known mutation in the SP-C gene (33), where a single base substitution results in the skipping of exon 4 in the mRNA, with the deletion of sequences corresponding to exon 4 in the proprotein. Such a mutation presumably results in misfolding of the proprotein, with interactions of misfolded SP-C with native SP-C accounting for the reduction of proSP-C and mature SP-C content in lung tissue, following possible aggresome formation, degradation, and secondary cellular injury.

SP-C is extensively postranslationally modified, including palmitoylation at cysteine residues at positions 5 and 6 of the mature peptide, and is thus a proteolipid (14, 23, 24). The mature SP-C peptide found in the airspaces contains 35 amino acids, and its amino acid sequence has been highly conserved across species (25). The protein contains a stretch of valine residues that form an  helix that is just long enough to span a lipid bilayer. The insertion of this helix into a lipid bilayer and interaction of the palmitic acid residues with lipids are thought to be important in SP-C's ability to augment surface tension lowering (5, 25).

As both SP-B and SP-C facilitate the adsorption of surfactant phospholipids to an air-liquid interface, and an effective surfactant for treating RDS in experimental animals can be made with either, it is unclear whether the roles of SP-B and SP-C are redundant, or whether each serves a unique function. Mice genetically engineered to produce SP-B do not inflate their lungs and die in the perinatal period, and human infants unable to make SP-B develop severe lung disease that is rapidly fatal in the first months of life despite maximal medical support (26). These observations clearly indicate that SP-B is essential for normal lung function and suggest that SP-C cannot compensate for the lack of SP-B in vivo. However, SP-B deficiency is also associated with abnormalities in the processing of SP-C. Specifically, in the absence of intracellular production of SP-B, proSP-C is not fully processed to the mature peptide, and aberrantly processed SP-C intermediates accumulate in the lung tissue and alveolar spaces (29, 30). Thus, it is likely that SP-B-deficient infants are also deficient in SP-C, and these observations do not completely answer whether SP-C can replace the role of SP-B in vivo. The role of these abnormal SP-C-related peptides in the pathophysiology of SP-B deficiency is also uncertain. They contain both hydrophilic and hydrophobic epitopes and are unlikely to be very surface-active, and are abundant enough to potentially inhibit surfactant function. The lung disease in SP-B deficiency is also complicated by other secondary changes in surfactant metabolism, including disruption of normal-appearing lamellar bodies (31). Thus, although hereditary SP-B deficiency demonstrates that intracellular SP-B expression is critical for normal type II cell metabolism, whether the roles of SP-B and SP-C are functionally redundant remains unknown.

To address the question of whether SP-C can compensate for the absence of SP-B in hereditary SP-B deficiency, Conkright and colleagues generated transgenic animals expressing the mature SP-C peptide with an HA tag, but without the flanking domains of proSP-C (32). Their goal was to have a mouse that could produce mature SP-C without the need for the normal post-translational processing of proSP-C. By crossbreeding with mice heterozygous for SP-B deficiency, they could be used to bypass the block in SP-C processing in SP-B-deficient mice to determine whether expression of mature SP-C could rescue the lethal SP-B-null phenotype. However, the few mice obtained that expressed the transgene had lungs that were markedly underdeveloped and the severity of the pulmonary phenotype correlated with the level of transgene expression. It is unclear why expression of mature SP-C without its flanking proprotein domains at the appropriate time in gestation for proSP-C expression should have resulted in arrested lung development in these animals, and the downstream events leading to this phenotype require further study. Although these results precluded the use of these animals for the original purpose, they raise important new questions concerning the role of SP-C in both normal lung function and lung disease.

Abnormalities in the SP-C gene have recently been related to human lung disease (33). A mutation in the donor splice site of exon 4 of the SP-C gene was found in a mother and daughter who had interstitial lung disease with its onset in infancy. The mutation resulted in the in-frame skipping of an exon in the SP-C mRNA, which led to the production of a shortened proprotein lacking 37 amino acids from the carboxyterminal domain of wild-type proSP-C (Figure 1). Both the abnormal mRNA and proprotein were detected in the lung tissue of the affected infant. Aggresome formation has been observed in A549 cells transfected with constructs containing mutated proSP-C sequences (34). It is thus plausible that the mutation resulted in misfolding of proSP-C, with exposure of hydrophobic epitopes and aggregation of abnormal proSP-C in the secretory pathway leading to cellular injury (35). Although the mutation was present on only one allele, the amount of normal proSP-C was reduced, and mature SP-C was undetectable in the affected child's lung tissue. Mature SP-C can self-associate, and an adenoviral vector expressing mature SP-C was able to direct SP-C secretion in wild-type but not SP-C-deficient mice, indicating that self-association may occur within the secretory pathway (21, 36). The abnormal proprotein may thus have had a dominant negative effect on the metabolism of wild-type proSP-C. Although the lack of mature SP-C could have also contributed to the lung disease associated with this mutation, the findings of Conkright and colleagues provide support for the hypothesis that lung disease was due to the expression of abnormal SP-C. SP-C genetic variants may thus contribute to a wider spectrum of disease, including neonates with arrested lung development.

The long-term consequences of a lack of SP-C production are uncertain. Unlike SP-B-deficient mice, genetically engineered SP-C-deficient mice did not develop perinatal lung disease. However, surfactant isolated from SP-C-deficient animals was unstable on a captive bubble apparatus, suggesting that at low lung volumes the lack of SP-C could be more critical (37). The role of SP-C in perinatal adaptation may also differ in other species. A lack of SP-C was observed in the lungs of calves dying from a form of RDS that is likely genetic in etiology (38). Similarly, a lack of proSP-C in some full-term human infants dying from RDS has also been noted, although the mechanisms responsible for the lack of proSP-C were not identified (39).

The consequences of overexpression of SP-C in other situations are unknown. Little is also known concerning SP-C metabolism, including its biologic half-life, whether it is recycled, and which cells are primarily responsible for its catabolism. SP-C may be a particularly important molecule whose abnormal expression may result in lung disease due to its relatively high level of expression in the alveolar type II cell, hydrophobicity, and other properties. The  helical domain of mature SP-C can undergo transformation into a  sheet conformation, leading to polymerization and formation of amyloid-like fibrils (40). Such fibrils have been isolated from the lung fluid of patients with alveolar proteinosis who have elevated levels of SP-C, as well as other surfactant components (41). Although the clinical significance of such fibrils is unknown, these observations indicate that conditions altering SP-C concentration and secondary structure could have adverse effects. As palmitoylation of SP-C slowed the formation of fibrils (42), mutations that interfere with normal post-translational modifications of proSP-C may predispose to fibril formation. If such fibrils form intracellularly, they are likely to be deleterious to the type II cell. It is not known whether SP-C derived from the transgene was normally post-translationally modified, but if not, it may have been more likely to form aggregates. Alveolar type II cells also serve as the progenitor cells for type I cells after nonspecific lung injury, and such hypertrophic type II cells stain intensely for proSP-C (43, 44). Misfolding of proSP-C in the reparative phase of lung injury due to either genetic or environmental influences could alter the balance between ongoing injury and repair, and factors that perturb SP-C expression, processing, and folding may thus be important in a variety of lung diseases.

Finally, the expression of any sufficiently hydrophobic or abnormal protein, or misfolding of any highly-expressed protein with hydrophobic epitopes within the secretory pathway of the type II, may be deleterious. The resulting phenotype, including disruption of normal lung development, may depend more upon the level, location, and timing of expression of the abnormal protein during development rather than the specific protein involved. This suggests a role for abnormal protein folding in the pathophysiology of altered lung development that has not previously been appreciated. Although disorders of disrupted lung development are rare, they have been reported, and their molecular causes are unknown. The etiology of fatal severe neonatal respiratory failure often remains unexplained (45). The concept that misfolding of secreted proteins within the type II cell could contribute to ongoing injury, inflammation, and abrogation of lung development is one that deserves further study.

In summary, the findings of Conkright and colleagues suggest a possible role for SP-C in lung development as well as its role in surfactant function in the airspaces (32). Their findings also provide support for the hypothesis that aberrant SP-C or other abnormally-folded proteins in the type II cell may be toxic and important in the pathogenesis of lung disease. Environmental factors, including drugs that either inhibit or facilitate the normal folding and processing of proSP-C or other secreted proteins, require further study. The role of genetic variations in the SP-C and other genes, including common polymorphisms or normally-occurring splice variants that influence the structure of proSP-C and its folding, or that affect the level and timing of SP-C expression during development, need to be better understood. Quantitative assays for both proSP-C and SP-C in lung tissue and bronchoalveolar lavage fluid are needed. The generation of animals containing SP-C variants and the ability to study the function of these variants in in vitro systems will be important in dissecting out the full roles of SP-C in normal and abnormal lung function.

	Footnotes

Address correspondence to: Lawrence M. Nogee, M.D., CMSC 6-104, Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, MD 21287-3200. E-mail: lnogee{at}jhmi.edu

(Received in original form December 19, 2001).

Acknowledgments: This work was supported by grants from the National Institutes of Health, HL-54703 and HL- 65174, and the Eudowood Foundation.

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