Nonspecific interstitial pneumonia, alveolar proteinosis, and abnormal proprotein trafficking resulting from a spontaneous mutation in the surfactant protein C gene.

Human surfactant protein C (hSP-C(1-197)) is synthesized as a 197 amino acid proprotein and cleaved to a mature 3.7 kD form. Although interstitial lung disease in patients with mutations of the hSP-C gene is becoming increasingly recognized, the mechanisms linking molecular events with clinical pathogenesis are not fully defined. We describe a full-term infant with respiratory insufficiency associated with a spontaneous heterozygous mutation resulting in a substitution of lysine for glutamic acid at position 66 (= E66K) of the proximal hSP-C COOH flanking propeptide. Lung histology and biochemical studies of the index patient (hSP-C(E66K)) revealed nonspecific interstitial pneumonia, increased alveolar total phospholipid lacking phosphatidylglycerol, and increased surfactant protein A. Localization of proSP-C from lung sections prepared from this patient using immunofluorescence and immunogold electron microscopy revealed abnormal proSP-C staining in endosomal-like vesicles of type II cells distinct from SP-B. To evaluate the effect of the E66K substitution on intracellular trafficking of proSP-C, fusion proteins consisting of enhanced green fluorescent protein (EGFP) and hSP-C(1-197) (wild type) or mutant hSP-C(E66K) were generated and transfected into A549 cells. EGFP/hSP-C(1-197) was expressed within CD-63-positive, EEA-1-negative vesicles, whereas EGFP/hSP-C(E66K) localized to EEA-1 positive vesicles. The E66K substitution is representative of a new class of SP-C mutation associated with interstitial lung disease that is diverted from the normal biosynthetic pathway. We propose that, similar to other storage disorders, lung injury results from induction of a toxic gain of function induced by the mutant product that is subject to genetic modifiers and environmental influences.

Processing of surfactant protein C requires a type II transmembrane topology directed by juxtamembrane positively charged residues.

Surfactant protein C (SP-C) is a lung-specific protein that is synthesized as a 21-kDa integral membrane propeptide (pro-SP-C) and proteolytically processed to a 3.7-kDa secretory product. Previous studies have shown that palmitoylation of pro-SP-C is dependent on two N-terminal juxtamembrane positively charged residues. We hypothesized that these residues influence modification of pro-SP-C by directing transmembrane orientation. Double substitution mutation of these juxtaposed residues from positive to neutral charged species resulted in complete reversal of transmembrane orientation of pro-SP-C and total abrogation of post-translational processing. Mutation of a single residue resulted in mixed orientation. Protein trafficking studies in A549 cells showed that while the double mutant was retained in the endoplasmic reticulum, single mutants produced a mixed pattern of both endoplasmic reticulum (double mutant-like) and vesicular (wild type-like) expression. Our study demonstrates the crucial role juxtamembrane positively charged residues play in establishing membrane topology and their influence on the trafficking and processing of pro-SP-C. Moreover this study provides a likely precedent for a mechanism in disorders associated with mutations in the membrane-flanking region of integral membrane proteins.

Biosynthesis of surfactant protein C (SP-C). Sorting of SP-C proprotein involves homomeric association via a signal anchor domain.

Rat surfactant protein C (SP-C) is synthesized as a 194-amino acid propeptide (SP-C-(1-194)) that is directed to the distal secretory pathway and proteolytically processed as an integral membrane protein to yield its mature form. We had shown previously that trafficking of proSP-C is mediated both by a signal anchor domain contained within the mature SP-C sequence and by a targeting domain in the NH(2)-flanking propeptide. Based on evidence from other integral membrane proteins, we hypothesized that proSP-C targeting is effected by oligomerization of proSP-C monomers. To evaluate this in vitro, cDNA constructs encoding for either wild type proSP-C (pcDNA3/SP-C-(1-194)) or heterologous fusion proteins containing green fluorescent protein (EGFP) linked to SP-C-(1-194) (EGFP/SP-C-(1-194)), to mutant proSP-C lacking the NH(2) targeting domain (EGFP/SP-C-(24-194)), or to mature SP-C alone (EGFP/SP-C-(24-58)) were produced. In transfected A549 cells, fluorescence microscopy revealed that pcDNA3/SP-C-(1-194) and EGFP/SP-C-(1-194) were each expressed in CD63 (+), EEA1 (-) cytoplasmic vesicles. Expression of EGFP/SP-C-(24-194) or EGFP/SP-C-(24-58) resulted in translocation but retention in early compartments. When co-transfected with pcDNA3/SP-C-(1-194), both EGFP/SP-C-(24-194) and EGFP/SP-C-(24-58) were directed to CD63 (+) vesicles that also contained SP-C-(1-194). In contrast, trafficking of a folding mutant that forms juxtanuclear aggregates, EGFP/SP-C(C122/186G), was not corrected by cotransfection with pcDNA3/SP-C-(1-194). Chemical cross-linking studies of transfected cell lysates with bismaleimidohexane produced multimeric forms of both EGFP/SP-C-(1-194) and EGFP/SP-C-(24-58). These results indicate that sorting involves oligomeric association of proSP-C monomers mediated by the mature SP-C domain. Heteromeric assembly allows wild type proSP-C to facilitate trafficking of SP-C mutants with intact transmembrane domains but lacking targeting signals. We speculate that heterotypic oligomerization of wild type with SP-C folding mutants produces a dominant negative thus contributing to the pathology of chronic lung disease associated with patients heterozygous for mutant SP-C alleles.