Abstract

Pulmonary surfactant protein A (SP-A) is synthesized by type II cells and stored intracellularly in secretory granules (lamellar bodies) together with surfactant lipids and hydrophobic surfactant proteins B and C (SP-B and SP-C). We asked whether the progressive decrease in pH along the exocytic pathway could influence the secondary structure and lipid binding and aggregation properties of porcine SP-A. Conformational analysis from CD spectra of SP-A at various pH values indicated that the percentage of alpha-helix progressively decreased and that of beta-sheet increased as the pH was reduced. The protein underwent a marked self-aggregation at mildly acidic pH in the presence of Ca2+, conditions thought to resemble those existing in the trans-Golgi network. Protein aggregation was greater as the pH was reduced. We also found that both neutral and acidic vesicles either with or without SP-B or SP-C bound to SP-A at acidic pH as demonstrated by co-migration during centrifugation. However, the binding of acidic but not neutral vesicles to SP-A led to 1) a striking change in the CD spectra of the protein, which was interpreted as a decrease of the level of SP-A self-aggregation, and 2) a protection of the protein from endoproteinase Glu-C degradation at pH 4.5. SP-A massively aggregated acidic vesicles but poorly aggregated neutral vesicles at acidic pH. Aggregation of dipalmitoylphosphatidylcholine (DPPC) vesicles either with or without SP-B and/or SP-C strongly depended on pH, being progressively decreased as the pH was reduced and markedly increased when pH was shifted back to 7.0. At the pH of lamellar bodies, SP-A-induced aggregation of DPPC vesicles containing SP-B or a mixture of SP-B and SP-C was very low, although SP-A bound to these vesicles. These results indicate that 1) DPPC binding and DPPC aggregation are different phenomena that probably have different SP-A structural requirements and 2) aggregation of membranes induced by SP-A at acidic pH is critically dependent on the presence of acidic phospholipids, which affect protein structure, probably preventing the formation of large aggregates of protein.

Highlights

  • Pulmonary surfactant protein A (SP-A) is synthesized by type II cells and stored intracellularly in secretory granules together with surfactant lipids and hydrophobic surfactant proteins B and C (SP-B and SP-C)

  • We found that both neutral and acidic vesicles either with or without SP-B or SP-C bound to SP-A at acidic pH as demonstrated by co-migration during centrifugation

  • We found that the secondary structure of SP-A was changed by lowering the pH and that the protein underwent a rapid aggregation at mildly acidic pH in the presence of Ca2ϩ, conditions thought to resemble those existing in the trans-Golgi network (TGN) and along the exocytic pathway

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Summary

POTENTIAL ROLE OF ACIDIFICATION ALONG ITS EXOCYTIC PATHWAY*

(Received for publication, November 4, 1997, and in revised form, March 30, 1998). From the Department of Biochemistry and Molecular Biology, Faculty of Biology, Complutense University of Madrid, 28040 Madrid, Spain. The association of SP-A with lipids appears essential for the conversion of lipid aggregates from multilamellar forms present in the exocytic granule to dispersed ordered arrays known as tubular myelin [29] This physical transformation requires the hydrophobic surfactant protein SP-B [30]. The present study analyzes the structural properties of SP-A and its interaction with phospholipids at acidic pH in the presence and absence of the hydrophobic surfactant proteins SP-B and SP-C. We found that the secondary structure of SP-A was changed by lowering the pH and that the protein underwent a rapid aggregation at mildly acidic pH in the presence of Ca2ϩ, conditions thought to resemble those existing in the TGN and along the exocytic pathway. Vesicle aggregation induced by SP-A at acidic pH was critically dependent on the presence of negatively charged phospholipids in the composition of the vesicle

EXPERIMENTAL PROCEDURES
RESULTS
The estimation of secondary structure fractions of porcine
DISCUSSION
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