Inhibition of Cellular Processing of Surfactant Protein C by Drugs Affecting Intracellular pH Gradients

Michael F Beers

doi:10.1074/jbc.271.24.14361

Abstract

Surfactant protein C (SP-C) is a hydrophobic protein synthesized and secreted exclusively by alveolar type II cells through proteolysis of a 21-kDa propeptide (SP-C21) to produce the 3.7-kDa surface active form. Previous studies from this laboratory have demonstrated that early processing of proSP-C involves extensive intracellular proteolysis of the COOH terminus of proSP-C21 in subcellular compartments, which include the acidic type II cell-specific subcellular organelle, the lamellar body. (Beers, M. F., Kim, C. Y., Dodia, C., and Fisher, A. B.(1994) J. Biol. Chem. 269, 20318-20328). The role of intracellular pH gradients in SP-C processing was studied in freshly isolated rat type II cells. Using vital fluorescence microscopy, the pH indicator acridine orange (AO) identified intense fluorescence staining of acidic cytoplasmic vesicles within fresh type II cells. The AO vesicular staining pattern was similar in cells labeled with the lamellar body marker phosphine 3R and the phospholipid dye nile red. AO fluorescence was quenched by the addition of a membrane-permeable weak base, methylamine. Immunoprecipitation of cell lysates with anti-proSP-C antisera following pulse-chase labeling (0-2 h) with 35S-Translabel demonstrated rapid synthesis of 35S-proSP-C21 with a time-dependent appearance of 16- and 6-kDa intermediates (SP-C16 and SP-C6). Tricine polyacrylamide gel electrophoresis analysis of organic extracts of cell lysates showed time-dependent appearance of mature SP-C3.7. The addition of 5 mM methylamine significantly blocked the post-translational processing of proSP-C resulting in disruption of normal precursor-product relationships and inhibition of SP-C3.7 formation. Methylamine-treated cells exhibited slow accumulation of SP-C16 and SP-C6, a persistence of SP-C21, and an absence of SP-C3.7 for the duration of the chase period. The lysosomotropic agent chloroquine, the proton ionophore monensin, and bafilomycin A1, a specific vacuolar H+-ATPase inhibitor, each caused inhibition of proSP-C processing in a similar manner. These results demonstrate that normal post-translational proteolysis of proSP-C occurs in acidic intracellular compartments, which include the lamellar body, and that complete processing to SP-C3.7 is dependent upon maintenance of transmembrane pH gradients by a vacuolar H+-ATPase.

Highlights

Pulmonary surfactant is a heterogeneous surface active complex composed of approximately 80% phospholipid, 10% other lipids, and 10% proteins synthesized and secreted exclusively by the alveolar type II cell (Beers and Fisher, 1992; Hawgood, 1989)
The lysosomotropic agent chloroquine, the proton ionophore monensin, and bafilomycin A1, a specific vacuolar H؉-ATPase inhibitor, each caused inhibition of proSP-C processing in a similar manner. These results demonstrate that normal posttranslational proteolysis of proSP-C occurs in acidic intracellular compartments, which include the lamellar body, and that complete processing to SP-C3.7 is depend
The surface tension lowering properties of surfactant are primarily due to the phospholipid components; enhancement of the biophysical properties of these lipids has been attributed to the presence of specific surfactant-associated proteins, SP-A,1 separate 35S-radiolabeled bands of 7 (SP-B), and Surfactant protein C (SP-C) (Hawgood, 1989; Mathalgian and Possmayer, 1990; Weaver and Whitsett, 1991)

Summary

Introduction

Pulmonary surfactant is a heterogeneous surface active complex composed of approximately 80% phospholipid, 10% other lipids, and 10% proteins synthesized and secreted exclusively by the alveolar type II cell (Beers and Fisher, 1992; Hawgood, 1989). Alveolar SP-C (“mature” SP-C3.7) is generated by synthesis of a larger primary translation product, proSP-C (Mr ϭ 21,000), post-translational addition of covalent palmitic acid residues, and intracellular proteolysis involving cleavage of NH2- and COOHterminal flanking domains of the precursor molecule to yield the secreted surface active form (Beers and Fisher, 1992; Beers et al, 1992, 1994a; Beers and Lomax, 1995; Fisher et al, 1989; Glasser et al, 1988; Vorbroker et al, 1992, 1995b) This laboratory had reported the production and characterization of several epitope-specific polyclonal antibodies directed against rat proSP-C using synthetic peptides corresponding to spatially distinct regions of the proSP-C primary sequence (Beers et al, 1992, 1994a; Beers and Lomax, 1995). Western blotting of lamellar bodies isolated from adult rat lungs showed that proSP-C6 was enriched in this subcellular fraction, and immunoprecipitation analysis of 35S-labeled lamellar bodies showed time-dependent appearance of SP-C6 and production of mature SP-C3.7 within this compartment (Beers et al, 1992, 1994a, 1994b; Beers and Lomax, 1995)

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