Synthetic Processing of Surfactant Protein C by Alevolar Epithelial Cells
THE COOH TERMINUS OF proSP-C IS REQUIRED FOR POST-TRANSLATIONAL TARGETING AND PROTEOLYSIS*
- From the ‡Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6068 and the §Pulmonary and Critical Care Division, Department of Medicine, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104
Abstract
Surfactant protein C (SP-C) is synthesized by alveolar type II cells as a 21-kDa propeptide (proSP-C21) which is proteolytically processed in subcellular compartments distal to the trans-Golgi network to yield a 35-residue mature form. Initial synthetic processing events for SP-C include post-translational cleavages of the COOH terminus of proSP-C21 yielding two intermediates (16 and 6 kDa). To test the role of specific COOH-terminal domains in intracellular targeting and proteolysis of proSP-C21, synthesis and processing of SP-C was evaluated using a lung epithelial cell line (A549) transfected with a eukaryotic expression vector containing either the full-length cDNA for rat SP-C (SP-Cwt) or one of six polymerase chain reaction (PCR)-generated COOH terminally truncated forms (SP-C1–185, SP-C1–175, SP-C1–147, SP-C1–120, SP-C1–72, and SP-C1–59). Using in vitrotranscription/translation, each of the seven constructs produced a35S-labeled product of appropriate length which could be immunoprecipitated by epitope specific proSP-C antisera. Immunoprecipitation of 35S-labeled A549 cell lysates from SP-Cwt transfectants demonstrated rapid synthesis of [35S]proSP-C21 with processing to SP-C16 and SP-C6 intermediates via cleavages of the COOH-terminal propeptide. Both the intermediates as well as the kinetics of processing in A549 cells were similar to that observed in rat type II cells. In contrast, constructs SP-C1–185, SP-C1–175, SP-C1–147, SP-C1–120, SP-C1–72, and SP-C1–59 were each translated but degraded without evidence of proteolytic processing. Fluorescence immunocytochemistry identified proSP-Cwt in cytoplasmic vesicles of A549 cells while all COOH-terminal deletional mutants were restricted to an endoplasmic reticulum/Golgi compartment identified by co-localization with fluorescein isothiocyanate-concanavalin A. We conclude that SP-Cwt expressed in A549 cells is directed to cytoplasmic vesicles where it is proteolytically processed in a manner similar to native type II cells and that amino acids Cys186-Ile194 located at the COOH terminus of proSP-C21 are necessary for correct intracellular targeting and subsequent cleavage events.
Footnotes
-
↵* This work was supported in part by National Institutes of Health Grants HL-02869, HL-19737, and P50-HL56401 (to M. F. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵¶ Recipient of a Clinician-Scientist Award from the American Heart Association. To whom correspondence should be addressed: Institute for Environmental Medicine, University of Pennsylvania School of Medicine, 1 John Morgan Bldg., 36th and Hamilton Walk, Philadelphia, PA 19104-6068. Tel.: 215-898-9100; Fax: 215-898-0868; E-mail:mfbeers{at}mail.med.upenn.edu.
-
↵1 The abbreviations used are: SP-B, pulmonary surfactant protein B (9 kDa); SP-C, pulmonary surfactant protein C (3.7 kDa); Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; ConA, concanavalin A; PCR, polymerase chain reaction; ER, endoplasmic reticulum.
-
↵2 M. F. Beers, unpublished observations.
-
↵3 H. Shuman, unpublished data.
-
- Received December 23, 1997.
- Revision received February 17, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
