CHARACTERIZATION OF EXPRESSION AND POST-TRANSLATIONAL PROCESSING OF HUMAN SP-C USING EPITOPE SPECIFIC ANTISERA. † 2085

In the adult rat, surfactant protein-C (SP-C) has been shown to be synthesized as a 21 kD propeptide (SP-C₂₁) and post-translationally processed to the 3.7 kD alveolar form with appearance of 16 kD and 6 kD intermediate forms. The goal of this study was to characterize expression and post-translational processing of SP-C in developing human lung. Anti-hCPRO(epitope=His⁵⁹-Ser⁷²) and anti-hCTERM(epitope=Gly¹⁶²-Gly¹⁷⁴) antisera were generated to complement a previously produced anti-NPROSP-C (epitope=Met¹⁰-Phe²³) antiserum. All 3 antisera recognized native proSP-C₂₁ produced byin vitro transcription and translation of human SP-C cDNA. Immunocytochemistry using anti-NPROSP-C revealed expression of proSP-C forms after 15 wks gestation which increased throughout the second trimester and was limited exclusively to epithelial cells. Fetal lung explants (20-24 wks) cultured with dexamethasone (10nM) for 5d were continously labeled with³⁵ S-met for up to 4h. Immunoprecipitation of explants with anti-NPROSP-C and anti-hCTERM each demonstrated appearance of SP-C₂₁, a 24 kD form and a 16 kD intermediate. Longer exposures of anti-NPROSP-C immunoprecipitates also identified 6-10 kD SP-C intermediates. The 24 kD form was not detected using anti-CPROSP-C. By pulse-chase labeling,³⁵ S-SP-C₂₄ preceded the appearance of lower M_r forms. Full length human SP-C cDNA, subcloned into the pcDNA3 eukaryotic expression vector, was transfected into A549 cells. A 21 → 24 → 16 → 6 kD processing pattern exhibited by these transfectants was identical to the pattern observed in a post-natal lung specimen from a patient with SP-B deficiency. These results indicate that in human lung, proSP-C is expressed by 16 wks of gestation but complete processing of proSP-C does not occur until after 24 wks gestation. Post-translational processing of proSP-C₂₁ involves modification to a 24 kD form and proteolysis with initial cleavage of C-terminal propeptide domains. We speculate that covalent modification of proSP-C₂₁ to produce proSP-C₂₄ results in conformational changes in amino acid residues 59-72 (hCPROSP-C epitope).