Involvement of Napsin A in the C- and N-terminal Processing of Surfactant Protein B in Type-II Pneumocytes of the Human Lung

Frank Brasch,Matthias Ochs,Thilo Kähne,Susan Guttentag,Vesna Schauer-Vukasinovic,Matthew Derrick,Georg Johnen,Nadine Kapp,Klaus-Michael Müller,Joachim Richter,Thomas Giller,Samuel Hawgood,Frank Bühling

doi:10.1074/jbc.m306844200

Abstract

Surfactant protein B (SP-B) is a critical component of pulmonary surfactant, and a deficiency of active SP-B results in fatal respiratory failure. SP-B is synthesized by type-II pneumocytes as a 42-kDa propeptide (proSP-B), which is posttranslationally processed to an 8-kDa surface-active protein. Napsin A is an aspartic protease expressed in type-II pneumocytes. To characterize the role of napsin A in the processing of proSP-B, we colocalized napsin A and precursors of SP-B as well as SP-B in the Golgi complex, multivesicular, composite, and lamellar bodies of type-II pneumocytes in human lungs using immunogold labeling. Furthermore, we measured aspartic protease activity in isolated lamellar bodies as well as isolated human type-II pneumocytes and studied the cleavage of proSP-B by napsin A and isolated lamellar bodies in vitro. Both, napsin A and isolated lamellar bodies cleaved proSP-B and generated three identical processing products. Processing of proSP-B by isolated lamellar bodies was completely inhibited by an aspartic protease inhibitor. Sequence analysis of proSP-B processing products revealed several cleavage sites in the N- and C-terminal propeptides as well as one in the mature peptide. Two of the four processing products generated in vitro were also detected in type-II pneumocytes. In conclusion, our results show that napsin A is involved in the N- and C-terminal processing of proSP-B in type-II pneumocytes.

Highlights

Surfactant protein B (SP-B) is a critical component of pulmonary surfactant, and a deficiency of active SP-B results in fatal respiratory failure
SP-B is synthesized by type-II pneumocytes as a 42-kDa propeptide, which is posttranslationally processed to an 8-kDa surface-active protein
This paper is available on line at http://www.jbc.org surfactant protein B [10], but cathepsin D itself was not detectable in type-II pneumocytes, and no specific activity was found in isolated lamellar bodies [11, 12]

Summary

Introduction

Surfactant protein B (SP-B) is a critical component of pulmonary surfactant, and a deficiency of active SP-B results in fatal respiratory failure. SP-B is synthesized by type-II pneumocytes as a 42-kDa propeptide (proSPB), which is posttranslationally processed to an 8-kDa surface-active protein. We measured aspartic protease activity in isolated lamellar bodies as well as isolated human type-II pneumocytes and studied the cleavage of proSP-B by napsin A and isolated lamellar bodies in vitro. Napsin A and isolated lamellar bodies cleaved proSP-B and generated three identical processing products. Hereditary alveolar proteinosis in babies without any detectable mutations in the SP-B gene as well as acquired pulmonary alveolar proteinosis in children and adults are characterized by an intraalveolar accumulation of mature surfactant proteins and abnormal SP-B precursors. Only SP-B precursors are detected in babies with congenital surfactant defects characterized by the absence of lamellar bodies in type-II pneumocytes.. It has been speculated that a cathepsin D-like protease is involved in the posttranslational processing of the hydrophobic

Methods

Results

Conclusion