Cysteine protease activity is required for surfactant protein B processing and lamellar body genesis.
Surfactant protein (SP)-B is essential for lamellar body genesis and for the final steps in proSP-C post-translational processing. The mature SP-B protein is derived from multistep processing of the primary translation product proSP-B; however, the enzymes required for these events are currently unknown. Recent ultrastructural colocalization studies have suggested that the cysteine protease Cathepsin H may be involved in proSP-B processing. Using models of isolated human type 2 cells in culture, we describe the effects of cysteine protease inhibition by E-64 on SP-B processing and type 2 cell differentiation. Pulse-chase labeling and Western immunoblotting studies showed that the final step of SP-B processing, specifically cleavage of SP-B(9) to SP-B(8), was significantly inhibited by E-64, resulting in delayed accumulation of SP-B(8) without adverse effects on SP-A or glyceraldehyde phosphate dehydrogenase expression. E-64 treatment during type 2 cell differentiation mimicked features of inherited SP-B deficiency in humans and mice, specifically disrupted lamellar body genesis, and aberrant processing of proSP-C. Reverse transcriptase-polymerase chain reaction and Western immunoblotting studies showed that Cathepsin H is induced during in vitro differentiation of type 2 cells and localizes with SP-B in multivesicular bodies, composite bodies, and lamellar bodies by immunoelectron microscopy. Furthermore, Cathepsin H activity was specifically inhibited in a dose-dependent fashion by E-64. Our data show that a cysteine protease is involved in SP-B processing, lamellar body genesis, and SP-C processing, and suggest that Cathepsin H is the most likely candidate protease.
Guttentag
Susan
S
Division of Neonatology, Department of Pediatrics, University of Pennsylvania School of Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104-4318, USA. guttentag@email.chop.edu
Robinson
Lauren
L
Zhang
Peggy
P
Brasch
Frank
F
Bühling
Frank
F
Beers
Michael
M
D001483:Base Sequence; D015153:Blotting, Western; D056655:Cathepsin H; D002403:Cathepsins / Q000378:metabolism*; D002454:Cell Differentiation; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008168:Lung / Q000187:drug effects / Q000196:embryology / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron / Q000379:methods; D011499:Protein Processing, Post-Translational; D037701:Pulmonary Surfactant-Associated Protein B / Q000378:metabolism*; D037721:Pulmonary Surfactant-Associated Protein C / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D037701:Pulmonary Surfactant-Associated Protein B; D037721:Pulmonary Surfactant-Associated Protein C; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534126:CTSH protein, human; D056655:Cathepsin H; C534127:Ctsh protein, mouse; D007930:Leucine; C024974:E 64