Intracellular Localization of Processing Events in Human Surfactant Protein B Biosynthesis

Surfactant protein B (SP-B) is essential to the function of pulmonary surfactant and to alveolar type 2 cell phenotype. Human SP-B is the 79-amino acid product of extensive post-translational processing of a 381-amino acid preproprotein. Processing involves modification of the primary translation product from 39 to 42 kDa and at least 3 subsequent proteolytic cleavages to produce the mature 8-kDa SP-B. To examine the intracellular sites of SP-B processing, we carried out immunofluorescence cytochemistry and inhibitor studies on human fetal lung in explant culture and isolated type 2 cells in monolayer culture using polyclonal antibodies to human SP-B8(Phe201-Met279) and specific epitopes within the N- (NFProx, Ser145-Leu160; NFlank Gln186-Gln200) and C-terminal (CFlank, Gly284-Ser304) propeptides of pro-SP-B. Fluorescence immunocytochemistry using epitope-specific antisera showed colocalization of pro-SP-B with the endoplasmic reticulum resident protein BiP. The 25-kDa intermediate was partially endo H-sensitive, colocalized with the medial Golgi resident protein MG160, and shifted into the endoplasmic reticulum in the presence of brefeldin A, which interferes with anterograde transport from endoplasmic reticulum to Golgi. The 9-kDa intermediate colocalized in part with MG160 but not with Lamp-1, a transmembrane protein resident in late endosomes and lamellar bodies. Brefeldin A induced a loss of colocalization between MG160 and NFlank, shifting NFlank immunostaining to a juxtanuclear tubular array. In pulse-chase studies, brefeldin A blocked all processing of 42-kDa pro-SP-B whereas similar studies using monensin blocked the final N-terminal processing event of 9 to 8 kDa SP-B. We conclude that: 1) the first enzymatic cleavage of pro-SP-B to the 25-kDa intermediate is in the brefeldin A-sensitive, medial Golgi; 2) cleavage of the 25-kDa intermediate to a 9-kDa form is a trans-Golgi event that is slowed but not blocked by monensin; 3) the final cleavage of 9 to 8 kDa SP-B is a monensin-sensitive, post-Golgi event occurring prior to transfer of SP-B to lamellar bodies. Surfactant protein B (SP-B) is essential to the function of pulmonary surfactant and to alveolar type 2 cell phenotype. Human SP-B is the 79-amino acid product of extensive post-translational processing of a 381-amino acid preproprotein. Processing involves modification of the primary translation product from 39 to 42 kDa and at least 3 subsequent proteolytic cleavages to produce the mature 8-kDa SP-B. To examine the intracellular sites of SP-B processing, we carried out immunofluorescence cytochemistry and inhibitor studies on human fetal lung in explant culture and isolated type 2 cells in monolayer culture using polyclonal antibodies to human SP-B8(Phe201-Met279) and specific epitopes within the N- (NFProx, Ser145-Leu160; NFlank Gln186-Gln200) and C-terminal (CFlank, Gly284-Ser304) propeptides of pro-SP-B. Fluorescence immunocytochemistry using epitope-specific antisera showed colocalization of pro-SP-B with the endoplasmic reticulum resident protein BiP. The 25-kDa intermediate was partially endo H-sensitive, colocalized with the medial Golgi resident protein MG160, and shifted into the endoplasmic reticulum in the presence of brefeldin A, which interferes with anterograde transport from endoplasmic reticulum to Golgi. The 9-kDa intermediate colocalized in part with MG160 but not with Lamp-1, a transmembrane protein resident in late endosomes and lamellar bodies. Brefeldin A induced a loss of colocalization between MG160 and NFlank, shifting NFlank immunostaining to a juxtanuclear tubular array. In pulse-chase studies, brefeldin A blocked all processing of 42-kDa pro-SP-B whereas similar studies using monensin blocked the final N-terminal processing event of 9 to 8 kDa SP-B. We conclude that: 1) the first enzymatic cleavage of pro-SP-B to the 25-kDa intermediate is in the brefeldin A-sensitive, medial Golgi; 2) cleavage of the 25-kDa intermediate to a 9-kDa form is a trans-Golgi event that is slowed but not blocked by monensin; 3) the final cleavage of 9 to 8 kDa SP-B is a monensin-sensitive, post-Golgi event occurring prior to transfer of SP-B to lamellar bodies. fluorescein isothiocyanate phosphate-buffered saline endoplasmic reticulum polyacrylamide gel electrophoresis N-[1-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 2-[bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-propane-1,3-diol Surfactant protein B is a 79-amino acid hydrophobic protein that is essential to the function of pulmonary surfactant, as illustrated by lethal SP-B deficiency in humans and the transgenic homozygous SP-B knock-out mouse (reviewed in Ref. 1.Whitsett J.A. Nogee L.M. Weaver T.E. Horowitz A.D. Physiol. Rev. 1995; 75: 749-757Crossref PubMed Scopus (162) Google Scholar). The 8-kDa protein is the result of extensive post-translational processing of a large 381-amino acid precursor within alveolar type 2 cells. Previous studies in cell lines, isolated rat type 2 cells, and human fetal lung (2.Weaver T.E. Lin S. Bogucki B. Dey C. Am. J. Physiol. 1992; 263: L95-L103Crossref PubMed Google Scholar, 3.O'Reilly M.A. Weaver R.E. Pilot-Matias T.J. Biochim. Biophys. Acta. 1989; 1011: 140-148Crossref PubMed Scopus (59) Google Scholar, 4.Hawgood S. Latham D. Borchelt J. Damm D. White T. Benson B. Wright J.R. Am. J. Physiol. 1993; 264: L290-L299PubMed Google Scholar, 5.Lin S. Phillips K.S. Wilder M.R. Weaver T.E. Biochim. Biophys. Acta. 1996; 1312: 177-185Crossref PubMed Scopus (70) Google Scholar, 6.Lin S. Akinbi H.T. Breslin J.S. Weaver T.E. J. Biol. Chem. 1996; 271: 19689-19695Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 7.Whitsett J.A. Weaver T.E. Clark J.C. Sawtell N. Glasser S.W. Korfhagen T.R. Hull W.M. J. Biol. Chem. 1987; 262: 15618-15623Abstract Full Text PDF PubMed Google Scholar) indicated that processing to the mature 8-kDa protein involves signal peptide cleavage and glycosylation of the C terminus, followed by cleavage of the N terminus and C terminus in succession. We have recently shown that cleavage of the N terminus occurs in two steps, leaving an approximately 10-amino acid remnant flanking mature SP-B which is removed in a final processing step that releases mature SP-B (8.Guttentag S.H. Beers M.F. Bieler B.M. Ballard P.L. Am. J. Physiol. 1998; 275: L559-L566PubMed Google Scholar). The subcellular location of these processing events and the enzymes necessary for processing SP-B are poorly understood. Previous work by Voorhout and colleagues (9.Voorhout W.F. Veenendaal T. Haagsman H.P. Weaver T.E. Whitsett J.A. van Golde L.M. Geuze H.J. Am. J. Physiol. 1992; 263: L479-L486PubMed Google Scholar) utilizing immunoelectron microscopy with antisera to mature SP-B and a synthetic pro-SP-B showed pro-SP-B in the endoplasmic reticulum and mature SP-B in lamellar bodies of adult human type 2 cells. Analysis of grain density over other organelles showed intermediate grain densities over multivesicular bodies and Golgi, indicating the involvement of these organelles in SP-B transport and/or processing. The extensive post-translational processing of SP-B is similar to the post-translational processing of the other hydrophobic surfactant protein, SP-C (10.Vorbroker D.K. Dey C. Weaver T.E. Whitsett J.A. Biochim. Biophys. Acta. 1992; 1105: 161-169Crossref PubMed Scopus (46) Google Scholar, 11.Beers M.F. Kim C.Y. Dodia C. Fisher A.B. J. Biol. Chem. 1994; 269: 20318-20328Abstract Full Text PDF PubMed Google Scholar, 12.Beers M. Shuman H. Liley H. Floros J. Gonzales L.W. Yue N. Ballard P.L. Pediatr. Res. 1995; 38: 668-695Crossref PubMed Scopus (62) Google Scholar, 13.Beers M.F. J. Biol. Chem. 1996; 271: 14361-14370Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The 21-kDa pro-SP-C undergoes sequential enzymatic cleavages resulting in a 3.7-kDa mature protein. Pro-SP-C is detected in endoplasmic reticulum and a 6-kDa intermediate is enriched in lamellar bodies. Inhibitors of intracellular trafficking and acidification in vitro disrupt all processing beyond the 16-kDa SP-C intermediate. Processing of SP-B and SP-C are linked, since in alveolar type 2 cells of patients with inherited SP-B deficiency SP-C is not processed beyond the 6-kDa intermediate (14.Beers M. Hamvas A. Moxley M. Gonzales L. Guttentag S. Solarin K. Longmore W. Nogee L. Ballard P. Am. J. Resp. Cell Mol. Biol. 2000; 22 (in press)Crossref PubMed Scopus (82) Google Scholar, 15.Vorbroker D.K. Profitt S.A. Nogee L.M. Whitsett J.A. Am. J. Physiol. 1995; 268: L647-L656Crossref PubMed Google Scholar). In this report, we use epitope-specific antisera and pulse-chase labeling studies with inhibitors of protein processing to show that most human pro-SP-B processing is in post-endoplasmic reticulum but pre-lamellar body compartments. Our data extend previous observations of pro-SP-B trafficking and processing to show that early N-terminal propeptide and C-terminal propeptide processing events occur within the Golgi apparatus with processing of the small vestigial N-terminal propeptide domain as a post-Golgi event. We speculate that the N-terminal remnant is involved in trafficking SP-B toward the lamellar body. Previous reports of these data have appeared elsewhere in abstract form (16.Korimilli A. Beers M.F. Ballard P.L. Guttentag S.H. Pediatr. Res. 1999; 45: 55ACrossref Google Scholar, 17.Guttentag S. Korimilli A.D. Xing B. Beers M.F. Ballard P.L. Pediatr. Res. 1998; 43: 49ACrossref Google Scholar). Express Protein Labeling Mix was obtained from NEN Life Science Products Inc. (Boston, MA). Protein A-agarose was obtained from Life Technologies, Inc. (Gaithersburg, MD). Dexamethasone, isobutylmethylxanthine, and 8-Br-cAMP were obtained from Sigma. Endoglycosidase H (endo H) and PNGase F were obtained from New England Biolabs (Beverly, MA). All other reagents were electrophoretic grade and were purchased from either Bio-Rad or Novex (San Diego, CA). Culture media were produced by the Cell Center Facility, University of Pennsylvania. The polyclonal antibody to BiP was supplied by StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada). The MG160 polyclonal antiserum was the generous gift of N. Gonatas, Division of Neuropathology, University of Pennsylvania. The Lamp-1 (H4A3) monoclonal antibody developed by J. T. August and J. E. K. Hildreth was obtained from the Developmental Studies Hybridoma Bank developed under the of the of and by The University of of Human fetal lung was obtained from under by the for Human of lung was of large into and in media on a as L.W. Ballard P.L. J. PubMed Scopus Google Scholar). and were to the media for the of the culture were and were on of 2 cells were isolated from human fetal lung in culture with using and to and on with of cells Am. J. Cell Mol. Biol. PubMed Scopus Google Scholar, L.W. S. A. Solarin Beers M.F. Ballard P.L. Pediatr. Res. 1999; 45: Scholar). of the final culture between and of cells. were in media with in for to these type 2 alveolar cells of and SP-B and and into surfactant for at least in culture L.W. S. A. Solarin Beers M.F. Ballard P.L. Pediatr. Res. 1999; 45: Scholar). on the of peptide were for of synthetic and antiserum (NFProx, NFlank, and Gly284-Ser304) as (8.Guttentag S.H. Beers M.F. Bieler B.M. Ballard P.L. Am. J. Physiol. 1998; 275: L559-L566PubMed Google Scholar) and illustrated in Human SP-B antiserum was using human isolated from patients with pulmonary alveolar as for SP-B antibody M. S. Fisher A. Am. J. Physiol. 1992; 262: Google Scholar). were for the peptide by The of NFlank, and antisera were isolated using the and to using Protein labeling labeling studies, cells were In labeling studies, isolated type 2 cells were on culture with or with brefeldin A for were of media in and in in followed by in with were in to followed by in for at to The cells were using in antisera were in and The primary antibody was as polyclonal MG160, at at Lamp-1, at in was at in the and for at in SP-B antisera were at and at were in and using to All immunostaining were in were obtained using a of the Facility, of were at the cell monolayer to the The and the of Golgi, and/or lamellar of labeling with and was at for all was at and were with a Culture media was with with or inhibitors for 2 in on a The inhibitors brefeldin A or monensin were at the of the and the and inhibitors was with inhibitors with Protein Labeling Mix which is of and Life Science Products a the media was to media with To that inhibitor media was at subsequent the of the were labeling and at 8 were in with inhibitors 2 and and in of with lung were by modification of previous (8.Guttentag S.H. Beers M.F. Bieler B.M. Ballard P.L. Am. J. Physiol. 1998; 275: L559-L566PubMed Google Scholar). were on acid using 3 of SP-B antibody or the first protein A-agarose were and the were in of gel A was for and a was to in polyacrylamide using a as (8.Guttentag S.H. Beers M.F. Bieler B.M. Ballard P.L. Am. J. Physiol. 1998; 275: L559-L566PubMed Google Scholar). were to at for transfer to were using the using and to from human fetal lung pulse-chase for to protein A-agarose were with either endo H or PNGase were in were in with of endo H PNGase were in with of PNGase All were at for 2 followed by a final the in with as the these were using a gel with as the Diego, transfer to for To SP-B precursor and intermediate within type 2 cells of human fetal we epitope-specific antisera developed within the N- and C-terminal Fluorescence cytochemistry of type 2 cells isolated from human fetal lung showed immunostaining for of the antisera The antiserum immunostaining a The antiserum a of the The NFlank antiserum showed a but in to lamellar bodies which have a by the antiserum to mature SP-B lamellar bodies and other to the of within lamellar bodies. The of antiserum was by the of synthetic peptide to the signal in not immunofluorescence labeling was to the subcellular location of with the epitope-specific antisera using of subcellular organelles in type 2 cells. were but were by of with the epitope-specific antisera for subcellular were obtained using the for primary SP-B to to NFlank to to the for were to which were for and to the antiserum as by the and for the epitope-specific antisera to 3 the of immunofluorescence cytochemistry of isolated type 2 cells using monoclonal antibody to BiP a of endoplasmic reticulum and by and SP-B epitope-specific antisera to of immunostaining with BiP in the endoplasmic reticulum was in 3 and 3 was colocalization of and NFlank antisera with whereas the of pro-SP-B in the endoplasmic reticulum was by was colocalization of BiP with immunofluorescence of isolated type 2 cells using polyclonal antibody to MG160, by and epitope-specific MG160 is a medial Golgi resident transmembrane which is as a of the Golgi apparatus of most cells A. J. J. Cell 1995; PubMed Google Scholar, A. N. Res. 1995; PubMed Scopus Google Scholar). not with and NFlank immunostaining colocalized with MG160 in a tubular in to the which is of the medial Golgi. this was to lamellar was colocalization of with immunofluorescence of isolated type 2 cells using Lamp-1 monoclonal by and epitope-specific Lamp-1 is a transmembrane protein which to late in alveolar type 2 cells, lamellar bodies K. 1994; PubMed Scopus Google Scholar). antisera colocalized with NFlank immunostaining to lamellar bodies in 2) which were the antiserum to within lamellar bodies these immunofluorescence data that at pro-SP-B is endoplasmic reticulum resident and the 25-kDa which is by and NFlank, is within the Golgi. In mature SP-B is within the lamellar body. To examine to Golgi transport of SP-B we carried out pulse-chase labeling of human fetal lung in the presence or of brefeldin A, an inhibitor of anterograde trafficking between endoplasmic reticulum and Golgi P. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). We showed that processing from pro-SP-B and 9-kDa to mature 8-kDa SP-B occurs within in human fetal lung (8.Guttentag S.H. Beers M.F. Bieler B.M. Ballard P.L. Am. J. Physiol. 1998; 275: L559-L566PubMed Google Scholar) In the presence of brefeldin A, processing of pro-SP-B was blocked with of kDa or over 8 of that the N-terminal cleavage of pro-SP-B occurs in a brefeldin or medial Golgi We of SP-B isolated by a The and 8-kDa SP-B are not and not with either PNGase F or endo H PNGase which all the 42-kDa pro-SP-B to kDa and the 25-kDa intermediate to endo H pro-SP-B and the 25-kDa intermediate to partially endo and 21-kDa and endo and 25-kDa The of pro-SP-B is endo but a small are endo a small of the 25-kDa intermediate is endo and the of this intermediate is endo with the brefeldin A studies, these data the N-terminal cleavage of pro-SP-B to 25-kDa intermediate in the medial Golgi. To the location of the first N-terminal cleavage from the subsequent cleavage of the C terminus, we the of SP-B in the presence of brefeldin A using immunofluorescence of isolated type 2 cells. In brefeldin cells were and using antibodies to BiP 8 or MG160 8 in with the epitope-specific cells the immunostaining illustrated in and immunostaining were by brefeldin A immunostaining to brefeldin A, as the of MG160, with antisera with BiP. immunostaining a of SP-B intermediate within a brefeldin A-sensitive, Golgi similar to NFlank an intermediate that at was to the brefeldin Golgi with the of NFlank immunostaining into tubular of trans-Golgi to To late processing we carried out pulse-chase labeling of lung in the presence and of an the of intracellular transport of with transfer Golgi and from the trans-Golgi Full Text PDF PubMed Scopus Google Scholar). pro-SP-B processing was monensin not the of and 9-kDa SP-B processing to 8-kDa mature SP-B was not to 8 that the final N-terminal cleavage of 9-kDa intermediate is a post-Golgi event. with the that NFlank immunostaining is not within lamellar this the cleavage event in a post-Golgi but pre-lamellar body The processing of pro-SP-B to mature SP-B in alveolar type 2 cells a of post-translational and proteolytic the of SP-B processing, the mature protein to the lamellar body is with SP-C and The the of lamellar body and the of these surfactant are poorly understood. To these to examine the intracellular of SP-B processing. Previous studies by using immunoelectron microscopy the primary translation to the endoplasmic reticulum with mature SP-B in lamellar bodies. The these observations with data that the proteolytic cleavage of the N-terminal propeptide is in the brefeldin A-sensitive, medial Golgi with a subsequent C-terminal cleavage in the trans-Golgi and a final N-terminal cleavage event in a post-Golgi but pre-lamellar body as in We type 2 cells isolated from human fetal lung for immunofluorescence in type 2 cell culture of type 2 cell for Am. J. Cell Mol. Biol. PubMed Scopus Google Scholar, L.W. S. A. Solarin Beers M.F. Ballard P.L. Pediatr. Res. 1999; 45: Scholar). isolated type 2 cells the lamellar bodies have a which of subcellular in to lamellar bodies. We have recently these cells in pulse-chase labeling studies and have in SP-B processing over with similar studies in human fetal lung W. Gonzales and S. H. culture a for studies lamellar body and as as in the surfactant protein processing. In the the of type 2 cell immunofluorescence cytochemistry with pulse-chase studies of human fetal lung of of SP-B with the of inhibitors and on SP-B processing. a from acid to and M. T. H. J. 1995; PubMed Scopus Google Scholar, E. M. Biol. PubMed Scopus Google Scholar). the of epitope-specific antisera were to within between Our immunofluorescence studies on the of these antisera to SP-B We have shown by that these antisera the SP-B the peptide and are by antiserum with peptide (8.Guttentag S.H. Beers M.F. Bieler B.M. Ballard P.L. Am. J. Physiol. 1998; 275: L559-L566PubMed Google Scholar). In using the epitope-specific antisera for studies, we that all epitopes and for antibody prior this all of the SP-B antisera pro-SP-B in the and to the was in post-translational processing immunofluorescence showed in the of with as in the and the to the which pro-SP-B and the N terminus by colocalized with the endoplasmic reticulum BiP M.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, L.M. M. M.R. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). pro-SP-B in the endoplasmic which is in with previous work by Voorhout and colleagues (9.Voorhout W.F. Veenendaal T. Haagsman H.P. Weaver T.E. Whitsett J.A. van Golde L.M. Geuze H.J. Am. J. Physiol. 1992; 263: L479-L486PubMed Google Scholar). NFlank, and which pro-SP-B by showed colocalization with BiP. observations by in the of the antisera for in the labeling of the primary in the of SP-B within the or by of the epitopes within the SP-B were for antiserum to to for in antiserum and labeling and not epitopes were the of and mature SP-B protein are not between organelles and of the immunostaining we showed that pro-SP-B is processed to the 25-kDa intermediate in human fetal lung with type 2 cells (8.Guttentag S.H. Beers M.F. Bieler B.M. Ballard P.L. Am. J. Physiol. 1998; 275: L559-L566PubMed Google Scholar). The of mature SP-B protein occurs at is 42-kDa pro-SP-B within the type 2 cell 25-kDa intermediate. using the and NFlank antisera of the of 25-kDa intermediate over the of pro-SP-B. mature SP-B and is within lamellar a of the a immunostaining using the antiserum of the lamellar body of mature SP-B over the intermediate Our previous studies showed that the N-terminal propeptide is in two steps, first a small vestigial propeptide that is in the final event SP-B (8.Guttentag S.H. Beers M.F. Bieler B.M. Ballard P.L. Am. J. Physiol. 1998; 275: L559-L566PubMed Google Scholar). We have shown that the N-terminal cleavage is a medial Golgi event. Brefeldin A, a small hydrophobic that and of the Golgi which to the endoplasmic reticulum in P. 1999; Full Text Full Text PDF PubMed Scopus Google all proteolytic processing of pro-SP-B. which on Golgi Full Text PDF PubMed Scopus Google not this first proteolytic cleavage event. In brefeldin shifted immunostaining out of a Golgi to with the BiP. by to examine the trafficking of to of the Golgi M. E. J. Cell Biol. PubMed Scopus Google Scholar, S. J. 1995; PubMed Scopus Google MG160 which brefeldin M. T. J. D. 1999; PubMed Scopus Google Scholar). the for an endo H of 42-kDa pro-SP-B and the 25-kDa intermediate is that the first N-terminal cleavage occurs in the medial Golgi the modification is endo which from the endo to the Golgi on cell type into the trans-Golgi A. L. J. Cell Biol. 1993; PubMed Scopus Google Scholar, P. T. N. J. Cell Biol. 1994; PubMed Scopus Google Scholar, C. N. T. J. Cell 1995; PubMed Google Scholar). endo pro-SP-B is from the to the medial Golgi endo with N-terminal The of the antiserum to with MG160 at that the of the of pro-SP-B is the the Golgi. The step in pro-SP-B processing is C-terminal propeptide Our that this occurs in a late Golgi most the cleavage was not by The immunostaining for and NFlank in the presence of brefeldin show that the of the 25-kDa and 9-kDa are in compartments. and NFlank antisera the 25-kDa previous studies showed that not a C-terminal propeptide which of these studies (8.Guttentag S.H. Beers M.F. Bieler B.M. Ballard P.L. Am. J. Physiol. 1998; 275: L559-L566PubMed Google Scholar). NFlank, but not antiserum the 9-kDa intermediate. immunostaining showed a to endoplasmic reticulum and colocalization with BiP brefeldin the in NFlank immunostaining was of other trans-Golgi resident the antiserum the 25-kDa intermediate within and medial Golgi the NFlank antiserum a trans-Golgi of 9-kDa SP-B with the cleavage event occurring in the The location of the final N-terminal cleavage event step to by in a post-Golgi NFlank immunostaining was not within lamellar in as with the small were in to Lamp-1 in the of the small within multivesicular bodies of type 2 cells K. 1994; PubMed Scopus Google Scholar). microscopy studies to NFlank and Lamp-1 antisera of within multivesicular bodies. The of pro-SP-B processing is of processing, in which are and to at of (reviewed in Ref. S. N. J. 1994; PubMed Scopus Google and of post-translational processing of surfactant protein C Ballard P.L. Guttentag S.H. Beers M.F. Pediatr. Res. PubMed Scopus (46) Google Scholar). SP-C is as a that is processed to a hydrophobic mature protein. studies of surfactant protein C processing showed that brefeldin and monensin blocked most pro-SP-C processing, indicating that post-Golgi multivesicular body and/or lamellar are for SP-C processing M.F. Kim C.Y. Dodia C. Fisher A.B. J. Biol. Chem. 1994; 269: 20318-20328Abstract Full Text PDF PubMed Google Scholar, 13.Beers M.F. J. Biol. Chem. 1996; 271: 14361-14370Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). inhibitor studies indicated that most pro-SP-B processing occurs in a pre-lamellar body The of the of SP-B processing The and between SP-B and have out by M. T. H. J. 1995; PubMed Scopus Google Scholar, A. A. M. B. A. P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). SP-B and A, and from large All from a precursor protein and are a of enzymatic cleavages T. A. E. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). of the a function in between the and the N- and C-terminal propeptides of to have to the SP-B studies have shown the in with processing that are we have for SP-B T. S. PubMed Scopus Google Scholar, K. A. J. Cell Biol. Google Scholar). the of the knock-out mouse have and show with the homozygous SP-B knock-out alveolar type 2 cells, lamellar bodies which are into the the to form a The homozygous knock-out have a that on microscopy The have lamellar bodies of the lamellar bodies of alveolar type 2 cells of the homozygous SP-B knock-out mouse and human with inherited SP-B The between and SP-B post-translational processing is necessary to these in an sites of In is that processing trafficking that the of these to final are trafficking within the acid of either or pro-SP-B. the small N-terminal peptide in the final step of SP-B processing is a Studies in transgenic homozygous SP-B the of SP-B have shown that the N terminus is essential for of this lethal S. Phillips K.S. Wilder M.R. Weaver T.E. Biochim. Biophys. Acta. 1996; 1312: 177-185Crossref PubMed Scopus (70) Google Scholar, 6.Lin S. Akinbi H.T. Breslin J.S. Weaver T.E. J. Biol. Chem. 1996; 271: 19689-19695Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, H.T. Breslin J.S. M. Clark J.C. Whitsett J.A. Weaver T.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In these studies the N terminus, the vestigial N-terminal the pro-SP-B was Our data show that this was a pre-lamellar body which a for this in trafficking SP-B toward lamellar bodies. studies to this peptide is necessary and for trafficking SP-B to similar within type 2 cells and other cell with We the of in lung and of isolated type 2 cells, as as the of and in the of this

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