In vitro translation, post-translational processing and secretion of pulmonary surfactant protein B precursors

Biochimica et Biophysica Acta, 1011 (1989) 140-148

140

Elsevier

BBA 12456

In vitro translation, post-translational processing and secretion of pulmonary surfactant protein B precursors Michael A. O ' R e i n y 1, T i m o t h y E. W e a v e r 1, T a m i J. P i l o t - M a t i a s 2, V i r e n d e r K. S a r i n 2, A d i F. G a z d a r 3 a n d J e f f r e y A. W h i t s e t t I Division of Pulmonary Biology, Children's Hospital, Cincinnati, OH 2 Corporate Molecular Biology, Abbott Laboratories, Abbott Park, IL and 3 National Cancer Institute, Bethesda, MD (U.S.A.)

(Received 27 September 1988) (Revised manuscript received 15 December 1988)

Key words: Pulmonary surfactant; Posttranslational processing; In vitro translation; (Human adenocarcinoma cell line)

Surfactant pmteolipid SP-B is a hydmphobic protein of Mr - 8000 identified in organic solvent extracts of pulmonary surfaetant. Analysis of the human SP.B RNA predicts that the active surfactant peptide is derived by proteolysis of an Mr m 40000 precursor. In the present work, characteristics of synthesis, secretion and processing of SP-B were demonstrated in a pulmonary adenecarcinoma cell line by inununoprecipitation of radiolabeHed precursors. Treatment of cells with tunicamycin resulted in synthesis and secretion of unglycosylated proSP-B of Mr - 39000. Immunoprecipitation of protein produced by in vitro translation of human lung poly(A) + RNA detected an M r - 4 0 0 0 0 protein; the size discrepancy is likely related to cleavage of a leader signal sequence. Endoglycosidase-H-sensitive precursors of M r = 41000-43000, pl = 5.1-5.4 were the first isoforms detected within the cells, and were processed to endoglycosidase-H-resistant isofonns and secreted. Neuraminidnse and endoglycosidase-F-sensitive forms of pmSP-B were first detected in the media at 60 rain as Mr - 42-46000 isoforms with p l - 4.6-5.1. Proteolytically processed isoforms of proSP-B were detected primarily in the media and were generated by cleavage of an amino-terminal M r - 16000 peptide resulting in M r - 27000-33000 isoforms (pH - 5 . 6 - 6 . 8 ) . The M r - 27000-33000 isoforms were sensitive to neuraminidnse, resulting in isoforms with pH ~=6.0-6.8. Digestion of the Mr - 27000-33 000 peptide with endoglycosidase-F resulted in isoforms of M r = 23000, pH = 6.0-6.8. The endoglycosidase-F-resistant peptide of M r - 16000, p l - 4 . 2 - 4 . 4 was identified with an antiserum generated against synthetic peptides derived from the amino-terminal domain, as deduced from the SP-B DNA sequence. Further pmteolytie processing of the M r = 27000-33000 isoforms to the M r - 8000 peptide detected in surfactant was not observed in this cell line. Thus, in the H441-4 cells (a cell line with mmpbologic features of Clara cells), SP-B is synthesized as a preproprotein which undergoes cleavage of a signal sequence and addition of asparagine-linked carbohydrate; proSP-B is secreted by processes which are independent of glycosylation. SP-B peptides of M r - 2 7 0 0 0 - 3 3 0 0 0 and M r - 1 6 0 0 0 , representing carboxy and amino-terminal domains, accumulate in the media.

Abbreviations: SP-A, surfactant protein of Mr = 28000-36000; SP-B, surfactant protein of M r ffi8000: surtactant proteolipid N-terminus phenylalanine, SPL(Phe); SP-C, surfactant protein of Mr = 4000: surfactant pmteolipid with polyvaline domain; SPL(pVal); 2D-IEFSDS-PAGE, two-dimensional isoelectric focusing sodium dodecylsulfate polyacrylamide gel electrophoresis; NEPHGE, nonequilibrium pH gradient electrophoresis. Correspondence: J.A. Whitsett, University of Cincinnati, College of Medicine, Department of Pediatrics, Division of Pulmonary Biology, 231 lkthesda Avenue, Cincinnati, OH 45267, U.S.A.

Introduction Pulmonary surfactant is a lipid-rich complex which reduces alveolar surface tension at minimal lung volumes [1]. Several surfactant-associated proteins have recently been identified which enhance the surface active properties of phospholipids. These include SP-A (glycoprotein M r --28000-36000) [2] and two hydrophobic peptides termed SP-B (Mr = 8000) [3,4] and SP-C (Mr = 4000) [5,6]. SP-B and SP-C have been identified in organic

0167-4889/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

141 solvent extracts of surfactant and are associated with surface active properties of replacement surfactants utilized for treatment of hyaline membrane disease [7,8]. Recently, cDNAs for both of these proteins, SP-B [3,4] and SP-C [5,6], have been isolated. The amino acid sequences derived from the cDNAs predict that both proteins are produced from larger protein precursors. cDNAs for human SP-B predict an Mr = 40000 precursor with consensus sequences for asparagine-linked glycosylation at amino acid 310. A second potential glycosylation site is predicted by annie,sis of the cDNAs at amino acid 129. Substitution of leucine for threonine in one class of cDNAs results in the loss of this consensus glycosylation site. In vitro translation of adult lung poly(A +) RNA has identified an Mr = 40 000 SP-B preproprotein [3,4]. An antiserum generated against SP-B identified endoglycosidase-F-sensitive proteins of approx. M r - 4 2 0 0 0 and Mr = 25000 and smaller peptides in human fetal lung tissue [9,10], although the precise identity of the smaller peptides was not established. While the active surfactant peptide of Mr = 8000 is detected in pulmonary surfactant, the routing and secretion of SP-B by pulmonary epithelial cells, the relationship among the various immunoreactive SP-B peptides and the nature of the post-translational processing and secretion of the SP-B peptides remain unknown. In the present stud3/, we demonstrate the characteristics of biosynthesis, secretion and proteolytic processing of SP-B precursor in a human lung adenocarcinoma cell line with morphologic properties similar to those of Clara cells. Methods

Cell culture Human adenocarcinoma cell line NCI-H441-4 was cultured in RPMI 1640 with 10% fetal calf serum until near confluency. Morphology and hormonal contrt~l of SP-B synthesis were recently reported [11]. Cells were cultured in 10 nM dexamethasone for 72 h prior to metabolic labelling in order to increase synthesis of proSP-B. Cells were placed in Dulbeeco's modified Eagles' medium with 1 m g / l unlabelled methionine and 0.1% bovine serum albumin for 1 h prior to the addition of 100 /LCi/ml [3SS]methionine (1000 Ci/mmol) from New England Nuclear, Boston, MA. After labelling, cells were extensively washed and chased in methioninerepleted (30 mg/l) media. At various times, media were collected and frozen at - 8 0 ° C. Cells were harvested in 150 mM NaC1/30 mM Tris (pH = 7.4)/10 mM EDTA/1 mM phenylmethanesulfonyl fluoride (PMSF) and centrifuged at 800 ×g. Cell pellets were immediately solubilized in solubilization buffer (68 mM "Iris (pH=7.4) 190 mM NaC1/6 mM EDTA/4% SDS/1 mM PMSF) with sonication, heated at 100 °C for 4 min and stored at 4°C after addition of an equal

volume of water. In experiments with tunicamycin, cells were pre-treated with 1 # g / m l tunicamycin (Boehringer Mannheim) for 12 h prior to labelling. Cells were then washed and incubated in labelling media with tunicamycin. Addition of dexamethasone to the cell culture did not alter cellular DNA content as previously described for this cell line [11].

Analysis of proteins lmmunoprecipitation was performed as described by Anderson and Blobel [12]. After normalizing cell lysates for incorporation of label, samples were diluted with 4 vol. of dilution buffer (50 mM Tris (pH - 7,4)/190 mM NaCI/6 mM EDTA/2.5% Triton X-100/1 mM PMSF). Media were evaporated to dryness and the pellet was resuspended in 50 btl of solubilization buffer, followed by addition of 50 ~1 of water and 4 vol. of dilution buffer. Immunoprecipitation reactions with 5 #1 of appropriate antiserum were performed overnight at 4" C. Immune complexes were precipitated with 30/~! of 1:1 protein A-Sepharose (Sigma) suspension in water at room temperature and washed four times with 50 mM Tris (pH=7.5)/150 mM NaCI/5 mM EDTA/0.1~ Triton X-100/0.2% SDS and then twice with same buffer without detergent. Immunoprecipitated proteins were released from protein A-Sepharose by heating in sample buffer for 1D-SDS-PAGE as described by Lammeli [13] or for 2D-IEF-SDS-PAGE as described by Garrison and Wagner [14]. Non-equilibrium pH gradient electrophoresis (NEPHGE) was initiated bv separation in the first dimension (pH--3.5-10) at 1600 VHr and 13% PAGE in the second dimension as described by O'Farrell et al. [15]. In 1D-SDS-PACE experiments, samples were separated on SDS polyacrylamide (10-20%) gels purchased from Integrated Separation Systems, Newton, MA. Gels were electrophoretically transferred to nitrocellulose and exposed to Kodak XAR2 film at - 8 0 " C. In experiments designed to examine the charge shift of proSP-B after glycosidase treatment, 10 #g of human alveolar proteinosis protein was added to sample buffer and gels were immunoblotted for SP-A as previously described [16], for use as a charge reference point. Glycosidase d~gestion Immunoprecipitated proteins bound to protein ASepharose ~ere washed in glycosidase buffer once and then incubated in 200 #1 of the same buffer with enzymes at 37°C with rotation for 16 h. Endoglycosidase-F (2 U/ml) from New England Nuclear, Boston, MA or neura,rninidase (1 U/ml) from Sigma Chemical Company, St. Louis, MO, were incubated in 50 mM sodium phosphate (pH=6.2)/0.1% NP-40/10 mM EDTA/1 ~tM leupeptin/1 #M pepstatin/1 mM PMSF. Endoglycosidase-H (1 #g/ml) from New England Nuclear, Boston, MA, was incubated in 50 mM sodium

142 the synthesis of these peptides and the bacterial expression system will be reported separately.

acetate ( p H - 5.5)/0.1~ NP-40/10 mM E D T A / 1 / t M leupeptin/1 /~M pepstatin/1 mM PMSF. The protein A-Sepharose pellets were dried under vacuum and processed for polyacrylamide gel electrophoresis as described above.

Results

Identification of proSP-B and proteolytic fragments produced by H441-4 cells Antiserum prepared against bovine surfactant proteolipid (R10) reacts with the M, = 6000-14000 surfactant proteolipids and immunoprecipitates SP-B precursors of Mr - 40 000 after in vitro translation of lung poly(A) + RNA [9,10,20]. Immunoprecipitation of media from [35S]methionine-labelled H441-4 cells with this antiserum detects proteins of M r - 42000-46000 and Mr-27000-33000 (Fig. 1A). Both proteins contain asparagine-linked carbohydrate, as demonstrated by their sensitivity to endoglycosidase-F, resulting in M r 39000 and Mr = 23000 proteins (Fig. 1A). Antiserum (R4633), generated against the entire recombinant human preproSP-B recognized identical M r - 4 2 0 0 0 46000 and Mr-27000-33000 proteins and an additional endoglycosidase-F-resistant protein of M r 16000 (Fig. 1A). In order to confirm that the Mr = 16000 protein was derived from proteolysis of M r 42 000-46 000 proSP-B, antiserum was prepared against a synthetic peptide corresponding to amino acids 143171 of preproSP-B (R97). This antiserum recognized an endoglycosidase-F-resistant M r = 16000 peptide by both immunoblot (data not shown) and by immunoprecipitation of [35S]methionine-labelled media (Fig. 1A). Antiserum was also prepared against a synthetic SP-B polypeptide comprising amino acids 200-260 of the human preproSP-B (R589). This anti-

Ii: uitro translation Total lung RNA was isolated from adult human lung at avtopsy as described by BraeU and Lodish [17], a modification of Chirgwin et al. [18]. Poly(A) + RNA was selected by affinity chromatography on oligo(dT) cellulose as described by Aviv and Leder [19]. In vitro translation in nuclease-treated rabbit reticulocytes was carried out according to specifications of the supplier (Promega Biotec). Generation of antiserum Surfactant proteolipids of Mr-6000-14000 were isolated from bovine surfactant and antiserum (R10) was generated in rabbits as previously described [20]. This antiserum recognized bovine, rat, canine and human surfactant proteolipids of Mr-6000-14000 by immunoblot analysis. Antiserum (R589) was prepared in rabbits by repeated injections of a human SP-B synthetic peptide (amino acids 200-260 of preproSP-B) [10]. Antiserum R97 was generated against synthetic peptides comprising the amino-terminal domain of preproSP-B (amino acids ~43-171) as deduced from the human eDNA [3,4]. The peptides were assembled on a resin support by stepwise solid phase synthesis according to the method of Merrifield [21]. Antiserum (R4633) was also prepared against the entire recombinant human preproSP-B after expression in E. coil. Details of

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Fig. 1. Immunologic identification of proSP-B and fragments. Cells were labelled with[35S]methionine for 12 h at which time media were collected and processed for immunoprecipitation. Proteins were separated on a 10-20~; polyacrylamide-SDS gel in the presence of/3-mercaptoethanol and blotted to nitrocellulose for autoradiography. (A) Proteins were immunoprecipitated with R10 serum (lanes 1, 2), R4633 serum (lanes 3, 4), R97 serum (lane 5) or with nonimmune rabbit serum (lane 6). Samples in lanes 2 and 4 were digested with endoglycosidase-F as described in Methods. Antiserum prepared against bovine surfactant SP-B (R10) recognized M r = 42000-46000 and M r = 27000-33000 endoglycosidase-F-sensitive proteins, whereas R4633 (generated against the recombinant preproSP-B) recognized the same peptides and an M r = 16000 peptide. In contrast, R97 (raised against a synthetic peptide amino-terminal to the active surfactant peptide) recognized the Mr = 42000-46000 (weakly) and M, = 16000 but not the M r = 27000-33000 peptide. In (B), labelled proteins were immunoprecipitated with R10 serum (lane 1), R4633 serum (lane 2), and with nonimmune rabbit serum (lane 3) and separated in the absence of/~-mercaptoethanol.

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Fig. 2. Linear map of human preproSP-B. SP-B of Mr = 8000 (hatched box) is contained within a larger precursor protein of 381 amino acids whose amino-terminus contains an approx. 23 amino acid consensus signal sequence. Consensus asparagine-linked glycosylation sites are present at amino acids 129 and 310. The amino-terminal glycosylation site is not present in some mRNAs. Proteolysis near amino acids 199 and 200 results in generation of two peptides of M r =16000 and M r = 23000 identified by SDS-PAGE.

serum recognized the Mr = 8000 SP-B peptide in bovine and human suri'actant (by inununoblot) and the M~ = 42000-46000 and M, = 27000-33000 proteins previously detected with R10 serum. (by immunoprecipitation) [10]. Like the R10 serum, R589 serum did not recognize the Mr = 16 000 peptide after immunoprecipitation of media from [a~S]methionine-labelled H441-4 cells (data not shown). Analysis of the SP-B precursors from the media under non-reducing conditions demonstrated that larger oligomers were not detected (Fig. 1B). Thus, these antisera have been utilized to identify endoglycosidase-F-sensitive SP-B precursors of Mr = 42 000-46 000 and Mr = 27 000-33 000 which contain the antigenic sites reacting with the surface active SP-B peptide of Mr = 8000 and an amino-terminal, endoglycosidase-F-resistant peptide of Mr = 16 000, which lacks the antigenic determinants contained in the surfactant peptide, (Fig. 2). The resistance of the Mr = 16000 peptide to endoglycosidase-F suggests that in the H441-4

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Fig. 3. Poly(A) + RNA from human lung was translated in vitro with rabbit reticulocyte iysate using [3~S]methionine as the labelled amino acid and SP-B was immunoprecipitated with R10 serum (lane 1). H441-4 cells were incubated with (lane 2) and without (lane 3) 1 p g / m l tunicamycin (for 1 h) prior to incubation with [~Slmethionine and immunoprecipitated with R10 antiserum. Samples were then separated on a 10-207o polyacrylamide-SDS gel in the presence of fl-mercaptoethanol and blotted onto nitroe gulose for autoradiography.

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Fig. 4. Identification of intracellular proSP-B. Cells were labelled with [35S]methionine for 15 min, extensively washed and in, uba'.'d with methionine-repleted media. Cells were harvested at 0, 15. 30 rain and 1, 3, 5 h of chase. ProSP-B was immunoprecipitated w i t RI0 serum after 1 h of chase and resolved by 2D-IEF-SDS-PAGE as isoforms of M r = 41000-43000, pl = 5.1-5.4, (A). (B) Autoradiogram of intracellular proSP-B in a pulse-chase experiment ( M r = 41000-43000) immunoprecipitated with R4633 serum and separated on a 10-20~ polyacrylamide-SDS gel. Between 1 and 3 h, lesser amoun ~,s of protein of M r = 27000-33000 and M r =16000 were also obselved (barely detectable). Lane (N) represents immunoprecipitation o~ cell lysate with nonimmune rabbit serum. (C) ProSP-B after immu~oprecipitation of labelled cells at 0 min (lanes 1, 2) and 1 h (lanes 3, 4). Lanes 6 and 7 represent immunoprecipitation of media at 5 h ol the chase period. Samples in lanes 2, 4. 7 were treated with endoglyo,sidase-H. Lane 5 represents immunoprecipitation t)[ the cell ly.~,ate with r,onimmune rabbit serum, lntracellular proSP-B was sensitive to eadoglycosidase-H with resulting M r = 39000 protein up through 1 h of the chase period. ProSP-B isoforms detected in the me(;a were resistant to endoglycosidase-H.

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Fig. 5. Identification of secreted proSP-B. Cells were labelled with [3SS]methionine for 15 min and chased with methionine-repleted media. Media were collected and proSP-B was immunoprecipitated with R10 serum and separated by 2D-SDS.PAGE. At 5 h of chase, secreted forms of proSP-B migrated as ,Mr = 42000-46000, p ! = 4.6-5.1 (A). Digestion with neuraminidase resulted in charge shift of M, = 42000-46000 isoforms to pl = 5.1-5.4. (B). Endoglycosidase-F treatment of these forms resulted in protein of M r = 39000, pl-5.1 and 5.4 isoforms (C). (D) Time-course of the appearance of proSP-B (M r = 42000-46000). The M r = 27000-33000 proteins were first readily identified in the media at 1-3 h of the chase period. cell line there is n o a s p a r a g i n e - l i n k e d c a r b o h y d r a t e a d d i t i o n to a m i n o acid 129, c o n s i s t e n t w i t h t h e SP-B c D N A s , w h i c h p r e d i c t allelic h e t e r o g e n e i t y o f t h e c o n s e n s u s s e q u e n c e s for a s p a r a g i n e - l i n k e d a d d i t i o n o f c a r b o h y d r a t e at this site [3].

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Evidence for proteolysis of a signal sequence An Mr=40000 p r o t e i n was i m m u n o p r e c i p i t a t e d f r o m 32S-labelled p r o t e i n s a f t e r in v i t r o t r a n s l a t i o n o f h u m a n l u n g m R N A u t i l i z i n g t h e R 1 0 a n t i s e r u m (Fig. 3, l a n e 1). T h e i n t r a c e U u l a r f o r m s o f p r o S P - B d e t e c t e d

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Fig. 6. Identification of M, 27000-33000 protein in the media. Cells were labelled with [35S]met,,ionine and media collected at 6 h. ProSP-B was immunoprecipitated with RIO serum and separated by NEPHGE for 1600 VHr in the first dimension followed by SDS-13~ PAGE and subjected to autoradiography. Proteins of M r = 27000-33000 migrated as distinct isoforms of pH = 5.6-6.8 (A). Digestion with neuraminidase resulted in a charge shift to Mr = 27000-33000, pH = 6.0-6.8 (B). Endoglycosidase-F treatment resulted in isoforms of M r = 23000, pH = 6.0-6.8 (C). =

145 after [35S]methionine labelling of H441-4 cells migrated as endoglycosidase-F-sensitive isoforms of Mr = 41000-43 000 (lane 3). When labelled in the presence of tunicamycin, the cells produced an unglycosylated proSP-B peptide of M r = 39000 (lane 2). The size discrepancy between the unglycosylated Mr = 39000 precursor detected in tunicamycin-treated cells and that detected after in vitro translation ( M r - - 40 000) may be attributable to the cleavage of an amino-terminal sequence in preproSP-B as predicted by c D N A sequence analysis [4].

Glycosylation and secretion of proSP-B Pulse-chase experiments were performed by labelling H441-4 cells with [35S]methionine for 15 rnin followed by chase with methionine-repleted media. After 1 h of chase, intracellular forms of proSP-B were detected as predominant isoforms of Mr -- 41000-43 000, pl= 5.1-5.4 (Fig. 4A). The M r - 41000-43000 forms predominated intracellularly up to 60 min (Fig. 4B). Glycosylated forms of proSP-B were secreted from the cell within 1-3 h and were no longer readily detected intracellularly after 3-5 h. Intracellular precursors of proSP-B at the beginning of the chase were sensitive to endoglycosidase-H resulting in M r = 39 000 protein (Fig. 4C). After 1 h of chase, endoglycosidase-H-resistant forms were detected and increased with time. Sensitivity to endoglycosidase-H demonstrated the presence of high-marmose-containing carbohydrate on the precursors. Between 30 rain and 5 h, proteins of Mr = 27000-33000 and 114,=16000 proteins were barely detected intracellularly and only after long exposure of autoradiograms. Immunoprecipitation of proSP-B from the media identified more acidic, endoglycosidase-H-resistant (Fig. 4C), heterogeneous isoforms of Mr= 42000-46000, p l = 4.6-5.1 with a minor component of p l = 5.4 (Fig. 5A). These secreted isoforms contained sialic acid, as demonstrated by their sensitivity to neuraminidase (Fig. 5B). Removal of the carbohydrate by digestion with endoglycosidase-F resulted in isoforms of M r = 39000, pl= 5.1 and 5.4 (Fig. 5C). The mechanisms involved in the heterogeneous migration of the M r - 39 000 protein is not known at present. ProSP-B of M r = 42000-46000 was first detected in the media 60 rain into the chase period, and increased in abundance up to 5 h (Fig. 5D).

Time-course of proteolytic processing of proSP-B Between 1 and 3 h of the chase period, immunoprecipitable protein of M, = 27000-33000 was detected in the media and its abundance increased with time (Fig. 5D). These isoforms were also detected intracellularly, but at very low abundance. The M, = 27000-33000 peptide in the media was resolved into isoforms of pH = 5.6-6.8 (Fig. 6A). The more acidic forms were also sensitive to neuraminidase, resulting in

a charge shift to more basic isoforms of pH = 6.0-6.8, thus demonstrating ;'he presence of sialic acid (Fig. 6B). These forms were aL:o sensitive to endoglycosidase-F, resulting in isoforms of M r = 23000, pH --- 6.0-6.8 (Fig. 6C). The detection of the M r = 27 000-33 000 peptide in the media and the shared immunoreactivity of SP-B epitopes in both M r = 42 000-46 000 and 27 000-33 000 proteins suggest that proteolytic cleavage of some of the Mr = 42 000-46 000 glycoprotein precursor generates two fragments: an SP-B containing M r = 27000-33000 peptide and an M r = 16000 peptide lacking both carbohydrate and the antigenic epitopes defining the active M r - 8 0 0 0 SP-B peptide. The peptide of M r =

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tected in the media at 60 rain of chase (Fig. 8B). The similarity of the kinetics of secretion of unglycosylated proSP-B and glycosylated proSP-B suggests that secretion of proSP-B is not dependent upon the presence of asparagine-linked carbohydrate. Tunicamycin-induced, unglycosylated precursors migrated similarly to the endoglycosidase-F-digested proSP-B in 2D-SDS-PAGE (data not shown). Proteolytic processing of the unglycosylated SP-B precursor to the nonglycosylated M r 23 000 peptide was moderately inhibited in the presence of tunicamycin (Fig. 8B).

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Fig. 8. Effects of tunicamyciuon synthesis and secretion of proSP-B. Cells were treated with tunicamycin as described in Methods and labelled with [35S]methionine for 15 min, followed by chase with methionine-repletedmedia. Cells and mediawerecollected at 0,15, 30 rain and 1, 3, 5 h of chase, immunoprecipitated with R10 serum, separated on a 10-20~ polyacrylamide-SDSgel and subjected to autoradiography. Intracellular forms of unglycosylated proSP-B of Mr = 39000weresecreted(A). Unglycosylatedpro~P-Bof Mr = 39000 was first detected in the media at 60 min and inclcaSedwith time (B). Some proteolysisto the unglycosylated Mr = 23000 isoform was also observed. 16000 identified in Fig. 1 was resolved as two distinct isoforms, a minor isoform of p l - - 4 . 2 and major isoform of p l = 4.4 (Fig. 7A). These forms do not contain asparagine-linked carbohydrate, as demonstrated by their resistance to endoglycosidase-F (Fig. 1). The mechanisms involved in the heterogenous migration of either the unglycosylated M r = 23 000 or the Mr = 16 000 peptide during isoelectric focusing are likewise unknown at present. The M r = 16000 protein was first identified in the media between 1 and 3 h, a time-course similar to that of the M r = 27000-33000 peptide (Fig. 7B). Extension of chase times through 60 h failed to detect complete proteolysis of the secreted M r = 42 000-46 000 precursor to the M r = 27 000-33 000 and M r = 16 000 peptides. In addition, the surfactant peptide of M r = 8000 was not detected by either immunoblot analysis of conditioned media or after metabolic labelling with radioactive amino acids followed by immunoprecipitation with appropriate antisera. Effects of tunicamycin Pulse-chase experiments in the presence of tunicamycin demonstrated the synthesis and secretion of unglycosylated proSP-B of M r = 39 000 (Fig. 8A). Secretion of unglycosylated proSP-B was rapid and occurred with a time-course similar to that of the glycosylated forms (Fig. 4A). Unglycosylated proSP-B was first de-

The present work demonstrates the biosynthesis, secretion and proteolytic processing of surfactant protein B (SP-B) by a continuous tumor cell line derived from a human pulmonary adenocarcinoma with Clara cell-fike morphology. PreproSP-B is rapidly glycosylated at the carboxy-terminal domain and a signal sequence is removed by intracellular proteolysis. ProSPB is then sialylated and secreted into the media. Proteolytically degraded fragments of Mr = 16000, representing a nonglycosylated amino-terminal peptide, and M r - 2 7 0 0 0 - 3 3 0 0 0 , representing a glycosylated carboxy-terminal peptide containing the M r - 8000 SP-B antigenic epitope, accumulate m the media of the tumor line. Surface active SP-B of M r - 8 0 0 0 identified in pulmonary surfactant [7,8] is not readily detected by immunoblot or metabolic labelling, suggesting that complete processing of the SP-B precursor is not accomplished by the cell fine. Identification of cDNA clones for both human and canine SP-B have predicted a preproSP-B precursor of M r - 4 0 0 0 0 from a single 2.0 kb RNA [3,4,22]. This precursor protein has been identified by both hybrid selected and arrested translation with SP-B cDNA [3,4]. In this work, tunicamycin-treated cells synthesized unglycosylated proSP-B of M r = 39000; the size discrepancy suggests cleavage of a signal sequence. Analysis of the amino-terminal sequence of the SP-B precursor, as deduced from the cDNA, reveals that the first 23 amino acids are compatible with the consensus structure of a signal sequence [23]. The central portion of this putative signal sequence contains a hydrophobic region of consecutive leucine residues, which precedes a more polar region, separated by a proline. Amino acids 21 and 23 are small neutral residues (Gly,Ala) which are usually observed at - 3 and - 1 to the cleavage site. A proline at the - 4 position of pre-fl-lactamase has been associated with assisting the formation of a ~8-turn [24]; the twentieth amino acid of preproSP-B is proline. Given these parameters, the signal sequence of preproSP-B is predicted to be approx. 23 amino acids. Definitive identification of the signal sequence cleavage site will require amino acid sequence analysis.

147 Two classes of human mRNAs for preproSP-B have been identified whose translation products differ at one amino acid, resulting in the loss of a consensus site for asparagine-linked glycosylation at amino acid 129 [3]. Both classes of mRNAs predict a similar glycosylation site at amino acid 310. The present study demonstrates that in the H441-4 cell line, proSP-B is glycosylated only at the carboxy-terminal site. The amino-terminal Mr = 16000 peptide was resistant to endoglycosidase-F. In contrast, the carboxy-terminal peptide of M r = 27000-33000 is sensitive tO endoglycosidase-F, with resultant Mr = 23 000 peptide. Sequence analysis of a canine SP-B cDNA has revealed that it lacks an aminoterminal glycosylation site, whereas the carboxy-terminal site is conserved with the two human cDNAs [22]. Addition of asparagine-linked carbohydrate is a rapid process which occurs during or shortly after translation [25]. Although the role of carbohydrate on surfactant proteins is not presently known, treatment of cells with tunicamycin does not result in inhibition of either SP-A or proSP-B secretion [26,27]. The present study demonstrates that proteolysis of proSP-B occurs at later time points during biosynthesis. ProSP-B of M r = 42 000-46 000, p l = 4.6-5.1 is the predominant form secreted from cells, first appearing in the media after 1 h of chase. Between 1 and 3 h proteolysis of some of the SP-B precursor to Mr 27000-33000, pH = 5.6-6.8 peptide and an acidic M r = 16000 , p l = 4.2-4.4 peptide is also observed. The Mr = 27000-33000 and 16000 forms are barely detectable intracellularly but are readily detected extracellulady. The precise site of proteolysis, whether extracellular or intracellular, has not been clarified, however. Tke lack of significant intracellular proteolytic fragments supports the concept that if proteolytic cleavage of proSP-B occurs intracellularly, it must be in close association with secretion. Whether this occurs before, during or after secretion remains to be clarified. The prolonged stability of proSP-B in the media (up to 60 h) suggests that ongoing proteolysis of the 42000 peptide in the media does not account for the Mr = 16 000 and M r --- 27 000-33 000 fragments. The site and nature of the production of the alveolar forms of SP-B remain to be clarified. The surface active SP-B peptide of M r = 8000 forms oligomers of Mr = 18000 and larger under non-reducing conditions. ProSP-B ( M r = 4~ 000-46 000) and both the Mr = 16 000 and Mr = 27 0C0-33 000 peptides fail to form such oligomers. Identification of the Mr = 16 000 peptide with antiserum directed against an amino-terminal peptide of proSP-B (amino acids 143-171) and demonstration of an acidic isoelectric point for the peptide support the likelihood that the proteolysis of proSP-B of M r = 42000-46000 occurs at or near the amino-terminus of the Mr = 8000 SP-B peptide. Antisera generated against synthetic peptides, corresponding to amino acids =

172-199 of preproSP-B, are likewise immunoreactive against the M r = 16 000 peptide by immunoblot analysis (data not shown). The amino-terminus of the M r = 8000 SP-B peptide is Phe-Pro-Ile-Pro .... and is preceded by two glutamine residues. Proteolysis at this site would necessitate cleavage at a Gln-Gln...Phe bond. This cleavage site is similar to the extracellular proteolytic site of proapolipoprotein A-I, which involves cleavage of G l n - G l n . . . A s p [28]. Processing of the SP-B precursors to the surface active M r - 8000 peptide was not detected in the H441-4 cell line. Thus, additional or distinct proteolysis of the SP-B precursors must occur to generate the M r = 8000 SP-B peptide observed in surfactant in vivo. The active Mr = 8000 may be generated by proteolytic processes unique to type II or other pulmonary cells in vivo. This work also demonstrates that the rate of synthesis and secretion of SP-B is distinct from that of SP-A previously reported in rat type 11 epithelial cells. Previous studies demonstrated that SP-A exists within type II cells as glycosylated isoforms both in vivo and in vitro [27]. SP-A is rapidly glycosylated to high-mannose endoglycosidase-H-sensitive precursors which are slowly processed to mature sialylated forms. SP-A is highly enriched in lamellar body fractions isolated from type II cells or lung tissue [27]. In rat type II cells in vitro, mature sialylated SP-A is secreted slowly, 50% of the SP-A being secreted within 16-20 h. Secretion of SP-B by the H441-4 line was more rapid than SP-A either in H441-4 or in rat type II cells. Thus, the secretory rates of SP-A and proSP-B appear to be distinct. in the H441-4 cell line, proSP-B is glycosylated and secreted primarily as the intact proprotein. Discrete proteolysis of some of the SP-B precursor occurs in close association with or after secretion, accounting for the production of Mr = 27 000-33 000 carboxy-terminal and M r = 16 000 amino-terminal peptides. Although the function of these peptides and their potential role as precursors of the active surfactant peptide have not been ascertained, the strong conservation of the nucleotide and amino acid sequences of the entire canine and human SP-B precursor supports the hypothesis that there may be functional roles for the precursor or its proteolytic fragments. Previous studies of SP-B synthesis in human fetal lung explants demonstrated production of the M r = 42000-46000 and M r = 27 000-33000 precursors [9,10]. In contrast to the present study, smaller peptides of M r = 8000 and 12 000 (migrating as M r = 18 000 in the absence of sulfhydryl reducing agents) were readily detected in the lung explant tissues. Secretion and processing of proSP-B were also detected in primary cultures of rat type II cells (Weaver and Whitsett, unpublished observations). In that study, secretion of the Mr = 42 000 forms and proteolysis to Mr = 25 000 and Mr = 16000 forms were also detected. However, in the rat primary type II cells, SP-B forms of Mr =

148 ~?000-12000 were identified which co-migrated in 2DIEF-SDS-PAGE with mature SP-B isolated from alveolar lavage fluid. It is not known whether the lack of complete processing of SP-B by the H441-4 cell line is related to its transformed cell phenotype or to a Clara cell-specific phenotype. Moreover, it is possible that addition of dexamethasone may alter the processing of SP-B precursors. However, since untreated cells do not synthesize detectable levels of proSP-B, it is technically impossible to test this hypothesis in this system. Recently, in situ hybridization studies of human lung demonstrated SP-B RNA synthesis in both distal bronchiolar epithelium and in Type II epithelial cells, supporting the hypothesis that SP-B synthesis is not specific for the type II cell [29]. Immunocytochemistry of human lung tissue demonstrated staining for surfacrant proteolipid (presumably SP-B and/or SP-C) in the distribution of both epithelial cell types [9]. Antisera specific for human SP-B stained primarily large granular inclusions in type II epithelial cells after explant culture of human fetal lung[10]. Thus, there is evidence that SP-B may be expressed by pulmonary epithelial cells with both Clara cell and type II cell features. The present work raises the possibility that the processing of the SP-B peptides may also be regulated in a cell-specific manner. The mechanisms of proteolytic processing invol,~¢d m the production of the M r - 8 0 0 0 peptide isolated from surfactant have not been elucidated. The secretion and proteolysis of SP-B presently reported may represent a major pathway of SP-B biosynthesis or alternatively represent a cell-specific pathway in transformed or Clara-related cells. Acknowledgements This work was supported by HL 38859 (J.W.) and HL 36055 (T.E.W.). M.A.O'R. is funded in part by a Molecular and Cellular Biology Training Grant HL 07527. References 1 King, R.J. (1982) J. Applied Physiol. 53, 1-8. 2 White, R.T., Datum, D., Miller, J., Spratt, K., Schilling, J., Hawgood, S., Benson, B. and CordeU, B. (1985) Nature 317, 361-363.

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