Lisa MitsockS, Joseph P. DoughertyS, H. William Taeuschn, and Joanna Florosn ..... Hawgood, S., Benson, B., and Cordell, B. (1985) Nature 317,. 47.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists,Inc.
Val. 262, No. 20, Issue of July 15,pp. 9808-9811, 1987 Printed in U.S.A.
Isolation of a cDNA Clone Encoding a High Molecular Weight Precursor to a 6-kDa Pulmonary Surfactant-associated Protein* (Received for publication, December 31, 1986)
Kenneth A. Jacobs$$, David S. Phelpsn, RandalSteinbrink$, Judith Fisch$, Ronald Kriz$, Lisa MitsockS, Joseph P. DoughertyS, H. William Taeuschn,and Joanna Florosn From $Genetics Institute, Inc.,Cambridge, Massachusetts 02140and the TDepartment of Pediatrics, Harvard Medical School, Boston, Massachusetts021 15
Mammalian surfactant is an incompletely defined mixture of lipids and associated proteins of molecular mass 35,000 Da and approximately 6,000 Da. Surfactant preparations which are highly effective in treating respiratory distress syndrome in premature infants lack the 35-kDa proteins, but contain the 6-kDa proteins. We isolated and partially sequenced one of these low molecular weight proteins from the lung lavage material of an alveolar proteinosis patient. Oligonucleotides deduced fromthe sequence were used as probes to isolate a human cDNA clone. The clone codes for a 42-kDa protein which contains the sequence of the 6kDa protein. Messenger RNA coding for the 42-kDa protein was identified in human lung RNA by in vitro translation and immunoprecipitationof the translation products with an antiserum against purified bovine surfactant 6-kDa proteins. Immunoprecipitationof the 42-kDa primary translation product is inhibited by the presence of the bovine 6-kDa protein. These observations suggest a precursor-product relationship of the 42-kDa protein to one of the 6-kDa proteins
Respiratory distress syndrome denotes the clinical condition of pulmonary dysfunction in prematureinfants. The disease is attributable to insufficient surface-active material (surfactant) which lines the air-liquid alveolar interface in the lung and prevents collapse of the alveoli during respiration (1).Recent clinical trials indicate that one promising therapy is the instillation of bovine-derived surfactant into the lungs of the neonate (2-5). The lipid composition of these replacement extractsis essentially the same as whole surfactant. The protein composition is substantially different (6-11). Analysis of the protein components of natural surfactant preparations has, in the past, revealed the presence of sialoglycoproteins of molecular mass 35,000 Da termedpulmonary surfactant-associated proteins (PSP-A)’ and of several hydrophobic proteins with molecular masses ranging from 5,000* This work was supported bythe National Heart, Lung, and Blood Institute Grants HL36004, HL34788, HL34616, and HL34597 and by Genetics Institute, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in thispaperhos been submitted totheGenBankTM/EMBLData Bank with accession numbeds) 502761. f To whom correspondence and reprint requests should be addressed. The abbreviationsused are: PSP-A, pulmonary surfactant protein A (35 kDa);PSP-B, pulmonary surfactant protein B (-6 kDa); SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.
20,000 Da(12-22). Experiments with purified PSP-A proteins suggested that they were the protein group responsible for the biophysical activities of surfactant (23-25). Clinical studies also showed the PSP-A as present in reduced amounts in amniotic fluid samples taken shortly before the birth of infants who developed respiratory distress syndrome (26-28). The lower molecular weight proteins were thoughtto be catabolic products of the PSP-A (29). The surfactant preparations used therapeutically to treat respiratory distress syndrome have been made by extracting natural surfactant with organic solvents. Surprisingly, biochemical analysis revealed that these extracts contained proteins of 6000 Da, but no PSP-A (7-11). Moreover, recent experiments by Fujiwara demonstrated that both in vitro and in uiuo surfactant activity were reconstituted from mixtures of a purified, bovine 6-kDa surfactant-associated protein and appropriate lipids (30). Biophysical analysis of one of these proteins has shown it to be different from thePSP-A in antigenicity, solubility in organic solvents, and in amino acid composition, but has notelucidated the precise nature of this protein (8-11, 22). To understand the biophysical properties of this protein and to provide substrate for reconstitution studies, we have isolated and characterized a cDNA clone coding for a human, low molecular weight surfactant-associated protein. MATERIALS AND METHOD$ RESULTSANDDISCUSSION
Hydrophobic proteins were extracted into n-butyl alcohol from the pulmonary lavage material of an alveolar proteinosis patient. A hydrophobic protein, which we refer to as PSP-B, was isolated and sequenced by amino-terminal degradation. Various combinations of oligonucleotides deduced from the amino acid sequence wereused as probes to isolate corresponding cDNA clones. The sequence of a full-length cDNA clone (Fig. 1) measures 2,014 nucleotides, which agrees with the size of the mRNA as measured by a Northern analysis (data not shown). This clone codes for a proteinof 381 amino acids with a predicted molecular mass of 42,122Da and contains two potential sites for N-linked glycosylation. As shown in Fig. 2, lanes A and B, the cDNA clone selects Portions of this paper (including “Materials and Methods,” Footnote 3, and Fig. 2) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-4514, cite the authors, and include a check or money order for $2.80 per set of photocopies. Full size photocopies are also included in themicrofilm edition of the Journal that isavailable from Waverly Press.
9808
cDNA Clones Encoding Pulmonary Surfactant Proteins 1ULPL[TC006GccAn:GcrGAGTCACACcrGcrGwnr.cn:cn:cn:cn;cn: M E T A l a G l u S e r H i s l e u L e u G l n h p L e U l a t ~ I . € U b Z U 14
9809
ular weight surfactant proteins (22). The specificity of the immunoprecipitation reaction is demonstrated by competition with an independent preparation of bovine 6-kDa protein FrozhruucyGlyPmGlyzhrAlaAlahpllu~SerSerleuAlacy 11oGccwGGcccTuy;Tx:~mcanpGccn;GAGcanGcATn:wm~ (Fig. 2, lune E ) . A protein of 42,000 Da hasalso been identified Ala G l n G l y Fro G l u Fhe hp Cys G l n Ser leu G l u G l n Ala lat G l n Cp m 50 in normal human lung tissue by radiolabeling and subsequent mmQ3zcATmmcnGGwLm~mcmGlGmmmG42m immun~precipitation.~ Ala Leu G l y Ki.9 Cys Leu G l n G l U V a l np G l y Hi6 V a l G l y Ala ASP Asp IeU A surfactant-associated protein of this size has not previ210rrc~GAGmG4z~c~~c&cATcCm~nW;ATGGccAM?.G4zGcc ously been reported. We postulate that 42,000-Da this protein cy ~ l Gnl u q% G l u ~ s Ile p val His Ile leu Asn Lys Mer Ala Lys G l u Ala 86 AlTTrc~G42mATGmAM?.TrccrGG4zcTGG4zmAxGlrmm is the precursor of PSP-B, a proteolipid (42), and that the Ile Fhe G l n Asp llu MeT Arg Lys Fhe Leu G l u G l n G l u Cy8 AQI V a l leu F m mature protein is formed by the proteolytic cleavage, probably the type I1 cell, of peptides from both its amino and 3 2 6 T n : f f i c r G ~ A n : ~ ~ ~ A x ( 7 A A G l G C m ' G A c ~ ? a C T T c ~ c rwithin G LeuIysleuleu~FroGlnCpAQIGlnValLeuAspAsplyrFheFroLeu122 carboxyl termini. Combining information on the amino acid mmG42?aCTrcc?GAxwAcrG42mAAcGGcmmAn:CACcn: composition with amino-terminal sequence data and themoValIleAspTyt-FheGln~~'IhrlAspSerAQIGlyIleCYs~nisleu lecular weight estimate, we can approximately assign a series 4 3 4 G G c c n : r r c ~ r r c a r , w n a ~ ~ u u : w G 4 z n a a r , A n : m ~ of residues that comprise the low molecular weight protein. G l y leu Cp Lys Ser Arg G l n Pm GlU Fro G l U G l n G l u Pm G l y WZC Ser ASP 158 mcn:mAAAmcn:~'GAcmcrGanG42mcIGcn;'GAcA?Gm These are between nucleotides 614 and 841, corresponding to PmLeuPmLy%FrouuArgASpPm~PmFtqmJleuLeuASPLy%Leu amino acids 201-276 in the unprocessed protein (Fig. 1). 5 4 2 ~ ~ m G l t 2 m m ~ ~ m w O a ; m ~ ~ m CAnalysis A C ~ of w the deduced amino acidsequence of the PSP-B V a l leu Fro V a l leu Fro G l y Ala Leu G l n Ala Arg Pro G l y P m His 'Ihr G l n 1 9 4 precursor provides insight into its potential properties. The mmlKsGKwarLTrcmAlTmmmTxTmnr.mmm using the algorithm A S P L e u S e r G l u G ~ G l n ~ F r o I l e F r o L e u F r o ~ C p ~ c y A r g hydropathicity of the protein, determined of Kyte and Doolittle (43), suggests a hydrophobic amino6 5 0 G c p c T c ~ f f i ~ A T c c A A G c T ~ ~ C a : n W ; o O r O a ; c E A ~ c I c . G c A A l & l Ile Lys Arg Ile G l n Ala Mer Ile Fm Lys G l y Ala Leu Ala V a l Ala 230 terminal "signal sequence" characteristic of secreted proteins (44). The region correspondingto "mature" PSP-B is exGTGGccwGlt2maxGlGmmmGIGOa;GGcGScmmcTGm ValAlaGlnValCpArgValValFroleuValAlaGlyGlyIle~Gln~ tremely hydrophobic. The remainder of the molecule is rela7 5 0 c n : ~ G 4 z a % ~ l K c m m c r G c T c G 4 2 ~ c r G c n : G G c ~ m c n : tively hydrophilic. These hydrophilic regions may mask the leu Ala G l u A q 'lyr Ser V a l Ile Leu leu Rsp 'Ihr Leu Leu G l y Arg MET Leu 266 hydrophobic portions, facilitating intracellular transport of mc?&mmma%cTcmmmmTo2mmwpGcGmGSc the protein to lamellar bodies. They may also be signals for FroGlnleuValCpArgleuValLeuArgcySer~AspAspSerAlaGlY steps. For example, a 0 6 6 a n ~ ~ ~ A ~ ~ ~ ~ c n : ~ ~ w ~ G various 4 z rpost-translational r c c & c ~ processing ~ FroArgSerFrolluGlyGluhpLeuProArgAspSerGluCpHisLeu~302 mitochondrial membrane proteolipid hasbeen shown to posAn:lKcmAo2mwGcc~AAc~pGcG4zwGccAmmwGa sess an amino-terminal sequence that directs it to the mitomr ~ e val r ?hr zhr ~ l n G l y l A s n ser s e r J G l u ~ l Ala n Ile pro G l n Ala chondria (45). ~ 7 4 A n : ~ ~ G c c ~ ~ ~ l K c ~ ~ ' G A c ~ ~ ~ m A M ? . ( 7 A A ~ The PSP-B precursor is not homologous to PSP-A. Unlike MeT Leu G l n Ala Cys V a l G l y Ser hp Leu Asp Arg G l u Lys Cy Iys G l n 338 the PSP-A, the PSP-B precursor hascollagen-like no regions GTGGGwc&cmmcTGcrGcrGmcn:mmmGGcn32mGcT V a l G l u G l n H i s llu Fro G l n Leu Leu zhr leu V a l Fro m G l y np Asp Ala (31, 46), nor do other parts of PSP-A share homology with of the genes coding forthese two proteins 1 0 8 2 ~ A ~ A o 2 ~ w G c c ~ ~ c I c . ~ ~ A o 2 A T G r r c PSP-B. p G c ~ mExpression ~ Hi6 zhr llu Cys G l n Ala Leu G l y V a l Cp G l y zhr MET Ser Ser F m Leu G l n 3 7 4 mayalso be regulated by different mechanisms. The gene IGTm~pu3mG42crrTGl\n;pGApc~" coding for the PSP-A protein contains a binding sequence for CpIleHisSermAepLeu the glucocorticoid receptor (46). It has recentlybeen demon1197 CIGAGpI0;GG CPCPGGGPCC A!mxGKm GGcpcmrrc cIwmxsm strated that the rate of accumulation of PSP-A mRNA is " " " increased by cortisol (47). Analysis of the DNAsequence of a 1317 C&XX@X C f X U C E I GIzucccITG TPXVDXZ PSP-B genomic clone 220 nucleotides upstream of the transcpcAAc(acA""GcAcpcoGAG lation initiation codon reveals neither a binding site for the glucocorticoid receptor nor any obvious homology with the 1 4 3 7 GW&XAWT TWXUXtX ?DDXAO3 OjAOCRCCA corresponding sequence of the PSP-Agene (data notshown). cKzAAAAAc""OGCCTCACRC Phizackerley et ul. (18) first suggested theexistence of 1 5 5 7 ccAcoaxaT WACXUSG AGW3WXI GcARTIcAAI\ multiple low molecular mass surfactant proteins. More re" " " cently, two other groups have alluded to the presence of two 1677 AorraAaxip GuLcAcpGcA OGcIcIGCcA distinct amino termini in organic extractable surfactant pro?WmxaTGcan ;oorcc" teins (10, 11).PSP-B has an aminoacid composition consist1 7 9 7 n%pITGma ing of nearly80% hydrophobicresidues. These values are comparable t o thosereported forbovine andcanine low " " " molecular mass protein samples (8, 9). The amino acid se1917 GGAOCCATIC C M Z G X E C AaWZWXA quence of PSP-B is different,however, from that obtainedfor "" m m rA x ln bovine PSP-B (22). It is not clearwhich of these proteins are FIG. 1. Sequence of human PSP-B cDNA clone 17 and declinically. Optimal surfacduced amino acid sequence. The deduced amino acid sequence of in the surfactant preparations used PSP-B is shown below the DNA sequence.The amino-terminal amino tant preparations may require both the 35-kDa and 6-kDa acid sequence determined by Edman degradation is underlined. The proteins, each of which may confer different surface activity boxed amino acids represent a potential site of N-linked glycosylation properties (48). T h e cDNA clone described here should be (Asn-X-Ser/Thr where X is any amino acid). useful in the study of PSP-B synthesis and regulation and facilitate studieson the role of PSP-B in surfactant function. by hybridization a human lung mRNA which upon i n vitro translation yields a protein of the predicted molecular mass Acknowledgments-We thank E. L. Brown for suggesting 2'-deox(-42 kDa). The same protein is observed when human lung ynebularine as a universal base for oligonucleotide probes, J. Brown RNA is translated and the translation products are immuno- and D. Van Stone for oligonucleotide synthesis, K. Kelleherfor DNA clone is sequencing, and L. Sultzman for the cDNA library. precipitated (Fig. 2, lune C), orwhenthecDNA expressed following transfection into COS cells (Fig. 2, lune F).The antiserum used herewas made to bovine, low molecD. Phelps, unpublished observations. mmmm~anGGcAmcxrGccnxmmm~mGcc~
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9810
cDNA Clones Encoding Pulimonary Surfactant Proteins
REFERENCES 1. Avery, M.E., and Mead, J. (1959) Am. J. Dis. Child. 97, 517523 2. Fujiwara, T., Maeta, H., Chida, S., Morita, T., Watabe, Y., and Abe, T. (1980) Lancet 1,55-59 3. Enhorning, G., Shennan, A., Possmayer, F., Dunn, M., Chen, C. P., and Milligan, J. (1985) Pediatrics 76, 145-153 4. Kwong,M. S., Egan, E. A., Notter, R. H., and Shapiro, D.L. (1985) Pediatrics 76,585-592 5. Gitlin, J. D., Soll, R. F., Parad, R. B., Horbar, H. D., Feldman, H. A., Lucey, J. F., and Taeusch, H. W. (1987) Pediatrics 79, 31-37 6. Tanaka, Y., Takei, T., and Kanazawa, Y. (1983) Chem. Pharm. Bull. (Tokyo) 31,4100-4109 7. Taeusch, H.W., Keough, K.M.W., Williams, M., Slavin, R., Steele, E., Lee, A. S., Phelps, D. S., Keriel, J., Floros, J., and Avery, M. E. (1986) Pediatrics 77,572-581 8. Takahashi, A., and Fujiwara, T. (1986) Biochem.Biophys.Res. Commun. 136,527-532 9. Whitsett, J. A., Ohning, B. L., Ross, G., Meuth, J., Weaver, T., Holm, B. A,, Shapiro, D. L., and Notter, R. H. (1986) Pediatr. Res. 20,460-467 10. Yu, S-H., and Possmayer, F. (1986) Biochem. J . 236, 85-89 11. Whitsett, J . A., Hull, W. M., Ohning, B., Ross, G., and Weaver, T. E. (1986) Pediatr. Res. 2 0 , 744-749 12. King, R. J., Klass, D. J., Gikas, E. G., and Clements, J. A. (1973) Am. J. Physiol. 224,788-795 13. Phelps, D. S., Taeusch, H. W., Jr., Benson, B., and Hawgood, S. (1984) Biochim. Biophys. Acta79 1,226-238 14. Phelps, D. S.,and Taeusch, H. W. (1985) Comp. Biochem. Physiol. 82B, 441-446 15. Whitsett, J. A., Hull, W., Ross, G., and Weaver, T. (1985) Pediatr. Res. 19,501-508 16. Wright, J. R., Benson, B. J., Williams, M.C., Goerke, J., and Clements, J. A. (1984) Biochim. Biophys. Acta7 9 1 , 320-332 17. Katyal, S. L., and Singh, G. (1981) Biochim. Biophys. Acta 6 7 0 , 323-331 18. Phizackerley, P. J. R., Town, M-H., and Newman, G. E. (1979) Biochem. J. 183, 731-736 19. Suzuki, Y. (1982) J. Lipid Res. 2 3 , 62-69 20. Suzuki, Y., Nakai, E., and Ohkawa, K. (1982) J. Lipid Res. 2 3 , 53-61 21. Ng, V. L., Herndon, V. L., Mendelson, C. R., and Snyder, J. M. (1983) Biochim. Biophys. Acta 754, 218-226 22. Phelps, D. S., Smith, L. M., and Taeusch, H. W. (1987) Am. Rev. Respir. Dis., in press 23. King, R. J., Carmichael, M.C., and Horowitz, P. M. (1983) J. Biol. Chem. 2 5 8 , 10672-10680
24. Benson, B. J., Hawgood, S., and Williams, M.C. (1984) Exp. Lung Res. 6,223-236 25. Hawgood, S., Benson, B. J., and Hamilton, R. L., Jr. (1985) Biochemistry 24,184-190 26. Katyal, S. L., Amenta, J. S., Singh, G., and Silverman, J. A. (1984) Am. J. Obstet. Gynecol. 148, 48-53 27. Shelley, S. A., Balis, J. U., Paciga, J. E., Knuppel, R. A., Ruffolo, E. H., and Bouis, P. J. (1982) Am. J . Obstet.Gynecol. 1 4 4 , 224-228 28. King, R. J., Ruch, J., Gikas, E. G., Platzker, A. C. G., and Creasy, R. K. (1975) J. Appl. Physiol. 39, 735-741 29. King, R. J., Martin, H., Mitts, D., and Holmstrom, F. M. (1977) J. Appl. Physiol. Respir. Enuiron. ExercisePhysiol. 48, 483491 30. Tanaka, Y., Takei, T., Aiba, T., Masuda, K., Kiuchi, A., and Fujiwara, T. (1986) J . Lipid Res. 27,475-485 31. Floros, J., Phelps, D. S., Taeusch, H. W., Steinbrink, R., Recny, R., Kriz, R., Sultzman, L., Jones, S., Jacobs, K., and Fritsch, E. (1986) J. Biol. Chem. 2 6 1 , 9029-9033 32. Laemmli, U. K. (1970) Nature 2 2 7 , 680-685 33. Hunkapiller, M. W., and Hood, L. E. (1983) Methods Enzymol. 9 1,486-493 34. Jacobs, K., Shoemaker, C., Rudersdorf, R., Neill, S. D., Kaufman, R. J., Mufson, A., Seehra, J., Jones, S. S., Hewick, R., Fritsch, E. F., Kawakita, J., Shimizu, T., and Miyake, T. (1985) Nature 313,806-810 35. Wood, W. I., Gitschier, J., Lasky, L. A., and Lawn, R. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1585-1588 36. Vieira, J., and Messing, J. (1982) Gene (Amst.) 1 9 , 259-268 37. Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,5436-5467 38. Kafatos, F., Jones, W. C., and Efstratiadis, A. (1979) Nucleic Acids Res. 7, 1541-1552 39, Toole, J. J., Pittman, D. D., Orr, E. C., Murtha, P., Wasley, L. C., and Kaufman, R. J. (1986) Proc. N ~ t lAcad. . Sci. U. S. A. 83,5939-5942 40. Gluzman, Y. (1981) Cell 23, 175-182 41. Kaufman, R. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,689-693 42. Schlesinger, M. J. (1981) Annu. Rev. Biochem. 50, 193-206 43. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 1 5 7 , 105-132 44. Heijne, G . (1985) Nucleic Acids Res. 14,4693-4690 45. Nicholas, J. G., and Locker, J. E. (1985) EMBO J. 4, 3519-3524 46. White, R. T., Damm, D., Miller, J., Spratt, K., Schilling, J., Hawgood, S., Benson, B., and Cordell, B. (1985) Nature 3 1 7 , 361-363 47. Mendelson, C.R., Chen, C., Boggaram, V., Zacharias, C., and Snyder, J. M. (1986) J. Biol. Chem. 261,9938-9943 48. Possmayer, F., Shou-Hwa, Y., Weber, J. M., and Harding, P. G. R. (1984) Can. J . Biochem. Cell Biol. 6 2 , 1121-1133
cDNA Clones Encoding Pulmonary Surfactant Proteins ISOlatiOn
Of CDNA Clone encodinga high mOleNlar weight preNrllOe to a 6kDa pulmonary surfactant-associated protein
-
Kenneth A. Jacobs, David 8 . Phelps, Randal Steinbrink, Judith Fimch,Ronald Kriz, Lisa Kitsock, JosephP.mugherty, 8. William TaBUmch,and Joanna Florom
n and characr.rirotion
v
of the L o w m e c u l a r W
S u b ~ ~ ~ O n t l yDNA , from two different Cl0n.a Wae subcloned into X13 for DNA mequence analysis. Thass clone- were c0mP10tely sequenced by generating an ordered mat of dsletiona with Bal 31 nuclease, rasloning into othsr 1113 vectors and Saquencing via the dideOXYnUcleOtide chain termination procedure (36,37). one clone corresponded to a full-length copy of tho type referred to as 17 (Figure l), the second began at nucleotide 148 of clone 17. The clones were identical throughout tho coding region. In the non-coding region, the only diffsrenose were a substitution of a G for the T at position 1 2 9 5 and a G for the C at position 1307. me latter substitutioncreates a ainf I sits. sequence of t h e 5 ' end of a third clone conrimed the sequence or the 5 ' end of clone 17. and I-"
Pulmonary lavage ( 5 0 m1) from an alveolar proteinomis patiant was centrifuged at 10.000 x g for 5 Din. The pallet was colleoted andwarnhad 5 times in 20 . I ITrisRCl.pH7.4, 0.5 M Hacl. to reduce the content of nonBurfactant protsina. The lipid* and lipid-a880Eiated proteins Were extracted f r o m the waahed pelletby shaking with50 ml 1-butanol for 1 h at room tamperatura. The butanol-ineolubla material yielded the PSP-A proteins previously described and cloned (31). The butanol soluble proteins were fractionated by cryoprecipitation. Storage Of the butanol extract at -20°C precipitated a previously Soluble protein. The precipitate was collected by centrifugation and dried under vacuum. The cold-butanol insoluble protein, which we refer to as PSP-B, was then diasolvedin Eh1orOfom:methanol (2:1, v/v), applied to a Sephadex U l 2 0 C O l W and eluted with Chlorof0rm:mBthanOl (2:l). The Column effluent Was monitored at 2 8 0 Dm, collected in fractions and analyzed by sodium dodecyl IIUlfate polyacrylamide gel electrophoresis (SDS-PAGE) (32). Protein containing fractions were pooled and evaporated to dryness for subsequent analysis.
translated
ind
A full-length =DNA clone (fig. 1) was cloned into the E.C.QLI site of COS orpression vector p m 2 (39). cos cells ( 4 0 ) were trsnnfectsd with 10 lg par 10 cm dish of either p m 2 or this vector containing a =DNA clone in 4 a1 Of DEAE-dextran Dultecco's modified Eagle's medium (41). Forty-sight hours post-transfection the aalle were labeled with (35s]methionina (0.5mCi/ml:spe~iric activity r800Ci/mmol) in som-fzee medium. Fifteen m i n l a t e r t h e m e d i u m wa- removed, and the cell- were lysed in immunoprecipitation buffer (31). Tha Samples Were immunopracipitated and analyzed by SDS-PAGE am dsasribad abcve.
D E
F G
Phe-Pro-Il~-Pro-L.u-Pro-Tyr-?-Trp-L.u-7-7-Al~-~u.
Pornitions denoted
5 of-the c
bya (7) indicate unidentified residues.
MW 2o
of ma-
Bamed on the first six amino acid. of th.s.qu.ncs shown above 380; oligonucleotide probeswere synthemized on an Applied Biosystsla Model On. set of probes consisted of eix pols of 17m.r. DNA synthesizer. divided according to tha major and minor leucinecodons, and including ali the coding possibilitisa. Three of the p o d s each omtained 128 different
piague purified, pooled 'into groups of 2 5 and screened wit6 ti& individual 17mer and 20mer pools in the same solvent. six phage which hybridized most intensely to one Of the 2"deoxynabularina containing 20mBT. OligOnUCleOtide probes andone of the corresponding 1 7 m r pools (pool 1447 containing 128 different sequences) were chosen. Thia 17mez pool was then divided into four pools of 32 different BBquOIICeS and hybridized to a dot blot of DNA prepared from these phage. Based on the hybridization intensity, DNA from one of these six phage was subcloned into 1113 for sequence analysis. A sequence corresponding to the m i n o acid sequence given above w a ~Obtained, confirming that the imolated clone aodsd for pulmonary swfaotant protein B (PSP-B). were
v
analyzed also as previbusly-described.
h i n o acid composition(data not ehan) warn detarmined by hydrolysis in6 N HC1 at llOoc for 22 h follarad by chrmtography on a Backman model6300 amino acid analyzer. N-terminal sequence Was determined on an Applied The phenylthiohydantoin amino acids were Biosylitem8 470A ssqu.ncer. The N-terminal analyzed by the method of Hunkapiller and Hood ( 3 3 ) . peptide sequence of the the cold-butanol, inmolubls proteinWas detamined to be:
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9811
14
€JLEL I ~ u n o P ~ i P i t a t etranslation d products of h-n lung RNA using against the low molecular Weight preilaule serum [lane C) or an anti.*The kunopracipitation in lane E was eurfactant proteins (lanos D,E). carried out in the preasanu of unlabeled lov molecular weight surfaotant proteins. F,G: Immunopracipitated intracellular proteins from cos cell containing a EDNA clone coding tranaractions of a recombinant pla-id for the 42 kDa Pr.NrmOr (lane F) or the vector alone (lane G)
.
3J.P. mugherty et al, in preparation.
