Jul 5, 2006 - Michael Recnyll, Lisa Sultzmanv, Simon Jonesll, H. William TaeuschS, ... Sueishi and Benson, 1981; King et al., 1973; Katyal and Singh,. 1981 ...
Vol. 261, No. 19, Issue of July 5,pp. 9029-9033,1986 Printed in U.S.A.
THEJOURNAL OF BIOLOGtCAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.
Isolation and Characterization ofcDNA Clones forthe 35-kDa Pulmonary Surfactant-associated Protein* (Received for publication, November 15,1985)
Joanna FlorosSS, Randal Steinbrinkll, Kenneth Jacobsll, David PhelpsS, Ronald Krizll, Michael Recnyll, Lisa Sultzmanv, Simon Jonesll, H. William TaeuschS, HowardA. Frank11 , and Edward F. Fritschll From the $Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, VGenetics Institute, Inc., Cambridge,Massachusetts 02140-2387, and the IlDepartment of Surgery, Harvard Medical School, Boston, Massachusetts 02115
A group of 35,000-dalton sialoglycoproteins is the lavage of an alveolar proteinosis patient, sequenced portions major non-serum protein component of pulmonary sur- of the proteins, and subsequently isolated the corresponding factant. Tryptic fragments ofthese proteins were se- cDNA clones. Clones corresponding to two different 35-kDa quenced, and oligonucleotide probes were synthesized translation products were identified. By in vitro transcriptionbasedontheaminoacidsequences.A human lung translation experiments, we show that these clones code for cDNA librarywas then screenedusing the oligonucle- proteins of 29 and 31 kDa. This is consistent with our earlier otide probes, and clones coding for these proteins were results identifying proteins of these molecular weights as the identified and characterized. By in vitro transcription- primary translation products in normal human lung tissue translation experimentswe have associated individual (Floros et al., 1985a). clones with particular proteins. The data suggest that co-translational modifications of two primary transEXPERIMENTAL PROCEDURES~ lation products account for many of the isoforms observed by two-dimensional gel electrophoresis in the RESULTS AND DISCUSSION precursors of35,000-dalton sialoglycoproteins. Using an enriched source (lavage material from alveolar proteinosis patients), we were able to purify PSP-A proteins on a C4 reverse phase high performance liquid chromatograSurface tension in the alveoli of the lung is lowered by a phy column. A peak containing predominately these proteins lipoprotein complex called pulmonary surfactant. This com- and a very small amount of the 60-kDa proteins (as detected plex consists of phospholipid and 5 1 0 % protein (King, 1982). by SDS-polyacrylamide gel electrophoresis analysis) was alThe protein fraction of the surfactant is composed of non- kylated and trypsinized. The tryptic fragments were chroserum and serum proteins. The major non-serum surfactant- matographed on a C4 reverse phase high performance liquid associated proteins are 35,000-dalton sialoglycoproteins chromatography column, and the eluted fragmentswere sub(PSP-A’) (Shelley et al., 1982; Bhattacharyya et al., 1975; jected to NH2-terminal Edman degradation. The amino acid Sueishi and Benson, 1981; King et al., 1973; Katyal and Singh, sequence of fragments T19, T26, and T28 is shown in Table 1981; Phelps et al., 1984). These proteins may be important I. for the normal function of the pulmonary surfactant (Kinget Based on the amino acid sequence of the tryptic fragment al., 1983; Hawgood et al., 1985). They are present in reduced T28, an oligonucleotide probe was synthesized and used to amounts in amniotic fluid samples taken shortly before the screen a X g t l O cDNA library prepared from human lung birth of infants who subsequently develop respiratory distress mRNA (Toole et al., 1984; Jacobs et al., 1985). Between 0.5 syndrome (Katyal et al., 1984; Shelley et al., 1982; King et al., and 1%of the phage clones hybridized with this probe, which 1975). agrees with our previous observations on the abundance of The 35-kDa proteins also accumulate in the lungs of pamRNA coding for PSP-A proteins(Floros et al., 1985a).DNAs tients with alveolar proteinosis (Bhattacharyya and Lynn, from two clones (PSAP-1 and PSAP-2) were subcloned into 1978, 1980). These proteins have the same electrophoretic M13 for DNA sequence analysis, generating the clones mobility, immunological determinants, and peptide map as MPSAP-1A and MPSAP-6A (Floros et al., 1985b). the 35-kDa proteins isolated from normal human bronchoalBy using the oligonucleotide mixture of Pool I1 as a seveolar lavage material (Phelps et al., 1984; Whitsett et al., quencing primer, the nucleotide sequence corresponding to 1985). We isolated en masse the 35-kDa proteins from the tryptic fragment T26 was identified in both MPSAP-1A and * The costs of publication of this article were defrayed in part by MPSAP-6A, confirming that the isolated clones code for the the payment of page charges. This article must therefore be hereby 35-kDa proteins found in the lavage material of alveolar marked “advertisement” in accordance with 18 U.S.C. Section 1734 proteinosis patients (see above). Further DNA sequence analsolely to indicate this fact. § T o whomcorrespondence and reprint requests should be addressed Joint Program in Neonatology, Brigham and Women’sHospital, 75 Francis St., Boston, MA 02115. The abbreviationsused are: PSP-A, pulmonary surfactant protein A (35 kDa); PSAP, cDNA clone for the pulmonary surfactant protein A; SDS, sodiumdodecyl sulfate; DTT, dithiothreitol; HPLC, high performance liquid chromatography;PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focusing; PIPES, piperazine-N,N”bis(2ethanesulfonic acid); kb, kilobase.
’
Portions of this paper (including Experimental Procedures,Figs. 2-4, Table I, and Acknowledgments) are presented in miniprint at the end of this paper. Miniprint is easily readwith the aidof a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-3760, cite the authors, and include a check or money order for$3.60 per set of photocopies. Full size photocopies are also includedin the microfilm edition of the Journal that is available from WLhrerly Press.
9029
cDNA Clones Encoding Pulmonary Surfactant Proteins
9030 -90 1A 6A
-60
-75 C CGGA GACCCAAGCA GCTCGACGCT
.....
5'......
-45
"GTGTGm
-15
-30
X
G
GTCGCTGAGT 'ITCTXGAGC CTGAAAAGAA AGAGCAGCGA CTGGACCCAG
15
-1 1
Thr
30
-
r AGCC ATG TGG CTG TGC CCT CTG GCC CTC AAC CTC ATC 7TC ATG HET Tt-p Leu Cys Pro Leu A l a Leu Asn Leu I l e Leu MFP
60 75 45 GCA GCC TCT GGT GCT GCG TGC GAA Cn; AAG GAC G l T TGT G l T Glu V a l Lys Asp Val Cys Val Ser G l y Ala A l a Cys AlaAla 120 90 105 GGA AGC CCT GGT ATC CCC GGC ACT CCT GGA TCC CAC GGC CTC G l y Ser P r o Gly I l e Pro G l y Thr Pro Gly Ser His Gly Leu 150 165 Va 1 G CCA GGC AGG GAC GGG AGA GAT GGT CTC AAA GCA GAC CCT GGC Fro G l y Arg Asp Gly Arg Asp G l y Leu Lys Gly Asp Pro Gly
135
-
195
180
Thr CA CCT CCA GGC CCC ATG GGT CCG CCT GGA GAA ATC CCA TCT CCT Pro Fro G l y Pro ME3 Gly Pro P r o Gly Glu HET P r o Cys Pro
240
225
210
Asn
Va1
G A G CCT GGA AAT GAT GGG CTC CCT GGA GCC CCT GGT ATC CCT GGA
P r o G l y Asn Asp Gly Leu Pro Gly Ala Pro Gly I l e Pro Gly 255
285
270
At-9
c
A
GAG TGT GGA GAG AAG GGC GAG CCT GCC GAG AGG GCC CCT CCA G l u Cys G l y C1u Lys G l y G l u Pro Gly G l u Acg Gly Pro Pro
330 300 315 GGC CTT CCA GCT CAT CTA GAT GAG GAG CTC CAA GCC ACA CTC
G l y Leu Pro Ala His Leu Asp Glu Glu Leu Gln Ala Thr Leu 360
345
375
C
TI" AGA CAT CAA ATC CTG CAG ACA AGC CCA GCC CTC His Asp Phe Arg His G l n I l e Leu Gln T h r Arg Gly A l a Leu CAC GAC
390 405 A ACT CTG CAG GGC TCC ATA ATG ACG GTA GGA GAG AAG GTC l T C S e r Leu Gln G l y Ser I l e MET Thr V a l G l y Glu Lys Val Phe
420 435 450 TCC AGC AAT GGG CAG TCC ATC ACT TIT GAT GCC A T T CAG GAG Ser S e r Asn G l y G l n Ser I l e Thr Phe Asp Ala I l e G l n G l u 495
480 465 GCA TGT GCC AGA GCA GGC GGC CGC A T T CCT G W CCA AGG AAT
Ala Cys Ala ArgAla
Gly Gly
A r q Ile Ala V a l Pro A r g k
525 540 510 CCA GAG G M AAT GAG GCC ATT GCA AGC TM: GTG AAG AAG TAC P r o G l u G l u Asn G l u Ala I l e Ala Ser Phe V a l Lys Lys Tyr
585 555 570 AAC ACA TAT GCC TAT GTA GGC CTG ACT GAG GGT CCC AGC CCT Asn Thr Tyr Ala Tyr Val Gly Leu Thr Glu Gly Pro Ser Pro
600 615 T GGA GAC T T C CGC TAC TCA GAC GGG ACC CCI' Gly Asp Phe Arg Tyr Ser Asp Gly T h r P r o 630
660
645
T AAC TGG TAC CGA GGG GAG CCC GCA GGT CGG GGA AAA GAG CAG Asn Trp Tyr Arg Gly Glu Pro A l a Gly A r q Gly Lys G l u Gln 705 675 690 TGT G T C GAG ATG TAC ACA GAT GGG CAG TGG AAT GAC AGG AAC Cys Val G l u M E T Tyr Thr Asp Gly Gln T r p Asn Asp Arg Asn 750 720 735 TGC CTG TAC TCC CGA CTG ACC ATC TGT GAG T E TGA GAGGCAT Ser T h r G l u Leu T y r Leu I l e Phe Cys Arg Cys 795 780 ." T C G A T P C ?TA,GGCCATGGGA CACGGAGGAC GCTCTCTGCC CTlTGGCCTC CATCCT
765
R10 825 840 C A GAGG CTCCACTIGG TCTGTCAGAT GCTAGAACTC C C " T K A A C A . . . . . 3 '
FIG. 1. Coding nucleotide sequence of MPSAP-1A and MPSAP-6A cDNAsand deduced amino acid sequence of clone
ysis identified the remaining major tryptic fragment (T19) in Table I, and confirmed that thetwo clones were highlysimilar but not identical (Fig. 1).Within the coding region, the two clones differ at seven nucleotides which lead to six amino acid changes and a t six nucleotides that do not change the encoded amino acid. Additional DNA sequencing of clone 1A at the3' end revealed a large untranslated region of about 1.3 kilobases. Clone 6A also has a large untranslated region at the 3' end. Comparison of the first 100 nucleotides of the 3' untranslated regions of clones MPSAP-1A and MPSAP-6A shows more variation (9%) than is found within the coding region (1.3%). The genomic clone for PSP-A reported by White et al. (1985) is similar to our 6A cDNA clone (Fig. l ) , and differences between the genomic and the 6A cDNA sequences are noted. Our sequence data show an extrauntranslated exon of 31 nucleotides (or 30 nucleotides in the genomic clone; one nucleotide is missing in this sequence compared to the 6A data). Thisexon in Fig. 1 is between nucleotide position -23 and -54 in 6A and should be between nucleotide position 535 and 565 in the genomic sequence presented by White et al. (1985). Comparison of the sequence data of the coding region for the 6A cDNA clone (Fig. 1)and the genomic clone shows seven nucleotide differences. Four of these differences result in three amino acid changes, alanine to valine, asparagine to histidine, and proline to leucine at nucleotide positions 56, 133,161, and 162, respectively (Fig. 1).Three of the nucleotide differences at positions 186,399, and 609 (Fig. 1) do not change the amino acid. Comparison of the coding sequences of the genomic clone in White et al. (1985) and coding sequences of the cDNA clone 1A results in 18 nucleotide differences with nine amino acid changes (as it can be deduced from the datain Fig. 1).Other nucleotide differences between the cDNA 6A or 1A sequence and the genomic sequence are also observed at their 5' and 3' end untranslated regions. Comparison of the first100 nucleotides of the 3' untranslated region of clones 1A and 6A with the genomic sequence shows more variation, 15 and 6%,respectively, than is found within the coding region (2.4% for 1A and 0.9% for 6A). Sequence analysis of both clones reveals an extensive homology with collagen as determined by the presence of 23 Gly-X-Y repeats between nucleotide positions 82-300 (Fig. 1) and described by White et al. (1985). Both clones also contain one possible glycosylation site, Asn-X-Thr, starting at nucleotide position 619 (Fig. l). This finding supports earlier observations in which each isoform from the human PSP-A primary translation products yielded (upon in uitro glycosylation) a single higher molecular weight isoform (Floros et al., 1985a). These data differ from results obtained from comparable experiments with rat in vitro glycosylated PSP-A. In this case each primary translation isoform yielded two higher molecular weight isoforms (Floros et al., 1986). We infer that the rat PSP-A protein has glycosylation sites in addition to the one identified in the human PSP-A cDNA sequence. The physiological significance of interspecies glycosylation differences is unclear. As shown in Fig. 2, the MPSAP-lasubclone selects human lung mRNA which upon in vitro translation results in two proteins at about 29-31 kDa (lane A3). This doublet is absent when MPSAP-lb (the orientation unable to hybridize PSPA mRNA) is used to select mRNA for translation (lane A4). MpSAp-GA. Differences in nucleotide and amino acid sequence Of clone MPSAP-1A are noted above the sequence of MPSAP-6A. Numbering of nucleotides is above the nucleotide sequence. The three tryptic peptide (T19, T26, T28) sequences by Edman degradation are underlined. The boxed amino acids represent a potential site of Nlinked glycosylation (Asn-X-Thr) where X is any amino acid.
cDNA Clones Encoding Pulmonary Surfactant Proteins These proteins (lane 3, Fig. 2) migrate at the same position as the immunoprecipitated 29- and 31-kDa proteins from translation of human lung RNA (lane A5). They are also immunoprecipitable (lane A6, Fig. 2) by antiserum that recognizes the natural 35-kDa surfactant proteins (Phelpset al., 1984). In addition, the immunoprecipitated products from either the hybrid translation selection (Fig. 2, panel B ) or from human lung RNA (Fig. 2, panel C ) appear identicalwhen analyzed by two-dimensional gel electrophoresis. When one of the M13 clones was used in a Northernanalysis of human lung RNA, one mRNA species of about 2.5 kilobases was detected (Fig. 3). To associate clones MPSAP-1A and MPSAP-6Awith particular proteins, the 5' EcoRI fragment of each clone, which included the entire coding sequence as shown in Fig. 1, was then subcloned into vector SP65 togenerate clones PSP-35k1A and PSP-35k-6A. RNA was prepared by transcription with SP6 RNA polymerase, translated, and analyzed by gel electrophoresis. Fig. 4 (lanes 2 and 3) shows the translation products of PSP-35k-6A and PSP-35k-lA, respectively, and the translation products from both clones (lane I). Since the DNA sequence predicts that the proteins encoded by clones 1A and 6A are indistinguishable by an SDS gel analysis (a molecular mass difference of only 16 daltons), itis surprising that the translated products have different mobilities. Both clones contain two in-frame methioninesat theNH2 terminus at nucleotide positions 1 and 37 (Fig. 1). To determine whether both clones start from the same methionine, in vitro synthesized RNA from each clone was translated using the metabolic trap technique (see below) in the presence of [3H] leucine and [35S]methionine.The translation products were purified by gel electrophoresis. The protein bands were identified by autoradiography, excised, eluted by equilibrium in 0.1 M NH4HC03, 0.02% SDS, and the NH, terminus was partially sequenced by Edman degradation. Scintillation counting of each residue of both proteins showed 35Scounts in cycles 1 and 13 and3H counts in cycles 3, 6, 8, 10, and 12, as expected by the predicted amino acid sequence (Fig. 1) of clones 1A and 6A. The data showed that both clones start from the firstmethionine. It is not clear at thispoint why the two primary translation productsderived from two non-identical but similar size cDNA clones migrate with different apparent mobilities on SDS gels. Perhaps amino acid differences between the two proteins may cause conformational changes that are reflected by differences in mobility. Panel B of Fig. 4 shows the two-dimensional gel analysis of the invitro transcription-translation products. Eachband (Fig. 4A) is resolved into more than one isoform (Fig. 4B), raising a question about the nature of these isoforms. In our laboratory we have found that theNH, terminus of the PSPA is b10cked.~N-acetylation, which appears to occur during translation (Pestana and Pitot, 1975), can result in blocking of the NHZ terminus (Rubenstein and Deuchler, 1979) and hence proteins with different isoelectric points. To investigate whether the N-acetylation contributes to the origin of some of the isoforms seen in Fig. 4B, we used the metabolic trap technique (Palmiter, 1977) to prevent NHz-terminal acetylation of PSP-A primary translation products. Oxaloacetate and citrate synthase were included during translation as deR. Steinbrink and M. Recny, unpublished observations.
9031
scribed by Palmiter (1983).Under these conditions the acetylCoA is used for the synthesis of citrate, and the pool of free acetyl-coA that could contribute to the NHz-terminal acetylation of proteins is reduced. The results of this experiment are shown in Fig. 4C. The basic isoform for each molecular weight group is increased (arrow) and the acidic isoforms decreased, a finding that indicates acetylation is contributing to the pattern of isoforms seen by two-dimensional analysis of the primary translation products. In conclusion, we have identified cDNA clones from a human lung library that code for two PSP-A proteins. We have sequenced these clones and found them to be similar but not identical (Fig. 1).The hybrid selected translation products and the translation products from in uitro synthesized RNA are identical to previously characterized immunoprecipitated translation products from human lung tissue (Floros et al., 1985a). N-acetylation duringin vitro translation accounts for some of the multiple isoforms seen in previous experiments. REFERENCES Bhattacharyya, S. N., and Lynn, W. S. (1978) Biochim. Biophys. Acta 5 3 7 , 329-335 Bhattacharyya, S. N., and Lynn, W. S. (1980) Biochim. Biophys. Acta 6 2 5 , 451-458 Bhattacha a, S. N., Passero, M.A., DiAugustine, R. P., and Lynn, W.D. (1975) J y l i n . Inuest. 55,914-920 Boedtker, H. (1971) Biochim. Biophys. Acta 240,448-453 Floros, J., Phelps, D. S., and Taeusch, H. W. (1985a) J. Biol. Chem. 260,495500 Floros, J., Phelps, D. S., Taeusch, W., Steinbrink, R., Recny, M., Kriz, R., Sultzman, L., Jones, S., Jacobs, K., and Fritsch, E. (1985b) J. Cell Biol. 101,
_" _
All%
Floros, J., Phelps, D. S., Kourembanas, S., and Taeusch, W. H. (1986) J. Biol. Chem. 261,828-831 Hawgood, S., Benson, B. J., and Hamilton, R. L.,Jr. (1985) Biochemistry 24, .~ 184-190 Hunkapiller, M. W., and Hood, L.E. (1983) Methods Enzymol. 91,486-493 Jacobs, K., Shoemaker, C., Rudersdorf, R., Neil, 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 Kafatos, F.,Jones, W. C., and Efstratiadis, A. (1979) Nucleic Acid Res. 7,15411552 Katyal, S. L., and Singh, G. (1981) Biochim. Biophys. Acta 670,323-331 Katyal, S. L., Amenta, J. S., Singh, G., and Silverman, J. A. (1984) Am. J. Obstet. Gynecol. 148,48-53 King, R. J. (1982) J. Appl. Physiol. Respir. Enuiron. Exercise Physiol. 6 3 , l - 8 King, R. J., Klass, D. J., Gikas, E. G., and Clements, J. A. (1973) Am. J. Physiol. 224,77a795 King, R. J., Ruch, J., Gikas, E. G., Platzker, A. C. G., and Creasy, R. K. (1975) J. Appl. Physiol. 39,735-741 King, R. J., Carmichael, M. C., and Horowitz, P. M. (1983) J. Biol. Chem. 2 5 8 , 10672-10680 Laemmli, U.K. (1970) Nature 227,680-685 Lehrach, H., Diamond, D., Wozney,J. M., and Boedtker, H. (1977)Biochemistry 16.4743-4751 Melton, D. A., Krieg, P. A., Rebagliati, M.R., Maniatis, T., Zinn, K., and Green, M. R. (1984) Nucleic Acids Res. 12,7035-7056 Miller, J. S., Paterson, B. M., Ricciardi, R. P., Cohen, L., and Roberts, B. E. (1983) Methods Enzymol. 101,650-674 Palmlter. R. D. (1977) J. Biol. Chem. .. .. 262.8781-878.1 Palmiter; R. D. (1983) Methods ~nzyGl-i)B;15O-.ii7 Pestana, A., and Pitot, H.C. (1975) Biochemistry 14,1397-1403 Phelps, D. S., Taeusch, W. H., Benson, B., and Hawgood, S. (1984) Biochim. Biophys. Acta 791,22&238 Rubenstein, P., and Deuchler, J. (1979) J. Biol. Chem. 254,11142-11147 Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,5436-5467 Shelley, S. A., Balis, J. U., Paciga, J. E., Knuppel, R. A., Ruffolo, E. H., and Bouis, P. J. (1982) Am. J. Obstet. Gynecol. 144,224-228 Sigrist, H., Sigrist-Nelson, K., and Gither, G. (1977) Biochern. Biophys. Res. Commun. 74,178-184 Sueishi, K., and Benson, B. J. (1981) Biochim. Biophys. Acta 6 6 5 , 442-453 Toole, J. J., Knopf, J. L., Wozney, J. M., Sultzman, L. A., Buecker, J. L., Pittman, D. D., Kaufman, R. J., Brown, E., Shoemaker, D., Orr, E. C., Amphlett, G. W.,Foster, W. B., Coe, M. L. Knutson, G. J., Gass, D. N., and Hewick, R. M. (1984) Nature 312,342-343 Vieira, J., and Messing, J. (1982) Gene (Amst.) 19,259-268 White, R. T., Damm, D., Miller, J., Spratt, K., Schilling, J., Hawgood, S., Benson, B., and Cordell, B. (1985) Nature 317,361-363 Whitsett, J. A., Hull, W., Ross, G., and Weaver, T. (1985) Pediotr. Res. 19, 501-508 I
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9032
cDNA Clones Encoding Pulmonary Surfactant Proteins Isolation and characterization of cnNA dones for the 35kD8 pulmonarvsurfactantassociatedprotein (PSP-A)
RNA Blotting Total human lung RNA (15up) was denatured and analyzed on an aparose formaldehvde -1 (Lehrachet a1 1917. Rnedtker 19111 in the nresrnce of ethidiumhmmide of the rRNA (1RS and (0.5urlmI). At the termination of therlectroohor*sisthepositions 28s) andthe 10s plohin R N A mnrkers were noted.The RNA WRS thentransferredonto nitmEellulose m p e r and hvhridized to an MI3 prohe venerated from clone IA.
RV
Joanna Floms. R ~ n d a lSteinhrink.Kenneth Jacobs. David Phelpr Ronald K r i z , Michael Rennv. Lisa Sultzmnn. Simon Jones H. lvillim Taewch. Howard A . Frank. and E d w w d F. Fritsch
SuhcloninK into SP65 vector and trmscriptian of the SP65 recomhinsnts
Exnerimentnl p r w e d u r e s The 5 ' EmRl fraflent COntaininF theentirecoding scouence of proteins 1A and 6A w88 subclonedinto SP65 (Melton e t SI, 1984) eeneratinrr elones PSP-35K-6A and PSP-35k-lA. The NH+rminus i-unitto nucleotide+57) and the COOH-terminus (fmm
Isolation of 35kDa and 60kDa Droteins l0.00Oxp. andthe Lavape from an ~ I V M I R F pmteinosis ostient W R S centrifupedat pH 7.4.Lipids W ~ P Pextrnctad with pellet was washed 5 times with 20mM Tris, 0.5M NRCI n-butanol (Sieriat et 81.. 1917). The hutmol insoluhlr material was Collected hv rentrifupation.dissolvedin 50mM sodium phosphatebuffer (pH 6 ) . 6.OM Gunnidinr HCI (GnHCI) and applied to a Vvdac C4 R ~ V B ~ Rnhsse C HP1.C column (4.6 x 250mm). The protein pmk elutinp st approximately 50% acetonitrile Cl13CN:N-Prooanol (?:I1 ( V I V I with 0.18 Trifluoreceticacid(TFA) was collected and Ivophilired. hv SDS-PAGE (Lnemmli. 1910).
Theproteins
were nnalvzed
nucleotide 504 totho .ston codon) ofthese clones were Peouenc-d hv usinp specific oligonuclmtides as sequencinp primers and run on sequencinp pels side hv side to eliminate any artifacts d u r i n ~ Subcloning into SP65 vector. Clones in the cmreet orientation W P ~ C linearized with Smal andtranscribed with SP6 RNA polvmernse aceordine tothe recommendations of the r-ier TechnicalBulletin K m b e r 001 of Promeca Riotec w i n e transcriptionvrotorolnumber two end materialssupplied hv the companv.The in vitrosvnthesired R.NA we8 translated as described above for RNA purifirdbvhvhrGj selection.
Alkvlatian andTrvDtieMapnine Tnhle I
Theprotein(npprox. 5 0 ~ 4 )was takrn up and reduced in 1nnm11 Tris. ImM EDTA. 6M GnHCI. 20mM DTT. p R 8.5 at 37-C for 2h. Solid iodoacetsmide was addedto a final concentrationof 60mM and the reaction incubated at O°C for 2h under arpon in the dark. Thoreaction was stopoed nnd the reapents removed bv dialvais into 0.111 NR4HC03. 50mM was dimsted with trvpsin (3% trvDSin 2-mercsptoethanol, pH 7.5. Thealkvlstedprotein 31oC for 16h end chromatopraphed ovev a C4 V - ~ ~ RReverse C phase RPLC hvweipht)at column (4.6x250mm). Thetrvpticneptides were eluted with B linenrmadicnt of95% acetonitrilennd TFA. collected nnd subjected N-terminal to Edmm deeradation Usinr Applied-Riosvstems Flodel 470A pmtcin sequencer. The phenvlthiohvdantoin (PTH) amino erids were anslvred hv the method of Hunkapillsrand H w ~(1983). Screeninp of tho cDNA lihrnrv and sequencine
Sequence
Observed Peptide ~
TI9
Asp Val Cvs Val Glv Scr Rvp Glv Ile Hvp Clv T h r Hyp Glv S w His ClV Leu Pro CIV
T?6
0.1%
an
of elones
Rased on the amino acid sequenrr of t r v p t ifcr a p e n t T18. (Table 1) an oli-nucleotide prohe was prepared on an Applied Riosvatems Plodel 38OA DNA prohe consisted of four D W ~ S of 20mers. and esch pool contained svnthesizer. The 31 differentsequences.
Asn Pro Glu GIu Asn Glu AIR IIe A h (-) Phe VII h s
T28
Sequences of trvptie pentides derived from the 35kna and 6OkDa surfwtnnt Rssociated proteins.Thetrvpticpeptides were suhiactedto N-termins1 Edman depradation end the PTH-amino acids were Rnalvzed hv the method of Hunkanillerand HWd (19R3).
1
2
3
4
5
6
-lEF
7
A CDNA library from human lune mRNA !vas orepared BB descrihcd in Toole et a l . , (1984) and screened with the totnlmixture of the four nmls usine trtrsmethvlsmmonium chloride as a hvhridirationSolvent (.lRCOhS e t a l . . 1985). DNA from twomndomlypicked clones (PSAP-1 and PSAP-2) was subclonedinto M13 for DNA sequence nnnlvses. Roth clones wore Sequenced hvpenerRtinP an ordered Set of deletionswith Ral 31 ~ U C I E B S B . recloning intoother M13 vectorsandrequenrine via the dideoxvnurleotidechainterminatorprocedure(Viera and Wessine. 1982: Saneer Ct g.. 1917). One clone c o n t a i n 4 B complete CODV of themrtinp repion of the clone referred to as 1A. Roth Strandsof clone 1A containingtheentire codin!? SeqUenEI: andnonrodine sequences at the 5' and 3' end 8s shown in FiKuure 1 w r ~ esequenced in their entiwtv. Onlv one strand of theremaininp 3' untranslated repion of clone IA was srouenced.The as 6A. second clone (PSAP-2)contained an incomolotc F O ~ Vof thetvpelaterreferredto This done (PSAP-?I rontainedsequencesbetweennucleotidepositions 244 tothe 3' end (Fis. 1). Roth Strands of this incomnlete clone were seouenc'ed as well as described above. Rv usinF an olifKm.ucleotide prohe Specificfor PSAP-2 tvpe. a clone containinp the complete coding redon was identified (6A) and one strand was sequencedinits entiretvbv "sine specificolieonocleotides RS sequencinpprimers.The second strand of the 6A clone containinp the complete ?"dine repinn was also sequenced hv USinC sequencinp primem atthe 5 ' end (-89) uptonucleotidrposition +51 (Fie. 1 ) . Theentire cnNA of 1 A is about1.4kh as determined hv nucleotidesequence and hoth clones mieratc similarly as determined hv R ( I B I O S ~ eel electrophoresis.
-36
The selected RNA W B R eluted hy hoilinp for one minutein 300ul of ImM RDTA p H 1 . 9 and l0uK of calf liver tRNA (Roehrinqer- Mannheim). The Dmcipitated RNA was translated.thetransletionproducts immunoprecipitated mr? subiected to one and two dimensional pel electrophoresis 8% dsscrihed in Floms et a l . . 19858.
MW
MW
X
x10-3
45-
-36
36-29 29-
C A
DNA binding. hybrid selection and in vitm translation in The 0.9 Kb EcoRl frSqFlent from PSAP-1 W R S Suhcloned into 1113 hoth Sinele stranded DNA (10-15 orientations. qeneratinllclones MPSP-la and MPSP-lh. uelml) from eithersuhrlone PIPSAP-Is or MPSAP-lh was anpliedtonitrncellulosrpaper vacuum (Kafntos et SI.. 1919). Enrh filter (lorn') W R S rutinto nine (louvlcm2)under Each reaction equal pieces. and eachpiece was used in a 20-3Oul hvhridimtionreaction. contained human lung RNA (5mrlml). 50% deionized formnmidr (Floka AC. Chemical Corn.). lnmM PIPES l(Finerazine-N. N'-his) (2-ethanesulfonic 8cid)l p H 6 . 4 and 0.4C1 NsCl (Miller et 81.. 1983). The source and preparation of the RNA hnvebeen rBpOrtPd praviouslv (Flomset 111. 1985a). Each hyhridizstionrenctian W R S routinelv incubatedat 50OC for 3h. A t the e k l of the incubation periodeachfilter was washed for five minutes with Iml lxssc ( O . I S M N ~ C I ,0 . 0 1 5sodium ~ citrate). 0.5% SDS fivetimesat firm. Thenit WRS washed for five minutes with l m l of 2mll EDTA. pH 1 . 9 at 6O0C three times.
-29
66-
Fimrre 2 ~
One and two-dim*nSiOnd re1 eleftmphoresis of hrrbridSelection. A.
%
trm8lRtion PrOdllCtS from
~ , a n e1.
The endorrenoustranslation
2.
Thetotaltranslntionoroducts
4.
The total translation products of selected humm lung MPSAP-la and the MPSAP-lb cloned DNA reSPeCtiv4v
5.
The immunoprecipitated pmducts of human lune RNA usine our oolvclonal antiserum (Phelps. et a l . . 1984) thatrecomizesthe major surfectnntassociatedproteins.
7.
The immunoprarinitnted products from lsnes 3 same antiscrum as Lane 5.
3
6 k
B and
c
DmdUCtS of rabbitreticulacvteIvsatesvstem of human lune RNA
k
RNA lrlinF the
4 respectivelv mine the
TWO dimensional eel electmnhoresis of immunODrecinitated translation fmm hvhrid selection (ompl R ) or human lune R N A (panelC).
9033
cDNA Clones EncodingPulmonary Surfactant Proteins 1
2
-lEF
3
-36
-28s
+ "2
66-
-29
MW B -36
-18s -29
20-
A -1os
Firnure 4
~
One and two-dimensional Keel electrophoresis of of PSP-A. A.
&
t=.RnScription-trBnSlation Drortucts
Lane 1.
The t=8nsC=iption-trRnSIRtiOn Droducts of FSP.35K.IA and PSP.35K.6A.
2 and 3.
The trRnE~:ription-trRnSlatiDn products of FSP.35K.6A resnectivelv.
B and C .
Two dinrnsional eel analysis of the transcription-tr~n~lationDmdUCts of PSP.35K.6A and PSP.35K.lA in the shsence or DPeSence of the metabolic t r m respectivelv.ArrowsPoint Rt the most basicisoform of each L ~ O U D .
md
PSP.35K.lA
AcknowIedePments
Fiwre 3
~
Northernblot of hunan lune RNA usine a sinRlc stranded MI3 robe. Theposition RNA markers of rRNA (18s end 1 8 s ) nnd of elobin mRNA f1OS1 are noted.
nf
We wish tothank
J. Fisch. C . B a s s l ~ r . T. Loh and. E. Orr for technicalsupport.
Thiswork was 6UpDOrted bv P m n t s H1.34188, Institutes of Health.
HL313956, H1.31394 from the National
