Jul 1, 1985 - Joanna FlorosS, David S. Phelps, Stella Kourembanas, and H. William Taeusch. From the Department of Pediatrics, Harvard Medical School, ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc
Vol. 261, No. 2, Issue of January 15, pp. 828-831, 1986 Printed in U.S.A.
Primary Translation Products, Biosynthesis, and Tissue Specificity of the Major Surfactant Protein in Rat* (Received for publication, July 1, 1985)
Joanna FlorosS, David S . Phelps, Stella Kourembanas, andH. William Taeusch From the Department of Pediatrics, Harvard Medical School, Boston,Massachusetts 02115
Rat lung tissue was labeled with [36S]methionineand the major surfactant-associated proteins immunoprecipitated using a specific antiserum. The protein pattern obtained wasvery similar to that seenin rat bronchoalveolar lavage. Rat lung mRNA was subsequently translated in an in vitro rabbit reticulocyte system, and surfactant-associated protein-related polypeptides were immunoprecipitated. A 26-kDa polypeptide was identified and characterized as follows. (a)Unlabeled surfactant proteins added to the immunoprecipitation mixture completely inhibited its immunoprecipitation.(6) Two-dimensional gel electrophoresis of the 26-kDa protein resolved it into 3 isoforms. (c) Inclusion of dog pancreatic microsomes in the translation mixture resulted in the formation of two distinct higher molecular weight groups of isoforms, suggesting that the 26-kDa protein is destined to becomea glycoprotein. Immunoprecipitationof [35S] methionine-labeled rat lung tissue proteins after tunicamycin treatment resulted in 3 isoforms, identical to the ones seen in the primary translation products. In addition, expression of the surfactant proteins appears specific to the lung.
Pulmonary surfactant, a lipoprotein complex, lowers the surface tension in the alveoli of the mammalian lung. However, the function of the protein component of the lipoprotein complex in surfactant is not yet completely understood, although a number of functions have been suggested. The best supported notion is that the proteins in surfactant permit formation of a three-dimensional lipoprotein complex, called tubular myelin, that is rapidly adsorbed at the air-liquid interface in alveoli (Hawgood et al., 1985; King and MacBeth, 1979; King and Clements, 1972; Suzuki, 1982). The major non-serumsurfactant associated proteins, with isoelectric points between PI 4.2-6.0 and molecular weights from 30,00040,000, appear to be similar in a number of species that have been studied (King et al., 1973; Bhattacharyya et al., 1975; Bhattacharyya et al., 1976; Sueishi and Benson, 1981; Ng et al., 1983; Weaver et al., 1985; Katyal and Singh,1984a;Phelps and Taeusch, 1985). These proteins have been called surfactant apoprotein A by King (King and Martin, 1980). In many species, the major surfactant-associated proteins or apoprotein A has three major forms. In this paper we refer to this protein as pulmonary surfactant-associated protein A * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ T o whom correspondence and reprint requests should be addressed: Joint Program in Neonatology, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115.
or PSP-A.’ In rat, in samples from lavage material, these forms have molecular weights of 26,000, 32,000, and 38,000 (Weaver et al., 1985; Katyal and Singh, 1984a). The 32- and 38-kDa proteins have the appearance of sialoglycoproteins with extensive charge and molecular weight heterogeneity (Katyal and Singh, 1984a; Weaver et al., 1985; Phelps and Taeusch, 1985; Sueishi and Benson, 1981). Recent studies with human and rat lung tissue indicate that the forms of PSP-A with higher molecular weights are modified forms of the primary translation product (the lowest molecular weight form) (Floros et al., 1985; Whitsett et al., 1985).However, not all reports concur with this belief (Katyal and Singh, 1985), and in this study additional information is presented on the synthesis, in vitro translation, andmodification of this protein group in rat. The tissue specificity of PSP-A has also been examined. EXPERIMENTAL PROCEDURES AND RESULTS’ DISCUSSION
In this report we have identified the primary translation products of PSP-A in theratand examined their tissue specificity. We have also studied the biosynthesis of PSP-A in rat lung tissue. Two previous reports have generated a controversy about the molecular weight of the primary translation products of PSP-A in the rat and consequently the interpretation of data in terms of which group of these proteins gives rise to theother groups. One group of investigators (Katyal and Singh, 1985) suggests that the primary translation product is a polypeptide of 35,000 daltons which is the common precursor of the 38-kDa, 32-kDa, and 26-kDa forms of PSP-A. They suggest that this 35-kDa precursor gives rise to other surfactant proteinsby partial proteolysis or by other post-translational modifications (Katyal and Singh, 1984b). Others have found that the primary translation products of rat PSP-A have a molecular mass of 26 kDa, and theysuggest thatthis precursor gives rise to higher molecularweight proteins by post-translational modifications such as N-linked glycosylation and the addition of sialic acid (Whitsett et al., 1985). Our findings agree with Whitsett et al. (1985). We first identified the primary translation products to be a polypeptide The abbreviation used is: PSP-A, pulmonary surfactant-associated protein A. Portions of this paper (including “Experimental Procedures,” “Results,” Figs. 1-4, and Acknowledgments) are presented in miniprint at theend 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. 85M-2164, cite the authors, and include a check or money order for $3.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
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processing, and the tissuespecificity of the major surfactantassociated protein (PSP-A) in the rat. Our data show that this protein is lungspecific, and the molecular weight of the primary translation product is about 26 kDa that can be resolved into three isoforms by two-dimensional gel electrophoresis. With in vitro glycosylation the primary translation products are partially converted into two distinct higher molecular weight groups. This kind of glycosylation appears to lane 4 ) . While the precursor forms of PSP-A consist of three iso- differ from the one described for the human primary translavage is quite lation products (Floros et al., 1985) in which each primary forms of 26 kDa, PSP-A in rat bronchoalveolar translation product in the presence of dog pancreatic memdifferent, consisting of many different isoforms ranging in molecular weight from 26,000 to 38,000 (Fig. 30). A similar branes gave rise only to oneglycosylated form and not totwo, as in the case of the rat. Moreover, the unglycosylated i n vivo pattern can be generated by immunoprecipitating secreted precursor forms of PSP-A from tunicamycin-treatedlung PSP-A after labeling rat lung tissue with [35S]methionine tissue are similar to the primary translation products of PSP(Fig. 1A). However, a slightly different picture is seen when A. intracellularlungtissueproteinsareimmunoprecipitated. The principal difference between intracellular and secreted REFERENCES PSP-A is the presence in intracellular PSP-A of isoforms Bhattacharyya, S. N., Passero, M. A., DiAugustine, R. P., and Lynn, with basicisoelectric points (Fig. l B , arrow). These basic W. S. (1975) J. Clin. Invest. 5 5 , 914-920 isoforms have similarisoelectric points as those of the primary Bhattacharyya, S. N.,Sahu, S., and Lynn, W. S. (1976) Biochim. Biophys. Acta 4 2 7 , 91-106 translation products buthigher molecular weights. Elhein, A. D. (1985) CRC Crit. Reu. Biochem. 1 6 , 21-49 We were apparently able toi n vitro glycosylate (Katz et al., Floros, J., Phelps, D. S., and Taeusch, H. W. (1985) J. Biol. Chem. 1977) the primary translation products for PSP-A (Fig. 3E) 260,495-500 Hawgood, S., Benson, B. J., and Hamilton, R. L. (1985) Biochemistry incontrasttoanearlierreportin which theseproducts 24,184-190 appeared unaltered in the presenceof dog pancreatic microS. L., and Singh, G. (1984a) Biochim.Biophys.Acta 7 9 4 , somes(Whitsett et al., 1985). In coelectrophoresis experi- Katyal, 411-418 ments (Fig. 3F) of the in vitro modified translation products Katyal, S. L., and Singh, G. (1984h) Exp. Lung Res. 6 , 175-189 (Fig. 3E) and surfactant proteinfrom rat lavage material (Fig. Katyal, S. L., and Singh, G. (1985) Biochem. Biophys. Res. Commun. 1 2 7 , 106-111 3 0 ) , the i n vitro glycosylated isoforms migrate approximately at the samemolecular weight as theA2 and ASgroups of PSP- Katz, F. N., Rothman, J. E., Lingapappa, V. R., Blobel, G., and Lodish, H. F. (1977) Proc. Natl. Acad. Sci. U. S. A . 7 4 , 3278-3282 A from lavage material. However, the in vitro glycosylated King, R. J., and Clements, J. A. (1972) A m . J.Physiol. 223,715-726 isoforms have a more basic isoelectricpoint than their mature King, R. J., and MacBeth, M. C. (1979) Biochim. Biophys. Acta5 5 7 , 86-101 in vivo (A2 and A3) secreted counterparts (Fig. 3F). The i n vitro glycosylated isoforms appear similar in isoelectric point King, R. J., and Martin, H. (1980) J. Appl. Physiol. Respir. Environ. Exercisee Physiol. 48,812-820 and molecular weight to the basic isoforms seen in intracel- King, R. J., Klass, D. J., Gikas, E. G., and Clements, J. A. (1973) A m , lular PSP-A (compare Fig. lB, arrow, and Fig. 3F). Perhaps J.Physiol. 2 2 4 , 788-795 the i n vitro glycosylated forms, after furtheri n vivo processing Mahoney, W. C., and Duksin, D. (1979) J. Biol. Chem. 2 5 4 , 65726576 (Whitsett et al., 1985), can result in the A2 and AB protein Ng, V. L., Herndon, V. L., Mendelson, C. R., and Snyder, J. M. (1983) groups identified in rat lavage materialandthesecreted Biochim. Biophys. Acta 754,218-226 proteins from rat lung tissue. Phelps, D. S., and Taeusch, H.W. (1985) Comp. Biochem. Physiol. When lung tissue is treated with the antibiotic tunicamycin, 82,441-446 Phelps, D. S., Taeusch, H. W . , Jr., Benson, B., and Hawgood, S. which inhibits glycosylation by the N-linked pathway (Ma(1984) Biochim. Biophys. Acta 7 9 1 , 226-238 honey and Duksin,1979), the complex pattern of intracellular Sueishi, K., and Benson, B. J. (1981) Biochim. Biophys. Acta 665, PSP-A is reduced to three isoforms (Fig. 3A) identical to the 442-453 primary translation products (Fig. 3, B and C). This in vivo Suzuki, Y. (1982) J. Lipid Res. 2 3 , 62-69 result provides additional evidence for our in vitro observa- Weaver, T. E., Hull, W. M., Ross, G. F., and Whitsett, J. A. (1985) Biochim. Biophys. Acta 8 2 7 , 260-267 tions about the size of the primary translation products and supports the notion that PSP-A contains N-linked oligosac- Whitsett, J. A., Weaver, T., Hull, W., Ross, G., and Dion, C. (1985) Biochim. Biophys. Acta 828. 162-171 charides (Floros et al., 1985; Whitsett et al., 1985). Williams, M. C., and Benson,'B. J. (1981) J. Histochem. Cytochem. In summary, we have examined the synthesis, the initial 29,291-305 of 26,000 molecular weight that, upon two-dimensional gel analysis, can be resolved into three isoforms (Fig. 3, B and C). Expression of the 26-kDa protein can onlybe detected in lung tissue, as determined by analysis of immunoprecipitates of in vitro translated RNA derived from various tissues (Fig. 4). Moreover, in the presence of unlabeled PSP-A, immunoprecipitation of this 26-kDa protein can be inhibited(Fig. 2,
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Primary Translation Productsof Rat Surfactant Proteins
830
PRIMARY TRANSLATION PRODUCTS, BIOSYNTHESIS. AND TISSUE SPECIFICITY OF THE MAJOR SURFACTANT PROTEIN IN RAT
Tiuur rprrifirily oJPSP-A
BY Joaoos Floror. David S. Phelp,. Stella Kourcmbnoas aod
Il.William Taeurcb E r r r r i m m t d Pmrrdurer
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-
Primary Translation Products Surfactant RatofProteins
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W e t h a n k Dr. M. Williams for the rat antibody. M. Pike and L. Smith for rcchniaal arsir~snce. T h i s work w a s supported by Grant%HL34788-01. HL313956. HL31384 from l h c Kalionsl I n ~ t i t u t cof~ llcallh sod American Thoracic Society American Lung A m v i a t i o n .
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