THE JOURNAL 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 the Major Surfactant

Products, Biosynthesis, Protein in Rat*

and Tissue Specificity of

(Received for publication, July 1, 1985)

Joanna FlorosS, David S . Phelps, Stella Kourembanas, and H. William Taeusch From the Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115

Rat lung tissue was labeled with [36S]methionine and the major surfactant-associated proteins immunopre- cipitated using a specific antiserum. The protein pat- tern obtained was very similar to that seen in rat bronchoalveolar lavage. Rat lung mRNA was subse- quently 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 fol- lows. (a) Unlabeled surfactant proteins added to the immunoprecipitation mixture completely inhibited its immunoprecipitation. (6) Two-dimensional gel electro- phoresis of the 26-kDa protein resolved it into 3 iso- forms. (c) Inclusion of dog pancreatic microsomes in the translation mixture resulted in the formation of two distinct higher molecular weight groups of iso- forms, suggesting that the 26-kDa protein is destined to become a glycoprotein. Immunoprecipitation of [35S] methionine-labeled rat lung tissue proteins after tuni- camycin 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. How- ever, the function of the protein component of the lipoprotein complex in surfactant is not yet completely understood, al- though 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-serum surfactant associated proteins, with isoelectric points between PI 4.2-6.0 and molecular weights from 30,000- 40,000, 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 surfac- tant 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 ad- dressed: Joint Program in Neonatology, Brigham and Women’s Hos- pital, 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, and modification 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 the rat and 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 trans- lation products of PSP-A in the rat and consequently the interpretation of data in terms of which group of these pro- teins gives rise to the other groups. One group of investigators (Katyal and Singh, 1985) suggests that the primary transla- tion 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 proteins by 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 they suggest that this precursor gives rise to higher molecular weight 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-associ- ated protein A.

Portions of this paper (including “Experimental Procedures,” “Results,” Figs. 1-4, and Acknowledgments) are presented in mini- print 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. 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.

828

Primary Translation Products of Rat Surfactant Proteins 829

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 only be 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, immuno- precipitation of this 26-kDa protein can be inhibited (Fig. 2, lane 4 ) .

While the precursor forms of PSP-A consist of three iso- forms of 26 kDa, PSP-A in rat bronchoalveolar lavage is quite different, consisting of many different isoforms ranging in molecular weight from 26,000 to 38,000 (Fig. 30). A similar pattern can be generated by immunoprecipitating secreted PSP-A after labeling rat lung tissue with [35S]methionine (Fig. 1A). However, a slightly different picture is seen when intracellular lung tissue proteins are immunoprecipitated. The principal difference between intracellular and secreted PSP-A is the presence in intracellular PSP-A of isoforms with basic isoelectric points (Fig. lB , arrow). These basic isoforms have similar isoelectric points as those of the primary translation products but higher molecular weights.

We were apparently able to i n vitro glycosylate (Katz et al., 1977) the primary translation products for PSP-A (Fig. 3E) in contrast to an earlier report in which these products appeared unaltered in the presence of dog pancreatic micro- somes (Whitsett et al., 1985). In coelectrophoresis experi- ments (Fig. 3F) of the in vitro modified translation products (Fig. 3E) and surfactant protein from rat lavage material (Fig. 30) , the i n vitro glycosylated isoforms migrate approximately at the same molecular weight as the A2 and AS groups of PSP- A from lavage material. However, the in vitro glycosylated isoforms have a more basic isoelectric point than their mature in vivo (A2 and A3) secreted counterparts (Fig. 3F). The i n vitro glycosylated isoforms appear similar in isoelectric point and molecular weight to the basic isoforms seen in intracel- lular PSP-A (compare Fig. lB, arrow, and Fig. 3F). Perhaps the i n vitro glycosylated forms, after further i n vivo processing (Whitsett et al., 1985), can result in the A2 and AB protein groups identified in rat lavage material and the secreted proteins from rat lung tissue.

When lung tissue is treated with the antibiotic tunicamycin, which inhibits glycosylation by the N-linked pathway (Ma- honey and Duksin, 1979), the complex pattern of intracellular PSP-A is reduced to three isoforms (Fig. 3A) identical to the primary translation products (Fig. 3, B and C). This in vivo result provides additional evidence for our in vitro observa- tions about the size of the primary translation products and supports the notion that PSP-A contains N-linked oligosac- charides (Floros et al., 1985; Whitsett et al., 1985).

In summary, we have examined the synthesis, the initial

processing, and the tissue specificity of the major surfactant- associated protein (PSP-A) in the rat. Our data show that this protein is lung specific, and the molecular weight of the primary translation product is about 26 kDa that can be resolved into three isoforms by two-dimensional gel electro- phoresis. With in vitro glycosylation the primary translation products are partially converted into two distinct higher mo- lecular weight groups. This kind of glycosylation appears to differ from the one described for the human primary trans- lation products (Floros et al., 1985) in which each primary translation product in the presence of dog pancreatic mem- branes gave rise only to one glycosylated form and not to two, as in the case of the rat. Moreover, the unglycosylated i n vivo precursor forms of PSP-A from tunicamycin-treated lung tissue are similar to the primary translation products of PSP- A.

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Continued on next page.

830 Primary Translation Products of Rat Surfactant Proteins PRIMARY TRANSLATION PRODUCTS, BIOSYNTHESIS. AND TISSUE SPECIFICITY

OF THE MAJOR SURFACTANT PROTEIN IN RAT

BY

Joaoos Floror. David S. Phelp,. Stella Kourcmbnoas aod Il.William Taeurcb

Errrr immtd Pmrrdurer

Tiuur rprrifirily oJPSP-A

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66- M W x~o-3 45-

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Primary Translation Products of Rat Surfactant Proteins 83 1

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Figure 4

1 2 3 4 5 6 7 8 9 1 0

This work was supported by Grant% HL34788-01. HL313956. HL31384 from l h c Kalionsl I n ~ t i t u t c ~ of We thank Dr. M. Williams for the rat antibody. M. Pike and L. Smith for rcchniaal arsir~snce.

llcallh sod American Thoracic Society - American Lung Amviat ion.