Purification and analysis of RTI40, a type I alveolar epithelial cell apical membrane protein

doi:10.1016/S0167-4838(98)00231-3

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 1429, Issue 1, 8 December 1998, Pages 208-216

Biochimica et Biophy...

https://doi.org/10.1016/S0167-4838(98)00231-3 Get rights and content

Abstract

RTI40 is a 40–42 kDa protein that, within the lung, is specific to the apical plasma membrane of the rat alveolar type I cell. Type I cells cover greater than 95% of the internal surface area of the lung. In this report, we describe some of the physical properties of RTI40, and its purification to homogeneity. By liquid phase isoelectric focusing, the pI of the protein is 3.0±0.5. In two-dimensional immunoblots, there is a 1.0 pH unit charge train, suggesting post-translational modification of the protein. We have purified the protein to homogeneity by the following method. A membrane preparation from perfused rat lungs was extracted with detergent and applied to an ion-exchange column. Immunoreactive fractions from the column were pooled, dialyzed and further fractionated by reverse phase high performance liquid chromatography (HPLC). Essentially all the antigenicity was recovered in one protein peak that was homogeneous both by spectral analysis and silver-stained polyacrylamide gels. Because the purified protein was N terminus blocked, we cleaved the protein with CNBr and fractionated peptide fragments by reverse phase HPLC. Fractions were pooled and concentrated. Direct amino acid sequencing of the major peptide fragment yielded a 15 amino acid peptide homologous to a mouse osteoblast protein, OTS-8. Analysis of purified RTI40 shows that the protein contains glycan, some of which is sialic acid. Characterization of RTI40 should facilitate future studies of the functional properties of RTI40.

Introduction

The alveolar epithelium is comprised of two morphologically distinct types of cells, type I and type II cells, both of which are thought to be critical for normal lung function. Type I cells are extremely large cells with very thin cytoplasmic extensions. Type II cells are cuboidal cells characterized by the presence of lamellar bodies, intracellular organelles that contain surfactant lipids and proteins. Type I cells are thought to play an important role in lung function because they cover more that 95% of the alveolar surface [1], [2], providing the very thin barrier between the air and blood compartments critical for gas exchange. Recent studies have demonstrated that water permeability of type I cells is extremely high, suggesting type I cells play an important role in the regulation of lung fluid [3]. Although the establishment and maintenance of a normal alveolar epithelium is essential for gas exchange, little is currently known about the factors which control the expression of the differentiated phenotype of its constituent cells. The topic has been difficult to study experimentally because there have been few established biochemical markers unique to the type I cells. By producing monoclonal antibodies specific for rat type I cells, we identified the first biochemical protein marker specific within the lung for type I cells, a 40–42 kDa protein localized within the lung to the apical plasma membrane of type I cells [4].

One of these monoclonal antibodies was used to screen a rat lung expression cDNA library. When three positive clones were identified and sequenced, the cDNA proved to be homologous to a previously described phorbol ester inducible osteoblast protein called OTS-8 [5], [6], [7]. In previous publications, supposed OTS-8 homologues have been variously referred to as gp38 [6], SF-1 [8], RTI40 [3], [9], [10], T1α [7] and gp40 [1]. OTS-8 was originally identified in thymus, spleen and peripheral lymphoid tissue [6]. Based on the cDNA, the deduced protein would have a molecular mass of 18 kDa [7]; when the cDNA was expressed in bacteria, the resultant protein matched the predicted size of 18 kDa. This finding raised the question whether the protein encoded by this cDNA was that initially identified by our monoclonal antibodies or whether it was a member of a larger family of proteins that shared common epitopes recognized by the antibodies. In order to answer this question and characterize the protein, we purified RTI40 from native rat lung tissue. In this communication, we report that the purified protein is acidic and extremely hydrophobic; it has the biochemical properties of an integral membrane protein. Direct amino acid sequencing of a 15 amino acid peptide fragment demonstrates that RTI40 and OTS-8 are similar. Glycosylation may account for the discrepancy between the predicted molecular weight and the observed molecular weight from electrophoretic analysis.

Section snippets

Assay of RTI40

We used a dot blot assay to measure RTI40. Samples were diluted 1/100 with 50 mM NaHCO₃, pH 9.0, and dot blotted onto nitrocellulose. Endogenous peroxidase activity was quenched by treatment with 15% hydrogen peroxide for 10 min and non-specific binding was blocked by a 1 h incubation in a solution of 1% non-fat dried milk, 0.4% gelatin, 0.1% bovine serum albumin (BSA), 0.9% NaCl and 10 mM Tris-base, pH 7.2 (blocking solution A). Primary antibody to RTI40 in blocking buffer (1:5000) was then

Two-dimensional gel electrophoresis and Western blotting

In two-dimensional Western blotting, RTI40 displays a variation in molecular weight and a charge train that are consistent with post-translational modification of the protein (Fig. 1). Fig. 2 shows a Western blot of RTI40 at various stages of purification. The less pure fractions contain an immunoreactive band at 80 kDa, in addition to the band at approx. 40 kDa. Under non-reducing conditions, the 80 kDa band was more prominent (data not shown). Because both protein purification and reduction

Discussion

We embarked on the present studies for two reasons. The first reason was to answer the question whether RTI40, the antigen recognized by a series of monoclonal antibodies raised against an apical plasma membrane protein of alveolar epithelial type I cells, was identical to OTS-8 or whether the two proteins were members of a family of similar proteins sharing common antigenic epitopes. The second reason was to study the biochemical characteristics of RTI40. The rationale for these studies was as

Acknowledgements

We thank Jean Nellenbogen for technical assistance, Ralph Reed for automated DNA sequencing and Lennell Allen for her assistance with preparation of the manuscript.

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Purification and analysis of RTI40, a type I alveolar epithelial cell apical membrane protein

Abstract

Introduction

Section snippets

Assay of RTI40

Two-dimensional gel electrophoresis and Western blotting

Discussion

Acknowledgements

Biochim. Biophys. Acta

Dev. Biol.

Anal. Biochem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Cell

J. Chromatogr.

J. Chromatogr.

J. Biol. Chem.

Anal. Biochem.

J. Chromatogr.

Biochem. J.

Am. J. Respir. Cell Mol. Biol.

Proc. Natl. Acad. Sci. USA

Cell Growth Differ.