The major APN transcript of the alveolar type II epithelial cell originates from a unique upstream promoter region.

Aminopeptidase N (APN, EC 3.4.11.2) is an ectopeptidase expressed in lung at the apical surface of alveolar type II epithelial cells. Its expression is upregulated during fetal lung development. To begin to understand the regulation of APN expression during lung development, we used the rapid modification of cDNA ends (RACE) to clone the 5' end of the major APN transcript in rat lung and alveolar type II cells. The cloned sequence revealed a unique 135 bp untranslated exon which genomic cloning and restriction mapping indicated was located more than 14 kb upstream from the coding sequence. A 172 bp genomic fragment flanking the untranslated exon produced a high level of expression of a reporter gene in transient transfection assays using a human lung adenocarcinoma cell line. The DNA fragment includes elements known to be important for expression of lung specific proteins, including the surfactant-associated proteins A, B, and C and the Clara cell specific protein. Comparison of the APN genomic sequences and gene structure from human and rat suggests that the exon present in the rat lung transcript may result from the use of a previously uncharacterized APN promoter.

Phosphatidylinositol transfer protein in lung: cellular and subcellular localization.

Determination of the cellular distribution of phosphatidylinositol transfer protein in rat lung by immunocytochemistry revealed that the protein is more readily observed in the nonciliated bronchial epithelial cells (Clara cells) than in other lung cells. By light microscopy, the phosphatidylinositol transfer protein (PtdIns-TP) was localized to the dome-shaped apical region of Clara cells that were identified by staining with an antibody to Clara cell protein. Further investigation by electron microscopy revealed that the PtdIns-TP accumulated at the limiting membrane surrounding secretory granules and at the apical plasma membrane. This localization is compatible with the proposed roles for PtdIns-TP in formation of vesicles and exocytosis of secretory granules and, when considered in the context of the proposed role of PtdIns-TP in phosphatidylinositide metabolism, suggests that phosphatidylinositides may be involved in the mechanisms regulating Clara cell secretion.

Regulation of expression of aminopeptidase N in fetal rat lung by dexamethasone and epidermal growth factor.

Aminopeptidase N (EC 3.4.11.2) (APN) is an ectopeptidase expressed in lung at the apical surface of alveolar type II epithelial cells. Its expression is up-regulated during fetal lung development. Recently several ectopeptidases have been recognized as possible regulators of growth and cell differentiation through their role in hydrolysis of autocrine and paracrine peptides that influence these processes. The studies reported here describe effects of factors known to promote lung development and differentiation of the alveolar epithelium on expression of aminopeptidase N during fetal lung development in organ culture. Fetal rat lung was placed in organ culture at the 15th gestational day and cultured for 6 days in the presence or absence of the synthetic glucocorticoid hormone dexamethasone or epidermal growth factor (EGF). Steady-state levels of APN mRNA increased approximately 10-fold during the 6-day culture. During this time the lung alveolar epithelium developed to the point where immature alveolar type II cells were recognized by the presence of lamellar bodies. Dexamethasone or EGF increased the levels of APN mRNA in the fetal lung 2-to 3-fold over control cultures by the third day in culture and concurrently accelerated the morphological development of the alveolar epithelium. Differences in treated and control cultures diminished after 6 days in culture when the epithelium appeared more mature, suggesting that the immature epithelium was more responsive to the treatments.

Expression of aminopeptidase N in fetal rat lung during development.

The ectopeptidase, aminopeptidase N, serves as a cell surface marker of the apical surface of the alveolar type II epithelial cell in adult lung. It is also present in fetal lung before differentiation of morphologically mature type II alveolar epithelial cells, suggesting that it is expressed by precursors of the type II cells. We have examined the mRNA coding for the aminopeptidase in adult and fetal lung and in mature type II cells and determined levels of mRNA and immunoreactive protein during fetal lung development. Comparison of the temporal patterns of steady-state levels of aminopeptidase mRNA and immunoreactive protein during development show that the expression of the protein is developmentally regulated and that expression is regulated, at least in part, at a pretranslational level. Both mRNA and immunoreactive protein levels increase severalfold on the final gestational day, suggesting that the function of the aminopeptidase may be associated with air breathing.

Cancer susceptibility in ataxia-telangiectasia.

Ataxia-telangiectasia (A-T) is a syndrome that has an extremely high incidence of cancer. Patients with the disease are homozygous for a mutant gene, the A-T gene, located at 11q23. Of these individuals, 30-40% develop cancer. Of these cancers, 80% are lymphoid. Those heterozygous for the A-T gene also have an increased frequency of cancer, the most notable being the 6.8-fold increase of breast cancer in females carriers. The syndrome is characterized cytogenetically by increased nonrandom chromosome breaks and rearrangements in lymphocytes involving the sites of the immunoglobulin and T-cell receptor genes. Clones of cells having the same rearrangements are often present in the blood of the A-T patients and if the rearrangements involve certain sites, especially a locus within 14q32, the propensity to progress to a malignant transformation is great. Sequencing the A-T gene and ascertaining its function should contribute significantly to our understanding of the molecular mechanisms underlying cancer susceptibility.

Ectopeptidases of alveolar epithelium: candidates for roles in alveolar regulatory mechanisms.

This commentary discusses the implications of recent observations indicating that at least one, and probably three, ectopeptidases are exhibited on the surface of alveolar epithelial cells. The unique biological feature of the ectopeptidases is that they are expressed on the surface of only selected cell types and at specific stages in the differentiation of those cells. Consequently they are positioned to exert their enzymatic activity in a very restricted locale. Drawing from information derived from studies of this activity on other cells, we develop the proposition that the ectopeptidases on alveolar epithelial cells regulate the biological activity of peptides encountered at this site. These may be peptides produced by the alveolar epithelial cells or cytokines that have the potential of influencing the alveolar cells. Awareness that these ectopeptidases are expressed on the luminal surface of these cells thus opens heretofore unrecognized avenues of investigation.

p146 type II alveolar epithelial cell antigen is identical to aminopeptidase N.

A prominent membrane protein of rat type II alveolar cells, p146, was originally identified by one of many mouse monoclonal antibodies that were produced to rat lung cells in the course of a search for differentiation antigens that might prove useful in studying lung differentiation. We report here results from analysis of the primary structure of this molecule and, based on this knowledge, the elucidation of the function of the protein. Amino acid sequencing of the NH2-terminal portion of the p146 protein, plus partial sequencing of several peptides obtained by limited proteolysis, indicates it is identical to aminopeptidase N. Further, the immunoaffinity purified p146 protein has aminopeptidase N activity. The discussion includes references to other molecules such as CD 13 and CD 10 (CALLA) that were recognized as differentiation antigens and subsequently found to be peptidases. The possible biological implications of such a peptidase on the luminal surface of type II alveolar cells are also considered.

Immunotargeting: a contemporary approach to the study of lung development.

This commentary discusses an immunological approach to lung development that has been termed immunotargeting. The approach involves the use of monoclonal antibody technology to identify cell membrane antigens unique to cells of particular lineage or stage of differentiation. Methods of using the antibodies to focus on important unresolved questions relating to development, particularly of the pulmonary epithelium, are discussed. Antigens and membrane molecules, which have already been identified and which may be useful in this type of approach to respiratory epithelial cell differentiation, are enumerated and briefly described.

Speculations on ataxia-telangiectasia: defective regulation of the immunoglobulin gene superfamily.

In this short article, Raymond Peterson and Jane Funkhouser develop the argument that the common molecular mechanism linking the various clinical manifestations of ataxia-telangiectasia (AT) is a defect in the regulation of the immunoglobulin (Ig) gene superfamily. They propose that the AT gene codes for a protein essential for the orderly expression of this gene family, perhaps regulating the gene rearrangement process that appears to be a unique characteristic of this system. Members of the Ig gene superfamily play a major role in the development and operation of the immune and nervous systems, and any perturbation of their expression would be anticipated to produce a panoply of signs and symptoms, such as those characterizing the AT phenotype.

Amino-terminal sequence of a phospholipid transfer protein from rat lung.

A phospholipid transfer protein from rat lung has been characterized in terms of the amino-terminal sequence. The sequence is Val-Leu-Leu-Lys-Glu-Tyr-Arg-Val-Ile-Leu-Pro-(Val)-His-Val-Asp-Glu-Tyr-Gln-Val- Gly. Comparison of the amino-terminal sequence of the protein from lung with sequences from phosphatidylcholine transfer protein and non-specific phospholipid transfer protein from bovine liver revealed no apparent sequence homology. The sequence showed no homology with fatty acid binding proteins or cellular retinoid binding proteins.

Monoclonal antibody isolation of type II pneumocytes.

A monoclonal antibody that identifies a membrane molecule unique in rat lung for type II alveolar epithelial cells was used to isolate these cells from enzymatically dispersed lung cells by fluorescence-activated cell sorting. Although multistep physical separation techniques have permitted the isolation of large quantities of these cells and flow cytometry has been used by others to isolate lamellar body-containing cells, the application of this antibody-directed sorting has distinct advantages. Because the marker molecule is expressed on immature type II cells prior to the development of lamellar bodies, the antibody will also permit their isolation and study.

Monoclonal antibody identification of a type II alveolar epithelial cell antigen and expression of the antigen during lung development.

A monoclonal antibody identifying an antigen expressed by rat type II alveolar epithelial cells, but not by type I epithelial cells or other mature lung cells, was produced by immunization of mice with cells of the rat L2 cell line. The antigen recognized by the antibody was present on the microvillous luminal surface of type II epithelial cells. In adult rat lung, only type II epithelial cells bound the antibody. During fetal development the antigen was expressed by cuboidal epithelial cells lining the respiratory ducts of the first divisions of the tracheal bud, but not by epithelial cells lining the esophagus or trachea. The antigen continued to be expressed by cuboidal epithelial cells lining the larger respiratory ducts until approximately 19 days gestational age. Thereafter, expression was increasingly limited to selected single cells or clusters of two to four cuboidal cells in the smallest ducts. By the 21st postnatal day, the antigen was expressed only by type II alveolar epithelial cells. Type II alveolar epithelial cells isolated from adult lung and the L2 cell line in culture expressed the antigen on the cell surface. A protein of approximately 146,000 Mr was isolated by immunoadsorption of the antigen from non-ionic detergent extracts of type II cells and L2 cells. Preliminary studies of the binding of the antibody to other rat tissues indicate that the antibody binds to renal proximal tubular epithelial cells of the kidney and the luminal surface of the small bowel epithelial cells.

Phospholipid transfer proteins from lung, properties and possible physiological functions.

Phospholipid transfer proteins have been found in lung just as they have in tissues throughout the body. There is speculation that the proteins are involved in membrane biogenesis and in determining the phospholipid composition of membranes. For this reason the lung, which contains subcellular organelles of distinct phospholipid composition, is of interest in terms of its complement of phospholipid transfer proteins. The lamellar bodies of pulmonary type II alveolar cells have a phospholipid composition unique in terms of the proportions of dipalmitoyl phosphatidylcholine and phosphatidylglycerol. Studies of the phospholipid transfer proteins in lung have demonstrated two molecular species of the transfer proteins that differ significantly from those found in liver and other tissues. These proteins show specificity for the transfer of dipalmitoyl phosphatidylcholine and phosphatidylglycerol.

Acyl-chain specificity and membrane fluidity. Factors which influence the activity of a purified phospholipid-transfer protein from lung.

A purified phospholipid-transfer protein from rat lung has been characterized in terms of the specificity of the protein for phosphatidylcholine molecules with different apolar moieties. The study demonstrated that the lung-phospholipid-transfer protein discriminates between dipalmitoylphosphatidylcholine and molecular species of phosphatidylcholine with unsaturated acyl chains. The initial rate of transfer of dipalmitoylphosphatidylcholine is 1.5-fold greater than the rate of transfer of dioleoylphosphatidylcholine, 1-palmitoyl-2- arachidonylphosphatidylcholine , or egg phosphatidylcholine under most assay conditions. Although the protein preferentially transfers dipalmitoylphosphatidylcholine, the incorporation of increasing mole percentages of dipalmitoylphosphatidylcholine into unilamellar phosphatidylcholine vesicles profoundly affects their effectiveness as donors for phosphatidylcholine transfer by the transfer protein. At 60 mol% dipalmitoylphosphatidylcholine, the rate of transfer is one-third that observed when vesicles are composed of 100% egg phosphatidylcholine. Decreases in membrane fluidity as estimated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene correlate with decreases in the effectiveness of the vesicles as donors in the phospholipid-transfer reaction. The conclusion from these studies is that the rate of transfer of phosphatidylcholine by the purified phospholipid-transfer protein from lung is determined by physical properties of membrane interfaces with which the protein interacts, as well as by the specificity of the phospholipid-transfer protein for different molecular species of phosphatidylcholine.

Properties of a non-specific phospholipid-transfer protein purified from rat lung.

The present report describes the purification and characterization of a non-specific phospholipid-transfer protein from rat lung. The protein is the major phospholipid-transfer protein in lung which transfers phosphatidylcholine. The transfer protein was purified 1200-fold, with a final yield of 3%. The activity of the protein was monitored by measuring the transfer of [14C]phosphatidylcholine from radioactively labeled liposomes to mitochondria. The purified proteins transfers phosphatidylcholine, phosphatidylinositol, phosphatidylserine and phosphatidylethanolamine from radioactively labeled microsomes to either mitochondria or liposomes. The transfer of each phospholipid is proportional to its content in the donor membrane. The protein was purified from a pH 5.1 supernatant preparation by fractionation on DEAE-cellulose, Sephadex G-75 and hydroxyapatite. The molecular weight of the purified protein was estimated as 35 000 by SDS-polyacrylamide gel electrophoresis. The amino acid analysis revealed a high content of glutamic acid (including glutamine) and glycine. The specificity of the purified protein for transfer of phospholipids suggests that it may be the phospholipid-transfer activity which is highly enriched in isolated type II alveolar cells of rat lung.

The lung lamellar body as a functioning membrane in protein-catalyzed phosphatidylcholine transfer.

Lung lamellar bodies and liver mitochondria were used to demonstrate that soluble phospholipid transfer proteins from lung transfer phosphatidylcholine to both of these acceptors. The initial rate of transfer to lung lamellar bodies is about half that of the rate of transfer to the liver mitochondria when both acceptor membranes are present at saturating concentrations. Phosphatidylcholine unilamellar vesicles were used to demonstrate that the fatty acyl composition of the membrane phosphatidylcholine is a significant determinant of the rate of phosphatidylcholine transfer catalyzed by these proteins. The lamellar bodies have a unique phosphatidylcholine composition, and these studies suggest that this is an important factor in determining the lower initial rate of transfer to lamellar bodies. The studies have also characterized two phospholipid transfer proteins in rat lung in terms of isoelectric point. Isoelectric points for the two proteins which transfer phosphatidylcholine were found to be 5.6 +/- 0.08 and 6.2 +/- 0.03.

Additional effects of monovalent cations on the lysine-sensitive aspartokinase of E. coli B.

The apparent specificity of activation of lysine-sensitive aspartokinase (E.C.2.7.2.4) from E. coli by monovalent cations differs depending on the assay used and on the Mg2+ concentration. Activity is nearly absolutely dependent on and is highly specific for a monovalent cation in the aspartate semialdehyde dehydrogenase coupled assay or the adenosine triphosphate-adenosine diphosphate exchange assay. Little specificity for monovalent cations is observed using the aspartyl hydroxamate assay. Activation and specificity are also altered by Mg2+ concentrations at a constant 5 mM nucleotide concentration. At a low (1.25 or 1.6 mM)Mg2+ concentration, monovalent cation activation and specificity are nearly absolute. Less dependence on monovalent cations and less specificity are observed at a higher Mg2+ concentration (6 mM). Li+ inhibits aspartokinase competitively with respect to either K+ or NH4+. Monovalent cations are also thermoprotective and differential thermal inactivation experiments at 56 degrees C reveal that NH4+ and K+, either of which will produce maximum catalytic activity, interact differently with aspartokinase. K+ interacts with positive cooperativity, whereas NH4+ does not. K+, NH4+, and Na+ are about equally effective in enhancing the dissociation of the aspartokinase-aspartylphosphate complex. Li+ is less effective.

On the suitability of organotypic cultures of fetal rat lung type II cells for biochemical studies concerning development.

When organotypic cultures of fetal rat lung epithelial cells are initiated with undifferentiated cells, the cells differentiate into type II cells (Douglas, W.H.J., McAteer, J. A., Smith, J.R. and Braunschweiger, W.R. (1979) Int. Rev. Cytol., Suppl. 10, 45-65). This conclusion was based only on morphologic studies. The present study was undertaken to investigate whether such maturation in culture could also be demonstrated biochemically. In organotypic cultures initiated with epithelial cells from fetal rat lungs at 17-days gestation, the amount of phospholipids increased for at least 10 days. However, no change took place in the percentage of phosphatidylglycerol nor in the ratio of disaturated to total phosphatidylcholine. In cultures initiated with cells obtained at day 17 of gestation the specific activity of cholinephosphate cytidylyltransferase reached a maximum after approximately 3 days, followed by a decrease. A similar profile was obtained, however, if the culture was started at day 20 of gestation. This indicates that the activity profiles obtained in the organotypic cultures reflect changes caused by the culture conditions rather than changes caused by maturation. From these investigations it is concluded that biochemical studies on type II cell development using organotypic cultures as model should be interpreted with caution.

Acylation of 1-palmitoyl-lysophosphatidylglycerol in a alveolar type II cells from rat lung.

1. Alveolar type II cells from adult rat lung have the enzymic capability to acylate 1-palmitoyl-lysophosphatidyl-glycerol, using palmitoyl-CoA to form dipalmitoylphosphatidylglycerol. 2. On a protein basis the acylation of 1-palmitoyl-lysophosphatidylglycerol is at least 2-fold more active in sonicated type II cells than in whole lung homogenates. 3. Both type II cells and whole lung homogenates show higher activity towards palmitoyl-CoA than towards oleoyl-CoA for acylation of 1-palmitoyl-lysophosphatidylglycerol. 4. Both in type II cells and in whole lung homogenates the rates of acylation of 1-palmitoyl-lysophosphatidylglycerol and 1-palmitoyl-lysophosphatidylcholine with palmitate are of the same order of magnitude, while the rate of acylation of 1-palmitoyl-lysophosphatidylethanolamine is much lower.