Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Alveolar type II pneumocytes have a central role in the assembly and secretion of lung surfactant lipid components, mainly dipalmitoyl phosphatidylcholine and cholesterol. These cells are able to synthesize all lipid classes, but the major part of cholesterol is taken up from plasma lipoproteins (1). All types of lipoproteins, high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL), are accepted by type II cells as lipid sources (2), but the interaction with HDL may be of major physiologic relevance for several reasons. The adsorption of HDL to cell surfaces leads to the selective, bidirectional transport of cholesterol and cholesterol ester without internalization of the lipoprotein particles (3). This process may contribute to the regulation of the cholesterol level in type II cells and of the cholesterol content of the surfactant. Furthermore, the major lipophilic antioxidant vitamin E is preferentially taken up by type II cells from HDL (4).

It is generally accepted that the interaction of HDL with cell surfaces and the HDL-dependent lipid exchange processes are mediated by specific receptors or binding proteins. Convincing evidence is accumulating that the scavenger receptor SR-BI functions as HDL receptor and selective transporter of cholesterol and cholesterol ester in vivo, at least in liver and steroidogenic glands (3). Recently, SR-BI was also detected on type II pneumocytes, and the regulation of the SR-BI expression by vitamin E was demonstrated (4). In addition, numerous attempts to purify and characterize other candidate HDL receptors have been reported, but structural information became available only in a few cases (5, 6). Two HDL-binding proteins, HB1 and HB2, were detected in rat lung in approximately the same high concentration as in liver (7). The physiologic role of HB2 in HDL-binding and HDL-mediated cholesterol uptake was demonstrated by transfection into HepG2 and COS (African green monkey kidney) cells (8). Together, these results are consistent with the view that a variety of HDL-binding proteins may exist, even on the same cell type. In agreement, Guendouzi and coworkers (9) and Morrison and colleagues (10) found on hepatocytes two binding sites differing in dissociation constant (K_d) by up to two orders of magnitude.

Most of the studies on HDL receptors dealt with the liver in relation to the reverse cholesterol transport and with steroidogenic tissues. However, the secretion of surfactant into lung alveoles is another example of a peripheral lipid-consuming process that requires interaction with lipoproteins for the supply with lipid constituents. The essential role of lipoproteins in the assembly of lung surfactant was already underlined by the early work of Hass and Longmore (11, 12) on the cholesterol metabolism in perfused rat lungs, and binding studies indicated the existence of HDL receptors (12). Later, the stimulation by HDL of signal transduction events and of the secretion of surfactant was demonstrated in type II cells, but the participating receptors were not identified (13). The present study therefore aims at the characterization of candidate HDL receptors in rat lung and type II pneumocytes. Two HDL-binding proteins were identified, the previously described HB2 (8) and a membrane-bound dipeptidase. The concentration of both proteins in type II cells increased when the plasma cholesterol concentration was reduced, consistent with the concept that both proteins are functionally involved in the lipid uptake into these cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials

Porcine kidney membrane dipeptidase (MDP) and rabbit polyclonal antibodies (immunoglobulin [Ig] G fraction) against mouse MDP with affinity to the rat enzyme were kind gifts from Dr. N. M. Hooper, University of Leeds, Leeds, United Kingdom. A rabbit antiserum raised against baculovirus/insect cell-expressed HB2 was generously donated by Dr. L. Pyle, Baker Medical Research Institute, Prahram, Victoria, Australia. Protease inhibitors, 4-aminopyrazolo[3,4-d]-pyrimidine (APP), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), lipoprotein lipase from bovine milk, chemicals for electrophoresis, and methyl--D-mannopyranoside were purchased from Sigma (Dreieich, Germany). Elastase was obtained from Boehringer (Mannheim, Germany), heparin-Na from Hoffmann-La Roche (Grenzach-Wyhlen, Germany), and heparin sepharose columns (5 ml HiTrap) from Amersham Pharmacia Biotech (Freiburg, Germany).

Animals

Throughout the study, male Wistar rats (120 to 150 g) had free access to food and water. APP-treated rats were injected intraperitoneally for three consecutive days with 10 mg/kg body weight of the reagent. The APP was suspended at 5 mg/ml in buffer (10 mM Na-phosphate, pH 7.4, 150 mM NaCl), and it was dissolved by titration with 1 M HCl to pH 3.6 (14). Control animals received an equivalent volume of acidified buffer. The concentration of vitamin E in rat plasma was reduced by feeding the rats a vitamin E-depleted diet over 5 wk as detailed elsewhere (4). A control group received standard rat chow, while a third group was treated in the same way as the first group, except that these rats were fed a vitamin E-enriched diet for 48 h before they were killed (4).

Type II Pneumocytes

Rats were anesthetized by intraperitoneal injection of 30 mg sodium pentobarbital, and the lungs were perfused five times with isotonic NaCl before they were excised and used for cell preparation. Type II pneumocytes were removed from lung tissue by digestion with porcine pancreas elastase and purified by panning on IgG-coated plates according to Dobbs and coworkers (15).

HDL-Binding Proteins in Pneumocytes

Freshly harvested cells (20 × 10⁶) were washed in Dulbecco's phosphate-buffered saline without NaHCO₃. The cell pellet was resuspended in 1 ml buffer A containing 10 mM Tris, 10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, pH 7.4, with HCl, 0.32 M sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA), 2.5 µg/ml leupeptin, 5 µg/ml aprotinin, and 2 mM phenylmethylsulfonyl fluoride (PMSF), and the pneumocytes were disrupted by three bursts of 20 s with a tip sonicator at 60% setting (Sonoplus HD60; Bandelin Electronics, Berlin, Germany) according to Fisher and colleagues (16). The suspension was centrifuged at 300 × g for 10 min. The supernatant was recentrifuged at 25,000 × g for 30 min, and the resulting membrane pellet was suspended in 200 µl of buffer B (20 mM Tris/HCl, pH 8.0, 20 mM CHAPS, plus protease inhibitors as in buffer A). The CHAPS-extractable proteins were obtained by overhead rotation for 1 h and by subsequent centrifugation at 10,000 × g for 10 min. The supernatant was stored for up to 4 wk at 80°C. All steps were carried out at zero to 4°C.

Purification of HDL-Binding Proteins from Rat Lung

The purification scheme is based on the procedure of Fidge and coworkers (17, 18) with some modifications. Briefly, lungs from two rats were perfused and lavaged with isotonic NaCl to remove blood cells and surfactant. The tissue was disrupted in the fivefold volume of buffer A by two treatments of 30 s with an Ultra-turrax (IKA Werke, Staufen, Germany) and by two bursts of 30 s with a tip sonicator (Labsonic U; B. Braun Biotech GmbH, Melsungen, Germany). Tissue debris was removed by centrifugation at 300 × g and, after another ultrasonic burst of 30 s, at 1,000 × g. Membrane fragments in the supernatant were collected by centrifugation at 25,000 × g for 30 min. Membrane proteins were extracted from the resuspended pellets by overhead rotation in CHAPS-buffer B for 1 h. Undissolved material was pelleted at 2,000 × g, and the supernatant was stored frozen at 80°C. After thawing, the extract was cleared by centrifugation at 10,000 × g for 10 min before the supernatant was subjected to anion exchange chromatography. Econo-Pac High Q (Bio-Rad, Hercules, CA) and HiTrap Q (Pharmacia Biotech, Uppsala, Sweden) cartridges were used with the same result. The columns were equilibrated and eluted in buffer C (50 mM Tris/HCl, pH 8.0, 10 mM CHAPS) until no ultraviolet-absorbing material was detectable in the effluent. Binding proteins were desorbed in a linear gradient (0 to 0.5 M) of NaCl in buffer C. Fractions that showed HDL-binding activity in ligand blots (100 to 250 mM NaCl) were combined and mixed with concanavalin A-sepharose 4B (Sigma, St. Louis, MO), bed volume 0.8 ml in buffer D (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 10 mM CHAPS). Binding of glycoproteins was achieved during overnight rotation at 4°C. The suspension was then transferred into a column and washed with buffer D before HDL-binding proteins were desorbed with 0.2 M methyl-- D-mannopyranoside in buffer D. Fractions that showed activity in ligand blots were combined, and the proteins were precipitated by a fourfold volume of acetone at 15°C for 16 h. The precipitate was redissolved in 200 µl of buffer C followed by 200 µl of electrophoresis sample buffer containing 10% mercaptoethanol. After heating for 10 min at 40°C, the solution was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 8% gels under a Laemmli buffer system (19). Protein bands were visualized by submerging the gels in 0.3 M KCl. The appropriate gel slices were transferred onto Centricon 10 chambers of an Amicon electroelutor (Millipore, Bedford, MA) and eluted at 200 V for 3 to 4 h. Proteins were concentrated at 2,000 × g at 8°C and stored at 80°C.

Carbohydrate Analysis

The electrophoretically purified binding proteins were deglycosylated in a medium containing 50 mM Na-phosphate, pH 7.5, 20 mM EDTA, 0.5% (wt/vol) CHAPS, 1 mM PMSF, 10 U/100 µl N-glycosidase F (Boehringer) and up to 10 µg/100 µl binding protein. After 16 h at 30°C, the reaction was stopped by adding an equal volume of twofold concentrated electrophoresis sample buffer and by heating at 95°C for 3 min. Proteins were electrophoresed on 8% gels and blotted to nitrocellulose for the detection of the carbohydrate moiety of the glycoproteins by an overlay assay with lectins and peroxidase as previously described (20).

Separation of Lipoproteins

Plasma was isolated from freshly drawn venous blood of normolipemic human males in tubes containing EDTA. Lipoproteins were isolated by KBr/NaCl density gradient ultracentrifugation for 24 h in a Beckman SW 41 Ti rotor at 35,000 rev/min as described by Chapman and colleagues (21), except that all gradient solutions contained 0.05% (wt/vol) NaEDTA and 0.02% (wt/vol) NaN₃. Fractions of 0.5 ml were removed from the meniscus down by aspiration, and the content of apolipoproteins was analyzed by SDS-PAGE to avoid cross-contamination of HDL by apoB and of LDL by apoA. Fractions of HDL with and without electrophoretically detectable apoE content were also identified in this way. In some instances, apoE-containing particles were removed from HDL fractions by affinity chromatography on heparin-sepharose according to Wilson and coworkers (22). The pooled density gradient fractions were desalted and transferred into Tris-buffered saline (TBS) (50 mM Tris/HCl, pH 7.4, 150 mM NaCl) by passage through a column of Sephadex G-25 (PD-10; Pharmacia).

Modification and Analysis of Lipoproteins

LDL was acetylated by acetic anhydride as described by Basu and associates (23) with slight modifications. Freshly centrifuged LDL was desalted via a PD-10 column in 150 mM NaCl. The total volume of the reaction mixture in Na-acetate was increased to 6 ml, whereas the protein concentration was reduced to 0.5 mg/ml, and acetic anhydride was added in aliquots of 0.7 to 0.8 µl. Electrophoretic analysis of acetylated LDL by SDS-PAGE on 6% gels revealed that the apoB-100 band was still detectable by silver staining, and the apoB-100 in acetylated LDL cross-reacted approximately to the same extent as in native samples with anti-apoB antibodies from rabbit (Pierce, Rockford, IL) as detected by dot blotting and visualization by peroxidase-conjugated second antibodies. Thiobarbituric acid-reactive substances as indicators of lipid oxidation were determined by fluorescence (24). The content of freshly prepared LDL samples, which were used as ligands for lipoprotein-binding proteins, was below 4 nmol/mg protein in all cases. The apoA1 content of HDL, as measured by a turbidimetric procedure in the University Hospital's Lab for Clinical Chemistry, amounted to 68% of the total HDL protein.

Electrophoresis and Ligand Blotting

Proteins were separated by electrophoresis in 8% (HDL-binding proteins), 6% (LDL-apoproteins), or 14% (HDL-apoproteins) polyacrylamide gels under a Laemmli buffer system containing 0.1% SDS (19). The sample buffer always comprised 0.5% SDS and 50 mM dithiothreitol (DTT) or 10% 2-mercaptoethanol if reducing conditions were required. Proteins were transferred to nitrocellulose by semidry blotting in medium containing 20% methanol. Protein bands with affinity to HDL, LDL, apoA1, apoB, or acetylated LDL were detected by an overlay assay as described by Hidaka and Fidge (18) with some modifications. Briefly, unspecific binding was blocked with 2% nonfat milk powder in TBS for 1 h. Incubations with lipoproteins and apolipoproteins (50 µg protein/ml) were run in 2 mM CHAPS and 2% milk powder in TBS for 16 h. The filters were washed in TBS and incubated for 1 h with rabbit antihuman apoA1 IgG or rabbit antihuman apoB-100 IgG at dilutions of 1:1,000 in 2% milk powder (both monospecific, purified IgG; Calbiochem, La Jolla, CA). The second antibody, donkey peroxidase-conjugated antirabbit IgG (Pierce), was used in a dilution of 1:2,000 in 2 mM CHAPS/ TBS. Bound peroxidase was detected with 4-chloro-1-naphthol (0.18 mg/ml in TBS). The same second antibodies and electrophoresis procedures were used to detect lipoprotein-binding proteins by immunoblotting. The nitrocellulose filters were blocked with 10% (wt/vol) milk powder in Tween TBS (TTBS) (Tris-buffered 150 mM NaCl, 0.05% Tween 20). Antisera against MDP and HB2 were used in 1:1,000 dilution and second antibodies in 1:2,000 dilution, all in 5% milk powder in TTBS. Bands were visualized with a peroxidase chemiluminescence substrate (Boehringer) on Kodak X-OMAT AR films. A different blotting procedure was used when protein bands were excised for N-terminal sequencing. Proteins were transferred to Immobilon-P membranes (Millipore), pore size 0.45 µm, in borate buffer, and bands were stained with coomassie (25).

Protein Sequence Analysis

N-terminal sequences of the deglycosylated lipoprotein-binding proteins were determined by Edman degradation using an Applied Biosystems sequencer, model Procise (Foster City, CA). The Swissprot database was used for searches of similarities to other known protein sequences. The analysis was performed by WITA GmbH (Teltow, Germany).

Solid Phase Binding Assay

The electrophoretically purified binding proteins were diluted about 20-fold in TBS and concentrated by ultrafiltration (Microcon YM-10; Millipore). The concentrate was again diluted in 400 µl TBS plus 1 mM CHAPS for a second filtration through the same filter. Finally, the concentrated proteins were diluted 50-fold over the initial concentration by addition of TBS. The wells of Immulon microtiter plates (Greiner, Frickenhaus, Germany) were coated with aliquots of 50 µl of the diluted binding proteins for 3 h at 25°C. The wells were washed four times with TBS before unoccupied sites were blocked with 5% nonfat milk powder in TTBS for 16 h at 4°C. All subsequent incubations were carried out in this solution, whereas TTBS was used for the intermediary washing steps. The ligands were incubated at 37°C for 3 h in aliquots of 100 µl/well. Bound ligands were detected by the same first and second antibodies that were used for ligand blotting. The incubations were run for 3 h at 37°C, and the dilutions were 1:1,000 (first antibody) and 1:2,000 (second antibody). Peroxidase activity was measured with a ready-to-use substrate solution containing 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS solution; Boehringer) in a microplate reader at 405 nm. All tests were carried out in triplicate and controls without binding protein coating were run in parallel. Binding data were calculated as described by Ashcom and coworkers (26), using the apoA1 content to define HDL concentrations, whereas the concentration of LDL was based on the protein content.

Other Analytical Procedures

Protein was quantified by Bradford's procedure (27) using an assay kit (Sigma). The cholesterol content of rat plasma and lipoproteins was determined by means of a cholesterol oxidase-based test kit (Merck, Darmstadt, Germany).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Detection and Purification of HDL-Binding Proteins

Ligand blots with HDL as ligand were used in this study as a screening procedure to detect proteins with affinity to lipoproteins. Several bands were detected by this procedure in CHAPS extracts from rat lung membrane preparations (Figure 1, lane a) and in complete protein extracts from rat lung type II pneumocytes (Figure 1, lane b). Some of these bands are hardly visible in the photographic reproduction of Figure 1 because of the rather low sensitivity of the ligand blotting technique. An efficient purification procedure was therefore necessary to detect and to characterize candidate HDL-binding proteins. Two bands, migrating at 53 and 104 kD, were enriched to apparent electrophoretic homogeneity (Figure 1, lanes c and d). Both showed strong HDL-binding activity as detected by ligand blotting (Figure 1, lanes e and f ). In a typical run, the yields from two rat lungs (wet weight, 5 to 5.5 g) were 22 µg protein of the upper band and 10 µg protein of the lower band. It should be noted that the 53-kD band was not detectable in the crude extracts (Figure 1, lane a and b) because this band is a dimeric protein linked by an S-S bridge (see subsequent discussion), and these separations were run without reductant in the sample buffer. A third band with HDL-binding activity migrating at 120 kD could also be purified by this procedure, but this protein was not further analyzed owing to its instability and major contamination with inactive bands (results not shown).

View larger version (80K): [in this window] [in a new window]   Figure 1.   HDL-binding proteins in rat lung membranes and type II pneumocytes. Membrane proteins from lung (lane a) and from type II cells (lane b) as well as purified binding proteins (lanes c-f ) were separated by SDS-PAGE. Bands were visualized by ligand blotting with an HDL overlay (lanes a, b, e, and f ) or by silver staining (lanes c and d).

Structural Properties

The binding to immobilized lectin during the purification suggests that both proteins are glycoproteins. In accordance, affinoblotting with concanavalin A and peroxidase showed positive responses (Figure 2, lanes a and b). Incubation with N-glycosidase F in the presence of CHAPS after heating the samples at 95°C led to the complete disappearance of the concanavalin A-detectable carbohydrates (Figure 2, lanes c and d). Silver staining of the glycosidase-digested, 104-kD band revealed that this band was contaminated with several other proteins, which showed different electrophoretic mobilities upon loss of their carbohydrates (Figure 2, lane e). Only one of these bands, migrating at 70 kD, consistently exerted HDL-binding activity (Figure 2, lane g). In some preparations, a second active band with lower electrophoretic mobility was detected (Figure 2, lane g). Because this band was reduced when the incubation with N-glycosidase F was extended, we assume that it is an incompletely deglycosylated form of the 70-kD band. Further structural studies were therefore carried out with the main band at 70 kD. In addition, it should be noted that the binding activity of the 70-kD band was not detectable when the samples were tested immediately after the incubation with N-glycosidase F, but the activity was restored by treating the samples with a strong reductant, usually DTT at 50 mM. The deglycosylation of the 53-kD HDL-binding protein led to a shift of the electrophoretic mobility to 40 kD (Figure 2, lanes f and h).

View larger version (63K): [in this window] [in a new window]   Figure 2.   Deglycosylation of HDL-binding proteins. Purified binding proteins were electrophoresed and detected by affinoblotting with a concanavalin A overlay (lanes a-d), by silver staining (lanes e and f ), or by ligand blotting with HDL (lanes g and h). The native proteins were applied to lanes a and b, whereas the deglycosylated forms were applied to lanes c-h. The position of the deglycosylated band with HDL-binding activity is indicated by an arrow on lane e.

To obtain structural information on the protein moiety and to evaluate relationships to previously described rat peptides, the deglycosylated proteins were subjected to N-terminal sequencing by Edman degradation. Both bands show 100% N-terminal sequence identity with other rat proteins (Figure 3). The sequence of the 104-kD band is identical to the complementary DNA (cDNA)-predicted sequence at positions 28 to 38 of HB2, a previously described HDL-binding protein from rats (8). Rat HB2 exerts 93% sequence homology with human activated leukocyte-cell adhesion molecule (ALCAM, CD166), an adhesion protein and CD6 ligand of the immunoglobulin superfamily (8). The ALCAM sequence as derived from cDNA contains an N-terminal 27-position signal peptide (28). Because our data show that the mature form of HB2 starts at position 28, it has to be concluded that a signal peptide of the same length is also present in HB2. The reason for the difference between the apparent size of the deglycosylated HB2 (70 kD) and the calculated molecular weight of 65 kD (8) is not known, but we assume that this discrepancy is due to residual carbohydrate, which was resistant to N-glycosidase F and which was not detectable by concanavalin A binding. Similar observations were reported for HB2 from liver (18).

View larger version (11K): [in this window] [in a new window]   Figure 3.   Sequencing by Edman degradation of HDL-binding proteins. The numbering refers to the complete sequences derived from cDNA (8, 29).

The first 18 positions of the N-terminal end of the 53-kD band equal the N-terminal sequence of the mature form of a rat MDP, also known as renal dipeptidase or dehydrodipeptidase, EC 3.4.13.19 (29). In all mammalian species and organs investigated so far, the enzyme was a cystinyl-linked homodimer of 47 to 59 kD subunits, which are each linked to membranes by a well-characterized glycosyl phosphatidylinositol (GPI) anchor (30). The apparent size of 40 kD as determined by electrophoresis with the deglycosylated and reduced rat lung MDP compares reasonably well with the value of 40.5 kD, which was found in other species. This result is therefore consistent with the observation that differences of the apparent size of mature MDP from several mammalian species are due to different glycosylation (30).

Together with the immunologic results (see subsequent text), we consider the evidence that was obtained by electrophoresis and sequencing as sufficient to identify both proteins as MDP and HB2, and these designations will be used in the subsequent parts of this report.

Binding Characteristics

Some recently described candidate HDL receptors, especially of the scavenger receptor group, accept a broad spectrum of ligands (33). As a first step to evaluate the potential physiologic function of the lung HDL-binding proteinsespecially of MDP, which has not been analyzed in this respectthe ligand specificity has to be determined. Two procedures were used for this purpose, ligand blotting and a solid phase assay with immobilized binding proteins on microtiter plates.

Using ligand blots, the affinity toward purified apoA1 in addition to HDL was demonstrated with both binding proteins, HB2 and MDP (Figure 4, lanes a-d). The binding is therefore not strictly dependent on the lipid content of the HDL particles. In addition, the apoE content of the HDL particles was without major influence on the binding because apoE-free and apoE-carrying HDL fractions showed the same binding (results not shown). The binding of HDL was not affected by EDTA (5 mM), CaCl₂ (2 mM), heparin (500 U/ml), or lipoprotein lipase in a concentration of 5 µg/ml (results not shown). Both lung binding proteins differed in their affinity to LDL. MDP was clearly detected in ligand blots with overlays containing LDL, whereas HB2 showed no staining under the same conditions (Figure 4, lanes e and f ). Furthermore, both binding proteins did not exert any affinity to VLDL and acetylated LDL (results not shown). Taken together, these results are in accordance with the investigations of Fidge and coworkers (17, 18) who found that HB2 is a highly specific binding protein for HDL and for its major apolipoproteins, A1 and A2. In contrast, MDP shows a broader ligand specificity with affinity to LDL. In a marked difference to typical scavenger receptors, both binding proteins do not accept acetylated LDL as ligand.

View larger version (52K): [in this window] [in a new window]   Figure 4.   Ligand specificity of rat lung HDL-binding proteins. Deglycosylated binding proteins were electrophoresed and visualized by ligand blotting with HDL (lanes a and b), apoA1 (lanes c and d), or LDL (lanes e and f ) as ligands.

To verify these observations by a different procedure and to measure binding data, the purified proteins were adsorbed to microtiter plate wells and incubated with lipoproteins before the bound apolipoproteins were detected with the appropriate antibodies. In this way, saturable high affinity binding of HDL to HB2 was demonstrated (Figure 5A). The apparent K_d was determined to be 2.1 µg apoA1/ml. As expected, hardly any binding of LDL was detected in this concentration range. The slight increase of the LDL-binding curve at high concentrations may be due to incomplete compensation of nonspecific binding. Figure 5B shows that the binding of HDL to MDP required higher concentrations of lipoprotein than did the HB2 binding. The data yielded an estimate for the apparent K_d of 25 µg apoA1/ml. In addition, the binding of LDL was detected, at least at lipoprotein concentrations above 40 µg/ml.

View larger version (18K): [in this window] [in a new window]   Figure 5.   Concentration dependence of lipoprotein binding. The binding proteins HB2 (A) and MDP (B) were adsorbed to microtiter plate wells. The binding of HDL (closed circles) and LDL (open circles) was quantified by incubation with the lipoproteins and with specific antibodies against apoA1 and apoB-100. The concentrations of HDL are indicated as µg apoA1/ ml, and the LDL concentrations are given as µg protein/ml. Binding of HDL to MDP was also measured in the presence of a twofold concentration of LDL (B, triangles).

To demonstrate mutual influences of the lipoproteins, the HDL binding to MDP was assayed in the presence of a twofold concentration of LDL (Figure 5B). Hardly any effects of LDL on the HDL binding were observed up to a lipoprotein concentration of 20 µl/ml HDL and 40 µg/ml LDL. At concentrations of more than 40 µg/ml HDL (80 µg/ml LDL), when a pronounced binding of LDL has to be expected, the amount of bound HDL was reduced. Only 18% of the maximal HDL binding was observed at an LDL concentration of 200 µg/ml. Obviously, both lipoproteins competed for binding sites on MDP, and HDL was released by LDL. This effect was only detectable at high concentrations of LDL owing to the binding characteristic of this lipoprotein.

In Vivo Investigations

Provided that the lung lipoprotein-binding proteins described here are involved in the lipid metabolism of type II pneumocytes, their expression may change on experimental manipulation of the supply with lipoproteins. In this study, the lipoprotein release in the liver was blocked by APP over 3 d to reduce the plasma cholesterol concentration from 495 to 74.5 µg/ml. The APP treatment led to a clearly detectable increase of both binding proteins, HB2 and MDP, in membrane extracts of type II pneumocytes (Figure 6).

View larger version (40K): [in this window] [in a new window]   Figure 6.   Effect of plasma cholesterol depletion on lipoprotein-binding proteins in vivo. The binding proteins HB2 and MDP were detected by immunoblotting in membrane proteins extracts from type II pneumocytes of control (lane a) and APP-treated rats (lane b). For the detection of HB2, 0.5 µg protein of each sample was applied to gels, whereas 10 µg protein were applied to detect MDP. The type II cells from three rats of each group were pooled. Essentially the same results were obtained in a second experiment with the same number of animals.

Another approach to demonstrate the regulation of these binding proteins is based on our recent observation that the HDL receptor SR-BI is upregulated upon long-term depletion of vitamin E (4). Figure 7 shows that feeding rats a vitamin E-reduced diet resulted in a dramatic increase in type II pneumocytes of both binding proteins. The effect is readily reversible because refeeding the rats a vitamin E-enriched diet for 48 h resulted in the reduction of the protein level of both binding proteins. HB2 was also present at a low level in type II cells of control rats, but the band is hardly visible in the photographic reproduction (Figure 7, upper panel, lane b). In contrast, no changes were detected in complete membrane protein extracts from rat lungs (Figure 7).

View larger version (32K): [in this window] [in a new window]   Figure 7.   Expression of lipoprotein-binding proteins in type II pneumocytes and lung tissue as affected by vitamin E. Equal amounts of membrane proteins from rat type II pneumocytes (T) and from whole lungs (L) were separated by electrophoresis, and the binding proteins HB2 and MDP were detected by immunoblotting. The rats were fed a vitamin E-depleted diet over 5 wk (lane a), the normal rat chow (lane b), or the vitamin E-depleted diet for 5 wk and subsequently a vitamin E-enriched diet for 48 h (lane c).

The level of the binding proteins was estimated by immunoblots using antibodies raised against rabbit kidney MDP with affinity to rat MDP and antibodies against rat HB2. It should be noted that the anti-MDP antibodies detected only the dimeric form of the protein in complete membrane extracts, but the low affinity to rat lung MDP in the monomeric form could be demonstrated with the highly purified protein (results not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Two candidate lipoprotein binding proteins were isolated by standard purification procedures from rat lung and identified by N-terminal sequencing and immunoblotting. One of them, HB2, was characterized before as an HDL-binding protein with high ligand specificity by Fidge and coworkers (8, 17, 18) using biochemical purification from liver, cloning, and transfection. The results of the present investigation on rat lung HB2 are in accordance with these results in respect to substrate specificity and other binding parameters.

The other protein from rat lung, MDP, has not yet been identified as a lipoprotein-binding protein. MDP was described as a metallodipeptidase with zinc in the active center (34). The enzymatic activity is readily blocked by chelators for zinc and strong reductants like DTT (35). Because EDTA and reductants in high concentrations were used during the purification, it is highly improbable that the enzymatic activity is related in any way to the lipoprotein-binding properties of MDP.

Further analysis of the structural properties by Keynan and colleagues (36) revealed that MDP is a homodimeric protein linked by a single S-S bridge. The participating cysteinyl residues are positioned near the C-terminus where the subunits carry GPI anchors, which are therefore positioned close to each other. The primary structures of MDP from several mammalian species were analyzed to reveal more than 80% sequence identity in all cases, but there is no sequence relationship with other mammalian proteins (34).

The feature of MDP as a GPI-anchored cell surface protein is well compatible with a role as a lipoprotein-binding protein, although nothing is known about the binding mechanism and the binding site. Interestingly, GPI-anchored proteins in fibroblasts and in human lung carcinoma SK-MES-1 cells also showed HDL-binding activity on ligand blots, but the identity of these bands was not investigated (37). A common property of GPI-anchored membrane proteins is their preferential caveolar localization in the plasma membrane. In accordance, Nion and associates (37) detected the GPI-linked bands with affinity to HDL, including a 104-kD band, in a caveolar fraction from plasma membranes of their cell lines. The caveolar localization could be of major physiologic relevance because these membrane areas may represent sites of cholesterol exchange with HDL (37).

The view that MDP exerts a nonenzymatic function at least in the lung is supported by contradictory reports on the physiologic role of the dipeptidase activity. Cysteinyl glycine and cystinyl bisglycine are readily hydrolyzed by MDP, and a role in the degradation of glutathione was therefore suggested (35). Analysis of the organ distribution in rat showed that the lung contains the highest level of MDP (29), but the enzyme to catalyze the first step of glutathione degradation, -glutamyl transpeptidase, was almost undetectable in lung tissue (38). Recently, some nonenzymatic functions of MDP were discussed. These include roles in cell differentiation (39), insulin-stimulated generation of second messengers from the GPI anchor (39), and a membrane-stabilizing role in the zymogen granule membrane of pancreatic acinar cells (40). Together, these results are compatible with the view that MDP is a multifunctional protein. In this report, evidence is presented that the binding of lipoproteins, especially of HDL, is one of these functions.

In contrast to HB2, MDP also showed affinity to LDL on ligand blots. The analysis by means of the solid phase assay revealed that substantial binding occurred only above a concentration of about 40 µg/ml. In the presence of both lipoproteins, competition between HDL and LDL resulted in the exclusive binding of HDL at low concentrations, whereas the binding of HDL at high concentrations was prevented by LDL (Figure 5B). Investigations of the distribution in lung tissue by immunohistochemical procedures (41) and by in situ hybridization (38) showed that MDP is not present in blood vessel endothelia but rather in interstitium-exposed cell types. Data on the lipoprotein concentration in rat lung interstitium are not available, but regarding the rather low LDL concentration in rat plasma of about 50 µg protein/ml (42) and the observation that the relation of LDL to HDL is much lower in human interstitium samples compared with plasma (43), the binding of LDL to MDP is probably not of any physiologic relevance in rat lung.

The analysis of the binding data showed that the affinity of HDL to both proteins from rat lung differed in K_d by an order of magnitude. The K_d of the HDL binding to MDP (25 µg apoA1/ml) is comparable to the value of about 30 µg/ml reported for SR-BI (44). In contrast, the affinity of HB2 to HDL with a K_d of 2.1 µg apoA1/ml is extremely low. To our knowledge, similarly low values were only reported for the scavenger receptor CD36 at 2.9 µg/ml (45) and by Guendouzi and coworkers (9) as well as Morrison and colleagues (10) for a high affinity site on hepatocytes (0.51 and 0.94 µg/ml). The latter investigators also reported that only one of four CNBr fragments of apoA1 showed high affinity binding to rat liver plasma membranes as well as affinity to liver HB2 on ligand blots (46). These results are consistent with the observations of the current study, and together they support the conclusion that HB2 is a high affinity binding protein for HDL in lung as well as liver. Differences of the K_d values (0.94 versus 2.1 µg/ml) may be due to the completely different methods used to determine lipoprotein binding. In addition, some modification of the structure and ligand affinity of the purified protein adsorbed to microtiter plates compared with native HB2 in membranes cannot be excluded.

Both rat lung binding proteins, MDP and HB2, were also detected in type II pneumocytes (Figure 6). These cells play an essential role in the assembly of the lamellar bodies, which are secreted into the alveolar space to replenish surfactant lipids and proteins. Type II cells are able to synthesize lipids to a certain extent, but the major part of cholesterol has to be taken up from external lipoprotein sources (1). Intercalated into the alveolar epithelium, type II cells do not have physical contact with the plasma, and the lipid exchange has to proceed at the low interstitial concentration of lipoproteins. Experimental reduction of the plasma lipoprotein level may be accompanied by a drop of the HDL concentration at the surface of type II cells below the necessary level to supplement the cells with lipids and other HDL-transported compounds like vitamin E. The reduction of the plasma cholesterol concentration to 15% of controls was achieved by treatment with APP. This lipid-lowering drug inhibits the synthesis and release of lipoproteins in the liver (47). Figure 6 shows that the cells respond to that situation with an induction of HB2 as well as MDP. These observations are in accordance with an essential role of the lipoprotein-binding proteins from rat lung in the lipid uptake in vivo. Interestingly, the increase of HDL binding upon treatment with APP was also observed with rat ovarian plasma membranes, but the binding sites were not characterized (48).

This view is supported by another set of in vivo experiments using a vitamin E-depleted diet to reduce the concentration of this compound in plasma and organs. Previous investigations showed that rat type II cells take up vitamin E preferentially from HDL (4). In addition, the reduction of the vitamin E content in rat plasma resulted in a pronounced upregulation in type II cells of SR-BI, the well-characterized HDL receptor (4). Both HDL-binding proteins on type II cells, HB2 and MDP, responded in the same way as SR-BI to the vitamin E depletion (Figure 7). This observation is compatible with a role of the binding proteins in the uptake of both compounds, vitamin E and cholesterol.

Type II pneumocytes constitute only a marginal part of the complete cell mass of the lung, and type II cell-specific changes of protein expression are therefore masked in whole lung extracts by the quantitatively more important cell types (Figure 7). In view of the importance of the lipid supply in type II cells owing to their role in the surfactant synthesis, the tight regulation of MDP and HB2 preferentially in this cell type is not surprising.