From the Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo 060, Japan and the § Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206
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ABSTRACT |
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Pulmonary surfactant proteins A (SP-A) and D (SP-D) are collectins in the C-type lectin superfamily. SP-A binds to dipalmitoylphosphatidylcholine and galactosylceramide, and it regulates the uptake and secretion of surfactant lipids by alveolar type II cells. In contrast, SP-D binds to phosphatidylinositol (PI) and glucosylceramide (GlcCer). We investigated the functional region in the carbohydrate recognition domain of rat SP-A and SP-D that is involved in binding lipids and interacting with alveolar type II cells by using chimeric proteins. Chimeras ad3, ad4, and ad5 were constructed with SP-A/SP-D splice junctions at Gly194/Glu321, Gln173/Thr300, and Met134/Cys261, respectively. All three chimeras lost SP-A-specific functions. Chimeras ad3, ad4, and ad5 bound to PI with increasing activity. In contrast, chimeras ad3 and ad4 did not bind to GlcCer, whereas ad5 avidly bound this lipid. From these results, we conclude that 1) the SP-A region of Glu195-Phe228 is required for lipid and type II cell interactions, 2) the SP-D region of Cys261-Phe355 is required for optimal lipid interactions, and 3) the structural requirement for the binding of SP-D to PI is different from that for GlcCer.
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INTRODUCTION |
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Pulmonary surfactant is a complex mixture of lipids and proteins that acts to keep alveoli from collapsing during expiration (1). Surfactant protein A (SP-A)1 is the most abundant hydrophilic surfactant protein (2). SP-A binds to dipalmitoylphosphatidylcholine (DPPC) (3), a lipid component essential for the biophysical function of surfactant, and initiates aggregation of liposomes containing DPPC in the presence of calcium (4). SP-A also binds to the glycosphingolipids galactosylceramide (GalCer), lactosylcermide, and asialo-gangliotriaosylceramide (5, 6). The specific interaction of SP-A with alveolar type II cells has also been documented. SP-A inhibits the secretion of surfactant phospholipids (7, 8) and enhances the uptake of liposomes by type II cells (9). In addition, SP-A binds to alveolar type II cells with high affinity (10, 11), and a receptor binding activity correlates well with the inhibitory effect of SP-A on lipid secretion (12).
The in vitro functions of SP-A have all of the characteristics expected of a regulatory protein involved in surfactant homeostasis within the alveolar compartment of the lung. However, mouse strains with null alleles for SP-A are viable and appear to have only modest alterations in surfactant properties (13). The possibility of functional redundancy between SP-A and other molecules remains to be elucidated. In addition to its interaction with lipid and type II cell ligands, SP-A interacts with macrophages (14) and a large spectrum of microorganism, including viruses (15) and Pneumocystis carinii (16), and is now thought to be an important component of the innate (antibody-independent) immune system of the lung. SP-A-deficient mice have been shown to be susceptible to group B streptococcal infection (17). A clear understanding of the interaction of SP-A with its multiple ligands is likely to provide important insight into its physiological roles in surfactant dynamics and host defense.
Surfactant protein D (SP-D) is also a hydrophilic protein of the extracellular alveolar compartment produced by type II cells (18, 19). It has a molecular mass of 43 kDa under denaturing and reducing conditions (20). SP-D also binds specific lipids, but its specificity is distinct from that of SP-A. SP-D binds phosphatidylinositol (PI) (21, 22) and glucosylceramide (GlcCer) (23). The role of SP-D in lipid metabolism in the surfactant system has not yet been clearly defined. A growing body of evidence also implicates SP-D as an important element of the innate immune system of the lung. The protein binds viruses (24), bacteria, fungi (25), and P. carinii (26). SP-D also binds to different classes of molecules on macrophages. One of the SP-D-binding proteins found on the surface of alveolar macrophages has been purified (27). A clear definition of the ligands for SP-D and their binding sites on the protein is essential for understanding how this protein functions within the alveolar compartment.
SP-A and SP-D belong to the collectin subgroup of the C-type lectin superfamily, along with mannose-binding protein (MBP), bovine conglutinin, and the protein CL43 (28). These proteins possess characteristic primary structures that are organized into four domains: 1) a cysteine-containing amino terminus, 2) a collagen-like domain, 3) a neck domain, and 4) a carbohydrate recognition domain (CRD) (2). SP-A forms a bouquet-like structure consisting of an octadecamer composed of six trimeric subunits (29), whereas SP-D is a dodecamer that forms a cruciform structure (30). Although lung collectins possess homologous structures and exhibit similar carbohydrate binding specificities, they show quite different properties of binding lipids and interacting with alveolar type II cells. Previous studies have suggested that the CRD of SP-A or SP-D plays a critical role in interactions with lipids and type II cells (31-34). However, the regions of the CRDs that are essential for lipid binding and type II cell interactions have not yet been fully identified. The purpose of this study was to investigate the functional region in the CRD of rat SP-A or SP-D for the interactions with lipids and type II cells by using chimeric proteins of SP-A and SP-D. We constructed SP-A/SP-D chimeras in which progressively longer carboxyl-terminal regions of SP-D were substituted for the corresponding SP-A regions and examined whether these chimeras retained the SP-A functions or acquired the SP-D functions. We also determined whether anti-rat SP-A monoclonal antibodies that affect the SP-A functions recognized the SP-A/SP-D chimeras. In this report, we show that the SP-A region of Glu195-Phe228 is required for the interaction of SP-A with lipids and type II cells and that the structural requirement for the binding of SP-D to PI is different from that for its GlcCer binding.
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EXPERIMENTAL PROCEDURES |
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Isolation and Purification of Rat SP-A-- Surfactant was isolated from lung lavage fluids of Sprague-Dawley rats (4) that had been given an intratracheal instillation of silica in saline 4 weeks before the lavage (35). The surfactant was delipidated by extraction with 1-butanol. SP-A was isolated and purified from the delipidated surfactant by mannose-Sepharose 6B column chromatography followed by gel filtration over Bio-Gel A5m as described previously (10).
Lipids-- DPPC, phosphatidylcholine from egg yolk, PI, phosphatidylglycerol from egg yolk, phosphatidylserine (PS) from bovine brain, GalCer, and GlcCer were purchased from Sigma. Cholesterol was obtained from Serdary Research Laboratories. The 1-palmitoyl-2-[3H]palmitoyl-L-3-phosphatidylcholine ([3H]DPPC) was purchased from NEN Life Science Products.
DNA Construction of SP-A, SP-D, and Chimeric Molecules-- The isolation and sequencing of the 1.6-kilobase cDNA for rat SP-A was described previously (36). The isolation of the 1.2-kilobase cDNA for rat SP-D was also previously reported (37).
We constructed three chimeric proteins with SP-A and SP-D in which the progressively longer carboxyl-terminal regions of the SP-D CRD were substituted for the corresponding CRD regions of SP-A. The chimeric molecules used in this study are schematically represented in Fig. 1. Chimera ad3 consists of Asn1-Gly194 of SP-A and Glu321-Phe355 of SP-D. Chimera ad4 consists of Asn1-Gln173 of SP-A and Thr300-Phe355 of SP-D. Chimera ad5 consists of Asn1-Met134 of SP-A and Cys261-Phe355 of SP-D. The cDNAs for chimeric molecules were constructed by the polymerase chain reaction and the overlap extension method (38) using the cDNAs for SP-A and SP-D as the templates. The two primers used at the SP-A/SP-D splicing junctions were 5'-AACTGGTACCCAGGAGAGCCCAACAACAAT-3' and 5'-ATTGTTGTTGGGCTCTCCTGGGTACCAGTT-3' for chimera ad3; 5'-ATGATTGAAGACCAGACGGAGGGCAAGTTC-3' and 5'-GAACTTGCCCTCCGTCTGGTCTTCAATCAT-3' for chimera ad4; 5'-ACCATTAAAGAGATGTGCAGGCAGGCCGGA-3' and 5'-TCCGGCCTGCCTGCACATCTCTTTAATGGT-3' for chimera ad5. An SP-A sense and an SP-D antisense primers used were 5'-TCTAGAGAATTCTGTGGCAGAAGCCACTGG-3' and 5'-TAACCCGGGGCTAAGCTCCTGGCCCAG-3', respectively. The EcoRI and XmaI sites were incorporated into the flanking 5' and 3' primers, respectively. All of the constructions were inserted into pVL1392 plasmid vector using the EcoRI and XmaI sites. The recombinant plasmids constructed were confirmed by a combination of restriction enzyme mapping and DNA sequencing.
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Expression and Isolation of Recombinant Proteins--
The
recombinant proteins were expressed in the baculovirus system using the
methods described by O'Reilly et al. (39). Monolayers of
Spodoptera frugiperda (Sf-9) cells were cotransfected with linearized virus DNA (BaculoGold, Phamingen) and the pVL 1392 plasmid
vector containing the cDNAs for SP-A, SP-D, and the chimeras. Recombinant plaques were isolated and viral titers were amplified to
approximately 5 × 107 to 5 × 108
plaque-forming units/ml. The recombinant viruses were used to infect
monolayers of Sf-9 cells in serum-free insect medium at a multiplicity
of 2. The culture medium was then collected and dialyzed against 5 mM Tris buffer (pH 7.4) containing 0.15 M NaCl and 1 mM EDTA to remove monosaccharides. The culture medium
was then applied to an affinity column of mannose-Sepharose 6B in the
presence of 5 mM CaCl2. The recombinant
proteins were eluted with 5 mM Tris buffer (pH 7.4)
containing 10 mM EDTA. The purified recombinant proteins
were dialyzed against 5 mM Tris buffer (pH 7.4) and stored
at 20 °C.
Antibody Binding Assay-- Monoclonal antibodies were prepared against rat SP-A as described previously (42). The monoclonal antibodies recognized epitopes in the polypeptide portion of SP-A and had nearly equivalent affinity for the SP-A antigen. The epitopes for antibodies 1D6 and 6E3 have been identified to be located at the CRD and the neck domain, respectively (32).
The binding of antibodies to SP-A, SP-D, or the chimeric proteins was examined by enzyme-linked immunosorbent assay (ELISA). Aliquots of the purified proteins (50 µl, 1 µg/ml) in 5 mM Tris buffer (pH 7.4) were coated onto the microtiter wells and incubated with polyclonal antibody to SP-A or SP-D, or monoclonal antibody 1D6 or 6E3 (50 µl, 10 µg/ml) at 37 °C for 1 h after the nonspecific binding sites were blocked with phosphate-buffered saline containing 0.1% (v/v) Triton X-100 and 3% (w/v) skim milk. The wells were washed and incubated with horseradish peroxidase-labeled anti-rabbit IgG or anti-mouse IgG. o-Phenylenediamine was used as the substrate for the peroxidase reaction. The antibody binding to the proteins was measured by absorbance at 492 nm.Binding of Chimeras to Lipids Coated onto Microtiter Wells-- Phospholipids (1 µg/well) in 20 µl of ethanol were coated on microtiter wells and air-dried. After the nonspecific binding was blocked with 50 mM Tris buffer (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl2 and 2% (w/v) bovine serum albumin, various concentrations of SP-A or the chimeric proteins (0-20 µg/ml) were added and incubated for 1 h at room temperature. After the incubation, the wells were washed three times with ice-cold buffer of 50 mM Tris (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl2, and 1 mg/ml of bovine serum albumin. Monoclonal antibody 6E3 (10 µg/ml) was then added and incubated for 1 h, followed by the incubation with horseradish peroxidase-labeled anti-mouse IgG. The binding of the proteins to lipids was determined by measuring the absorbance at 492 nm using o-phenylenediamine as a substrate for the peroxidase reaction. The binding of SP-D to the lipids was examined in the same manner as described using anti-SP-D polyclonal antibody and horseradish peroxidase-labeled anti-rabbit IgG.
Binding of Chimeras to Multilamellar Liposomes-- DPPC, PI, or the lipid mixture composed of GalCer:PS:cholesterol (7:2:1, w/w/w) or GlcCer:PS:cholesterol (3:4:3, w/w/w) was dried under nitrogen and hydrated in 20 mM Tris buffer (pH 7.4) containing 0.1 M NaCl at 37 °C for 1 h and vortexed vigorously for 5 min to prepare multilamellar liposomes. The protein solutions (0.2 µg/tube) were prepared in 50 µl of 20 mM Tris buffer (pH 7.4) containing 0.1 M NaCl, 5 mM CaCl2, and 20 mg/ml of bovine serum albumin (binding buffer). Then the multilamellar liposomes (100 µg) and the protein solutions were separately centrifuged at 12,000 rpm at room temperature for 10 min. The supernatant of the protein solution (50 µl) was added to the liposome pellet. The mixtures were suspended and incubated for 1 h at room temperature. The mixture was then put on ice for 15 min and then centrifuged at 12,000 rpm at 4 °C for 10 min. The supernatant was stored, and the resultant pellet was once washed with ice-cold binding buffer (50 µl/tube) and centrifuged again. The supernatant was then combined, and the pellet was suspended with 100 µl of the binding buffer. The amount of the proteins in each fraction was determined by competitive ELISA as described below. Liposome binding was defined as percentage of sedimentation (the proteins in pellet/the proteins in pellet plus supernatant × 100). Control experiments without liposomes were also performed.
Competitive ELISA-- Because monoclonal antibody 6E3 binds all three chimeras to the same extent (see "Results"), we developed a competitive ELISA to determine the amount of chimeras in liposome pellets and the supernatants. The wild type (wt) recombinant SP-A (0.5 µg/ml, 100 µl/well) was coated onto microtiter wells. After the nonspecific binding was blocked with phosphate-buffered saline containing 3% (w/v) skim milk, 0.1% (v/v) Triton X-100 (blocking buffer), the various dilutions of samples or standard wt SP-A (50 µl/well), and antibody 6E3 (1 µg/ml, 50 µl/well) were added to the wells and simultaneously incubated at 37 °C for 90 min. After the wells were washed, horseradish peroxidase-labeled anti-mouse IgG (1:1000, 100 µl/well) was added and incubated for 37 °C for 60 min. The peroxidase reaction was finally developed using o-phenylenediamine and the absorbance was measured at 492 nm.
To determine the amount of SP-D, wt SP-D (0.2 µg/ml) as the solid phase antigen, anti-SP-D polyclonal antibody (1 µg/ml), and anti-rabbit IgG were used instead of solid phase wt SP-A, antibody 6E3, and anti-mouse IgG, respectively.Liposome Aggregation-- Liposome aggregation was performed by a modified method based on that described by Hawgood et al. (4). Unilamellar liposomes were prepared from the multilamellar liposomes composed of DPPC:egg phosphatidylcholine:phosphatidylglycerol (7:2:1, w/w/w) by probe sonication for 5 min. Unilamellar liposomes (200 µg/ml) and the purified proteins (20 or 80 µg/ml) in 20 mM Tris buffer (pH 7.4) containing 0.15 M NaCl were preincubated for 5 min. After equilibration, absorbance was measured at 400 nm using a Hitachi U-2000 spectrophotometer at room temperature. Following the initial absorbance reading, CaCl2 was added to a final concentration of 5 mM after 3 min, and the absorbance was further measured until a time of 10 min.
Isolation of Alveolar Type II Cells and Uptake of Phospholipid Liposomes-- Alveolar type II cells were isolated from the lungs of male Sprague-Dawley rats by tissue dissociation with elastase digestion and purification on metrizamide gradients (43). Uptake of phospholipids by freshly isolated type II cells was performed by the method described by Wright et al. (9). Briefly, freshly isolated type II cells (2 × 106 cells) were incubated with radiolabeled phospholipid liposomes (100 µg/ml) composed of DPPC:egg phosphatidylcholine:phosphatidylglycerol (7:2:1, w/w/w) and a trace amount of [3H]DPPC, in the presence of the indicated amounts of proteins (5 or 20 µg/ml) at 37 °C for 1 h in 0.5 ml of Dulbecco's modified Eagle's medium containing Hepes (10 mM) (pH 7.4). Following the incubation, cells and media were separated by centrifugation at 1500 rpm for 5 min at 4 °C. The cells were resuspended with 1 ml of ice-cold phosphate-buffered saline containing 1 mg/ml bovine serum albumin. The washing steps were repeated three times. Before the final centrifugation, the cell suspension was transferred to a fresh tube. The final cell pellet was analyzed for radioactivity.
Secretion of Phosphatidylcholine from Alveolar Type II
Cells--
Type II cells (2 × 106 cells) were
incubated overnight with [3H]choline (0.5 µCi/ml) in
Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf
serum. Secretion of radiolabeled phosphatidylcholine was performed
using 12-o-tetradecanoylphorbol-13-acetate (TPA) (107 M) as the stimulant for the surfactant
secretion and the purified proteins as antagonists, as described
previously (42). Following a 3-h incubation, radiolabeled phospholipids
were extracted from the cells and media, and counted. The lipid
secretion from type II cells was expressed as percentage of secretion
(radioactivity in medium/radioactivity in medium plus cells × 100).
Recptor Binding--
The binding of chimeras to type II cells
was performed by determining the ability of chimeric proteins to
compete with rat 125I-SP-A for receptor occupancy on
alveolar type II cells, as described previously (12, 44). Briefly,
monolayers of freshly isolated type II cells (2 × 106/dish) were cultured overnight, and then were incubated
with 0.5 µg/ml rat [125I]SP-A in the presence of 20 or
40 µg/ml unlabeled proteins for 3 h at 37 °C. The cells were
washed four times on ice with 50 mM Tris buffer (pH 7.4)
containing 0.1 M NaCl, 2 mM CaCl2,
and 1 mg/ml bovine serum albumin. The cells were then dissolved in 2 ml
of 0.1 N NaOH and the radioactivity associated with the
cells was determined using a -counter.
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RESULTS |
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Characterization of Chimeric Recombinant Proteins-- Wild type SP-A, SP-D, and SP-A/SP-D chimeras were expressed using the baculovirus expression system. All three chimeras used in this study were purified by affinity chromatography using a matrix of mannose-Sepharose 6B. The preservation of lectin activity confirms a significant degree of fidelity in the protein folding. The purified recombinant proteins and native rat SP-A were analyzed by SDS-polyacrylamide gel electrophoresis. Native SP-A migrated as a triplet at 26, 32, and 38 kDa (Fig. 2A, lane nat/A). In contrast, recombinant wt SP-A migrated as a broad band at 27-32 kDa under reducing conditions (Fig. 2A, lane wt/A). The differences in molecular mass between native and recombinant protein is attributable to the differences in glycosylation between mammalian cells and insect cells and to the lack of hydroxylation of proline residues, as described previously (45). The recombinant wild type SP-A retains all of the functions of its native counterpart (45). When analyzed under reducing conditions, SP-A/SP-D chimeras migrated as broad bands at 27-35 kDa (Fig. 2A, lanes A/D chimeras 3-5). Analysis under nonreducing conditions revealed that the wild type SP-A and all of the chimeric molecules migrated as oligomers (Fig. 2B, lanes A/D chimeras 3-5). Under nonreducing conditions the recombinant proteins (SP-A and SP-A/SP-D chimeras) showed significant levels of monomer that are a consequence of incomplete covalent oligomerization. wt SP-D migrated as a band at 43 kDa under reducing condition. wt SP-D appeared mainly as a monomer with smaller populations of oligomers under nonreducing conditions (Fig. 2, A and B, lanes wt/D), which is consistent with the result observed in the previous study (33).
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Binding of Anti-SP-A Monoclonal Antibodies to SP-A/SP-D Chimeras-- Previous studies revealed that the epitopes for anti-SP-A monoclonal antibodies 1D6 and 6E3 are located at the CRD and the neck domain, respectively (32). Antibody 1D6 has been reported to block the binding of SP-A to lipids, the SP-A-induced liposome aggregation, the SP-A-mediated lipid uptake into type II cells, the inhibitory activity of SP-A on lipid secretion, and the receptor binding (12, 32). Antibody 6E3 partially blocked SP-A-receptor interactions and completely blocked lipid uptake by alveolar type II cells. To examine whether monoclonal antibodies to SP-A recognized SP-A/SP-D chimeras, antibody binding to the purified proteins was measured by ELISA. Antibody 1D6 failed to bind to chimera ad3, ad4, or ad5 (Fig. 3). In contrast, antibody 6E3 bound to all of the chimeras, as well as to wild type SP-A. When monoclonal antibodies were tested for the reactivity to purified proteins applied to nitrocellulose membranes, the results were consistent with those obtained by ELISA (data not shown). Anti-SP-A polyclonal antibody bound to chimera ad3 at nearly one-half the level of that of wt SP-A, and it bound very weakly to chimeras ad4 and ad5. Anti-SP-D polyclonal antibody exhibited almost no binding to chimeras ad3 and ad4 but recognized ad5 at a level equivalent to that of wt SP-D. The results indicate that the substitution of the SP-D region Glu321-Phe355 for the SP-A region Glu195-Phe228 disrupts an epitope for anti-SP-A monoclonal antibody 1D6 and are consistent with our previous results that antibody 1D6 failed to recognize antigen in Sf-9 cells infected with the recombinant virus directing the synthesis of Cys204-Cys218 deletion within the CRD (32). The nearly equivalent binding of 6E3 to chimeras and to wt SP-A suggests that antibody 6E3 is useful as a probe for the detection of chimeras in further experiments. We next directly evaluated whether the SP-A/SP-D chimeras possess the functions of either SP-A or SP-D.
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Interaction of Chimeras with Lipids-- We first examined the ability of chimeras to bind lipids coated onto microtiter wells using antibody 6E3 as a probe (Fig. 4). Native SP-A bound to DPPC and GalCer strongly. wt SP-A also bound to these lipids in a concentration-dependent manner (Fig. 4, A and B). The binding of wt SP-A to DPPC was approximately 30% of that of native SP-A at 20 µg/ml, whereas wt SP-A showed binding to GalCer at a level nearly equivalent to native SP-A. The wt SP-D and the chimeras ad3, ad4 and ad5 did not exhibit any binding to DPPC or GalCer (Fig. 4, A and B). The binding of the recombinant proteins to multilamellar liposomes containing DPPC or GalCer was also examined by the sedimentation method (Fig. 5, A and B). As much as 63 or 40% of the wild type SP-A was cosedimented with liposomes containing DPPC or GalCer, respectively. None of the chimeras cosedimented with the DPPC or GalCer liposomes. These results clearly indicate that the chimeras do not show any of lipid binding specificity exhibited by SP-A. Furthermore, the SP-D region of Glu321-Phe355 cannot replace the SP-A region of Glu195-Phe228 with respect to the binding to DPPC or GalCer. The data demonstrate that the SP-A region of Glu195-Phe228 is required for the interaction with DPPC and GalCer.
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Interaction of Chimeras with Alveolar Type II Cells-- Another measure of the SP-A-specific interaction with lipids is the ability to mediate liposome uptake by freshly isolated alveolar type II cells (9). Therefore, we examined whether the chimeras mediated liposome uptake. The wild type SP-A enhanced the uptake of liposomes containing DPPC into type II cells in a concentration-dependent manner (Fig. 7); 3.4 and 7.9% of liposomes were taken up in the presence of 5 and 20 µg/ml of wild type SP-A, respectively. When type II cells were incubated with chimeras or wild type SP-D in the presence of radiolabeled phospholipids, the protein-mediated increases of cell-associated radioactivities were not observed.
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DISCUSSION |
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The purpose of this study was to investigate the CRD regions of SP-A and SP-D involved in binding lipids and interacting with alveolar type II cells by using chimeric proteins of SP-A and SP-D. Although structurally related, these collectins exhibit distinct functions. SP-A binds DPPC and GalCer (3, 5), whereas SP-D binds PI and GlcCer (21-23). SP-A also interacts with alveolar type II cells (10, 11). Previous studies have identified the CRDs as essential for the interactions of lung collectins with lipids and type II cells (31-33). In this study, we focused on the CRD regions of SP-A and SP-D, and progressively longer carboxyl-terminal regions of the SP-D CRD were substituted for the corresponding SP-A CRD regions. The rationale for this approach is based on the conserved primary structure organization observed in SP-A, SP-D, and other collectins. The SP-A/SP-D chimeras were expressed in insect cells and purified by affinity chromatography of mannose-Sepharose 6B. The preservation of lectin activity unambiguously demonstrates that carbohydrate recognition and binding are retained in the chimeras and that global folding defects are unlikely.
The present work shows that the SP-A region of Glu195-Phe228 is essential for the interaction of SP-A with lipids and type II cells. The work also indicates that the SP-D region of Glu321-Phe355 is essential for the binding to PI, and that the binding of SP-D to GlcCer is critically dependent upon the SP-D region of Cys261-Phe355. This demonstrates that the structural requirement for the binding of SP-D to PI is different from that for the binding of SP-D to GlcCer. Some evidence has accumulated to understand the structure-function relationships of surfactant proteins. Site-directed mutagenesis of SP-A (45) reveals that the N-linked oligosaccharide moiety is not required for type II cell interaction or for phospholipid vesicle aggregation. Altered carbohydrate binding specificity engineered into SP-A or SP-D is accompanied by changes of the functions of these proteins (44, 46). Although the amino-terminal domain and the collagenous domain are essential for the oligomerization of the protein (29, 34, 47) to stabilize these macromolecules, the CRDs have been shown to be functional domains of SP-A and SP-D (31-33). The present study specifies the regions within the CRDs that are critical for the functions of SP-A and SP-D. Although the work in this report is largely limited to well defined low molecular weight ligands, it may have important consequences for unraveling the interactions of SP-A and SP-D with the surfaces of microorganisms that contain both carbohydrate and lipid determinants available for recognition.
A recent study from this laboratory (48) showed that the SP-A/MBP-A chimera (AM3) composed of the SP-A region Asn1-Gly194 and the MBP-A region Glu185-Ala221 retained SP-A functions, although MBP-A does not possess the ability to interact with DPPC, GalCer, or type II cells as SP-A does. This demonstrates that the MBP-A region of Glu185-Ala221 can functionally replace the SP-A region of Glu195-Phe228. However, the present report reveals that the SP-D region of Glu321-Phe355 cannot replace the homologous SP-A region of Glu195-Phe228 without loss of the SP-A functions. It is unknown why different results are obtained from the replacement studies between the homologous proteins, although lung collectins and MBP-A show similarities in carbohydrate binding specificity and oligomeric structure and 48-53% sequence identity between the SP-A region of Glu195-Phe228 and the corresponding regions of MBP-A or SP-D. An understanding of this discrepancy awaits the resolution of the crystal structures of lung collectins.
Anti-SP-A monoclonal antibody 1D6 did not bind to any of the ad chimeras. Because antibody 1D6 blocks SP-A interactions with lipids and type II cells (12, 32, 42), the loss of the 1D6 epitope by ad chimeras correlates with the dysfunction of the ad chimeras with respect to the SP-A properties. Furthermore, the results from antibody binding and functional assays of chimera ad3, in which the small disulfide loop region (Cys204-Cys218) of SP-A has been replaced with that of SP-D, are consistent with our previous finding that 1D6 binds to epitopes in this region (32). Collectively, these data are consistent with the conclusion that the SP-A region of Glu195-Phe228 is required for binding DPPC and GalCer, causing phospholipid liposome aggregation, enhancing lipid uptake, and inhibiting lipid secretion from type II cells.
Antibody 6E3 did not block the binding of SP-A to lipids but did inhibit the SP-A-mediated lipid uptake by type II cells (32). Because antibody 6E3 recognizes the neck domain of SP-A (32), the neck domain may also be involved in the process of the SP-A-mediated uptake of phospholipids. In this study, the chimeras ad3-ad5, which contain the SP-A neck, failed to bind DPPC and to augment lipid uptake by type II cells. Thus, these results indicate that the neck domain of SP-A cannot account for all of the process of SP-A-mediated lipid uptake and support the idea that the binding of SP-A to lipids is an initial step for lipid uptake by type II cells.
A previous study (33) using SP-A/SP-D chimeras revealed that the A1A2A3D4 chimera, in which the whole SP-A CRD (Gly115-Phe228) was replaced with the corresponding domain of SP-D, weakly bound to DPPC liposomes. These findings suggested a positive role for the neck domain in DPPC binding by SP-A. In the present study, the chimeras ad3-ad5 did not bind to DPPC or GalCer. The only structural difference between the A1A2A3D4 chimera and ad chimeras is that the whole SP-D CRD is directly connected to the SP-A neck domain in the A1A2A3D4 chimera, but the ad3-ad5 chimeras contain the SP-A neck plus a portion of the SP-A CRD. These observations support the ideas that the CRD of SP-A is the most important domain for the lipid binding property of the protein and that the hydrophobic neck region may contribute to some aspect of lipid binding but cannot account for all of the interaction. These studies are also consistent with the epitope localization and effect of monoclonal antibodies 1D6 and 6E3 on the lipid binding of SP-A (32).
The current results narrow the minimal SP-D structure required for PI binding to the Glu321-Phe335 portion of the CRD. In contrast, the minimal region of SP-D required for GlcCer binding extends from Cys261 to Phe355. These conclusions are consistent with those made previously (46) suggesting that there are distinct differences between the PI and GlcCer binding sites. The findings highlight chimeras ad3-ad5 as important new tools for examining the mechanism of lipid binding by SP-D. The structures composed of chimera ad3 or ad4 appear to be specific for PI binding without the property of GlcCer binding. It will certainly be very interesting to determine how these chimeric proteins interact with complex biological targets such as bacteria, viruses, and fungi.
In summary, we focused on the CRDs of lung lectins with different lipid binding specificities, expressed novel proteins of SP-A/SP-D chimeras and examined the interaction of the chimeras with lipids and alveolar type II cells. All of the chimeras lacked the specific SP-A interactions with lipids and type II cells. In contrast, the chimeras exhibited partial to complete interaction with the SP-D ligand PI depending upon the length of the SP-D sequence. Only one of the chimeras, ad5, retained the GlcCer binding activity of SP-D. From these data, we conclude that 1) the SP-A region of Glu195-Phe228 is required for the SP-A functions; 2) the SP-D region of Glu321-Phe355 is essential for the binding to PI, and the binding increases with the extension of the region to Cys261; 3) the binding of SP-D to GlcCer is critically dependent upon the SP-D region of Cys261-Phe355; and 4) the structural requirement for SP-D binding to PI is different from that for GlcCer.
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ACKNOWLEDGEMENTS |
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We thank Drs. Hirofumi Chiba and Masaki Saitoh for helpful discussion and graphic works.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan and from ONO Medical Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Department of
Biochemistry, Sapporo Medical University School of Medicine, South-1 West-17 Chuo-ku, Sapporo 060, Japan. Tel.: 81-11-611-2111; Fax: 81-11-612-5861; E-mail: kurokiy{at}sapmed.ac.jp.
1 The abbreviations used are: SP, surfactant protein; wt, wild type; CRD, carbohydrate recognition domain; DPPC, dipalmitoylphosphatidylcholine; PI, phosphatidylinositol; GalCer, galactosylceramide; GlcCer, glucosylceramide; PS, phosphatidylserine; ELISA, enzyme-linked immunosorbent assay; MBP, mannose-binding protein; TPA, 12-o-tetradecanoylphorbol-13-acetate.
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