Epitope Mapping for Monoclonal Antibodies Identifies Functional Domains of Pulmonary Surfactant Protein A That Interact with Lipids*

Pulmonary surfactant protein A (SP-A) contains 4 domains: a disulfide forming amino terminus, a colla-gen-like region, a neck region, and a carbohydrate rec- ognition region. The protein binds the lipids dipalmitoylphosphatidylcholine and galactosylceramide and induces aggregation of phospholipid vesicles. SP-A also inhibits lipid secretion and enhances the uptake of phospholipid by alveolar type I1 cells. Previously described monoclonal antibody 1D6 blocks the inhibitory effect of SP-A on lipid secretion by type I1 cells, but antibody 6E3 has no effect. In the present study we mapped the epitopes for monoclonal antibodies 1D6 and 6E3 by enzyme-linked immunoassay of recombinant pro- teins expressed using the baculovirus system, and investigated the domain that is responsible for the SP-A interactions with lipid. Monoclonal antibody 1D6 bound to mutant S P A in which the neck portion of the molecule was deleted or substituted with that of mannose-binding protein A, but 6E3 failed to bind to these mutants. In contrast, 1D6 did not bind to a chimera in which the

Pulmonary surfactant protein A (SP-A) contains 4 domains: a disulfide forming amino terminus, a collagen-like region, a neck region, and a carbohydrate recognition region. The protein binds the lipids dipalmitoylphosphatidylcholine and galactosylceramide and induces aggregation of phospholipid vesicles. SP-A also inhibits lipid secretion and enhances the uptake of phospholipid by alveolar type I1 cells. Previously described monoclonal antibody 1D6 blocks the inhibitory effect of SP-A on lipid secretion by type I1 cells, but antibody 6E3 has no effect. In the present study we mapped the epitopes for monoclonal antibodies 1D6 and 6E3 by enzyme-linked immunoassay of recombinant proteins expressed using the baculovirus system, and investigated the domain that is responsible for the SP-A interactions with lipid. Monoclonal antibody 1D6 bound to mutant S P A in which the neck portion of the molecule was deleted or substituted with that of mannose-binding protein A, but 6E3 failed to bind to these mutants. In contrast, 1D6 did not bind to a chimera in which the carbohydrate recognition domain (CRD) was substituted with that of surfactant protein D (SP-D). In addition, 1D6 failed to recognize antigen in cells infected with the recombinant virus directing the synthesis of a C y~~~-C y s~'~ (small disulfide loop) deletion within the CRD. Antibody 1D6 completely blocked the binding of SP-A to dipalmitoylphosphatidylcholine and galactosylceramide and liposome aggregation. By comparison, 6E3 failed to completely attenuate the interactions of SP-A with lipids. However, both 6E3 and 1D6 blocked the uptake of lipid by type I1 cells that is caused by S P A From these data, we conclude that: 1) the epitope for antibody 6E3 is located at the neck domain of SP-A and that for antibody 1D6 is at the small loop region in the CRD; 2) the CRD is essential for the SP-A functions of lipid binding, liposome aggregation, the inhibitory effect on lipid secretion, and the augmentation of lipid uptake by type I1 cells, and these activities are largely attributable to amino acid residues within the steric inhibitory footprint of 1D6 bound to the small disulfide search Grants HL45286, HL02423, and HL29891. The costs of publica-* This research was supported by National Institutes of Health Retion of this article were defrayed in part by the payment of page in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
charges. This article must therefore be hereby marked "advertisement" loop region; and 3) the neck domain of SP-A may also be involved in the process of SP-A-mediated uptake of phospholipids by alveolar type I1 cells.
Pulmonary surfactant is a complex mixture of lipids and proteins that function to keep alveoli from collapsing at the end of expiration. Surfactant protein A (SP-A)' is a major glycoprotein component of surfactant, with a reduced denatured molecular mass of 26-38 kDa in the rat (1). The polymorphism of this protein is mainly due to the differential glycosylation of 26-kDa species (2). The c D N h for SP-A from human, rat, dog, rabbit, and mouse have been isolated and sequenced (3)(4)(5)(6). In vitro studies with SP-A have provided compelling evidence to demonstrate that it can function as an inhibitor of phospholipid secretion by alveolar type I1 cells (7,8 ) via interaction with a high affinity cell surface receptor (9)(10)(11)(12). SP-A (13) binds to dipalmitoylphosphatidylcholine (DPPC) and galactosylceramide (GalCer) (14)(15)(16)(17) and facilitates phospholipid uptake by type I1 cells (18). SP-Apreferentially enhances DPPC uptake by type I1 cells, and it facilitates the incorporation of this lipid into lamellar bodies (19). Since DPPC is the principal component responsible for the biophysical properties of surfactant, SP-A may play a n important role in phospholipid homeostasis in the alveolar space. SP-A can also accelerate calcium-induced aggregation of phospholipid vesicles (20) and form tubular myelin-like structures in concert with SP-B when these proteins are added to artificial phospholipid mixtures (21).
SP-A is a member of the C-type lectin superfamily and along with mannose-binding proteins A and C, surfactant protein D (SP-D), conglutinin (221, and CL43 (23) it forms the collectin (Group 111) subgroup (24). These proteins possess characteristic structural features of: 1) a n NH,-terminal domain containing cysteine(s1 involved in interchain disulfide bond formation; 2) a collagenous domain that is rich in hydroxyproline; 3) a neck domain; and 4) a carbohydrate recognition domain (CRD). The minimal CRD has been identified as the COOH terminus from Gly'07 to Alazz1 m ' rat mannose-binding protein A (MBP-A) (25, 261, which corresponds to that from Gly1I5 to PheZz8 in the rat SP-A. The structural determinants of SP-A responsible for its multiple functions have not been fully mapped, although some chemical modification and proteolytic degradation studies have provided important insights. The integrity of disulfide bonds is required for SP-A to inhibit surfactant secretion (1) and to The abbreviations used are: SP-A, surfactant protein A: DPPC, dipalmitoylphosphatidylcholine; GalCer, galactosylceramide; CRD, carbohydrate recognition domain; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; MBP-A, mannose-binding protein A PG, phosphatidylglycerol; PC, phosphatidylcholine.

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aggregate phospholipid (27). Removal of the amino terminus and collagen-like domain markedly attenuates but does not completely eradicate the inhibition of secretion (28) or the binding of the protein to lipids (14).
In an effort to more precisely map structural domains of SP-A that are responsible for specific functions, we have undertaken a program of site-directed mutagenesis and heterologous expression of SP-A using baculovirus vectors. Recent studies reveal that SP-A produced in insect cells by recombinant baculovirus constructs retains significant inhibitory activity upon surfactant secretion by type I1 cells, and induces lipid aggregation despite imperfect post-translational modification to the protein (29). In this report we have utilized site-directed mutagenesis in conjunction with previously isolated monoclonal antibodies for the purpose of both epitope mapping and structure-function analysis of the protein. Five monoclonal antibodies recognizing peptide epitopes have been prepared against Four out of five monoclonal antibodies blocked the inhibitory activity of SP-A on lipid secretion by type I1 cells, but one antibody (6E3) failed to alter the inhibitory effect of this protein (1). The purpose of this study was to map epitopes for monoclonal antibodies that exhibited different effects upon SP-A functions using recombinant proteins expressed in the baculovirus system. This approach has enabled us to map domains involved in lipid binding, liposome aggregation, and lipid uptake by alveolar type I1 cells.

EXPERIMENTAL PROCEDURES
Purification and Iodination of Rat SP-A-Surfactant was isolated from Sprague-Dawley rats given an intratracheal instillation of 10 mg of silica in saline, 4 weeks before lung lavage (30). Native SP-A was isolated and purified from surfactant by mannose-Sepharose 6B column chromatography followed by gel filtration over a Bio-Gel A5m column as described previously (9).
The lZ5I-SP-A was prepared by the method of Bolton and Hunter (31) using the Bolton-Hunter reagent (Amersham) as described previously (9). More than 95% of the radioactivity was precipitated by treatment with 10% (w/v) trichloroacetic acid. The specific activity of '251-SP-A used ranged between 270 and 360 cpm/ng and the major forms of iodinated protein correlated well with the major forms of unlabeled protein as described previously (9).
Monoclonal Antibodies to Rat SP-A-Monoclonal antibodies to rat SP-A were prepared as reported previously (1). Antibodies were purified by affinity chromatography on protein A-Sepharose CL-4B (Pharmacia Biotech Inc.). All monoclonal antibodies recognized epitopes in the polypeptide portion of SP-A, and had nearly equivalent affinity for the SP-A antigen as described previously (1). Monoclonal antibodies 1D6 and 6E3 were used in this study.
Monoclonal Antibody Binding Assay-The binding of monoclonal antibodies, 1D6 and 6E3, to the purified recombinant mutant proteins was examined by ELISA. Aliquots of the mutant proteins (50 pl, 1 pg/ml) in 5 m M Tris buffer, pH 7.4, were coated onto the microtiter wells (Dynatech Laboratories). After adsorption of antigen onto the wells, nonspecific binding sites were blocked with phosphate-buffered saline (PBS) containing 0.1% (v/v) Triton X-100 and 3% (w/v) skim milk (PBS-TS). The wells were incubated with increasing concentrations (0.01,0.1, 1.0, and 10 pg/ml) of 1D6,6E3, or anti-SP-Apolyclonal antibody at 37 "C for 1 h followed by horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG. 0-Phenylenediamine was used as the substrate for the peroxidase reaction. Monoclonal antibody binding to mutant proteins was measured by absorbance at 490 nm. The absorbances of antibody binding at 10 pg/ml are presented.
The monoclonal antibody binding assay for SP-Ahyp~AcY*2~~cyaz18 was carried out by dot-blot analysis since the level of the production of this mutant protein expressed by Spodoptera frugiperda (Sf9) cells was extremely low. Sf9 cells infected with a recombinant virus stock of SP-Ahy~~ACY8ztLI-cY*z18 were harvested and the cell suspension was sonicated with a microprobe. After centrifugation at 7,000 x g for 10 min, an aliquot (5 pg of total protein) from the supernatant was applied onto a nitrocellulose sheet. The nitrocellulose sheet was treated with PBS-TS to block nonspecific binding and subsequently with anti-SP-A antibodies followed by horseradish peroxidase-conjugated goat anti-IgG. Dia-minobenzidine was used as the substrate for the reaction.
DNA Constructs-The 1.6-kilobase cDNA for rat SP-A was isolated and sequenced as described previously (4) and inserted into pVL 941 plasmid vector (29). The 1.2-kilobase cDNA for rat SP-D was isolated (32) and inserted into pVL 1393 plasmid vector as described in the accompanying paper (43). The cDNA for rat MBP-A in pUC8 plasmid vector was the generous gift from Dr. Kurt Drickamer. The mutant cDNAs were produced by the polymerase chain reaction and the technique of overlapping extension (33) using the cDNh for SP-A, SP-D, and MBP-A as templates. The DNA constructs for the deletion mutants of SP-A and the chimera with SP-A and MBP-A, and the chimeras with SP-A and SP-D were inserted into pVL 1392 and pVL 1393 plasmid vectors. Mutations were confirmed by sequencing the entire coding region of each mutant cDNA using the Sequenase kit based on the dideoxynucleotide termination method of Sanger et al. (34).
Expression and Isolation of Recombinant Proteins-The expression of recombinant proteins in the baculovirus system was carried out as described by O'Reilly et al. (35). Monolayers of S. frugiperda (SB) cells were co-transfected with linearized virus DNA (Baculogold, Pharmingen) and the pVL plasmid vectors containing the cDNh for SP-A and mutant proteins. Recombinant baculovirus was purified by three rounds of plaque purification. Viral titers were amplified to approximately 10' plaque-forming unitdml. For protein expression, the recombinant virus stock was used to infect Sf9 cells at a multiplicity of 2-5 at 28 "C for 1 h, and the infective media was then removed and the cells were incubated in Excel1 400 media. Media was harvested and the recombinant SP-A and the chimeras of SP-A and SP-D were purified by affmity chromatography on mannose-Sepharose 6B by adsorption in the presence of 1-5 m~ CaCl, and elution with 2-10 m~ EDTA as described previously (29). For the purification of the deletion mutants of SP-Aand the chimera with SP-A and MBP-A, a protein A-Sepharose CL-4B column covalently coupled with monoclonal antibody 1D6 was used, since these mutants fail to bind to the mannose-Sepharose 6B column.
Lipids-DPPC, PG, and GalCer were purchased from Avanti Polar Lipids, Inc. Egg PC was purchased from Sigma. 1-palmitoyl 2-[3Hlpalmitoyl-L3-phosphatidylcholine ([3HlDPPC) was obtained from DuPont NEN. To prepare liposomes, DPPC:egg PC:PG, 7:2:1, was dried under nitrogen and hydrated in 20 m M Tris buffer, pH 7.4, containing 0.15 M NaCl (1.0 mg of phospholipid/ml) at 37 "C for 1 h. Unilamellar liposomes were prepared by sonication of the lipid mixture above with a microprobe as described previously (29). For the experiments examining liposome uptake by alveolar type I1 cells, the liposomes were radiolabeled with trace amounts of L3H1DPPC (1600 cpdnmol, final specific activity).

Binding of 1251-SP-A to Lipids-The direct binding of SP-A to DPPC
and GalCer on thin-layer chromatograms was visualized using lZ5I-SP-A as a probe as described previously (15,16). DPPC (3.5 pg) and GalCer (3.5 pg) were separated by thin-layer chromatography (TLC) on Polygram Si1 G (Macherey-Nagel, Postfach, Germany) with a solvent system of chloroform:methanol:water (65:35:5, v/v). After development with the organic solvent, the plate was air-dried and soaked in 50 m M Tris buffer, pH 7.4, containing 0.1 M NaCl, 2 m M CaCI,, and 20 mg/ml bovine serum albumin (the binding buffer). Next, the TLC plate was incubated with lZ5I-SP-A (0.25 pg/ml) in the presence or absence of monoclonal antibodies (20 pg/ml) in the binding buffer for 30 min at room temperature. The plate was then washed with gentle shaking on ice in the washing buffer (50 m~ Tris buffer, pH 7.4, containing 0.1 M NaC1,2 m M CaCl,, and 1 mg/ml bovine serum albumin). The TLC plate was finally air-dried and exposed to x-ray film at -70 "C overnight.
The binding of lZ5I-SP-A to lipids coated onto microtiter wells was also performed as described previously (15). DPPC or GalCer (1 pglwell) in 20 pl of ethanol was put into the microtiter well (Immulon Removawells, Dynatech Laboratories) and air-dried. After preincubation with binding buffer to block nonspecific adsorption, 50 pl of '=I-SP-A (1 pg/ml) in the same buffer containing the indicated concentrations (1-400 pg/ml) of monoclonal antibodies was incubated for 1 h at room temperature. The wells were washed three times with the ice-cold washing buffer and then the radioactivity of each well was measured using a y radiation counter.
Liposome Aggregation-Liposome aggregation was determined using the method of Hawgood et al. (20). Unilamellar liposomes (200 pg/ml), SP-A (5 pg/ml), and monoclonal antibodies (50 pg/ml) in 20 m M Tris buffer, pH 7.4, containing 0.15 M NaCl were preincubated in a cuvette for 3 min. After equilibration, turbidity was measured at 400 nm using a Beckman DU-64 spectrophotometer at room temperature. Following the initial absorbance readings, CaCl, was added to a final concentration of 5 m~ at 30 s and turbidity was further measured for 5 min. In some experiments, after inducing liposome aggregation in the presence  Type I1 cells (lo6 cells) were incubated with 0.5 ml of Dulbecco's modified Eagle's medium containing 10 m~ HEPES, pH 7.4, radiolabeled phospholipid liposomes (100 pg/ml), SP-A (5 pg/ml), and monoclonal antibodies (50 pg/ml) at 37 "C for 1 h. After incubation, cells and media were separated by centrifugation at 160 x g for 5 min at 4 "C. The medium was removed and the cells were gently resuspended in 1 ml of ice-cold phosphate-buffered saline containing 1 mg/ml bovine serum albumin. The washings were done three times. Before the final centrifugation, the cell suspension was transferred to a fresh tube. The final cell pellet was analyzed for radioactivity. Other Methods--Protein contents were estimated by the bicinchoninic protein assay kit (BCA) (Pierce) using bovine serum albumin as a standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed by the method of Laemmli (37).

Construction of Mutant Proteins and Proteins Expressed in
Insect Cells-Group I11 C-type lectins such as MBP-A, SP-A, and SP-D are composed of four characteristic domains which are: 1) a n NH,-terminal domain involved in interchain disulfide formation; 2) a collagenous domain; 3) a neck domain; and 4) a carbohydrate recognition domain (CRD) (4,32,38,39). For simplicity these domains are denoted A1-A4 in SP-A, Ml"4 in MBP-A, and Dl-D4 in SP-D. All proteins expressed in baculovirus-infected insect cells lack proline hydroxylation and hence are denoted SP-AhyP (29). In this study we constructed three deletion mutants of SP-A, a chimeric molecule of SP-A/MBP-A and three chimeric molecules of SP-NSP-D as follows: 1) mutants in whichAla81 to Glylo3, Leu"' to Leu"', or Cys'04 to Cys218 to Lys105 of MBP-A, and Gly113 to PheZz8 of SP-A (230 amino acids); or 3) chimeras AlA2D3D4 (233 amino acids), AlA2A3D4 (229 amino acids), and DlD2A3A4 (351 amino acids) as described in the accompanying paper (43). The recombinant proteins used in this study are schematically represented in Fig. 1.
Analysis of native SP-A by SDS-PAGE reveals the protein migrates as a triplet at 26, 32, and 38 kDa (Fig. 2, lune a). In contrast, SP-A produced by the baculovirus expression system migrates as a set of bands at 25-32 kDa on SDS-PAGE under reducing conditions (SP-AhYP) (Fig. 2, lune b). The difference in molecular weight between native and recombinant protein is related to the glycosylation pattern by insect cells and the lack of hydroxylation of proline residues as described previously chimera used for the monoclonal antibody binding assay were also analyzed by electrophoresis (Fig. 2, lunes c, d, and e, respectively). These mutant proteins also migrated as broad bands as observed in the SP-AhYP. The protein bands evident after Coomassie Blue staining of the electrophoretic gels are identical to those reactive with polyclonal antibody upon immunoblot analysis indicating that the purified proteins are isoforms of SP-A. The electrophoretic profiles also demonstrate the purity of the protein preparations and the minimal degree of proteolysis. Electrophoretic characterization of chimeras of SP-A and SP-D also employed in this study is described in the accompanying paper (43).
Epitope Mapping-Previous experiments from this laboratory have described the isolation and properties of two monoclonal antibodies 1D6 and 6E3 (1). These antibodies have  markedly different actions when they bind SP-A. Antibody 1D6 completely blocks the action of SP-A as an inhibitor of secretion and prevents SP-A binding to its high affinity receptor (1,12). Antibody 6E3 fails to alter the action of SP-A as an inhibitor of secretion and partially blocks interaction of SP-A with its receptor (1,12). The mutant proteins and chimeras described above were analyzed for their reactivity to the antibodies 1D6 and 6E3 for the purpose of mapping the epitopes and correlating domain structure with protein function. To examine these epitopes, monoclonal antibody binding to recombinant proteins was measured by ELISA (Fig. 3). Polyclonal anti-SP-A IgG was used as a positive control and it bound to all mutant proteins used. Non-immune mouse IgG was used as a negative control and it failed to bind any of the proteins (data not shown). Both monoclonal antibodies, 1D6 and 6E3, bound to SP-AhYP as well as DlD2A3A4, but neither antibody bound to AlA2D3D4, indicating these antibodies recognize epitopes located in the A3 + A4 regions (neck domain + CRD) of SP-A. Antibody 1D6 bound cating that all these proteins contain the relevant epitope. In contrast, 6E3 bound to none of the neck-deletion mutants or the chimera in which the neck of SP-A is substituted with the neck of MBP-A. Antibody 1D6 failed to bind to AlA2A3D4 but 6E3 bound to this chimera to nearly the same extent as the polyclonal antibody. These results clearly indicate that these monoclonal antibodies recognize different epitopes of SP-A and that 1D6 binds to the A4 domain and 6E3 binds to the A3 domain.
We further investigated the epitope for 1D6 by dot-blot analysis using cell lysates from Sf9 cells infected with the recombinant virus encoding SP-Ah~P~Jc~s204~c~sz'8 as the antigen (Fig. 4). Both 6E3 and polyclonal anti-SP-A IgG bound to SP-AhYP and SP-Ah~~~~c~s204-c~s2'8. In contrast, 1D6 exhibited no binding to SP-AhYP~scYa2~~cYsz'8. Control mouse IgG did not bind any of these antigens. In addition, lysates from non-infected Sf9 cells failed to bind any antibodies. Analysis of cell lysates was necessary because SP-Ahyp~ACyrzo4.CYs2'8 was not secreted. Antibody 1D6 also recognizes reduced and alkylated SP-A that has been transferred to nitrocellulose indicating that the three-dimensional arrangement of the small disulfide loop is not required for antibody binding. Collectively, these data are consistent with the conclusion that the epitope for 1D6 is localized at Cys204-Cys2I8 in the A4 domain (CRD) and that for 6E3 is at the A3 (neck) domain.
Effect of Monoclonal Antibodies upon the Binding of SP-A to L i p i d s S P -A binds the lipids DPPC and GalCer. We next sought to determine what structural domains of the protein were involved in these processes and how these domains were to sp-AhYPp~'~81-~~Y103 Sp_AhYPs~Ul05-~Ull2, and AlMM3A4, indi-related to the epitopes for 1D6 and 6E3. The results presented in Fig. 5 demonstrate the binding of '251-SP-A (0.25 pg/ml) to DPPC and GalCer on TLC plates in either the absence or presence of monoclonal antibodies (20 pg/ml). Antibody 1D6 and polyclonal anti-SP-A antibody blocked the binding of SP-A to both DPPC and GalCer. In contrast, antibody 6E3 showed almost no effect on the lipid-binding property of SP-A.
To further characterize the effect of monoclonal antibodies, 1251-SP-A binding (1 pg/ml) to lipids coated onto microtiter wells at 1 pg/well was performed in the presence of various concentrations ( 1 4 0 0 pg/ml) of antibodies. When '251-SP-A was incubated with DPPC coated onto the wells in the absence of antibodies, 16.6 2 3.2 ng/well (mean 2 S.E., n = 3) of SP-A bound to DPPC. 1D6 diminished the binding of SP-A to DPPC in a concentration-dependent manner (Fig. 6A). 1D6 completely blocked the SP-A binding to DPPC at 10 pg/ml, while inclusion of control mouse IgG a t 1 0 4 0 0 pg/ml in the binding solution failed to eliminate 12sII-SP-A binding to DPPC. 6E3 reduced the binding of SP-A to DPPC by approximately 55% at high concentrations but also failed to completely block the binding of Iz5I-SP-A to DPPC. Next, the effect of antibodies on the binding of SP-A to GalCer was also examined (Fig. 6B). The amount of lZ5I-SP-A binding to GalCer in the absence of antibodies was 18.6 2 2.3 ng/well (mean 2 S.E., n = 3). The results of antibody effects on GalCer binding were similar to that on DPPC binding. Antibody 1D6 completely blocked the binding of SP-A to GalCer but 6E3 failed to completely block SP-A binding to GalCer. These results indicate that the CRD (A41 domain is directly involved in the binding of SP-A to phospholipid and glycolipid. Epitope-specific Inhibition of SP-A-induced Liposome Aggregation by Monoclonal Antibodies-In addition to binding lipid, SP-A can induce aggregation of DPPC liposomes. We next sought to determine the effects of the monoclonal antibodies 1D6 and 6E3 upon this process. SP-A and phospholipid liposomes were preincubated in the presence of monoclonal antibodies, and the turbidity caused by lipid aggregation that occurs with the addition of CaCl, was measured (Fig. 7). SP-A aggregated phospholipid vesicles in a time-dependent manner. When SP-A, liposomes, or CaCl, was deleted, negligible light scattering was observed (data not shown). Control mouse IgG showed no effect on liposome aggregation. Antibody 1D6 completely prevented the liposome aggregation effected by SP-A. The results obtained using 1D6 at 50 pg/ml were identical to those found using the antibody a t 100 pg/ml (data not shown). In contrast, SP-A retained the ability to induce liposome aggregation even in the presence of 6E3, although 6E3 reduced the turbidity by approximately 35% with respect to the final extent of aggregation. Increasing the 6E3 concentration to 200 pg/ml failed to cause any further reduction in lipid aggregation (data not shown). Next, the effect of monoclonal antibodies on preformed SP-A-vesicle aggregates was investigated. SP-A was mixed with phospholipid liposomes and the aggregation was initiated by the addition of 5 mM CaCl,. Monoclonal antibodies were added after 5 min and the turbidity was further measured for 10 min (Fig. 8). Addition of 6E3 into the cuvette in which SP-A had caused liposome aggregation decreased the turbidity to a new equilibrium absorbance that was 81% of the control value. By comparison, 1D6 decreased the equilibrium absorbance to 19% of the control value. These results demonstrate that 1D6 but not 6E3 can reverse the liposome aggregation induced by SP-A.
Since the epitope for 1D6 is associated with the small disulfide loop ( C y~~~~-C y s~'~) , the data further indicate that this region of the CRD (A41 domain plays a critical role in aggregation of phospholipid vesicles by SP-A. IgG (control) a t 5 pg/ml followed by anti-rabbit IgG or anti-mouse IgG conjugated with horseradish peroxidase, as described under "Experimental Procedures."

Effect of Monoclonal Antibodies on SP-A-mediated Phospho-
lipid Uptake by Alveolar Type ZZ Cells-Another measure of the interaction of SP-A with phospholipid is the ability of the protein to augment liposome uptake by freshly isolated alveolar type I1 cells. The basis of this uptake is poorly understood but it is thought to be related to the phospholipid recycling documented to occur within the alveolus (40,411. When type I1 cells were incubated with phospholipid liposomes containing L3H1 DPPC in the presence of SP-A (5 pg/ml), the protein enhanced the uptake of liposomes by approximately 5 times the levels found without SP-A (Fig. 9). We examined the effect of monoclonal antibodies on the SP-A-mediated liposome uptake by type I1 cells. The radioactivity sedimented in the presence of SP-A and monoclonal antibodies in the absence of cells was less than 2% of that occurring in the presence of cells. When 1D6 and 6E3 were incubated with cells and liposomes in the presence of SP-A, the protein-mediated uptake of lipid was essen- tially reduced to basal levels. These data implicate the neck domain of SP-A as well as the CRD as an important structural domain involved in the process of SP-A-mediated uptake of phospholipid vesicles by alveolar type I1 cells. DISCUSSION SP-A has been shown to inhibit secretion of surfactant lipids by primary cultures of alveolar type I1 cells (7,8) and to bind to a high affinity receptor expressed on type I1 cells (1,10,11). Interactions of SP-A with phospholipids have also been reported. SP-A specifically binds to DPPC (15) and GalCer (16,17), and causes the aggregation of phospholipid liposomes con-  taining DPPC (20). This protein can also enhance the uptake of phospholipid liposomes by type I1 cells. We previously reported the preparation of several monoclonal antibodies recognizing peptide epitopes on SP-A(l). Monoclonal antibody ID6 blocked the inhibitory effect of SP-A upon surfactant lipid secretion by type I1 cells. In contrast, monoclonal antibody 6E3 failed to alter the inhibitory effect of SP-A on lipid secretion. Our previous studies indicated that monoclonal antibodies to SP-A, which exhibited different effects on functions of the protein, ultimately should prove useful for structurally mapping these domains. The purpose of this study was to determine the relationship of SP-A structure and biological functions by mapping epitopes for monoclonal antibodies using recombinant proteins expressed in the baculovirus system. We examined the effects of monoclonal antibodies on the functions of SP-A in lipid binding, phospholipid vesicle aggregation, and liposome uptake by type I1 cells. Antibody 1D6 completely blocked all of the SP-A functions examined in this study. Antibody 6E3 failed to abrogate the SP-A activities of lipid binding and phospholipid vesicle aggregation. Interest-ingly, 6E3 blocked the effect of SP-A on lipid uptake by type I1 cells to the same extent as antibody 1D6. This observation is in marked contrast to the disparate effects of the two antibodies upon SP-A-mediated inhibition of surfactant secretion (1D6 prevents SP-Afrom inhibiting secretion and 6E3 has no effect). These results imply that the interaction of SP-A with the cell surface receptor involved in regulating secretion is different from the interaction with components of the type I1 cell surface involved in lipid uptake. Such a result suggests that type I1 cells may have multiple receptors for SP-A. Previous work with the antibody 6E3 (12) and recombinant forms of SP-A (29) have also been consistent with the idea that there are multiple receptors for SP-A.
The data presented in this report also demonstrate that the A4 domain (CRD) is essential for the inhibitory activity of SP-A on lipid secretion and the stimulatory activity of SP-A upon lipid uptake by type I1 cells. Furthermore, the results suggest that the A3 (neck) domain also plays an important role in the process of SP-A-mediated uptake of phospholipids. Taken together, these observations suggest that some interaction be- -+ + + + + + + tween domains A3 and A4 is likely to be required for the lipid uptake phenomenon. We have shown in the accompanying paper (44) that SP-Ahyp,G1n195,hp197 mutant, in which GluIg5 and ArgIg7 of SP-A have been converted to Gln and Asp, respectively, fails to inhibit lipid secretion and to compete with 1251-SP-A for cell surface binding although it retains the ability to bind DPPC. These data are consistent with the conclusion that GluIg5 and ArgIg7 are essential for the inhibitory effect of SP-A on lipid secretion. Although antibody 1D6 failed to bind to SP-Ahyp~AC~sza4-Cys218 it did bind to for antibody 1D6 is localized to the loop region (residues 204-218) but probably not to the adjacent containing region of the protein. Our preferred interpretation of these results is that antibody binding to the epitope contained in the region sterically prevents receptor access t o critical residues GluIg5 and Since l'D6 recognizes reduced and alkylated SP-A after transfer from denaturing electro-Sp-Ahyp,Gln195.A"pl97 (data not shown), indicating that the epitope phoretic gels to nitrocellulose, it is probable that this antibody does not require a specific three-dimensional conformation of the region for binding. Both monoclonal antibodies, 1D6 and 6E3, blocked the SP-A-mediated uptake of lipids by type I1 cells, while only 1D6 blocked the binding of SP-A to lipids and the SP-A-induced liposome aggregation. The antibody 1D6 appears to alter SP-A interaction with lipids primarily by preventing the initial binding. The binding of SP-A to lipids is considered as the essential first step to initiate SP-A-mediated liposome aggregation and lipid uptake by type I1 cells. The hydrophobic and amphipathic region (neck domain) of SP-A may be involved in the uptake process of lipids by type I1 cells at a step subsequent to lipid binding, since antibody 6E3 allows SP-A to bind lipids.
Ross et al. (14) described that the collagenase-resistant fragment of dog SP-A (CRF) could bind to phospholipids and that an acidic COOH-terminal fragment comprising residues Gly115 to PheZ3' did not bind to lipids, indicating the importance of the corresponding hydrophobic amino acid residues Gly78 to Val1I4 in the rat.
We expressed the recombinant proteins of SP-, , and the AlA2M3A4 chimera to examine the role of the neck domain in SP-A function. These mutant proteins all failed to bind to a mannose-Sepharose affinity column in spite of retaining the minimal CRD (25, 26). The neck domain might be important for the folding of SP-A, but this indicates that we are unable to determine the lipid binding property using these inactive mutant proteins. Spissinger et al. (42) reported that deletion mutants of SP-A expressed in COS cells, lacking small parts of the non-collagenous domain, interfered with the correct folding and assembly of the molecule. Our results are consistent with these previous findings. Without suitable deletion mutants, we must rely upon the results obtained with antibody 6E3 to infer the function of the neck domain. The results indicate that the neck region accounts for some but not all of the lipid binding of SP-A. This is consistent with the result shown in the accompanying paper (43). The AlA2A3D4 chimera bound DPPC a t 25% of the level of SP-AhYP, whereas the AlA2D3D4 chimera failed to bind any DPPC. Thus, the hydrophobic region (A3 domain) of SP-A may contribute to lipid binding but it does not appear responsible for all of the lipid binding properties of SP-A.
In summary, we found that the epitope for antibody 6E3 is located at the neck domain of SP-A and that for antibody 1D6 is at the small loop region in the CRD. Antibody 1D6 completely blocked the binding of SP-A to lipids, SP-A-induced liposome aggregation, and SP-A-mediated lipid uptake into type I1 cells. In contrast, antibody 6E3 did not block the binding of SP-A to lipid or SP-A-mediated lipid aggregation. However, antibody 6E3, like antibody 1D6, did inhibit the SP-A mediated lipid uptake into type I1 cells.
These results demonstrate that the CRD is an important protein domain involved in SP-A-lipid interactions and that the region of the small disulfide loop is either part of or close to (as defined by the antibody inhibitory footprint) residues essential for lipid binding, aggregation, and cellular uptake. Our findings also demonstrate that the neck domain plays a role in lipid uptake phenomena.