Pulmonary surfactant protein A (SP-A) specifically binds dipalmitoylphosphatidylcholine.

Phospholipids are the major components of pulmonary surfactant. Dipalmitoylphosphatidylcholine is believed to be especially essential for the surfactant function of reducing the surface tension at the air-liquid interface. Surfactant protein A (SP-A) with a reduced denatured molecular mass of 26-38 kDa, characterized by a collagen-like structure and N-linked glycosylation, interacts strongly with a mixture of surfactant-like phospholipids. In the present study the direct binding of SP-A to phospholipids on a thin layer chromatogram was visualized using 125I-SP-A as a probe, so that the phospholipid specificities of SP-A binding and the structural requirements of SP-A and phospholipids for the binding could be examined. Although 125I-SP-A bound phosphatidylcholine and sphingomyeline, it was especially strong in binding dipalmitoylphosphatidylcholine, but failed to bind phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. Labeled SP-A also exhibited strong binding to distearoylphosphatidylcholine, but weak binding to dimyristoyl-, 1-palmitoyl-2-linoleoyl-, and dilinoleoylphosphatidylcholine. Unlabeled SP-A readily competed with labeled SP-A for phospholipid binding. SP-A strongly bound dipalmitoylglycerol produced by phospholipase C treatment of dipalmitoylphosphatidylcholine, but not palmitic acid. This protein also failed to bind lysophosphatidylcholine produced by phospholipase A2 treatment of dipalmitoylphosphatidylcholine. 125I-SP-A shows almost no binding to dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylethanolamine. The addition of 10 mM EGTA into the binding buffer reduced much of the 125I-SP-A binding to phospholipids. Excess deglycosylated SP-A competed with labeled SP-A for binding to dipalmitoylphosphatidylcholine, but the excess collagenase-resistant fragment of SP-A failed. From these data we conclude that 1) SP-A specifically and strongly binds dipalmitoylphosphatidylcholine, 2) SP-A binds the nonpolar group of phospholipids, 3) the second positioned palmitate is involved in dipalmitoylphosphatidylcholine binding, and 4) the specificities of polar groups of dipalmitoylglycerophospholipids also appear to be important for SP-A binding, 5) the phospholipid binding activity of SP-A is dependent upon calcium ions and the integrity of the collagenous domain of SP-A, but not on the oligosaccharide moiety of SP-A. SP-A may play an important role in the regulation of recycling and intra- and extracellular movement of dipalmitoylphosphatidylcholine.


Pulmonary Surfactant Protein A (SP-A) Specifically Binds
Dipalmitoylphosphatidylcholine* (Received for publication, September 13, 1990) Yoshio KurokiS and Toyoaki Akino From the Department of Biochemistry,Sapporo Medical College,Sapporo 060,Japan Phospholipids are the major components of pulmonary surfactant. Dipalmitoylphosphatidylcholine is believed to be especially essential for the surfactant function of reducing the surface tension at the air-liquid interface. Surfactant protein A (SP-A) with a reduced denatured molecular mass of 26-38 kDa, characterized by a collagen-like structure and N-linked glycosylation, interacts strongly with a mixture of surfactant-like phospholipids. In the present study the direct binding of SP-A to phospholipids on a thin layer chromatogram was visualized using 1251-SP-A as a probe, so that the phospholipid specificities of SP-A binding and the structural requirements of SP-A and phospholipids for the binding could be examined. Although L251-SP-A bound phosphatidylcholine and sphingomyeline, it was especially strong in binding dipalmitoylphosphatidylcholine, but failed to bind phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. Labeled SP-A also exhibited strong binding to distearoylphosphatidylcholine, but weak binding to dimyristoyl-, l-palmitoyl-2-linoleoyl-, and dilinoleoylphosphatidylcholine. Unlabeled SP-A readily competed with labeled SP-A for phospholipid binding. SP-A strongly bound dipalmitoylglycerol produced by phospholipase C treatment of dipalmitoylphosphatidylcholine, but not palmitic acid. This protein also failed to bind lysophosphatidylcholine produced by phospholipase Az treatment of dipalmitoylphosphatidylcholine. 12'I-SP-A shows almost no binding to dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylethanolamine. The addition of 10 m M EGTA into the binding buffer reduced much of the '"I-SP-A binding to phospholipids. Excess deglycosylated SP-A competed with labeled SP-A for binding to dipalmitoylphosphatidylcholine, but the excess collagenase-resistant fragment of SP-A failed. From these data we conclude that 1) SP-A specifically and strongly binds dipalmitoylphosphatidylcholine, 2) SP-A binds the nonpolar group of phospholipids, 3) the second positioned palmitate is involved in dipalmitoylphosphatidylcholine binding, and 4) the specificities of polar groups of dipalmitoylglycerophospholipids also appear to be important for SP-A binding, 5) the phospholipid binding activity of SP-A is dependent upon calcium ions and the integrity of the collagenous domain of SP-A, but not on the oligosaccharide moiety of SP-A. SP-A may play an important role in the regulation of recycling and intra-and extracellular move-* This research was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Japan and a Grant from the Akiyama Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed. ment of dipalmitoylphosphatidylcholine.
Alveolar type I1 cells produce and secrete a complex mixture of lipids and proteins called pulmonary surfactant, which functions to keep the alveoli from collapsing at the end of expiration (1, 2). The major protein component of surfactant, surfactant protein A (SP-A),' is a glycoprotein with a reduced denatured molecular mass of 26-38 kDa in the rat (3). The cDNAs of the human, rat, and dog SP-A molecules have been isolated. This protein possesses the striking feature of collagen-like sequences. SP-A has been identified as a potent negative regulator of surfactant phospholipid secretion by primary cultures of alveolar type I1 cells (4,5). Recent studies (6, 7) have demonstrated the presence of a high affinity receptor for SP-A expressed on alveolar type I1 cells.
Phospholipids are the major components of pulmonary surfactant, making up 80-90% of its weight. Two major classes of surfactant phospholipids are phosphatidylcholine and phosphatidylglycerol, which constitute 70-80% and 5-10% of phospholipids, respectively. Disaturated species, especially with fatty acids being palmitic acid (2), form about 60% of the phosphatidylcholine. Dipalmitoylphosphatidylcholine is believed to be essential for the surfactant function of reducing surface tension at the air-liquid interface. SP-A interacts with surfactant-like lipids, such as dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (8-ll), causes phospholipid aggregation (12), and promotes the rapid formation of stable surface films of phospholipid cooperating with the hydrophobic surfactant proteins (13). SP-A has also been demonstrated to enhance the uptake of phospholipid liposome by alveolar type I1 cells (14). However, whether SP-A binds all phospholipids, which classes of lipids it binds, and how it binds are not fully understood. In the present study the direct binding of SP-A to phospholipids was visualized using '*'I-SP-A as a probe, and the structural requirements of SP-A and phospholipids for the binding was examined. This work provides direct evidence that SP-A specifically binds dipalmitoylphosphatidylcholine.

MATERIALS AND METHODS
Purification of SP-A-Surfactant was isolated from rats given intratracheal instillation of 10 mg of silica in saline 4 weeks prior to lung lavage (15). The surfactant was purified as described by Hawgood et al. (12). SP-A was purified by the method described previously (6).
Briefly, surfactant was delipidated with 1-butanol and the organic solvent soluble lipids were separated from the protein when precipitated by centrifugation a t 2,000 X g for 30 min at room temperature. After the residual butanol was evaporated under a gentle stream of The abbreviations used are: SP-A, surfactant-associated protein A CRF, collagenase-resistant fragment of SP-A; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid TLC, thin layer chromatography; ANSA, 8-anilino-1-naphthalenesulfonic acid. nitrogen, the protein was suspended in 5 mM Tris buffer (pH 7.4) and dialyzed against the same buffer. The suspension was then centrifuged at 150,000 X g. , for 1 h at 4 "C and the supernatant was applied to a mannose-Sepharose 6B (16) column. The SP-A bound to the affinity matrix in the presence of Ca2+ and was eluted with 2 mM EDTA. Further purification and removal of EDTA was accomplished by gel filtration over Bio-Gel A5m (Bio-Rad). The human SP-A was purified from lung lavage obtained from patients with alveolar proteinosis in the same fashion as the rat SP-A. The protein content was estimated by the method of Lowry et al. (17) using bovine serum albumin as the standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to the method of Laemmli (18).
Iodination of Rat SP-A-Rat SP-A was iodinated by the method of Bolton and Hunter (19) using the Bolton-Hunter reagent (Amersham Corp.) as described previously (6). The specific activity of the "'I-SP-A used ranged between 140 and 432 cpm/ng. In all experiments more than 90% of the radioactivity was precipitated by treatment with 10% (w/v) trichloroacetic acid.
Preparation of Modified SP-A-Deglycosylated rat SP-A was prepared based on the method described previously (3). Briefly, the rat SP-A (0.5 ml of 1.78 mg/ml) in 5 mM Tris buffer (pH 7.4) containing 1 mM EDTA was incubated with N-glycanase (Genzyme Corp.) at a concentration of 2.5 units/mg of SP-A at 37 "C for 24 h. The reaction mixture was applied to a Bio-Gel A5m (Bio-Rad) column (1.5 X 59 cm) to separate free oligosaccharide from the deglycosylated form of the protein. The deglycosylated SP-A was eluted with 5 mM Tris buffer (pH 7.4) containing 1 mM EDTA.
The noncollagenous carboxyl-terminal domain of SP-A was prepared as described below. Human SP-A (0.5-1.0 mg) was suspended with 50 mM Tris buffer (pH 7.4) containing 2 mM CaCI2. Collagenase (Advance Biofactures Corp.) was added (500 units/mg protein) to the SP-A preparation and incubated at 37 "C for 24 h. The collagenaseresistant fragment (CRF) of SP-A was isolated by gel filtration using Sephacryl S-300 (Pharmacia Fine Chemicals) equilibrated with 10 mM Tris buffer (pH 7.4) containing 50 mM NaCI. Human CRF was used for the phospholipid binding experiment described below since manipulations that denature the protein such as reduction, boiling and the addition of detergent were required to completely digest the rat SP-A.
Thin Layer Chromatography of Phospholipids-Phosphatidylcholine from egg yolk, phosphatidylglycerol from egg yolk, phosphatidylinositol from egg yolk, sphingomyelin from egg yolk, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, dilinoleoylphosphatidylcholine, l-palmitoyl-2linoleoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and dipalmitoylphosphatidylethanolamine were purchased from Sigma. Phosphatidylethanolamine and phosphatidylserine were isolated from rat lung. Phospholipid concentration was calculated from the phosphorus content determined by the method of Bartlett (20). Lipids were separated by one dimensional thin layer chromatography (TLC) on Polygram si1 G (Macherey-Nagel, Postfach, Federal Republic of Germany) with a solvent system of ch1oroform:methanol:water (7030:5, v/v) or (85:151, v/v). Each sample was applied on a plate as a band less than 8 mm wide in each lane (1 X 9 cm). Lipids were visualized with iodine vapor or ANSA under ultraviolet light.
Phospholipase Treatment of Phospholipids-Dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and dipalmitoylphosphatidylethanolamine were treated with phospholipase C (from Bacillus cereus, Toyo Jozo) (8 units/pmol phospholipid) in the reaction mixture (3 ml of diethyl ether and 0.5 ml of 0.1 M Tris buffer (pH 6.5) containing 25 mM CaCIJ. The reaction was carried out with vigorous shaking for 3 h at room temperature and then the ether phase was collected. After drying the solvent, diacylglycerol was extracted by the method of Bligh and Dyer (21). Sphingomyelin was also treated with sphingomyelinase (from Streptomyces sp. A9107, Toyo Jozo) in the same fashion as phospholipase C. Dipalmitoylphosphatidylcholine was treated with phospholipase A2 (from Naja naja venom, Sigma). Dried lipid was suspended with 0.1 M Tris buffer (pH 8.5) containing 10 mM CaCI2 and 2 mM sodium deoxycholate (Katayama Chemical) and incubated with phospholipase A2 (25 pg/ pmol phospholipid) at 37 "C for 2 h. The reaction was stopped by the addition of chloroform and lysophosphatidylcholine was extracted by the method of Bligh and Dyer (21). Control reactions were carried out without phospholipases in the same fashion as described above.
Direct Binding of l2'I-SP-A to Phospholipids on Thin Layer Chromatogram-The methods used in our binding studies were adapted from those described for binding of cholera toxin to ganglioside (22).
After development with the organic solvent, the plate of a TLC was air-dried completely and soaked in 50 mM Tris buffer (pH 7.4) containing 0.1 M NaCI, 2 mM CaC12, and 20 mg/ml bovine serum albumin (the binding buffer) and incubated for 30 min at room temperature. Next, the TLC plate was put on the parafilm, and l2'Ilabeled SP-A (0.5 or 1.0 pg/ml) in the binding buffer was carefully overlayered on the plate and then incubated for 1 h at room temperature. The plate was finally washed with gentle shaking on ice for 1 h with at least four changes of ice-cold buffer (50 mM Tris buffer (pH 7.4) containing 0.1 M NaCI, 2 mM CaC12, and 1 mg/ml bovine serum albumin). The air-dried plate was then exposed to Fuji Xray film at -80 "C for 3-7 days. In some experiments "'1-SP-A binding was performed in the presence of 100 pg/ml unlabeled proteins or 10 mM EGTA.

Electrophoretic Analysis of Iodinated and Modified SP-A-
Purified rat SP-A and l2'I-SP-A were analyzed by electrophoresis (Fig. 1B, lanes c and d, and Fig. IA, lanes a and b, respectively). The major forms of iodinated protein correlated well with the major forms of unlabeled protein. Rat deglycosylated SP-A is shown in Fig. lB, lane e. The majority of the oligosaccharide was removed. Human SP-A and human CRF were also electrophoresed as shown in Fig. lB, lanes f and g. Collagenase treatment of human SP-A created an apparent PO-kDa peptide.
Phospholipid Specificity of "'I-SP-A Binding-Each phospholipid (7 nmol) was developed on a TLC plate and stained with iodine vapor (Fig. 2A ). The same amount of phospholipid was developed and '251-SP-A (1 pg/ml) binding on the TLC plate was performed in the absence (Fig. 2, B and D ) or presence (Fig. 2C) of 100 pg/ml native rat SP-A and then autoradiographed and visualized. l2'I-SP-A bound phosphatidylcholine, derived from egg and rat lung, and sphingomyelin, and especially bound dipalmitoylphosphatidylcholine strongly (Fig. 2, B and D), but did not bind 7 nmol of phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, nor phosphatidylinositol. Excess unlabeled SP-A clearly reduced lZ5I-SP-A binding to phospholipids (Fig. ZC)  FIG. 3. 9 -S P -A binding to various amounts of phospholipids. 10 and 50 nmol of dipalmitoylphosphatidylcholine (DPPC) and 10-500 nmol of egg phosphatidylcholine (PC), phosphatidylgly-cero1 ( P C ) , phosphatidylethanolamine (PE), and phosphatidylinosi-to1 ( P I ) were developed with ch1oroform:methanol:water (70305, v/ v), and "'1-SP-A (1 pg/ml) binding was performed as described under "Materials and Methods." Each arrow indicates the apparent position of each phospholipid detected with iodine vapor on TLC plates. phospholipids. 1251-SP-A binding increased as the amount of phosphatidylcholine increased (Fig. 3). The result demonstrates that SP-A binding is dependent upon the amount of phosphatidylcholine. SP-A bound dipalmitoylphosphatidylcholine far more strongly than egg phosphatidylcholine. The protein, however, failed to bind in the presence of a large amount of phosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol.

Specific Binding of SP-A to Dipalmitoylphosphatidykholine
Next, the question of which molecular species of phosphatidylcholine I2'1-SP-A binds was tested using 10 nmol of dimyristoyl-, dipalmitoyl-, distearoyl-, 1-palmitoyl-2-linoleoyl-, and dilinoleoylphosphatidylcholine. The result is shown in Fig. 4. Labeled SP-A bound dipalmitoyl-and distearoylphosphatidylcholine strongly, but bound dimyristoyl-, 1palmitoyl-2-linoleoyl-, and dilinoleoylphosphatidylcholine weakly (Fig. 4). The data show that I2'1-SP-A binding to phosphatidylcholine is molecular species-specific. These results demonstrate that SP-A specifically and strongly binds dipalmitoylphosphatidylcholine. water (70:305, v/v), and "'1-SP-A (1 pg/ml) binding was performed as described under "Materials and Methods." The molecular species of phosphatidylcholine are: 14:0/140, dimyristoyl; 160/160, dipalmitoyl; 180/180, distearoyl; 16:0/182, 1-palmitoyl-2-linoleoyl; 182/ 182, dilinoleoyl. plates with a control lipid (7 nmol/lane). Palmitic acid (15 nmol/lane) was also developed and "'I-SP-A binding was performed. The data are shown in Fig. 5. SP-A bound dipalmitoylglycerol very strongly, but did not bind palmitic acid in an amount almost equivalent to that of dipalmitoylphosphatidylcholine. SP-A also bound ceramide very weakly. These results indicate that SP-A binds a nonpolar group of the phospholipid molecule and that the binding site of SP-A in the phosphatidylcholine molecule is different from that in the sphingomyelin molecule.

Structural Requirement of Phospholipid for SP-A Binding-
Next, dipalmitoylphosphatidylcholine was treated with phospholipase A2 and developed on a TLC plate. Phospholipase A2 treatment of dipalmitoylphosphatidylcholine produced lysophosphatidylcholine (Fig. 6A). 10 nmol of phosphatidylcholine and the same amount of lysophosphatidylcholine were subjected to 12'I-SP-A binding. SP-A failed to bind lysophosphatidylcholine (Fig. 6 B ) . The  SP-A Binding to Dipalmitoylphosphatidylglycerol and Dipalmitoylphosphutidykthumlurnine-We speculate that 1251-SP-A may bind the dipalmitoyl species of glycerophospholipids, since the protein binds dipalmitoylglycerol very strongly as shown in Fig. 5. Dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylethanolamine were tested. '251-SP-A binding to these phospholipid is very weak (Fig. 7A), albeit there is almost no binding in this picture. But SP-A strongly bound dipalmitoylglycerol derived from dipalmitoylglycerophospholipids after treatment with phospholipase C (Fig. 7B), which is essentially the same result as shown in Fig. 5. The data demonstrate that SP-A binding to dipalmitoylglycerophospholipid is polar group specific.
Ionic and Structural Requirement of SP-A for Phosphuti- dykholine Binding-The role of calcium ions, and that of the oligosaccharide moiety and the collagenous domain of SP-A for binding to dipalmitoylphosphatidylcholine were also examined. When '*'I-SP-A binding to lipid was carried out in the presence of 10 mM EGTA in a binding buffer that contained 2 mM CaC12, SP-A binding was much reduced compared with control binding (Fig. 8, lanes A and B). This result demonstrates that calcium ions are required for SP-A binding to phosphatidylcholine. The band did not disappear completely in spite of the presence of 10 mM EGTA, perhaps because the TLC plate itself contains calcium or there may exist some calcium-independent binding.
Next, 0.5 pg/ml of rat '*'II-SP-A was incubated with 100 pg/ ml of native or deglycosylated rat SP-A, native human SP-A, or collagenase-resistant fragment of human SP-A (CRF) on TLC plates. The electrophoretic analysis of SP-A used for the competition experiments is shown in Fig. 1B. Deglycosylated SP-A as well as native rat SP-A competed for lZ5I-SP-A binding to dipalmitoylphosphatidylcholine (Fig. 8, lanes C   and D). Human SP-A also much reduced the binding of labeled protein (Fig. 8, lane E ) . However, in the presence of excess human CRF, lZ5I-SP-A was capable of binding dipalmitoylphosphatidylcholine (Fig. 8, lane F ) , though the band visualized is thinner than in the absence of unlabeled protein.
These results show that the full size of oligosaccharide moiety of SP-A is not required for phospholipid binding, that human SP-A is able to compete with rat SP-A for phospholipid binding and that the noncollagenous domain of SP-A does not appear to compete with labeled SP-A for lipid binding.

DISCUSSION
SP-A has been thought to contribute to the biophysical and physiological activity of surfactant (1, 2 ) . Several studies (8ll), in which interactions of SP-A with mixtures of surfactant-like phospholipids, such as dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol, were analyzed mainly by sedimentation methods which separated associated and free constituents, suggest that SP-A binds phospholipids. However, which classes of phospholipids SP-A binds and how this protein binds lipids have not been demonstrated. Using '"I-SP-A as a probe the direct binding of SP-A to phospholipids was visualized. The results demonstrate that SP-A binds phosphatidylcholine and sphingomyelin, and especially exhibits a strong binding to dipalmitoylphosphatidylcholine. This may be important in a physiological sense (as described below) although the significance of SP-A binding to sphingomyelin is unclear. The protein also shows a strong binding to distearoylphosphatidylcholine, but a weak binding to dimyristoyl-, l-palmitoyl-2-linoleoyl-, and dilinoleoylphosphatidylcholine. SP-A appears to bind saturated phosphatidylcholine with longer fatty acid acyl chains more strongly. The binding study after enzyme treatment of lipids reveals that SP-A binds the nonpolar group of dipalmitoylphosphatidylcholine and that the palmitate at the second position is involved in the binding of SP-A, but SP-A does not bind an equivalent amount of palmitic acid. Iodinated SP-A exhibits almost no binding to dipalmitoylphosphatidylglycerol nor dipalmitoylphosphatidylethanolamine. This may be due to the differences of structures and charges which are dependent on polar groups of dipalmitoylglycerophospholipids. Polar groups of glycerophospholipids also appear to be important for SP-A binding.
The ionic and structural requirements of SP-A for phosphatidylcholine binding were studied. The results demonstrate that phospholipid binding of SP-A is dependent upon calcium ions as well as some other functions of SP-A, i.e. the aggregation of phospholipid liposomes (12), the inhibitory effect of lipid secretion by type I1 cells (4, 5), and receptor binding activity (6, 7). The full size of oligosaccharide moiety in the SP-A molecule does not appear to be a required structural feature of SP-A binding to phosphatidylcholine. In contrast, CRF fails to compete with lZ5I-SP-A for binding to dipalmitoylphosphatidylcholine, but the band seen in Fig. 8, lane F, appears to be less dark than that seen in Fig. 8, lane A. These findings suggest that CRF competes with labeled SP-A incompletely. CRF, in which the amino terminus starts at Gly-75, Gly-78 or Ala-81 of SP-A and in which the carboxyl terminus of SP-A is preserved (data not shown), may bind only weakly. This is consistent with the result described by Ross et al. (11). They emphasized the importance of hydro-phobic amino acid residues Leu-102 to Val-117 and the aminoterminal collagenous domain in the interaction of SP-A with phospholipids. Phospholipid binding activity of this protein appears to be dependent upon the integrity of the collagenous domain.
Several calcium-dependent phospholipid binding proteins have been isolated from lungs (23)(24)(25). In the presence of micromolar Ca2+, these phospholipid binding proteins specifically bind acidic phospholipids, usually found at the cytoplasmic face of the membranes (26). These proteins and SP-A possess different specificities for phospholipid. Thus SP-A appears to differentiate from a family of so called calciumdependent phospholipid (and membrane)-binding proteins. Wright et al. (14) found that SP-A enhances the uptake of phospholipid liposomes labeled with ['4C]dipalmitoylphosphatidylcholine by alveolar type I1 cells and that only 86% of the radioactivity is recovered in phosphatidylcholine and 7% is recovered in phosphatidylglycerol in the absence of added SP-A, whereas more than 97% of the cell-associated radioactivity is recovered in phosphatidylcholine when SP-A is included in the incubation buffer. Wright et al. (14) suggested one possible mechanism that SP-A may direct lipids to lamellar bodies where they are not catabolized. The results from the present study are on the lines of this hypothesis. Lamellar bodies are secreted by exocytosis, form tubular myelin (27, 28) and then can generate a surface film (29), which is believed to be enriched in dipalmitoylphosphatidylcholine, which is probably the most important factor in reducing surface tension at the air-liquid interface. SP-A may selectively bind dipalmitoylphosphatidylcholine, an essential factor of pulmonary surfactant, and lead it back to the lamellar body, preventing futile recycling and reutilization of surfactant.
In summary, the present study provides direct evidence that SP-A specifically and strongly binds dipalmitoylphosphatidylcholine. SP-A binding to phosphatidylcholine is specific for the molecular species of the fatty acid acyl chain. SP-A binds the non-polar group of phospholipids. The second positioned palmitate is also involved in dipalmitoylphosphatidylcholine binding. The specificities of polar groups of dipalmitoylglycerophospholipids also appear to be important for SP-A binding. The phospholipid binding activity of SP-A is dependent upon calcium ions and the integrity of the collagenous domain of SP-A, but not on the oligosaccharide moiety of SP-A.