From the The Lord and Taylor Laboratory for Lung
Biochemistry, National Jewish Medical and Research Center, Denver,
Colorado 80206, the § Department of Biochemistry, Sapporo
Medical University, School of Medicine, Sapporo, Japan, and the
¶ Division of Pulmonary and Critical Care Medicine, University of
Cincinnati School of Medicine, Cincinnati, Ohio 45267
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ABSTRACT |
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In previous studies, tandem mutagenesis of Glu195 and Arg197 of surfactant protein A (SP-A) has implicated both residues as critical participants in the interaction of the molecule with alveolar type II cells and phospholipids. We substituted Ala, Lys, His, Asp, and Asn mutations for Arg to evaluate the role of a basic amino acid at position 197 in SP-A action. Unexpectedly, Ala197 retained complete activity in the SP-A functions of carbohydrate binding, type II cell binding, inhibition of surfactant secretion, lipid binding, lipid aggregation, and lipid uptake by type II cells. The results unambiguously demonstrate that Arg197 is not mechanistically essential for SP-A function. The Lys197 mutation displayed all functions of the wild type protein but exhibited a 2-fold increase in lipid uptake activity. The His197 mutation displayed all SP-A functions studied except for lipid uptake. The results obtained with the His197 mutation clearly demonstrate that lipid aggregation alone by SP-A is insufficient to promote lipid uptake by type II cells. These findings indicate that specific interactions between type II cells and SP-A are involved in the phospholipid uptake processes.
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INTRODUCTION |
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Pulmonary surfactant protein A (SP-A)1 is a hydrophilic C-type mammalian lectin with a subunit molecular mass of 26-38 kDa that is synthesized primarily by alveolar type II and Clara cells (1, 2). SP-A is composed of four distinct domains (1): 1) an amino-terminal domain that forms interchain disulfide bonds; 2) a collagen-like domain containing multiple Gly-X-Y repeats; 3) a neck region composed of an amphipathic helix and a hydrophobic domain; and 4) a carbohydrate recognition domain (CRD). The mature protein forms an oligomeric bouquet structure composed of six trimeric subunits which are covalently joined via disulfide bonds (1). SP-A binds and aggregates phospholipid vesicles at physiological concentrations of Ca2+ (3, 4). The protein also facilitates uptake of phospholipid by isolated alveolar type II cells (5). These in vitro properties are thought to reflect some of the functions of SP-A in vivo that include structural reorganization and recycling of surfactant lipids. Other important Ca2+-dependent functions of SP-A include high affinity binding to specific receptors on alveolar type II cells and negative regulation of surfactant secretion (6-9). SP-A also plays a role in immunoglobulin independent host defense in the lung (10). The protein stimulates macrophage chemotaxis and can enhance phagocytosis of Herpes simplex virus type 1 (11), Staphylococcus aureus (12), and Mycobacterium tuberculosis (13). SP-A also enhances FcR- and CR1-mediated phagocytosis (14). Several proteins that interact with SP-A have been identified on alveolar type II cells and macrophages (15-17).
Mutations in the CRD affect specific type II cell receptor binding, phospholipid binding and aggregation, and inhibition of surfactant secretion as well as augmentation of phospholipid uptake by alveolar type II cells (18, 19). The simultaneous substitutions E195Q and R197D of the CRD alter carbohydrate binding specificity, prevent SP-A-mediated phospholipid uptake and aggregation of phospholipid, and destroy the ability of SP-A to compete with its radiolabeled counterpart for surface receptors on alveolar type II cells. These results implicate the Glu195 and Arg197 as essential amino acids in SP-A function. This previous conclusion for SP-A is consistent with results first obtained with the closely related MBP-A that defined the active site for carbohydrate binding (20, 21). The work with MBP-A followed solution of the crystal structure and unambiguously defined the carbohydrate and Ca2+-binding sites of the protein. Work with SP-A using site-directed mutagenesis has largely followed from extrapolation of the MBP-A data.
In this study, we examined the specific role of amino acid Arg197 of the SP-A in the interaction of the protein with the type II cell receptor and lipids. Position 197 was replaced with amino acids Ala, Lys, His, Asp, and Asn. These mutants were prepared using the baculovirus expression system in invertebrate (Sf9) cells. Recombinant rat SP-A has been shown to have all of the functions of native SP-A with respect to lipid interactions and regulation of surfactant secretion by type cells (22). The primary objective of this study was to understand how essential the basic residue at position 197 is for multiple SP-A functions.
Our results demonstrate unexpectedly that a basic residue at position 197 is not required for SP-A function. Substitutions at position 197 further reveal that SP-A-mediated lipid aggregation is insufficient to promote lipid association and uptake by type II cells. In addition, we show that mutant R197H is defective exclusively in the lipid uptake process.
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EXPERIMENTAL PROCEDURES |
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Purification and Iodination of Native Rat SP-A-- Surfactant was isolated from Sprague-Dawley rats 28 days after an intratracheal instillation of 10 mg of silica in saline (23). Native SP-A was isolated and purified from surfactant extracted from bronchoalveolar lavage as described previously by Kuroki et al. (6).
The 125I-SP-A was prepared using the method of Bolton-Hunter as described previously (6). The 125I-SP-A (552-690 cpm/ng) used in the receptor-binding experiments was greater than 95% precipitable, 10% (w/v) trichloroacetic acid.DNA Constructs-- Mutant SP-A cDNAs (R197A, R197K, R197H, R197N, and R197D) were synthesized using a 1.6-kilobase SP-A cDNA template (22) and mutagenic oligonucleotide primers by the overlap extension method of polymerase chain reaction (24). The polymerase chain reaction products were digested with Bsu36I and MscI before ligation into Bsu36I/MscI-digested pGem7zf/SP-A vector. The entire polymerase chain reaction-amplified region of each mutant was sequenced to confirm the presence of only a single mutation. The 1.6-kilobase SP-A cDNA containing the mutation was digested and ligated into Bsu36I and XmaI-digested pVL1392/SP-A vector.
Expression and Isolation of the Mutant Proteins-- The mutant proteins were expressed in the baculovirus system as described by O'Reilly (25). Recombinant baculoviruses were produced by cotransfection of Spodoptera frugiperda (Sf9) cells with the pVL1392/SP-A vector and linearized Autographa californica virus (Baculogold, Pharmingen). Plaques containing recombinant baculoviruses were isolated and amplified to 107-108 plaque-forming units/ml before protein expression. Recombinant proteins were secreted from infected Trichoplusia ni cells and purified from contaminants in serum-free Excell 400 medium by absorption to mannose-Sepharose 6B, as described previously (22).
Primary Culture of Alveolar Type II Cells and Inhibition of
Phosphatidylcholine Secretion--
Alveolar type II cells were
isolated by tissue dissociation with elastase and purification on
metrizamide gradients (26). The type II cells were seeded at 2 × 106/35-mm dish in 2 ml of the Dulbecco's modified Eagle's
medium (DMEM) containing 10% fetal calf serum. For measurement of
phosphatidylcholine secretion, the type II cells were cultured with
DMEM, 10% fetal calf serum containing [3H]choline (0.5 µCi/ml) for 20 h, washed to remove unincorporated precursor and
subjected to agonist/antagonist treatment as described (27). Surfactant
lipid secretion was induced with 107 M
12-O-tetradecanoylphorbol-13-acetate (TPA). Wild type and
mutant forms of SP-A were added to cultures to antagonize TPA
action.
Receptor Binding--
Isolated type II cells were cultured in
DMEM, 10% fetal calf serum for 20 h. The assay was carried out as
described previously (28). The cell monolayers were washed with DMEM
and incubated with 1 µg/ml 125I-SP-A and varying
concentrations of unlabeled SP-A (either wild type or mutant) in DMEM
containing 10% fetal calf serum, for 4 h at 37 °C.
Subsequently, each dish was placed on ice, and the monolayers were
washed 3 times with ice-cold buffer B (50 mM Tris buffer
(pH 7.4), 0.15 M NaCl, 2 mM CaCl2,
and 2 mg/ml bovine serum albumin) and 1 time with ice-cold buffer A (50 mM Tris buffer (pH 7.4), 0.15 M NaCl, 2 mM CaCl2) before solubilization in 1 ml of 0.1 N NaOH. The amount of bound 125I-SP-A was
quantified using a -counter. Nonspecific binding was determined as
the amount of 125I-SP-A bound in the presence of 50 µg/ml
unlabeled native rat SP-A.
Lipids--
Dipalmitoyl phosphatidylcholine (DPPC),
L--lecithin (egg phosphatidylcholine) (egg PC),
L-
-phosphatidylglycerol (egg sodium salt) (egg PG), and
1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (DPPG) were purchased from Avanti Polar Lipids, Inc. 1-Palmitoyl 2-[3H]palmitoyl-L3-phosphatidylcholine
([3H]DPPC) was obtained from NEN Life Science Products
Inc. For phospholipid uptake experiments, liposome mixtures (DPPC:egg
PC:DPPG or DPPC:egg PC:egg PG (7:2:1)) containing 2 µCi of
[3H]DPPC (specific activity of 50 Ci/mmol) per mg of
liposomes were dried under nitrogen and hydrated (concentration of 1 mg/ml) in 20 mM HEPES (pH 7.4) containing 0.15 M NaCl at 48 °C for 1 h. Unilamellar liposomes were
prepared by sonication of the lipid mixture with a microprobe at 25 watts for 10 min while the tube was immersed in an external waterbath
(45 °C), followed by centrifugation at 100,000 × g
for 1 h to remove multilamellar liposomes.
Phospholipid Uptake by Type II Cells-- Freshly isolated type II cells were washed one time in DMEM containing 10 mM HEPES (pH 7.4) (DMEM/HEPES), and resuspended in DMEM/HEPES at 107/ml. The phospholipid uptake experiment was carried out using 1 × 106 cells per tube as described previously (19).
Liposome Aggregation-- The unilamellar liposomes (100 µg/ml) were mixed with 10 µg/ml of the SP-A variants in a cuvette in 500 µl of 20 mM Tris buffer (pH 7.4) containing 0.15 M NaCl and 0.5 mM EDTA. After 3 min, CaCl2 was added to a final concentration of 5 mM. Light scattering (A400) was measured for 7 min in a Beckman DU-64 spectrophotometer. All measurements were made at ambient temperature.
Liposome Binding-- Multilamellar liposomes (100 µg) were mixed with 200 ng of SP-A or mutants in 50 µl of 10 mM Tris buffer (pH 7.4) containing 50 mM NaCl, 5 mM CaCl2, and 20 mg/ml bovine albumin (fatty-acid free). For control conditions, multilamellar liposomes and SP-A or mutants were mixed in 50 µl of 10 mM Tris buffer (pH 7.4), 5 mM EGTA, and 20 mg/ml bovine albumin (fatty-acid free). After a 1-h incubation at 25 °C, binding was determined by centrifugation at 14,000 × g for 10 min at room temperature followed by enzyme-linked immunosorbent assay (29) to determine the SP-A concentration of the supernatant and pellet fractions. Percent binding was calculated from [SP-Apellet]/[SP-Asupernatant + SP-Apellet] × 100.
Protein Analysis and Assays-- The SP-A proteins were characterized by electrophoresis in 8-16% polyacrylamide gels in the presence of SDS (30) as well as immunoblotting with anti-SP-A IgG (31). Protein concentrations were determined with the bicinchoninic protein assay kit (BCA) (Pierce, Rockford, IL).
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RESULTS |
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Expression and Characterization of Position 197 Mutants-- In an effort to understand the role of Arg197 in SP-A structure and function, this position was substituted with Ala, Lys, His, Asp, and Asn and the recombinant proteins expressed using baculovirus vectors. We have previously shown that rat SP-A expressed in insect cells (S. frugiperda and T. ni), yields a modified form of the protein with altered glycosylation, hydroxylation of prolines, and oligomerization (22). Despite these structural alterations to the recombinant protein, it exhibits all of the functions of its counterpart purified from lung lavage. The wild type recombinant protein recovered from insect cells is designated SP-Ahyp because of its reduced hydroxyproline content. In SP-A proteins, Arg197 was substituted to give R197A, R197K, R197H, R197N, and R197D variants that were expressed in T. ni cells and purified from culture media by affinity chromatography using mannose-Sepharose. The proteins were recovered in high yield and the success of the affinity chromatography demonstrates that all of the mutants retained the Ca2+-dependent lectin property of the native protein. Previously, we reported that the tandem substitution of Glu195 and Asp197 altered the carbohydrate binding specificity. The binding specificity of the Ala197 mutant was examined in detail, using mannose- and galactose-Sepharose, and found to be identical to wild type recombinant protein.
The electrophoretic properties of the proteins were also determined (data not shown). All of the mutant proteins display electrophoretic properties under both reducing and nonreducing conditions that are virtually identical to SP-Ahyp. Further analysis of the recombinant proteins using immunoblotting (data not shown) also demonstrates that the purified proteins are genuine SP-A variants and reveals that they oligomerize to the same extent as previously shown for SP-Ahyp (19, 22). The results from the above experiments clearly establish the lectin activity of the mutant recombinant proteins and demonstrate that their covalent organization is the same as that of the wild type recombinant protein.Receptor Binding-- SP-A interacts with a specific receptor on type II cells and the binding site appears to reside within the CRD (19). Receptor binding is required for inhibition of surfactant secretion and enhancement of phospholipid uptake activities by SP-A (18, 19, 32). To determine whether Arg197 of the CRD is essential for this function, we examined the ability of the R197A, R197H, R197K, R197N, and R197D mutants to compete with 125I-native rat SP-A for high affinity binding to isolated type II cells. Surprisingly, the Ala197 mutation competed for receptor binding as well as the wild type recombinant protein. This result was unexpected since previous work had implicated Arg197 as an essential residue in multiple SP-A functions (19). As shown in Fig. 1, the IC50 for the Ala197 mutant is approximately 8 µg/ml compared with that of 9 µg/ml for its wild type counterpart. Both His197 and Lys197 substitutions gave weaker competition for 125I-rat SP-A binding, with the IC50 values greater than 20 µg/ml for both mutants. The activities for Asp197 and Asn197 mutants were lower than those shown for the preceding mutants with slight inhibition observed at concentrations of 40 µg/ml (data not shown). The results demonstrate that a basic amino acid at position 197 is not required for high affinity binding of SP-A to its receptor. In addition, charge reversal is not tolerated at that position.
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Inhibition of Phosphatidylcholine Secretion by Type II Cells-- SP-A inhibits phosphatidylcholine secretion by cultured type II cells after interaction with a specific receptor (28). We examined the effect of the Arg197 substitution on this biological activity. The Ala197 and Lys197 mutants were the strongest inhibitors of secretion with the IC50 values of 0.05 and 0.06 µg/ml, respectively, compared with both native rat SP-A and recombinant SP-A which had IC50 values of 0.075 and 0.1 µg/ml, respectively (Fig. 2). The His197 mutant was a weaker inhibitor with an IC50 value of 0.2 µg/ml (Fig. 2). Consistent with the finding for receptor binding, the Asp197 and Asn197 mutations were significantly weaker requiring 10 µg/ml levels to approach maximal inhibition of secretion (Fig. 3).
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Augmentation of Phospholipid Uptake into Type II Cells-- SP-A has been implicated in phospholipid recycling in the lung. The protein may enhance phospholipid uptake into type II cells via receptor-mediated endocytosis (33). In this experiment, we examined the effect of the five mutants at position 197 upon SP-A-mediated phospholipid uptake. The Lys197 mutation facilitated the highest phospholipid uptake (15.12 ± 2.5% at 10 µg/ml, n = 5), whereas the His mutation yielded barely detectable activity (3.08 ± 0.8% at 10 µg/ml, n = 5), as shown in Fig. 4. The Ala197 mutation had the same potency as both native rat SP-A and the wild type recombinant protein. Neither the Asp nor Asn mutants were capable of augmenting lipid uptake (not shown). These results indicate that a basic amino acid at position 197 is not required for lipid uptake. Moreover, the conservative substitution of Lys for Arg clearly enhances lipid uptake 2-fold (Fig. 4). In contrast, the His mutation yields a protein that is relatively inactive in promoting lipid uptake by type II cells despite near normal interactions with the cells otherwise.
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Phospholipid Binding and Aggregation-- The CRD of SP-A participates in both phospholipid binding and aggregation activities (18) (19). The role of Arg197 in both phospholipid binding and aggregation was also examined. Both lipid binding and aggregation by wild type and mutant SP-A's requires calcium as shown in Figs. 5 and 6. The phospholipid binding of the Ala, Lys, His, Asp, and Asn197 mutants was comparable and about 30% less potent (50% binding) than that of native SP-A (81.6% binding) (Fig. 5). However, the binding ability of these mutants was not significantly different from the wild type recombinant protein. Examination of Ca2+-dependent lipid aggregation revealed that the R197A, R197H, and R197K mutants were comparable in activity to the SP-A isolated from lung lavage and of slightly higher activity than the wild type recombinant protein (Fig. 6). In contrast, the Asp197 replacement yielded a protein that was very weak at lipid aggregation and the Asn197 protein was inactive. The results provide clear evidence that a basic amino acid at position 197 of SP-A is not required for either phospholipid binding or aggregation.
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DISCUSSION |
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The results from previous studies of SP-A structure and function implicated both Glu195 and Arg197 as essential residues in carbohydrate binding, receptor binding, and lipid aggregation and binding (19). In the present study, we focused on the basic residue, Arg197. Our initial hypothesis was that the positive charge due to Arg197 was an important factor for the CRD functions. This hypothesis was predicated on previous studies using Arg modifying reagents with SP-A (28) and site-directed mutagenesis (19). We, therefore, examined the effect of the charged group by substitution with Ala (uncharged, short side chain), His (imidazole side chain, uncharged at pH 7.4), or Lys (amino alkyl side chain, positively charged), Asp (charge reversal), or Asn (uncharged, medium side chain). If the positive charge group of Arg197 is essential for the CRD functions, one would anticipate that the Ala substitution would abolish all activities, His substitution would show pH-dependent acquisition of function, Lys substitution may retain some or all functions, and Asp and Asn substitutions would be inactive.
All five mutants were purified on mannose-Sepharose affinity columns, indicating that correct folding and the lectin properties of the protein were preserved. All mutants were shown by both gel electrophoresis and immunoblot analysis to be comparable in denatured molecular size and covalent oligomerization to the wild type recombinant protein expressed in insect cells (22).
Three mutants (Ala, His, and Lys197) competed for binding to the high affinity receptor present on isolated type II cells. The Ala197 mutant was as effective as its wild type counterpart and more effective than the His197 and the Lys197 mutants. These results unambiguously demonstrate that the positive charge at position 197 is not an essential factor in receptor occupancy. The Asp and Asn197 substitutions were essentially inactive ligands for the specific high affinity receptor. The Asp197 result demonstrates that charge reversal is not tolerated at this position. The Asn197 substitution indicates that a neutral medium side chain group is also unfavored.
The Ala, Lys, and His substitutions of Arg197 did not
significantly change the inhibitory action of SP-A upon TPA-stimulated surfactant secretion. We conclude that these SP-A variants recognized the high affinity receptor because the inhibition of secretion was not
reversed by -methylmannoside which competes for the nonspecific concanavalin A-binding sites. These findings also demonstrate that
basic residues at position 197 are not required for inhibition of
secretion. The Asn197 protein failed to demonstrate
specific inhibition of surfactant lipid secretion and the
Asp197 protein was a very weak inhibitor.
Interestingly, the results from the receptor binding competition assay showed that the His197 and Lys197 mutants did not compete very well (IC50 > 20 µg/ml) against 125I-native SP-A for the specific receptor on type II cells surface. However, in the surfactant secretion measurements, the Lys197 mutant was nearly twice as effective as the wild type Arg197 form but the His197 mutant was about 50% as potent. This disparity between the specific receptor binding and the inhibition of surfactant secretion is similar to results from studies by Kuroki et al. (28) and McCormack et al. (22) but is not well understood. There is clearly not an exact correlation between the receptor binding results and the inhibition of secretion. The difference between the two activities may occur as a consequence of a more efficient coupling of inhibition of secretion to specific receptors, as proposed by Kuroki et al. (28). Alternatively, two different receptor populations could account for the differences between binding and inhibition of surfactant secretion.
The uptake of surfactant lipid by type II cells, mediated by SP-A, may be one of several phospholipid clearance mechanisms in the lung (5). The results from the double substitution of Glu195 and Arg197 (19) indicated that both amino acids are involved in the uptake process. However, our current findings show that Arg197 is unlikely to be essential for this activity. As the results clearly show, the Ala197 mutation readily enhanced the uptake of phospholipid similar to native SP-A and surpassed that of recombinant wild type SP-A. This latter finding effectively ruled out an obligatory role for a positive charge at position 197. By comparison, the His197 mutant containing an uncharged imidazole group was essentially inactive at 10 µg/ml. The results indicate that a positive charge is not required at position 197 and that a bulky uncharged or weakly protonated group is ineffective. Consistent with their relative inactivity in other SP-A functions, the Asn and Asp197 mutants failed to promote lipid uptake.
Despite the absence of a requirement for a positive charge at position 197 for function, a Lys substitution clearly enhances lipid uptake almost 2-fold at a protein concentration of 10 µg/ml. The action of the Lys197 protein highlights an inconsistency in the data. Although the Lys197 protein is a poorer competitor of 125I-labeled SP-A binding to the cell surface than the native or wild type recombinant protein, it is more effective than the native or wild type recombinant protein at facilitating lipid uptake. The results suggest that either the interaction with lipid or the specific process of lipid uptake (distinct from receptor binding) is enhanced by Lys197. Our preferred interpretation is that Lys197 functions more efficiently than other proteins in the lipid uptake process. Comparatively, the Lys197 mutant does not compete for receptor occupancy, bind, or aggregate lipid any better than the His197 protein. However, the difference between His and Lys197 mutants at lipid uptake is dramatic, with the His substitution being essentially inactive. The results clearly indicate that the amino acid at position 197 can play a unique role in the lipid uptake reaction.
Phospholipid aggregation is another method used to measure SP-A lipid interactions. This process may reflect the role of SP-A in lipid reorganization and formation in the alveolus (34). The exact mechanism of aggregation is still unknown. It is unlikely that lipid aggregation occurs via a lectin carbohydrate interaction between SP-A oligosaccharides and the CRD's in conjunction with phospholipid binding (22, 35). Although previous work implicated Arg197 in lipid aggregation, the current study shows that the Ala, Lys, and His mutants at this position aggregated unilamellar liposomes better than wild type recombinant SP-A. The findings presented in this report also provide strong evidence that lipid aggregation and lipid uptake by type II cells are not completely interdependent. The distinction between lipid aggregating and uptake phenomena is important since the work of Horowitz et al. (36) has suggested that SP-A promotes the association of lipid aggregates with the surface of type II cells. Specifically, the results with the His197 mutant clearly demonstrate that lipid aggregation activity, alone, is not sufficient to ensure uptake of lipid by type II cells. This important observation suggests that lipid uptake requires specific interplay between SP-A and the type II cell as well as lipid. The results clearly argue against a mechanism of lipid uptake by type II cells that is merely the nonspecific uptake of lipoprotein aggregates. The findings highlight the His197 and Lys197 proteins as important new tools for examining the lipid uptake process. The structural alteration imposed by His197 appears quite specific for reducing the lipid uptake reaction without eliminating other SP-A functions.
The in vitro functions measured in these studies are thought to reflect the interactions of SP-A with lipids and alveolar type II cells that occur in vivo. Mice homozygous for null alleles of SP-A have been produced, and they exhibit no evidence for gross defects in surfactant secretion, recycling, or biophysical surfactant function (37, 38). The genetic experiments indicate that SP-A is not essential and imply the presence of redundant mechanisms for its functions. The silent phenotype of the SP-A null animals currently makes the exact relationship between the in vitro experiments and the in vivo activity of SP-A unclear.
In summary, this study demonstrates that Arg197 of SP-A is not essential for binding to carbohydrate, binding to the high affinity receptor on type II cells, inhibition of surfactant secretion, enhancement of phospholipid uptake, and phospholipid aggregation and binding. The positive charge of this residue is not involved in mediating the functions of the CRD. The results also show that the SP-A-mediated augmentation of phospholipid uptake by type II cells can be specifically enhanced and inhibited and is not directly correlated with the phospholipid aggregation.
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ACKNOWLEDGEMENTS |
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We thank Amanda Evans for excellent technical assistance and Kathy Ryan Morgan for excellent secretarial assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL45286 (to D. R. V.).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: National Jewish
Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Tel.:
303-398-1300; Fax: 303-398-1806; E-mail: voelkerd{at}njc.org.
1 The abbreviations used are: SP-A, surfactant protein A; CRD, carbohydrate recognition domain; DMEM, Dulbecco's modified Eagle's medium; TPA, 12-O-tetradecanoylphorbol-13-acetate; DPPC, 1,2-dipolmitoyl-sn-glycero-3-phosphocholine; DPPG, 1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-(1-glycerol)); PG, phosphatidylglycerol; PC, phosphatidylcholine.
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REFERENCES |
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