Deletion Mapping of N-terminal Domains of Surfactant Protein A
THE N-TERMINAL SEGMENT IS REQUIRED FOR PHOSPHOLIPID AGGREGATION AND SPECIFIC INHIBITION OF SURFACTANT SECRETION*

Francis X. McCormackDagger , Mamatha Damodarasamy, and Baher M. Elhalwagi

From the Division of Pulmonary/Critical Care Medicine, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

    ABSTRACT
Top
Abstract
Introduction
References

The objective of the current study was to examine the functional importance of the N-terminal domains of surfactant protein A (SP-A) including the N-terminal segment from Asn1 to Ala7 (denoted domain 1), the N-terminal portion of the collagen domain from Gly8 to Gly44 (domain 2), and the C-terminal portion of the collagen-like domain from Gly45 to Pro80 (domain 3). Wild type recombinant SP-A (SP-Ahyp; where hyp indicates hydroxyproline-deficient) and truncated mutant (TM) SP-As containing deletions of domain(s) 1 (TM1), 2 (TM2), 1 and 2 (TM1-2), and 1, 2, and 3 (TM1-2-3) were synthesized in insect cells and purified by mannose-Sepharose affinity chromatography. N-terminal disulfide-dependent dimerization was preserved at near wild type levels in the TM1-2 (at Cys-1) and TM2 proteins (at Cys-1 and Cys6), and to a lesser extent in TM1 (at Cys-1), but not in TM1-2-3. Cross-linking analyses demonstrated that the neck + CRD was sufficient for assembly of monomers into noncovalent trimers and that the N-terminal segment was required for the association of trimers to form higher oligomers. All TM proteins except TM1-2-3 bound to phospholipid, but only the N-terminal segment containing TM proteins aggregated phospholipid vesicles. The TM1, TM1-2, and TM2 but not the TM1-2-3 inhibited the secretion of surfactant from type II cells as effectively as SP-Ahyp, but the inhibitory activity of each mutant was blocked by excess alpha -methylmannoside and therefore nonspecific. TM1 and TM1-2-3 did not enhance the uptake of phospholipids by isolated type II cells, but the TM1-2 and TM2 had activities that were 72 and 83% of SP-Ahyp, respectively. We conclude the following for SP-A: 1) trimerization does not require the collagen-like region or interchain disulfide linkage; 2) the N-terminal portion of the collagen-like domain is required for specific inhibition of surfactant secretion but not for binding to liposomes or for enhanced uptake of phospholipids into type II cells; 3) N-terminal interchain disulfide linkage can functionally replace the N-terminal segment for lipid binding, receptor binding, and enhancement of lipid uptake; 4) the N-terminal segment is required for the association of trimeric subunits into higher oligomers, for phospholipid aggregation, and for specific inhibition of surfactant secretion and cannot be functionally replaced by disulfide linkage alone for these activities.

    INTRODUCTION
Top
Abstract
Introduction
References

Pulmonary surfactant lines the distal airspaces and stabilizes the gas exchanging alveolar units of the lung by reducing surface tension (1). Surfactant is composed of phospholipids and proteins that are organized as lattice-like arrays (e.g. tubular myelin) and vesicular aggregates within the alveolar lining fluid, and as a film at the alveolar air-liquid interface. Surfactant protein A (SP-A)1 is a hydrophilic, Ca2+-dependent phospholipid-binding protein that is secreted into the airspace by alveolar type II cells (2). The function of SP-A is not fully understood, but reports that SP-A binds to a high affinity receptor on the surface of alveolar type II cells (3, 4), is required for the formation and/or stability of tubular myelin (5, 6) and large surfactant aggregates (7), and protects surfactant surface activity in the presence of foreign protein contamination (8) suggest one or more roles in surfactant function. The notion that SP-A is also a pulmonary host defense protein is supported by membership in the structurally homologous collectin family of antibody-independent opsonins (including mannose-binding protein A) (9, 10), reports that SP-A binds to, aggregates, and opsonizes multiple microorganisms (for review see Ref. 11), and the finding that the SP-A gene-targeted mouse is susceptible to infection with group B streptococcus (12).

Rat SP-A is a large oligomer composed of 18 very similar subunits that are distinguished by variable glycosylation at one or both N-linked carbohydrate attachment sites (13, 14) and microheterogeneity in the N-terminal amino acid sequences. The latter most probably arises by alternative site cleavage by signal peptidase, which results in a slightly elongated variant containing an Ile-Lys-Cys (IKC) sequence upstream of the predicted N terminus of Asn1 (15). The primary structure of the SP-A subunits are otherwise identical, composed of several discrete domains including the following (16, 17): 1) a short N-terminal segment, 2) a collagen-like sequence of Gly-X-Y repeats (where X is any amino acid and Y is often Pro or hydroxyproline) containing a midpoint interruption (Gly44), 3) a hydrophobic neck domain, and 4) a carbohydrate recognition domain (CRD). Trimeric association of monomeric subunits occurs by the folding of the collagen-like domains into triple helices (18). The role of the neck domain in the oligomeric assembly of SP-A has not been reported, but in related collectins, the formation of alpha -helical coiled coil bundles in this region is critical for trimerization (19). Fully assembled SP-A is a hexamer of trimers that are laterally associated through the N-terminal segment and the first half of the collagen-like domain and stabilized by inter- and intratrimeric interchain disulfide bonds at Cys-1 and Cys6 (18, 20).

The domains of SP-A that interact with type II cells and surfactant phospholipids have been mapped by site-directed mutagenesis. The CRD of the molecule contains binding sites for carbohydrate, the major surface glycoprotein of the pulmonary pathogen Pneumocystis carinii, the high affinity SP-A receptor on type II cells, and surfactant phospholipids, especially dipalmitoylphosphatidylcholine (DPPC) (21-24). We have recently reported that the N-terminal domains that contribute to oligomeric assembly, including the interchain disulfide bond at Cys6 and the collagen-like region, are essential for SP-A function (25). The collagen-like region was required for competitive binding to the SP-A receptor and the specific inhibition of surfactant secretion from type II cells. Disruption of the Cys6 interchain disulfide bond, by substitution of serine for cysteine, blocked the aggregation of liposomes by SP-A and reduced the inhibitory effect of SP-A on surfactant secretion from isolated alveolar type II cells. Recent analyses indicate that the N-terminal IKC sequence, and specifically the interchain disulfide-forming cysteine at Cys-1, is not essential for the interactions of SP-A with type II cell or surfactant phospholipids (26).

In the current study, we used truncated mutant forms of SP-A to examine the role of the N-terminal segment and defined regions of the collagen-like domain in oligomeric assembly, phospholipid binding and aggregation, and SP-A receptor-mediated functions.

    EXPERIMENTAL PROCEDURES

Production of Mutant Recombinant Proteins-- The synthesis of the mutant recombinant proteins TM1, TM2-3, and TM1-2-3 has been previously reported (15). The TM2 protein was generated by similar methods. Briefly, the TM2 mutant cDNA encoding the deletion of amino acids Gly8-Gly44 of rat SP-A was produced using mutagenic oligonucleotides by overlapping extension polymerase chain reaction amplification of a 1.6-kilobase pair rat SP-A cDNA (27, 28). The TM2 cDNA was ligated into the EcoRI site of PVL 1392 (Invitrogen), and the correct orientation was confirmed using KpnI. Nucleotide sequencing of the Gly8-Gly44 coding region of the TM2 cDNA confirmed the intended deletion and the absence of spurious mutations (29). A recombinant baculovirus containing the TM2 cDNA for SP-A was produced by homologous recombination in Spodoptera frugiperda (Sf-9) cells following contransfection with linear viral DNA and the PVL 1392/TM2 construct (BaculoGold, PharMingen). Fresh monolayers of 107 Trichoplusia ni (T. ni) cells were infected with plaque-purified recombinant virus at a multiplicity of infection of 10 and then incubated with serum-free media (IPL-41) supplemented with 0.4 mM ascorbic acid and antibiotics for 72 h. Recombinant SP-A was purified from the culture media by adsorption to mannose-Sepharose 6B columns in the presence of 1 mM Ca2+ and elution with 2 mM EDTA (30). The purified recombinant SP-A was dialyzed against 5 mM Tris (pH 7.4) and stored at -0 °C.

Purification/Modification of Native SP-A-- Surfactant was isolated by bronchoalveolar lavage of silica-pretreated Sprague-Dawley rats (31) and floated on NaBr gradients (32, 33). SP-A was isolated and purified from the surfactant pellicle by delipidation, mannose-Sepharose affinity chromatography (30), and gel permeation chromatography with Bio-Gel A-5m.

Protein Assays-- The SP-A content of tissue culture media containing recombinant SP-A was determined with a rabbit polyclonal IgG against rat SP-A using a sandwich enzyme-linked immunosorbent assay (34). The lower limit of sensitivity of the assay was 0.20 ng/ml, and the linear range extended from 0.16 to 10.0 ng/ml. Routine protein concentrations were determined with the bicinchoninic protein assay kit (BCA) (Pierce) using bovine serum albumin as a standard.

Analysis of Recombinant SP-A-- The effects of mutations on the oligomeric structure of the recombinant proteins were assessed by chemical cross-linking. Variant SP-As (10 µg) were incubated with the 5 mM disuccinimidyl glutarate (DSG, Pierce) in 20 mM HEPES containing 0.1 mM EGTA, 0.15 M NaCl, and 100 mM KCl at room temperature for 30 min. The reaction was stopped by addition of reducing sample buffer, and the proteins were size-fractionated on 8-16% gradient SDS-PAGE gels and stained with Coomassie Blue.

Primary Culture of Alveolar Type II Cells and Secretion of Phosphatidylcholine-- Experiments to assess the effect of N-terminal deletions on the SP-A-mediated inhibition of surfactant secretion were performed as described previously (14). Briefly, alveolar type II cells were isolated from male Sprague-Dawley rats by tissue dissociation with elastase and purification on metrizamide gradients (35). The type II cells were seeded into tissue culture flasks and incubated overnight in [3H]choline (0.5 µCi/ml) containing Dulbecco's modified Eagle's medium and 10% fetal calf serum (D10) at 37 °C in a 10% CO2 atmosphere. After washing, SP-A variants were tested for their ability to antagonize 12-O-tetradecanoylphorbol-13-acetate (10-7 M)-stimulated surfactant secretion by coincubation with the monolayer for 3 h. In some experiments, 0.125 M alpha -methylmannoside was added simultaneously with the 12-O-tetradecanoylphorbol-13-acetate and SP-A to determine if the SP-A effect was reversible with excess monosaccharides. Secretion was measured using [3H]phosphatidylcholine (PC) as a marker for surfactant and expressed as percent secretion (radioactivity in the media/radioactivity in the cells + media).

Receptor Binding-- A whole cell competitive binding assay was performed to determine the ability of various recombinant forms of SP-A to compete with 125I-rat SP-A for receptor occupancy on the surface of isolated type II cells (36). Following isolation, 2 × 106 type II cells/35-mm dish were incubated overnight in D10 at 37 °C in a 10% CO2 atmosphere. The following morning, the nonadherent cells were removed by washing the monolayers three times at 4 °C with 10 ml of Dulbecco's modified Eagle's medium containing 1 mg/ml BSA. The monolayers were then incubated with 1 µg/ml 125I-rat SP-A and various recombinant SP-As in D10 for 3 h at 37 °C in a 10% CO2 atmosphere. After washing three times on ice with buffer containing 50 mM Tris (pH 7.4), 100 mM NaCl, 2 mM CaCl2, and 1 mg/ml BSA, the cells were solubilized in 0.1 N NaOH, and radioactivity was quantified in a gamma radiation counter.

Liposome Aggregation and Lipid Binding-- Liposome binding and aggregation experiments were performed using lipids purchased from Avanti Polar Lipids, as described previously (14). Unilamellar vesicles were produced by probe sonication of lipid mixtures composed of DPPC:egg PC:phosphatidylglycerol (PG), 9:3:2, and equilibrated with SP-As (lipid:protein ratio 20:1 by weight) in 50 mM Tris, 150 mM NaCl buffer (buffer A) at 20 °C. Aggregation was determined by measuring light scattering (A400 nm) at 1-min intervals after the addition of 5 mM Ca2+ (final). For lipid binding, multilamellar liposomes produced by vigorous vortexing of the same lipid mixture were incubated with 10 µg/ml SP-A (lipid:protein ratio 50:1) in buffer A containing 2.0% BSA and 5 mM Ca2+. Following incubation for 1 h at room temperature, the reaction mixture was centrifuged at 14,000 × gav for 10 min and washed once, and the SP-A content of the pellet and pooled supernatant fractions were determined by enzyme-linked immunosorbent assay. Percent binding was defined as SP-Apellet/(SP-Apellet + supernatant) × 100. Control experiments in which liposomes or Ca2+ were individually deleted were also performed.

Phospholipid Uptake by Type II Cells-- Uptake of phospholipid liposomes by type II cells was performed according to the method of Wright et al. (37) with minor modifications (38). Freshly isolated alveolar type II cells (1 × 106/tube) were incubated with unilamellar liposomes (100 µg/ml) composed of [3H]DPPC (1600 cpm/nmol):egg PC:PG, 7:2:1, and SP-A variants in 0.5 ml of Dulbecco's modified Eagle's medium, 10 mM HEPES (pH 7.4) for 1 h at 37 °C. The media and cells were separated by centrifugation at 160 × g for 5 min at 4 °C, and the cells were washed three times in ice-cold phosphate-buffered saline containing 1 mg/ml BSA. An additional volume of 0.5 ml of phosphate-buffered saline was added to each tube, and the cells and media were transferred to separate liquid scintillation vials and counted. Percent uptake was calculated according to the equation: [3H]DPPCcells/([3H]DPPCcells + 3[H]DPPCmedia) × 100.

    RESULTS

Characterization of the Recombinant Proteins-- The structures of the mutant recombinant proteins used in this study are shown in Fig. 1. Recombinant SP-A that is overproduced in insect cells has functional characteristics that are comparable to the natural protein, despite incomplete hydroxylation of prolines in the collagen-like region (designated "hyp" for hydroxyproline-deficient) (14). For the purposes of this study we have designated the domains of SP-A from N to C terminus as the follows: I-K-C domain, domain 1 (N-terminal segment), domain 2 (N-terminal half of the collagen-like domain), domain 3 (C-terminal half of the collagen-like domain), neck, and CRD. The synthesis and physical characterization of the mutant recombinant SP-As containing telescoping deletions from Asn1 through the end of the N-terminal segment (TM1 or Delta Asn1-Ala7), the midpoint of the collagen-like region (TM1-2 or Delta Asn1-Gly44), and end of the collagen region (TM1-2-3 or Delta Asn1-Pro80) have been previously reported (15). For this study, an additional mutant SP-A containing a nested deletion of the proximal collagen-like region (TM2 or Delta Gly8-Gly44) was produced using similar methods. The TM2 migrated slightly more rapidly than the SP-Ahyp on SDS-PAGE gels under reducing conditions, consistent with the deletion of 37 amino acids (Fig. 2). Like the SP-Ahyp, the TM2 formed disulfide-linked multimers at least as large as tetramers under non-reducing conditions. This migration profile is consistent with interchain disulfide bond formation at both sulfhydryls which are available for interchain linkage, at Cys-1 and Cys6. We have previously reported that disulfide bond formation at Cys-1 in TM1 and TM1-2 results in 17 and 51% dimers, respectively, based on densitometry of nonreducing SDS-PAGE gels. SP-Ahyp, by comparison, is 60% dimers and higher oligomers (15). The TM1-2-3 is monomeric due to the near-absence of the Cys-1-containing tripeptide sequence at the N terminus (<3% of polypeptide chains), presumably because the mutations introduced lead to exclusive signal peptidase cleavage site at the Cys-1-Ala81 bond rather than the alternative site cleavage that occurs in SP-Ahyp, TM1, and TM1-2 (15). The preservation of partial and near wild type levels of Cys-1-dependent disulfide cross-linking at the N terminus of TM1 and TM1-2, respectively, allows for evaluation of the functional consequences of the peptide sequence deletions that are distinct from those that are due to loss of covalent interchain linkage. Noncovalent interactions that also contribute to the assembly of the SP-A oligomer were analyzed by DSG cross-linking, and the results are shown in Fig. 3. Treatment of the SP-Ahyp with DSG followed by size fractionation on reducing SDS-PAGE resulted in the appearance of a series of 8-9 distinct bands, indicating that the same number of polypeptide chains were closely associated in the soluble protein, most likely as three trimers. Previously reported gel filtration chromatography data are also consistent with nonameric assembly of SP-Ahyp (25). Cross-linking analyses further revealed that the TM1-2-3 was a noncovalent trimer, demonstrating that collagen-like region and interchain disulfide bond formation are not required for triple-stranded folding of SP-A during synthesis or to maintain the association of three chains in the fully processed protein. The TM1 and TM1-2 did not form detectable oligomers greater than trimers, but the TM2 was at least hexameric. These results indicate that N-terminal interchain disulfide linkage is not sufficient for the association of trimers and that either the N-terminal segment itself or two interchain bridge-forming cysteines are required for the interaction. We have recently reported that individual disruption of Cys6 or Cys-1 by substitution with Ser did not prevent the assembly of large SP-A oligomers, based on cross-linking analyses2 and gel exclusion chromatography (25, 26). Taken together, these data suggest that the neck + CRD are sufficient for trimerization of SP-A, and that the N-terminal segment plays an important role in the association of trimeric subunits in solution that is not solely attributable to disulfide bond formation.


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Fig. 1.   Schematic of TM SP-As. Wild type recombinant SP-A, SP-Ahyp, contains an N-terminal extension composed of amino acids Ile-Lys-Cys (IKC), an N-terminal segment (NTS) denoted domain 1, a collagen-like region divided by a midpoint interruption at Gly44 into domains 2 and 3, a neck region, and a carbohydrate recognition domain (A). Truncated mutant (TM) recombinant SP-As TM1, TM1-2, TM1-2-3, and TM2 are shown (B). Branched structures represent N-linked carbohydrate.


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Fig. 2.   Electrophoretic analysis of TM2 SP-A. Rat SP-A, wild type recombinant SP-A (SPAhyp), and TM2 SP-As were subjected to 8-16% SDS-PAGE under reducing and nonreducing conditions and stained with Coomassie Brilliant Blue. hyp, hydroxyproline-deficient.


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Fig. 3.   Cross-linking analysis of oligomeric association of polypeptide chains of TM SP-As. The wild type recombinant SP-A (SP-Ahyp), TM1, TM1-2, TM1-2-3, or TM2 were incubated with the cross-linking reagent DSG for 30 min at room temperature and then size-fractionated on 8-16% reducing SDS-PAGE gels and stained with Coomassie Brilliant Blue. hyp, hydroxyproline-deficient.

Direct Binding of TM SP-As to Multilamellar Liposomes-- The effects of N-terminal deletions on the direct binding of TM SP-As to multilamellar liposomes was assessed by determining the percent of added protein that cosedimented with the phospholipid pellet upon centrifugation, as described previously (14). Specific binding was defined by subtracting Ca2+-independent sedimentation of SP-A (i.e. in the presence of EDTA) from total Ca2+-dependent binding. As shown in Fig. 4, 53.8 ± 2.2% of SP-Ahyp specifically bound to the liposomes, compared with 32.2 ± 2.5% of TM1 and 43.8 ± 1.6% of TM1-2. The TM2 protein exhibited wild type levels of phospholipid binding activity (54.5 ± 2.6%), but binding by TM1-2-3 was negligible (2.1 ± 1.3%). We have previously reported that the binding of TM2-3 to lipid is about half that of SP-Ahyp (25). These data indicate that domain 2 of SP-A does not contribute to lipid binding and that domain 3 is required for wild type levels of lipid binding activity. The finding that TM1-2 bound to lipid approximately 80% as effectively as SP-Ahyp demonstrates that N-terminal disulfide linkage can functionally replace the N-terminal segment with respect to lipid binding.


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Fig. 4.   Direct binding of TM SP-As to phospholipid liposomes. Multilamellar liposomes were incubated with SP-Ahyp, TM1, TM1-2, TM1-2-3, or TM2 at room temperature for 1 h. Following centrifugation, SP-A in the pellet and supernatant were quantified by enzyme-linked immunosorbent assay. Specific binding was determined by subtraction of SP-A sedimentation in the presence of EDTA from total binding. The data shown are mean ± S.E., n = 3. hyp, hydroxyproline-deficient.

Aggregation of Phospholipid Liposomes by TM SP-As-- We have previously determined that the Cys6 interchain disulfide bond but not the collagen-like domain of rat SP-A is required for aggregation of phospholipid liposomes (25). The role of the N-terminal segment in aggregation was examined by incubation of truncated mutant forms of SP-A with unilamellar liposomes in the presence of Ca2+ and measurement of light scattering. As shown in Fig. 5, the maximal end point for light scattering induced by SP-Ahyp was 0.0965 ± 0.0003 A400 units. The TM1 retained only minimal aggregation activity, approaching an end point for light scattering of 0.0233 ± 0.0007 A400 units. The TM1-2 and TM1-2-3 were essentially nonfunctional in the assay with end points of 0.0062 ± 0.003 and 0.0030 ± 0.0001 A400 units, respectively. Comparison of the complete loss of function in TM1-2-3 and the wild type aggregation activity of the previously reported TM2-3 (also known as Delta G8-P80) (25) indicates that covalent interchain linkage, or the N-terminal segment peptide sequence, or both, are required for aggregation. At least one (25), but not both N-terminal interchain bonds are required, since disruption or deletion of Cys-1 that forms interchain bonds does not block aggregation (26). The aggregation activity of TM2 was nearly identical to SP-Ahyp, approaching a maximal end point of 0.0965 ± 0.0008 A400 units. Comparison of the TM1-2 and TM2 results indicate that the N-terminal segment, and not simply N-terminal interchain disulfide-linkage (present in near wild type levels in TM1-2), is required for lipid aggregation by SP-A. Collectively, these data are consistent with a role for SP-A self-association via the N-terminal segment polypeptide domain in the aggregation of phospholipid vesicles.


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Fig. 5.   Calcium-dependent aggregation of phospholipid liposomes by TM SP-As. Unilamellar liposomes were incubated with SP-Ahyp, TM1, TM1-2, TM1-2-3, or TM2 in Tris buffer (pH 7.4) in a quartz cuvette. After equilibration for 2 min at 20 °C, 5 mM Ca2+ was added and light scattering (A400 nm) was measured in a spectrophotometer. The data shown are mean ± S.E., n = 3.

Competition of TM SP-As with 125I-Rat SP-A for Binding to the SP-A Receptor on Type II Cells-- The binding of deletion mutant SP-As to the receptor on the surface of isolated type II cells was assessed in a competitive binding experiment, and the results are shown in Fig. 6. SP-Ahyp competed with 125I-rat SP-A for receptor occupancy in a dose-dependent manner with an IC50 of 9.25 µg/ml, similar to what has been reported previously (14, 21, 38). The TM1 and TM1-2-3, which have limited interchain linkage at the N terminus, did not compete for receptor occupancy. The TM1-2 and the TM2 both competed with 125I-rat SP-A for receptor binding with IC50 values that were approximately 76 µg/ml (estimated) and 35.5 µg/ml. These data suggest that N-terminal interchain disulfide bond formation is critical for the activity of SP-A to compete for receptor occupancy and that domain 2 and the N-terminal segment are not absolutely required. In addition, comparison of the well preserved activity of TM2 and the loss of competitive binding by the previously characterized TM2-3 protein (25) suggests that domain 3 may play an important role in the binding of SP-A to the receptor.


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Fig. 6.   Competition of TM SP-As with 125I-rat SP-A for receptor occupancy on isolated alveolar type II cells. Primary cultures of alveolar type II cells were incubated with 0.5-1 µg/ml 125I-rat SP-A and various concentrations of rat SP-A, SP-Ahyp, TM1, TM1-2, TM1-2-3, or TM2 for 3 h at 37 °C. The monolayers were washed, dissolved in 0.1 N NaOH, and counted in a gamma radiation counter as described under "Experimental Procedures." hyp, hydroxyproline-deficient.

The Inhibition of Surfactant Secretion from Type II Cells by the TM SP-As-- We have previously reported that disruption of the Cys6 interchain disulfide bridge markedly impairs all inhibition of surfactant secretion by SP-A, but deletion of the collagen-like domain blocks only specific (monosaccharide-reversible) inhibition (25). In this study, we examined the role the N-terminal segment and subdivisions of the collagen domain in inhibition of secretion by SP-A (Fig. 7). The concentration of SP-Ahyp required for half-maximal inhibition of secretion (IC50) was approximately 5.5 µg/ml, which was somewhat higher than previously reported values of 0.1-1.0 µg/ml (14, 21, 25, 38). Inhibition by TM1, TM1-2, and TM2 was comparable with that seen with SP-Ahyp, with IC50 values of 1.10, 0.18, and 0.63 µg/ml, respectively. In contrast to the potent inhibition by these proteins and the previously characterized TM2-3 protein (25), the TM1-2-3 was a poor inhibitor of surfactant secretion (IC50 >65 µg/ml). This result indicates that interchain disulfide linkage is critical for inhibition of surfactant secretion. To assess the specificity of inhibition by the TM proteins, the experiment was also carried out in the presence of alpha -methylmannoside, a monosaccharide that has been shown to block the nonspecific inhibition of surfactant secretion by lectins such as concanavalin A (36). The inhibition by all of the TM proteins was at least partially blocked by excess alpha -methylmannoside, which is never seen for rat SP-A or SP-Ahyp (Fig. 8). Collectively, these results indicate that interchain disulfide bond formation at Cys-1 can functionally replace the N-terminal segment and/or the Cys6 interchain disulfide bond with respect to nonspecific inhibition of surfactant secretion. However, specific inhibition of surfactant secretion requires the N-terminal segment and both the N-terminal and C-terminal portions of the collagen-like domain.


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Fig. 7.   Inhibition of phospholipid secretion from alveolar type II cells by TM SP-As. Isolated alveolar type II cells in primary culture were incubated overnight with [3H]choline to label cellular phosphatidylcholine. The secretagogue 12-O-tetradecanoylphorbol-13-acetate (TPA) and SP-Ahyp, TM1, TM1-2, TM1-2-3, or TM2 were added and incubated for 3 h at 37 °C. Media and cells were harvested and counted in a scintillation counter. Percent secretion was defined as count in the media/count in the media + cells. The data shown are mean ± S.E., n = 3. hyp, hydroxyproline-deficient.


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Fig. 8.   Assessment of specificity of inhibition of secretion by TM SP-As. Inhibition of secretion by SP-Ahyp, TM1, TM1-2, TM1-2-3, TM2, or TM2-3 was performed as outlined in Fig. 7 except that 0.125 M alpha -methylmannoside was added to the media. hyp, hydroxyproline-deficient.

Enhanced Uptake of Surfactant Liposomes by TM SP-As-- We have previously reported that deletion of the collagen-like domain and disruption of the Cys6 disulfide bond blocked the SP-A-mediated uptake of lipid vesicles by type II cells (25). In the absence of added SP-A, approximately 0.43 ± 0.09% of the [3H]DPPC label was taken up by the cells (Fig. 9). In the presence of 50 µg/ml SP-Ahyp, there was a more than a 4-fold increase in lipid uptake, to 1.88 ± 0.28%. The two truncated proteins with the greatest degree of N-terminal disulfide linkage, TM1-2 and TM2, enhanced liposome uptake by 3-fold to 1.36 ± 0.22% and 3.5-fold to 1.55 ± 0.20%, respectively. The TM1, which is only 17% dimeric, had a weak effect on lipid uptake (1.7 fold enhancement to 0.710 ± 0.07%) and the TM1-2-3, which is monomeric with respect to disulfide bond formation, was even less active (1.4-fold enhancement to 0.61 ± 0.09%). We conclude that interchain disulfide linkage but not domain 1 or domain 2 is essential for SP-A-mediated lipid uptake by type II cells. The finding that TM2 is nearly as active as SP-Ahyp and that TM2-3 is inactive in the assay (25) may indicate that the sequences within domain 3 may play a role in lipid uptake by SP-A.


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Fig. 9.   Enhanced association of phospholipid liposomes with alveolar type II cells by TM SP-As. Unilamellar liposomes labeled with trace [3H]DPPC were incubated with monolayers of isolated alveolar type II cells in the presence of 50 µg/ml SP-Ahyp, TM1, TM1-2, TM1-2-3, or TM2. Following incubation for 1 h, the cells were washed, dissolved in 0.1 N NaOH, and counted in a scintillation counter. The data shown are mean ± S.E., n = 3.


    DISCUSSION

The purpose of this study was to examine the individual functional contributions of the major N-terminal domains of SP-A including the interchain disulfide bonds, the N-terminal segment, and the N-terminal and C-terminal portions of the collagen-like domain. The strategy employed was to introduce telescoping N-terminal deletions through the beginning, middle, and end of the collagen-like region of SP-A and characterize the effects of the truncations on the interactions of SP-A with type II cells and surfactant lipids. Pairwise comparisons of TM proteins with complementary mutants that contained the N-terminal segment (TM1 and SP-Ahyp, TM1-2 and TM2, and TM1-2-3 and TM2-3) permitted dissection of the function of each of the N-terminal domains. Partial preservation of N-terminal disulfide cross-linking at Cys-1 in TM1 and TM1-2 facilitated examination of the functional consequences of loss of the N-terminal segment and the N-terminal half of the collagen-like domain without the confounding influences of complete loss of covalent interchain linkage. Data from this study and previous reports that indicate the domains of SP-A that are essential or not essential for several SP-A functions are outlined in Table I.

                              
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Table I
Domains of SP-A that are individually essential (+) or nonessential (-) for individual functions

The assembly of SP-A can be conceptualized in two parts, the folding of monomeric subunits into trimers and the association of trimers into an octadecamer. Three domains of SP-A have potential roles in the trimerization of SP-A as follows: the neck domain, the collagen-like region, and interchain disulfide bonds. The observation that collagenase-digested SP-A remains associated as a noncovalent trimer indicates that intermolecular forces in the neck + CRD are sufficient to support the oligomeric association of three strands (39). Crystallographic analyses of related collectins demonstrate that the CRDs fold independently and that the neck regions form tightly associated triple-stranded bundles of alpha -helical coiled coils (19, 40, 41). Data from this study are consistent with that model. Cross-linking analyses indicated that the TM1-2-3 was composed of three strands, indicating that the neck domain is sufficient for trimeric assembly. This result further indicates that folding of the neck + CRD into a functional conformation (as judged by competence to bind mannose-Sepharose) does not require the collagen-like region, the N-terminal segment, or interchain disulfide bonds and that the folding of SP-A likely proceeds from the C terminus to the N terminus, as has reported in other soluble collagens and collectins (42).

In the second step of oligomeric assembly, six trimeric subunits become associated through the N-terminal domains including the collagen-like domain, the N-terminal segment, and the N-terminal interchain disulfide bonds. We previously characterized mutant proteins containing disruption of the Cys6 interchain disulfide bond, the Cys-1 interchain disulfide bond, or deletion of the collagen-like domain (25, 26). Neither interchain disulfide bridges or the collagen-like region were found to be individually required for assembly of SP-A into a nonameric oligomer, the predicted structure of the wild type recombinant protein SP-Ahyp (25). In the current study, the oligomeric assembly of SP-A was investigated by chemical cross-linking experiments. The largest oligomer of TM1-2 that was detected by the cross-linking analyses was a trimer, indicating that the C-terminal half of the collagen-like domain and extensive disulfide cross-linking at the N terminus were not sufficient to support the association of trimeric subunits into higher oligomers. The complementary mutant protein TM2 was a hexamer, however, which strongly implicates the N-terminal segment in the interaction between trimeric subunits of SP-A. This property of the N-terminal segment does not appear to be attributable to the availability of two (rather than one) interchain disulfide bonds flanking this domain, since cross-linking analyses2 and gel filtration chromatography indicated that the SP-Ahyp, C-1S and SP-Ahyp, C6S were also composed of multiple trimeric subunits (25, 26). Collectively, the findings that reduced and alkylated SP-A (33), SP-Ahyp, C6S (25), and SP-Ahyp, C-1S (26) remain associated as high molecular weight oligomers are consistent with the notion that strong noncovalent intermolecular forces in the neck, collagen-like region, and the N-terminal segment, and not interchain disulfide bonds, are the major determinants of SP-A assembly into trimeric and supratrimeric oligomers.

SP-A binds to surfactant lipids and to phospholipid liposomes that contain DPPC. Lipid binding can be blocked by point mutations in the CRD, providing strong evidence that the major lipid-binding site resides in that domain (21). However, mutations in the N-terminal region also reduce binding to lipid vesicles. We have previously reported that the disruption of the Cys6 interchain disulfide bond reduced binding to liposomes by 66% compared with SP-Ahyp (25). In this study, deletion of the N-terminal segment which includes Cys6 reduced lipid binding by only 40%. Greater retention of lipid binding by TM1 may have been related to more extensive dimerization by disulfide bridging at Cys-1 in TM1 (17% dimeric) compared with the SP-Ahyp, C6S (<5% dimeric). Lipid binding by the TM1-2 mutant, which is extensively dimerized, was about 81% of that of SP-Ahyp, and wild type levels of lipid binding were exhibited by TM2. These data eliminate the N-terminal half of the collagen-like domain as an essential element for lipid binding and demonstrate that N-terminal interchain disulfide linkage can functionally replace the N-terminal segment with respect to lipid binding. Two interchain disulfide bonds are not required for lipid binding since deletion of Cys-1 was silent (26), but the greater lipid binding activity of TM2 compared with TM1-2 suggests that other subdomains of the N-terminal segment (e.g. hydrophobic residues, carbohydrate moiety) may contribute to lipid interactions. The characterization of the TM proteins also provides some information about the role of the C-terminal half of the collagen-like region in lipid binding. The TM1-2-3, which contains only the neck + CRD of SP-A and is not disulfide-bridged, did not exhibit detectable lipid binding in our assay, but the N-terminal segment containing TM2-3 had lipid binding activity that was about 1/2 that of SP-Ahyp (25). Comparison of lipid binding activities of TM2 and TM2-3 indicates that the C-terminal half of the collagen-like domain may contribute to lipid binding (25).

Lipid aggregation is blocked by disruption of the Cys6 interchain disulfide bond (25) and by point mutations in the CRD (21). In this study, deletion of the N-terminal segment which contains Cys6 also greatly reduced lipid aggregation, and the larger N-terminal deletions contained in TM1-2 and TM1-2-3 completely eliminated the activity. Addition of the N-terminal segment to the the TM1-2-3 protein (to produce the previously characterized TM2-3 (25)) restores aggregation activity, suggesting that covalent interchain disulfide bridging, the N-terminal segment polypeptide sequence, and/or the N-terminal oligosaccharide moiety are required for the function. Functional comparison of the TM1-2 and TM2 proteins elucidated the role of these three N-terminal domains in lipid aggregation. Unlike the lipid binding function of SP-A, extensive disulfide bond formation at Cys-1 in the TM1-2 mutant could not functionally replace the N-terminal segment in the aggregation assay. However, the N-terminal segment containing TM2 aggregated liposomes as well as SP-Ahyp. The availability of a second interchain disulfide-forming cysteine (at Cys6 within the N-terminal segment) is not a likely explanation for difference in function between TM1-2 and TM2, since we have recently shown that only one interchain disulfide bridge is sufficient (26). Another model for SP-A-mediated liposome aggregation that has been proposed is self-association by a lectin interaction between the CRD and SP-A-associated carbohydrate (43), which is attached to Asn1 of the aggregation-competent TM2 but is absent from the N terminus of the nonfunctional TM1-2. We believe that this mechanism is unlikely, based on our previously reported finding that recombinant SP-A which contains no carbohydrate, produced by synthesis in the presence of tunicamycin or genetic ablation of both the Asn1 and Asn187 consensus sequences for glycosylation, aggregates lipid vesicles (14). Collectively, these data indicate that the N-terminal segment is required for liposome aggregation and that the N-terminal interchain disulfide cross-linking is required but is not sufficient for the activity. We propose a model for liposome aggregation in which SP-A binds to phospholipid vesicles via the CRD and links adjacent liposomes by interactions between N-terminal segments.

Specific binding to the SP-A receptor requires Ca2+ and is not sensitive to the presence of excess monosaccharides (36). Mutagenesis experiments indicate that SP-A receptor binding requires cooperative interactions between the major receptor recognition site in the CRD and domains involved in oligomeric assembly, including the collagen-like domain and Cys6 interchain disulfide bonds. Specifically, we have shown point mutations in the CRD (21, 38) and deletion of the collagen-like domain (25) eliminates competitive binding to the SP-A receptor. Analysis of the TM1-2 and TM2 proteins was used to map domains within the collagen-like region that are required for receptor binding. Competition for receptor occupancy by the TM2 protein was less effective than SP-Ahyp at low SP-A concentrations but was comparable to SP-Ahyp at the highest concentrations tested. The TM1-2 protein, which is 51% dimeric, also competed for binding, albeit at a level that was less than SP-Ahyp or TM2. The TM1 did not compete for receptor occupancy, most probably because of the limited degree of interchain disulfide linkage at the N terminus (17% dimeric). Our interpretation of these data is that the N-terminal segment is not absolutely essential for receptor binding and can be functionally replaced by interchain disulfide linkage at the N terminus. The C-terminal half of the collagen-like domain likely contributes to competitive receptor binding, however, since the TM1-2 and TM2 proteins were effective competitors and the TM2-3 protein was not.

Binding to the SP-A receptor on type II cells is coupled to inhibition of secretion of surfactant. Although several lectins can inhibit surfactant secretion, their activities are blocked by excess carbohydrate and are therefore nonspecific (36). In contrast, inhibition of secretion by SP-A is insensitive to the presence of monosaccharide competitors. We recently reported that deletion of the collagen-like domain (TM2-3) of SP-A blocks specific inhibition of surfactant secretion (25). The TM1 data presented in Fig. 8 indicates that, in addition to the collagen-like domain, the N-terminal segment is also required for specific inhibition of surfactant secretion. Monosaccharide-sensitive inhibition by the TM2 protein demonstrates that at least one determinant of specific inhibition within the collagen-like region maps to the N-terminal half of the domain. The integrity of the CRD is also important for specific inhibition, since a mutant protein containing the substitutions Glu195 to Gln and Arg197 to Asp produces weak, monosaccharide-reversible inhibition (38). Collectively, these data indicate that the Ca2+ and carbohydrate binding region of the CRD, the N-terminal segment, and the collagen-like domain are critical for specific inhibition of surfactant secretion by SP-A (Table I). Further studies will be required to determine if sequences within the C-terminal half of the collagen-like domain and the neck region contribute to specific inhibition of surfactant secretion.

The finding that the TM1 and TM1-2 were potent nonspecific inhibitors of surfactant secretion (monosaccharide-sensitive) indicates that the N-terminal segment is not required for this activity. Some degree of disulfide cross-linking is required, however, since the TM2-3 is potent nonspecific inhibitor of surfactant secretion, whereas the TM1-2-3 is a very weak nonspecific inhibitor. The data suggest that the minimal structural requirements for potent nonspecific inhibition of secretion by SP-A include the neck + CRD and disulfide-linkage at the N terminus.

The results of this study and prior studies indicate that SP-A-enhanced uptake of [3H]DPPC into isolated type II cells requires the collagen-like domain, interchain disulfide linkage, and the CRD. In general, the activity of the TM mutants in the uptake assay paralleled the extent of disulfide linkage at the N terminus. The TM1-2 and TM2 proteins which have near wild type levels of interchain linkage at the N terminus enhanced lipid uptake to an extent that was comparable to SP-Ahyp. On the other hand, the TM1 protein which is less extensively disulfide-bridged, had little activity in the assay. The previously characterized interchain disulfide-deficient mutant SP-Ahyp, C6S was also only a weak promoter of lipid uptake (25). Comparison of the well preserved functional activity of TM2 and the minimal activity of the previously described TM2-3 suggests that the Gly45-Pro80 but not the Gly8-Gly44 portion of the collagen-like domain of SP-A contain sequences that are critical for the property of SP-As to enhance the association of klipids into type II cells.

In summary, deletion mutagenesis of the N-terminal domains of SP-A reveals that lipid binding requires interchain disulfide linkage, lipid aggregation requires the N-terminal segment and interchain bridging, receptor competition and SP-A-modulated uptake of surfactant by type II cells requires N-terminal interchain bonds and the C-terminal half of the collagen-like region, and specific inhibition of surfactant secretion requires all N-terminal domains except the IKC region. Functional mapping of the molecule may ultimately reveal the minimum SP-A structural requirements for the formation of tubular myelin, for improving the resistance of surfactant to protein inhibition, and for antimicrobial defense.

    FOOTNOTES

* This research was supported by National Institutes of Health Grant P30ES-0609606, a Career Investigator Award from the American Lung Association, and the Medical Research Service of the Department of Veteran Affairs.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.

Dagger To whom correspondence should be addressed. Tel.: 513-558-0480; Fax: 513-558-4858; E-mail: frank.mccormack{at}uc.edu.

The abbreviations used are: SP-A, surfactant protein A; TM, truncated mutant; CRD, carbohydrate recognition domain; IKC, isoleucine-lysine-cysteine; BSA, bovine serum albumin; DSG, disuccinimidyl glutarate; DPPC, dipalmitoylphosphatidylcholine; PC, phosphatidylcholine; PG, phosphatidylglycerol; PAGE, polyacrylamide gel electrophoresis.

2 F. X. McCormack, unpublished data.

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Top
Abstract
Introduction
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