Interactions of Surfactant Protein D with Fatty Acids - ATS Journals

Interactions of Surfactant Protein D with Fatty Acids Nihal S. DeSilva, Itzhak Ofek, and Erika C. Crouch Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri; and Department of Human Microbiology, Tel Aviv University, Tel Aviv, Israel

Surfactant Protein D (SP-D) plays important roles in antimicrobial host defense, inflammatory and immune regulation, and pulmonary surfactant homeostasis. The best-characterized endogenous ligand is phosphatidylinositol; however, this lipid interaction at least in part involves the carbohydrate moiety. In this study we observed that SP-D binds specifically to saturated, unsaturated, and hydroxylated fatty acids (FA). Binding of biotinylated-SP-D to FAs or biotinylated FA to SP-D was dosedependent, saturable, and specifically competed by the corresponding unlabeled probe. Specific binding to FA chains was also demonstrated by solution phase competition for FA binding to acrylodan-labeled FA binding protein (ADIFAB), and by overlay of thin layer chromatograms with SP-D. Maximal binding to FA was dependent on calcium, and binding was localized to the neck and carbohydrate recognition domains (CRD) using recombinant trimeric neck⫹CRDs. Saccharide ligands showed complex, dose-dependent effects on FA binding, and FAs showed dose- and physical state–dependent effects on the binding of SP-D to mannan. In addition, CD spectroscopy suggested alterations in SP-D structure associated with binding to monomeric FA. Together, the findings indicate specific binding of FA to one or more sites in the CRD. We speculate that the binding of SP-D to the fatty acyl chains of surfactant lipids, microbial ligands, or other complex lipids contributes to the diverse biological functions of SP-D in vivo.

Surfactant proteins (SPs)-A and -D, and serum mannose binding lectin, play diverse and important roles in innate immunity (1–3). In particular, there is growing evidence that the lung collectins, SP-A and SP-D, contribute to antimicrobial host defense, inflammatory and immune regulation within the lung, as well as the regulation of pulmonary surfactant homeostasis. SP-D is predominantly assembled as tetramers of trimeric subunits (dodecamers) with terminal lectin domains (4, 5). As for the other collectins, each trimeric subunit consists of an amino-terminal crosslinking domain, a triple helical collagen domain, a coiled–coil linking peptide or neck domain, and a C-type lectin carbohydrate recognition

(Received in original form May 8, 2003 and in revised form June 13, 2003) Address correspondence to: Erika C. Crouch, M.D., Ph.D., Dept. of Pathology and Immunology, Barnes-Jewish Hospital, North campus, Box 8118 216 S. Kingshighway, St. Louis, MO 63110. E-mail: [email protected] Abbreviations: acrylodan-labeled fatty acid binding protein ADIFAB; bovine serum albumin, BSA; circular dichroism, CD; critical micellar concentration, CMC; carbohydrate recognition domain, CRD; dimethylsulfoxide, DMSO; fatty acid, FA; fatty acid–free BSA, FAF-BSA; Hepes-buffered saline, HBS; molecular weight cutoff, MWCO; phosphate-buffered saline, PBS; phosphatidylinositol, PI; recombinant rat surfactant protein D, RrSP-D; surfactant protein A, SP-A; surfactant protein D, SP-D; submicellar, sub-CMC. Am. J. Respir. Cell Mol. Biol. Vol. 29, pp. 757–770, 2003 Originally Published in Press as DOI: 10.1165/rcmb.2003-0186OC on June 19, 2003 Internet address: www.atsjournals.org

domain (CRD). Trimerization of CRDs, which is dependent on the neck domain, appears to be necessary and sufficient for high-affinity interactions with complex multivalent carbohydrate ligands, whereas the higher order oligomerization of trimeric subunits is required for the aggregation of particulate ligands. SP-D, like many other effectors of innate immunity, is a pattern recognition molecule with a variety of potential ligands (1, 6). Most known interactions involve complex oligosaccharides or glycoconjugates expressed by various microorganisms or other organic particles. However, SP-D also shows a restricted range of interactions with lipidcontaining molecules, particularly phosphatidylinositol (PI), its major surfactant-associated ligand (7), and glucosylceramide (8). The mechanism of SP-D binding to lipids is incompletely understood. SP-D shows high-affinity binding to PI in solid phase assays or when presented in liposomal complexes (7, 9, 10). The interactions of SP-D with PI and glucosylceramide are calcium-dependent and inhibited by competing sugars, including glucose and myoinositol, but not by inefficient saccharide competitors such as galactose. In addition, SP-D binds to glucosylceramide, but not to galactosylceramide (8). Thus, these lipid interactions appear to involve—to a significant degree—carbohydrate recognition. On the other hand, there is indirect evidence that PI also interacts with regions of the molecule that are not directly involved in saccharide binding. Site-directed mutagenesis of the saccharide-binding site with simultaneous substitutions of Gln for Glu321 and Asp and Asn323 (RrSPDQPD) altered the saccharide preference from glucose/mannose to galactose sugars (11). Although the QPD mutation blocked interactions with glucosylceramide, there was only partial inhibition of binding to PI-containing liposomes, suggesting that other interactions contribute to PI binding. In addition, limited interactions of recombinant rat SP-D with monomolecular layers of phospholipids were observed in surface balance studies (12). Because there was no detectable preference for specific phospholipid head-groups, hydrophobic interactions with the acyl chains were suggested. Lastly, PI and other phospholipids have been reported to bind to recombinant neck domains (13). In the current studies, we explored the interactions of SP-D with selected fatty acids (FA) and examined the effects of these interactions on binding to two well-characterized ligands, mannan and PI. Given other ongoing studies relating to bacterial interactions, many of our experiments used 2- or 3-hydroxymyristate (2-OH-myristate or 3-OHmyristate), FA that are highly represented in gram-negative lipopolysaccharides. These FA also proved convenient for many experiments because of their relatively high solubility

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and high critical micellar concentrations, which facilitated uniform adsorption to surfaces and use as solution phase, monomeric competitors. For many studies, we also examined palmitate, which is highly represented in the airspaces of the lung, both as a substituent of surfactant phospholipids and as free FA. Our experiments demonstrated specific, high-affinity binding to FA. Binding was predominantly mediated by calcium-dependent interactions with the neck⫹CRD domain of SP-D. However, the interactions of SP-D with carbohydrate and lipid ligands were complex and functionally interdependent.

Materials and Methods Chemicals and Biological Reagents Most chemicals and biologicals were obtained from Sigma-Aldrich (St. Louis MO), including: mono- and disaccharides, mannan, EGTA and EDTA, bovine hemoglobin (H-2500), human fatty acid–binding protein (FABP, FO677), sodium metaperiodate, soybean phosphatidylinositol, and FA. The latter included 2- and 3hydroxy lauric (12:0), myristic (14:0), and palmitic (16:0) acids; saturated myristic, palmitic, and stearic (18:0) acids; and unsaturated oleic (18:1) and linoleic (18:2) acids. For a few experiments we also used soybean PI (Avanti Polar Lipids, Alabaster, AL). EZ-Link-Biotin-LC-hydrazide, EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride), EZ-Link-Sulfo-NHS-LCBiotin, biotinylated secondary antibodies, streptavidin-peroxidase, and TMB Substrate were from Pierce Chemical (Rockford, IL). Acrylodan-labeled fatty acid–binding protein (ADIFAB) was obtained from Molecular Probes (Eugene, OR). All organic solvents were reagent grade and obtained from Fisher Scientific Co. (Hanover Park, IL). Glass- and aluminum-backed TLC plates coated with silica gel GF were from Analtech (Newark, DE). Ultrafiltered, endotoxin-free Milli-Q water was used for the preparation of all buffers and solutions, and all solutions were stored at 4⬚C.

Bovine Serum Albumin For some preliminary solid-phase assays we used bovine serum albumin (BSA), Fraction V (A4503; Sigma-Aldrich, St. Louis, MO) in the blocking and binding buffers. This BSA, which is often used as a blocking agent for enzyme-linked immunoassays and other binding assays, contains 0.1–1% (wt/vol) free FA (0.3–9 mol of FA per mole of BSA). Thus, for a 0.1% (wt/vol) BSA solution with 1% contamination of free acids there is a total FA concentration of ⵑ 40 ␮M, assuming an average FA mass of 260 Da. For convenience, this BSA will be referred to simply as BSA. For reasons discussed in the Results, subsequent experiments used BSA, Fraction V, FA-free, low endotoxin (A8806; Sigma-Aldrich). This preparation contains ⬍ 0.005% FA, giving a final concentration of ⬍ 200 nM FA. For a few preliminary experiments we also used FA-free BSA (FAF-BSA) from JRH Biosciences (Lenexa, KS). The results were essentially identical for these two FAF-BSA samples. For convenience, we will refer to the latter preparations as FAF-BSA.

Fatty Acids For most experiments, lipids were reconstituted at 1 mg/ml in chloroform/methanol (1:4, vol/vol). When the lipids were required in aqueous phase, they were reconstituted in Blocking/Binding buffer (see Binding Assays below), vortexed vigorously, and then sonicated at 30-s bursts for a total of 3 min in a water bath at 24⬚C

(W 220, Sonicator; Ultrasonic Inc., Farmingdale, NY). The resulting lipid solutions or dispersions were temporarily stored at 4⬚C. Before each experiment the lipids were warmed to room temperature and vortexed three times for several seconds at the maximum setting on a Vortex Genie to obtain a homogenous preparation.

Determination of Critical Micellar Concentration FA monomers show concentration-dependent association to form micelles. Published values for CMCs for specific FA are quite varied, reflecting dependence on a variety of variables including the ionic strength and calcium concentration of the buffer, and the incubation temperature (14). For the present experiments CMCs of FA were empirically determined under the specific conditions of assay, i.e., at room temperature (24.5⬚C) in binding/blocking buffer containing calcium and 0.1% FAF-BSA. The CMC was estimated based on the minimum concentration of FA, resulting in a change in the measured conductivity (15). It was recognized as a break in the slope of the molar conductivity versus concentration curve and defined by linear extrapolation to the abscissa. This point reproducibly occurred at a lower concentration than the appearance of turbidity. For this assay we used a microelectrode (MI 900 series; Microelectrodes, Inc., Bedford, NH) with a cell constant of 1.13 micromhos/cm.

Recombinant Proteins Recombinant rat SP-D dodecamers and single-arm trimeric subunits (RrSP-Dser15, 20) were expressed by stably transfected CHO-K1 cells (16). Proteins were collected in the absence of serum and purified by sequential maltosyl-agarose affinity chromatography and gel filtration chromatography. The reduced proteins migrated as a single band of 43-kD on silver-stained gels, contained very low levels of endotoxin as measured by chromogenic assays, and were fully functional in their interactions with specific strains of gram-negative bacteria and influenza virus. Trimeric rat neck⫹CRD domains were expressed in bacteria as recently described (17), and are functional as lectins in a variety of assay systems. Protein concentrations were determined using the Coomassie Plus-200 protein assay reagent (Pierce Chemical). The assay was adapted to the micro protocol test tube version with a working range of 1–25 ␮g/ml, using BSA as standard.

Biotinylation of SP-D and Trimeric neck⫹CRD Domains For most assays using SP-D dodecamers the protein was biotinylated with EZ-Biotin-LC-Hydrazide (Pierce Chemical) at the single site of asparagine-linked glycosylation at Asn70. This oligosaccharide is located within the collagen domain, remote from the neck or CRD. Because deletion of this consensus has no discernable effect on SP-D oligomerization or function, it is unlikely that biotinylation of this site will alter protein function. For studies using the neck⫹CRD, which lacks carbohydrate attachments, and control experiments with RrSP-D, the protein was labeled at primary amino-groups with EZ-link-Sulfo-NHS-LC-Biotin (Pierce Chemical). Reactions were performed using limited modifications of the manufacturer’s recommended procedures and described as follows. Biotin-hydrazide coupling method. Five hundred micrograms of SP-D in 1 ml of 5 mM HEPES, 150 mM NaCl, 10 mM EDTA, pH 7.4 (HBS/EDTA) was mixed with 1 ml freshly prepared 20 mM sodium periodate solution prepared in 0.1 M sodium acetate, pH 5.5, and incubated in the dark for 30 min. After terminating the

DeSilva, Ofek, and Crouch: SP-D and Fatty Acids

reaction with 300 ␮l of 100 mM glycerol, the sample was vortexed, transferred to a 3-ml Slide-A-Lyser cassette with a MWCO of 10,000 (Pierce Chemical), and dialyzed overnight against 0.1 M sodium acetate, pH 5.5. Biotin-LC-hydrazide was dissolved in DMSO (50 mM) and then added to the dialyzed sample in a sterile 15-ml tube to a final concentration of 5 mM. After vortexing, the reaction mixture was incubated for 2 h at room temperature with end-overend rotation. The biotinylated SP-D was dialyzed against HBS/ EDTA, pH 7.4 buffer at room temperature overnight. The extent of biotinylation was determined using the HABA/Avidin reaction (Pierce Chemical) according to the manufacturer’s instructions, and the final protein concentration was determined using the dyebinding assay. The final specific activity was in the range of 40–45 pmoles biotin/␮g SP-D. Aliquots (200 ␮l) of the biotinylated SP-D were transferred to 1-ml vials and stored at –80⬚C. Sulfo-NHS-LC-Biotin. SP-D was diluted to 10–20 ␮g/ml with phosphate-buffered saline (PBS), pH 7.4. A 1-mg/ml solution of Sulfo-NHS-Biotin was prepared fresh in distilled water, and 10–20 ␮l of this solution was added to the protein. The reactants were mixed and transferred to a 500-␮l Slide-A-Lyser cassette (Pierce Chemical) and incubated with end-over-end rotation for 2 h at room temperature. The cassette was then dialyzed against PBS for 2–4 h. The extent of biotinylation was determined by the HABA/Avidin reaction and the biotinylated protein was stored at –80⬚C. The specific activity was ⵑ 50 pmoles biotin/␮g SP-D.

Biotinylation of FA FA were biotinylated at the carboxyl group with EZ-Link-BiotinLC-Hyrazide using EDC (Pierce Chemical). A fresh solution of 50 mM Biotin-LC-hydrazide was prepared in DMSO. The FA (1 mg/ml) were suspended in MES buffer (0.1 M MES [(2-Nmorpholino) ethanesulfonic acid], pH 5.5), and vortexed before sonication. A fresh solution of EDC (100 mg/ml) in MES buffer was prepared, and aliquots of the biotin (35 ␮l) and of EDC solution (20 ␮l) were added to the FA. The sample was vortexed and then mixed with end-over-end rotation for 16 h at room temperature. After centrifugation for 5 min at 4,000 rpm, the supernatant was transferred to a Spectra/Por Irradiated DispoDialyser (capacity 2 ml, MWCO 500 or 100) and dialyzed against PBS for 48 h at room temperature. The extent of biotinylation was determined with the HABA/Avidin reaction as before. The specific activity of the biotinylated FA was determined, and the identity of the biotinylated species was confirmed, by quantifying the amount of biotinylated FA using a thin-layer chromatographic (TLC) assay. Two 100-␮l aliquots of biotinylated FA were transferred to glass centrifuge tubes and extracted using 0.37 ml of Bligh and Dyer reagent (CHCl3/MeOH/H2O: 1:2:0.8, vol/ vol). The mixture was vortexed before the addition of 100 ␮l CHCl3 and 100 ␮l H2O, and then centrifuged for 3 min at 1,000 rpm. The lower phase was dried under nitrogen, dissolved in 50 ␮l of CHCl3, and spotted on a TLC plate before chromatography in CHCl3/ MeOH/H2O:100:42:8, vol/vol (18). The dried TLC plate was either sprayed with DPH (1,6-diphenyl-2,3,5-hexatriene, Sigma) and viewed under ultraviolet light to visualize FA. For visualization of lipid mixtures the plates were stained with iodine vapor or sprayed with a concentrated sulfuric acid:water, 70:30 (vol/vol) before charring on a hot plate. A standard curve was prepared by spotting increasing amounts of a standard lipid (e.g., 3-OH-myristic acid). The biotinylated lipid migrated as a single spot under ultraviolet but with a higher Rf than the starting material (ⵑ 0.94). The spot areas of replicate samples were quantified and compared with a standard curve (19). The curve was linear up to ⵑ 20 ␮g FA. The specific activity was ⵑ 260 pmoles biotin/␮g FA.

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Solid Phase Microtiter Binding Assays The binding of biotin-labeled SP-D to surface adsorbed lipids and mannan, and the binding of labeled FA to surface adsorbed proteins, were assessed using 96-well plates and a streptavidinperoxidase detection system. Buffers for binding assays were prepared as follows: coating buffer, 15 mM Na2CO3, 35 mM NaHCO3, 0.05% (wt/vol) NaN3, pH 9.6; wash buffer, 20 mM Tris-HCl, 140 mM NaCl, 5 mM CaCl2, 0.05% (wt/vol) NaN3, pH 7.4; blocking/ binding buffer, 20 mM Tris-HCl, 140 mM NaCl, 5 mM CaCl2, 0.05% (wt/vol) NaN3, pH 7.4 and containing 0.1% (wt/vol) BSA with or without free FA as specified. For some studies washing, blocking, and binding were performed in the presence of 0.05% (vol/vol) Tween-20. Tween-20 only slightly decreased the binding of FA to SP-D (data not shown). Solid phase lipid-binding assay. Lipid-binding assays were performed using Immulon 4HBX hydrophilic plates or IB hydrophobic plates (Dynex Technologies, Chantilly, VA) as indicated in the legends. For the experiments shown, wells were coated with lipids dissolved in chloroform/methanol at the indicated concentration, dried under nitrogen, and hydrated in coating buffer overnight at 4⬚C. The wells were rinsed three times with washing buffer, and then incubated for 2 h with Blocking Buffer. Biotinylated, recalcified SP-D was diluted as required in binding buffer and added to the wells in a final volume of 100 ␮l. The plates were incubated for 1 h at room temperature. After washing, bound biotin–SP-D was reacted with streptavidin-peroxidase (1:5,000) for 1 h at room temperature, and color was developed by incubation for 30 min with TMB Substrate prepared according to the manufacturer’s instructions. Absorbance was measured at 450 nm. All samples were in duplicate or triplicate. Some experiments were performed with hydration of the lipids in the blocking buffer; the absorbance values were slightly lower than when the coating buffer was used. Solid phase protein-binding assay. The binding of labeled lipids to surface adsorbed proteins was assayed using Immulon 4HBX plates. Wells were coated with protein in coating buffer for 2 h at room temperature, followed by washes and incubations with washing and blocking buffer as above. Biotinylated FA was diluted as required in binding buffer and added to the wells in a final volume of 100 ␮l, and binding was detected as described above. Mannan-binding assays. To assess the binding of SP-D to mannan, the wells of 4HBX plates were coated with 10 ␮g/ml mannan in coating buffer. After washing, coated plates were incubated with SP-D at the indicated concentration in the absence or presence of various competitors, and bound biotin-labeled protein was detected as described above. Data analysis. Unless otherwise stated, all values are given as the mean ⫾ SD of triplicate determinations, and the data are representative of at least two independent experiments. All binding data were plotted and analyzed using Sigma Plot 8.0 (SPSS Inc., Chicago, IL). When data for representative experiments were derived from two determinations, the variation around the mean was calculated. For all experiments, this variation was ⬍ 5% of the plotted mean value.

TLC Binding Assays TLC overlay binding experiments were performed using aluminum-backed plastic TLC plates (Analtech). Twenty microliters (1 mg/ml in chloroform:methanol, 1:4, vol/vol) of FA solution was spotted on the plates and chromatographed in an ascending solvent system consisting of chloroform/methanol/water, 100:42:8, vol/vol). Replicate plates were chromatographed in parallel; one plate was stained with iodine vapor or charred to visualize the predominant lipid species, the other was incubated with 10 ␮g/ml RrSP-D for 60 min at 37⬚C in blocking/binding buffer, and washed three times with

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wash buffer. Bound SP-D was visualized by incubating with rabbit anti-rat SP-D (1:500) for 16 h at 4⬚C. The plates were then washed, and bound antibody was detected using an indirect immunoperoxidase assay with goat anti-rabbit horseradish peroxidase (Bio-Rad) and 4-chloro-1-napthol as substrate. Incubations with antibody but without RrSP-D were performed as a negative control.

ADIFAB Competitive Binding Assay To further examine FA binding in the “aqueous phase” and in the absence of a protein blocking agent, we used a competition assay employing ADIFAB (Molecular Probes). ADIFAB responds to FA binding by undergoing a shift in fluorescence emission from 432 to 505 nm. As a consequence, the free FA concentration can be determined from the ratio of 505 to 432 nm in absence (Ro) or presence (R) of FA (20), and FA binding to other proteins can be detected by competing with the binding of FA to ADIFAB (21). Measurements of the 505/432 intensity ratio (R value) were obtained using a modified PicoFluor fluorometer (Turner Biosystems, Sunnyvale, CA) with excitation set at 390nm and methacrylate cuvettes (Turner Biosystems). Initial calibration of the ADIFAB fluorophore was accomplished by titration with increasing amounts of 3-OH-myristate or oleate using the recommended concentration of ADIFAB. Both FA gave linear increases in R up to a concentration of ⵑ 2 ␮M, consistent with findings by others (22). The ability of SP-D to compete for binding of FA to ADIFAB was measured using increasing amounts of SP-D or trimeric neck⫹CRDs in the presence of 0.1 ␮M FA and 0.2 ␮M ADIFAB. All incubations were performed in 20 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 20 mM calcium, pH 7.4 and a final assay volume of 2 ml. Preliminary experiments confirmed that equilibrium binding was achieved in less than 15 min. Incubations were routinely performed for 60 min at room temperature. All measurements were corrected for background fluorescence. Human FABP was used as a positive control.

Circular Dichroism Spectral Analysis The CD spectra of RrSP-D dodecamers and the RrSP-Dser15,20 single arm mutant were determined in PBS, pH 7.2, containing 20 mM CaCl2, in the absence and presence of submicellar concentrations of 3-OH-myristate. The spectra were recorded at 24⬚C using a Jasco J-50 spectropolarimeter interfaced with a computer and a quartz cell with a pathlength of 0.10 cm.

Results SP-D Binds to FA, but Binding Is Dependent on the Type and Purity of the Blocking Agent In preliminary experiments we observed the binding of SP-D to solid-phase 2-hydroxy and 3-hydroxymyristate (2OH-myristate and 3-OH-myristate, respectively) or palmitate, when using microplate assays similar to those routinely employed for studying the binding of collectins to carbohydrate ligands. However, we noted that the extent of binding was highly dependent on the source and amount of the BSA that was used as a blocking protein and included during incubations with labeled SP-D. In particular, there was very low binding of SP-D to 2-OH-myristate in the presence of the BSA that we routinely use for immunologic and saccharide binding assays (Albumin, Bovine, Fraction V; Sigma) (Figure 1A, open circles). By contrast, enzyme-linked binding assays performed in the presence of FAF-BSA obtained from two different sources (see Materials and Methods) gave strong

Figure 1. SP-D specifically binds to 2-OH- and 3-OH-myristate. (A ) The binding of biotinylated SP-D dodecamers to 2-OH-myristate was examined in solid-phase binding assays as described in Materials and Methods. FA was adsorbed to the wells of Immulon 1B microtiter plates at a concentration of 10 ␮g/ml. Blocking buffers contained BSA (open circles) or FAF-BSA (closed circles). The mean of duplicates is shown; the data are representative of at least two separate experiments; the range of values of the average did not exceed 5%. There was no detectable binding in the presence of BSA containing FFA (open circles). (B ) The binding of biotinylated SP-D dodecamers to solid phase 3-OH-myristate was examined as above. FA was adsorbed to the wells of Immulon 1B microtiter plates at a concentration of 20 ␮g/ml, and the blocking buffer contained FAF-BSA. Specific binding is indicated (closed circles). The mean of duplicates is shown; the data are representative of three separate experiments. Similar results were also obtained with palmitic acid (16:0). (C ) The specificity of binding of biotinylated SP-D dodecamers to solid phase 3-OH-myristate was further examined in competition assays using increasing amounts of unlabeled RrSP-D mixed with biotinylated SP-D (3 ␮g/ml). Specific binding (closed circles) and nonspecific binding (open circles) are indicated. The mean of duplicates is shown; the data are representative of two separate experiments.


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signals (Figure 1A, Sigma FAF-BSA, closed circles). Thus, it appeared that SP-D was binding to surfaces coated with fatty acids, but that binding was markedly reduced by competition with fatty acids present in the relatively unpurified Fraction V BSA. Labeled SP-D Shows Specific Binding to Solid-Phase FA Recombinant rat SP-D labeled with biotin at sugars in the collagen domain showed concentration-dependent, saturable binding to microtiter wells coated with 3-OH-myrisate in the presence of 0.1% FAF-BSA (Figure 1B). Preliminary studies confirmed a dose-dependent increase in binding as a function of the coating concentration of FA (data not shown), and showed no significant binding of SP-D in the absence of coated FA (Figure 1C, open circles). Binding to FA was blocked by the addition of excess FA to the solution phase (data not shown), indicating that the protein and lipid components can interact in the aqueous phase. Specific binding was further demonstrated by efficient, dose-dependent competition with unlabeled SP-D (Figure 1C, closed circles). A 2-fold excess of unlabeled SP-D (6 ␮g/ml) decreased the binding by nearly 80%. Virtually identical results were obtained in preliminary experiments using SP-D biotinylated at primary amino-groups (data not shown). Labeled FA Show Specific Binding to Solid-Phase SP-D Soluble, biotin-labeled 3-OH-myristate at sub-micellar (sub-CMC) concentrations (10–35 ␮g/ml) bound in a dosedependent and saturable manner to surface immobilized SP-D (Figure 2A). Binding of SP-D was blocked by subCMC concentrations of unlabeled 3-OH-myristate (Figure 2B, closed squares). Thus, competition experiments confirmed that both components are able to interact in the solution phase. Notably, 2-OH-myristate, which has a CMC comparable to 3-OH-myristate, was a significantly less effective competitor, suggesting differences in the affinity of binding among structurally different FA. For example, in similar experiments 100 ␮g/ml 2-OH-myristate gave ⬍ 20% inhibition, as compared with ⬎ 90% for 3-OH-myristate (data not shown). As for the experiments shown in Figure 1B, binding was dependent on the concentration of surface adsorbed SP-D (Figure 2C, open circles), and there was no significant binding of the labeled FA in the absence of ligand (Figure 2C, closed circles). Binding was also efficiently competed with a 3-fold excess of unlabeled SP-D (Figure 2C, diamond). Because BSA can bind to specific FA, with 6–12 sites of varying affinity (minimum Kd of 4–8nM) (23), the presence of 0.1% FAF-BSA (15 ␮M) in blocking/binding buffer could potentially reduce free FA concentrations by as much as 90–180 ␮M. It is, therefore, notable that soluble labeled FA (38–133 ␮M) showed dose-dependent binding to SP-D even in the presence of 15 ␮M FAF-BSA. No more than a 10–15% increase in maximal binding was observed when hemoglobin was substituted for FAF-BSA in the blocking/binding buffer (data not shown). Thus, the data suggest low binding of the biotinylated hydroxy-FA to BSA in this system, and/or efficient competition of SP-D for bound FA. In this regard, Scatchard analysis of the binding of biotinylated SP-D to solid phase 3-OH-myristate gave an estimated Kd of less than 1 ⫻

Figure 2. Labeled 3-OH-myristate binds to SP-D. (A ) The binding of biotinylated 3-OH-myristate to adsorbed SP-D dodecamers (50 ng/ml) was examined in the presence of FAF-BSA. The mean and SD of triplicates is shown; the data are representative of three experiments. (B ) RrSP-D was adsorbed to the wells of Immulon 4HBX microtiter plates at a concentration of 3 ␮g/ml and incubated with of biotinylated 3-OH-myristic acid (5 ␮g/ml) in the presence of increasing amounts of unlabeled 3-OH-myristate. There was efficient and dose-dependent inhibition of binding by sub-CMC concentrations of unlabeled FA presented in the aqueous phase (closed squares), and minimal binding in the absence of ligand (open squares). The mean of duplicate samples is shown. The data are representative of two separate experiments. Comparable results were also obtained with biotinylated 2-OH-myristate. (C ) The binding of biotinylated 3-OH-myristate (10 ␮g/ml) to solid phase RrSP-D is dependent on the coating concentration of SP-D (open circles). There was minimal binding to the substrate in the absence of SP-D (closed circles).

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10⫺9, and binding of biotinylated 3-OH-myristate monomers to solid-phase RrSP-D gave a Kd of less than 1 ⫻ 10⫺11, each in two independent experiments (data not shown). Binding to FA Is Calcium-Dependent All of the known biological activities of SP-D are dependent on calcium, which is coordinated at the carbohydratebinding site and participates in ligand binding. Accordingly, we examined the effects of added calcium and a calcium chelator on the binding of SP-D dodecamers to FA. Maximal binding required the presence of added calcium, and binding was markedly decreased in the presence of a molar excess of EGTA at a final pH of 7.4 (Figure 3). Recombinant Neck⫹CRD Domains Bind to FA Given the calcium dependency of binding, we hypothesized that FAs bind to the carboxy-terminal C-type lectin domain. Accordingly, we examined the ability of recombinant, trimeric neck⫹CRD domains to bind to FA. The trimeric neck⫹CRD of rat SP-D was labeled at primary amine groups using N-hydroxysulfosuccinimide because of the absence of N-linked oligosaccharides. Consistent with studies by others, the mutant showed calcium-dependent lectin activity in solid-phase binding assays using surface-adsorbed mannan or lipopolysaccharide (data not shown), and in its interactions with Pneumocystis carinii (24). As predicted, the biotin-labeled trimeric neck⫹CRDs bound to surfaceimmobilized FA, including 3-OH-myristate, in the presence of calcium (Figure 4A). Binding was concentration dependent and saturable, as described for SP-D dodecamers (data not shown); and inhibited by EGTA (Figure 4A, bar 2). In addition, binding was completely blocked in the presence of a 10-fold excess of the unlabeled recombinant protein (Figure 4A, bar 3). Sub-CMC concentrations of biotin-labeled 3-OHmyristate also showed specific binding to surface-adsorbed neck⫹CRD or RrSP-Dser15,20 (data not shown).

Figure 3. Binding of SP-D to fatty acids requires calcium and is inhibited by chelation with EGTA. The binding of biotinylated 3OH-myristate to solid phase SP-D dodecamers was examined as described for Figure 2. SP-D was adsorbed to the wells of Immulon 4HBX microtiter plates at a concentration of 5 ␮g/ml, and the blocking/binding buffer contained FAF-BSA. Binding reactions were performed in the absence or presence of added calcium, or with recalcified SP-D in the presence of 10 mM EGTA, as indicated in the figure. The mean ⫾ SD of triplicate determinations are shown.

The molar dose–response of binding of the biotinylated trimeric neck⫹CRD (3 ⫻ 17 kD ⫽ 51 kD) to solid-phase 3-OH-myristate was compared with that obtained for biotinylated wild-type SP-D dodecamers (12 ⫻ 43 kD ⫽ 516 kD). The trimeric neck⫹CRD showed ⵑ 400-fold lower binding to the FA (data not shown). Similar differences have been described in relation to carbohydrate binding (25), and are consistent with the loss of cooperative binding among the four trimeric CRDs of a dodecamer. Competing Saccharides Show Dose-Dependent Effects on the Binding of the Carboxy-Terminal Domain to FA We also examined the effects of known saccharide ligands on the binding of trimeric neck⫹CRDs to solid-phase FA.

Figure 4. Recombinant, trimeric neck⫹CRD binds to fatty acids and binding is influenced by saccharide ligands. (A ) The binding of biotinylated SP-D neck⫹CRD trimers (10 ␮g/ml) to solid phase 3-OH-myristate was examined as described in Materials and Methods. FA was adsorbed to the wells of Immulon 1B microtiter plates at a concentration of 20 ␮g/ml, and the blocking/binding buffer contained FAF-BSA. Assays were performed in the absence or presence of excess unlabeled recombinant neck⫹CRD, as indicated. Binding was inhibited in the presence of 10 mM EGTA. The mean ⫾ SD of triplicate determinations are shown; the data are representative of two separate experiments. (B ) The binding of labeled neck⫹CRD trimers to solid phase 3-OH-myristate was examined as in A. However, incubations were performed in the presence of the indicated concentrations of monosaccharides. The maximum absorbance at 450 nm (corresponding to 100% binding) was 1.2 OD.


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D-glucose, an effective competitor of SP-D binding to a variety of carbohydrate ligands (4), inhibited SP-D binding to 3-OH-myristate at concentrations of 0.1–30 mM (Figure 4B). Similar inhibition was observed for palmitate or myristate (data not shown). Specificity was demonstrated by performing parallel incubations in the presence of D-galactose (Figure 4B), a relatively inefficient competitor of SP-D binding to carbohydrate ligands (26); significant inhibition required nearly 100-fold greater concentrations of galactose. Surprisingly, lower concentrations of D-glucose, enhanced FA binding in a dose-dependent manner. Similar enhancement was observed with RrSP-D; in these experiments the stimulatory effects were also biphasic with decreased activity at sugar concentrations ⬍ 10 ␮M (data not shown). Specificity of the phenomenon was again suggested by the absence of enhancement in the presence of low concentrations of D-galactose. SP-D Binds to Hydroxylated, Saturated, and Unsaturated FA of Various Chain Lengths To confirm that SP-D was binding to FA in our lipid preparations, and to assess the interactions with structurally different FAs, we resolved equimolar amounts of chloroform/ methanol solubilized FA on thin layer plates and examined binding to unlabeled RrSP-D dodecamers using overlay binding assays and an indirect immunodetection system. This approach permits quantitative adsorption of lipids to the solid phase and allows direct visualization of components reacting with the SP-D. Charring of parallel chromatograms revealed only minor contaminants near the solvent front in a few lipid preparations. As shown in Figure 5, SP-D reacted with the major lipid species in all FA preparations (Rfs in the range of 0.6–0.8). In particular, SP-D bound to 12, 14, and 16 carbon 2-hydroxy FAs (Figure 5A); 12, 14, and 16 carbon 3-OH-FAs (Figure 5B); 14, 16, and 18 carbon saturated FAs (Figure 5C); and 18:1 and 18:2 unsaturated FA (Figure 5D). In other experiments, SP-D bound to 12 carbon saturated FAs, as well as certain other neutral lipids including esterified cholesterol, tripalmitin, and dipalmitoyl-phosphatidylcholine (data not shown). There was no visible reactivity in the absence of SP-D (data not shown). SP-D Can Compete for FA Binding to ADIFAB Although the various assays described above indicate specific binding of SP-D to FA, they all employed a solid-phase component, and most were performed in the presence of albumin. To further confirm these findings we implemented an “aqueous phase” competition assay using acrylodanlabeled FA-binding protein in the absence of BSA or other blocking proteins (ADIFAB; see Materials and Methods). As shown in Figure 6, we observed dose-dependent binding of SP-D to 3-OH-myristate as measured by the ability of soluble and unlabeled SP-D to compete with soluble ADIFAB for soluble FA binding, thereby decreasing the ratio of emission at 505/432 nm (Figure 6). The ratio in the absence of added FA (Ro) was ⵑ 0.5, which was only slightly higher than published values (21), presumably reflecting differences in the buffer systems and instrumentation. The R value increased to ⵑ 0.9 in the presence of 0.1 mM 3-OH-myristate, reflecting the binding of the FA to

Figure 5. SP-D binds specifically to FA resolved by TLC. FA were resolved by TLC and visualized by charring (charred) or reacted with SP-D and detected by indirect immunoassay (SP-D) as described in Materials and Methods. (A ) 2-OH FA. (B ) 3-OH FA. (C ) Saturated FA. (D ) Unsaturated FA. The acyl chain length is indicated at the top of each panel, the Rf is indicated at the left. In every case, SP-D binding co-localized with the major species visualized by charring. The data are representative of two independent experiments.

ADIFAB. However, the addition of 3 ␮g/ml SP-D (ⵑ 6 nM) to this solution significantly decreased the ratio, and the R-value decreased to slightly less than Ro in the presence of 10 ␮g/ml SP-D. Comparable inhibition was achieved with 3 ␮g/ml (60nM) of recombinant neck⫹CRD or 3 ␮g/ml (200 nM) of the positive control, heart FA-binding protein (FABP) (Figure 6). The addition of 10 ␮g/ml SP-D or 3 ␮g/ml of FABP to ADIFAB in the absence of FA resulted in a slight decrease in Ro, consistent with R-values slightly less than Ro in the presence of these concentrations of competitor (data not shown). Interactions of SP-D with PI Given these findings and the known interactions of SP-D with PI, the major known surfactant lipid ligand, we examined the effect of FA on the binding of SP-D to PI in the presence of FAF-BSA. Consistent with previous data, 20 mM inositol markedly inhibited the binding of SP-D neck⫹CRD to PI presented on the solid phase (Figure 7, bar 2). However, purified FAs also showed dose-dependent

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FA Monomers and Micelles Can Modify the Interactions of SP-D with Mannan

Figure 6. SP-D binds to 3-OH-myristate as detected using the ADIFAB binding assay. The binding of SP-D dodecamers to 3-OHmyristate was examined using the ADIFAB assay as described in Materials and Methods. Changes in the ratio of the absorbance at 505–432 nm were used to examine the ability of SP-D, recombinant trimeric neck⫹CRDs (N-CRD), and human FABP to compete for FA binding to ADIFAB. Bar 1 corresponds to Ro; the maximal ratio of 505/432 nm obtained in the presence of FA alone is shown in bar 2. SP-D shows dose-dependent competition of SP-D for fatty acid binding to ADIFAB (bars 3 and 4 ).

inhibition of binding. For example, a nominal concentration of 50 ␮M palmitate markedly inhibited binding (bar 3), and there was no detectable binding in the presence of both competitors (bar 4). Less effective inhibition was observed in comparable assays using equal weight concentrations of wild-type RrSP-D dodecamers (two experiments, data not shown), presumably reflecting the loss of cooperative binding among trimeric CRDs.

Figure 7. The binding of SP-D to PI is competed by saccharide and FA ligands. The binding of biotinylated SP-D neck⫹CRD trimers to PI was examined in solid-phase assays. PI was adsorbed to the wells of Immulon 1B microtiter plates at a concentration of 2 ␮g/ml, and the blocking buffer contained FAF-BSA. Incubations were performed in the absence of competitor, in the presence of 20 mM inositol or a nominal concentration of 50 ␮M palmitic acid, and in the presence of these amounts of both competitors. The mean and SD of triplicates is shown; the data are representative of three experiments. Similar results were also obtained in two experiments using RrSP-D dodecamers.

Given the effects of saccharides on binding of SP-D to FA, we hypothesized that FA might modify the interactions with saccharide ligands. Consistent with numerous previously published studies, labeled SP-D showed dose-dependent binding to mannan or maltosyl-BSA, and 10 mM glucose or EGTA completely inhibited this binding (data not shown). However, in preliminary experiments we observed that binding to mannan was significantly decreased when assays were performed in the presence of BSA that contains FA. Accordingly, we examined the effects of added competing FA on the binding of labeled SP-D to surface-adsorbed yeast mannan, a well-characterized polysaccharide ligand. Both 3-OH-myristate and palmitate showed dose-dependent inhibition of SP-D binding to mannan in the presence of FAF-BSA. In the case of 3-OH-myristate (Figure 8A, open circles), inhibition was evident at a nominal concentration of ⵑ 10 ␮M, and increased to 30–40% by 100 ␮M. Inhibition reached a broad plateau as the concentration approached the measured CMC, which was ⬇1 mM under the conditions of our assay (i.e., in the presence of calcium and FAF-BSA). Significantly higher concentrations of 3OH-myristate (ⵑ 250 mM) showed complete inhibition, but at these concentrations the FA sample was visibly turbid, indicating higher order organization of the lipid. In the case of palmitate (Figure 8A, closed circles), there was ⬍ 10–20% inhibition at concentrations below the measured CMC of ⬇ 2 ␮M (see inset). However, inhibition increased dramatically above the CMC, with a reproducible inflection at a nominal palmitate concentration of ⵑ 30 ␮M. As for OH-myristate, the final phase of increasing inhibition approximately coincided with increased turbidity of the lipid dispersion; complete inhibition was only observed at concentrations ⬎ 1 mM. We hypothesized that lower concentrations of FA would be required to inhibit the binding of the biotinylated neck⫹CRD to mannan because of the loss of cooperative binding among trimeric CRDs. For these experiments the dodecamers and neck⫹CRDs were labeled at primary amines. Consistent with our hypothesis, the inhibition curve for neck⫹CRDs was shifted to the left with ⵑ 40–50% inhibition of binding at concentrations below the measured CMC (Figure 8A, triangles). In these assays, palmitate was at least 10-fold more potent as an inhibitor of SP-D binding to mannan than maltose or glucose, which are prototypical competitive inhibitors of SP-D CRD binding to saccharide ligands (Figure 8B). However, the inhibition curve was reproducibly broader and more complex, and complete inhibition was achieved only observed at concentrations considerably above the CMC. Although binding of FA to albumin could influence the shapes of the curves and the relative inhibitory effectiveness of the two FA, changes in slope approximately corresponded to transitions from monomers to micelles, as reflected by the measured CMC, and from micelles to more highly aggregated complexes or multimers, as reflected by turbidity of the lipid mixture.


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Figure 8. FA inhibit the binding of SP-D and recombinant neck⫹CRDs to mannan. (A ) The binding of biotinylated SP-D (5 ␮g/ml) to mannan was assessed in the presence of increasing concentrations of 3-OH-myristate (open circles) or palmitate (closed circles). In addition, binding of recombinant trimeric neck⫹CRDs (1 ␮g/ml) is shown in the presence of various concentrations of palmitate (closed triangles). The conductivity profile used to assess the CMC of palmitate in the same buffer system is shown in the inset. The maximum absorbance at 450 nm (corresponding to 100% binding) for palmitate with the neck⫹CRD was 0.38 OD; the maximum for palmitate or 3-OH-myristate with dodecamers was 2.6 OD. (B ) In a separate experiment, the inhibitory effect of palmitate (open triangles) on the binding of labeled SP-D to mannan was compared with maltose (open circles), glucose (closed triangles), or galactose (closed circles), as examples of efficient and inefficient saccharide competitors, respectively. The maximum absorbance at 450 nm (corresponding to 100% binding) was 2.6 OD.

FA Show Concentration-Dependent Effects on SP-D Binding to Mannan in the Presence of Competing Saccharide Ligands As indicated above, competing sugars, such as glucose and maltose, can efficiently inhibit SP-D binding to mannan (e.g., Figure 8B). However, sub-CMC concentrations of 3-OH-myristate gave no more than 30% inhibition at nominal FA concentrations up to ⵑ 100 mM (Figure 8A, open circles). To further characterize the fraction of binding resistant to inhibition by 3-OH-myristate, SP-D was incubated

with mannan in the presence of an inhibitory, but sub-CMC, concentration of 3-OH-myristate (82 ␮M) and increasing concentrations of saccharide competitor. The extent of inhibition achieved in the presence of both classes of monomeric FA and glucose was approximately additive (Figure 9A). Similar results were obtained with maltose (data not shown). Because most effects of palmitate on mannan binding occurred above the CMC, we further examined the effects of higher concentrations of 3-OH-myristate, approaching its measured CMC. As shown in Figures 8B and 9B, 10 mM

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dependent increase in the affinity of the SP-D for the immobilized polysaccharide ligand, which increases as the FA concentration approaches the measured CMC of ⵑ 1 mM. As indicated in Figure 8A, much higher concentrations of FA, which were associated with obvious turbidity of the FA, resulted in marked inhibition.

Figure 9. FA show dose-dependent effects on the binding of SP-D to mannan in the presence of competing sugars. (A ) The binding of biotinylated SP-D to mannan was assessed in the presence of increasing concentrations glucose in the absence or presence of 20 ␮g/ml 3-OH-myristate. Binding was normalized to that observed in the absence of competing glucose or FA. The data are representative of three independent experiments. The maximum absorbance at 450 nm (corresponding to 100% binding) was 2.4 OD. (B ) The binding of biotinylated SP-D to mannan was assessed in binding reactions performed in the presence of the indicated concentrations of maltose and/or 3-OH-myristate. Binding was normalized to that observed in the absence of competing saccharide or FA. The data are representative of two separate experiments. The maximum absorbance at 450 nm (corresponding to 100% binding) was 2.5 OD.

maltose inhibited binding to mannan by ⬎ 95%. However, 3OH myristate gave a dose-dependent decrease in inhibition by maltose such that the combination of 10 mM maltose and 819 ␮M 3-OH-myristate showed ⵑ 20% inhibition (Figure 9B, bars 3–6). Notably, nearly complete inhibition was restored by simply increasing the maltose concentration to 40 mM (Figure 9B, bar 7). Thus, the FA caused a dose-dependent decrease in the inhibitory effectiveness of the competing sugar that was reversed by a 4-fold increase in the concentration of competing saccharide ligand. Similar results were obtained for glucose (data not shown). The findings suggest a FA-

FA Binding Alters the CD Spectrum of SP-D We used CD spectroscopy to look for conformational changes in the SP-D molecule in the presence of 3-OHmyristate monomers, and in the absence of albumin. To exclude possible effects relating to aggregation or changes in quaternary structure (12), we examined single arm trimeric subunits (RrSP-Dser15,20). This mutant has substitutions of serine for cysteine at sites of interchain crosslinking within the amino-terminus, remote from the neck and CRD domains (16). It is fully functional as a lectin (16), and binds to FA in solid-phase assays (data not shown). Circular dichroism of RrSP-Dser15,20 revealed a relatively simple spectrum with a maximal negative ellipticity in the range of 200–216 nm and a shallow peak of negative ellipticity in the region of 215–230 nm. The spectrum, although lacking obvious features of defined secondary structure and resembling a coiled-coil, is very similar to those previously published for native wild-type RrSP-D dodecamers (12), and resemble those obtained for natural SP-A (12, 27) and mannose binding lectin (MBL) (28). By contrast with SP-D, SP-A shows only a small shoulder and considerably less negative ellipticity in the region of 215–225 nm. Thermal denaturation analysis and protease digestion assays have confirmed that this shoulder in SP-A reflects contributions of the short collagen domain (27, 29). Spectroscopic analysis of isolated trimeric neck domains of SP-D, which included only eight triplets of contiguous collagen sequence, showed an ␣-helical profile with a maximal negative ellipticity at ⵑ 205 nm, and only a very small shoulder in the region of 210–220 (30). Consistent with these observations, subtractive analysis of SP-D dodecamers with and without prior incubation with bacterial collagenase unmasked a region of positive ellipticity with a maximum in the range of 220–230, consistent with the collagen domain (data not shown). Thus, the spectrum of RrSP-Dser15,20 results from the averaging of the ellipticities of at least three domains: the triple helical collagen domain, which is considerably larger in SP-D than SP-A, but much smaller than a matrix collagen; the coiled-coil neck domain; and the CRD, which contains ␣ helical coils and ␤ structures similar to those found in other C-type lectins. As shown in Figure 10, the presence of sub-CMC concentrations of 3-OH-myristate increased the negative ellipticity at 200–216 nm. Incubation of RrSP-D dodecamers (0.5 ␮M) with 108 ␮M 3-OH-myristate also resulted in an increase in the negative ellipticity at 200–216 nm, and in one parallel experiment a similar increase in negative ellipticity was observed following the addition of 10 mM maltose (data not shown). Previous studies have shown that the addition of calcium to RrSP-D (12), natural human SP-A (29), or the isolated collagenase-resistant neck⫹CRD domain of SP-A (31), results in a dose-dependent decrease in negative ellipticity in the region of 200–210 nm. Thus, the findings


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(Kd of less than 1 ⫻ 10⫺9M). Relatively high affinity binding is also suggested by the ability of SP-D to compete with FABP and BSA for FA binding. In any case, solution-phase studies in the absence of albumin and using dodecamers, trimeric neck⫹CRDs, and monomeric CRDs are required.

Figure 10. FA binding can alter the CD spectrum of SP-D. Purified single arm trimeric mutants of SP-D (RrSP-Dser15,20) were incubated at a concentration of 0.6 ␮M in the presence (closed squares) or absence (open triangles) of 108 ␮M 3-OH-myristate, a concentration well below the measured CMC. All CD spectra were recorded in PBS, pH 7.2, and containing 20 mM calcium chloride. The data are representative of two separate experiments.

suggest that binding of SP-D to FA is accompanied by alterations in secondary structure, potentially near the known sites of calcium coordination and saccharide binding within the neck⫹CRD.

Discussion SP-D Binding to FA We have shown that SP-D specifically binds to saturated, unsaturated, and hydroxylated FA through calcium-dependent interactions with the carboxy-terminal domains of SP-D. Although SP-D, SP-A, and MBL have all been shown to interact with various phospholipids or glycolipids, to our knowledge this is the first direct demonstration of collectin binding to FA chains, or to lipids lacking one or more carbohydrate moieties. The binding of labeled SP-D to surface-adsorbed FA was dose-dependent, saturable, and blocked in a dosedependent manner by unlabeled SP-D or by sub-CMC concentrations of monomeric FA presented in the solution phase. Binding of labeled FA presented in the aqueous phase to immobilized SP-D was similarly dose-dependent and completely blocked with SP-D or sub-CMC concentrations of unlabeled FA. There was no significant binding of labeled SP-D or labeled FA to the solid-phase in the absence of ligand, and binding was linearly related to the amount of ligand on the plate. SP-D and the FA can efficiently interact in the solution phase, as indicated by the competition assays. Binding of soluble SP-D to soluble FA was further confirmed with a solution phase assay using acrylodan-labeled FABP, which demonstrated that SP-D can compete for FA binding to a known FA-binding protein. In addition, conformational changes were suggested by changes in the CD spectrum of SP-D in the presence of FA monomers. Analysis of the available binding data suggests an impressively high affinity of SP-D dodecamers for 3-OH-myristate

Interactions of FA with the CRD We have not yet defined the precise site(s) of binding to FA. However, FA binding probably occurs at one or more sites in close proximity to the calcium/saccharide binding sites within the CRD. This is consistent with the inhibitory effect of EGTA, the ability of competing sugars to completely inhibit FA binding by trimeric neck⫹CRDs, the effects of competing sugars on FA binding, and the effects of FA on binding to mannan. Such effects could indicate overlapping binding sites, steric effects at the ligand binding interface, and/or conformational changes secondary to ligand binding at distinct sites within the neck ⫹ CRD. With regard to the first possibility, proteins with C-type lectin domains recognize diverse ligands, and there is increasing evidence that residues outside the primary binding site can contribute to ligand recognition (32). For example, recent molecular docking studies of SP-D identified additional amino-acid residues near the calcium-binding site that can participate in hydrogen bonding to the internal sugars of polysaccharide chains (33). In addition, the cys-2 to cys-3 loop of the CRD, which is in spatial proximity to the sugar binding site, participates in binding to PI (34). Fatty acid binding sites have been directly visualized for relatively few proteins including intestinal FABP and human serum albumin (23). FA binding by human serum albumin was shown to involve interactions of basic amino acid residues with the carboxylate group, hydrogen bonds, as well as interactions of the methylene chain with hydrophobic moieties associated with paired amphipathic ␣-helical coils (23, 35). SP-D specifically bound to hydroxylated or saturated FAs, as well as to both classes of FA when biotinylated at their carboxylate group. Together, these observations suggest that hydrogen bonding with 2- or 3-hydroxyl groups, or electrostatic interactions with the carboxylate group, is not essential for binding, at least under the conditions of our assays. They further suggest the potential importance of hydrophobic interactions with the acyl chains. This possibility is consistent with the findings of Taneva and coworkers, which suggested that the interactions of subphase SP-D with phospholipid monolayers are headgroup-independent (12). Saccharide-Induced Enhancement of FA Binding The biphasic enhancing effects of low concentrations of Dglucose and maltose on FA binding are intriguing. The effects appear specific given that D-galactose, a poorly competing sugar of this mannose-type C-type lectin, showed very little enhancement. It is possible that even transient binding of sugar ligands enhances or stabilizes binding of FA at one or more binding sites through conformational changes. The C-type lectin CRD is stabilized by intrachain disulfide bonds and conserved proline residues that could limit conformational changes on ligand binding (36). However, Kimura and coworkers reported that monosaccharide bind-

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ing can cause a conformational change in the CRD of a macrophage C-type lectin that induced the formation of an epitope for a monoclonal antibody under conditions of low calcium; in addition, antibody binding altered the affinity for calcium (37). Conformational changes have also been reported to accompany calcium binding to SP-D (12), and we observed changes in the CD spectrum of SP-D in the presence of saccharide ligand or 3-OH-myristate, suggesting conformational changes in response to FA binding. Because there are hydrogen bonds between the neck of one chain of a trimer and the globular region of another (38), ligand binding might also permit conformational “cross-talk” between the monomeric subunits of a trimeric CRD, particularly as a result of interactions with multivalent ligands or with ligands adsorbed to surfaces. Although interactions between the FA and sugar binding sites are probably important, it is unclear whether or not enhancement of binding per se is of physiologic significance, given the comparatively high concentrations of glucose and other potential saccharide ligands in the airspace in vivo. Influence of SP-D Valency and FA Monomer-CMC Transitions on Mannan Binding: a Model Trimeric neck⫹CRDs showed lower apparent affinity for immobilized FA than SP-D dodecamers, and considerably lower concentrations of FA were required to inhibit the binding of neck⫹CRDs to mannan. Although binding of SP-D to FA was observed below the CMC, effects on mannan binding appeared to be enhanced by the higher order association of FA chains. For such reasons, we propose that the complex, concentration-dependent effects of FA on mannan binding reflect the effects of monomer-CMC transitions on the interactions with the multivalent binding protein. At concentrations well below the CMC, the binding of 3-OH-myristate monomers to SP-D CRDs significantly but incompletely inhibited mannan binding (e.g., Figure 9A). However, at FA concentrations approaching the measured CMC (see Figure 9B), the apparent affinity of SP-D for mannan was increased, as reflected by decreased inhibition in the presence of 10 mM maltose. The latter phenomenon could be explained if the association of lipid monomers permits the formation of fatty acyl/SP-D complexes containing uncomplexed CRDs that remain available for interaction with the mannan. Under these conditions, FA might enhance SP-D binding to mannan by permitting higher affinity cooperative interactions among several or many trimeric CRDs. Such interactions could reasonably be prevented by simply increasing the concentration of saccharide competitor (e.g., Figure 9B, 40 mM maltose). FA micelles range up to ⵑ 200 angstroms in maximum diameter. However, the approximate separation of ligand binding sites within a trimeric CRD is ⵑ 50 angstroms, and the trimeric CRDs of an SP-D molecule are separated by ⵑ 100 nm. Thus, at sufficiently high FA micellar concentrations, there is a potential for bridging interactions between FA micelles or micellar aggregates and the trimeric subunits of SP-D dodecamers. This possibility seems consistent with the ability of SP-D to aggregate PI-containing liposomes (11). Under such conditions, CRDs may no longer be avail-

able for interaction with solid-phase mannan leading to marked inhibition of binding (Figure 8A). Within the context of this model, the apparently greater inhibitory potency of palmitate could be largely explained by its lower CMC. Binding to Phosphatidylinositol As expected, the binding of SP-D to PI was inhibited with inositol (7), but when assays were performed in FA-free buffers we also observed efficient inhibition by FA. The findings are consistent with the previous suggestion that interactions with PI involve more than one recognition mechanism (11). However, given the complex effects of FA on binding to mannan discussed below, indirect FAdependent effects involving altered interactions with the inositol moiety are very possible. Implications for SP-D Binding Assays We found that the binding of SP-D to FA chains is highly dependent on the purity of the albumin included in the reaction mixture. More importantly, FA show concentration-dependent effects on the interactions of SP-D with nonlipid ligands. Binding to complex ligands such as mannan and PI was lower when using crude, but low endotoxin, preparations of Fraction V BSA containing the usual levels of contaminating lipids (0.1–1% free FA). Thus, it is possible that the range of potential ligand interactions has been influenced by the presence of contaminating FA in the albumin preparations used for various published binding assays. Potential Interactions with FA In Vivo Relatively high concentrations of FA, as well as potential acylated ligands, such as PI, are present in the airspaces in vivo. Although accurate determinations of lavage FFA are complicated by potential hydrolysis of phospholipids, palmitic acid, the major FFA in cell-free lavage, was reported to be present at a concentration of ⵑ 11 ␮M, and the measured total FFA concentration was ⬎ 21 ␮M (39). Because the lavage is diluted by 100-fold or more relative to the alveolar lining fluid, SP-D could encounter millimolar concentrations of free FA in the airspace. Interestingly, qualitative and quantitative alterations in FA have been described in association with cystic fibrosis (39, 40), asthma (40), and in a rodent model of bleomycin-induced fibrosis (41). A significant fraction of human SP-D is recovered from the lung in association with surfactant, and the soluble protein co-isolates with lipids during the initial stages of purification (42). In this regard, we have observed that human proteinosis SP-D dodecamers can also bind to fatty acids (data not shown). Because binding to FA is markedly decreased in the absence of calcium, associated lipids are largely removed during subsequent purification, which includes exposure to EDTA or EGTA. Given the high concentrations of acylated molecules and FA in the airspaces, we suspect that FA-binding sites are at least partially occupied in situ, and that bound FA or other lipid or hydrophobic moieties contribute to the physiologic function of this molecule. Potential Physiologic Role(s) of FA Binding The dramatic abnormalities in surfactant lipid homeostasis observed with transgenic models of SP-D deficiency are


unexplained (43, 44). It is possible that SP-D deficiency contributes to abnormal lung lipid metabolism through direct interactions with FA or with acyl chains of surfactant lipids, thereby modifying cellular uptake or catabolism. Lipid-dependent mechanisms might also influence the interactions of SP-D with microbial ligands such as bacterial lipopolysaccharides, gram-positive lipoteichoic acids, and mycobacterial lipoglycans, or with some organic antigens. Lastly, there is considerable interest in the roles of SP-D in modulating various aspects of innate and acquired immunity, including inhibition of lymphocyte proliferation (45) and antigen presentation to immature dendritic cells (46). FA have also been implicated in various aspects of immune regulation (47), thus these interactions could also be important for the immunomodulatory functions of SP-D and lipids in the lung. Acknowledgments: The authors thank Dr. James G. Bann, Dept. of Biochemistry and Molecular Biophysics for assistance with the CD spectral analyses. The comments and advice of Drs. Morris Kates (University of Ottawa, ON, Canada), Arnis Kuksis (University of Toronto, ON, Canada), and Alan M. Kleinfeld (Torrey Pines Institute for Molecular Studies, San Diego, CA) are gratefully acknowledged. The authors also thank Janet North for excellent administrative assistance. This study was supported by NIH grants HL-44015 and HL-29594.

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