©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of the Amino-terminal Domain and the Collagenous Region in the Structure and the Function of Rat Surfactant Protein D (*)

(Received for publication, April 20, 1995; and in revised form, May 19, 1995)

Yoshinori Ogasawara Dennis R. Voelker (§)

From theLord and Taylor Laboratory for Lung Biochemistry and the Anna Perahia Adatto Clinical Research Center, Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206 and the Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado Health Science Center, Denver, Colorado 80262

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Surfactant protein D (SP-D) is a member of the C-type lectin superfamily with four distinct structural domains: an amino terminus involved in forming intermolecular disulfides, a collagen-like domain, a neck region, and a carbohydrate recognition domain. A collagen domain deletion mutant (CDM) of SP-D was created by site-directed mutagenesis. A second variant lacking both the amino-terminal region and the collagen-like domain was generated by collagenase treatment and purification of the collagenase-resistant fragment (CRF). The CDM expressed in CHO-K1 cells formed the covalent trimers, but not the noncovalent dodecamers, typical of native SP-D. The CRF derived from recombinant SP-D formed only monomers. The CDM bound mannose-Sepharose and phosphatidylinositol (PI) as well as SP-D, but the binding to mannosyl bovine serum albumin and glucosylceramide was diminished by approximately 60%. The CRF displayed weak binding to mannose-Sepharose and PI and essentially no binding to mannosyl bovine serum albumin and glucosylceramide. Both SP-D and CDM altered the self-aggregation of PI-containing liposomes. SP-D reduced the density and the light scattering properties of PI aggregates.

These results demonstrate that the collagen-like domain is required for dodecamer but not covalent trimer formation of SP-D and plays an important, but not essential, role in the interaction of SP-D with PI and GlcCer. Removal of the amino-terminal domain of SP-D along with the collagen-like domain diminishes PI binding and effectively eliminates GlcCer binding.


INTRODUCTION

Pulmonary surfactant is an extracellular complex of lipids and proteins that stabilize the alveoli at the air-liquid interface(1) . Surfactant protein D (SP-D) (^1)is a hydrophilic glycoprotein that is synthesized and secreted by alveolar type II cells (2, 3) and is loosely associated with pulmonary surfactant. The protein can also be produced by non-pulmonary cells (4) . SP-D is a member of the collectin subgroup of the C-type lectin superfamily(5) , and its structure is characterized by four distinct domains comprised of: 1) an intermolecular disulfide containing amino-terminal region, 2) a collagen-like domain, 3) a neck region, and 4) a carbohydrate recognition domain (CRD)(6) . Electron microscopy reveals that the protein exists as a dodecamer which is assembled into a cruciform structure composed of four identical trimers(5, 7) . In addition to cruciform-shaped oligomers, large radial aggregates composed of 10 or more trimeric subunits have also been reported. The basic cruciform structure shares similarity with bovine conglutinin and CL-43(5, 8, 9) .

SP-D is known to bind the glycolipids phosphatidylinositol (PI) and glucosylceramide (GlcCer)(10, 11, 12) , and previous studies revealed that the lipid binding properties map to the carbohydrate recognition domain (13, 14) . SP-D also binds to several microorganisms and alveolar macrophages (15, 16, 17) via carbohydrate recognition. The interaction of SP-D and other collectins with microorganisms may constitute an important component of immunoglobulin-independent host defense. In contrast to the CRD, the role of other SP-D domains in measurable protein function is largely uncharacterized.

The role of the collagenous region of surfactant protein A (SP-A), another hydrophilic surfactant-associated glycoprotein that is structurally related to SP-D, has been studied using collagenase-resistant protein fragments (denoted CRF)(18, 19) . These studies revealed that the collagenous domain is not essential for lipid binding(19) , inhibition of lipid secretion from alveolar type II cells (18) , and binding to the high affinity receptor on type II cells(18) . Although the SP-A CRF retained several activities of its intact counterpart, its affinity for different ligands was reduced compared with its native form(18, 19) . Extrapolating from these observations, it is probable that the domains other than the CRD of SP-D can contribute to its interactions toward multiple ligands.

The purpose of this study was to 1) construct and purify SP-D lacking the collagen domain (collagen deletion mutants (CDM)), 2) prepare and purify SP-D lacking the collagen domain and the amino terminus (CRF), 3) characterize some of the physical properties of the SP-D variants, and 4) determine the effects of the structural interactions upon lipid binding. The results demonstrate that the collagen-like region is required for dodecamer formation but not for trimer formation. In addition, the CDM variant retains 75% of phosphatidylinositol binding and 35% of GlcCer binding, whereas the CRF retains about 35% of the phosphatidylinositol binding but loses GlcCer binding.


MATERIALS AND METHODS

DNA Construction of SP-D and Its Collagen Deletion Mutant

The 1.2-kb cDNA for rat SP-D (5) was isolated and ligated into a pEE14 plasmid vector using XbaI and HindIII sites as described previously(5, 14) . A mutant cDNA of SP-D with a deletion at nucleotides corresponding to the region Leu to Pro (CDM) was produced using the polymerase chain reaction and the overlapping extension method(20) . This construct was also ligated into pEE14 vector using XbaI and HindIII sites. Defined mutations were confirmed by sequencing the entire cDNA using Sequenase and the method of Sanger et al.(21) .

Expression and Isolation of Recombinant Proteins

The recombinant proteins were expressed using the glutamine synthetase amplification system as described previously(14) . Briefly, the pEE14 plasmid vectors containing the cDNAs for wild type SP-D and CDM were transfected into CHO-K1 cells using lipofectamine (Life Technologies, Inc.). Transfected cells were incubated in glutamine-free GMEM (22) supplemented with 10% fetal bovine serum (complete GMEM-10) in the presence of 25 µM methionine sulfoxamine for 7-10 days at 37 °C in a humidified air, 5% CO(2) atmosphere. The surviving colonies were transferred to medium containing a higher concentration of methionine sulfoxamine (100-500 µM) and incubated for another 2 weeks. The clones which produced the highest amount of recombinant proteins were selected after screening by SP-D enzyme-linked immunosorbent assay. To isolate recombinant proteins, the confluent cells incubated in complete GMEM-10 were transferred to serum-free medium. Forty-eight hours later, the medium was harvested and dialyzed against TBS at 4 °C. The dialyzed medium was then applied to a mannose-Sepharose affinity matrix (23) in the presence of 5 mM calcium, and the recombinant proteins were eluted using 2 mM EDTA. The eluted proteins were dialyzed against TBS again and stored at -20 °C.

Metabolic Labeling of Recombinant Proteins

The recombinant proteins were metabolically labeled with [S]methionine and [S]cysteine using TranS-label (ICN) as described previously(14) . The labeled proteins were isolated and stored at -20 °C. The specific activity of S-rSP-D and -CDM used ranged between 1500 and 4500 cpm/ng and 400 and 600 cpm/ng, respectively.

Enzyme Digestion of Wild Type Recombinant SP-D

Wild type recombinant SP-D and S-labeled recombinant SP-D were incubated with collagenase (5 units/µg of protein) (Advanced Biofactures Corp.) in 50 mM Tris, pH 7.2, containing 150 mM NaCl, 10 mM CaCl(2), and 0.2 mM phenylmethylsulfonyl fluoride at 37 °C for 48 h. The reaction was stopped by adding 20 mM EDTA. The CRFs were isolated by high performance liquid chromatography using a C8 column and an acetonitrile gradient (20-80% in water containing 0.1% trifluoroacetic acid). The purified CRF was subjected to Edman degradation, and a partial sequence of CRF was determined using a model 492 automated amino acid sequencer (Applied Biosystems, Inc.). The specific activity of S-CRF used was 800 cpm/ng.

Lipids

Dipalmitoylphosphatidylcholine (DPPC), PI, phosphatidylglycerol, phosphatidylserine (PS), and cholesterol were purchased from Avanti Polar Lipids, Inc. Glucosylceramide (GlcCer) was purchased from Sigma. Multilamellar and unilamellar liposomes were prepared as described previously (13, 14) and used for the lipid binding and self-association assay described below.

Direct Binding of Recombinant Proteins to Immobilized Sugars

Recombinant S-labeled proteins (0.1 µg) were incubated with 25 µl of a mannose-Sepharose or galactose-Sepharose affinity matrix in 500 µl of TBS (5 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 5 mM CaCl(2) and 0.2% BSA (binding buffer) for 1 h at 4 °C. The Sepharose matrices were centrifuged at 5000 g for 5 min, and the resultant pellet was washed with ice-cold binding buffer. This step was repeated three times, and the radioactivity in the precipitated Sepharose matrix was counted and expressed as percent sedimentation (radioactivity in pellet/radioactivity of total). We determined that Ca-dependent binding of rSP-D, CDM, and CRF to underivatized Sepharose was 4.2, 3.8, and 0.3% of the respective proteins (n = 2). In the absence of Ca the binding of all proteins was never higher than 0.1% (n = 2). The binding of S-labeled proteins to solid phase carbohydrate was performed using microtiter plates. Mannosylated BSA (E-Y Laboratories) was adsorbed to microtiter wells by incubating the protein (100 µg/ml) in TBS buffer overnight. After removing unadsorbed protein the wells were blocked with 5 mM Tris-HCl, 150 mM NaCl, and 2% BSA, pH 7.4 (blocking buffer), for 1 h. Next, the S-labeled variants of SP-D in the blocking buffer were added to the wells (0-5 µg/ml, 40 µl/well) and incubated for 1 h at room temperature. The wells were washed five times with ice-cold blocking buffer, and the radioactivity in each well was quantified using liquid scintillation spectrometry. None of the recombinant proteins bound to underivatized solid phase BSA under any conditions (n = 2). In the absence of Ca the binding of the recombinant proteins never exceeded 0.1% of the added proteins (n = 3).

Direct Binding of Recombinant Proteins to Multilamellar Liposomes

Multilamellar liposomes (100 µg) were incubated with 0.1 µg of S-labeled recombinant proteins in TBS containing varying amounts of calcium (as indicated in the figures) and 2% BSA (binding buffer) for 1 h at room temperature followed by incubation at 0 °C for 10 min. The incubation mixtures were then centrifuged at 10,000 g at 4 °C for 10 min. The resultant pellet was washed once with ice-cold binding buffer, and the amount of protein sedimented with liposomes was measured using liquid scintillation spectrometry and expressed as percent sedimentation (sedimented radioactivity/total radioactivity added).

Direct Binding of Recombinant Proteins to Lipids on Thin Layer Chromatograms (TLC)

Binding of S-labeled recombinant proteins to solid phase lipids on TLC plates was performed as described previously(10) . Briefly, 5 µg of lipids were developed on the TLC plates with organic solvent, and the plates were air-dried and soaked in the blocking buffer (TBS containing 5 mM CaCl(2), 2% BSA, 1% polyvinylpyrrolidone) for 1 h. Next, the plates were overlaid with S-labeled proteins in blocking buffer for 90 min at room temperature. Finally the plates were washed with ice-cold blocking buffer, air-dried, and exposed to x-ray film at -80 °C for 1-5 days.

Light Scattering Measurement of Calcium-induced Phospholipid Self-agglutination

Unilamellar liposomes (100 µg/ml) containing PI and DPPC (50:50, w/w) or PI and DPPC (30:70, w/w) were preincubated with native and recombinant protein (5-20 µg/ml) for 1 min in TBS. Subsequently calcium was added to a final concentration of 5 mM, and the absorbance at 400 nm was measured for 5 min using a Beckman DU-64 spectrophotometer at 20 °C.

Percoll Gradients of Unilamellar Liposomes and Recombinant Proteins

Unilamellar liposomes of PI/DPPC (50:50, w/w) (100 µg/ml), labeled with [^3H]DPPC, and 20 µg/ml of rSP-D, CDM, or CRF were incubated in TBS containing 5 mM CaCl(2) at 20 °C for 5 min. Subsequently, the lipid and protein mixtures were overlaid on 12 ml of TBS containing 5 mM CaCl(2) and 40% Percoll. The gradients were formed by centrifugation at 30,000 g for 1 h at 20 °C. Fractions were collected from the top of the tubes, and the amount of lipid and protein in each fraction was measured by liquid scintillation spectrometry and enzyme-linked immunosorbent assay, respectively.

Other Methods

Rat SP-D was isolated from the bronchoalveolar lavage fluids of silica-treated rats as described previously(10) . Protein concentrations were estimated by the BCA assay (Pierce) using bovine serum albumin as a standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli(24) . Size fractionations of native and recombinant proteins was performed by gel filtration chromatography using Bio-gel A-1.5m (1.5 100-cm column) and Sephacryl S-200 (1.5 120 cm) in the presence of 10 mM EDTA at room temperature.


RESULTS

Construction of Recombinant Proteins

SP-D is a member of the C-type lectin superfamily composed of four characteristic domains which are: 1) an NH(2)-terminal domain involved in interchain disulfide formation, 2) a collagenous domain, 3) a neck domain, and 4) a CRD(6) . For simplicity, each domain was designated as D1 to D4 as described previously(13) . As shown in Fig.1, recombinant wild type SP-D is composed of D1 to D4, and CDM is composed of D1, D3, and D4. This mutant contains the deletion Leu to Pro. The CRF obtained from collagenase digestion of rSP-D is composed of D3 and D4, with the amino terminus beginning at Gly (the last Gly-X-Y in the collagenous domain) as determined by amino acid sequencing.


Figure 1: Schematic representation of recombinant surfactant proteins. The domain structures of the monomeric subunit of recombinant wild type (rSP-D), the collagen domain deletion mutant form (CDM), and the collagenase-resistant fragment of recombinant SP-D (CRF) are shown. The four major domains of rSP-D are denoted D1 to D4, and the correponding amino acid sequences are: D1, Ala^1-Asn; D2, Gly-Pro; D3, Asp-Val; and D4, Gly-Phe.



Characterization of the CDM and the CRF of SP-D

The CDM of rat SP-D was expressed using the glutamine synthetase system described under ``Materials and Methods.'' The production of CDM ranged from 1 to 2 mg/48 h/liter culture medium when confluent cells were incubated in 150-mm dishes with 25 ml of medium. The production of CDM was lower than that of wild type recombinant SP-D (3-6 mg/48 h/liter). The results clearly demonstrate that the collagenous region is not required for the secretion of SP-D from CHO-K1 cells. Fig.2shows the SDS-PAGE analysis of recombinant wild type SP-D (rSP-D), CDM and CRF (lanes a-f). As we reported previously(14) , rSP-D appeared at approximately 43 kDa under reducing and denaturing conditions and formed disulfide bonded trimers under nonreducing conditions and appears identical to native rat SP-D. CDM migrated at 22 kDa under reducing conditions, and most of the protein appeared as trimer (61 kDa), although minor amounts of monomer and dimer were detected by autoradiography under nonreducing conditions. CRF obtained after collagenase digestion of rSP-D appeared at 18 kDa under reducing conditions and also appeared as a monomer migrating at 17 kDa under nonreducing conditions. Lanes g-l in Fig.2show the autoradiography of S-labeled rSP-D, CDM, and CRF. Radiolabeled recombinant proteins were identical to their unlabeled counterparts, and little degradation of the labeled proteins was observed. Fig.3shows the results of the size fractionation of the proteins by gel chromatography. The S-labeled rSP-D and CDM were analyzed using a Bio-Gel A-1.5m column (Fig.3A), and the radioactivity of each fraction was quantified using liquid scintillation spectrometry. The S-rSP-D eluted mostly at the position of 1200 kDa as reported previously(14) . In contrast, most of the CDM appeared later than aldolase (158 kDa). To evaluate the size of CDM and CRF more accurately, we also performed the chromatography using Sephacryl S-200 (Fig.3B). Using this latter matrix, the CDM migrated at the position of 67 kDa. The CRF appeared at the position of 18 kDa. These results are consistent with the conclusion that CDM forms a disulfide-bonded trimer, and CRF exists as a monomer in solution.


Figure 2: Electrophoretic analysis of recombinant proteins. Proteins were subjected to 8-16% SDS-PAGE under reducing (lanes a, b, c, g, h, and i) and nonreducing (lanes d, e, f, j, k, and l) conditions and visualized by Coomassie staining (lanes a-f) or autoradiography (lanes g-l). rSP-D (lanes a, d, g, and j), CDM (lanes b, e, h, and k), and CRF (lanes c, f, i, and l) are shown.




Figure 3: Gel chromatography of recombinant proteins. S-rSP-D (), CDM (bullet), and CRF () were subjected to chromatography using a Bio-Gel A-1.5m column (A) and a Sephacryl S-200 column (B). The radioactivity of each fraction was quantified using liquid scintillation spectrometry. The arrows show the peak position of eluted standards: a, blue dextran; b, thyroglobulin; c, aldolase; d, bovine serum albumin; e, ribonuclease A.



Direct Binding of Recombinant Proteins to Immobilized Carbohydrates

Direct binding of S-labeled recombinant proteins to mannose-Sepharose or galactose-Sepharose affinity matrices was performed, and the results are shown in Table1. Nearly 100% of rSP-D and CDM bound to the mannose-Sepharose matrix. By comparison, only 20% of CRF bound to the matrix. The binding of CRF to the mannose-Sepharose matrix was not increased when the binding was performed in the presence of up to 20 mM calcium (data not shown). With a galactose-Sepharose matrix, only 20% of rSP-D bound with the matrix, which is identical to the binding of CDM. However, CRF failed to show significant binding to a galactose-Sepharose matrix. Fig.4shows the results of direct binding of labeled proteins to solid phase mannosylated BSA. rSP-D and CDM bound to solid phase mannosyl-BSA in a dose-dependent manner, but the binding of CDM at comparable levels of protein was significantly reduced. The CRF did not show any significant binding to mannosyl-BSA. These data suggest that the complete oligomerization of the molecule is an important determinant for the carbohydrate binding activity of SP-D.




Figure 4: Direct binding of recombinant proteins to solid phase mannosyl-BSA. Aliquots ranging from 0 to 5 µg/ml of S-rSP-D (), CDM () and CRF () were incubated with mannosyl-BSA that had been previously adsorbed to microtiter wells. The amount of recombinant protein bound to each well was measured by liquid scintillation spectrometry. Values are mean ± S.E. of three experiments.



Direct Binding of Recombinant Proteins to Glycolipids

Direct binding of S-labeled recombinant proteins was performed using multilamellar liposomes containing PI:PS:cholesterol (40:30:40) or GlcCer:PS:cholesterol (40:30:40), and the results are shown in Fig.5. SP-D and the variants studied do not bind PS:cholesterol liposomes. Approximately 90% of S rSP-D bound to PI liposomes as described previously(13, 14) . Binding of CDM to PI liposomes (75%) was similar to that of rSP-D, but significantly higher than that of CRF (35%). Binding of the three SP-D proteins to GlcCer liposomes showed greater differences among the three molecules. Only 32% of CDM sedimented with GlcCer liposomes, whereas 80% of the wild type protein sedimented with the glycosphingolipid. No significant binding between CRF and GlcCer liposomes was detected. Fig.6shows the direct binding of recombinant proteins to glycolipids on TLC plates, and the results parallel those described in Fig.5. The rSP-D bound PI most strongly, followed by CDM and CRF. Likewise, rSP-D and CDM showed significant binding to GlcCer; however, CRF again failed to bind to this lipid. These data suggest that the collagenous region is not absolutely required for the glycolipid binding property of SP-D; however, it clearly contributes to the extent of binding found for membrane and solid phase interactions. In addition, although the lipid binding domain of SP-D maps to the CRD, the amino-terminal region containing the intermolecular disulfides appears essential for GlcCer binding.


Figure 5: Direct binding of S recombinant proteins to multilamellar liposomes. Aliquots of 0.1 µg of S-rSP-D, [S]CDM and [S]CRF were incubated with 100 µg of multilamellar liposomes and the sedimentation of each protein with PI (PI:PS:cholesterol, 30:40:30) (white column) and GlcCer (GlcCer:PS:cholesterol, 30:40:30) (gray column) liposomes was measured. Results are expressed as percent sedimentation (sedimented radioactivity/total activity added). Values are mean ± S.E. of three experiments.




Figure 6: Direct binding of S recombinant proteins to lipids on TLC plates. 5 µg of PI and GlcCer were subjected to thin layer chromatography in the solvent system chloroform:methanol:water (70:30:5). After drying the plates and blocking nonspecific binding sites, S-rSP-D (lane a), S-CDM (lane b), or S-CRF (lane c) were incubated with the TLC plates for 90 min. Binding of the radiolabeled proteins was visualized by autoradiography after washing and air drying.



Calcium Requirement for the Binding of Recombinant Proteins to Glycolipids

The calcium requirement for binding of the S-labeled recombinant proteins to glycolipids was examined using multilamellar liposomes and the results are shown in Fig.7. As reported previously, rSP-D exhibited maximum binding of PI liposomes at approximately 2.0 mM calcium (Fig.7A). CDM also showed its maximum binding to PI at 2.0 mM calcium, although the absolute amount of the binding of this protein was lower than that of rSP-D at each concentration of calcium examined. The maximum binding of CRF to PI appeared to occur at approximately 5 mM calcium. Fig.7B shows the calcium dependence of the binding of the recombinant proteins to GlcCer liposomes. Maximum binding of rSP-D and CDM was observed at approximately 5 mM calcium. CRF failed to demonstrate any significant binding, even at 10 mM calcium.


Figure 7: Calcium requirements for S recombinant protein binding to PI and GlcCer liposomes. Direct binding of S-rSP-D (), CDM (), and CRF () to PI (A) or GlcCer (B) liposomes was performed at the indicated concentrations of calcium, and the results are expressed as percent sedimentation. Values are mean ± S.E. of three experiments.



Light Scattering Measurements of Calcium-induced Association of Unilamellar Liposomes

Unilamellar liposomes containing high concentrations of PI aggregate in the presence of Ca and this process can be altered by lipid binding proteins such as SP-D. The self-association of unilamellar liposomes composed of PI and DPPC (50:50 or 30:70) was measured in the presence of the recombinant proteins to determine how structural alterations to SP-D affect lipid-lipid and lipid-protein interactions. Fig.8, A-C, shows the effect of rSP-D, CDM, and CRF upon the self-association of PI/DPPC (50:50) unilamellar liposomes. As reported previously(14) , rSP-D decreased the maximum A of 50% PI-containing liposomes in a concentration-dependent manner (Fig.8A). In contrast, CDM increased the maximum A effected by calcium (Fig.8B). The absorbance at 6 min in the presence of 0, 5, and 20 µg/ml of CDM was 0.09, 0.12, and 0.14, respectively. The effect of CRF on the light scattering change induced by calcium was different from the other two molecules (Fig.8C). CRF reduced the initial rate of change in A at the beginning of reaction induced by calcium, but the absorbance at 6 min (or longer times) in the presence of CRF was not significantly different from that in the absence of the protein (control value). Fig.8, D-F, shows the effects of the molecules on the association of 30% PI containing liposomes induced by calcium. Compared with 50% PI containing liposomes, the self-association of 30% PI containing liposomes induced by calcium was minimal and approached the limits of detection. The absorbance of 50 and 30% PI containing liposomes effected by calcium alone at 6 min was 0.09 and 0.007, respectively. The rSP-D slightly increased the change of absorbance (Fig.8D). However, CDM significantly increased the maximal absorbance in a concentration-dependent manner (Fig.8E). The absorbance at 6 min in the presence of 0, 5, and 20 µg/ml of CDM was 0.007, 0.023, and 0.054, respectively. The CRF slightly reduced the change of A400 (Fig.8F); however, the difference from the control value was not significant. These results suggest that different forms of SP-D affect liposome-liposome interactions in significantly different ways.


Figure 8: The effects of recombinant proteins on the calcium-induced self-association of unilamellar liposomes. Aliquots of 5 or 20 µg/ml of rSP-D (A and D), CDM (B and E), or CRF (C and F) were preincubated with 100 µg/ml of unilamellar liposomes containing PI:DPPC (50:50) (A-C) or PI:DPPC (30:70) (D-F) for 1 min at room temperature. Next, calcium was added to final concentration of 5 mM and the change in absorbance at 400 nm was measured. ``Controls'' are the values in the absence of proteins. Data are from a representative one of three or four experiments.



The Distribution of Lipids and Recombinant Proteins in Percoll Gradients

Self-forming Percoll gradients were used to further analyze the types of interactions between lipids and recombinant proteins. More than 70% of PI unilamellar liposomes were located in the fractions 1-4 in the absence of calcium (Fig.9A, white triangle). In contrast, 60% of the liposomes were located in the fractions 10-13 in the presence of 5 mM calcium (Fig.9A, white circle), demonstrating that PI-containing liposomes undergo self-association and a change in buoyant density in the presence of calcium. Fig.9B shows the distribution of lipids and proteins when the unilamellar liposomes were incubated with rSP-D in the presence of calcium. Only 20% of lipids were located in fractions 10-13, and more than 40% of lipids were co-distributed with proteins in fractions 7 and 8 in the presence of rSP-D. Nearly 80% of rSP-D was located in fractions 1-3 in the absence of lipids, but the protein quantitatively co-migrated with the shifted lipid peak in the presence of lipid and Ca. This latter result is important, because it demonstrates that the association of SP-D with liposomes causes a marked alteration in the buoyant density of the latter. By comparison, CDM behaved differently from wild type SP-D with the lipids (Fig.9C). When the lipids were incubated with CDM, both the molecules were sedimented in fractions 10-13, and the amount of sedimentable lipid increased 50% over that found for lipid plus calcium. CDM without lipids was distributed in fractions 1-3. Fig.9D shows the distribution of lipids and CRF. In the presence of CRF, the distribution of liposomes was almost identical to that in the absence of the protein; however, the amount of lipids located in fractions 10 and 11 was slightly reduced compared with that in the absence of the proteins. In addition, the amount of the lipids and the proteins located in fraction 1 was increased when both the molecules were co-incubated. Collectively these results indicate 1) rSP-D and liposomes formed protein-lipid complexes whose buoyant density is lower than that of self-associated liposomes, 2) CDM also formed protein-lipid complexes with PI unilamellar liposomes and the buoyant density is nearly identical to that of self associated lipids, and 3) the affinity of CRF for PI unilamellar liposomes was significantly lower than that of rSP-D and CDM, and very little CRF formed a stable complex with lipids.


Figure 9: The distributions of SP-D variants and PI containing unilamellar liposomes within self-forming Percoll gradients. The sedimentation of lipid alone is shown in A. 20 µg/ml of rSP-D (B), CDM (C), or CRF (D) were preincubated with 100 µg/ml of unilamellar liposomes composed of PI:DPPC (50:50) in the presence of 5 mM calcium for 5 min and overlaid on TBS containing 5 mM calcium and 40% Percoll. The gradients were formed by centrifugation at 30,000 gav 1 h, and the amount of protein and liposome in each fraction was measured as described under ``Materials and Methods.'' The distributions of liposomes in the absence of protein (), liposomes in the absence of protein and calcium (), liposome in the presence of protein (bullet), protein in the absence of liposome (), and protein in the presence of liposome (black square) are shown.




DISCUSSION

The purpose of this study was to examine the roles of the intermolecular disulfide bond containing NH(2) terminus and the collagenous region of rat SP-D in the structure and the function of the protein. SP-D is a member of the C-type lectin superfamily that forms oligomeric structures due to its NH(2)-terminal disulfide bonds and its collagenous domains(5, 7) . The macromolecular organization of SP-D is based upon a reiterated trimerization of a single polypeptide. In electron micrographs, each trimer forms a radial arm ending in a globular domain. Trimerization within the arm is a consequence of interaction of collagen-like domains that form triple helices. Four trimeric subunits are covalently coupled to form cruciform structures. However, higher order oligomers containing more than four trimeric subunits have also been described(7, 9) . In order to examine the relationship of SP-D structure to function, we expressed wild type recombinant rat SP-D and a variant in which the collagenous domain was deleted using CHO-K1 cells. The rat rSP-D was also treated with bacterial collagenase to obtain a proteolytic fragment.

The wild type recombinant rat SP-D was identical in its size and interaction with lipids, to its native form as reported previously (14) . The macromolecular structure of rSP-D expressed in CHO cells has also been shown to be similar to that of native form by Crouch et al.(7) . For these reasons, the recombinant molecules expressed in mammalian cells are ideal to study the relationship between structure and function.

The CRF obtained from the collagenase digestion of rSP-D in these studies appeared as monomer in nonreducing SDS-PAGE and as an 18-kDa polypeptide when examined by gel filtration chromatography. In contrast, the CDM appeared primarily as trimers in nonreducing SDS-PAGE and three times the size of the CRF by gel filtration chromatography. Because both the CDM and the CRF possess no long arms (D2 domain), the molecular sizes of both proteins obtained by gel filtration chromatography using globular proteins as standards should be very close to the actual values. Accordingly, the CRF should exist as a monomer and CDM as a trimer in solution. These observations indicate that the two cysteine residues in the NH(2) terminus (D1 domain) are likely to be the only cysteine residues involved in the intertrimeric disulfide bond formation of rSP-D, and these findings are consistent with observations using enzymatically digested native rat SP-D(25) . Furthermore, the fact that the CDM does not form higher order oligomers than trimers suggests that the D2 domain (collagenous domain) is important not only for the triple helical structure but also necessary for the formation of the dodecameric structure of SP-D.

The oligomeric state of the CRF of SP-D utilized in these studies was different from that of other collectin family CRFs reported previously. The CRF of recombinant human mannose-binding protein appears as dimers and trimers as well as monomers(26) , and rat mannose-binding protein's CRF forms trimers(27) . The CRF of native rat SP-D has also been reported to form trimers in the presence of Ca(25) . However, we have been unable to obtain reliable gel filtration results of CRF in the presence of Ca. These differences may occur as a consequence of disulfide interchange among CRDs that convert intramolecular pairings to intermolecular pairings. Such interchanges may be stabilized by noncovalent interactions among CRDs.

The rSP-D utilized in these studies behaved identically to its native form in its interaction with the carbohydrates. The CDM bound to mannose-Sepharose as efficiently as rSP-D; however, its affinity toward solid phase mannosyl-BSA was significantly reduced compared to wild type. Furthermore, only 20% of the CRF bound to a mannose-sepharose affinity matrix and it failed to bind to solid phase mannosyl-BSA. These findings indicate that the degree of oligomerization of the molecules is a critical determinant of the high affinity binding to immobilized carbohydrates.

The binding properties of recombinant molecules toward glycolipids were also studied. In direct binding to multilamellar liposomes, the activity of CDM toward PI liposomes was quite close to that of its wild type counterpart; however, the affinity for GlcCer liposomes was decreased to about 30% of that of rSP-D. The CRF bound to PI liposomes with approximately 30% of the efficiency of rSP-D, and it failed to show significant binding to GlcCer liposomes. The results obtained from the direct binding assay using solid phase glycolipids on TLC plates were essentially the same as those from the liposome binding studies. These findings indicate that the oligomerization of the molecule contributes to the high affinity binding to glycolipids, especially to GlcCer, despite the fact that the binding domain of SP-D for glycolipids has been mapped to its CRD (D4 domain)(13, 14) . We have previously identified important differences between PI binding and GlcCer binding based upon site-directed mutagenesis(14) . The finding that CRF coordinately lost solid phase carbohydrate and GlcCer binding activity, but retained significant PI binding, suggests that the structural determinants of SP-D required for GlcCer binding and PI binding are different. This latter conclusion is consistent with previous findings in which engineering of altered specificity of carbohydrate binding demonstrated different mechanisms for PI and GlcCer binding(14) .

The interactions of PI-containing unilamellar liposomes and the recombinant proteins demonstrate that the cruciform structure of SP-D can significantly alter lipid-lipid interaction. rSP-D reduced the calcium-dependent self-association process, whereas CDM enhanced it. The effect of CRF was much smaller than those of rSP-D and CDM; however, it reduced the initial change of the calcium-induced self-aggregation of liposomes. Our preferred interpretation of these results is that liposomes containing 50% PI will form tightly packed aggregates in which Ca bridges apposing liposomes. In the presence of rSP-D the liposomes interact with the SP-D and are effectively held apart by as much as the end to end distance between the globular heads which is 114 nm, thereby effectively creating an open lattice. This arrangement would account for the lower buoyant density of rSP-D-liposome complexes compared with Ca-liposome complexes. In contrast to the wild type protein, the CDM is likely to be composed of only trimeric globular domains which effectively cross-link liposomes with a spacing of as little as 9 nm. The increased A occurring with CDM may be due to recruitment of more liposomes into aggregates than with Ca alone. Evidence for this latter interpretation is found in Fig.9C in which the amount of lipid in high buoyant density aggregates significantly increases in the presence of CDM. When liposomes contain 30% PI, their aggregation in response to Ca alone is barely detectable, but the inclusion of CDM greatly enhances the process. Such enhancement by CDM illustrates that it is a more effective aggregating agent than Ca alone and is entirely consistent with the above interpretations. The apparent monomeric structure of CRF in conjunction with its PI binding may function to weakly interfere with Ca bridging of liposomes and principally reduce the rate of lipid aggregation.

In summary, we expressed, purified and characterized rSP-D and its CDM and CRF variants. The CDMs appeared as trimers and the CRF as monomers, indicating that the collagenous domain is essential for the assembly of the dodecameric structure of SP-D. Both CRF and CDM retained significant PI binding activity. The GlcCer binding activity was reduced in CDM and lost in CRF. These findings further substantiate important structural differences between GlcCer and PI binding sites in SP-D.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants HL45286 and HL29891. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 303-398-1300; Fax: 303-398-1806; voelkerd{at}njc.org.

^1
The abbreviations used are: SP-D, surfactant protein D; CRD, carbohydrate recognition domain; PI, phosphatidylinositol; GlcCer, glucosylceramide; SP-A, surfactant protein A; CRF, collagenase-resistant fragment; CDM, collagen domain deletion mutant; GMEM, Glasgow minimal essential medium; TBS, Tris-buffered saline; DPPC, Dipalmitoylphosphatidylcholine; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary.


ACKNOWLEDGEMENTS

We thank Peggy Hammond for excellent secretarial assistance. This work was performed in the Anna Perahia Adatto Clinical Laboratories.


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