©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Altered Carbohydrate Recognition Specificity Engineered into Surfactant Protein D Reveals Different Binding Mechanisms for Phosphatidylinositol and Glucosylceramide (*)

Yoshinori Ogasawara , Dennis R. Voelker (§)

From the (1)Lord and Taylor Laboratory for Lung Biochemistry, Anna Perahia Adatto Clinical Laboratories, 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

Pulmonary surfactant protein D (SP-D) is a member of the collectin subgroup of the C-type lectin superfamily that binds glycosylated lipids such as phosphatidylinositol (PI) and glucosylceramide (GlcCer). We have previously reported that the carbohydrate recognition domain of SP-D plays an essential role in lipid binding. However, it is unclear how the carbohydrate binding property of SP-D contributes to the lipid binding. To clarify the relationship between the lectin property and the lipid binding activity of rat SP-D, we expressed wild-type recombinant rat SP-D (rSP-D) and a mutant form of the protein with substitutions Glu-321 Gln and Asn-323 Asp (SP-D) in CHO-K1 cells. The indicated mutations have previously been shown to change the carbohydrate binding specificity of surfactant protein A and mannose-binding protein from mannose > galactose to the converse. rSP-D expressed in mammalian cells was essentially identical to native rat SP-D in its lipid and carbohydrate binding properties. In contrast, SP-D was unable to bind GlcCer, but retained binding activity toward PI liposomes and solid-phase PI. The efficiency of SP-D binding to PI liposome was 50% of that of rSP-D in the presence of 5 mM Ca, but equivalent at 20 mM Ca. Carbohydrates competed for SP-D binding to PI such that maltose > galactose for rSP-D, and the order was reversed for SP-D. Furthermore, SP-D could bind to digalactosyldiacylglycerol more effectively than rSP-D. These results suggest the following. 1) The carbohydrate binding specificity of SP-D was changed from a mannose-glucose type to a galactose type; 2) the GlcCer binding property of SP-D is closely related to its sugar binding activity; and 3) the PI binding activity is not completely dependent on its carbohydrate binding specificity.


INTRODUCTION

Pulmonary surfactant protein D (SP-D)()is a hydrophilic glycoprotein synthesized and secreted by alveolar type II cells(1, 2) . The primary structure of rat SP-D is characterized by four distinct domains: 1) an N-terminal region involved in intermolecular disulfide bonding, 2) a collagenous domain composed of 59 Gly-X-Y repeats, 3) a neck domain, and 4) a carbohydrate recognition domain (CRD)(3) . In solution, SP-D forms a 12-mer composed of four identical trimers(4) . The protein can also form higher order oligomers(4) . SP-D is a member of the C-type lectin superfamily and shares significant structural homology with conglutinin, CL43, and surfactant protein A (3, 5). While SP-A has been implicated as an important molecule in surfactant lipid metabolism(6, 7, 8) , the function of SP-D is not well understood. Several studies suggest that SP-D may play a role in immunoglobulin-independent host defense mechanisms in the lung(9, 10, 11, 12, 13) . SP-D also binds several glycolipids, including phosphatidylinositol (PI) (14, 15) and glucosylceramide (GlcCer)(16) . Under some conditions, SP-D can antagonize the inhibitory effect of SP-A upon lipid secretion by alveolar type II cells(17) . Collectively, these observations suggest that interactions of SP-D with lipid are likely to be important to its functions within the lung.

We have previously reported that the domains responsible for the lipid binding properties of SP-A and SP-D are located in the CRDs of the molecules(18, 19, 20) . The lipid binding specificity of SP-D is quite different from that of SP-A, which binds to dipalmitoylphosphatidylcholine (DPPC)(21) , galactosylceramide (GalCer), lactosylceramide, and asialo-G(22, 23) . However, the mechanism of lipid binding by the proteins and the relationship between the lectin property and the glycolipid binding activity are unclear.

Comparison of the sequence at the carboxyl-terminal portions of C-type lectin CRDs has demonstrated that SP-D can be categorized as a mannose- and glucose-type lectin similar to serum mannose-binding proteins (MBPs) and SP-A(24) . These studies also revealed that Glu-187 and Asn-189 in mannose-binding protein A (MBP-A) are essential for the sugar binding specificity of the molecule(24, 25) . Mutations of Glu-187 Gln and Asn-189 Asp in MBP-A altered the carbohydrate binding specificity of the molecule from a mannose type to a galactose type(24) . Similar results were also reported for analogous mutations in SP-A(20) .

In an effort to understand the contribution of the lectin property of SP-D to glycolipid binding activity, we introduced mutations into rat SP-D (Glu-321 Gln and Asn-323 Asp) to alter the sugar binding specificity of the molecule. Using this mutant form of SP-D (SP-D) expressed in CHO-K1 cells, we show that the carbohydrate binding specificity is essential for GlcCer binding by SP-D, but is not critical for the PI binding property of the molecule.


MATERIALS AND METHODS

Purification of Rat SP-D

Rat SP-D was isolated from bronchoalveolar lavage fluids of Sprague-Dawley rats by the method previously described(14) . Briefly, bronchoalveolar lavage fluids of silica-treated rats were centrifuged at 33,000 g at 4 °C for 16 h to sediment surfactant, and the resultant supernatant was applied to a mannose-Sepharose affinity matrix in the presence of 5 mM calcium. The SP-D-containing fraction was then eluted in the presence of 2 mM EDTA, followed by gel filtration in the presence of 10 mM EDTA using a Bio-Gel A-15m column (Bio-Rad). The protein was dialyzed against TBS (5 mM Tris-HCl, 150 mM NaCl, pH 7.4) at 4 °C and stored at -20 °C.

DNA Construction of SP-D and SP-D

The 1.2-kilobase pair cDNA for rat SP-D was isolated as reported previously (3) The SP-D cDNA, ligated into pGem-7Zf(+) at the EcoRI site, was digested completely with HindIII and partially with XbaI, and the resultant full-length insert was subsequently ligated into a pEE14 plasmid vector (26) using the same restriction sites. The mutant cDNA of SP-D with substitutions Glu-321 Gln and Asn-323 Asp (SP-D) was produced using the polymerase chain reaction and the overlapping extension method (27) and the cDNA of SP-D as the template. This construct was also ligated into a pEE14 vector using a unique XbaI site, and the orientation of the insert was confirmed by restriction enzyme digestions using EcoRI and NotI. Mutations were confirmed by DNA sequencing using the method of Sanger et al.(28) and Sequenase.

Expression and Isolation of Recombinant Proteins

The recombinant proteins were expressed using the glutamine synthetase amplification system(26) . The pEE14 plasmid vectors containing the cDNAs for wild-type SP-D and SP-D were transfected into CHO-K1 cells using Lipofectamine (Life Technologies, Inc.). Transfected cells were incubated in glutamine-free Glasgow minimum essential medium (GMEM) supplemented with 10% dialyzed fetal bovine serum (complete GMEM-10(26) ) in the presence of 25 µM methionine sulfoxamine for 7-10 days. Colonies were isolated using cloning cylinders, transferred to complete GMEM-10 containing higher concentrations of methionine sulfoxamine (100-500 µM), and incubated for another 10-15 days. The colonies isolated from the highest methionine sulfoxamine concentration were then cloned by limiting dilution. The clones that produced the highest amount of recombinant proteins were identified using an SP-D enzyme-linked immunosorbent assay. To isolate rSP-D, cloned cells (4 10/150-mm dish) were incubated in complete GMEM-10 for 4-5 days until reaching confluence (40 10/150-mm dish) and then changed to serum-free medium. 48 h later, the medium was harvested and centrifuged to remove cell debris, followed by dialysis (3 10 volume) against TBS at 4 °C. The dialyzed SP-D solution was then applied to a mannose-Sepharose affinity matrix (bed volume of 5 ml) in the presence of 5 mM calcium, and rSP-D was eluted using 2 mM EDTA. The eluted protein was dialyzed against TBS again and stored at -20 °C. For SP-D, which failed to bind to mannose-Sepharose, the medium containing expressed protein was directly applied to a protein A-Sepharose affinity column covalently coupled with polyclonal anti-rat SP-D IgG (29). Immunoreactive SP-D was eluted from the affinity matrix with 0.1 M glycine, pH 3.0; immediately neutralized with 1 M Tris base; and then dialyzed against TBS and stored at -20 °C.

Metabolic Labeling of Recombinant Proteins

The recombinant proteins were metabolically labeled with TranS-label (ICN). Confluent cells were washed twice with phosphate-buffered saline and incubated in methionine- and cysteine-free GMEM for 2 h. Next, the cells were incubated in the same medium containing 20 µCi/ml TranS-label for 48 h. The labeled proteins were isolated and stored as described above for nonlabeled proteins. The specific activity of S-labeled rSP-D and SP-D used ranged between 1500 and 4500 cpm/ng.

Lipids

DPPC, PI, phosphatidylglycerol (PG), phosphatidylserine (PS), and cholesterol were purchased from Avanti Polar Lipids, Inc. GlcCer, GalCer, galactosyldiacylglycerol (GalDAG), and digalactosyldiacylglycerol (GalDAG) were purchased from Sigma. Multilamellar and unilamellar liposomes were prepared as described previously (18) and used for the lipid binding and self-association assay described below.

Direct Binding of Recombinant Proteins to Multilamellar Liposomes

100 µg of multilamellar liposomes were incubated with 0.1 µg of S-labeled recombinant proteins in TBS containing 5 mM calcium and 2% bovine serum albumin (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 sedimenting with liposomes was quantified using liquid scintillation spectrometry. In some experiments, all the procedures described above were performed at room temperature. In carbohydrate competition experiments, the binding was performed in the presence of 0-200 mM carbohydrate as indicated.

Direct Binding of Recombinant Proteins to Lipids on Thin-layer Chromatograms

Binding of S-labeled recombinant proteins to solid-phase lipids on TLC plates was performed as described previously (14) Briefly, 5-50 µg of lipids were separated on TLC plates with organic solvent. Subsequently, the plates were air-dried and soaked in blocking buffer (TBS containing 5 mM CaCl, 2% bovine serum albumin, 1% polyvinylpyrrolidone) for 1 h. Then 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.

Measurement of Calcium-induced Phospholipid Self-agglutination

100 µg/ml unilamellar liposomes containing PI and DPPC (50:50, w/w) or PS and DPPC (50:50, w/w) was preincubated with native and recombinant proteins (5-20 µg/ml) for 1 min in TBS. Subsequently, calcium was added to a final concentration of 5 mM, and the change in absorbance at 400 nm was measured for 5 min using a Beckman DU-64 spectrophotometer at 20 °C. In the absence of lipid, SP-D does not exhibit any light scattering in the presence of calcium.

Other Procedures

Protein concentrations were measured by the bicinchoninic acid assay (Pierce) using bovine albumin as a standard. SDS-polyacrylamide gel electrophoresis (PAGE) was performed according to the method of Laemmli(30) . Immunoblotting of native and recombinant proteins was performed by the method of Towbin et al.(31) using polyclonal anti-rat SP-D IgG as a probe. Size fractionation of native and recombinant proteins was performed by gel filtration chromatography using Bio-Gel A-15m (1.5 100-cm column) in the presence of 10 mM EDTA, 0.15 M NaCl at room temperature.


RESULTS

Characterization of Wild-type Recombinant SP-D and SP-DExpressed in CHO-K1 Cells

Wild-type rSP-D and SP-D were expressed using glutamine synthetase amplification under methionine sulfoxamine selection. Production of these recombinant proteins ranged from 3 to 6 mg/48 h/liter of culture medium when confluent cells in 150-mm dishes were incubated with 25 ml of medium. rSP-D was purified using mannose-Sepharose 6B affinity chromatography, and >95% of the protein was recovered. In contrast, SP-D failed to bind the mannose-Sepharose affinity matrix, requiring that the protein be isolated using an anti-rat SP-D IgG affinity column. The recovery of the mutant protein was >90% after affinity column purification. Fig. 1shows the SDS-PAGE analysis of native rat SP-D, rSP-D, and SP-D (lanes a-f). Native rat SP-D appeared at 43 kDa under reducing and denaturing conditions and formed disulfide-bonded trimers (120 kDa) under nonreducing conditions. rSP-D and SP-D appeared identical to each other and formed disulfide-bonded trimers under nonreducing conditions. The molecular size of both recombinant proteins was slightly smaller than that of native rat SP-D (<0.5 kDa under reducing conditions). The difference in molecular size between native and recombinant molecules was not observed when the proteins were treated with N-glycanase (data not shown). This latter result suggests that a minor difference in oligosaccharide structure may exist between native SP-D and its recombinant form expressed in CHO-K1 cells. Fig. 1also shows the autoradiography of S-labeled rSP-D and SP-D (lanes g-j). The radioactive proteins appeared essentially identical to their unlabeled counterparts, and little degradation of the labeled proteins was observed. Fig. 2shows the immunoblotting of the native and recombinant proteins. Each protein (100 ng) was subjected to SDS-PAGE and transferred to a nitrocellulose sheet, and the reactive antigens were detected using anti-rat SP-D IgG as a probe. In spite of the mutations, SP-D reacted to the antibody as well as native and wild-type recombinant SP-D. Under reducing conditions, all three proteins yield identical immunoreactive forms. Examination of the proteins under nonreducing conditions indicates that the major forms are nearly identical, although several minor bands appear in the native SP-D lane, suggesting that a small fraction of the preparation has been proteolyzed.


Figure 1: Electrophoretic analysis of recombinant proteins. Proteins were subjected to 8-16% SDS-PAGE under reducing (lanes a-c, g, and h) and nonreducing (lanes d-f, i, and j) conditions and visualized by Coomassie Blue staining (lanes a-f) or autoradiography (lanes g-j). Native rat SP-D (lanesa and d), rSP-D (lanesb, e, g, and i), SP-D (lanesc, f, h, and j) are shown.




Figure 2: Immunoblotting analysis of recombinant proteins. 100 ng of native rat SP-D, rSP-D, and SP-D were subjected to SDS-PAGE under reducing (lanes a-c) and nonreducing (lanes d-f) conditions and transferred to nitrocellulose sheets. The immunoreactive materials were detected using polyclonal antibody against rat SP-D. Native rat SP-D (lanesa and d), rSP-D (lanesb and e), and SP-D (lanesc and f) are compared.



The results in Fig. 3show the size fractionation of the proteins by gel chromatography. S-Labeled rSP-D (Fig. 3A) and SP-D (Fig. 3B) were analyzed using a Bio-Gel A-15m column, and the radioactivity of each fraction was quantified by liquid scintillation spectrometry. Most of the S-labeled rSP-D eluted at the position of 1200 kDa (peakb), which corresponds to the main peak of native rat SP-D(18) . There also appeared small peaks at the positions of 2100 kDa (peaka) and 600 kDa (peakc), and both peaks were also observed in the elution of native rat SP-D. The radioactivity present in each peak fraction was 4% at 2100 kDa, 73% at 1200 kDa, and 13% at 600 kDa. The size distribution of the recombinant proteins is similar to that of native SP-D, although up to 10% of the native protein may be present in the 2100-kDa fraction in some preparations. The elution pattern of S-labeled SP-D was identical to that of rSP-D (Fig. 3B). While the results in Fig. 1indicate some minor heterogeneity in N-linked glycosylation between native and recombinant proteins, the oligomerization of rSP-D as well as SP-D expressed in CHO-K1 cells is essentially the same as that of native rat SP-D.


Figure 3: Gel chromatography of rSP-D and SP-D. S-Labeled rSP-D (A) and SP-D (B) were subjected to chromatography using a Bio-Gel A-15m column. The radioactivity of each fraction was quantified using a liquid scintillation spectrometer as described under ``Materials and Methods.'' The arrows show the peak positions corresponding to 2100 kDa (arrowa), 1200 kDa (arrowb), and 600 kDa (arrowc).



Direct Binding of Recombinant Proteins to Multilamellar Liposomes

Direct binding of S-labeled recombinant proteins to multilamellar liposomes composed of PI, DPPC, GlcCer, or GalCer was performed, and the results are shown in Fig. 4. In these experiments, 0.1 µg of S-labeled rSP-D or SP-D was incubated with 100 µg of the different liposomes in the presence of 5 mM calcium, and sedimentable radioactivity was measured. In Fig. 4A, >90% of the rSP-D sedimented with PI liposomes, while none appeared with DPPC liposomes. In addition, nearly 90% of the rSP-D coprecipitated with GlcCer liposomes, and none was found with GalCer. These observations are essentially the same as the result obtained using native rat SP-D reported previously(18) . However, SP-D behaved quite differently from rSP-D. Only 50% of the SP-D bound to PI liposomes and was sedimentable. Strikingly, there was no binding of SP-D to GlcCer liposomes. SP-D also failed to sediment with GalCer and DPPC liposomes, similar to its wild-type counterpart. This latter result with GalCer is important since the mutations introduced were expected to switch carbohydrate specificity to favor galactose binding. The experiments described in Fig. 4A were repeated using populations of SP-D that were purified by gel filtration to correspond only to the dodecamer population, and the results are shown in Fig. 4B. The data clearly indicate that there are no significant differences between the selected dodecamer population of SP-D and the total population in the interaction of the proteins with liposomes.


Figure 4: Direct binding of S-labeled recombinant proteins to multilamellar liposomes. Aliquots of 0.1 µg of S-labeled rSP-D (blackcolumns) or SP-D (graycolumns) were incubated with 100 µg of multilamellar liposomes, and the sedimentation of each protein with PI (PI:PS:cholesterol, 30:40:30), DPPC (DPPC:PS:cholesterol, 30:40:30), GlcCer (GlcCer:PS:cholesterol, 30:40:30), and GalCer (GalCer:PS:cholesterol, 30:40:30) liposomes was measured. Results are expressed as percent sedimentation (sedimented radioactivity/total activity added). Values are means ± S.E. of three experiments. A contains data for all size oligomers of recombinant and mutant SP-D, and B contains data for purified dodecameric oligomers of recombinant and mutant SP-D.



Direct Binding of Recombinant Proteins to Lipid on TLC Plates

Direct binding of S-labeled recombinant proteins (2 µg/ml) to lipids (5 µg/lane) on TLC plates was also performed as described under ``Materials and Methods,'' and the results are shown in Fig. 5. S-Labeled rSP-D bound to PI and GlcCer, but not to PG or GalCer (Fig. 5, laneb). In contrast, S-labeled SP-D bound to PI, but not to PG, GlcCer, or GalCer (Fig. 5, lanec). Increasing the amount of either SP-D (10 µg/ml) or GlcCer (50 µg) had no effect upon binding. These results are essentially the same as those derived from the direct binding assay using multilamellar liposomes (described above) and indicate that Glu-321 and Asn-323 of SP-D are important for the GlcCer binding property and partially affect the PI binding activity of SP-D.


Figure 5: Direct binding of S-labeled recombinant proteins to lipids on TLC plates. 5 µg of PI, PG, GlcCer, and GalCer were developed in the solvent system chloroform:methanol:water (70:30:5). Lipids were visualized with iodine vapor (lanea). The same amount of lipids was also developed as described above, and S-labeled rSP-D (laneb) or SP-D (lanec) binding was performed on TLC plates and visualized by autoradiography as described under ``Materials and Methods.''



Characterization of PI Binding Property of Recombinant Proteins

The direct binding of recombinant proteins to multilamellar liposomes was also examined at different concentrations of calcium, and the results are shown in Fig. 6. rSP-D bound to PI liposomes in a Ca-dependent manner, and the maximum binding was observed at 2.5 mM calcium. The binding was reduced slightly as the calcium concentration was increased above 2.5 mM. The binding of rSP-D to PI liposomes at 2.5 and 20 mM calcium was 96.4 and 83.3% of the maximum attainable binding, respectively. The SP-D binding to PI liposomes was also Ca-dependent; however, the binding below 2.5 mM Ca was significantly lower than that of rSP-D. The binding of the mutant protein to PI liposomes increased to the level of the wild-type protein at 20 mM Ca. The binding at 2.5 and 20 mM calcium was 40.3 and 80.3% of the theoretical maximum, respectively. Next, we studied the effect of temperature upon binding (Fig. 7). The PI liposome binding of recombinant proteins was performed at 20 °C for 1 h, and the incubation mixtures were transferred to 0 °C or left at 20 °C for an additional 10 min as described under ``Materials and Methods.'' There was no difference in the binding of rSP-D to PI liposomes at 20 and 0 °C; however, the PI binding of SP-D at 20 °C was significantly reduced compared with that at 0 °C (52% at 0 °C and 21% at 20 °C). These results suggest that Glu-321 and Asn-323 affect the stability of SP-D/PI interactions at low Ca concentrations and elevated temperatures.


Figure 6: Calcium requirement of S-labeled recombinant protein binding to PI liposomes. Direct binding of S-labeled rSP-D () and SP-D () to PI liposomes (100 µg of PI) was performed with varying concentrations of calcium, and the results are expressed as percent sedimentation as described under ``Materials and Methods.'' Values are means ± S.E. of three experiments.




Figure 7: Temperature dependence of S-labeled recombinant protein binding to PI liposomes. S-Labeled rSP-D (blackcolumns) or SP-D (graycolumns) was incubated with PI liposomes (100 µg of PI) for 1 h at 20 °C, and the incubation mixtures were transferred to 0 °C or left at 20 °C for another 10 min. The results are expressed as percent sedimentation as described under ``Materials and Methods.'' Values are means ± S.E. of three experiments.



Light Scattering Measurement of Calcium-induced Self-association of Unilamellar Liposomes

Self-association of unilamellar liposomes composed of either PI and DPPC (50:50) or PS and DPPC (50:50) was measured in the presence of native and recombinant proteins as described under ``Materials and Methods.'' Liposomes that contain a high percentage of negatively charged phospholipids such as PI and PS self-associate in the presence of calcium. Fig. 8A shows the effect of native rat SP-D on the self-association of PS containing unilamellar liposomes. SP-D has no significant effect upon either the rate or extent of PS vesicle aggregation as monitored by an increase in A. Mutation-bearing SP-D gave the same result as rSP-D in its interaction with PS vesicles. Next, we studied the effects of the proteins on the self-association of PI liposomes. rSP-D reduced the A change effected by Ca in a concentration-dependent manner (Fig. 8B). The effect of 20 µg/ml rSP-D was almost the same as that of 10 µg/ml rSP-D. The absorbance at 6 min in the presence of 0, 5, 10, and 20 µg/ml rSP-D was 0.063, 0.046, 0.033, and 0.031, respectively, and these values correspond well to those found with native SP-D (data not shown). SP-D also reduced the change in A of PI liposomes (Fig. 8C); however, the effect was significantly less than that of rSP-D. The absorbance at 6 min in the presence of 20 µg/ml mutant protein was 0.041. These results show that rSP-D and SP-D specifically interact with unilamellar PI liposomes and that the efficiency of SP-D binding to PI and capacity to alter liposome aggregation are lower than those of rSP-D.


Figure 8: SP-D variants alter calcium-induced self-association of unilamellar liposomes. Proteins (5-20 µg/ml) were preincubated with 100 µg/ml unilamellar liposomes for 1 min at room temperature. Next, calcium was added to a final concentration of 5 mM, and the change in absorbance (ABS) at 400 nm was measured. Controls are the values in the absence of proteins. A, PS liposomes (PS:DPPC, 50:50) with native rat SP-D; B, PI liposomes (PI:DPPC, 50:50) with rSP-D; C, PI liposomes (PI:DPPC, 50:50) with SP-D. Data are from a representative one of three to five experiments.



Carbohydrate Competition for SP-D Binding to PI Liposomes

Direct binding of rSP-D or SP-D to PI vesicles was performed in the presence of 25-100 mM sugar (inositol, maltose, glucose, mannose, galactose, and N-acetylgalactosamine), and the results are presented in Fig. 9. The values are expressed as the percentage of the control value obtained in the absence of the sugars. Inositol was the most effective inhibitor of PI binding of rSP-D (Fig. 9A). The order of the competition for rSP-D binding to PI vesicles was inositol > maltose > glucose > mannose > galactose, and it corresponded well with the order of the carbohydrate binding affinity of native rat SP-D(32) . N-Acetylgalactosamine showed no effect even at a concentration of 200 mM (data not shown). By comparison, the binding of SP-D to PI vesicles was most effectively inhibited by inositol; however, galactose became the second most effective competitor in the binding, consistent with the hypothesis that the introduced mutations should increase the affinity of the protein for galactose. The order of the competition for mutant protein binding to PI vesicles was inositol > galactose > maltose > mannose > glucose. The most dramatic changes in rank order affinity for carbohydrate competition were found for maltose and galactose. For rSP-D, the IC values of maltose and galactose were 18.5 and >200 mM, respectively. For mutant SP-D, the IC values of maltose and galactose were 38.5 and 27.5 mM, respectively. These results clearly indicate the sugar binding specificity of the mutant protein, SP-D, changed from a mannose-glucose type to a galactose type; however, inositol remained the most effective ligand for SP-D.


Figure 9: Carbohydrate competition for S-labeled rSP-D and SP-D binding to PI multilamellar liposomes. Direct binding of S-labeled rSP-D (A) or SP-D (B) to PI liposomes (100 µg of PI) was performed in the presence of 0-100 mM concentrations of the indicated sugars, and the results are expressed as percent sedimentation as described under ``Materials and Methods.'' Inositol (), maltose (), glucose (), mannose (), galactose (), and N-acetylgalactosamine (GalNac) () were used as competitors. Values are means ± S.E. of three experiments.



Direct Binding of Recombinant Proteins to Other Glycolipids

To examine further changes in the ligand binding specificity of the mutant protein, we investigated the direct binding of the recombinant proteins to GalDAG and GalDAG. Fig. 10shows the direct binding of the S-labeled recombinant proteins to GalDAG and GalDAG on a TLC plate. Both rSP-D and SP-D bound to GalDAG, but not to GalDAG, and the binding of SP-D to GalDAG was significantly greater than that of rSP-D. Next, we performed the direct binding of the proteins to GalDAG and GalDAG liposomes in the presence of 5 mM calcium (Fig. 11). No significant binding was observed for either protein toward GalDAG liposomes; however, GalDAG liposomes bound significantly more SP-D than rSP-D. The difference in binding to GalDAG between rSP-D and SP-D became more significant when the reactions were performed in the presence of 20 mM calcium (16% in rSP-D and 38% in SP-D; data not shown).


Figure 10: Direct binding of S-labeled recombinant proteins to GalDAG and GalDAG on TLC plates. 5 µg of GalDAG and GalDAG were developed on TLC plates in chloroform:methanol:water (70:30:5) and visualized by iodine vapor (lanea). Identical amounts of lipids were developed as described above, and direct binding of S-labeled rSP-D (laneb) and SP-D (lanec) was performed as described under ``Materials and Methods.''




Figure 11: Direct binding of S-labeled recombinant proteins to GalDAG and GalDAG liposomes. S-Labeled rSP-D (blackcolumns) or SP-D (graycolumns) was incubated with 100 µg of multilamellar liposomes, and the sedimentation of each protein with GalDAG (GalDAG:PS:cholesterol, 30:40:30) or GalDAG (GalDAG:PS:cholesterol, 30:40:30) liposomes was measured. The results are expressed as percent sedimentation as described under ``Materials and Methods.'' Values are means ± S.E. of three experiments.




DISCUSSION

The purpose of this study was to examine the relationship between the carbohydrate binding property and the lipid binding activity of SP-D. We have previously reported that the region of SP-D responsible for the lipid binding property is located in the CRD(18) . Both lipid ligands for SP-D (PI and GlcCer) are glycolipids, an observation that raises the question of whether the lipid binding property of SP-D is completely due to its sugar binding specificity. To address this problem, we expressed wild-type recombinant SP-D and the mutant protein with substitutions Glu-321 Gln and Asn-323 Asp in an effort to alter the sugar binding specificity of the molecule.

The substitutions Glu-321 Gln and Asn-323 Asp were chosen based upon the results of a study conducted with serum MBP-A that demonstrated that analogous mutations resulted in the alteration of the sugar binding specificity of the molecule from a mannose-glucose type (24) to a galactose type. The homology of the amino acid sequence at corresponding regions between rat SP-D and MBP-A (3, 24) suggests the possibility that identical substitutions in rat SP-D may also change the carbohydrate binding specificity of the molecule. This same approach has recently been used with the related protein SP-A(20) .

rSP-D expressed in CHO-K1 cells appeared essentially identical to native rat SP-D. This result is in agreement with the recent observations by Crouch et al.(33) . The difference of molecular size on SDS-PAGE between native and recombinant molecules was <0.5 kDa (which was due to the heterogeneity of N-linked glycosylation of recombinant molecules), and rSP-D eluted at exactly the same positions as native rat SP-D on gel filtration and showed equal reactivity to the antibody raised against native protein. In addition, the rSP-D used in this study interacted with lipids in essentially the same manner as native rat SP-D. In direct binding assays using either TLC plates or multilamellar liposomes, there were no differences between the lipid binding specificity of rSP-D and that of native SP-D: both the molecules bound to PI and GlcCer, but not to DPPC, PG, or GalCer. The sedimentation of rSP-D with PI liposomes was also identical to the value observed with native rat SP-D(18) . Furthermore, rSP-D and native SP-D were indistinguishable in competition with S-labeled rSP-D for binding to solid-phase PI (data not shown).

Unlike its wild-type counterparts, SP-D was unable to bind to a mannose-Sepharose affinity matrix. Mutant SP-D was isolated using an antibody affinity column, and it appeared to be identical to rSP-D when analyzed by SDS-PAGE, gel filtration, and immunoblotting. However, SP-D behaved quite differently from rSP-D in its interactions with lipids. The sedimentation of SP-D with PI liposomes at 2.5 mM Ca was about half of that of rSP-D. In addition, SP-D required higher concentrations of calcium (20 mM) as well as lower temperature conditions than rSP-D to achieve equivalent binding efficiency. Experiments examining the disruption of PI self-aggregation also indicated that the affinity of SP-D for this lipid was lower than that of rSP-D. These observations demonstrate that Glu-321 and Asn-323 play an important role in the expression of the PI binding property of SP-D, but are not absolutely required.

The results obtained in experiments examining the binding to GlcCer were more striking. rSP-D retained the GlcCer binding property of the native molecule, while SP-D did not bind to GlcCer. These results indicate that Glu-321 and Asn-323 of SP-D are essential for the GlcCer binding activity of the molecule.

We also studied the effects of various carbohydrates on the PI vesicle binding of the recombinant molecules. The affinity of SP-D for PI is much higher than that for inositol(14) . Inositol was the most effective competitor of PI binding by rSP-D and SP-D; however, the rank order for competition by other carbohydrates was markedly altered. Maltose was the second most effective competitor for rSP-D binding to PI, and galactose was insignificant as a competitor. In contrast, for SP-D binding to PI, galactose was the second most effective competitor. These results show that SP-D still possessed the carbohydrate binding properties, but that its binding specificity had been altered to a galactose > mannose-glucose type. Furthermore, SP-D bound to GalDAG with higher affinity than rSP-D. These observations demonstrate clear evidence of altered carbohydrate binding specificity by SP-D.

The studies presented also demonstrate that the binding mechanisms of SP-D to PI and GlcCer are not identical. Glu-321 and Asn-323 are essential for the GlcCer binding, but are not critical for the PI binding. Furthermore, the relative affinity of the protein for inositol was not affected by the alteration of carbohydrate binding specificity (data not shown). These observations suggest that the recognition domains of SP-D for PI and GlcCer may overlap, but are not identical. While the binding of rSP-D and the mutant protein to the diacylglycerol-containing lipids (GalDAG and PI) is similar, the binding to ceramide-containing lipids (i.e. GlcCer) is markedly different. The results suggest that the carbohydrate specificity of SP-D may play a more dominant role in interactions of the protein with ceramide-containing lipids than with diacylglycerol-containing lipids.

In addition to carbohydrate, the hydrophobic components of glycolipids appear to play an important role in SP-D binding reactions. Although SP-D binds PI and GlcCer, it fails to bind lyso-PI (14) and glucosylsphingosine on TLC plates (data not shown). Thus, the nature of the hydrophobic moiety influences the reactivity and affinity of SP-D for the ligand. Consistent with this idea is the observation that rSP-D and SP-D bind GalDAG, but not GalCer ( Fig. 4and Fig. 5), ceramide dihexoside, and ceramide trihexoside (data not shown).

The interaction of SP-D with unilamellar PI liposomes reveals yet another aspect of lipid interaction by the protein. SP-D diminishes the Ca-dependent self-aggregation process by an unknown mechanism. Our speculation is that SP-D may form an ordered lattice in which the protein is interposed between liposomes and thus reduces the absorption of light that occurs when liposomes are agglutinated by Ca. In the experiments described in Fig. 8, the highest levels of SP-D give a liposome:SP-D ratio of 1. The cruciform structure of the SP-D oligomer and the location of the CRDs at the extreme ends of the molecules constitute a favorable structural arrangement for preventing liposome/liposome interactions.

In summary, we expressed wild-type rSP-D and the mutant with substitutions Glu-321 Gln and Asn-323 Asp (SP-D) using the glutamine synthetase system and CHO-K1 cells. Both of the recombinants appeared to be identical to native rat SP-D in terms of the size and extent of oligomerization. rSP-D behaved essentially the same as native rat SP-D in the interactions with carbohydrates and lipids. The affinity of SP-D for PI was significantly reduced compared with that of rSP-D, and the GlcCer binding property was completely ablated in the mutant. The carbohydrate binding specificity of the mutant was altered to a galactose type from a mannose-glucose type. Glu-321 and Asn-323 are therefore essential for the GlcCer binding property of SP-D, but are not absolutely required for PI binding.


FOOTNOTES

*
This work 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; E-mail: voelkerd@njc.org.

The abbreviations used are: SP-D, surfactant protein D; rSP-D, recombinant SP-D; SP-A, surfactant protein A; CRD, carbohydrate recognition domain; PI, phosphatidylinositol; GlcCer, glucosylceramide; DPPC, dipalmitoylphosphatidylcholine; GalCer, galactosylceramide; MBP, mannose-binding protein; TBS, Tris-buffered saline; GMEM; Glascow minimum essential medium; PG, phosphatidylglycerol; PS, phosphatidylserine; GalDAG, galactosyldiacylglycerol; GalDAG, digalactosyldiacylglycerol; PAGE, polyacrylamide gel electrophoresis; G, IlNeuAc-GgOseCer.


ACKNOWLEDGEMENTS

We thank Peggy Hammond for excellent secretarial assistance.


REFERENCES
  1. Persson, A., Rust, K., Chang, D., Moxley, M., Longmore, W., and Crouch, E.(1988) Biochemistry27, 8576-8584 [Medline] [Order article via Infotrieve]
  2. Persson, A., Chang, D., Rust, K., Moxley, M., Longmore, W., and Crouch, E.(1989) Biochemistry28, 6361-6367 [Medline] [Order article via Infotrieve]
  3. Shimizu, H., Fisher, J. H., Papst, P., Benson, B., Lau, K., Mason, R. J., and Voelker, D. R.(1992) J. Biol. Chem.267, 1853-1857 [Abstract/Free Full Text]
  4. Crouch, E., Persson, A., Chang, D., and Heuser, J.(1994) J. Biol. Chem.269, 17311-17319 [Abstract/Free Full Text]
  5. Holmskov, U., Teisner, B., Willis, A. C., Reid, K. B. M., and Jensenius, J. C.(1993) J. Biol. Chem.268, 10120-10125 [Abstract/Free Full Text]
  6. Kuroki, Y., Mason, R. J., and Voelker, D. R.(1988) J. Biol. Chem.263, 3388-3394 [Abstract/Free Full Text]
  7. Rice, W. R., Ross, G. F., Singleton, F. M., Dingle, S., and Whitsett, J. A.(1987) J. Appl. Physiol.63, 692-698 [Abstract/Free Full Text]
  8. Wright, J. R., Wager, R. E., Hawgood, S., Dobbs, L., and Clements, J. A.(1987) J. Biol. Chem.262, 2888-2894 [Abstract/Free Full Text]
  9. van Iwaarden, J. F., Shimizu, H., Van Golde, P. H. M., Voelker, D. R., and Van Golde, L. M. G.(1992) Biochem. J.286, 5-8 [Medline] [Order article via Infotrieve]
  10. Kuan, S. F., Rust, K., and Crouch, E.(1992) J. Clin. Invest.90, 97-106 [Medline] [Order article via Infotrieve]
  11. Hartshorn, K. L., Crouch, E., White, M. R., Eggleton, P., Tauber, A. I., Chang, D., and Sastry, K.(1994) J. Clin. Invest.94, 311-319 [Medline] [Order article via Infotrieve]
  12. Kuan, S. F., Persson, A., Parghi, D., and Crouch, E.(1994) Am. J. Respir. Cell Mol. Biol.10, 430-436 [Abstract]
  13. Miyamura, K., Leigh, L. E. A., Lu, J., Hopkin, J., Lopez Bernal, A., and Reid, K. B. M.(1994) Biochem. J.300, 237-242 [Medline] [Order article via Infotrieve]
  14. Ogasawara, Y., Kuroki, Y., and Akino, T.(1992) J. Biol. Chem.267, 21244-21249 [Abstract/Free Full Text]
  15. Persson, A. V., Gibbons, B. J., Shoemaker, J. D., Moxley, M. A., and Longmore, W. J.(1992) Biochemistry31, 12183-12189 [Medline] [Order article via Infotrieve]
  16. Kuroki, Y., Gasa, S., Ogasawara, Y., Shiratori, M., Makita, A., and Akino, T.(1992) Biochem. Biophys. Res. Commun.187, 963-969 [Medline] [Order article via Infotrieve]
  17. Kuroki, Y., Shiratori, M., Murata, Y., and Akino, T.(1991) Biochem. J.279, 115-119 [Medline] [Order article via Infotrieve]
  18. Ogasawara, Y., McCormack, F. X., Mason, R. J., and Voelker, D. R. (1994) J. Biol. Chem.269, 29785-29792 [Abstract/Free Full Text]
  19. Kuroki, Y., McCormack, F. X., Ogasawara, Y., Mason, R. J., and Voelker, D. R.(1994) J. Biol. Chem.269, 29793-29800 [Abstract/Free Full Text]
  20. McCormack, F. X., Kuroki, Y., Stewart, J. J., Mason, R. J., and Voelker, D. R.(1994) J. Biol. Chem.269, 29801-29807 [Abstract/Free Full Text]
  21. Kuroki, Y., and Akino, T.(1991) J. Biol. Chem.266, 3068-3073 [Abstract/Free Full Text]
  22. Kuroki, Y., Gasa, S., Ogasawara, Y., Makita, A., and Akino, T.(1992) Arch. Biochem. Biophys.299, 261-267 [Medline] [Order article via Infotrieve]
  23. Childs, R. A., Wright, J. R., Ross, G. F., Yuen, C.-T., Lawson, A. M., Chai, W., Drickamer, K., and Feizi, T.(1992) J. Biol. Chem.267, 9972-9979 [Abstract/Free Full Text]
  24. Drickamer, K.(1992) Nature360, 183-186 [CrossRef][Medline] [Order article via Infotrieve]
  25. Weis, W. I., Drickamer, K., and Hendrickson, W. A.(1992) Nature360, 127-134 [CrossRef][Medline] [Order article via Infotrieve]
  26. Ausbel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, L. G., Smith, J. A., and Struhl, K.(1992) Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York
  27. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59 [CrossRef][Medline] [Order article via Infotrieve]
  28. Sanger, F., Nicklen, S., and Coulson, A. R.(1977) Proc. Natl. Acad. Sci. U. S. A.74, 5463-5467 [Abstract]
  29. Harlow, E., and Lane, D.(1988) in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  30. Laemmli, U. K.(1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  31. Towbin, H., Staehelin, T., and Gordon, J.(1979) Proc. Natl. Acad. Sci. U. S. A.76, 4350-4353 [Abstract]
  32. Persson, A., Chang, D., and Crouch, E.(1990) J. Biol. Chem.265, 5755-5760 [Abstract/Free Full Text]
  33. Crouch, E., Chang, D., Rust, K., Persson, A., and Heuser, J.(1994) J. Biol. Chem.269, 15808-15813 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.