Glycoprotein-340 Binds Surfactant Protein-A (SP-A)

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Glycoprotein-340 Binds Surfactant Protein-A (SP-A) and Stimulates Alveolar Macrophage Migration in an SP-A–Independent Manner Michael James Tino and Jo Rae Wright Department of Cell Biology, Duke University Medical Center, Durham, North Carolina

Glycoprotein-340 (gp-340) was first identified as a surfactant protein (SP)-DÐbinding molecule purified from lung lavage of patients with alveolar proteinosis (Holmskov, et al., J. Biol. Chem. 1997;272:13743). In purifying SP-A from proteinosis lavage, we isolated a protein that copurifies with SP-A and SP-D and that was later found by protein sequencing to be gp-340. We have shown that soluble gp-340 binds SP-A in a calcium-dependent manner independent of the lectin activity of SP-A. To examine the functional significance of this interaction, we tested the ability of soluble gp-340 to block SP-A binding to and stimulation of the chemotaxis of alveolar macrophages. We found that gp-340 does not affect the binding of SP-A to alveolar macrophages over a wide range of SP-A concentrations, nor does it inhibit the ability of SP-A to stimulate macrophage chemotaxis. We also found that gp-340 alone stimulates the random migration (chemokinesis) of alveolar macrophages in a manner independent of SP-AÐstimulated chemotaxis. These results suggest that gp-340 is not a cell-surface receptor necessary for SP-A stimulation of chemotaxis, and show that gp-340 can directly affect macrophage function. Tino, M. J., and J. R. Wright. 1999. Glyco- protein-340 binds surfactant protein-A (SP-A) and stimulates alveolar macrophage migration in an SP-A–independent manner. Am. J. Respir. Cell Mol. Biol. 20:759–768.

Glycoprotein-340 (gp-340), first identified by Holmskov proteins of unknown function produced in the intestinal and colleagues (1) as a surfactant protein (SP)-D–binding crypts and taste buds, respectively. protein in lung lavage, is a large, highly glycosylated pro- SP-A, the most abundant of the pulmonary surfactant tein that migrates on sodium dodecyl sulfate–polyacryla- proteins, has been implicated in various pathways of pul- mide gel electrophoresis (SDS–PAGE) gels as a doublet, monary host defense through its interaction with alveolar and on gel filtration columns as a complex apparently macrophages. Among the effects of this interaction are the larger than 1,000 kD. It belongs to a family of proteins stimulation of phagocytosis of bacteria (2–4), the produc- with homology to the scavenger receptor, all of which con- tion of reactive oxygen (5, 6), the regulation of cytokine tain scavenger receptor cysteine-rich (SRCR) domains. production (7, 8), the directional polymerization of actin Several subsets of this family exist; the proteins in this (9), and the stimulation of directional cell migration (che- family that are most homologous to the published se- motaxis) (10). quences of gp-340 include CRP-ductin and ebnerin (1), Several proteins have been identified that bind SP-A, including a receptor for complement protein C1q (11) and a protein named the 210-kD SP-A receptor (12). Previous studies have suggested that these proteins play a role in (Received in original form June 1, 1998 and in revised form September 8, 1998) mediating several functions of SP-A, including the stimu- Address correspondence to: Dr. Jo Rae Wright, Box 3709, Dept. of Cell lation of phagocytosis by peripheral blood monocytes (13) Biology, Duke University Medical Center, Durham, NC 27710. E-mail: and bone marrow–derived macrophages (3), and the inhi- [email protected] bition of phospholipid secretion in phorbol ester–stimu-

Abbreviations: absorbance at 492 nm, A492; analysis of variance, ANOVA; lated alveolar epithelial type II cells (12). The precise role alveolar proteinosis, AP; human alveolar proteinosis, AP–SP-A; bovine of these receptors in regulating macrophage chemotaxis is serum albumin, BSA; Dulbecco’s phosphate-buffered saline, DPBS; eth- not clear. ylenediaminetetraacetic acid, EDTA; ethyleneglycol-bis-(␤-aminoethyl ether)-N,NЈ-tetraacetic acid, EGTA; Gey’s balanced salt solution, GBSS; In our ongoing studies of SP-A–binding proteins, we glycoprotein-340, gp-340; horseradish peroxidase, HRP; dissociation con- identified gp-340 as a protein that co-isolates with SP-A stant, Kd; oil immersion fields, OIF; recombinant rat surfactant protein-D, and SP-D from an ethylenediaminetetraacetic acid (EDTA) rSP-D; sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS– extract of lung lavage from patients with alveolar pro- PAGE; surfactant protein, SP; scavenger receptor cysteine-rich, SRCR; Tris-buffered saline, TBS; Tris-buffered saline ϩ 0.05% Tween-20 and 2 teinosis (AP). Our goals in this study were to determine mM CaCl2, TBS-C; Tris-buffered water (5 mM Tris), TBW. whether gp-340 binds SP-A, to characterize the effect of Am. J. Respir. Cell Mol. Biol. Vol. 20, pp. 759–768, 1999 gp-340 on SP-A binding to and stimulation of alveolar Internet address: www.atsjournals.org macrophages, and to attempt to determine the function of

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gp-340 in an environment in which SP-A is the predomi- Fast Flow column (Pharmacia, Uppsala, Sweden) eluted nant protein. We have focused our attention on the role of with a gradient of 0 to 1 M NaCl in solution A. The gp- gp-340 in the stimulation of chemotaxis. The results show 340–containing fractions were pooled, diluted 1:10 with that gp-340 itself is a potent stimulator of alveolar mac- solution A, and applied to a 1-ml Source 15Q anion ex- rophage migration, although this stimulation is consistent change column (Pharmacia). Proteins were eluted with so- with the stimulation of random migration (chemokinesis) lution A, containing various concentrations of NaCl in four and not directed migration (chemotaxis). In addition, steps: a 0 to 0.5 M gradient, a 0.5 M wash, a 0.5 to 1 M gra- whereas gp-340 binds SP-A in a calcium-dependent and dient, and a 1 M wash. gp-340–containing fractions were carbohydrate-independent manner, SP-A stimulates chemo- pooled, concentrated on a Centriplus-100 (Amicon, Bev- taxis in the presence of gp-340, and gp-340 has no effect on erly, MA), and separated on a Superose-6 FPLC sizing SP-A binding to alveolar macrophages over a wide con- column using modified solution A (with no added deter- centration range. gent). gp-340–containing fractions were pooled and dia- A preliminary report of this study has been published lyzed into DPBS, pH 7.4, without calcium or magnesium. as an abstract (14). Protein content was quantified using the BCA reagent (Pierce Chemical Co., Rockford, IL). Materials and Methods Anti–gp-340 Antibody Media and Chemicals Polyclonal antibodies were prepared by Bio-Synthesis, Dulbecco’s phosphate-buffered saline (DPBS) and Gey’s Inc. (Lewisville, TX) against a peptide of gp-340 consisting balanced salt solution (GBSS) were from GIBCO-BRL of the following amino acid sequence: NH2-GCKQLGCG- (Grand Island, NY). All other chemicals (except as noted) WATSAPGNAR-COOH. The serum reacted specifically were from Sigma Chemical Co. (St. Louis, MO). Bovine with purified gp-340 and proteins of similar size in samples serum albumin (BSA; also from Sigma) for buffers in con- of human lavage. The antibody was used at a dilution of tact with isolated cells was from Fraction V, fatty acid- 1:1,000 for Western blots. free, Ͻ 0.1 ng/mg endotoxin, and cell-culture tested. Isolation of Human gp-340 Isolation of Human AP SP-A (AP–SP-A) gp-340 was initially isolated from a human AP lavage pellet, SP-A was purified from the bronchoalveolar lavage fluid using the method of Suwabe and associates for the purifi- of patients with AP via butanol extraction as previously cation of SP-A without the use of butanol extraction (15). described (16). SP-A was stored in 5 mM Tris, pH 7.4 In this method, lung lavage from patients with AP was al- (Tris-buffered water [TBW]), at Ϫ20ЊC. SP-A prepara- lowed to settle overnight, and the resulting pellet was re- tions were tested for the presence of bacterial endotoxin suspended in a minimal amount of deionized water and using a Limulus amebocyte lysate assay (Bio-Whittaker, stored in aliquots at Ϫ20ЊC. Thawed aliquots were homog- Walkersville, MD); all SP-A preparations used contained enized with a Dounce homogenizer in Tris-buffered saline no detectable endotoxin (Ͻ 0.25 EU/ml, or, depending on (TBS; 50 mM Tris, pH 7.4, and 150 mM NaCl) containing the exact concentration of SP-A in each preparation, Ͻ 0.006–0.04 pg endotoxin/␮g SP-A). 1 mM CaCl2, and centrifuged at 100,000 ϫ g in a swinging bucket rotor for 30 min at 10ЊC. The supernatant was dis- carded, and the pellet was rehomogenized and washed Purification of Recombinant Rat Surfactant Protein-D four times by centrifugation. The calcium-extracted pellet The full-length rat SP-D (rSP-D) complementary DNA was then homogenized in TBS containing 1 mM MgCl2 was provided by Dr. J. H. Fisher, and has been previously and 2 mM ethyleneglycol-bis-(␤-aminoethyl ether)-N,NЈ- described by Shimizu and coworkers (17). This construct tetraacetic acid (EGTA), and spun as above. The superna- was subcloned into the pEE14 vector, and the construct tant (containing SP-A, SP-D, and gp-340) was applied to a was transfected into CHO K1 cells (American Type Cul- Biogel A5 (Bio-Rad, Hercules, CA) gel filtration column ture Collection, Rockville, MD). Consequent selection, (1.5 ϫ 170 cm) and eluted at a flow rate of 4.8 ml/h with production of a single SP-D–producing subclone, and pro- buffer containing 5 mM Tris (pH 7.4), 50 mM NaCl, and duction of rSP-D were done via a slight modification of 2 mM EGTA. gp-340–containing fractions were pooled the procedures of Crouch and associates (18). Changes to and dialyzed into DPBS, pH 7.4, without calcium or mag- this protocol were as follows: Transfected cells were origi- nesium. nally grown in ␣ minimal essential medium containing 10% Subsequent isolation of gp-340 was done via a slight dialyzed fetal bovine serum (both from GIBCO-BRL); modification of the method of Holmskov and coworkers rSP-D was purified from serum-free HB-CHO medium (1). Briefly, the lavage from a patient with AP was spun at (Irvine Scientific, Santa Ana, CA) containing 50 ␮g/ml 10,000 ϫ g and the resulting pellet was extracted overnight ascorbate and 500 ␮M methionine sulfoximine. Briefly, with a buffer containing 50 mM Tris (pH 7.5), 150 mM rSP-D–containing medium was dialyzed against maltosyl- NaCl, 10 mM EDTA, and 0.05% emulphogene (polyoxy- Sepharose loading buffer (2 mM Tris [pH 7.8], 5 mM CaCl2, ethylene 10-tridecyl ether). The 10,000 ϫ g supernatant and 100 mM NaCl) and applied to a maltose-Sepharose col- from the extraction was dialyzed into 10 mM Tris (pH 7.5), umn. rSP-D was eluted using buffer containing 5 mM Tris 10 mM EDTA, and 0.05% emulphogene (solution A). The (pH 7.8), 2 mM EDTA, and 100 mM NaCl (maltose elu- protein was then purified using a three-column protocol. tion buffer). rSP-D–containing fractions were dialyzed The first purification column was on a 30-ml Q-Sepharose into DPBS without calcium, pH 7.2, and stored at 4ЊC.

Tino and Wright: Glycoprotein-340 Stimulates Alveolar Macrophage Migration 761

Iodination of SP-A at 4ЊC with rotation. SP-A–Sepharose beads and bound Human AP–SP-A was labeled with 125I using Iodo Beads proteins were pelleted in a microfuge at maximum speed (Pierce) and Na125I (DuPont–NEN, Boston, MA). In pre- for 1 min, and resuspended in TBS containing 1 mM CaCl2 vious studies, SP-A labeled in a similar manner retained to wash. The SP-A–Sepharose was washed three times the ability to stimulate lipid uptake by isolated lung cells with Ca-containing buffer, and then resuspended in TBS (19). Two Iodo Beads were washed with TBW, added to containing 2 mM EDTA. Beads were once again pelleted 1 mCi Na125I with 25 ␮l TBW, and allowed to react for 5 min. and the EDTA extraction was repeated. Thirty microli- A total of 500 ␮g of SP-A was added and allowed to react ters of the supernatant from each of the washes and elu- for 12 min. The sample was desalted using an Exocellulose tions was separated on 6% SDS–PAGE under nonreduc- GF-5 column (Pierce) in TBW. Samples from each frac- ing conditions; proteins were visualized by silver stain. tion were precipitated with 10% trichloroacetic acid (TCA) and 400 ␮g BSA as a carrier, and pellets and supernatants Microtiter Plate SP-A and SP-D Binding Assay were counted using a ␥-counter. Fractions with Ͼ 90% Immulon-2 96-well microtiter plate wells were coated TCA-precipitable counts were pooled and assayed for pro- overnight at 4ЊC in 0.1 M sodium bicarbonate, pH 10, with tein concentration using the BCA protein assay (Pierce). either human gp-340 (300 ng/well), human AP–SP-A (as a Iodinated protein was stored at 4ЊC and used within 2 wk positive control for SP-D binding, at 100 ng/well [20]), rab- of iodination. The specific activity was 0.12 ␮Ci/␮g. bit skeletal muscle myosin (as a positive control for SP-A binding, at 550 ng/well [21]), or nothing (BSA-only wells). Biotinylation of SP-A and rSP-D Wells were washed twice with TBS containing 0.05% Tween- 20 and 2 mM CaCl (TBS-C) and blocked for 2 h at room SP-A was prepared for biotinylation via the butanol ex- 2 temperature with 1 mg BSA/ml in TBS-C. Wells were then traction method described previously, with the exception washed twice with either TBS-C or TBS containing 0.05% that the final dialysis was into 5 mM N-2-hydroxyethylpip- Tween-20 and 2 mM EDTA (TBS-E), and incubated over- erazine-NЈ-ethane sulfonic acid, pH 6.5. Sulfo-NHS-Biotin night at 4ЊC with a 1:200 dilution of biotinylated SP-A or (Pierce) was added to a 2:1 weight ratio, and the mixture SP-D in either TBS-C or TBS-E, in some wells in the pres- was incubated 20 min at room temperature with gentle ro- ence of 100 mM maltose or mannose, or with BSA (1 mg/ tation. The reaction was stopped by adding glutamine to a ml) in TBS-C (as a negative control for the secondary in- final concentration of 20 mM (to react with free NHS- cubation step). Wells were then washed three times with biotin) and incubating the resulting mixture on ice for 10 TBS-C and incubated 4.5 h at room temperature with a min. The reaction was dialyzed against TBW (2 liters ϫ 1:500 dilution of biotinylated streptavidin-HRP complex three changes) to remove excess biotin and glutamine. To in TBS-C containing 1 mg BSA/ml. Wells were subse- confirm biotinylation, the proteins were resolved by SDS– quently washed three times with TBS-C, and color was de- PAGE, transferred to nitrocellulose, and detected by blot- veloped using 100 ␮l of a solution of H O and o-phe- ting with streptavidin-biotinylated horseradish peroxidase 2 2 nylenediamine dihydrochloride (Pierce) in 0.1 M citric acid, (HRP) (Amersham, Little Chalfont, Buckinghamshire, UK). adjusted to pH 5 with saturated dibasic sodium phosphate. rSP-D was biotinylated using the same method, with the The color reaction was stopped using 50 ␮l of 4 N H SO , exception that SP-D was stored in and dialyzed into DPBS 2 4 and the color intensity was measured in a microtiter plate without calcium, pH 7.2. spectrophotometer at 492 nm.

SP-A Coupling to Sepharose 4B Animals SP-A–coupled Sepharose 4B was prepared as follows: Male Sprague–Dawley rats (200 to 250 g) were obtained CNBr-activated Sepharose 4B (Pharmacia) was reconsti- from Charles River (Raleigh, NC). tuted in 1 mM HCl and allowed to rehydrate for 15 min. The swollen, activated gel was washed thoroughly with Isolation of Rat Alveolar Macrophages 1 mM HCl and then with 0.01 M sodium bicarbonate, pH Rat alveolar macrophages were isolated as previously de- 8.3 (coupling buffer). The beads were then resuspended in scribed (16). Briefly, rat lungs were lavaged six times with coupling buffer containing 2.5 mg SP-A per gram of DPBS, pH 7.2, containing 0.2 mM EGTA, and then twice Sepharose, and incubated overnight at 4ЊC on a rotator. with DPBS, pH 7.2, containing 1 mM CaCl2. Cells were The next day, the SP-A–Sepharose was washed and incu- pelleted at 228 ϫ g and resuspended in the appropriate bated for 2 h with 0.2 M glycine, pH 8, to block uncoupled medium. Alveolar macrophages constituted Ͼ 95% of the sites on the beads. The beads were then washed thor- cells obtained as measured by differential staining with the oughly with 0.2 M glycine, rinsed with coupling buffer, and Hemacolor differential blood stain kit (EM Diagnostic washed thoroughly with TBW containing 0.02% sodium Systems, Gibbstown, NJ). azide. The gel was stored at 4ЊC as a 50% slurry. Binding of SP-A to Alveolar Macrophages Binding of gp-340 to SP-A–Sepharose SP-A binding to alveolar macrophages was measured as A total of 16 ␮g gp-340 was added to 0.5 ml of SP-A–cou- previously described for SP-D binding assays (22). Briefly, pled Sepharose (or, as a control, Sepharose prepared in tubes were blocked with 1% BSA in DPBS overnight at parallel without the addition of SP-A) in a total volume of 4ЊC, and 2.5 ϫ 106 alveolar macrophages in DPBS contain- 1 ml TBS containing 1 mM CaCl2 and incubated overnight ing 1% BSA were added to tubes containing 0.13, 1.3, or

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13 ␮g 125I-labeled SP-A/ml, in the presence of either 1 mM for normal distribution, and values were considered signif- CaCl2 or 10 mM EDTA, and with or without 1.3 ␮g gp- icant when P Ͻ 0.05. 340/ml, in a total volume of 500 ␮l. This mixture was incubated at 4ЊC for 4 h with rotation. Cells were pelleted at 250 ϫ g for 10 min in a refrigerated microcentrifuge, and Results 2ϩ resuspended in DPBS containing either Ca or EDTA A Large Protein Doublet that Co-Isolates (as appropriate) to wash. This was repeated twice, and the with SP-A and SP-D Is gp-340 cell suspension was transferred to new microfuge tubes after the second wash. The final cell pellets were resus- The treatment of the surfactant pellet from the lung lavage pended in 200 ␮l cold lysis buffer (0.1 M sodium phos- of AP patients with EGTA and magnesium releases pro- phate, 30 mM NaCl, 1% NP-40, and 4 mM EDTA), and teins bound to the AP pellet in a calcium-dependent man- this mixture was incubated overnight at 4ЊC. A total of 150 ner, including a large amount of SP-A. This property of ␮l of lysate was used for ␥ counting, and 1 ␮l for a micro- SP-A was employed by Suwabe and coworkers (15) to de- titer plate BCA protein assay (Pierce). No cell control velop a purification protocol for SP-A that does not use counts, which averaged approximately 4% of the signal detergents or organic solvents. In purifying SP-A by this obtained with cells, were subtracted from each sample, protocol, the three major proteins that were visible in and corrected counts were normalized to total cell protein Coomassie blue–stained SDS–PAGE separations of the present. sizing column eluate were SP-A, SP-D, and a large protein that barely entered a 15% polyacrylamide gel during elec- Cell Migration Assay trophoresis (Figure 1A). Fractions containing this protein, Directed migration (chemotaxis) and random migration which eluted at fraction number 45, were further analyzed (chemokinesis) of cells were measured using a modified using 6% SDS–PAGE in the presence and absence of the Boyden chamber, as previously described (10). Briefly, 50 reducing agent dithiothreitol (DTT) (Figure 1B), and visu- ␮l of alveolar macrophages, suspended at 2.5 ϫ 106 cells/ alized by silver stain. The lower-percentage gel made it ml in GBSS containing 0.1% BSA, in the presence or ab- clear that the large protein was a doublet with an apparent sence of gp-340, were placed in the upper wells of a 48-well molecular weight of greater than or equal to 300 kD. Fur- microchemotaxis chamber (Neuro Probe, Cabin John, thermore, the large protein appeared to have the unusual MD), in the presence or absence of additional protein. characteristic of having a faster mobility in its unreduced The lower chambers contained 28 ␮l of test solution, con- state, consistent with the possibility that it contains at least sisting of GBSS containing 0.1% BSA and either nothing, one intrachain disulfide bond. varying amounts of SP-A and/or gp-340, equivalent amounts of their storage buffers, or 10% zymosan-activated serum Microsequence Analysis of the Isolated Protein (prepared according to the method of Snyderman and Pike Results from microsequence analysis of the upper band of [23] as a positive control). All test solutions were used in the doublet are shown in Figure 2. The upper band of the triplicate in each assay. A polyvinylpyrrolidone-free poly- unknown protein doublet was excised from a 15% SDS– carbonate filter with 5-␮m pores (Poretics, Livermore, polyacrylamide gel and sent to the Harvard Microchemis- CA) was placed between the wells, along with the assem- try Laboratory (Cambridge, MA) for analysis. The whole bly’s rubber gasket. The chamber was incubated at 37ЊC protein was digested with trypsin and separated by high- with 5% CO2 for 2 h, then disassembled. Nonmigrating performance liquid chromatography; two peptides from cells were scraped from the upper surface, and the migrat- this separation were chosen for microsequence analysis. ing cells were stained using the Hemacolor stain kit. The The sequences of the peptides were as follows: peptide 1, filter was placed on a glass coverslip and mounted with im- VEVLYR; peptide 2, QLGCWATSAPGNAR. Both pep- mersion oil onto a glass slide. Cells that migrated through tides match the consensus sequence for SRCR domains the filter were counted in 10 randomly selected oil immer- from bovine gall bladder mucin (24), mouse CRP-ductin sion fields (OIF) in each well at ϫ1,000 magnification. (25), and rat ebnerin (26), as well as sequences for seven Data, expressed as cells per OIF for the three wells used peptides derived from gp-340 (1). In aligning these pep- for each solution, were either converted to a percentage of tides with those from gp-340, only one amino acid was the negative (no added protein) control (percentage of noted to be different (the P residue at position 10 in pep- control migration) or normalized to an experimental treat- tide 2); this residue is completely conserved in SRCR do- ment group (relative migration), as specified. mains in this subfamily, and likely to be a proline in gp-340 as well (1). On the basis of this information, as well as the Statistical Analysis purification of the large molecular-weight doublet from When the response of cells to different treatments was ex- AP lavage and its unusual electrophoretic mobilities when pressed as percentage of the control response, the data reduced and unreduced, we concluded that the isolated were analyzed using a multiplicative model that results in protein was gp-340. The results of all further studies were a value ␦ (where the control value, Po, is related to the confirmed, with both this protein and gp-340 purified ac- treatment value, Po␦, by this factor) which, when multi- cording to the method of Holmskov and coworkers (1). A plied by 100, is equivalent to the percentage of the control silver-stained SDS–PAGE gel and a Western blot analysis response. These ␦ values were then analyzed using analysis of the purified gp-340 are shown in Figure 3. No contami- of variance (ANOVA) and t tests with the null hypothesis nating proteins were detectable by silver stain. In addition, that ␦ ϭ 1 (or 100% of control). Data were also analyzed neither SP-A nor SP-D could be detected by Western blot Tino and Wright: Glycoprotein-340 Stimulates Alveolar Macrophage Migration 763

In addition, gp-340 immobilized on a microtiter plate binds both SP-A and SP-D in a calcium-dependent manner (Table 1). Biotinylated SP-A and SP-D were incubated in the presence of calcium or EDTA; wells were subsequently probed with a biotinylated streptavidin-HRP complex and developed and measured as detailed in MA- TERIALS AND METHODS. SP-A clearly bound to the immobilized gp-340 in the presence of calcium: The absorbance at 492 nm of the resulting solution (A492) was increased 5-fold over the absorbance that resulted from background binding (binding of SP-A to wells treated only with BSA to block). No SP-A bound to gp-340 in the presence of EDTA, resulting in an A492 approximately equal to that of background binding. In addition, in this assay SP-D bound to gp-340 in a calcium-dependent manner, confirming the ob- servation of Holmskov and associates (1).

gp-340 Binding to SP-A Is Independent of the Lectin Activity of SP-A Neither 100 mM mannose, which binds the carbohydrate recognition domain of SP-A, nor 100 mM maltose, which does not bind SP-A with high affinity, blocks the binding of SP-A to gp-340 (Figure 5). Indeed, binding of SP-A to gp-340 in the microtiter plate assay described above was significantly enhanced (P Ͻ 0.05) in the presence of either maltose or mannose and Ca2ϩ. Binding of biotinylated SP-A to gp-340 in the absence of competing sugars corre- lated to an A492 of 1.00 Ϯ 0.08 in these assays; in the presence of 100 mM mannose, the A492 of the final colorimet- ric reaction was increased by 80% and in the presence of 100 mM maltose it was increased by 125% over values without sugars. When biotinylated SP-A was exposed to gp-340 in the presence of both mannose and EDTA, the Figure 1. A large protein doublet co-isolates with SP-A and SP-D. (A) Proteins were extracted from human AP lavage A492 of the reaction was decreased to a level not distin- through the nonbutanol extraction method and applied to a Bio- guishable from either binding to gp-340 alone in the pres- Gel A5 sizing column as detailed in MATERIALS AND METHODS. ence of EDTA or background binding (without gp-340 on Fractions, 2.5 ml, were collected, and aliquots of each fraction the plate). were separated by 15% SDS–PAGE under reducing conditions. Gels were stained with Coomassie blue, destained, and dried. gp-340 Has No Effect on the Binding of Fractions 44–54 are shown, and bands corresponding to SP-A and SP-A to Alveolar Macrophages SP-D are labeled accordingly. A large molecular-weight band that co-isolates with SP-A and SP-D is labeled “unknown.” (B) A 125I–SP-A was incubated with alveolar macrophages at 4ЊC 6% SDS–PAGE gel, silver-stained as described in M ATERIALS for 4 h in the presence and absence of 1.3 ␮g gp-340/ml AND METHODS, of sizing column fraction 45 (F45), human AP– (Figure 6). Because neither the native molecular weight of SP-A (SP-A), and rat SP-D (SP-D) in their unreduced state, and gp-340 nor the stoichiometry of the SP-A–gp-340 interac- fraction 45 after reduction with DTT. tion is known, three different concentrations of SP-A were tested: 0.13 ␮g SP-A/ml (a concentration equal to 0.2 nM SP-A, or one-tenth the published dissociation constant analysis of the purified gp-340 that was used in the func- [Kd] of SP-A binding to alveolar macrophages [27]), 1.3 ␮g tional assays (data not shown). SP-A/ml (2 nM SP-A, its Kd), and 13 ␮g SP-A/ml (20 nM SP-A, or 10 times its Kd). At none of these concentrations gp-340 Binds to SP-A was the binding of SP-A to alveolar macrophages signifi- In two different assays, gp-340 was shown to bind to SP-A cantly different in the presence of gp-340 than in the ab- in a calcium-dependent manner. In the first assay, gp-340 sence of gp-340. In addition, the binding of SP-A at 1.3 ␮g/ was added, in the presence of calcium, to SP-A–coupled ml was examined in the presence or absence of 10 mM Sepharose. The gp-340 bound to the column and was not EDTA. The addition of EDTA eliminated approximately released by repeated washes with calcium-containing 90% of SP-A binding in both the presence and absence of buffer. EDTA-containing buffer released the gp-340 from gp-340; without gp-340, the addition of EDTA reduced the SP-A–Sepharose beads (Figure 4). Without the addi- binding of SP-A at 1.3 ␮g/ml from 266 Ϯ 108 ng SP-A/mg tion of SP-A, gp-340 did not bind to Sepharose prepared cell protein to 21.1 Ϯ 8.2 ng SP-A/mg protein; and in the in parallel with the SP-A–Sepharose (data not shown). presence of gp-340, binding was reduced by EDTA from 764 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 20 1999

Figure 2. Protein sequence analysis of the large protein in fraction 45 reveals it to be gp-340, a protein previously described as an SP-D–binding protein. The large molecular-weight doublet shown in Figure 1 was further purified using anion exchange chromatography, as detailed in MATERIALS AND METHODS. The protein was separated on a 6% SDS–PAGE gel under nonreducing conditions and lightly stained with Coomassie blue. The upper band was cut from the gel and sent to the Harvard Microchemistry Laboratory for microsequencing analysis. Two peptides were chosen for sequencing; their amino acid sequences are denoted F45 sequence 1 and F45 sequence 2. These sequences are aligned with two of the seven sequences (numbers 1 and 7) of human gp-340 (1), as well as sequences from the second SRCR domain of bovine gall bladder mucin (24), the first SRCR domain of murine CRP-ductin (25), the fourth SRCR domain of rat ebnerin (26), and the SRCR consensus sequence derived from these (h ϭ hydrophobic residue).

178 Ϯ 84 ng SP-A/mg protein to 20.4 Ϯ 7.2 ng SP-A/mg ulated cells over a wide range of gp-340 concentrations protein. (Figure 7). At 0.1 ␮g gp-340/ml, alveolar macrophage migration was stimulated to 173% of control (buffer-only) gp-340 Stimulation of Alveolar Macrophage Migration levels. Increasing concentrations of gp-340 stimulated in- Is Concentration-Dependent creasing levels of alveolar macrophage migration: At 1 ␮g Alveolar macrophages stimulated with gp-340 migrate in a gp-340/ml, migration was 331% of control levels (a level microchemotaxis chamber significantly more than unstim- significantly greater than both the control and 0.1 ␮g/ml levels, P Ͻ 0.05), and at 6 ␮g gp-340/ml, migration was increased to 637% of control levels, a level often as high as that of the positive control (10% zymosan-activated serum, data not shown). gp-340 Stimulation of Alveolar Macrophage Migration Is Consistent with Chemokinesis To determine whether this stimulation is due to a directed migratory stimulus (chemotaxis) or an increase in random cell motility (chemokinesis), we tested the ability of gp-340 to stimulate alveolar macrophage migration in the presence and absence of a concentration gradient (Table 2),

Figure 4. gp-340 binds to SP-A coupled to Sepharose 4B. gp-340 was incubated overnight with SP-A–Sepharose in a calcium-con- Figure 3. Silver stain and Western blot of purified gp-340. (A) A taining buffer, as detailed in M ATERIALS AND METHODS. The 6% SDS–PAGE gel, silver-stained as described in M ATERIALS SP-A–Sepharose beads were washed three times with calcium- AND METHODS of unreduced, purified gp-340. (B) Western blot containing buffer and twice with EDTA-containing buffer, and analysis of purified gp-340. Samples were resolved in 6% SDS– aliquots from each wash were separated on 6% SDS–PAGE un- PAGE gels as described in MATERIALS AND METHODS, and trans- der nonreducing conditions and silver-stained. Shown are puri- ferred to nitrocellulose incubated with a 1:1,000 dilution of poly- fied gp-340 (negatively stained because of high protein content), clonal rabbit anti–gp-340 antibody and a 1:10,000 dilution of goat the initial supernatant from the overnight incubation (SUP), sam- antirabbit IgG-HRP conjugated. The reaction was visualized us- ples of each of the three Ca-containing washes (Washes), and ing the ECL detection system. samples of each of the two EDTA elutions (Fractions Eluted). Tino and Wright: Glycoprotein-340 Stimulates Alveolar Macrophage Migration 765

TABLE 1 6 ␮g gp-340/ml, 10 ␮g SP-A/ml stimulates alveolar mac- Binding of biotinylated SP-A and SP-D to gp-340 rophage migration by 34% over the level stimulated by gp- immobilized on microtiter plates 340 alone, a significant increase (P Ͻ 0.05), and a level Protein BSA ϩ Ca2ϩ gp-340 ϩ Ca2ϩ gp-340 ϩ EDTA comparable to the 31% stimulation exhibited by the same concentration of SP-A in the absence of gp-340 (to 49% of SP-A 0.21 Ϯ 0.04 1.01 Ϯ 0.10* 0.16 Ϯ 0.02 the gp-340 control from 18% of control in the buffer-only SP-D 0.06, 0.09 2.57, 2.82 0.36, 0.21 treatment). All four treatment groups (buffer only, SP-A gp-340 or BSA was coated onto Immulon-2 microtiter plates, which were only, gp-340 only, and SP-A ϩ gp-340) were significantly then blocked with BSA and incubated with biotinylated SP-A or SP-D followed different from one another (P Ͻ 0.05 by ANOVA and by biotinylated streptavidin-HRP complex, and developed as described in MA- TERIALS AND METHODS. Data are expressed as the absorbance of the developed two-tailed t tests). reaction at 492 nm. SP-A data are means Ϯ SE for n ϭ 3. *Statistically significant from BSA only, P Ͻ 0.005 by one-way ANOVA; SP-D data are raw data from two experiments. Discussion These results show that gp-340, a protein isolated from lung lavage, binds SP-A in a calcium-dependent manner, and that it stimulates the random migration (chemokine- with gp-340 at a concentration of 6 ␮g/ml in either the bot- sis) of alveolar macrophages in a concentration-dependent tom well alone or both the bottom and top wells. In the manner. The functional consequences of the gp-340–SP-A presence of a concentration gradient (with gp-340 in only interaction are still unclear; gp-340 neither affects the the bottom well), migration was stimulated to 637 Ϯ 166% binding of SP-A to alveolar macrophages at the ratios and of control levels, versus 546 Ϯ 155% of control with gp-340 concentrations tested nor blocks the ability of SP-A to in both wells. Because these figures are not significantly stimulate the chemotaxis of alveolar macrophages, even at different from one another, these data are consistent with relatively low SP-A concentrations. gp-340 stimulating chemokinesis rather than chemotaxis. gp-340 as a Receptor for SP-A and SP-D SP-A Stimulates Chemotaxis of Alveolar Macrophages in the Presence of gp-340 The precise mechanism of SP-A binding to gp-340 has not been elucidated, because several characteristics of gp-340 gp-340 does not affect the stimulation of alveolar macrophage chemotaxis by SP-A (Figure 8). In the presence of

Figure 5. SP-A binding to gp-340 is not inhibited by co-incuba- Figure 6. gp-340 has no effect on SP-A binding to alveolar mac- tion with sugars. gp-340 was coated onto Immulon-2 microtiter rophages. Binding of 125I-labeled SP-A to alveolar macrophages plates, which were then blocked with BSA and incubated with bi- was examined as described in MATERIALS AND METHODS, in the otinylated SP-A alone or in the presence of 100 mM mannose or absence (open circles) or presence (filled diamonds) of 1.3 ␮g gp- maltose, followed by biotinylated streptavidin-HRP complex, 340/ml. Binding was converted to nanograms of SP-A bound per and developed as described in MATERIALS AND METHODS. Data milligram of cell protein, based on the specific activity of the are expressed as the absorbance of the developed reaction at 492 SP-A. Values shown are the means Ϯ SE for n ϭ 3 experiments; nm. SP-A data are means Ϯ SE for at least three experiments none of the values in the presence of gp-340 is significantly differ- (*Statistically significant from gp-340 only, P Ͻ 0.005 by t test). ent from those in the absence of gp-340, as analyzed by ANOVA. 766 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 20 1999

Figure 7. Stimulation of alveolar macrophage migration by gp- 340 is concentration-dependent. Chemotaxis assays were per- formed as described in MATERIALS AND METHODS, with varying concentrations of gp-340 in the bottom well. Data were converted to percentage of the buffer-only control, and are expressed as means Ϯ SE for at least three experiments. *Significantly greater than control; **significantly greater than both control and 0.1 ␮g gp-340/ml treatment, SE too small to appear on graph (SE ϭ Figure 8. Stimulation of alveolar macrophage migration by gp- Ϯ 16% of control). Significance indicates P Ͻ 0.05 by t test. 340 is independent of stimulation of chemotaxis by SP-A. Chemotaxis assays were conducted as described in M ATERIALS AND METHODS, in the presence or absence of 10 ␮g SP-A/ml and/ could mediate its binding to SP-A. For example, gp-340 is or 6 ␮g gp-340/ml. Raw data were normalized to a multiplicative scale, with the gp-340–only wells assigned the value of 1. Data are a highly glycosylated molecule. N-glycosidase treatment of expressed as means Ϯ SE for n ϭ 4 experiments. *Significantly gp-340 results in a protein with an apparent electro- greater than buffer-only control; **significantly different from phoretic molecular weight of approximately 300 kD (1). control, SP-A–only, and gp-340 ϩ SP-A treatments; ***signifi- Holmskov and colleagues (1) demonstrated that the bind- cantly greater than control, SP-A–only, and gp-340–only treat- ing of gp-340 to SP-D was not inhibited in the presence of ments (P Ͻ 0.05 by ANOVA and two-tailed t test). maltose; we have shown here that neither mannose nor maltose inhibits the binding of SP-A to gp-340, suggesting that neither SP-A nor SP-D bind to oligosaccharides on must be elucidated. Clues to this might be taken from the gp-340. Despite this, the possibilities still exist that gp-340 unique characteristics of proteins that are homolgous to contains sugars that bind to SP-A with a higher affinity gp-340, including ebnerin, CRP-ductin, and a newly identi- than does mannose, and that SP-A and SP-D interact with fied member of this family, DMBT1 (28), deletions in gp-340 in different manners. which are found in many brain-tumor cell lines. Ebnerin In addition, it is unclear whether gp-340 is a membrane- and CRP-ductin appear to share a characteristic unique to associated or secreted protein (or if isoforms of each ex- the SRCR family in that each seems to have several forms, ist). Furthermore, if it is indeed found to be membrane- including at least one that has no apparent transmembrane associated, the nature of its interaction with the membrane domain; in fact, evidence for the existence of membrane- bound forms of either protein is not compelling. Ebnerin has localized by Western blot to both particulate and solu- TABLE 2 ble fractions of von Ebner’s gland cells, leading Li and gp-340 stimulates the migration of alveolar macrophages in Snyder to suggest that two forms exist: one membrane- a manner consistent with chemokinesis and not chemotaxis bound and one soluble and secreted (26). Two forms of gp-340 in Top Well gp-340 in Bottom Well Migration the CRP-ductin messenger RNA were cloned; they appear (␮g/ml) (␮g/ml) (% control) to result from alternative splicing, and the ␤ clone (which 0 0 100 is not complete) has an extra C-terminal portion that has a 0 6 637 Ϯ 166* putative transmembrane domain (the complete ␣ clone 6 6 546 Ϯ 155* has no such domain) (25). DMBT1 appears to encode no transmembrane domain (28). Holmskov and colleagues gp-340 was added to the bottom and/or top (along with alveolar macrophages) wells of a microchemotaxis chamber at the concentrations shown as detailed in (1) suggested that the use of detergent in the initial solubi- MATERIALS AND METHODS. Cells migrating through the filter separating the wells lization of gp-340 indicated that it was membrane-associ- were stained and 10 randomly chosen OIF were counted in each well. Data are ated; evidence presented here suggests that at least some expressed as percentage of control (buffer-only) wells and are means Ϯ SE for at least 4 experiments. gp-340 is liberated from AP lavage by the simple chelation *Significantly greater than control (P Ͻ 0.05 by t test). of calcium ions. Alternatively, the gp-340 could be a mem- Tino and Wright: Glycoprotein-340 Stimulates Alveolar Macrophage Migration 767

brane-associated protein that is liberated from the cell sur- the surface of alveolar macrophages, more precise local- face by proteolytic or other mechanisms. In any case, a se- ization of both gp-340 protein (on cellular and subcellular creted form of gp-340 could be a cofactor for SP-A and/or levels) and message is necessary to understand fully the SP-D binding to cells, though the finding that gp-340 does physiologic functions of gp-340. not affect SP-A binding to alveolar macrophages argues Acknowledgments: The authors thank Daniel Zlogar for the initial identifica- against such a role for SP-A binding. tion of a high molecular-weight co-isolate in preparations of SP-A; Julie Taylor for subsequent purification of SP-A; and Joel Herbein, Omar Quintero, Trista gp-340 as a Secreted Protein in Lung Lavage: Schagat, and Drs. Bill Mariencheck, Qun Dong, Marc Caron, Arturo De Loz- Potential Functions and Interactions anne, Christopher Nicchitta, Yusuf Hannun, and Stephen Young for their criti- cal evaluations of the results and discussion contained in this manuscript. This The finding that gp-340 stimulates chemokinesis of alveo- work was supported by National Institutes of Health grant HL-51134. lar macrophages drives speculation into possible functions for gp-340 in the lung that are diverse yet intriguing. Known References protein stimulators of chemokinesis can be grouped into three main categories: growth factors, such as platelet- 1. Holmskov, U., P. Lawson, B. Teisner, I. Tornøe, A. C. Willis, C. Morgan, K. Koch, and K. B. M. Reid. 1997. 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