Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Glycoprotein-340 (gp-340), first identified by Holmskov and colleagues (1) as a surfactant protein (SP)-D-binding protein in lung lavage, is a large, highly glycosylated protein that migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels as a doublet, and on gel filtration columns as a complex apparently larger than 1,000 kD. It belongs to a family of proteins with homology to the scavenger receptor, all of which contain scavenger receptor cysteine-rich (SRCR) domains. Several subsets of this family exist; the proteins in this family that are most homologous to the published sequences of gp-340 include CRP-ductin and ebnerin (1), proteins of unknown function produced in the intestinal crypts and taste buds, respectively.

SP-A, the most abundant of the pulmonary surfactant proteins, has been implicated in various pathways of pulmonary host defense through its interaction with alveolar macrophages. Among the effects of this interaction are the stimulation of phagocytosis of bacteria (2), the production of reactive oxygen (5, 6), the regulation of cytokine production (7, 8), the directional polymerization of actin (9), and the stimulation of directional cell migration (chemotaxis) (10).

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 mediating several functions of SP-A, including the stimulation of phagocytosis by peripheral blood monocytes (13) and bone marrow-derived macrophages (3), and the inhibition of phospholipid secretion in phorbol ester-stimulated alveolar epithelial type II cells (12). The precise role of these receptors in regulating macrophage chemotaxis is not clear.

In our ongoing studies of SP-A-binding proteins, we identified gp-340 as a protein that co-isolates with SP-A and SP-D from an ethylenediaminetetraacetic acid (EDTA) extract of lung lavage from patients with alveolar proteinosis (AP). Our goals in this study were to determine whether gp-340 binds SP-A, to characterize the effect of gp-340 on SP-A binding to and stimulation of alveolar macrophages, and to attempt to determine the function of gp-340 in an environment in which SP-A is the predominant protein. We have focused our attention on the role of gp-340 in the stimulation of chemotaxis. The results show that gp-340 itself is a potent stimulator of alveolar macrophage migration, although this stimulation is consistent with the stimulation of random migration (chemokinesis) and not directed migration (chemotaxis). In addition, whereas gp-340 binds SP-A in a calcium-dependent and carbohydrate-independent manner, SP-A stimulates chemotaxis in the presence of gp-340, and gp-340 has no effect on SP-A binding to alveolar macrophages over a wide concentration range.

A preliminary report of this study has been published as an abstract (14).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Media and Chemicals

Dulbecco's phosphate-buffered saline (DPBS) and Gey's balanced salt solution (GBSS) were from GIBCO-BRL (Grand Island, NY). All other chemicals (except as noted) were from Sigma Chemical Co. (St. Louis, MO). Bovine serum albumin (BSA; also from Sigma) for buffers in contact with isolated cells was from Fraction V, fatty acid-free, < 0.1 ng/mg endotoxin, and cell-culture tested.

Isolation of Human gp-340

gp-340 was initially isolated from a human AP lavage pellet, using the method of Suwabe and associates for the purification of SP-A without the use of butanol extraction (15). In this method, lung lavage from patients with AP was allowed to settle overnight, and the resulting pellet was resuspended in a minimal amount of deionized water and stored in aliquots at 20°C. Thawed aliquots were homogenized with a Dounce homogenizer in Tris-buffered saline (TBS; 50 mM Tris, pH 7.4, and 150 mM NaCl) containing 1 mM CaCl₂, and centrifuged at 100,000 × g in a swinging bucket rotor for 30 min at 10°C. The supernatant was discarded, and the pellet was rehomogenized and washed four times by centrifugation. The calcium-extracted pellet was then homogenized in TBS containing 1 mM MgCl₂ and 2 mM ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), and spun as above. The supernatant (containing SP-A, SP-D, and gp-340) was applied to a Biogel A5 (Bio-Rad, Hercules, CA) gel filtration column (1.5 × 170 cm) and eluted at a flow rate of 4.8 ml/h with buffer containing 5 mM Tris (pH 7.4), 50 mM NaCl, and 2 mM EGTA. gp-340-containing fractions were pooled and dialyzed into DPBS, pH 7.4, without calcium or magnesium.

Subsequent isolation of gp-340 was done via a slight modification of the method of Holmskov and coworkers (1). Briefly, the lavage from a patient with AP was spun at 10,000 × g and the resulting pellet was extracted overnight with a buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 10 mM EDTA, and 0.05% emulphogene (polyoxyethylene 10-tridecyl ether). The 10,000 × g supernatant from the extraction was dialyzed into 10 mM Tris (pH 7.5), 10 mM EDTA, and 0.05% emulphogene (solution A). The protein was then purified using a three-column protocol. The first purification column was on a 30-ml Q-Sepharose Fast Flow column (Pharmacia, Uppsala, Sweden) eluted with a gradient of 0 to 1 M NaCl in solution A. The gp-340-containing fractions were pooled, diluted 1:10 with solution A, and applied to a 1-ml Source 15Q anion exchange column (Pharmacia). Proteins were eluted with solution A, containing various concentrations of NaCl in four steps: a 0 to 0.5 M gradient, a 0.5 M wash, a 0.5 to 1 M gradient, and a 1 M wash. gp-340-containing fractions were pooled, concentrated on a Centriplus-100 (Amicon, Beverly, MA), and separated on a Superose-6 FPLC sizing column using modified solution A (with no added detergent). gp-340-containing fractions were pooled and dialyzed into DPBS, pH 7.4, without calcium or magnesium. Protein content was quantified using the BCA reagent (Pierce Chemical Co., Rockford, IL).

Anti-gp-340 Antibody

Polyclonal antibodies were prepared by Bio-Synthesis, Inc. (Lewisville, TX) against a peptide of gp-340 consisting of the following amino acid sequence: NH₂-GCKQLGCGWATSAPGNAR-COOH. The serum reacted specifically with purified gp-340 and proteins of similar size in samples of human lavage. The antibody was used at a dilution of 1:1,000 for Western blots.

Isolation of Human AP SP-A (AP-SP-A)

SP-A was purified from the bronchoalveolar lavage fluid of patients with AP via butanol extraction as previously described (16). SP-A was stored in 5 mM Tris, pH 7.4 (Tris-buffered water [TBW]), at 20°C. SP-A preparations were tested for the presence of bacterial endotoxin using a Limulus amebocyte lysate assay (Bio-Whittaker, Walkersville, MD); all SP-A preparations used contained no detectable endotoxin (< 0.25 EU/ml, or, depending on the exact concentration of SP-A in each preparation, < 0.006-0.04 pg endotoxin/µg SP-A).

Purification of Recombinant Rat Surfactant Protein-D

The full-length rat SP-D (rSP-D) complementary DNA was provided by Dr. J. H. Fisher, and has been previously described by Shimizu and coworkers (17). This construct was subcloned into the pEE14 vector, and the construct was transfected into CHO K1 cells (American Type Culture Collection, Rockville, MD). Consequent selection, production of a single SP-D-producing subclone, and production of rSP-D were done via a slight modification of the procedures of Crouch and associates (18). Changes to this protocol were as follows: Transfected cells were originally grown in  minimal essential medium containing 10% dialyzed fetal bovine serum (both from GIBCO-BRL); rSP-D was purified from serum-free HB-CHO medium (Irvine Scientific, Santa Ana, CA) containing 50 µg/ml ascorbate and 500 µM methionine sulfoximine. Briefly, rSP-D-containing medium was dialyzed against maltosyl-Sepharose loading buffer (2 mM Tris [pH 7.8], 5 mM CaCl₂, and 100 mM NaCl) and applied to a maltose-Sepharose column. rSP-D was eluted using buffer containing 5 mM Tris (pH 7.8), 2 mM EDTA, and 100 mM NaCl (maltose elution buffer). rSP-D-containing fractions were dialyzed into DPBS without calcium, pH 7.2, and stored at 4°C.

Iodination of SP-A

Human AP-SP-A was labeled with ¹²⁵I using Iodo Beads (Pierce) and Na¹²⁵I (DuPont-NEN, Boston, MA). In previous studies, SP-A labeled in a similar manner retained the ability to stimulate lipid uptake by isolated lung cells (19). Two Iodo Beads were washed with TBW, added to 1 mCi Na¹²⁵I with 25 µl TBW, and allowed to react for 5 min. A total of 500 µg of SP-A was added and allowed to react for 12 min. The sample was desalted using an Exocellulose GF-5 column (Pierce) in TBW. Samples from each fraction were precipitated with 10% trichloroacetic acid (TCA) and 400 µg BSA as a carrier, and pellets and supernatants were counted using a -counter. Fractions with > 90% TCA-precipitable counts were pooled and assayed for protein concentration using the BCA protein assay (Pierce). Iodinated protein was stored at 4°C and used within 2 wk of iodination. The specific activity was 0.12 µCi/µg.

Biotinylation of SP-A and rSP-D

SP-A was prepared for biotinylation via the butanol extraction method described previously, with the exception that the final dialysis was into 5 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, pH 6.5. Sulfo-NHS-Biotin (Pierce) was added to a 2:1 weight ratio, and the mixture was incubated 20 min at room temperature with gentle rotation. The reaction was stopped by adding glutamine to a final concentration of 20 mM (to react with free NHS- biotin) and incubating the resulting mixture on ice for 10 min. The reaction was dialyzed against TBW (2 liters × three changes) to remove excess biotin and glutamine. To confirm biotinylation, the proteins were resolved by SDS- PAGE, transferred to nitrocellulose, and detected by blotting with streptavidin-biotinylated horseradish peroxidase (HRP) (Amersham, Little Chalfont, Buckinghamshire, UK). rSP-D was biotinylated using the same method, with the exception that SP-D was stored in and dialyzed into DPBS without calcium, pH 7.2.

SP-A Coupling to Sepharose 4B

SP-A-coupled Sepharose 4B was prepared as follows: CNBr-activated Sepharose 4B (Pharmacia) was reconstituted in 1 mM HCl and allowed to rehydrate for 15 min. The swollen, activated gel was washed thoroughly with 1 mM HCl and then with 0.01 M sodium bicarbonate, pH 8.3 (coupling buffer). The beads were then resuspended in coupling buffer containing 2.5 mg SP-A per gram of Sepharose, and incubated overnight at 4°C on a rotator. The next day, the SP-A-Sepharose was washed and incubated for 2 h with 0.2 M glycine, pH 8, to block uncoupled sites on the beads. The beads were then washed thoroughly with 0.2 M glycine, rinsed with coupling buffer, and washed thoroughly with TBW containing 0.02% sodium azide. The gel was stored at 4°C as a 50% slurry.

Binding of gp-340 to SP-A-Sepharose

A total of 16 µg gp-340 was added to 0.5 ml of SP-A-coupled Sepharose (or, as a control, Sepharose prepared in parallel without the addition of SP-A) in a total volume of 1 ml TBS containing 1 mM CaCl₂ and incubated overnight at 4°C with rotation. SP-A-Sepharose beads and bound proteins were pelleted in a microfuge at maximum speed for 1 min, and resuspended in TBS containing 1 mM CaCl₂ to wash. The SP-A-Sepharose was washed three times with Ca-containing buffer, and then resuspended in TBS containing 2 mM EDTA. Beads were once again pelleted and the EDTA extraction was repeated. Thirty microliters of the supernatant from each of the washes and elutions was separated on 6% SDS-PAGE under nonreducing conditions; proteins were visualized by silver stain.

Microtiter Plate SP-A and SP-D Binding Assay

Immulon-2 96-well microtiter plate wells were coated overnight at 4°C in 0.1 M sodium bicarbonate, pH 10, with either human gp-340 (300 ng/well), human AP-SP-A (as a positive control for SP-D binding, at 100 ng/well [20]), rabbit skeletal muscle myosin (as a positive control for SP-A binding, at 550 ng/well [21]), or nothing (BSA-only wells). Wells were washed twice with TBS containing 0.05% Tween-20 and 2 mM CaCl₂ (TBS-C) and blocked for 2 h at room temperature with 1 mg BSA/ml in TBS-C. Wells were then washed twice with either TBS-C or TBS containing 0.05% Tween-20 and 2 mM EDTA (TBS-E), and incubated overnight at 4°C with a 1:200 dilution of biotinylated SP-A or SP-D in either TBS-C or TBS-E, in some wells in the presence of 100 mM maltose or mannose, or with BSA (1 mg/ ml) in TBS-C (as a negative control for the secondary incubation step). Wells were then washed three times with TBS-C and incubated 4.5 h at room temperature with a 1:500 dilution of biotinylated streptavidin-HRP complex in TBS-C containing 1 mg BSA/ml. Wells were subsequently washed three times with TBS-C, and color was developed using 100 µl of a solution of H₂O₂ and o-phenylenediamine dihydrochloride (Pierce) in 0.1 M citric acid, adjusted to pH 5 with saturated dibasic sodium phosphate. The color reaction was stopped using 50 µl of 4 N H₂SO₄, and the color intensity was measured in a microtiter plate spectrophotometer at 492 nm.

Animals

Male Sprague-Dawley rats (200 to 250 g) were obtained from Charles River (Raleigh, NC).

Isolation of Rat Alveolar Macrophages

Rat alveolar macrophages were isolated as previously described (16). Briefly, rat lungs were lavaged six times with DPBS, pH 7.2, containing 0.2 mM EGTA, and then twice with DPBS, pH 7.2, containing 1 mM CaCl₂. Cells were pelleted at 228 × g and resuspended in the appropriate medium. Alveolar macrophages constituted > 95% of the cells obtained as measured by differential staining with the Hemacolor differential blood stain kit (EM Diagnostic Systems, Gibbstown, NJ).

Binding of SP-A to Alveolar Macrophages

SP-A binding to alveolar macrophages was measured as previously described for SP-D binding assays (22). Briefly, tubes were blocked with 1% BSA in DPBS overnight at 4°C, and 2.5 × 10⁶ alveolar macrophages in DPBS containing 1% BSA were added to tubes containing 0.13, 1.3, or 13 µg ¹²⁵I-labeled SP-A/ml, in the presence of either 1 mM CaCl₂ or 10 mM EDTA, and with or without 1.3 µg gp-340/ml, in a total volume of 500 µl. This mixture was incubated at 4°C for 4 h with rotation. Cells were pelleted at 250 × g for 10 min in a refrigerated microcentrifuge, and resuspended in DPBS containing either Ca²⁺ or EDTA (as appropriate) to wash. This was repeated twice, and the cell suspension was transferred to new microfuge tubes after the second wash. The final cell pellets were resuspended in 200 µl cold lysis buffer (0.1 M sodium phosphate, 30 mM NaCl, 1% NP-40, and 4 mM EDTA), and this mixture was incubated overnight at 4°C. A total of 150 µl of lysate was used for  counting, and 1 µl for a microtiter plate BCA protein assay (Pierce). No cell control counts, which averaged approximately 4% of the signal obtained with cells, were subtracted from each sample, and corrected counts were normalized to total cell protein present.

Cell Migration Assay

Directed migration (chemotaxis) and random migration (chemokinesis) of cells were measured using a modified Boyden chamber, as previously described (10). Briefly, 50 µl of alveolar macrophages, suspended at 2.5 × 10⁶ cells/ ml in GBSS containing 0.1% BSA, in the presence or absence of gp-340, were placed in the upper wells of a 48-well microchemotaxis chamber (Neuro Probe, Cabin John, MD), in the presence or absence of additional protein. The lower chambers contained 28 µl of test solution, consisting of GBSS containing 0.1% BSA and either nothing, varying amounts of SP-A and/or gp-340, equivalent amounts of their storage buffers, or 10% zymosan-activated serum (prepared according to the method of Snyderman and Pike [23] as a positive control). All test solutions were used in triplicate in each assay. A polyvinylpyrrolidone-free polycarbonate filter with 5-µm pores (Poretics, Livermore, CA) was placed between the wells, along with the assembly's rubber gasket. The chamber was incubated at 37°C with 5% CO₂ for 2 h, then disassembled. Nonmigrating cells were scraped from the upper surface, and the migrating cells were stained using the Hemacolor stain kit. The filter was placed on a glass coverslip and mounted with immersion oil onto a glass slide. Cells that migrated through the filter were counted in 10 randomly selected oil immersion fields (OIF) in each well at ×1,000 magnification. Data, expressed as cells per OIF for the three wells used for each solution, were either converted to a percentage of the negative (no added protein) control (percentage of control migration) or normalized to an experimental treatment group (relative migration), as specified.

Statistical Analysis

When the response of cells to different treatments was expressed as percentage of the control response, the data were analyzed using a multiplicative model that results in a value  (where the control value, P_o, is related to the treatment value, P_o, by this factor) which, when multiplied by 100, is equivalent to the percentage of the control response. These  values were then analyzed using analysis of variance (ANOVA) and t tests with the null hypothesis that  = 1 (or 100% of control). Data were also analyzed for normal distribution, and values were considered significant when P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

A Large Protein Doublet that Co-Isolates with SP-A and SP-D Is gp-340

The treatment of the surfactant pellet from the lung lavage of AP patients with EGTA and magnesium releases proteins bound to the AP pellet in a calcium-dependent manner, including a large amount of SP-A. This property of SP-A was employed by Suwabe and coworkers (15) to develop a purification protocol for SP-A that does not use detergents or organic solvents. In purifying SP-A by this protocol, the three major proteins that were visible in Coomassie blue-stained SDS-PAGE separations of the sizing column eluate were SP-A, SP-D, and a large protein that barely entered a 15% polyacrylamide gel during electrophoresis (Figure 1A). Fractions containing this protein, which eluted at fraction number 45, were further analyzed using 6% SDS-PAGE in the presence and absence of the reducing agent dithiothreitol (DTT) (Figure 1B), and visualized by silver stain. The lower-percentage gel made it clear that the large protein was a doublet with an apparent molecular weight of greater than or equal to 300 kD. Furthermore, the large protein appeared to have the unusual characteristic of having a faster mobility in its unreduced state, consistent with the possibility that it contains at least one intrachain disulfide bond.

View larger version (35K): [in this window] [in a new window]   Figure 1.   A large protein doublet co-isolates with SP-A and SP-D. (A) Proteins were extracted from human AP lavage through the nonbutanol extraction method and applied to a Bio-Gel A5 sizing column as detailed in MATERIALS AND METHODS. Fractions, 2.5 ml, were collected, and aliquots of each fraction were separated by 15% SDS-PAGE under reducing conditions. Gels were stained with Coomassie blue, destained, and dried. Fractions 44-54 are shown, and bands corresponding to SP-A and SP-D are labeled accordingly. A large molecular-weight band that co-isolates with SP-A and SP-D is labeled "unknown." (B) A 6% SDS-PAGE gel, silver-stained as described in MATERIALS AND METHODS, of sizing column fraction 45 (F45), human AP- SP-A (SP-A), and rat SP-D (SP-D) in their unreduced state, and fraction 45 after reduction with DTT.

Microsequence Analysis of the Isolated Protein

Results from microsequence analysis of the upper band of the doublet are shown in Figure 2. The upper band of the unknown protein doublet was excised from a 15% SDS- polyacrylamide gel and sent to the Harvard Microchemistry Laboratory (Cambridge, MA) for analysis. The whole protein was digested with trypsin and separated by high-performance liquid chromatography; two peptides from this separation were chosen for microsequence analysis. The sequences of the peptides were as follows: peptide 1, VEVLYR; peptide 2, QLGCWATSAPGNAR. Both peptides match the consensus sequence for SRCR domains from bovine gall bladder mucin (24), mouse CRP-ductin (25), and rat ebnerin (26), as well as sequences for seven peptides derived from gp-340 (1). In aligning these peptides with those from gp-340, only one amino acid was noted to be different (the P residue at position 10 in peptide 2); this residue is completely conserved in SRCR domains 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 purification of the large molecular-weight doublet from AP lavage and its unusual electrophoretic mobilities when reduced and unreduced, we concluded that the isolated protein was gp-340. The results of all further studies were confirmed, with both this protein and gp-340 purified according to the method of Holmskov and coworkers (1). A silver-stained SDS-PAGE gel and a Western blot analysis of the purified gp-340 are shown in Figure 3. No contaminating proteins were detectable by silver stain. In addition, neither SP-A nor SP-D could be detected by Western blot analysis of the purified gp-340 that was used in the functional assays (data not shown).

View larger version (21K): [in this window] [in a new window]   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 = hydrophobic residue).

View larger version (35K): [in this window] [in a new window]   Figure 3.   Silver stain and Western blot of purified gp-340. (A) A 6% SDS-PAGE gel, silver-stained as described in MATERIALS AND METHODS of unreduced, purified gp-340. (B) Western blot analysis of purified gp-340. Samples were resolved in 6% SDS- PAGE gels as described in MATERIALS AND METHODS, and transferred to nitrocellulose incubated with a 1:1,000 dilution of polyclonal rabbit anti-gp-340 antibody and a 1:10,000 dilution of goat antirabbit IgG-HRP conjugated. The reaction was visualized using the ECL detection system.

gp-340 Binds to SP-A

In two different assays, gp-340 was shown to bind to SP-A in a calcium-dependent manner. In the first assay, gp-340 was added, in the presence of calcium, to SP-A-coupled Sepharose. The gp-340 bound to the column and was not released by repeated washes with calcium-containing buffer. EDTA-containing buffer released the gp-340 from the SP-A-Sepharose beads (Figure 4). Without the addition of SP-A, gp-340 did not bind to Sepharose prepared in parallel with the SP-A-Sepharose (data not shown).

View larger version (37K): [in this window] [in a new window]   Figure 4.   gp-340 binds to SP-A coupled to Sepharose 4B. gp-340 was incubated overnight with SP-A-Sepharose in a calcium-containing buffer, as detailed in MATERIALS AND METHODS. The SP-A-Sepharose beads were washed three times with calcium-containing buffer and twice with EDTA-containing buffer, and aliquots from each wash were separated on 6% SDS-PAGE under nonreducing conditions and silver-stained. Shown are purified gp-340 (negatively stained because of high protein content), the initial supernatant from the overnight incubation (SUP), samples of each of the three Ca-containing washes (Washes), and samples of each of the two EDTA elutions (Fractions Eluted).

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 MATERIALS 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 observation 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 < 0.05) in the presence of either maltose or mannose and Ca²⁺. Binding of biotinylated SP-A to gp-340 in the absence of competing sugars correlated to an A₄₉₂ of 1.00 ± 0.08 in these assays; in the presence of 100 mM mannose, the A₄₉₂ of the final colorimetric 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 A₄₉₂ of the reaction was decreased to a level not distinguishable from either binding to gp-340 alone in the presence of EDTA or background binding (without gp-340 on the plate).

View larger version (21K): [in this window] [in a new window]   Figure 5.   SP-A binding to gp-340 is not inhibited by co-incubation with sugars. gp-340 was coated onto Immulon-2 microtiter plates, which were then blocked with BSA and incubated with biotinylated SP-A alone or in the presence of 100 mM mannose or maltose, followed by biotinylated streptavidin-HRP complex, and developed as described in MATERIALS AND METHODS. Data are expressed as the absorbance of the developed reaction at 492 nm. SP-A data are means ± SE for at least three experiments (*Statistically significant from gp-340 only, P < 0.005 by t test).

gp-340 Has No Effect on the Binding of SP-A to Alveolar Macrophages

¹²⁵I-SP-A was incubated with alveolar macrophages at 4°C for 4 h in the presence and absence of 1.3 µg gp-340/ml (Figure 6). Because neither the native molecular weight of gp-340 nor the stoichiometry of the SP-A-gp-340 interaction is known, three different concentrations of SP-A were tested: 0.13 µg SP-A/ml (a concentration equal to 0.2 nM SP-A, or one-tenth the published dissociation constant [K_d] of SP-A binding to alveolar macrophages [27]), 1.3 µg SP-A/ml (2 nM SP-A, its K_d), and 13 µg SP-A/ml (20 nM SP-A, or 10 times its K_d). At none of these concentrations was the binding of SP-A to alveolar macrophages significantly different in the presence of gp-340 than in the absence of gp-340. In addition, the binding of SP-A at 1.3 µg/ ml was examined in the presence or absence of 10 mM EDTA. The addition of EDTA eliminated approximately 90% of SP-A binding in both the presence and absence of gp-340; without gp-340, the addition of EDTA reduced binding of SP-A at 1.3 µg/ml from 266 ± 108 ng SP-A/mg cell protein to 21.1 ± 8.2 ng SP-A/mg protein; and in the presence of gp-340, binding was reduced by EDTA from 178 ± 84 ng SP-A/mg protein to 20.4 ± 7.2 ng SP-A/mg protein.

View larger version (16K): [in this window] [in a new window]   Figure 6.   gp-340 has no effect on SP-A binding to alveolar macrophages. Binding of 125I- labeled SP-A to alveolar macrophages was examined as described in MATERIALS AND METHODS, in the absence (open circles) or presence ( filled diamonds) of 1.3 µg gp-340/ml. Binding was converted to nanograms of SP-A bound per milligram of cell protein, based on the specific activity of the SP-A. Values shown are the means ± SE for n = 3 experiments; none of the values in the presence of gp-340 is significantly different from those in the absence of gp-340, as analyzed by ANOVA.

gp-340 Stimulation of Alveolar Macrophage Migration Is Concentration-Dependent

Alveolar macrophages stimulated with gp-340 migrate in a microchemotaxis chamber significantly more than unstimulated cells over a wide range of gp-340 concentrations (Figure 7). At 0.1 µg gp-340/ml, alveolar macrophage migration was stimulated to 173% of control (buffer-only) levels. Increasing concentrations of gp-340 stimulated increasing levels of alveolar macrophage migration: At 1 µg gp-340/ml, migration was 331% of control levels (a level significantly greater than both the control and 0.1 µg/ml levels, P < 0.05), and at 6 µg 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).

View larger version (12K): [in this window] [in a new window]   Figure 7.   Stimulation of alveolar macrophage migration by gp-340 is concentration-dependent. Chemotaxis assays were performed 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 ± SE for at least three experiments. *Significantly greater than control; **significantly greater than both control and 0.1 µg gp-340/ml treatment, SE too small to appear on graph (SE = ± 16% of control). Significance indicates P < 0.05 by t test.

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), with gp-340 at a concentration of 6 µg/ml in either the bottom well alone or both the bottom and top wells. In the presence of a concentration gradient (with gp-340 in only the bottom well), migration was stimulated to 637 ± 166% of control levels, versus 546 ± 155% of control with gp-340 in both wells. Because these figures are not significantly different from one another, these data are consistent with gp-340 stimulating chemokinesis rather than chemotaxis.

SP-A Stimulates Chemotaxis of Alveolar Macrophages in the Presence of gp-340

gp-340 does not affect the stimulation of alveolar macrophage chemotaxis by SP-A (Figure 8). In the presence of 6 µg gp-340/ml, 10 µg SP-A/ml stimulates alveolar macrophage migration by 34% over the level stimulated by gp-340 alone, a significant increase (P < 0.05), and a level comparable to the 31% stimulation exhibited by the same concentration of SP-A in the absence of gp-340 (to 49% of the gp-340 control from 18% of control in the buffer-only treatment). All four treatment groups (buffer only, SP-A only, gp-340 only, and SP-A + gp-340) were significantly different from one another (P < 0.05 by ANOVA and two-tailed t tests).

View larger version (16K): [in this window] [in a new window]   Figure 8.   Stimulation of alveolar macrophage migration by gp-340 is independent of stimulation of chemotaxis by SP-A. Chemotaxis assays were conducted as described in MATERIALS AND METHODS, in the presence or absence of 10 µg SP-A/ml and/ or 6 µg gp-340/ml. Raw data were normalized to a multiplicative scale, with the gp-340-only wells assigned the value of 1. Data are expressed as means ± SE for n = 4 experiments. *Significantly greater than buffer-only control; **significantly different from control, SP-A-only, and gp-340 + SP-A treatments; ***significantly greater than control, SP-A-only, and gp-340-only treatments (P < 0.05 by ANOVA and two-tailed t test).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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 (chemokinesis) of alveolar macrophages in a concentration-dependent manner. The functional consequences of the gp-340-SP-A interaction are still unclear; gp-340 neither affects the binding of SP-A to alveolar macrophages at the ratios and concentrations tested nor blocks the ability of SP-A to stimulate the chemotaxis of alveolar macrophages, even at relatively low SP-A concentrations.

gp-340 as a Receptor for SP-A and SP-D

The precise mechanism of SP-A binding to gp-340 has not been elucidated, because several characteristics of gp-340 could mediate its binding to SP-A. For example, gp-340 is a highly glycosylated molecule. N-glycosidase treatment of gp-340 results in a protein with an apparent electrophoretic molecular weight of approximately 300 kD (1). Holmskov and colleagues (1) demonstrated that the binding of gp-340 to SP-D was not inhibited in the presence of 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 gp-340. Despite this, the possibilities still exist that gp-340 contains sugars that bind to SP-A with a higher affinity than does mannose, and that SP-A and SP-D interact with gp-340 in different manners.

In addition, it is unclear whether gp-340 is a membrane-associated or secreted protein (or if isoforms of each exist). Furthermore, if it is indeed found to be membrane- associated, the nature of its interaction with the membrane must be elucidated. Clues to this might be taken from the unique characteristics of proteins that are homolgous to gp-340, including ebnerin, CRP-ductin, and a newly identified member of this family, DMBT1 (28), deletions in which are found in many brain-tumor cell lines. Ebnerin and CRP-ductin appear to share a characteristic unique to the SRCR family in that each seems to have several forms, including at least one that has no apparent transmembrane 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 soluble fractions of von Ebner's gland cells, leading Li and Snyder to suggest that two forms exist: one membrane-bound and one soluble and secreted (26). Two forms of the CRP-ductin messenger RNA were cloned; they appear to result from alternative splicing, and the  clone (which is not complete) has an extra C-terminal portion that has a putative transmembrane domain (the complete  clone has no such domain) (25). DMBT1 appears to encode no transmembrane domain (28). Holmskov and colleagues (1) suggested that the use of detergent in the initial solubilization of gp-340 indicated that it was membrane-associated; evidence presented here suggests that at least some gp-340 is liberated from AP lavage by the simple chelation of calcium ions. Alternatively, the gp-340 could be a membrane-associated protein that is liberated from the cell surface by proteolytic or other mechanisms. In any case, a secreted form of gp-340 could be a cofactor for SP-A and/or SP-D binding to cells, though the finding that gp-340 does not affect SP-A binding to alveolar macrophages argues against such a role for SP-A binding.

gp-340 as a Secreted Protein in Lung Lavage: Potential Functions and Interactions

The finding that gp-340 stimulates chemokinesis of alveolar macrophages drives speculation into possible functions for gp-340 in the lung that are diverse yet intriguing. Known protein stimulators of chemokinesis can be grouped into three main categories: growth factors, such as platelet- derived growth factor-AB, vascular endothelial growth factor, fibroblast growth factors, and epidermal growth factors (29); extracellular matrix (ECM) and adhesion molecules, such as fibronectin, laminin, thrombin, and collagen (32); and immune and inflammatory regulators (such as C1q; interleukins 2, 4, 5, and 8; and platelet-activating factor) (35).

Chemokinesis is an important function of cells in development and immune responses to infection and tissue damage. Growth factor-stimulated chemokinesis events have been demonstrated to be important in such areas as angiogenesis (29); the development of embryonic tissues in the limb (30); the proper localization of melanocytes in the skin (41); and the recruitment of microglial cells, the macrophages of the brain, to lesion sites (31). ECM-stimulated chemotaxis has been implicated in the recruitment of neutrophils to inflammatory sites (32) and the development of an organized vascular system in the mouse embryo (34). Similarly, the induction of random migration by immune activators is thought to be a first step in cell locomotion (37), an important step in the initiation of the extravasation of neutrophils and other inflammatory cells (35), the homing of lymphocytes and natural killer cells (38, 40), and the initiation of eosinophil- and mast cell-based inflammation (37, 39). These roles for chemokinetic proteins lead to the possibility that gp-340 is involved in such activities as the regulation of lung inflammation, the recruitment of monocytes into the lung and/or the regulation of their differentiation, the recruitment of macrophages and/ or fibroblasts to sites of lung injury, the embryonic development of the highly vascularized lung, or the adhesion of macrophages to the epithelium.

The elucidation of gp-340 function will be possible only when the entire sequence and structure of the protein is known. Inevitably, the cloning of gp-340 will lead to the production of genetically altered mice lacking gp-340 expression, which may help delineate the in vivo role of gp-340. Furthermore, both the native molecular weight and oligomerization of gp-340 and the stoichiometry of the interactions between gp-340 and SP-A and SP-D must be determined in order to perform meaningful binding assays. Antibodies raised against gp-340 and peptides from its sequence may be useful in investigating the nature of the association of gp-340 with both lipids and proteins in AP lavage. Finally, the precise localization of gp-340 is still unclear; although immunohistochemical studies by Homlskov and colleagues (1) found immunoreactive protein on the surface of alveolar macrophages, more precise localization of both gp-340 protein (on cellular and subcellular levels) and message is necessary to understand fully the physiologic functions of gp-340.