Isolation and Characterization of a New Member of the Scavenger Receptor Superfamily, Glycoprotein-340 (gp-340), as a Lung Surfactant Protein-D Binding Molecule*

(Received for publication, November 7, 1996, and in revised form, January 22, 1997)

Uffe Holmskov Dagger §, Peter Lawson , Børge Teisner Dagger , Ida Tornøe Dagger , Antony C. Willis , Cliff Morgan par , Claus Koch ** and Kenneth B. M. Reid

From the Dagger  Department of Medical Microbiology, Institute of Medical Biology, University of Odense, DK-5000 Odense, Denmark, the  MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom, the par  Department of Anaesthesia and Intensive Care, Royal Brompton National Heart & Lung Hospital, London SW3 6NP, United Kingdom, and ** Statens Seruminstitut, Copenhagen, DK-2300, Denmark

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

We have purified a previously unknown glycoprotein (designated gp-340) from human bronchioalveolar lung washings from a patient with alveolar proteinosis. gp-340 was identified by its calcium-dependent binding to lung surfactant protein D (SP-D) and by its molecular mass of 340 kDa in the reduced state on SDS-polyacrylamide gel electrophoresis (PAGE). gp-340 was purified from the 10,000 × g pellet of the lavage fluid by ion-exchange and gel permeation chromatography. On SDS-PAGE, gp-340 showed an apparent molecular mass of 290 kDa in the unreduced state. On gel chromatography under non-dissociating conditions, the apparent molecular mass of gp-340 was >1000 kDa. The presence of N-linked glycosylation was shown by digestion with N-glycosidase F, which reduced the apparent molecular mass of gp-340 under reducing condition to about 300 kDa. Partial amino acid sequence data showed the presence of scavenger-receptor type domains. Monoclonal and polyclonal antibodies were raised against gp-340, and their specificities were confirmed by Western blotting. The antibodies were used for immunohistochemical localization of gp-340 in the lung, where it was found on the surface of and within alveolar macrophages. Direct binding between gp-340 and SP-D took place at physiological ionic strength, required the presence of calcium, and was not inhibited by maltose. The binding between SP-D and mannan also required the presence of calcium, but this interaction was completely inhibited by maltose. The same binding pattern was seen between gp-340 and recombinant human SP-D composed of the trimeric neck region and three carbohydrate recognition domains. These findings indicate that the binding between gp-340 and SP-D is a protein-protein interaction rather than a lectin-carbohydrate interaction and that the binding to gp-340 takes place via the carbohydrate recognition domain of SP-D. We conclude that gp-340 is a new member of the scavenger-receptor superfamily and likely to be a truncated form of a receptor for SP-D.


INTRODUCTION

Lung surfactant protein D (SP-D)1 belongs to a group of C-type lectins called collectins (1, 2). Five collectins have been described, mannan-binding lectin (MBL), conglutinin and collectin-43 (CL-43), which are serum proteins produced by the liver; and lung surfactant proteins A and D (SP-A and SP-D), which have until recently been described as surfactant-associated proteins synthesized by alveolar type II cells and by non-ciliated bronchial epithelial cells but both have now been found to be present in cells lining the gastrointestinal tract (3, 4). Like the other collectins, SP-D is an oligomeric lectin with subunits composed of three identical polypeptides held together by disulfide bonds and non-covalent interactions at the N-terminal ends of the chains. Each polypeptide chain consists of a short N-terminal segment, followed by a collagen-like sequence, a short alpha -helical sequence, and the carbohydrate recognition domain (CRD) (5-8). The subunits are linked into oligomers consisting of four to eight subunits (9, 10). SP-D has been shown to bind directly to carbohydrates on the surface of various bacteria as well as to influenza A virus and to Cryptococcus neoformans (11-13). SP-D also interacts with surfactant-associated phospholipids in vitro, suggesting a role in the turnover of pulmonary surfactant in vivo (14, 15). SP-D binds directly to alveolar macrophages (16, 17) and is thereby able to enhance the production of oxygen radicals (18). SP-D can also be internalized by alveolar macrophages in vivo (19). However, the receptor responsible for the SP-D binding remains to be defined.

Two molecules have so far been found to interact with collectins, and both were first described as receptors for the complement protein C1q. One was found to bind MBL, conglutinin, CL-43, and SP-A, and this receptor is now referred to as the collectin receptor (20-22). This molecule, which has a high degree of homology with calreticulin, has been observed on many cell types, including macrophages and alveolar type II cells. The other receptor is referred to as the C1q receptor with a molecular mass of 126 kDa. A monoclonal antibody that recognizes this cell surface molecule inhibited the MBL-mediated enhancement of phagocytosis (23). However, neither of these receptors has been shown to bind SP-D.

In the present paper, we describe the purification and characterization of a 340 kDa glycoprotein obtained from lung lavage from a patient with alveolar proteinosis, and we provide data consistent with this molecule being a secreted form of a putative receptor for SP-D.


MATERIALS AND METHODS

Buffers and Reagents

The buffers and reagents used were Tris-buffered saline (TBS) containing 140 mM NaCl, 10 mM Tris, 7.5 mM NaN3, pH 7.2; TBS/Tw consisting of TBS containing 0.05% (v/v) Tween 20 (polyoxyethylene sorbitan monolaurate, Merck-Schuchardt, FRG); TBS/E consisting of TBS containing 0.05% (v/v) emulphogene (polyoxyethylene tridecyl ether, BC 720, from Sigma); coating buffer containing 0.1 M sodium carbonate, pH 9.6; diethanolamine buffer containing 10% (v/v) diethanolamine, 0.5 mM MgCl2, 7.5 mM NaN3, pH 9.8; carbazole buffer containing 0.04% 3-amino-9-ethylcarbazole (Sigma A-5754) in 0.015% H2O2, 50 mM sodium acetate buffer, pH 5.0; and buffer A containing 10 mM Tris, 10 mM EDTA, 0.05% (v/v) emulphogene, pH 7.5. TSK-maltose gel was prepared by coupling 100 mg of maltose/ml of divinylsulfone-activated Fractogel TSK HW/75(F) (14985, Merck, Darmstadt, Germany) (24). Rabbit anti-human Ig (Code A 190, Dakopatts A/S, Copenhagen, Denmark) was coupled to CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.) at a concentration of 2.5 mg of IgG/ml of gel in 0.1 M citrate buffer, pH 7.5, for 2 h at 4 °C.

SDS-PAGE and Western Blotting

Electrophoresis was performed on 4-20% (w/v) polyacrylamide gradient gels with discontinuous buffers (25). Samples were reduced by heating at 100 °C for 3 min in 40 mM dithiothreitol, 1.5% (w/v) SDS, 5% (v/v) glycerol, 0.1 M Tris, pH 8.0, and were carboxamidated by the addition of iodoacetamide to 90 mM. Unreduced samples were heated in sample buffer with 90 mM iodoacetamide. Protein bands were detected by silver staining (26).

For Western blotting, the FAST system (Pharmacia, Uppsala, Sweden) was used. Proteins were separated on 8-14% (w/v) polyacrylamide gradient gels with the discontinuous buffer system and blotted onto polyvinylidene difluoride membranes (Immobilon P, Millipore, Bedford, MA).

The membrane was incubated with primary antibody (monoclonal anti-gp-340 antibody, 50 ng/ml, or polyclonal rabbit anti-gp-340 antiserum diluted 1/1000) and secondary alkaline-phosphatase-coupled rabbit anti-mouse Ig or alkaline-phosphatase-coupled goat anti-rabbit Ig in TBS/Tw containing 0.5 M NaCl. The membranes were washed and developed with nitro blue tetrazolium and potassium 5-bromo-4-chloro-3-indolylphosphate.

Human Bronchioalveolar Lavage (BAL)

BAL was obtained from a patient suffering from pulmonary alveolar proteinosis. Both lungs were lavaged for therapeutic purposes at an interval of one week, using a heat exchanger and a modified perfusion apparatus to improve the efficiency of the lavage. The result was monitored by analysis of total protein content. BAL was also obtained from four patients undergoing lung washings for diagnostic purposes other than alveolar proteinosis.

Purification of SP-D

One liter of human BAL was centrifuged at 300 × g for 15 min and then at 10,000 × g for 30 min. The 10,000 × g supernatant was made 5 mM in CaCl2 and 10 mM in Tris and applied to a 50-ml maltose-TSK column. After washing with TBS/E containing 5 mM CaCl2, the column was eluted with the same buffer containing 100 mM maltose. The eluate was applied to a column of rabbit anti-human Ig Sepharose (15 ml) to remove contaminating anti-carbohydrate antibodies and then passed through a Superose 6 FPLC column (Pharmacia) equilibrated with TBS containing 10 mM EDTA and 0.05% (v/v) emulphogene.

Purification of gp-340

Purification of gp-340 was performed on a computer-monitored FPLC system (FPLCdirector Version 1.3, Pharmacia). One liter of human BAL was centrifuged at 300 × g for 10 min. After separation of the cell pellet, the supernatant was centrifuged at 10,000 × g for 30 min. The precipitate was dissolved in 500 ml of TBS/E containing 10 mM EDTA and incubated for 18 h at 4 °C with gentle stirring. Insoluble material was removed by centrifugation at 10,000 × g for 30 min. The supernatant was dialyzed against buffer A and applied to a column of Mono-Q Fast Flow (100 ml, Pharmacia), and retained proteins were eluted with a linear gradient from 0 to 1 M NaCl in buffer A. Fractions containing gp-340 were diluted in buffer A, applied to a Resource Q column (1-ml prepacked column, Pharmacia) and eluted with a stepped gradient from 0 to 1 M NaCl in buffer A. The gp-340-containing fractions were further separated by gel permeation chromatography on a Superose 6 prepgrade column (1.6 × 57 cm, Pharmacia) with TBS, pH 7.4, containing 10 mM EDTA, at a flow rate of 30 ml/h. The purity of the resulting gp-340 preparation was analyzed by gel permeation chromatography on a Superose 6 column (1 × 30 cm, Pharmacia) and by SDS-PAGE.

The amount of gp-340 was estimated by measurement of absorption at 280 nm assuming that E280 of 1 mg/ml for gp-340 was 1.0.

Amino Acid Sequencing and Amino Acid Analysis

The procedures were as described in (27). Amino acid analysis was performed directly on purified gp-340 using an Applied Biosystems 420A amino acid analyzer (Perkin-Elmer, Warrington, UK). Attempts at N-terminal sequencing of intact gp-340 were unsuccessful. To obtain internal sequence data, partially pure gp-340 was run on 7.5% SDS-PAGE, and protein bands were detected with Coomassie Brilliant Blue R-250. The two bands of gp-340 (A = upper band and B = lower band) were excised from the SDS-PAGE gel, homogenized in 100 mM Tris, pH 8.0, and digested with trypsin (Modified Trypsin-Sequencing Grade, Promega UK Ltd.) at an estimated enzyme:substrate ratio of 1:20 for 16 h at 37 °C. The supernatants were then run on by reverse phase chromatography using a Brownlee BU-300 VC4 column (100 × 1 mm). Peaks were collected and sequenced using an Applied Biosystems 470A protein sequencer (Perkin-Elmer). Purified gp-340 was also digested directly with trypsin digestion and purification of peptides as above. Sequences similar to gp-340 were searched for in GenBankTM/EBI data bank plus DDBI and Protein Data Bank data bases using the BLAST program. Alignment of multiple sequences were carried out using the Clustal method with PAM250 residue weight table.

Expression of Recombinant Neck-CRD of Human SP-D (rSP-D)

A recombinant polypeptide composed of the alpha -helical neck region and the CRD of human SP-D was expressed in Escherichia coli using the glutathione S-transferase gene fusion vector (Pharmacia) and purified as described in (8).

Preparation of Anti-gp-340 Antiserum

Antisera were raised in rabbits by subcutaneous immunization with an emulsified mixture of equal volumes of gp-340 (approximately 20 µg per dose) and Freund's complete adjuvant (Statens Seruminstitut, Copenhagen, Denmark). The rabbits were boosted at 1 month intervals with gp-340 in Freund's incomplete adjuvant. Irrelevant antibodies were removed by absorption; the antiserum was incubated with 1/10 volume of normal human serum and with 80 µg of SP-A/ml for 18 h at 4 °C followed by a centrifugation at 10,000 × g for 30 min. The IgG fraction of the rabbit antiserum against gp-340 was purified on a 1-ml HiTrapTM Protein G column (Pharmacia, Uppsala, Sweden), and the specificity was confirmed by Western blotting.

Production of Monoclonal Antibody Against gp-340

CF1 × BALB/c female mice were immunized with 10 µg of purified gp-340 in 0.25 ml of 0.1 M sodium phosphate, adsorbed onto 1 mg of Al(OH)3 gel (Alhydrogel®, Superfoss, Denmark) per injection. The injections were repeated at intervals of 14 days. Mice with a high antibody titer against gp-340, as determined by direct enzyme-linked immunosorbent assay on gp-340 coated microtiter plates, were selected for fusion of spleen cells. Fusions were performed as described by Köhler and Milstein (28) and modified by Reading (29) with the cell line X63Ag8.6.5.3 and with polyethylene glycol 1500 as the fusogen. Hybridoma clones were screened by direct enzyme-linked immunosorbent assay on gp-340-coated microtiter plates. Cells from wells with antibodies against gp-340 were recloned three times by the limiting dilution method (30). The monoclonal antibody selected was of IgG 1 subclass, and its specificity was tested by Western blotting.

Deglycosylation of gp-340

The amount of N-linked saccharides in gp-340 was evaluated by enzymatic digestion with N-glycosidase F for 18 h at 37 °C using the PGNase F kit (New England Biolabs, Beverly, MA) followed by SDS-PAGE.

Immunohistochemistry

The indirect immunoperoxidase staining technique was used. Briefly, the procedure was as follows. The dried frozen sections were fixed in acetone for 10 min, washed in TBS, preincubated in 2% (w/v) BSA in TBS for 10 min, incubated for 30 min with the monoclonal or polyclonal antibody against gp-340 diluted in 1% (w/v) BSA in TBS, washed with TBS, incubated for 1/2 h with peroxidase-labeled goat anti-mouse Ig (P 447, Dakopatts) or peroxidase-labeled goat anti-rabbit Ig (P 448, Dakopatts) diluted 1:75 in TBS, washed with TBS and H2O, incubated with carbazole buffer for 20 min, washed, counterstained with Mayer's hematoxylin for 2 min, and mounted in Aquamount (BDH Limited Poole, UK). Staining was also performed with a monoclonal anti-CD68 antibody as primary antibody (Code M 718, Dakopatts).

Conventional staining controls were performed as well as replacing the primary antibody with an unrelated monoclonal antibody of the same subclass as the anti-gp-340 antibody, or preabsorbing the primary polyclonal antibody with highly purified gp-340 .

Analysis of SP-D and rSP-D binding to gp-340

Microtiter plates (Polysorp, Nunc, Kamstrup, Denmark) were coated with gp-340 (300 ng/ml in coating buffer) by overnight incubation. This incubation and all the following steps were carried out in a volume of 100 µl/well and at room temperature, and all washes and incubations were carried out with TBS/Tw containing 5 mM CaCl2 unless otherwise stated. The plates were washed and incubated with 200 µg of human serum albumin (Statens Seruminstitut) in 200 µl of TBS for 2 h. After washing, the plates were incubated with dilutions of SP-D or with SP-D containing 100 mM maltose. As a control, the SP-D was also applied in buffer containing 10 mM EDTA instead of calcium. The same experiments were carried out using rSP-D. The plates were incubated overnight at 4 °C and washed. They were then incubated for 2 h with rabbit anti-human SP-D (a generous gift from Dr. Jinhua Lu, Department of Biochemistry, University of Singapore, Singapore) diluted 1:500 in TBS/Tw. After washing, the plates were incubated for 2 h with alkaline-phosphatase-coupled goat anti-rabbit Ig diluted 1:2000. After a final wash, the bound enzyme was estimated by adding 1 mg/ml of para-nitrophenyl phosphate disodium salt in diethanolamine buffer. The absorbance of the 96 wells was read at 405 nm by means of a multichannel spectrophotometer.


RESULTS

During the purification of human SP-D from the 10,000 × g supernatant from BAL of a patient suffering from alveolar proteinosis, a molecule with an apparent molecular mass of 340 kDa was observed by SDS-PAGE in reducing conditions. When the supernatant was applied to a maltose-TSK column in the presence of calcium, the component was bound and could be eluted with maltose together with SP-D and anti-carbohydrate antibodies. The component could then be separated from SP-D and immunoglobulins by gel permeation chromatography in the presence of EDTA (data not shown). Rechromatography of the recovered component on a maltose-TSK column in the presence of calcium showed no binding, indicating that the initial retention of the component on the column must be mediated by one of the other proteins bound to the maltose. Because of low yields, another purification procedure was developed, utilizing the 10,000 × g pellet from BAL as starting material for ion-exchange and gel permeation chromatography. The component was named gp-340 because of its glycoprotein nature and because of its molecular mass of 340 kDa in the reduced state on SDS-PAGE (see below).

Purification of gp-340

gp-340 was eluted from the 10,000 × g pellet in the presence of EDTA and purified by ion-exchange and gel permeation chromatography. Fig. 1 shows the elution profile from ion-exchange on Mono-Q Fast Flow (Fig. 1A), on Resource Q (Fig. 1B), and the gel permeation chromatography on Superose 6 Prepgrade (Fig. 1C). Purified gp-340 was analyzed by chromatography on Superose 6 where it emerged close to the void volume, corresponding to an apparent molecular mass >1000 kDa (Fig. 1D). Approximately 500 µg of gp-340 (estimated by E280) was obtained from 1 liter of washings.


Fig. 1. Purification of gp-340 from the redissolved 10,000 × g pellet from BAL. A, elution profile of 500 ml of starting material applied to a Mono-Q Fast Flow column (100-ml bed volume) eluted with a 0-1 M NaCl gradient. The horizontal bar indicates the gp-340-containing fractions selected for further purification. B, elution profile of gp-340-containing fractions (from panel A) applied to a Resource Q column (1 ml) and eluted with a stepped NaCl gradient. C, elution profile of gp-340 (from panel B) fractionated by gel permeation chromatography on a Superose 6 Prepgrade column (1.6 × 57 cm). The horizontal bar indicates the gp-340 containing fractions, which were pooled and further analyzed by gel permeation chromatography and SDS-PAGE. D, analytical gel permeation chromatography of purified gp-340 on a Superose 6 column (1 × 30 cm). The column was calibrated with blue dextran (BD), purified human IgM (950 kDa), and IgG (150 kDa). A280, solid line; ionic strength, dotted line.
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Fig. 2 shows a comparison by SDS-PAGE of gp-340 reduced (lane 1), unreduced (lane 2), and reduced after treatment with N-glycosidase F (lane 3). gp-340 showed two closely spaced bands with a mobility corresponding to a molecular mass of approximately 340 kDa when reduced and an apparent molecular mass of 290 kDa in its unreduced state. When glycosidase-digested gp-340 was examined on SDS-PAGE in its reduced state, the two closely spaced bands appeared at a position corresponding to a molecular mass of approximately 300 kDa.


Fig. 2. SDS-PAGE of purified gp-340. Lanes 1 and 2 show gp-340 with and without the reduction of disulfide bonds, respectively. Lane 3 shows gp-340 in the reduced state after treatment with N-glycosidase F.
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Amino Acid Sequencing

Amino acid sequences of different fragments obtained by tryptic digestion of gp-340 are shown in Fig. 3. Peptide 5 obtained from the upper band of gp-340 (band A) was found to have an identical N-terminal sequence to that of peptide 6 obtained from the lower band of gp-340 (band B).


Fig. 3. Amino acid sequences from gp-340. Sequences were obtained either from peptides isolated after direct digestion of purified gp-340 with trypsin (peptides 1 and 2), or from peptides derived from tryptic digestion of band A (upper) and band B (lower) cut from the gel after SDS-PAGE of partially purified gp-340 (peptides 3-9). Tryptic peptides were separated by reversed phase chromatography.
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Amino Acid Composition

The amino acid composition of gp-340 is shown in Table I. The presence of 0.3% hydroxyproline indicated that gp-340 might contain collagen-like sequences. However, gp-340 was not digested by collagenase (data not shown), and the appearance of hydroxyproline in the amino acid composition of gp-340 may be due to small amounts of SP-A or SP-D in the gp-340 preparation.

Table I. Amino acid composition of gp-340

ND, not determined. ND, not determined.

Composition Mole percent

Ala 6.3
Cys 2.4
Asx 12.7
Glx 6.1
Phe 3.2
Gly 11.0
His 2.4
Ile 3.2
Lys 4.3
Leu 8.5
Met ND
Pro 6.2
Arg 6.1
Ser 10.7
Thr 6.0
Val 7.0
Trp ND
Tyr 3.4
Hyp 0.3

Characterization of Antibodies

The specificities of the selected monoclonal and polyclonal antibodies against gp-340 was analyzed by Western blots of both purified gp-340 and crude lung lavage separated on SDS-PAGE in the reduced and unreduced state (Fig. 4). The monoclonal antibody directed against gp-340 reacted with both bands of 340 kDa equally well in the reduced and unreduced state, and it also reacted with deglycosylated gp-340 (Fig. 4A). The rabbit anti-gp-340 antibody reacted with both bands of 340 kDa, but, in contrast to the monoclonal antibody, it displayed less binding after reduction of the antigen and no binding when the antigen was reduced and deglycosylated (Fig. 4B).


Fig. 4. Specificities of monoclonal (A) and polyclonal (B) anti-gp-340 antibodies tested by Western blotting. Lane 1, reduced purified gp-340; lane 2, unreduced purified gp-340; lane 3, reduced purified gp-340 after N-glycosidase F treatment; lane 4, reduced crude lung washing; lane 5, unreduced crude lung washing. The blots were incubated either with monoclonal anti-gp-340 antibody (A) (50 ng/ml), or polyclonal anti-gp-340 antiserum diluted 1:1000 (B). Bound antibody was visualized by means of alkaline-phosphatase-coupled rabbit anti-mouse Ig (A) or alkaline-phosphatase-coupled goat anti-rabbit Ig (B) and substrate.
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Western blot analysis revealed that the monoclonal antibody also reacted with gp-340 in lung washings from four patients undergoing diagnostic bronchioalveolar lavage for other reasons than alveolar proteinosis (data not shown).

1mmunohistochemistry

Staining of normal human lung revealed gp-340 immunoreactivity on and within the alveolar macrophages (Fig. 5). The staining was distinctly granular with both types of antibody. However, staining with the monoclonal antibody was distinctly weaker than that obtained with the polyclonal antibody. Staining of serial sections with monoclonal anti-CD68 antibody identified the gp-340 stained cells as cells belonging to the macrophage cell lineage (data not shown). Staining, absorption, and substitution controls were all negative.


Fig. 5. Immunohistochemical localization of gp-340 in normal human lung. The sections were counterstained with Mayer's hematoxylin. A, section of human lung stained with the monoclonal antibody against gp-340 in which a macrophage shows distinct staining corresponding to the membrane region. B, section of human lung stained with the polyclonal antibody against gp-340. C, Human lung stained with control monoclonal antibody.
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Binding of SP-D and rSP-D to gp-340 and Mannan

In the presence of calcium, human SP-D binds to microtiter plates coated with gp-340 (Fig. 6). The binding was saturable and dependent on the concentration of SP-D and on the amount of gp-340 coated on the plate. In the presence of calcium, human SP-D binds to microtiter plates coated with mannan (Fig. 7A). The binding was also dependent on the concentration of SP-D and could be totally inhibited by EDTA or by the addition of 100 mM maltose. The binding of human SP-D to microtiter plates coated with gp-340 was also inhibited by EDTA (Fig. 7B), but, when 100 mM maltose was added in the presence of calcium, no inhibition of binding was observed.


Fig. 6. Binding of SP-D to gp-340. Microtiter plates coated with different concentrations of gp-340 were incubated with serial dilutions of SP-D. Binding was carried out at physiological ionic strength in the presence of calcium. Coating contractions of gp-340 were 300 ng/ml (bullet -bullet ); 150 ng/ml (open circle -open circle ); 75 ng/ml (black-square-black-square):19 ng/ml (black-triangle-black-triangle); 2.3, ng/ml (black-down-triangle -black-down-triangle ); and no coating (black-diamond -black-diamond ).
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Fig. 7. Effect of maltose and EDTA on the binding of SP-D to gp-340 and mannan. A, mannan-coated microtiter plates were incubated with serial dilutions of SP-D in the presence of calcium (bullet -bullet ), calcium and 100 mM maltose (black-triangle-black-triangle), or with 10 mM EDTA (black-square-black-square). B, gp-340-coated microtiter plates were incubated with serial dilutions of SP-D in the presence of calcium (bullet -bullet ), calcium and 100 mM maltose (black-triangle-black-triangle), or with 10 mM EDTA (black-square-black-square). Bound SP-D was detected by means of rabbit anti-human SP-D, followed by alkaline-phosphatase-coupled goat anti-rabbit Ig and substrate.
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Like native SP-D, rSP-D also bound mannan in the presence of calcium, and the binding was inhibited by EDTA or by the addition of 100 mM maltose (Fig. 8A). rSP-D also bound gp-340 in the presence of calcium, and the binding was inhibited by EDTA (Fig. 8B). When 100 mM maltose was added in the presence of calcium, only minor inhibition of binding was seen.


Fig. 8. Binding of rSP-D to gp-340 and mannan. A, mannan-coated microtiter plates were incubated with serial dilutions of rSP-D in the presence of calcium (bullet -bullet ), calcium and 100 mM maltose (black-triangle-black-triangle), or with of 10 mM EDTA (black-square-black-square). B, gp-340-coated microtiter plates were incubated with serial dilutions of SP-D in the presence of calcium (bullet -bullet ), calcium and 100 mM maltose (black-triangle-black-triangle), or with 10 mM EDTA (black-square-black-square). Bound SP-D was detected by means of rabbit anti-human SP-D, followed by alkaline-phosphatase-coupled goat anti-rabbit Ig and substrate.
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The IgG fraction of the polyclonal antiserum against gp-340 completely abolished the SP-D binding to solid phase gp-340 when plates coated with gp-340 were incubated with the IgG fraction prior to addition of SP-D (data not shown).


DISCUSSION

The present report describes the identification and purification of an SP-D-binding protein from BAL obtained from a patient with alveolar proteinosis. The SP-D-binding molecule has provisionally been termed gp-340 since it is a glycoprotein of apparent molecular mass of 340 kDa on SDS-PAGE.

In contrast to the molecular mass of 340 kDa seen in the reduced state, gp-340 showed an apparent molecular mass of 290 kDa when analyzed in the unreduced state. This difference suggests that intra-molecular disulfide bonds have been broken by reduction. gp-340 always appeared as two closely spaced but distinct bands. Since gp-340 may be a membrane protein requiring proteolytic cleavage for release from the surface of macrophages, the difference in size could be explained by two different, closely spaced cleavage sites within each polypeptide chain of gp-340.

The apparent molecular weight of native gp-340 was estimated by gel filtration to be greater than 1000 kDa. This suggests that gp-340 is either a very extended molecule, thereby leading to an overestimation of its molecular mass on gel filtration, or that it exists as an oligomer of identical or closely related non-covalently linked polypeptide chains.

Partial amino acid sequences showed the presence of scavenger-receptor type domains in both bands A and B, and identical sequences were obtained from the two bands. The different sizes of the components of these bands could also be due to alternative splicing of the molecule since size heterogeneity due to alternative splicing has been described for closely related members of the scavenger receptor superfamily (31). The spacing of the cysteines in the domains showed that gp-340 belongs to group B of the scavenger-receptor superfamily (32). Members of this group are all transmembrane proteins and many of them are located on immunocompetent cells such as B-lymphocytes, T-lymphocytes or macrophages. Although no direct function has been described for these molecules, a role in the primary immune system has been suggested on the basis of their localization on such cells (32). Three members of the scavenger-receptor superfamily, bovine gallbladder mucin, Ebnerin, and CRP-ductin-alpha, showed an extremely high homology with gp-340, and an alignment between domains from these molecules and peptides 1-4, 6-7 from gp-340 is shown in Fig. 9. Bovine gallbladder mucin is a densely glycosylated molecule which is the primary secretory product of the gallbladder epithelium (33). Four SRCR domains are found in bovine gallbladder mucin along with short tandem repeating sequences found in mucins. Ebnerin, which was cloned from a rat taste bud gland cDNA library (34), is also a mosaic protein containing four SRCR, two bone morphogenic domains, a zona pellucida domain, a transmembrane region, and a short cytoplasmic tail. CRP-ductin-alpha (35) has an overall domain organization similar to Ebnerin, but it has eight SRCR domains instead of four and five bone morphogenic domains instead of two. The cDNA of CRP-ductin-alpha, which is the largest of the three molecules, predicts a protein of 2083 amino acid residues with a molecular mass of 227 kDa and shows an apparent molecular mass of 260 kDa in the reduced state on SDS-PAGE. The molecular mass of reduced and deglycosylated gp-340 was estimated to be 300 kDa using reduced and unreduced fibronectin (molecular mass = 220 kDa and 440 kDa, respectively), purified alpha -2 macroglobulin-receptor (molecular mass = 500 kDa, a gift from Søren Moestrup, Aarhus University, Denmark), together with standard molecular weight markers (data not shown). A very high degree of homology was found both between the SRCR domains of mouse CRP-ductin-alpha and bovine gallbladder mucin and peptides 1-7 of gp-340 (100% amino acid sequence identity between SRCR 1, 3, and 4 of bovine gallbladder mucin, and with SRCR 3 of CRP-ductin-alpha and the 19 amino acid residues in peptide 6 of gp-340) (Fig. 9). However, as the same high degree of homology is seen on comparison of the different proteins of SRCRs and between species, and, in view of the size difference between gp-340 and the gall-bladder mucin, Ebnerin, and the CRP-ductin-alpha proteins, it seems unlikely that gp-340 is the human analogue of these proteins. Moreover, CRP-ductin-alpha mRNA was only expressed in cells lining the small and large intestine, the pancreatic duct system, and the larger hepatic ducts, and no CRP-ductin-alpha mRNA was detected in the lung (35).


Fig. 9. Alignment of peptides 1-4, 6, and 7 from gp-340 with SRCR domains of members of group B of the scavenger-receptor superfamily. The domains are aligned by the Crustal method with the PAM250 residue weight table. The sequences shown are bovine gallbladder mucin domains 1-4 (33); CRP-ductin-alpha domains 1-8 (35); and Ebnerin domains 1-4 (34).
[View Larger Version of this Image (36K GIF file)]

Purified gp-340 was used to immunize mice and rabbits, and the resulting monoclonal and polyclonal antibodies were used for the immunohistochemical localization of gp-340. In the lung, both antibodies revealed a distinct granular staining of alveolar macrophages, with the most intense staining at the cell membrane, but in some cells, organelles considered to be phagolysosomes were also stained. This staining pattern is in agreement with what would be expected for a receptor for SP-D. It has earlier been shown that SP-D is found in the endocytic compartment of rat and human alveolar macrophages, and a receptor for SP-D will probably be internalized together with SP-D and material bound via the CRD of SP-D (19, 36). It is also worth noting that, to date, all the proteins containing SRCRs of type B are known to be membrane bound, and both CRP-ductin and Ebnerin are known to have both a membrane-bound form and a free form due to either cleavage of the molecules or alternative splicing of the molecules (34, 35)

Direct binding of SP-D to purified gp-340 was observed at physiological ionic strength. The binding was saturable, and dependent on the presence of calcium ions. No inhibition was observed on the addition of the disaccharide maltose. In contrast, binding of mannan to SP-D was completely inhibited by maltose. The same binding pattern was seen between rSP-D (consisting of the neck-region and CRD of SP-D) and gp-340. Maltose would be expected to inhibit any binding of the CRD of SP-D to carbohydrates on gp-340. The result thus suggests that the binding between SP-D and gp-340 is a protein-protein interaction rather than a lectin-carbohydrate interaction, and the fact that the rSP-D is also bound shows that the binding site is located within the neck-CRD structure.

The crystal structure of the CRD of rat MBL-A complexed with an oligosaccharide revealed two calcium binding sites (37, 38). The calcium ion in site 2 forms coordination bonds with the carbohydrate ligand while no such bonds were found for the calcium ion in site 1. All the amino acid residues involved in calcium and carbohydrate binding in rat MBL-A are present in human SP-D. No function of the first calcium binding site has so far been suggested for any CRD. We, therefore, suggest that this calcium binding site may be important for recognizing ligands other than carbohydrates. It is interesting to note that CD72, which has one extracellular CRD, is the ligand for CD5 which has three extracellular SRCRs (39).

It has earlier been shown that the direct binding of SP-D to alveolar macrophages (and neutrophil granulocytes) can be completely abolished by maltose, indicating that SP-D in these experiments bound to the cells via a carbohydrate-lectin interaction (19). SP-D also enhances the respiratory burst in rat alveolar macrophages (18), a reaction which was also inhibited by carbohydrates. These observations suggest that more than one ligand or receptor for SP-D is present on alveolar macrophages.

We conclude that gp-340 is the soluble form of a molecule that binds SP-D in a calcium-dependent manner. The gp-340 molecule is thus a potential receptor for SP-D that has been shed or cleaved from the surface of the alveolar macrophages.


FOOTNOTES

*   This work was supported by the Danish Medical Research Council, Michaelsen Fonden, the Novo Nordisk Foundation, Fonden til Lægevidenskabens Fremme, the Carlsberg Foundation, and Nationalforeningens Fond.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom all correspondence should be addressed: Dept. of Medical Microbiology, Institute of Medical Biology, Odense University, DK-5000, Denmark. Tel.: 45 65 57 37 75; Fax: 45 65 91 52 67; E-mail: holmskov{at}imbmed.ou.dk.
1   The abbreviations used are: SP-D, lung surfactant protein D; SP-A, lung surfactant protein A; CRD, carbohydrate recognition domain; MBL, mannan-binding lectin; CL-43, collectin-43; BAL, bronchioalveolar lavage; SRCR domain, scavenger receptor cystein-rich domain; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TBS/Tw, TBS-Tween 20; TBS/E, TBS-emulphogene; rSP-D, recombinant neck-CRD of human SP-D.

ACKNOWLEDGEMENT

We thank Mr. Ole Nielsen and Annette Kliem for skilled technical assistance.


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