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

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 ofN-linked glycosylation was shown by digestion withN-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. 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 ofN-linked glycosylation was shown by digestion withN-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. Lung surfactant protein D (SP-D) 1The 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. belongs to a group of C-type lectins called collectins (1Drickamer K. Taylor M.E. Annu. Rev. Cell Biol. 1993; 9: 237-264Google Scholar, 2Holmskov U. Malhotra R. Sim R.B. Jensenius J.C. Immunol. Today. 1994; 15: 67-74Google Scholar). 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 (3Rubio S. Lacaze-Masmonteil T. Chailley-Heu B. Kahn A. Bourbon J.R. Ducroc R. J. Biol. Chem. 1995; 270: 12162-12169Google Scholar, 4Fisher J.M. Mason R. Am. J. Respir. Cell Mol. Biol. 1995; 12: 13-18Google Scholar). 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 α-helical sequence, and the carbohydrate recognition domain (CRD) (5Persson A. Chang D. Rust K. Moxley M. Longmore W. Crouch E. Biochemistry. 1989; 28: 6361-6367Google Scholar, 6Rust K. Grosso L. Zhang V. Chang D. Persson A. Longmore W. Cai G-Z. Crouch E. Arch. Biochem. Biophys. 1991; 290: 116-126Google Scholar, 7Lu J. Willis A.C. Reid K.B.M. Biochem. J. 1992; 284: 795-802Google Scholar, 8Hoppe H-J. Barlow P.N. Reid K.B.M. FEBS Lett. 1994; 344: 191-195Google Scholar). The subunits are linked into oligomers consisting of four to eight subunits (9Lu J. Wiedermann H. Holmskov U. Thiel S. Timpl R. Reid K.B.M. Eur. J. Biochem. 1993; 215: 793-799Google Scholar, 10Crouch E. Persson A. Chang D. Heuser J. J. Biol. Chem. 1994; 269: 17311-17319Google Scholar). 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 (11Kuan S.F. Rust K. Crouch E. J. Clin. Invest. 1992; 90: 97-106Google Scholar, 12Hartshorn K.L. Crouch E. White M.R. Eggleton P. Tauber A.I. Chang D. Sastry K. J. Clin. Invest. 1994; 94: 311-319Google Scholar, 13Schelenz S. Malhotra R. Sim R . B. Holmskov U. Bancroft G.J. Infect. Immun. 1995; 63: 3360-3366Google Scholar). SP-D also interacts with surfactant-associated phospholipids in vitro, suggesting a role in the turnover of pulmonary surfactant in vivo (14Kuroki Y. Gasa S. Ogasawara Y. Shiratori M. Makita A. Akino T. Biochem. Biophys. Res. Commun. 1992; 187: 963-969Google Scholar,15Persson A. Gibbons B.J. Shoemaker J.D. Moxley M.A. Longmore W.J. Biochemistry. 1992; 31: 12183-12189Google Scholar). SP-D binds directly to alveolar macrophages (16Miyamura K. Leigh L.E.A. Lu J. Hopkin J. Bernal A.L. Reid K.B.M. Biochem. J. 1994; 300: 237-242Google Scholar, 17Kuan S.F. Persson A. Parghi D. Crouch E. Am. J. Respir. Cell Mol. Biol. 1994; 10: 430-436Google Scholar) and is thereby able to enhance the production of oxygen radicals (18Van Iwaarden J.F. Shimizu H. Van Golde P.H.M. Voeller D.R. Van Golde L.M.G. Biochem. J. 1992; 286: 5-8Google Scholar). SP-D can also be internalized by alveolar macrophages in vivo(19Crouch E. Kuan S.F. Persson A. Müller B. von Wichert P. Lung Surfactant: Basic Research in the Pathogenesis of Lung Disorder. Karger, Basel1994: 123-140Google Scholar). 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 (20Malhotra R. Thiel S. Reid K.B.M. Sim R.B. J. Exp. Med. 1990; 172: 955-959Google Scholar, 21Malhotra R,. Haurum J. Thiel S. Sim R.B. Eur. J. Immunol. 1992; 22: 1437-1445Google Scholar, 22Holmskov U. Laursen S.B. Malhotra R. Wiedemann H. Timpl R. Stuart G.R. Tornøe I. Madsen P.S. Reid K.B.M. Jensenius J.C. Biochem. J. 1995; 305: 889-896Google Scholar). 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 (23Tenner A.J. Robinson S.L. Ezekowitz R.A. Immunity. 1995; 3: 485-493Google Scholar). 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. 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 0.1m sodium carbonate, pH 9.6; diethanolamine buffer containing 10% (v/v) diethanolamine, 0.5 mmMgCl2, 7.5 mm NaN3, pH 9.8; carbazole buffer containing 0.04% 3-amino-9-ethylcarbazole (Sigma A-5754) in 0.015% H2O2, 50 mmsodium 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) (24Fornstedt N. Porath J. FEBS Lett. 1975; 57: 187-191Google Scholar). 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. Electrophoresis was performed on 4–20% (w/v) polyacrylamide gradient gels with discontinuous buffers (25Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar). 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 (26Merril C.R. Goldman D. Van-Keuren M.L. Methods Enzymol. 1983; 96: 230-239Google Scholar). 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 50 polyclonal and Ig Ig in TBS/Tw containing 0.5 m The membranes were and with and was obtained from a patient from pulmonary alveolar proteinosis. were for at an of a and a to the of the The was by of protein was also obtained from four lung washings for other than alveolar proteinosis. One of human was at 300 × g for min and at 10,000 for The 10,000 × g was mm in and 10 mm in and to a with TBS/E containing mm the was with the same buffer containing 100 mm maltose. The was to a of anti-human Ig Sepharose to antibodies and a with TBS containing 10 mm and 0.05% (v/v) of gp-340 was performed on a system One of human was at 300 for 10 of the cell the was at 10,000 × g for The was in of TBS/E containing 10 mm and incubated for h at 4 °C with was by at 10,000 × g for The was against buffer A and to a of and proteins were with a gradient from to m in buffer A. containing gp-340 were in buffer to a and with a gradient from to m in buffer A. The were separated by gel permeation chromatography on a × with TBS, pH containing 10 mm EDTA, at a of The of the gp-340 was by gel permeation chromatography on a × and by The of gp-340 was by of at that of for gp-340 was The were as described in Willis A.C. Biochem. J. 1990; Scholar). acid was performed directly on purified gp-340 an amino acid at N-terminal of gp-340 were sequence gp-340 was on SDS-PAGE, and protein bands were detected with The bands of gp-340 and were from the in 100 mm Tris, pH 8.0, and with at an of for h at °C. The were on by chromatography a × were and an protein gp-340 was also directly with digestion and purification of as to gp-340 were for in data and Protein data the of were the with A recombinant polypeptide composed of the α-helical neck region and the of human SP-D was in the and purified as described in H-J. Barlow P.N. Reid K.B.M. FEBS Lett. 1994; 344: 191-195Google Scholar). were raised in by with an of of gp-340 20 and Copenhagen, The were at with gp-340 in antibodies were by the was incubated with of human serum and with of for h at 4 °C followed by a at 10,000 × g for The of the against gp-340 was purified on a Protein (Pharmacia, Uppsala, and the was confirmed by Western blotting. × were with 10 of purified gp-340 in of 0.1 m sodium onto mg of gel Denmark) The were at of with a high antibody against gp-340, as by on gp-340 were for of cells. were performed as described by and Nature. 1975; Scholar) and by J. Immunol. Scholar) with the cell and with as the were by on from with antibodies against gp-340 were three by the J. Monoclonal and Scholar). The monoclonal antibody was of and its was by Western blotting. The of in gp-340 was by digestion withN-glycosidase for h at °C the followed by The staining was used. the was as The were in for 10 in TBS, in (w/v) in TBS for 10 incubated for min with the monoclonal polyclonal antibody against gp-340 in (w/v) in TBS, with TBS, incubated for h with Ig Ig in TBS, with TBS and incubated with carbazole buffer for 20 with for 2 and in was also performed with a monoclonal antibody as primary antibody (Code staining were performed as well as the primary antibody with an monoclonal antibody of the same as the the primary polyclonal antibody with purified gp-340 . Denmark) were with gp-340 in coating by This and the were in a of 100 and at and and were with TBS/Tw containing mm The were and incubated with of human serum in of TBS for 2 the were incubated with of SP-D with SP-D containing 100 mm maltose. a the SP-D was also in buffer containing 10 mm of The same were The were incubated at 4 °C and were incubated for 2 h with anti-human SP-D from of of in the were incubated for 2 h with Ig a the was by of in diethanolamine The of the was at by of a the purification of human SP-D from the 10,000 from of a patient from alveolar proteinosis, a molecule with an apparent molecular mass of 340 kDa was observed by in reducing the was to a in the presence of calcium, the was and be with together with SP-D and The be separated from SP-D and by gel permeation chromatography in the presence of not of the on a in the presence of showed that the of the on the be by of the other proteins to the maltose. of purification was the 10,000 × g pellet from as for ion-exchange and gel permeation chromatography. The was gp-340 of its glycoprotein and of its molecular mass of 340 kDa in the reduced state on gp-340 was from the 10,000 × g pellet in the presence of and purified by ion-exchange and gel permeation chromatography. the from ion-exchange on on and the gel permeation chromatography on gp-340 was by chromatography on where it to the to an apparent molecular mass >1000 kDa of gp-340 by was obtained from of 2 a by of gp-340 reduced unreduced and reduced with gp-340 showed bands with a to a molecular mass of 340 kDa reduced and an apparent molecular mass of 290 kDa in its unreduced state. gp-340 was on in its reduced the bands at a to a molecular mass of 300 kDa. acid of obtained by digestion of gp-340 are shown in obtained from the of gp-340 was found to have an identical N-terminal sequence to that of obtained from the of gp-340 The amino acid of gp-340 is shown in I. The presence of that gp-340 collagen-like However, gp-340 was not by not and the of in the amino acid of gp-340 be to of SP-D in the gp-340 acid of not in a new not The specificities of the monoclonal and polyclonal antibodies against gp-340 was by Western of both purified gp-340 and lung lavage separated on in the reduced and unreduced state The monoclonal antibody against gp-340 with both bands of 340 kDa well in the reduced and unreduced and it also with gp-340 The antibody with both bands of 340 in to the monoclonal it binding of the and binding the was reduced and 4 Western that the monoclonal antibody also with gp-340 in lung washings from four bronchioalveolar lavage for other than alveolar not of human lung gp-340 on and within the alveolar macrophages The staining was with both of However, staining with the monoclonal antibody was than that obtained with the polyclonal of with monoclonal antibody identified the gp-340 cells as cells to the cell not and were In the presence of calcium, human SP-D binds to with gp-340 The binding was and on the concentration of SP-D and on the of gp-340 on the In the presence of calcium, human SP-D binds to with mannan The binding was also on the concentration of SP-D and be inhibited by by the addition of 100 The binding of human SP-D to with gp-340 was also inhibited by 100 mm was in the presence of calcium, of binding was of and on the binding of SP-D to gp-340 and were incubated with of SP-D in the presence of and 100 mm with 10 mm were incubated with of SP-D in the presence of and 100 mm with 10 mm SP-D was detected by of anti-human SP-D, followed by Ig and Like SP-D, also mannan in the presence of calcium, and the binding was inhibited by by the addition of 100 mm also gp-340 in the presence of calcium, and the binding was inhibited by 100 mm was in the presence of calcium, of binding was The of the polyclonal against gp-340 completely the SP-D binding to gp-340 with gp-340 were incubated with the to addition of SP-D not The present the and purification of an protein from obtained from a patient with alveolar proteinosis. The molecule has been gp-340 it is a glycoprotein of apparent molecular mass of 340 kDa on In to the molecular mass of 340 kDa seen in the reduced gp-340 showed an apparent molecular mass of 290 kDa in the unreduced state. This that disulfide bonds have been by gp-340 as but gp-340 be a membrane protein for from the surface of the in be by within polypeptide chain of The apparent molecular of gp-340 was by gel to be than kDa. This that gp-340 is a molecule, thereby to an of its molecular mass on gel that it as an of identical linked polypeptide chains. Partial amino acid showed the presence of scavenger-receptor type in both bands A and and identical were obtained from the The of the of these bands also be to of the molecule to has been described for of the scavenger receptor superfamily J.M. Zhang Y. Willis A.C. Mason Eur. J. Immunol. 1993; Scholar). The of the in the showed that gp-340 belongs to group of the scavenger-receptor superfamily D. A. M. Biochem. 1994; 5-8Google Scholar). of this group are proteins and many of are on cells as macrophages. has been described for these a role in the primary system has been on the of their localization on cells D. A. M. Biochem. 1994; 5-8Google Scholar). of the scavenger-receptor and showed an high homology with gp-340, and an between from these molecules and from gp-340 is shown in is a molecule which is the primary of the A.C. Biochem. J. 1995; Scholar). SRCR are found in with short found in which was from a J. Biol. Chem. 1995; 270: is also a protein containing four a domain, a and a short H. M. H. Scholar) has an domain to but it has eight SRCR of four and of The of which is the of the three a protein of amino acid with a molecular mass of kDa and an apparent molecular mass of kDa in the reduced state on The molecular mass of reduced and gp-340 was to be 300 kDa reduced and unreduced mass kDa and purified mass a from together with molecular not A high degree of homology was found both between the SRCR of and and of gp-340 amino acid sequence between SRCR and 4 of and with SRCR 3 of and the amino acid in of gp-340) However, as the same high degree of homology is seen on of the proteins of and between in of the between gp-340 and the and the it that gp-340 is the human of these was in cells lining the and the and the and was detected in the lung H. M. H. Scholar). gp-340 was used to and and the monoclonal and polyclonal antibodies were used for the immunohistochemical localization of In the lung, both antibodies a staining of alveolar with the staining at the cell but in to be were also This staining pattern is in with be for a receptor for SP-D. has been shown that SP-D is found in the of and human alveolar and a receptor for SP-D be internalized together with SP-D and via the of SP-D E. Kuan S.F. Persson A. Müller B. von Wichert P. Lung Surfactant: Basic Research in the Pathogenesis of Lung Disorder. Karger, Basel1994: 123-140Google Scholar, T. Y. Ogasawara Y. Van Golde L.M.G. J. 1992; Scholar). is also to the proteins containing of type are to be membrane and both and are to have both a form and a form to of the molecules of the molecules J. Biol. Chem. 1995; 270: Scholar, H. M. H. Scholar) Direct binding of SP-D to purified gp-340 was observed at physiological ionic The binding was and on the presence of was observed on the addition of the maltose. In binding of mannan to SP-D was completely inhibited by maltose. The same binding pattern was seen between of the and of and be to binding of the of SP-D to carbohydrates on The that the binding between SP-D and gp-340 is a protein-protein interaction rather than a lectin-carbohydrate and the that the is also that the binding is within the neck-CRD The of the of with an binding Kahn R. R. K. 1991; Scholar, K. Nature. 1992; Scholar). The in 2 bonds with the carbohydrate bonds were found for the in the amino acid in and carbohydrate binding in are present in human SP-D. of the first binding has so far been for that this binding be for other than is to that which has CRD, is the for which has three H. von I. W. J.R. K. Nature. 1991; Scholar). has been shown that the binding of SP-D to alveolar macrophages can be completely by that SP-D in these to the cells via a interaction E. Kuan S.F. Persson A. Müller B. von Wichert P. Lung Surfactant: Basic Research in the Pathogenesis of Lung Disorder. Karger, Basel1994: 123-140Google Scholar). SP-D also the in alveolar macrophages (18Van Iwaarden J.F. Shimizu H. Van Golde P.H.M. Voeller D.R. Van Golde L.M.G. Biochem. J. 1992; 286: 5-8Google a which was also inhibited by These that than receptor for SP-D is present on alveolar macrophages. We conclude that gp-340 is the form of a molecule that binds SP-D in a calcium-dependent The gp-340 molecule is a receptor for SP-D that has been from the surface of the alveolar macrophages. We and for