Isolation and characterization of a new member of the scavenger receptor superfamily, glycoprotein-340 (gp-340), as a lung surfactant protein-D binding molecule.
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 x 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.
Holmskov
U
U
Department of Medical Microbiology, Institute of Medical Biology, University of Odense, DK-5000 Odense, Denmark. holmskov@imbmed.ou.dk
Lawson
P
P
Teisner
B
B
Tornoe
I
I
Willis
A C
AC
Morgan
C
C
Koch
C
C
Reid
K B
KB
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry*; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D004492:Edetic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008320:Maltose / Q000378:metabolism; D008351:Mannans / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011649:Pulmonary Alveolar Proteinosis / Q000378:metabolism; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*
D000906:Antibodies; D002352:Carrier Proteins; D006023:Glycoproteins; D008351:Mannans; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants; D011971:Receptors, Immunologic; C116303:glycoprotein 340; D008320:Maltose; D004492:Edetic Acid; D002118:Calcium