Use of factorial experimental design to delineate the strong calcium- and pH-dependent changes in binding of human surfactant protein-A to neutral glycosphingolipids--a model for studies of protein-carbohydrate interactions.
Human surfactant protein-A (SP-A) is a C-type lectin belonging to the collection supergroup of mammalian lectins. It is produced by alveolar type II cells and has been shown to bind to lactosylceramide (R. A. Childs, J. R. Wright, G. F. Ross, C-T Yuen, A. M. Lawson, C. Chai, K. Drickamer, and T. Feizi (1992) J. Biol. Chem. 267, 9972-9979), galactosylceramide, and gangliotriaosylceramide (Y. Kuroki, S. Gasa, Y. Ogasawara, A. Makita, and T. Akino (1992) Arch. Biochem. Biophys. 299, 261-267). To evaluate the pH- and calcium-dependent binding of SP-A to neutral glycosphingolipids we employed a microtiter plate technique and performed a series of full factorial design experiments using lactosylceramide, galactosylceramide, and gangliotetraosylceramide and native nonderivatized SP-A. The optimal binding conditions were drastically different for these three receptors. At pH 7.4 and at 5 mM Ca concentration the binding affinity of SP-A followed the order galactosylceramide > lactosylceramide > gangliotetraosylceramide. However, this was close to optimal conditions for binding of SP-A to lactosylceramide and at minimum for binding to gangliotetraosylceramide. Binding of SP-A to galactosylceramide was relatively insensitive to pH but increased significantly with increasing Ca concentrations. These experiments using factorial experimental design emphasize the importance of critical interpretation of all earlier studies on protein-carbohydrate interactions especially when transferring experimental data into complex biological systems.
Hynsjö
L
L
Department of Clinical Chemistry and Transfusion Medicine, Göteborg University, Sahlgren's Hospital, Sweden.
Granberg
L
L
Haurum
J
J
Thiel
S
S
Larson
G
G
D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000737:chemistry; D002855:Chromatography, Thin Layer; D004285:Dogs; D006028:Glycosphingolipids / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D011485:Protein Binding; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D017186:Titrimetry / Q000379:methods
D001059:Apoproteins; D006028:Glycosphingolipids; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; C046616:pulmonary surfactant apoprotein; D002118:Calcium