Altered carbohydrate recognition specificity engineered into surfactant protein D reveals different binding mechanisms for phosphatidylinositol and glucosylceramide.
Pulmonary surfactant protein D (SP-D) is a member of the collection subgroup of the C-type lectin superfamily that binds glycosylated lipids such as phosphatidylinositol (PI) and glucosylceramide (GlcCer). We have previously reported that the carbohydrate recognition domain of SP-D plays an essential role in lipid binding. However, it is unclear how the carbohydrate binding property of SP-D contributes to the lipid binding. To clarify the relationship between the lectin property and the lipid binding activity of rat SP-D, we expressed wild-type recombinant rat SP-D (rSP-D) and a mutant form of the protein with substitutions Glu-321-->Gln and Asn-323-->Asp (SP-DE321Q,N323D) in CHO-K1 cells. The indicated mutations have previously been shown to change the carbohydrate binding specificity of surfactant protein A and mannose-binding protein from mannose > galactose to the converse. rSP-D expressed in mammalian cells was essentially identical to native rat SP-D in its lipid and carbohydrate binding properties. In contrast, SP-DE321Q,N323D was unable to bind GlcCer, but retained binding activity toward PI liposomes and solid-phase PI. The efficiency of SP-DE321Q,N323D binding to PI liposome was approximately 50% of that of rSP-D in the presence of 5 mM Ca2+, but equivalent at 20 mM Ca2+. Carbohydrates competed for SP-D binding to PI such that maltose > galactose for rSP-D, and the order was reversed for SP-DE321Q,N323D. Furthermore, SP-DE321Q,N323D could bind to digalactosyldiacylglycerol more effectively than rSP-D. These results suggest the following. 1) The carbohydrate binding specificity of SP-DE321Q,N323D was changed from a mannose-glucose type to a galactose type; 2) the GlcCer binding property of SP-D is closely related to its sugar binding activity; and 3) the PI binding activity is not completely dependent on its carbohydrate binding specificity.
Ogasawara
Y
Y
Lord and Taylor Laboratory for Lung Biochemistry, Anna Perahia Adatto Clinical Laboratories, USA.
Voelker
D R
DR
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D050260:Carbohydrate Metabolism; D002855:Chromatography, Thin Layer; D006224:Cricetinae; D005963:Glucosylceramides / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008027:Light; D008081:Liposomes; D010716:Phosphatidylinositols / Q000378:metabolism*; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012542:Scattering, Radiation
D005963:Glucosylceramides; D006023:Glycoproteins; D008081:Liposomes; D010716:Phosphatidylinositols; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants; D011994:Recombinant Proteins