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 collectin 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-D-E321Q,D-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-D-E321Q,D-N323D was unable to bind GlcCer, but retained binding activity toward PI liposomes and solid-phase PI. The efficiency of SP-D-E321Q,D-N323D binding to PI liposome was similar to 50% of that of rSP-D in the presence of 5 mM Ca2+, but equivalent at 20 mM Ca2+. Carbohydrats competed for SP-D binding to PI such that maltose > galactose for rSP-D, and the order was reversed for SPDE321Q,N323D. Furthermore, SP-D-E321Q,D-N323D could bind to digalactosyldiacylglycerol more effectively than rSP-D. These results suggest the following. 1) The carbohydrate binding specificity of Sp-D-E321Q,D-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.