Altered carbohydrate recognition specificity engineered into surfactant protein D reveals different binding mechanisms for phosphatidylinositol and glucosylceramide

Abstract

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 wildtype 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,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,N323D) was unable to bind GlcCer, but retained binding activity toward PI liposomes and solid-phase PI. The efficiency of SP-D(E321Q,N323D) binding to PI liposome was ~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- D(E321Q,N323D). Furthermore, SP-D(E321Q,N323D) could bind to digalactosyldiacylglycerol more effectively than rSP-D. These results suggest the following. 1) The carbohydrate binding specificity of SP-D(E321Q,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.