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 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.

Interactions of Surfactant Proteins A and D withSaccharomyces cerevisiaeandAspergillus fumigatus

“…mutagenesis study confirmed that by changing amino acids Glu 185 and Asn 187 to Gln and Asp respectively, the protein's monosaccharide affinity could be changed from mannose/glucose Ͼ galactose to galactose Ͼ mannose/glucose (7). Analogous mutagenesis work has been done with SP-D, which also shows higher affinity for glucose than for galactose (29), with similar results (27). Together, the carbohydrate recognition specificity, mutagenesis results, and structural similarity strongly suggests that SP-D binds carbohydrates by a mechanism similar to that used by MBP-A.…”

Crystal Structure of Trimeric Carbohydrate Recognition and Neck Domains of Surfactant Protein A

“…Lipid Binding by SP-A-Mutagenesis studies have revealed that the phospholipid binding domain of SP-A overlaps with the carbohydrate binding domain (40,(43)(44)(45). Findings that SP-A discriminates between saturated and unsaturated acyl chains and exhibits specificity for the choline head group (26) imply that discrete binding sites exist for these moieties on the SP-A molecule.…”

A 50-kDa variant form of human surfactant protein D

“…SP-D has been reported to increase the aggregation and phagocytosis of bacteria and viruses [15]. In addition, SP-D has moderate affinity for phosphatidylinositol and glucosylceramide [9,16]. SP-D is synthesized by both type II and nonciliated bronchiolar cells in the rodent lung.…”

“…Mature multimeric SP-D has 12 carbohydrate recognition domains and is thought to function as a multivalent lectin [3]. The carbohydrate recognition domain of SP-D has specificity for maltose, mannose and glucose residues [9]. By means of this lectin property SP-D has been reported to bind a variety of viruses, bacteria, mycobacteria and fungi [10][11][12][13][14].…”