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.
Highlights
The primary structure of rat SP-D is characterized by four distinct domains: 1) an N-terminal region involved in intermolecular disulfide bonding, 2) a collagenous domain composed of 59 Gly-X-Y repeats, 3) a neck domain, and 4) a carbohydrate recognition domain (CRD) [3]
The purpose of this study was to examine the relationship between the carbohydrate binding property and the lipid binding activity of SP-D
We have previously reported that the region of SP-D responsible for the lipid binding property is located in the CRD [18]
Summary
In an effort to understand the contribution of the lectin property of SP-D to glycolipid binding activity, we introduced mutations into rat SP-D (Glu-321 ~ GIn and Asn-323 ~ Asp) to alter the sugar binding specificity of the molecule Using this mutant form of SP-D (SP_DE321Q.N323D) expressed in CHO-Kl galactosyldiacylglycerol; PAGE, polyacrylamide gel electrophoresis; GM2, II"NeuAc-GgOsesCer. Using this mutant form of SP-D (SP_DE321Q.N323D) expressed in CHO-Kl galactosyldiacylglycerol; PAGE, polyacrylamide gel electrophoresis; GM2, II"NeuAc-GgOsesCer Direct Bin d ing of Recom bina nt Protein s 10 Mult ilam ellar Lipo - s(J/l/es- IOO /l g of mu lti la mella r liposomes wer e in cub ated with 0.1 /l g of :l"S-la heled recom hinant protein s in TIl S conta in ing 5 mM ca lcium a nd 2'h bovine se ru m a lhumin (bind ing buffer ) for I h at room temperature. Si ze fra ct ion ati on of nati ve a nd recombina nt proteins WIlS performed by g-el filtrat ion chroma tog-ra ph y using Bie-Ge l A-15m ( 1.5 ... 100-cm colum n) in thr pr esence of 10 rnxt EDTA. 0.15 ~t Na CI a t room temperature
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