We investigated the cellular and subcellular distribution of surfactant protein D (SPD) by immunogold l a w g in lungs of adult rats that had been given bovine s e " albumin coupled to 5-nm gold (BSAG) for 2 hr to visualize the endocytotic pathway. Specific gold labeling for SPD was found in alveolar Type II cells, Clara cells, and alveolar macrophages. In Type II cells abundant labeling was observed in the endoplasmic reticulum, whereas the Golgi complex and multivesicular bodies were labeled to a limited extent only. Lamellar bodies did not seem to contain SPD. Gold labeling in alveolar macrophages was restricted to structures containing endocytosed BSAG. In Clara ceh labeling was found in the endoplasmic reticulum, the Golgi complex, and was most ,ntroductionPulmonary surfactant is a complex mixture of lipids and proteins (1,2) which prevents the alveoli from collapsing at end expiration (3). Surfactant is synthesized by alveolar Type I1 cells, in which it is stored in lamellar bodies. On fusion of the limiting membrane of lamellar bodies with the apical plasma membrane, pulmonary surfactant is secreted into the alveolar space, where it is transformed into tubular myelin (4). Some surfactant constituents are not synthesized solely by the alveolar Type I1 cell but also by non-ciliated broncheolar epithelial (Clara) cells (5,6).Surfactant protein D (SP-D) is a collagenous surfactant-associated glycoprotein which is synthesized and secreted by freshly isolated rat Type I1 cells and has been isolated from rat bronchoalveolar lavage fluids (7-9). In rat lung it is synthesized as a 39.3 KD primary translation product that undergoes post-translational N-linked glycosylation, as well as hydroxylation and glycosylation of lysine residues within the collagenous domain (10). SP-D is secreted as a 43 KD protein and is isolated from bronchoalveolar lavage as a prominent in granules present in the apical domain of the cell. Double labeling experiments with anti-surfactant protein A (SPA) showed that both SPA and SPD were present in the same granules. However, SPA was distributed throughout the granule contents, whereas SPD was confined to the periphery of the granule. The Clara cell granules are considered seaetory granules and not lysosomes, because they were not labeled for the lysosomal markers cathepsin D and LGP120, and they did not contain endocytosed BSAG. multimeric complex of disulfide-bonded trimers composed of apparently identical subunits. SP-D is a calcium-dependent carbohydrate binding protein (9) which is structurally related to surfactant protein A (SP-A), conglutinin, and other collagenous C-type lectins (11,12). The structural similarities of SP-D and SP-A suggest that these two proteins have similar functions. Both SP-A (13) and SP-D (14) have been found immunohistochemically in alveolar Type I1 cells and Clara cells of the rat lung at the light microscopic (LM) level, and recently SP-D has been localized in Type I1 cells and in the airspace of silica-treated rats at the electron microscopic (EM) level (...

Pulmonary surfactant proteins A (SP-A

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.

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