Abstract
Pulmonary surfactant proteins A (SP-A) and D (SP-D) are collectins in the C-type lectin superfamily. SP-A binds to dipalmitoylphosphatidylcholine and galactosylceramide, and it regulates the uptake and secretion of surfactant lipids by alveolar type II cells. In contrast, SP-D binds to phosphatidylinositol (PI) and glucosylceramide (GlcCer). We investigated the functional region in the carbohydrate recognition domain of rat SP-A and SP-D that is involved in binding lipids and interacting with alveolar type II cells by using chimeric proteins. Chimeras ad3, ad4, and ad5 were constructed with SP-A/SP-D splice junctions at Gly194/Glu321, Gln173/Thr300, and Met134/Cys261, respectively. All three chimeras lost SP-A-specific functions. Chimeras ad3, ad4, and ad5 bound to PI with increasing activity. In contrast, chimeras ad3 and ad4 did not bind to GlcCer, whereas ad5 avidly bound this lipid. From these results, we conclude that 1) the SP-A region of Glu195-Phe228 is required for lipid and type II cell interactions, 2) the SP-D region of Cys261-Phe355 is required for optimal lipid interactions, and 3) the structural requirement for the binding of SP-D to PI is different from that for GlcCer.
Highlights
Pulmonary surfactant is a complex mixture of lipids and proteins that acts to keep alveoli from collapsing during expiration [1]
We show that the Surfactant protein A (SP-A) region of Glu195–Phe228 is required for the interaction of SP-A with lipids and type II cells and that the structural requirement for the binding of Surfactant protein D (SP-D) to PI is different from that for its GlcCer binding
Under nonreducing conditions the recombinant proteins (SP-A and SP-A/SP-D chimeras) showed significant levels of monomer that are a consequence of incomplete covalent oligomerization. wt SP-D migrated as a band at 43 kDa under reducing condition. wt SP-D appeared mainly as a monomer with smaller populations of oligomers under nonreducing conditions (Fig. 2, A and B, lanes wt/D), which is consistent with the result observed in the previous study [33]
Summary
Pulmonary surfactant is a complex mixture of lipids and proteins that acts to keep alveoli from collapsing during expiration [1]. SP-A and SP-D belong to the collectin subgroup of the C-type lectin superfamily, along with mannose-binding protein (MBP), bovine conglutinin, and the protein CL43 [28] These proteins possess characteristic primary structures that are organized into four domains: 1) a cysteine-containing amino terminus, 2) a collagen-like domain, 3) a neck domain, and 4) a carbohydrate recognition domain (CRD) [2]. Lung collectins possess homologous structures and exhibit similar carbohydrate binding specificities, they show quite different properties of binding lipids and interacting with alveolar type II cells. The purpose of this study was to investigate the functional region in the CRD of rat SP-A or SP-D for the interactions with lipids and type II cells by using chimeric proteins of SP-A and SP-D. We show that the SP-A region of Glu195–Phe228 is required for the interaction of SP-A with lipids and type II cells and that the structural requirement for the binding of SP-D to PI is different from that for its GlcCer binding
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