Structural and Functional Determinants of Rodent and Human Surfactant Protein A: A Synthesis of Binding and Computational Data.

Surfactant protein A (SP-A) provides surfactant stability, first line host defense, and lung homeostasis by binding surfactant phospholipids, pathogens, alveolar macrophages (AMs), and epithelial cells. Non-primates express one SP-A protein whereas humans express two: SP-A1 and SP-A2 with core intra- and inter-species differences in the collagen-like domain. Here, we used macrophages and solid phase binding assays to discern structural correlates of rat (r) and human (h) SP-A function. Binding assays using recombinant rSP-A expressed in insect cells showed that lack of proline hydroxylation, truncations of amino-terminal oligomerization domains, and site-directed serine (S) or alanine (A) mutagenesis of cysteine 6 (C6S), glutamate 195 (E195A), and glutamate 171 (E171A) in the carbohydrate recognition domain (CRD) all impaired SP-A binding. Replacement of arginine 197 with alanine found in hSP-A (R197A), however, restored the binding of hydroxyproline-deficient rSP-A to the SP-A receptor SP-R210 similar to native rat and human SP-A. calculation of Ca coordination bond length and solvent accessibility surface area revealed that the "humanized" R197A substitution alters topology and solvent accessibility of the Ca coordination residues of the CRD domain. Binding assays in mouse AMs that were exposed to either endogenous SP-A or hSP-A1 (6A) and hSP-A2 (1A) isoforms revealed that mouse SP-A is a functional hybrid of hSP-A1 and hSP-A2 in regulating SP-A receptor occupancy and binding affinity. Binding assays using neonatal and adult human AMs indicates that the interaction of SP-A1 and SP-A2 with AMs is developmentally regulated. Furthermore, our data indicate that the auxiliary ion coordination loop encompassing the conserved E171 residue may comprise a conserved site of interaction with macrophages, and SP-R210 specifically, that merits further investigation to discern conserved and divergent SP-A functions between species. In summary, our findings support the notion that complex structural adaptation of SP-A regulate conserved and species specific AM functions in vertebrates.