Abstract
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. In silico 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 (6A2) and hSP-A2 (1A0) isoforms in vivo 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.
Highlights
Surfactant protein A (SP-A) is the most abundant lipid binding and immune-surveillance component of pulmonary surfactant
We used deletion and point mutants of insect cell expressed rat SP-A to map SP-A binding to macrophages and recombinant SPR210 compared to native human or rat SP-A isolated from bronchoalveolar lavage (BAL)
Point mutation of cysteine 6 to serine in the amino terminal peptide diminished Bmax with similar binding affinity to both macrophages and SP-A receptor 210 (SP-R210) compared to rSP-Ahyp
Summary
Surfactant protein A (SP-A) is the most abundant lipid binding and immune-surveillance component of pulmonary surfactant. Deletion, sitedirected mutagenesis, and ligand competition studies showed that the amino-terminal and collagen-like domains influence receptor occupancy and functional responses in alveolar type II epithelial cells and macrophages, and interaction of the CRD domain with surfactant phospholipids [10,11,12,13,14,15,16]. SP-A binds different pathogen ligands such as the lipid A portion of lipopolysaccharide on Gram-negative bacteria [7], surface proteins and glycolipids on gram positive bacteria [22], mycobacteria [23, 24], and fungi [25, 26] These interactions facilitate pathogen clearance through agglutination, opsonization, and direct killing [22, 26,27,28,29]. SP-A enhances opsonic and non-opsonic phagocytosis [22, 30,31,32], and shapes pathogen-dependent polarization of inflammatory responses through the SP-R210 SP-A receptor (aka Myo18A or CD245) [22, 27, 33] in macrophages
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