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

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.

Keywords: alveolar macrophages; binding; lung; receptor; surfactant protein A.

Figure 1

Partial sequence alignment of the CRD (A) and CDM (B) domains of rodent and human SP-A. (A) Alignment of the CRD domains of human SP-A2 1A⁰ variant (hSP-A2) and rat SP-A carbohydrate recognition domain. Amino acid numbering is based on the mature peptide of rat SP-A. Arrows point to Ca⁺⁺-coordination residues. (B) Alignment of the collagen-like domains of human SP-A1 (6A²) (hSP-A1) and hSP-A2 1A⁰ variant, mouse SP-A (mSP-A), and rat SP-A (rSP-A). All alignments are made to hSP-A2 (1A⁰). The aligned sequences shown encompass the kink peptide (bolded residues 66–50) to the end of the CDM. Arrows point to core amino acids in the CDM that distinguish SP-A1 and SP-A2; the numbering is based on the precursor molecule. Residues shown in italicized red font show the sequence of embedded integrin binding motifs in the CDM. Sequence alignments were performed using ClustalW.

Figure 2

Impact of domain deletion and site directed mutagenesis on SP-A binding to macrophages. Binding assays used Raw264.7 macrophages in suspension. Cells were incubated with increasing concentrations of either radiolabeled recombinant rat SP-A proteins (A–D) or human SP-A (E,F) purified from alveolar proteinosis lavage. Binding data were fitted by non-linear regression to generate binding isotherms (A,C,E) or transformed into double reciprocal plots to evaluate SP-A binding behavior (B,D,F). Assays were carried out at 4°C for 1.5 h. at the 50,0000 cells/assay. Bound SP-A was separated by centrifugation over a silicon oil mixture to separate bound from free SP-A. Assays were performed in triplicate and data pooled from 2 independent experiments. Data shown are Means ± SE.

Figure 3

Impact of domain deletion and site-directed mutagenesis on SP-A binding to SP-R210. The recombinant SP-A binding domain of SP-R210 was coated onto microtiter well plates, and incubated with increasing concentration of indicated recombinant rat SP-A proteins (A–D), or native human or rat SP-A proteins (C,D). Bound protein was detected using an HRP-conjugated SP-A antibody. Binding data were fitted by non-linear regression to generate binding isotherms (A,C) or transformed into double reciprocal plots to evaluate SP-A binding behavior (B,D). Assays were performed in duplicate or triplicate and data pooled from 2 independent experiments. Data shown are Means ± SE.

Figure 4

Dynamics of CRD and neck-CRD domains. Plot of root mean square deviation (RMSD) of α-Carbon atoms over time for CRD (red trace) and neck-CRD (black trace).

Figure 5

The E195A substitution alters the Ca⁺⁺-coordination mode in the CLL binding pocket. Ribbon diagrams depicting the geometry of Ca⁺⁺-coordination site of the native (A) and mutant E195A NCRD (B). Red arrows point to the carbonyl oxygen in the R197 residue.

Figure 6

Murine and human SP-A variants are paracrine regulators of SP-A binding to alveolar macrophages in postnatal lung. Binding assays were carried out with alveolar macrophages obtained from either murine lung (A–D) or human lung (E,F) using radiolabeled human SP-A1 or SP-A2 in the presence (A,C,E) or absence of calcium (B,D,F). Alveolar macrophages from murine lungs (A–D) were obtained from WT (SP-A^+/+), SP-A-deficient (SP-A^−/−), or humanized mice expressing either SP-A1 variant 1A⁰ (SP-A^{−/−,hSFTPA2(1A0)} ), or SP-A2 variant 6A² (SP-A^{−/−,hSFTPA2(6A2)} ) in the absence of endogenous murine SP-A (Sftpa). Human alveolar macrophages were obtained from 6 month (E) and 20 years old (F) rejected transplant lungs. Assays were carried out at 4°C for 1.5 h at 50,0000 cells/assay. Bound SP-A was separated by centrifugation over a silicon oil mixture. Assays were performed in duplicate and data pooled from 2 independent experiments. Alveolar macrophages from mouse lungs were pooled from 10 mice per genotype to obtain sufficient number of cells per experiment. Binding isotherms were generated using Prizm software by non-linear regression analysis using the Hill equation. Data shown are Means ± SE.