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.

Human Surfactant Protein SP-A1 and SP-A2 Variants Differentially Affect the Alveolar Microenvironment, Surfactant Structure, Regulation and Function of the Alveolar Macrophage, and Animal and Human Survival Under Various Conditions

“…Consistent with this, SP-A2 is shown to exhibit a higher binding capacity to AM compared to SP-A1. Moreover, SP-A1, in contrast to SP-A2, showed no binding to AMs that were not previously exposed to SP-A (i.e., in KO AMs) (113). Collectively, these indicate potential complexities of SP-A interactions with AMs.…”

“…Collectively, these indicate potential complexities of SP-A interactions with AMs. SP-A1 may have an inhibitory effect on AM binding by SP-A2 since the SP-A2 binding capacity was reduced when AMs were exposed continually to SP-A1 prior to the assay (113).…”

Sex-Specific Regulation of Gene Expression Networks by Surfactant Protein A (SP-A) Variants in Alveolar Macrophages in Response to Klebsiella pneumoniae

“…A recent study showed that SP-A1 and SP-A2 differentially bind to AM. The maximal binding (Bmax) of SP-A2 to AM was shown to be 2-3 times higher than that of SP-A1 binding (127). Moreover, SP-A2 exhibits a higher ability to bind phagocytic cells, such as AMs and THP-1 cells than SP-A1, but neither SP-A1 or SP-A2 bound CHO cells, a non-phagocytic cell line (32), further supporting a differential cell-specific receptor binding.…”

Differences in the alveolar macrophage toponome in humanized SP-A1 and SP-A2 transgenic mice

“…Moreover, a higher degree of AM binding has been shown by SP-A2 than SP-A1, with no difference in binding between SP-A1 and SP-A2 observed when a nonphagocytic cell line was used ( 24 ), indicating that macrophage-specific cell surface proteins are involved in the differential binding. Furthermore, when binding kinetics were investigated ( 36 ), SP-A2 exhibited a markedly higher binding capacity to AM compared with SP-A1. SP-A1, in contrast to SP-A2, showed no binding to AMs that were not previously exposed to SP-A (i.e., in KO AMs), underscoring potential complexities of SP-A interactions with AMs.…”