Alanine Mutagenesis of Surfactant Protein A Reveals That Lipid Binding and pH-Dependent Liposome Aggregation Are Mediated by the Carbohydrate Recognition Domain

Abstract

The carbohydrate recognition domain (CRD) of surfactant protein A (SP-A) is critical for the modulation of surfactant secretion from isolated type II cells and for the Ca(2+)-dependent aggregation of surfactant liposomes, but the domains of SP-A that mediate lipid binding have not been precisely mapped. To determine the role of the CRD in lipid interactions and other functions, the conserved amino acids of the putative Ca2+ and carbohydrate binding site (Glu195, Glu202, Asn214, Asp215) were substituted with alanine. The wild-type recombinant protein, SP-Ahyp, and mutant SP-As SP-Ahyp,E195A, SP-Ahyp,E202A, SP-Ahyp,N214A, and SP-Ahyp,D215A, were expressed in insect cells using baculovirus vectors and compared functionally. The Ca(2+)-dependent binding and aggregation of liposomes at pH 7.0 by SP-Ahyp,N214A were comparable to SP-Ahyp, but these activities were blocked in SP-Ahyp,E195A, SP-Ahyp,E202A, and SP-Ahyp,D215A. In contrast, the SP-Ahyp,D215A but not the other mutant proteins induced the Ca(2+)-independent aggregation of phospholipid liposomes at pH 4.0. The mutant recombinant proteins did not compete with 125I-labeled rat SP-A for high-affinity receptor occupancy on isolated type II cells and were much less potent than SP-Ahyp as regulators of surfactant secretion and uptake from type II cells. We conclude that (1) lipid binding and pH-dependent liposome aggregation are mediated by the CRD of SP-A, (2) distinct but overlapping domains within the CRD are required for pH- and Ca(2+)-dependent liposome aggregation, and (3) conserved acidic and polar residues of the carbohydrate binding site of SP-A are essential for interactions with type II cells.

Crystal Structure of Trimeric Carbohydrate Recognition and Neck Domains of Surfactant Protein A

“…In contrast, in the SP-A complex with samarium, where both metal binding sites are occupied, the Glu-202 side chain can be clearly identified as one of the samarium-coordinating ligands. The finding that alanine substitution of Glu-202 blocks binding to calcium, phospholipid, and carbohydrate ligands is consistent with an important ligand-binding role for this residue (40,41) and suggests that the fully functional state contains calcium bound at both sites.…”

“…Lipid Binding by SP-A-Mutagenesis studies have revealed that the phospholipid binding domain of SP-A overlaps with the carbohydrate binding domain (40,(43)(44)(45). Findings that SP-A discriminates between saturated and unsaturated acyl chains and exhibits specificity for the choline head group (26) imply that discrete binding sites exist for these moieties on the SP-A molecule.…”

Deletion Mapping of N-terminal Domains of Surfactant Protein A

“…The CRD of the molecule contains binding sites for carbohydrate, the major surface glycoprotein of the pulmonary pathogen Pneumocystis carinii, the high affinity SP-A receptor on type II cells, and surfactant phospholipids, especially dipalmitoylphosphatidylcholine (DPPC) (21)(22)(23)(24). We have recently reported that the N-terminal domains that contribute to oligomeric assembly, including the interchain disulfide bond at Cys 6 and the collagen-like region, are essential for SP-A function (25).…”

Pulmonary Surfactant Protein-A (SP-A) Restores the Surface Properties of Surfactant after Oxidation by a Mechanism That Requires the Cys6 Interchain Disulfide Bond and the Phospholipid Binding Domain

“…Although initially controversial, considerable evidence has accrued indicating that the CRD of SP-A possesses both the carbohydrate-binding and PL-binding motifs (20). The two CRD mutants E195A and D215A used here reportedly lack the ability to bind or aggregate PL liposomes and exhibit reduced binding of carbohydrate beads or calcium (28). Nevertheless, some carbohydrate binding activity must remain, because the SP-As are purified on the basis of calcium-dependent binding on mannose columns.…”

“…The mutant rSP-A hyp-C6S (C6S), which has Cys 6 replaced by a serine, forms trimers but not larger oligomeric forms (29). The two CRD mutant proteins, rSP-A hyp-E195A (E195A) and rSP-A hyp-D215A (D215A), bear point mutations in amino acids predicted to contribute to coordination of a calcium ion involved in PL and carbohydrate binding (28). Although these SP-As have reduced ligand binding, they are isolated by mannose chromatography and consequently retain some carbohydrate binding activity (Fig.…”