Alanine mutagenesis of surfactant protein A reveals that lipid binding and pH-dependent liposome aggregation are mediated by the carbohydrate recognition domain.

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.