THE STRUCTURE AND FUNCTION OF SURFACTANT PROTEIN-A - HYDROXYPROLINE-DEFICIENT AND CARBOHYDRATE-DEFICIENT MUTANT PROTEINS

Pulmonary surfactant protein A (SP-A) regulates the uptake and secreti on of phospholipid by alveolar type II cells and is an important compo nent of surfactant lipid aggregates. In an attempt to understand how s pecific structural domains of SP-A relate to the function of the prote in, we used site-directed mutagenesis of the cDNA for SP-A and heterol ogous expression with baculovirus vectors. Synthesis of the wild type SP-A in insect cells resulted in a form of the protein in which prolin e residues were not hydroxylated and that is denoted Sp-A(hyp). Three mutant SP-As with substitutions in the consensus sequences for glycosy lation (Sp-A(hyp,glc)) to prevent N-linked oligosaccharide attachment at Asn(1) and Asn(187) were produced, individually and in tandem. The Sp-A(hyp) was glycosylated at both the Asn(1) and Asn(187) positions, demonstrated partial sulfhydryl-dependent oligomerization, and formed incomplete oligomers in solution. The Sp-A(hyp) and Sp-A(hyp,glc) boun d to immobilized carbohydrate and to phospholipid liposomes and partia lly competed for occupancy of a plasma membrane receptor for SP-A. The SP-A(hyp) and the Sp-A(hyp,glc) were equally effective inhibitors of the secretion of surfactant lipids from isolated type II cells (IC50 = 0.5 mu g/ml) and aggregated phospholipid liposomes at 20 degrees C. A ll of the recombinant SP As demonstrated markedly reduced aggregation of lipid at 37 degrees C. We conclude that the hydroxylation of prolin e residues is required for perfect oligomerization of SP-A and for the rmal stability in the interaction with lipid. Furthermore, recombinant SP-A is able to inhibit the secretion of phospholipid from isolated t ype II cells and to aggregate lipid vesicles independent of the presen ce of N-linked carbohydrate or the site of glycosylation.