Comparison of lipid aggregation and self-aggregation activities of pulmonary surfactant-associated protein A

1. We compared the Ca2+ dependence of the self-aggregation of surfactant protein A (SP-A) with that of vesicle aggregation induced by SP-A. The Ca2+ concentration required for half-maximal activity of lipid aggregation was 0.74 +/- 0.29 microM (n = 4) for pig SP-A and 98 +/- 5 microM (n = 2) for dog SP-A. In contrast, the threshold concentration of Ca2+ required to induce self-association of both pig and dog SP-A was 0.5 mM. The Ca2+ concentration needed for half-maximal self-association was 2.36 +/- 0.15 mM (n = 4) and 0.70 +/- 0.06 mM (n = 2) for pig and dog SP-A respectively. 2. We also compared the effect of Ca2+ on the trypsin sensitivity of lipid-free and membrane-bound SP-A. At 1 microM Ca2+, the tryptic digestion patterns of dog and pig lipid-free SP-A were quite different. Dog SP-A was very sensitive to proteolysis, being almost completely digested by 30 min, while pig SP-A was very resistant, even after 12 h. After protein aggregation of lipid-free SP-A (at 5 mM Ca2+), the accessibility of the trypsin cleavage targets of the protein depended on the SP-A species (self-aggregated pig SP-A became more sensitive to degradation than its non-aggregated form, whereas self-aggregated dog SP-A was less susceptible). In contrast, membrane-bound SP-A, from either pig or dog, was clearly protected from trypsin degradation at both low (1 microM) or high (1 mM) Ca2+ concentrations. The protection was slightly higher at 1 mM Ca2+ when the extent of lipid/SP-A aggregates was maximal. 3. On the other hand, vesicle aggregation activity of SP-A was decreased by 30-40% by removing the oligosaccharide moiety of the protein, whereas self-aggregation was not influenced by deglycosylation. The presence of mannan (at concentrations not lower than 10 micrograms/microliters) decreased vesicle aggregation induced by dog and pig SP-A by a mechanism that is independent of the binding of mannan to the carbohydrate-binding domain of SP-A. Self-aggregation of SP-A was not affected by the presence of sugars. 4. From these results, we conclude that: (1) the process of lipid aggregation induced by SP-A cannot be correlated with that of self-association of the protein occurring at supramillimolar concentrations of Ca2+; and (2) the N-linked carbohydrate moiety of SP-A and the ability of SP-A to bind carbohydrates are not involved in lipid aggregation.