Interactions of pulmonary surfactant protein SP-A with monolayers of dipalmitoylphosphatidylcholine and cholesterol: roles of SP-A domains

Pulmonary surfactant protein A (SPA) is an oligomeric glycoprotein that binds dipalmitoylphosphatidylcholine (DPPC), Interactions of rat SP-A and recombinant SP-As with pure and binary monolayers of DPPC and cholesterol were studied using a rhomboid surface balance at 37 degrees C. A marked inflection at equilibrium surface tension (23 mN/m) in surface tension-area isotherm of a pure DPPC film was abolished by rat SP-A. The inflection was decreased and shifted to 18 mN/m with wild-type recombinant SP-A (SP-A(hyp)). Both rat SP-A and SP-A(hyp) decreased surface area reduction required for pure DPPC films to reach near zero surface tension from 30 to 25%. SP-A(hyp,E195Q,R197D), mutated in carbohydrate recognition domain (CRD) known to be essential for SP-A-vesicle interactions, conveyed a detrimental effect on DPPC surface activity. SP-A(Delta G8-P80), With deletion of collagen-like domain, had little effect. Both SP-A(hyp,C6S) (Ser substitution for Cys(6)) and SP-Ahyp,(Delta N1-A7) (N-terminal segment deletion) which appear mainly as monomers on non-reducing SDS-PAGE analysis, increased required surface area reduction for minimal surface tension. All SP-As reduced collapse surface tension of a pure cholesterol film from 27 to 23 mN/m in the presence of Ca2+. When mixed films were formed by successive spreading of DPPC/SP-A/cholesterol, rat SP-A, SP-A(hyp), Or SP-A(Delta G8-P80) blocked the interaction of cholesterol with DPPC; SP-A(hyp,E195Q,R197D) could not impede the interaction; SP-A(hyp,C6S) or SP-A(hyp,Delta N1-A7) only partially blocked the interaction, and cholesterol appeared to stabilize SP-A(hyp,C6S)-DPPC association. These results demonstrate the importance of CRD and N-terminal dependent oligomerization in SP-A-phospholipid associations. The findings further indicate that SP-A-cholesterol interactions differ from SP-A-DPPC interactions and may be nonspecific.