Original Article

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

Correspondence to: Shou-Hwa Yu

Pulmonary surfactant protein A (SP-A) 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°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^G8-P80, with deletion of collagen-like domain, had little effect. Both SP-A^hyp,C6S (Ser substitution for Cys⁶) and SP-A^hyp,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^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,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.—Yu, S-H., F. X. McCormack, D. R. Voelker, and F. Possmayer. Interactions of pulmonary surfactant protein SP-A with monolayers of dipalmitoylphosphatidylcholine and cholesterol: roles of SP-A domains. J. Lipid Res. 1999. 40: 920–929.

Supplementary key words: air/water interface, monolayer, DPPC, SP-A, cholesterol, L-B film

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