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

Shou-Hwa Yu,Francis X Mccormack,Dennis R Voelker,Fred Possmayer

doi:10.1016/s0022-2275(20)32127-1

Abstract

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-Ahyp). Both rat SP-A and SP-Ahyp decreased surface area reduction required for pure DPPC films to reach near zero surface tension from 30 to 25%. SP-Ahyp,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-AΔG8-P80, with deletion of collagen-like domain, had little effect. Both SP-Ahyp,C6S (Ser substitution for Cys6) and SP-Ahyp,ΔN1-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-Ahyp, or SP-AΔG8-P80 blocked the interaction of cholesterol with DPPC; SP-Ahyp,E195Q,R197D could not impede the interaction; SP-Ahyp,C6S or SP-Ahyp,ΔN1-A7 only partially blocked the interaction, and cholesterol appeared to stabilize SP-Ahyp,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.

Highlights

Pulmonary surfactant protein A (SP-A) is an oligomeric glycoprotein that binds dipalmitoylphosphatidylcholine (DPPC)
In this study four additional variants of SP-A were used: 1) SPAhyp,E195Q,R197D was produced by substitution of Glu195 with Gln and Arg197 with Asp to alter the carbohydrate binding specificity of SP-Ahyp from mannose to galactose [36]; 2) SP-A⌬G8-P80 has a deletion of the collagen-like domain [27]; 3) SP-Ahyp,C6S, serine substitutes for Cys6 to prevent the formation of interchain-disulfide bonds [27]; 4) SP-Ahyp,⌬N1-A7 has a deletion of the amino terminal segment [28]
The surface tension remained constant upon addition of SP-Ahyp as shown in Table 1, rows 3 and 5. These results demonstrate the possible alteration in conformation of DPPC monolayers upon the addition of rat SP-A at 30 mN/m

Summary

MATERIALS AND METHODS

Dipalmitoyl-1-[14C]phosphatidylcholine was purchased from New England Nuclear (Markham, Ontario, Canada). Purified rat SP-A and recombinant SP-As, originally stored in aqueous media at Ϫ20ЊC, were thawed and dissolved in water– 2-propanol 2:1 before experiments. L-B films of DPPC in the presence of rat SP-A or recombinant SP-As were deposited on a 1 ϫ 1 cm microscope glass cover slip at equilibrium surface tension (ϳ23 mN/m) using the rhomboid surface balance at 37ЊC as described previously [24]. 30 ␮l of 0.4 mg/ml SP-A in water–2-propanol 2:1 was applied onto several locations of a 14C-labeled DPPC monolayer with a surface tension of 30–31 mN/m. An L-B film was deposited on the glass plate, which was lowered into the subphase of saline, 1.5 mm CaCl2 prior to the spreading of DPPC, by lifting the plate up slowly at 1 mm/min when the surface tension reached 23 mN/m. Autoradiographs of L-B films were produced by exposing the plates to Xray films for about 40 h at 4ЊC

RESULTS AND DISCUSSION

DPPC ϩ rat SP-A

CHOL ϩ SP-Ahyp