Surfactant protein-C enhances lipid aggregation activity of surfactant protein-A.

Pulmonary surfactant is a heterogeneous complex of lipids and proteins that serves to stabilize the alveoli and distal airways at low lung volumes. Dipalmitoylphosphatidylcholine (DPPC) is widely accepted to be the major lipid component of pulmonary surfactant. In addition to DPPC, phosphatidylglycerol (PG) and specific proteins are required for the full biological activity of surfactant. At least three of these proteins (SP-A, SP-B, and SPC) are involved in the organization of surfactant phospholipids (see (11 for references). One of the effects of SP-A on phospholipid vesicles is that it induces vesicle aggregation in the presence of millimolar concentrations of Ca'+ [2]. The present study was undertaken to determine the effect of the hydrophobic surfactant proteins (SP-B and SP-C) on the lipid aggregation activity of SP-A. Pulmonary surfactant was prepared from bronchoalveo iar lavage from pig lungs and separated from Mood components 131. Porcine SP-A was purified from isdated surtactant [4] and the hydrophobic proteins SP-B and SP-C were isdated from a lipid extract from surfactant (LES) IS]. Quantification of SP-A. SP-B and SP-C was routinely performed by amino acid analysis. The amount of phospholipids (PL) from LES as well as its composition were determined, as described previously 131. The amounts of hydrophobic surfactant proteins from LES were also determined by amino acid analysis. In our LES preparation we found 0.7t0.1 nmol SPB/pmol PL and 1.8t0.3 nmol SP-C/pmol PL, (n=7). The hydrophobic protein (SP-BtSP-C)/PL weight ratio was 1,6*0.3 pg prot/lOO pg PL. Unilamelar vesicles of DPPC, DPPC/PG (7:3. w/w), and DPPC/phosphatidyiinositd (PI) (7:3, w/w) or vesicles from E S were prepared at a phosphdipid concentration of 0.5 mg/ml in a buffer containing 1OOmM NaU and 50 mM Tris/HU, pH 7.4. as described previously [4]. We chose DPPC/PI and DPPC/PG vesicles because both PI and PG are the major acidic lipids of pig lung surfactant representing 8.5i2.196 (n=5) and 4.2i1.796 (n=5) of all phosphdiplds respectively. Reconstitution of either SP-B or SPC In DPPC. DPPC/PG of DPPC/PI vesicles was performed at a protein to lipid weight ratio of 1 : l O 151. Assay of SP-A-induced lipid aggregation was performed at 31C, as fdlows: phosphdipid vesicles (30 pg) with or without SP-B or SP-C, were added to both the sample and the reference cwette in a total vdume of 0.3 ml of 50 mM TrislHCI buffer, pH 7.4, 100 mM NaCI. After equilibration for 10 min at 37'C. 3 pg of SP-A were added to the sample cuvette and the change in optical density at 400 nm was monitored in I-min intervals over 10 min. Next, Ca2' (1 mM final conc.) was added to both the sample and the reference cwene and the change in absorbance was monitored for 10 min. EDTA was used for either preventing (1 mM) or reverting (5 mM) vesicle aggregation induced by SP-A. Figure 1 shows that addition of SP-A to different types of vesicles (prepared with or without SP-B or SPC) or LESliposomes resulted in an immediate increase in light absorbance due to lipid aggregation. The presence of 1 mM EDTA in the assay buffer prevented lipid aggregation induced by SP-A. SP-A is a calcium binding protein and undergoes a conformational change in the presence of EDTA [4,6]. The Ca" concentration in our experimental aqueous systems was about 15 pM as measured by atomic absorption spectroscopy. At this concentration, the SP-A binding sites for Ca2+ are expected to be saturated [6] thus, stimulating the lipid aggregation activity of SPA. After addition of Ca" (1 mM), the light absorbance of SPA/vesicle aggregates (without SP-B or SP-C) increased by an additional 20-25%. This additional increase could be due to selfaggregation of the lipid-associated SP-A. Higher concentrations of Ca" did not increase light absorbance. When vesicles were prepared with SP-B or SP-C the additional increase caused by Ca2+ (1 mM) was 515%. In the case of LES, no increase was observed (Fig. 1). Interestingly, the extent of vesicle aggregation. did not depend on the lipid composition of the vesicles but it was markedly influenced by the presence of SP-C in the lipid preparation. The change in light absorbance caused by the 0.25