Human surfactant protein A with two distinct oligomeric structures which exhibit different capacities to interact with alveolar type II cells

The lung lavage fluids from patients with pulmonary alveolar proteinosis have been generally used as a source for human surfactant protein A (SP-A). We have recently found that a multimerized form of SP-A oligomer (alveolar proteinosis protein-I, APP-I) exists besides the normal-sized octadecamer (APP-II) in SP-As isolated from the patients. When analysed by Bio-Gel A15m column chromatography in 5 mM Tris buffer (pH 7.4), the apparent molecular masses of APP-I and APP-II were 1.65 MDa and 0.93 MDa, respectively. Gel-filtration analysis also revealed that APP-II is clearly separated from APP-I in the presence of 2 mM Ca2+ and 150 mM NaCl. We investigated the abilities of both SP-A oligomers to regulate phospholipid secretion and to bind to alveolar type II cells. Although APP-I inhibited lipid secretion, it was clearly a less effective inhibitor than APP-II. IC50 for inhibition of lipid secretion was apparently 0.23 +/- 0.08 mu g/ml (0.14 +/- 0.05 nM) and 0.055 +/- 0.019 mu g/ml (0.059 +/- 0.020 nM) for APP-I and APP-II, respectively. Both proteins bound to monolayers of type II cells in a concentration-dependent manner; however, APP-I clearly had a lower affinity to bind to type II cells. The apparent dissociation contants were, K-d = 2.31 +/- 0.70 mu g/ml (1.40 +/- 0.43 nM) and 0.89 +/- 0.22 mu g/ml (0.95 +/- 0.24 nM) for APP-I and APP-II, respectively. Excess unlabelled rat SP-A replaced 45% of I-125-APP-I and 77% of I-125-APP-II for type II cell binding. Although I-125-APP-II competed with excess unlabelled APP-I or APP-II, I-125-APP-I failed to compete and instead its binding rather increased in the presence of unlabelled APPs. The biotinylated APP-I bound to APP-I and APP-II coated on to microtitre wells in a concentration-dependent manner, indicating that APP-I interacts with APPs. This study demonstrates that the multimerized form of human SP-A oligomer exhibits the following attributes: (1) the reduced capacity to regulate phospholipid secretion from type II cells, and (2) lower affinity to bind to type II cells, and that the integrity of a flower-bouquet-like octadecameric structure of SP A oligomer is important for the expression of full activity of this protein, indicating the importance of the oligomeric structure of mammalian lectins with collagenous domains.