CA2-DEPENDENT BINDING OF ANNEXIN-IV TO SURFACTANT PROTEIN-A AND LAMELLAR BODIES IN ALVEOLAR TYPE-II CELLS()

Surfactant protein A (SP-A), a lung-specific glycoprotein in pulmonary surfactant, is synthesized and secreted from the alveolar type LI cel ls. It has been shown that SP-A is a Ca2+- binding protein with severa l binding sites and that the high-affinity site(s) is located in the C -terminal region of SP-A. In the present study we isolated the protein s from bovine lung soluble fraction that bind to SP-A in a Ca2+-depend ent manner using DEAE-Sephacel and SP-A-conjugated Sepharose 4B. At le ast three different protein bands with molecular masses of 24.5, 32, a nd 33 kDa were observed on SDS/PAGE. The main protein, with molecular mass of 32 kDa, was identified as annexin IV by the partial-amino-acid -sequence analyses and an immunoblot analysis with anti-(annexin IV) a ntiserum. We also found from the immunoblot analysis that the cytosoli c fraction of isolated rat alveolar type II cells contains annexin IV. In addition, when rat lung cytosol was loaded on to the lung lamellar body-conjugated Sepharose 4B in the presence of Ca2+, two proteins, w ith molecular masses of 32 and 60 kDa on SDS/PAGE respectively, were e luted with EGTA. The 32 kDa protein was shown to be annexin IV by an i mmunoblot analysis with the antiserum against annexin IV. The lung ann exin IV augmented the Ca2+-induced aggregation of the lung lamellar bo dies from rats. However, the augmentation of aggregation of the lung l amellar bodies by annexin IV was attenuated when the lamellar bodies w ere preincubated with polyclonal anti-SP-A antibodies. SP-A bound to a nnexin IV under conditions where contaminated lipid was removed. These results suggest that SP-A bound to annexin TV based on protein-protei n interaction, though both proteins are phospholipid-binding proteins. All these findings suggest that the interaction between SP-A and anne xin IV may have some role in alveolar type II cells.