Ca(2+)-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 II cells. It has been shown that SP-A is a Ca(2+)-binding protein with several 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 proteins from bovine lung soluble fraction that bind to SP-A in a Ca(2+)-dependent manner using DEAE-Sephacel and SP-A-conjugated Sepharose 4B. At least three different protein bands with molecular masses of 24.5, 32, and 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) antiserum. We also found from the immunoblot analysis that the cytosolic 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, with molecular masses of 32 and 60 kDa on SDS/PAGE respectively, were eluted with EGTA. The 32 kDa protein was shown to be annexin IV by an immunoblot analysis with the antiserum against annexin IV. The lung annexin IV augmented the Ca(2+)-induced aggregation of the lung lamellar bodies from rats. However, the augmentation of aggregation of the lung lamellar bodies by annexin IV was attenuated when the lamellar bodies were preincubated with polyclonal anti-SP-A antibodies. SP-A bound to annexin IV under conditions where contaminated lipid was removed. These results suggest that SP-A bound to annexin IV based on protein-protein interaction, though both proteins are phospholipid-binding proteins. All these findings suggest that the interaction between SP-A and annexin IV may have some role in alveolar type II cells.
D000595:Amino Acid Sequence; D000818:Animals; D017314:Annexin A4 / Q000378:metabolism*; D015153:Blotting, Western; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry*; D002118:Calcium / Q000494:pharmacology*; D002449:Cell Aggregation / Q000187:drug effects; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D011510:Proteolipids / Q000378:metabolism*; D011649:Pulmonary Alveolar Proteinosis / Q000378:metabolism; D011650:Pulmonary Alveoli / Q000378:metabolism*; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D017314:Annexin A4; D006023:Glycoproteins; D010446:Peptide Fragments; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D002118:Calcium