Partial truncation of the NH 2-terminus affects physical characteristics and membrane binding, aggregation, and fusion properties of annexin A7

Abstract

Annexin A7 (synexin, annexin VII), a member of the annexin family of proteins, causes aggregation of membranes in a Ca 2+-dependent manner and has been suggested to promote membrane fusion during exocytosis of lung surfactant, catecholamines, and insulin. Although annexin A7 (A7) was one of the first annexin proteins described, limited studies of its physical characteristics or of structural domains affecting any of its proposed functions have been conducted. As postulated for other annexin proteins, the unique NH 2-domain possibly determines the functional specificity of A7. Therefore, we evaluated the effects of segmental deletions in the NH 2-terminus on several characteristics associated with the COOH-terminus of A7. The COOH-terminus contains the only tryptophan residue, and all potential trypsin sites, and the Ca 2+ and phospholipid binding sites. Recombinant rat A7 and its deletion mutants were expressed using constructs based on the cDNA sequence obtained by screening a rat lung cDNA library. Ca 2+ increased the tryptophan fluorescence of A7 and caused a small red shift in the emission maximum (λmax), which was further increased in presence of phospholipid vesicles (PLV). NH 2-terminal deletions of 29, 51, and 109 residues affected the peak width of fluorescence and λmax, surface-exposure of tryptophan residue, and caused a smaller Ca 2+-dependent red shift in λmax of membrane-bound protein in comparison to A7. Limited proteolysis with trypsin showed that Ca 2+ increased the proteolysis of all proteins, but the deletions also affected the pattern of proteolysis. The presence of PLV protected against Ca 2+-dependent increase in proteolysis of all proteins. The deletion of first 29 residues also caused decreased membrane binding, aggregation, and fusion, when compared with A7. Collectively, these results suggest that specific NH 2-terminus domains can alter those properties of A7 that are normally associated with the COOH-terminus. We speculate that interactions between the NH 2- and COOH-termini are required for membrane binding, and aggregation and fusion properties of annexin A7.