title: Characterization of the Ca2+-dependent binding of annexin IV to surfactant protein A.
abstract: We have shown previously that surfactant protein A (SP-A) binds to annexin IV in a Ca2+-dependent manner [Sohma, Matsushima, Watanabe, Hattori, Kuroki and Akino (1995) Biochem. J. 312, 175-181]. Annexin IV is a member of the annexin family having four consensus repeats of about 70 amino acids and a unique N-terminal tail. In the present study, the functional site of both annexin IV and SP-A for the Ca2+-dependent binding was investigated using mutant proteins. SP-A bound in a Ca2+-dependent manner to an annexin-IV truncation mutant consisting of the N-terminal domain and the first three domains (T(N-1-2-3)). SP-A also bound to T3-4, but this interaction was not Ca2+-dependent. SP-A bound weakly to the other truncation mutants (T(N-1-2), T(2-3) and T(2-3-4)). Each consensus repeat of annexin IV possesses a conserved acidic amino acid residue (Glu70, Asp142, Glu226 and Asp301) that putatively ligates Ca2+. Using annexin-IV DE mutants in which one, two or three residues out of the four Asp/Glu were altered to Ala by site-directed mutagenesis [Nelson and Creutz (1995) Biochemistry 34, 3121-3132], it was revealed that Ca2+ binding in the third domain is more important than in the other Ca2+-binding sites. SP-A is a member of the animal lectin group homologous with mannose-binding protein A. The substitution of Arg197 of rat SP-A with Asp or Asn eliminated binding to annexin IV, whereas the substitution of Glu195 with Gln was silent. These results suggest that the Ca2+ binding to domain 3 of annexin IV is required for the Ca2+-dependent binding by SP-A and that Arg197 of SP-A is important in this binding.
authors.affiliation: Department of Biochemistry, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo, 060-8556 Japan. sohma@sapmed.ac.jp
authors.forename: H
authors.initials: H
authors.lastname: Sohma
authors.forename: C E
authors.initials: CE
authors.lastname: Creutz
authors.forename: M
authors.initials: M
authors.lastname: Saitoh
authors.forename: H
authors.initials: H
authors.lastname: Sano
authors.forename: Y
authors.initials: Y
authors.lastname: Kuroki
authors.forename: D R
authors.initials: DR
authors.lastname: Voelker
authors.forename: T
authors.initials: T
authors.lastname: Akino
mesh_terms: D000818:Animals; D017314:Annexin A4 / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D009154:Mutation; D011485:Protein Binding; D011510:Proteolipids / Q000235:genetics / Q000378:metabolism*; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000235:genetics / Q000378:metabolism*; D051381:Rats
chemical_list: D017314:Annexin A4; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D001224:Aspartic Acid; D018698:Glutamic Acid; D002118:Calcium