Characterization of the Ca2+-dependent binding of annexin IV to surfactantprotein A

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 a nnexin IV and SP-A for the Ca2+-dependent binding was investigated using mu tant proteins. SP-A bound in a Ca2+-dependent manner to an annexin-IV trunc ation mutant consisting of the N-terminal domain and the first three domain s (TN-1-2-3). SP-A also bound to T3-4, but this interaction was not Ca2+-de pendent. SP-A bound weakly to the other truncation mutants (TN-1-2, T2-3, a nd T2-3-4). Each consensus repeat of annexin IV possesses a conserved acidi c amino acid residue (Glu(70), Asp(142), Glu(226) and Asp(301)) that putati vely ligates Ca2+, Using annexin-IV DE mutants in which one, two or three r esidues out of the four Asp/Glu were altered to Ala by site-directed mutage nesis [Nelson and Creutz (1995) Biochemistry 34, 3121-3132], it was reveale d 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 Arg(197) of rat SP-A w ith Asp or Asn eliminated binding to annexin IV, whereas the substitution o f Glu(195) with Gin 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 Arg(197) of SP-A is important in this binding.