Annexin A2 (ANXA2) is a Ca-dependent multifunctional protein containing five Ca-binding domains, but their functional significance and difference remain unclear. Herein, glutamic acid (E) or aspartic acid (D) in five Ca-binding domains of canine ANXA2 (98.82 % and 96.76-99.41 % identical to ANXA2 from human and other mammals, respectively) was substituted by alanine (A) using site-directed mutagenesis. Recombinant ANXA2 wild-type (WT) and E53A, E96A, D162A, E247A and D322A mutants were constructed and expressed using a bacterial expression system followed by high-affinity purification using nickel-nitrilotriacetic acid (Ni-NTA) matrix. Efficacies of their expression and purification were confirmed by SDS-PAGE and Western blotting. Their amino acid sequences were verified by nanoLC-ESI-Qq-TOF tandem mass spectrometry. ATR-FTIR spectroscopy revealed that their secondary structure significantly differed (α-helix decreased but random coil increased in all mutants). Analyses of physicochemical properties revealed that molecular weight slightly decreased, whereas isoelectric point, aliphatic index, grand average of hydropathicity, electrostatic potential and molecular hydrophobicity potential slightly increased in all the mutants compared with WT. Interestingly, Ca-binding capability of these mutants (particularly E96A and D322A) significantly decreased from that of WT. In summary, secondary structure, physicochemical properties, and Ca-binding capability of E53A, E96A, D162A, E247A and D322A mutants of ANXA2 significantly differed from its WT, consistent with the loss of negatively charged E/D. In particular, E96A and D322A exhibited the lowest Ca-binding capability. These data and recombinant proteins would be useful for further investigations of the Ca-dependent functions of individual Ca-binding domains in ANXA2.

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http://dx.doi.org/10.1016/j.abb.2024.110267DOI Listing

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