CHARACTERIZATION OF HUMAN RECOMBINANT ANNEXIN-II TETRAMER PURIFIED FROM BACTERIA - ROLE OF N-TERMINAL ACETYLATION

Annexin II tetramer (AIIt) is a Ca2+-dependent, phosphatidylserine-bin ding, and F-actin-bundling phosphoprotein which is localized to both t he extracellular and cytoplasmic surfaces of the plasma membrane. The tetramer is composed of two p36 heavy chains and two p11 light chains. We have produced prokaryotic cDNA expression constructs for both p36 and p11. Both proteins were expressed in large amounts in Escherichia coli upon induction with IPTG. Electrospray ionization mass spectromet ry and amino acid sequence analysis of purified recombinant p36 (rp36) and recombinant p11 (rp11) suggested that the recombinant proteins we re identical to their native counterparts except for the lack of N-ter minal acetylation of rp36. Furthermore, the non-acetylated rp36 bound rp11 and formed AIIt. The circular dichroism spectra and urea denatura tion profiles of acetylated AIIt and non-acetylated rAIIt were identic al. In addition, both the acetylated AIIt and non-acetylated rAIIt wer e similar in their Ca2+ dependence and concentration dependence of pho spholipid liposome aggregation, chromaffin granule aggregation, and F- actin bundling. These results suggest that N-terminal acetylation of p 36 is not in fact necessary for binding of the protein to pll and that N-terminal acetylation does not affect the conformational stability o f AIIt or the in vitro activities of AIIt. The availability of large a mounts of rAIIt will facilitate further characterization of the struct ure-function relationships of the protein.