Characterization of human recombinant annexin II tetramer purified from bacteria: role of N-terminal acetylation.
Annexin II tetramer (AIIt) is a Ca2+-dependent, phosphatidylserine-binding, and F-actin-bundling phosphoprotein which is localized to both the 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 spectrometry and amino acid sequence analysis of purified recombinant p36 (rp36) and recombinant p11 (rp11) suggested that the recombinant proteins were identical to their native counterparts except for the lack of N-terminal acetylation of rp36. Furthermore, the non-acetylated rp36 bound rp11 and formed AIIt. The circular dichroism spectra and urea denaturation profiles of acetylated AIIt and non-acetylated rAIIt were identical. In addition, both the acetylated AIIt and non-acetylated rAIIt were similar in their Ca2+ dependence and concentration dependence of phospholipid liposome aggregation, chromaffin granule aggregation, and F-actin bundling. These results suggest that N-terminal acetylation of p36 is not in fact necessary for binding of the protein to p11 and that N-terminal acetylation does not affect the conformational stability of AIIt or the in vitro activities of AIIt. The availability of large amounts of rAIIt will facilitate further characterization of the structure-function relationships of the protein.
Kang
H M
HM
Cell Regulation Research Group, Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Kassam
G
G
Jarvis
S E
SE
Fitzpatrick
S L
SL
Waisman
D M
DM
D000107:Acetylation; D017306:Annexin A2 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
D017306:Annexin A2; D018076:DNA, Complementary; D011994:Recombinant Proteins