Collagen binding activity of recombinant and N-terminally modified annexin V (anchorin CII).
We have cloned the full coding cDNA sequence of chicken annexin V and of a mutant lacking 8 amino acid residues of the N-terminal tail for prokaryotic expression. Both proteins were synthesized in Escherichia coli upon induction with isopropyl thio-beta-D-galactoside, and were purified following two different protocols: one based on the ability of these proteins to interact reversibly with liposomes in the presence of calcium, and the other based on two sequential ion-exchange chromatographic steps. Spectroscopical analysis of recombinant annexin V revealed that binding of calcium did not change the circular dichroism spectra indicating no significant changes on the secondary structure; however, a conformational change affecting the exposition to the solvent of the tryptophan residue 187 was detected by analysis of fluorescence emission spectra. Recombinant annexin V binds with high affinity to collagen types II and X, and with lower affinity to collagen type I in a calcium-independent manner. Heat denaturing of collagen decreases this interaction while pepsin-treatment of collagen almost completely abolishes annexin V binding. Mutated annexin V interacts with collagen in a similar way as the nonmutated recombinant protein, indicating that the N-terminal tail of annexin V is not essential for collagen binding.
Turnay
J
J
Max-Planck Society, Medical Clinic III, University of Erlangen-Nürnberg, Germany.
Pfannmüller
E
E
Lizarbe
M A
MA
Bertling
W M
WM
von der Mark
K
K
D000818:Animals; D017304:Annexin A5 / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism*; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009154:Mutation; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet
D017304:Annexin A5; D010743:Phospholipids; D011994:Recombinant Proteins; D003094:Collagen