Collagen binding activity of recombinant and N-terminally modified annexin V (anchorin CII)

J Cell Biochem. 1995 Jun;58(2):208-20. doi: 10.1002/jcb.240580210.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Annexin A5 / genetics
  • Annexin A5 / isolation & purification
  • Annexin A5 / physiology*
  • Base Sequence
  • Chickens
  • Cloning, Molecular
  • Collagen / metabolism*
  • Escherichia coli
  • Molecular Sequence Data
  • Mutation
  • Phospholipids / metabolism
  • Protein Binding
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Spectrophotometry, Ultraviolet

Substances

  • Annexin A5
  • Phospholipids
  • Recombinant Proteins
  • Collagen