Conformational Changes of the Recombinant Extracellular Domain of E-cadherin Upon Calcium Binding

Eur J Biochem. 1994 Aug 1;223(3):1019-26. doi: 10.1111/j.1432-1033.1994.tb19080.x.

Abstract

The cell-adhesion protein E-cadherin/uvomorulin exhibits a calcium-dependent homoassociation. The effect of Ca2+ on the extracellular fragment of E-cadherin was studied using the recombinant protein expressed in the baculovirus expression system. The recombinant and native fragment of E-cadherin were found to be similar by many biochemical criteria [Herrenknecht, K. & Kemler, R. (1993) J. Cell Sci. 17, 147-154]. A large and reversible conformational transition was observed upon Ca2+ depletion. A change from a rod-like structure, 22 nm in length, to a more globular assembly of the five subdomains became evident by electron-microscopical analysis. In the presence of Ca2+, the circular dichroic spectra indicated predominantly beta-structure but a more negative ellipticity was observed in the absence of Ca2+. The intrinsic tryptophan fluorescence decreased by 12% upon Ca2+ depletion. Both effects were used for calcium titrations which indicated calcium binding to several sites with average K(d) values of 45-150 microM. Cleavage of the protein fragment by trypsin occurred only at low Ca2+ concentrations and from the calcium-dependence of cleavage rates, a K(d) value of 24 microM was derived. The major site of cleavage was identified by partial sequencing to be located between the two putative calcium-binding sites in the third subdomain from the N-terminus. In agreement with earlier results with the native fragment, the recombinant protein did not associate in the presence or absence of Ca2+. We suggest the calcium-dependent homoassociation therefore depends on additional effects connected with the cell surface association of E-cadherin.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cadherins / chemistry*
  • Cadherins / genetics
  • Cadherins / metabolism
  • Cadherins / ultrastructure
  • Calcium / metabolism*
  • Circular Dichroism
  • Mice
  • Microscopy, Electron
  • Molecular Sequence Data
  • Negative Staining
  • Peptide Fragments / chemistry*
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / ultrastructure
  • Trypsin / metabolism

Substances

  • Cadherins
  • Peptide Fragments
  • Recombinant Proteins
  • Trypsin
  • Calcium