The C-terminal Ca2+-binding domain of SPARC confers anti-spreading activity to human urothelial cells

J Cell Physiol. 2006 Jan;206(1):211-20. doi: 10.1002/jcp.20462.


The anti-spreading activity of secreted protein acidic and rich in cysteine (SPARC) has been assigned to the C-terminal third domain, a region rich in alpha-helices. This "extracellular calcium-binding" (EC) domain contains two EF-hands that each coordinates one Ca2+ ion, forming a helix-loop-helix structure that not only drives the conformation of the protein but is also necessary for biological activity. Recombinant (r) EC, expressed in E. coli, was fused at the C-terminus to a His hexamer and isolated under denaturing conditions by nickel-chelate affinity chromatography. rEC-His was renatured by procedures that simultaneously (i) removed denaturing conditions, (ii) catalyzed disulfide bond isomerization, and (iii) initiated Ca2+-dependent refolding. Intrinsic tryptophan fluorescence and circular dichroism spectroscopies demonstrated that rEC-His exhibited a Ca2+-dependent conformation that was consistent with the known crystal structure. Spreading assays confirmed that rEC-His was biologically active through its ability to inhibit the spreading of freshly plated human urothelial cells propagated from transitional epithelium. rEC-His and rSPARC-His exhibited highly similar anti-spreading activities when measured as a function of concentration or time. In contrast to the wild-type and EC recombinant proteins, rSPARC(E268F)-His, a point substitution mutant at the Z position of EF-hand 2, failed to exhibit both Ca2+-dependent changes in alpha-helical secondary structure and anti-spreading activity. The collective data provide evidence that the motif of SPARC responsible for anti-spreading activity was dependent on the coordination of Ca2+ by a Glu residue at the Z position of EF-hand 2 and provide insights into how adhesive forces are balanced within the extracellular matrix of urothelial cells. .

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Amino Acid Sequence
  • Calcium / metabolism
  • Cell Movement / physiology*
  • Escherichia coli
  • Female
  • Humans
  • Infant
  • Molecular Sequence Data
  • Osteonectin / chemistry
  • Osteonectin / genetics
  • Osteonectin / metabolism*
  • Protein Conformation
  • Protein Folding
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Urothelium / cytology*


  • Osteonectin
  • Recombinant Fusion Proteins
  • Calcium