Abstract
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 α‐helices. This “extracellular calcium‐binding” (EC) domain contains two EF‐hands that each coordinates one Ca^2+^ 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 Ca^2+^‐dependent refolding. Intrinsic tryptophan fluorescence and circular dichroism spectroscopies demonstrated that rEC‐His exhibited a Ca^2+^‐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 Ca^2+^‐dependent changes in α‐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 Ca^2+^ 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. © 2005 Wiley‐Liss, Inc.