The C-terminal Ca(2+)-binding domain of SPARC confers anti-spreading activity to human urothelial cells

Delostrinos CF, Hudson AE, Feng WC, Kosman J, Bassuk JA.

J Cell Physiol. 2006 Jan;206(1):211-20.

Program in Human Urothelial Biology, Children’s Hospital and Regional Medical Center, Seattle, Washington.

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 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 ® 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:

removed denaturing conditions
catalyzed disulfide bond isomerization
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 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 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.