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

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 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. .

SPARC Represses E-Cadherin and Induces Mesenchymal Transition during Melanoma Development

“…Interestingly, in contrast to its effect on E-cadherin expression, this mutant failed to inhibit cell spreading and attachment, suggesting that SPARC induces E-cadherin repression independently of its well-established counteradhesive property (results not shown). This observation allows us to distinguish this function from E-cadherin suppression, which is consistent with a recent study showing the role of EC domain in anti-spreading activity of SPARC in urothelial cells (41). Further work is clearly required to explain fully how SPARC functions to trigger signaling pathways that functionally regulate E-cadherin in melanocytes.…”

Molecular cloning of a novel calcium-binding protein in the secreted saliva of the green rice leafhopper Nephotettix cincticeps

“…Protein motif search for NcSP84 using PROSITE predicted the existence of five EF-hand motifs despite the low scores. Domain search against Pfam also predicted a SPARC Ca bdg (379e431), which contains two EF-hands (Delostrinos et al, 2006) and another independent EFhand motif (481e499). The EF-hand structure is found in a large set of Ca 2þ -binding proteins, such as calbindin D9K, parvalbumin, and calmodulin, which contain two, three, and four EF-hands, respectively (Kawasaki et al, 1998;Lewit-Bentley and Rety, 2000).…”

The role of albumin receptors in regulation of albumin homeostasis: Implications for drug delivery

“…As the gene for SPARC has been cloned [76], the protein may be recombinantly produced to study its interaction with albumin. For such studies it may be of interest to address the effect of calcium, as the structure and function of SPARC has been shown to be modulated by the presence of calcium [77][78][79]. However, one study exists where the binding affinity for the SPARC-albumin complex was estimated to be as weak as 700 μM [80].…”