The C-terminal Ca2+-binding domain of SPARC confers anti-spreading activity to human urothelial cells.
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. .
Delostrinos
Catherine F
CF
Program in Human Urothelial Biology, Children's Hospital and Regional Medical Center, Seattle, Washington 98105-0371, USA.
Hudson
Amber E
AE
Feng
Waldo C
WC
Kosman
Jeffrey
J
Bassuk
James A
JA
D000293:Adolescent; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology*; D004926:Escherichia coli; D005260:Female; D006801:Humans; D007223:Infant; D008969:Molecular Sequence Data; D015676:Osteonectin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019459:Urothelium / Q000166:cytology*
D015676:Osteonectin; D011993:Recombinant Fusion Proteins; D002118:Calcium