| Fluoride Reduces Binding of MMP-20 to Amelogenin | ||
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K. TANIMOTO, T.Q. LE, Z. LI, P. HWANG, W. LI, and P. DENBESTEN, University of California, San Francisco, USA During enamel formation, hydrolysis of amelogenin by matrix metalloproteinase-20 (MMP-20) is critical to enamel biomineralization. Excessive fluoride during enamel formation results in a delay in the removal of amelogenin, possibly related to an effect on MMP-20 hydrolysis. In this study, we mutated MMP-20 to prevent autolysis, allowing us to measure binding between amelogenin and MMP-20, and to determine whether fluoride alters this enzyme/substrate interaction. Objectives: To measure the binding affinity of amelogenin to MMP-20, and to determine the effect of fluoride on binding affinity. Methods: A point mutation was introduced in recombinant human MMP-20 (rhMMP-20), resulting in a substitution of A at position 227 to E. This substitution prevented MMP-20 autolysis. rhMMP-20 and inactive rhMMP-20 (E227A) were synthesized from E. coli, purified by His-tag affinity chromatograph, and characterized by SDS-PAGE and Western blot analysis. Enzymatic activities of these proteases were analyzed using zymography and fluorescent quenched-peptide assay. Biacore analysis was used to quantify the affinity between an inactive rhMMP-20 and rH174 by immobilizing inactive rhMMP-20 onto a Ni-containing NTA sensor chip via His-tag, followed by injections of solutions containing rH174 with various concentration of NaF (0-50 µM F-). Results: Wild-type rhMMP-20 was autolysed- into smaller fragments; the predominant active form having a molecular weight of 25-kDa protein. However, inactive rhMMP-20 had no catalytic activity, and remained as a 50-kDa protein. Biacore analysis revealed that inactive rhMMP-20 bound specifically to rH174 with a Kd = 5.16 x 10-8 M. Fluoride inhibited the binding of inactive rhMMP-20 to amelogenin in a concentration-dependent manner. Conclusions: MMP-20 specifically binds to amelogenin. This binding is significantly reduced by micromolar levels of fluoride. This effect of fluoride on enzyme/substrate binding may have a role in the delayed removal of amelogenins, resulting in enamel fluorosis. Support: NIH/NIDCR R01-DE015821 | ||
| Seq #0 - Late Breaking News - Session I 9:00 AM-10:30 AM, Thursday, March 22, 2007 Ernest N. Morial Convention Center 287 | ||
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