Mutations in the Catalytic Domain of Human Matrix Metalloproteinase-1 (MMP-1) That Allow for Regulated Activity through the Use of Ca2
Conditionally active proteins regulated by a physiological parameter represent a potential new class of protein therapeutics. By systematically creating point mutations in the catalytic and linker domains of human MMP-1, we generated a protein library amenable to physiological parameter-based screen...
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Veröffentlicht in: | The Journal of biological chemistry 2013-03, Vol.288 (9), p.6629-6639 |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | Conditionally active proteins regulated by a physiological parameter represent a potential new class of protein therapeutics. By systematically creating point mutations in the catalytic and linker domains of human MMP-1, we generated a protein library amenable to physiological parameter-based screening. Mutants screened for temperature-sensitive activity had mutations clustered at or near amino acids critical for metal binding. One mutant, GVSK (Gly159 to Val, Ser208 to Lys), contains mutations in regions of the catalytic domain involved in calcium and zinc binding. The in vitro activity of GVSK at 37 °C in high Ca2+ (10 mm) was comparable with MMP-1 (wild type), but in low Ca2+ (1 mm), there was an over 10-fold loss in activity despite having similar kinetic parameters. Activity decreased over 50% within 15 min and correlated with the degradation of the activated protein, suggesting that GVSK was unstable in low Ca2+. Varying the concentration of Zn2+ had no effect on GVSK activity in vitro. As compared with MMP-1, GVSK degraded soluble collagen I at the high but not the low Ca2+ concentration. In vivo, MMP-1 and GVSK degraded collagen I when perfused in Zucker rat ventral skin and formed higher molecular weight complexes with α2-macroglobulin, an inhibitor of MMPs. In vitro and in vivo complex formation and subsequent enzyme inactivation occurred faster with GVSK, especially at the low Ca2+ concentration. These data suggest that the activity of the human MMP-1 mutant GVSK can be regulated by Ca2+ both in vitro and in vivo and may represent a novel approach to engineering matrix-remodeling enzymes for therapeutic applications. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M112.364729 |