[No authors listed]
Doppel (Dpl) is a glycosylphosphatidylinositol-anchored protein expressed in the testis. It exhibits 26% sequence identity with the prion protein (PrP) but lacks the octarepeat region implicated as the major copper-binding domain. Contrary to expectations, Cu(II) induced a 26% reduction in the intrinsic fluorescence of Dpl(27-154) and a calculated K(d) for a single-site model of 0.16 +/- 0.08 microm. Other metals had minimal effects on fluorescence quenching. Matrix-assisted laser desorption ionization mass spectrometry of a Dpl peptide revealed binding of copper (but not other metals) to the helical alphaB/B'-loop-alphaC subregion of Dpl. Fluorescence quenching and equilibrium dialysis analyses of this Dpl(101-145) peptide were compatible with a binding site of K(d) = 0.4 microm. Diethylpyrocarbonate footprinting (Qin, K., Yang, Y., Mastrangelo, P., and Westaway, D. (2002) J. Biol. Chem. 277, 1981-1990) of Dpl(27-154) defined one residue/molecule was protected by copper from diethylpyrocarbonate adduct formation, and reiteration of this analysis with Dpl(101-145) suggested that His(131) may contribute to Cu(II) binding. Taken together, our data indicate that the alpha-helical region of mouse Dpl possesses a selective copper-binding site with a submicromolar K(d) and perhaps one or more lower affinity sites. Although metallated forms of Dpl might exist in vivo, analyses of Tg(Dpl)10329 mice were inconsistent with reports that Dpl expression is associated with increased carbonylation and nitrosylation of brain proteins. Thus, rather than comprising an important source of free radical damage, copper binding may serve to modulate the activity, stability, or localization of the Dpl protein.
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