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J. Biol. Chem., Vol. 278, Issue 11, 8888-8896, March 14, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/11/8888    most recent M210875200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Qin, K. Articles by Westaway, D. Search for Related Content PubMed PubMed Citation Articles by Qin, K. Articles by Westaway, D. Social Bookmarking                      What's this?

The PrP-like Protein Doppel Binds Copper^*

Kefeng Qin, Janaky Coomaraswamy, Peter Mastrangelo, Ying Yang^§, Stan Lugowski^¶, Chris Petromilli, Stanley B. Prusiner, Paul E. Fraser^**, Jonathan M. Goldberg, Avijit Chakrabartty^**, and David Westaway^§§¶¶

From the  Centre for Research in Neurodegenerative Diseases, the ^§ Mass Spectrometry Laboratory, Molecular Medicine Research Centre, the ^¶ Institute for Biomaterials and Biomechanical Engineering, the ^** Department of Medical Biophysics, and the ^§§ Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 3H2, Canada, the  Institute for Neurodegenerative Diseases, University of California, San Francisco, California 94143, and the  Boston Biomedical Research Institute, Watertown, Massachusetts 02472

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 µM. 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 B/B'-loop-C 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 µM. 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 His¹³¹ may contribute to Cu(II) binding. Taken together, our data indicate that the -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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