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J. Biol. Chem., Vol. 279, Issue 35, 36497-36503, August 27, 2004

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Copper(II) Binding to the Human Doppel Protein May Mark Its Functional Diversity from the Prion Protein^*

Grazia M. Cereghetti, Alessandro Negro¶, Evi Vinck||**, Maria L. Massimino, Maria C. Sorgato, and Sabine Van Doorslaer||¶¶

From the Dipartimento di Chimica Biologica, ^¶C.R.I.B.I. and Istituto CNR di Neuroscienze, Università di Padova, Viale G. Colombo 3, I-35121 Padova, Italy and the ^||Department of Physics, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium

Doppel (Dpl) is the first described homologue of the prion protein, the main constituent of the agent responsible for prion diseases. The cellular prion protein (PrP^C) is predominantly present in the central nervous system. Although its role is not yet completely clarified, PrP^C seems to be involved in Cu²⁺ recycling from synaptic clefts and in preventing neuronal oxidative damage. Conversely, Dpl is expressed in heart and testis and has been shown to regulate male fertility by intervening in gametogenesis and sperm-egg interactions. Therefore, despite a high sequence homology and a similar three-dimensional fold, the functions of PrP^C and Dpl appear unrelated. Here we show by electron paramagnetic resonance and fluorescence spectroscopy that the in vitro binding of copper(II) to human recombinant Dpl occurs with a different pattern from that observed for recombinant PrP. At physiological pH values, two copper(II)-binding sites with different affinities were found in Dpl. At lower pH values, two additional copper(II)-binding sites can be identified as follows: one complex is present only at pH 4, and the other is observed in the pH range 5–6. As derived from the electron paramagnetic resonance characteristics, all Dpl-copper(II) complexes have a different coordination sphere from those present in PrP. Furthermore, in contrast to the effect shown previously for PrP^C, addition of Cu²⁺ to Dpl-expressing cells does not cause Dpl internalization. These results suggest that binding of the ion to PrP^C and Dpl may contribute to the different functional roles ascribed to these highly homologous proteins.

Received for publication, April 20, 2004 , and in revised form, June 21, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a post-doctoral fellowship from the University of Padova.

^** Supported by New Research Initiative (NOI-BOF) Ph.D. grant from the University of Antwerp.

Supported by Telethon Onlus Grant E.0945, Ministero dell'Istruzione, dell'Universitá e della Ricerca Grant RBNE01S29H, and Italian Ministry of Health Grant 1AA/F,2001.

^¶¶ To whom correspondence should be addressed: Dept. of Physics, University of Antwerp, Campus Drie Eiken, Universiteitsplein 1, B-2610 Antwerp, Belgium. Tel.: 0032-3-8202461; Fax: 0032-3-8202470; E-mail: Sabine.VanDoorslaer{at}ua.ac.be.

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