Eukaryotic DING Proteins Are Endogenous: An Immunohistological Study in Mouse Tissues Jean-Marc Collombet 1 , Mikael Elias 2¤ , Guillaume Gotthard 2 , Elise Four 1 , Fre ´de ´ rique Renault 1 , Aure ´ lie Joffre 3 , Dominique Baubichon 1 , Daniel Rochu 1 , Eric Chabrie `re 2 * 1 De ´ partement de Toxicologie, Institut de Recherche Biome ´ dicale des Arme ´ es, Centre de Recherche du Service de Sante ´ des Arme ´ es, La Tronche, France, 2 Architecture et Fonction des Macromole ´ cules Biologiques, Centre National de la Recherche Scientifique-Aix Marseille Universite ´ , Marseille, France, 3 Service de Microscopie et d’Imagerie Me ´ dicale, Institut de Recherche Biome ´dicale des Arme ´es, Centre de Recherche du Service de Sante ´ des Arme ´es, La Tronche, France Abstract Background: DING proteins encompass an intriguing protein family first characterized by their conserved N-terminal sequences. Some of these proteins seem to have key roles in various human diseases, e.g., rheumatoid arthritis, atherosclerosis, HIV suppression. Although this protein family seems to be ubiquitous in eukaryotes, their genes are consistently lacking from genomic databases. Such a lack has considerably hampered functional studies and has fostered therefore the hypothesis that DING proteins isolated from eukaryotes were in fact prokaryotic contaminants. Principal Findings: In the framework of our study, we have performed a comprehensive immunological detection of DING proteins in mice. We demonstrate that DING proteins are present in all tissues tested as isoforms of various molecular weights (MWs). Their intracellular localization is tissue-dependant, being exclusively nuclear in neurons, but cytoplasmic and nuclear in other tissues. We also provide evidence that germ-free mouse plasma contains as much DING protein as wild- type. Significance: Hence, data herein provide a valuable basis for future investigations aimed at eukaryotic DING proteins, revealing that these proteins seem ubiquitous in mouse tissue. Our results strongly suggest that mouse DING proteins are endogenous. Moreover, the determination in this study of the precise cellular localization of DING proteins constitute a precious evidence to understand their molecular involvements in their related human diseases. Citation: Collombet J-M, Elias M, Gotthard G, Four E, Renault F, et al. (2010) Eukaryotic DING Proteins Are Endogenous: An Immunohistological Study in Mouse Tissues. PLoS ONE 5(2): e9099. doi:10.1371/journal.pone.0009099 Editor: Art J. Lustig, Tulane University Health Sciences Center, United States of America Received September 29, 2009; Accepted January 20, 2010; Published February 8, 2010 Copyright: ß 2010 Collombet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by grants to E.C. by De ´le ´ gation Ge ´ne ´rale pour l’Armement (CO nu010807/03-10) and by the C.N.R.S. D.R. is under contract with the German Bundesministerium der Verteidigung (M/SABX/8A001). M.E. is a post-doctoral fellow supported by the F.E.B.S. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: eric.chabriere@afmb.univ-mrs.fr ¤ Current address: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel Introduction DING proteins, named according to their four conserved N- terminal amino-acid residues, encompass a recently discovered protein family [1]. Intriguingly, eukaryotic DING genes are consistently missing from genomic databases although proteins belonging to this family seem to be ubiquitous in eukaryotes: they have been identified in animals (human, monkey, rat, turkey), plants (Hypericum perforatum, Arabidopsis thaliana, potato, tobacco) and fungi (Candida albicans, Ganoderma lucidum) [2,3] mostly as a 40 kDa protein or higher molecular weight DING proteins [4]. Although no complete eukaryotic DING coding sequence is available in databases, few partial DNA sequences have been either cloned [3] or identified in non-annoted parts of genomes [2]. These few pieces of sequences show that DING proteins are strongly conserved [2]. DING proteins have also been isolated from several prokaryotes [5,6], and their coding genes are in some cases available, such as the gene encoding PfluDING, the protein isolated from P. fluorescens [7,8,9]. The high sequence identity between known eukaryotic DING sequences and available prokaryotic DING sequences (.70% sequence identity) and the systematic absence of eukaryotic DING genes raised a controversy about their prokaryotic [10] or eukaryotic origins [11]. DING proteins have been mostly isolated by virtue of a biological function. One of the most striking examples as such, remains the search for a new HIV inhibitor in St John’s Wort that led to the characterization of a novel DING protein named p27 sj [3]. In humans, several DING proteins have been identified from different tissues [2,12,13]. The human phosphate binding protein (HPBP) is a serendipitously discovered plasma apolipoprotein that binds phosphate and is isolated from human plasma [14,15]. HPBP structure was solved [16] and its physiological function, i.e. its association with paraoxonase (HPON1), an enzyme involved in atherosclerosis [17], has been extensively studied [18,19,20]. The complete protein sequence of HPBP was recently determined by a tandem use of mass spectrometry and X-ray crystallography [21]. Such a sequence stands out as the single complete eukaryotic sequence of a DING representative, known to date. Three other PLoS ONE | www.plosone.org 1 February 2010 | Volume 5 | Issue 2 | e9099