Localization and distribution of wolframin in human tissues Maria De Falco 1 , Lucrezia Manente 2 , Angela Lucariello 2 , Gianluca Baldi 3 , Paola Fiore 4 ,Vincenza Laforgia 1 , Alfonso Baldi 4 , Alessandro Iannaccone 5 , Antonio De Luca 2 1 Department of Biological Sciences, Section of Evolutionary and Comparative Biology, University of Naples "Federico II", Naples, Italy, 2 Department of Medicine and Public Health, Section of Human Anatomy, Second University of Naples, Naples, Italy, 3 Hospital of Nocera Inferiore" Umberto I", Service of Ophthalmology, Nocera Inferiore, Italy, 4 Department of Biochemistry “F. Cedrangolo”, Section of Pathologic Anatomy, Second University of Naples, Naples, Italy, 5 Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, USA TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Materials and methods 3.1. Generation of a polyclonal antibody against wolframin 3.2.Dot blot 3.3. Human cell lines 3.4. Protein extraction and western blotting analysis 3.5. Normal foetal and adult human tissues 3.6. Immunohistochemistry 4. Results 4.1.Antibody characterization 4.2. Western blot analysis 4.3. Wolframin localization in foetal human tissues 4.4. Wolframin localization in adult human tissues 5. Discussion 6. Acknowledgments 7. References 1. ABSTRACT Wolframin is a transmembrane glycoprotein of 890 aminoacids, encoded by WFS1 gene. WFS1 mutations are responsible for Wolfram syndrome, an autosomal recessive disorder. In the present paper, we first characterized the polyclonal wolframin antibody by dot blot. Secondly, we verified antibody specificity by western blotting using different human cell lines. Thirdly, we studied wolframin localization in human foetal (14-35 weeks) and adult tissues by immunohistochemistry. Wolframin expression was distributed in many organs, with different tissue and cell localization and expression levels. In foetal systems, wolframin expression was faint at 14-16 weeks and increased when development proceeded. In adult human tissues a variable positive staining was observed in both simple and stratified epithelia. A moderate wolframin expression was observed in liver and in the endocrine portion of the pancreas. In conclusion, our data suggest that this protein may have important roles in a number of different tissues, including many that are not known to be affected by WFS1-linked diseases. The immunopositivity in adult human tissues suggests that it may function maintaining physiological cellular homeostasis. 2. INTRODUCTION Wolfram Syndrome (WS), also called with the acronym DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy and deafness; 1-3) is a rare autosomal recessive disorder characterized by juvenile onset insulin deficient diabetes mellitus, neurodegeneration (1) and optic atrophy (1, 4). About 60% of the patients shows various degrees of hearing impairment by 20 years of age (5, 6), and frequently endocrinological, psychiatric, and urological symptoms (5, 7). The variety and multitude of symptoms are consistent with the disease representing a progressive neurodegenerative disorder affecting the central and peripheral nervous systems, such as ataxia, nystagmus, peripheral neuropathies and mental retardation (8, 9). WS patients usually die from central respiratory failure as a result of brainstem atrophy and/or from complication of urinary tract atony in their third or fourth decade (1, 10). The WSF1 gene, discovered in 1998 (2, 3), maps to chromosome 4p16.1 and contains eight exons spanning 33.4 kb of genomic DNA, of which exon 1 is noncoding (11). The product of the WFS1 gene, wolframin, is an integral, endoglycosidase H-sensitive membrane