Distribution of new human -defensin genes clustered on chromosome 20 in functionally different segments of epididymis Francisco-Javier Rodrı ´guez-Jime ´nez, Alexander Krause, Sandra Schulz, Wolf-Georg Forssmann,* Jose ´-Ramo ´n Conejo-Garcia, Rainer Schreeb, and Dirk Motzkus IPF PharmaCeuticals GmbH, Feodor-Lynen-Str. 31, 30625 Hannover, Germany Received 12 August 2002; accepted 5 December 2002 Abstract Human -defensins are a family of cationic peptides that share a pattern of six conserved cysteine residues. We describe the cloning and characterization of the cDNAs of five novel -defensin genes (DEFB25–DEFB29) clustered on chromosome 20p13, which were identified using a bioinformatics approach. Expression analysis revealed the occurrence of the transcripts in only a few organs, with the highest abundance in the male genital tract. Examination of -defensin expression in human epididymis by real-time quantitative RT-PCR revealed a distribution along the functionally different segments of the epididymal duct. In situ hybridization for one of the cDNAs shows mRNA restriction to the epithelial cell layer of the epididymis, known to secrete factors responsible for sperm maturation. We suggest that the novel peptides carry out physiological functions in the male genital tract that may not be directly related to bacterial growth inhibition in host defense. © 2003 Elsevier Science (USA). All rights reserved. Keywords: -defensin; Chromosome 20; Cluster analysis; Expression; TaqMan; Epididymis Introduction Mammalian defensins are a family of secreted cationic and highly disulfide-bonded peptides. On the basis of the spatial distribution of the cysteine residues, the family is subdivided into -, -, and -defensins, which are predom- inantly considered to act as antimicrobial peptides forming an early barrier in host defense [1]. To date, four human -defensins are known and described to be involved in innate immunity. Recently, two more -defensins termed hBD-5 and hBD-6 were described [2]. In addition, three human -defensin-like peptides sharing their characteristic cysteine pattern have been identified and found to represent epididymis-specific secretory proteins. hBD-1 was origi- nally isolated from hemofiltrate [3], whereas hBD-2 and hBD-3 were purified from skin [4,5]. The gene encoding hBD-1 (DEFB1) is constitutively expressed in kidney, pan- creas, the female reproductive tract, and the airways [6,7]. Whereas DEFB2 is expressed in epithelia such as skin and lung [4], DEFB3 expression was found in nonepithelial tissues such as the heart or skeletal muscle [8,9]. In contrast, DEFB4 has a distribution pattern with the highest abun- dance in the male genital tract, and a much lower expression in pancreas and kidney [10]. In the group of -defensin-like peptides, HE2 (HE2 and variants are referred to as EP2 [11]) was described as a human sperm antigen that was initially identified during a screening for epididymis-specific transcripts [12]. Two of the splicing variants, EP2D and EP2E, share the conserved cysteine pattern of -defensins [11]. They are secreted to the epididymal lumen and at least EP2D specifically binds to the sperm surface [13]. A homolog of another human -defensin-like peptide, ESP13.2, was reported to be lo- cated in the principal cells of the epididymal epithelium of Macaca fascicularis [14]. Once spermatozoa leave the tes- tis, they acquire the capacity to bind and fertilize eggs within the epididymis by a process known as maturation [15]. In the absence of maturation, the spermatozoa remain * Corresponding author. Fax: +49-511-5466-422. E-mail address: wgforssmann@ipf-pharmaceuticals.de (W.-G. Forss- mann). R Available online at www.sciencedirect.com Genomics 81 (2003) 175–183 www.elsevier.com/locate/ygeno 0888-7543/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0888-7543(02)00034-4