Gene 481 (2011) 83–92 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Claudin-18 gene structure, regulation, and expression is evolutionary conserved in mammals Özlem Türeci a,⁎, Michael Koslowski b, Gerd Helftenbein a, John Castle b, Christoph Rohde a, Karl Dhaene c, Gerhard Seitz d, Ugur Sahin e,b a Ganymed Pharmaceuticals AG, Freiligrathstr. 12, 55131 Mainz, Germany b TRON—Translational Oncology and Immunology GmbH, Langenbeckstr. 1, 55131 Mainz, Germany c Algemeen Stedelijk Ziekenhuis Aalst, Department of Pathology, Merestraat 80, B-9300 Aalst, Belgium d Hospital Bamberg, Department of Pathology, Buger Str. 80, 96049 Bamberg, Germany e III. Department of Internal Medicine, Division of Translational and Experimental Oncology, Johannes Gutenberg University, Obere Zahlbacherstr. 63, 55131 Mainz, Germany article info abstract Article history: Claudin-18 isoform 2 (CLDN18.2) is one of the few members of the human claudin family of tight junction Accepted 15 April 2011 molecules with strict restriction to one cell lineage. The objective of the current study was to compare Available online 4 May 2011 molecular structure and tissue distribution of this gastrocyte specific molecule in mammals. We show here that the CLDN18.2 protein sequence is highly conserved, in particular with regard to functionally relevant Received by A.J. van Wijnen domains in mouse, rat, rabbit, dog, monkey and human and also in lizards. Moreover, promoter regions of orthologs are highly homologous, including the binding site of the transcription factor cyclic AMP–responsive Keywords: Tight junction element binding protein (CREB), which is known to regulate activation of human CLDN18.2. Employing RT- Claudins PCR and immunohistochemistry, we found that, analogous to the human gene, all orthologous CLDN18.2 Ortholog transcripts and proteins are exclusively expressed in differentiated gastric cells. Gene structure, promoter Conservation elements and RNA expression pattern of the lung-tissue specific Claudin-18 isoform 1 (CLDN18.1) as well, are homologous across species. These findings exemplify phylogenetic conservation of lineage-specific members of a multigene family. Given that CLDN18.2 is a novel drug target candidate, our data is also relevant for drug development as it reveals all six investigated mammalian species as suitable models for testing safety of CLDN18.2 targeting regimen. © 2011 Elsevier B.V. All rights reserved. 1. Introduction (Smetana, 1947). The paracellular aspect of this control is mainly attributed to cell–cell contact sites known as tight junction (TJ) In multicellular organisms, maintenance of compartments that strands (Farquhar and Palade, 1963). TJs have additional roles, such as differ in fluid and solute composition is ensured by epithelial and maintaining cell polarity by forming a barrier that prevents lateral endothelial cell layers, which control the passage of water and solutes diffusion of membrane proteins and lipids and recruitment of signaling molecules for regulation of proliferation, differentiation, motility and other cellular functions (Stevenson et al., 1988). Abbreviations: CLDN18.2, Claudin-18 isoform 2; CLDN18.1, Claudin-18 isoform 1; Tight junctions require claudin molecules for their formation CREB, cyclic AMP response element-binding protein; RT-PCR, reverse transcription (Furuse et al., 1998). The claudin family comprises more than two polymerase chain reaction; TJ, tight junction; BLAST, basic local alignment search tool; dozens of four-pass transmembrane proteins with similar sequence nr, non-redundant protein database; RefSeq, NCBI Reference Sequence database; T/EBP/NKX2.1, homeodomain transcription factor; EBI, European Bioinformatics and structure, but divergent tissue distribution. Whereas most Institute; IgG, immunoglobulin G; HRP, horseradish peroxidase; FITC, fluorescein claudins are active in multiple tissues or even expressed ubiquitously, isothiocyanate; FFPE, formalin fixed paraffin-embedded; min, minutes; PBS, phosphate a few claudins are restricted to single tissues (reviewed in (Krause ′ buffered saline; DAB, 3,3 -diaminobenzidine; kb, kilobases; UCSC, University of et al., 2008)). Some cell types express unique claudin species (Fujita California Santa Cruz; aa, amino acids; bp, base pairs; TM, transmembrane domain; fi ECL, extracellular loop; PDZ, PDZ protein domain; MUPP-1, multi-PDZ domain protein et al., 2006). For correct tissue-speci city of transcriptional activation, 1; ICH, The International Conference on Harmonisation of Technical Requirements for individual claudin genes are controlled by unique regulatory Registration of Pharmaceuticals for Human Use; CPMP, Committee for Proprietary mechanisms. Medicinal Products; ID, identifier; IHC, immunohistochemistry; NTC, no template Tight junction strands generally contain multiple claudin species control. that may interact in homo- and heterophilic ways (Gonzalez-Mariscal ⁎ Corresponding author at: Ganymed Pharmaceuticals AG, Freiligrathstr. 12, 55131 Mainz, Germany. Tel.: +49 6131 101; fax: +49 6131 114. et al., 2003; Morita et al., 1999). It is hypothesized that the distinct E-mail address: [email protected] (Ö. Türeci). composition of claudins plays a major role in variable physiological 0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.04.007 84 Ö. Türeci et al. / Gene 481 (2011) 83–92 properties of TJs in different tissues (Anderson, 2001; Mitic et al., targets (Sahin et al., 2008). We showed that expression of CLDN18.2 in 2000; Rahner et al., 2001). Furthermore, due to their crucial function normal tissues is strictly confined to differentiated epithelial cells of the in compartment separation, TJs are an evolutionary well conserved gastric mucosa and is absent from the gastric stem cell zone. CLDN18.2 is component of vertebrate cytoarchitecture (Kollmar et al., 2001). retained upon malignant transformation and is expressed in a However, phylogenetic analysis of claudins has been largely done significant proportion of primary gastric cancers and metastases thereof. in the context of evolutionary aspects concerning tight junction Moreover, we found frequent ectopic activation of CLDN18.2 in conservation (Krause et al., 2008) and little is known about the pancreatic, esophageal, ovarian, and lung tumors. The closely related evolutionary conservation of single claudins. splice variant isoform 1 (CLDN18.1), in contrast, is exquisitely restricted Recently, a member of the claudin multigene family, isoform 2 to cells of lung tissue (Niimi et al., 2001). of claudin-18 (CLDN18.2), appeared among the hits in a combined The strict lineage specificity of both CLDN18 variants in normal in silico data mining and wet bench strategy to identify gastrocyte human tissues prompted us to consider this gene as a model to in- lineage–specific cell surface molecules for use as therapeutic antibody vestigate how gene structure, regulation and expression of highly Fig. 1. Phylogenetic conservation of gene structure, transcript sequence and cis-acting regulatory promoter elements of CLDN18. (A) Genomic organization of the human CLDN18 gene locus on chromosome 3q22, which was used as reference. CLDN18.1 and CLDN18.2 result from alternative splicing of the first exon. (B) Screenshot of the UCSC genome browser shows an alignment of the human CLDN18 gene locus against genomic sequences of primate, mammals, and vertebrates. (Top) RefSeq transcripts feature alternative first exons for transcripts encoding isoform 1 or isoform 2 (Kent et al., 2002). (Middle) Summarized conservation across 46 vertebrates by PhastCons, with taller bars indicating higher conservation (Siepel et al., 2005). (Lower) Cross-species alignments for selected species, with bars indicating conservation (Blanchette et al., 2004). (C) Alignment of the Claudin18.1 and Claudin18.2 promoter sequences. Cis-acting regulatory elements described for the human CLDN18 isoforms are boxed. The genomic sequence of O. cuniculus for the Claudin18.1 promoter was not available. Ö. Türeci et al. / Gene 481 (2011) 83 – 92 Fig. 1 (continued). 85 86 Ö. Türeci et al. / Gene 481 (2011) 83–92 selective members of multigene families with housekeeping function 2.4. First and second antibodies evolve across species. For immunohistochemistry, four different antibodies with IgG 2. Materials and methods constant regions from three different species were available. This facilitated detection of CLDN18 across different species. Generally 2.1. Sequence retrieval and analysis speaking, we chose secondary antibodies to be used from different species as the first antibody they were combined with. Rabbit antisera Orthologs of human CLDN18 isoforms were detected by BLAST aCLDN18.2/n-term (generated at Eurogentec) and anti-CLDN18/mid searches either in the non-redundant protein database (nr) at NCBI (purchased from Zymed) were used on fixed paraffin embedded (http://blast.ncbi.nlm.nih.gov/Blast.cgi) or in all available species data- tissue in combination with the second antibody Powervision Poly HRP bases at the Ensembl server site (http://www.ensembl.org/Multi/ goat-anti-rabbit (ImmunoLogic). anti-CLDN18/mid was the only one blastview). In each case the human CLDN18.2 protein sequence RefSeq among the four antibodies which was cross-reactive with CLDN18.1. ID: NP_001002026 served as query. Failing RefSeq NM transcripts and Mouse IgG2a muMAB362 and its chimerized human IgG1 variant NP proteins, we selected Ensembl identifier.ABLASTsearchofthe iMAB362,