Organization of the rat Tage4 gene and herpesvirus entry activity of the encoded protein
Introduction
The immunoglobulin (Ig) superfamily encompasses a growing number of molecules that share a common structural homology unit (Williams and Barclay, 1988). These molecules mediate a variety of homotypic and heterotypic cellular interactions playing a general role in cell-surface recognition (Halaby and Mornon, 1998). Several members of the Ig family are overexpressed in cancer cells. The CEA (carcinoembryonic antigen) and the NCA (non-specific cross-reacting antigen) genes are overexpressed in human colorectal carcinoma (Chi et al., 1991, Mafune et al., 1992). The human B-CAM (B-cellular adhesion molecule) gene is highly expressed in ovarian carcinomas in vivo (Campbell et al., 1994). Muc18 has been identified as a marker of tumor progression in human malignant melanoma (Lehmann et al., 1989) and de novo expression of ICAM-1 (intercellular CAM-1) correlates with increased risk of metastasis in melanoma (Johnson et al., 1989).
The Tage4 gene (Tumor Associated Glycoprotein E4) codes for a rat colon-carcinoma-associated antigen that we previously identified as a novel member of the Ig superfamily (Chadéneau et al., 1994). This antigen is a transmembrane glycoprotein (Chadéneau et al., 1991) containing, in its extracellular region, three Ig-like domains (V-type, C2-type and C2-type). The Tage4 gene is overexpressed by chemically induced rat colon tumors (Denis, 1998) and in Min mouse intestinal adenomas (Chadéneau et al., 1996a) as compared to normal adult tissues. The Tage4 cDNA has been isolated (Chadéneau et al., 1994) and sequence analysis using the ALIGNn software revealed a 59.1% identity between the coding sequence of rat Tage4 and human CD155 (α transcript), another member of the Ig superfamily (Mendelsohn et al., 1989). The Tage4 gene has been mapped to rat chromosome 1q22 (Chadéneau et al., 1997) and mouse 7A2–B1 (Chadéneau et al., 1996b), regions that are homologous to the long arm of human chromosome 19 where is located the CD155 gene (Koike et al., 1990). CD155 serves as the receptor for human poliovirus (Mendelsohn et al., 1989) and can also mediate the entry into cells of porcine pseudorabies virus (PRV) and bovine herpesvirus 1 (BHV-1) (Geraghty et al., 1998), two animal herpesviruses that can infect human cells. Physiological roles of CD155 have not yet been identified. CD155 belongs to a small subfamily of the immunoglobulin superfamily that includes nectin-1, nectin-2 and nectin-3, all three of which have been shown to mediate cell-cell adhesion and to localize to sites of cadherin-based cell junctions (Lopez et al., 1998, Takahashi et al., 1999, Satoh-Horikawa et al., 2000).
All attempts to isolate and identify the human homolog of the rat Tage4 gene were unsuccessful: rat and mouse Tage4 probes did not hybridize human genomic DNA and PCR amplifications using a number of oligonucleotides derived from the rat Tage4 cDNA sequence did not allow us to amplify human genomic DNA or cDNA from various tissues and cell lines. Because of the sequence identity between Tage4 and CD155 cDNAs and the location of the corresponding genes on homologous chromosomes, we cloned and characterized the rat Tage4 gene and compared organization of this gene with that of the human CD155 gene. We also determined whether Tage4 had the ability to mediate entry of viruses known to use CD155 as receptor. On the basis of these findings we propose that the rat Tage4 gene is the ortholog of the human CD155 gene.
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Cell lines
WB2054M and PROb rat colon carcinoma cell lines and HCT8R human colon carcinoma cells were grown in RPMI-1640 containing l-Glutamine and sodium bicarbonate, supplemented with 10% fetal calf serum, penicillin (100 units/ml) and streptomycin (0.1 mg/ml). Chinese hamster ovary cells (CHO-K1) (American Type Culture Collection # CCL-61) were grown in Ham's F12 medium supplemented with 10% fetal bovine serum. Cells were maintained at 37°C in humidified atmosphere of 5% carbon dioxide and passaged
Isolation of rat Tage4 genomic clones and analysis of the Tage4 gene structure
A probe corresponding to the coding region of the Tage4 cDNA was used to probe a rat PAC DNA library. This allowed us to isolate and identify eight clones. One of these clones (RPCIP-N22604Q2; http://resource.rzpd.de) was selected and studied further.
PCR amplifications were performed on both cDNA and DNA of the selected PAC clone with several sets of primers in order to localize and amplify intronic sequences. These oligonucleotides were designed based on the Tage4 cDNA sequence. They are
Discussion
The rat Tage4 gene and the human CD155 gene code for proteins that are members of the Ig superfamily. Members of this superfamily contain one or many Ig-like constant (C) domains and Ig-like variable (V) domains. The most characteristic feature of the gene structure of Ig superfamily molecules is that each Ig-like domain is encoded within one exon as illustrated by the CD22 gene, the ICAM-1 gene and the CEA gene (Williams and Barclay, 1988). However, exceptions to the ‘one Ig-like domain – one
Acknowledgments
This work was supported by grants from the Ligue Départementale (44) contre le Cancer (M.G.D.) and from the National Institutes of Health, R37 AI36293 (P.G.S.). B.B. is a recipient of a fellowship from the Ligue Départementale (44) contre le Cancer. R.J.G. was supported by National Research Service Award F32 AI09471. We wish to thank Carl Pavel and Vincent Racaniello for the results on poliovirus susceptibility and for critical reading of the manuscript.
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