Molecular characterisation of mouse and human TSSC6: evidence that TSSC6 is a genuine member of the tetraspanin superfamily and is expressed specifically in haematopoietic organs

Abstract

Previous analyses of the murine and human TSSC6 (also known as Phemx) proteins were not carried out using the full length sequence. Using 5′-RACE and cDNA library screening, we identified an additional 5′ sequence for both the murine Tssc6 cDNA and its human homologue TSSC6. This novel sequence encodes a 5′ exon encoding an in frame, upstream start codon, an N-terminal cytoplasmic domain and a transmembrane domain. The deduced, and now full length, murine and human TSSC6 proteins contained four hydrophobic regions together with other features characteristic of the tetraspanin superfamily. Computational analyses of the full length sequences show that TSSC6 is a genuine, albeit relatively divergent member of this superfamily. Using RNA from a number of mouse tissues, we identified seven splice variants of Tssc6. Splice variants of the human gene were also detected. Tssc6 expression was detected early in embryogenesis in primitive blood cells and was confined to haematopoietic organs in the adult mouse. Tssc6 expression was detected in many haematopoietic cell lines and was highest in cell lines of the erythroid lineage.

Introduction

Haematopoietic cells communicate with their environment via molecular interactions mediated principally by membrane proteins expressed at the cell surface. Members of the tetraspanin superfamily class of membrane proteins are frequently found on haematopoietic cells. These proteins are characterised by four highly conserved transmembrane domains. They have short cytoplasmic domains at the N- and C-termini of the polypeptide, and two relatively divergent extracellular domains [1], [2]. Two broad themes in the literature hint at tetraspanin function. Firstly, it is clear from experiments where monoclonal antibodies cross-link tetraspanin molecules at the cell surface that tetraspanins are molecules which can couple to signal transduction pathways, and cells can respond to these stimuli by activating tyrosine phosphorylation and mobilising intracellular calcium stores [3], [4], [5], modulating cellular proliferation [4], [6], [7] and even regulating cell motility [8], [9], [10]. Secondly, immunoprecipitation experiments using cells lysed with detergents that can preserve protein–protein interactions have shown that tetraspanins exist at the cell surface in multi-molecular complexes [1], [2], [11]. Indeed the tetraspanins would appear to be exceptionally promiscuous in this regard, and a strikingly large variety of other proteins have been reported in the literature to be associated with tetraspanins including integrins [11], lymphocyte co-receptor molecules such as CD4, CD8, and CD19 [12], [13], MHC [14], [15], the EGF receptor [16] and a claudin [17]. It is also remarkable that all the tetraspanins tested to date can apparently co-precipitate any other tetraspanin [18]. The specificity and functional implications of these tetraspanin multimolecular complexes await further dissection. However, these observations have led to the proposal that tetraspanins are ‘molecular facilitators’, i.e. their primary role is one of organising other proteins into multimolecular signal transduction complexes [2].

In this paper we demonstrate that the mouse and human TSSC6 proteins are genuine members of the tetraspanin superfamily. The human TSSC6 (tumour-suppressing STF cDNA 6) gene was first identified as a novel cDNA located within a 2.5 Mb subchromosomal transferable fragment (STF) from human chromosome 11p15 which suppressed the growth of a rhabdomyosarcoma cell line [19]. The TSSC6 locus was located in the centre of a 1 Mb imprinted domain, although it was shown not to be imprinted. The predicted protein did not have close similarity to known proteins, other than a weak similarity to rat CD81/TAPA1 [19]. The human CD81/TAPA1 gene was found to lie 60 kb centromeric to TSSC6, suggesting that one of the genes may have arisen by gene duplication. A second group also sequenced human EST clones encoding splice variants of TSSC6 (designated PHEMX1 and PHEM2) [20]. The murine Tssc6 locus was found to lie at the distal end of mouse chromosome 7 in a region syntenic with human chromosome 11p15 [20], [21]. Like TSSC6, the gene did not appear to be imprinted in embryonic or adult tissues [20], [21]. A 250 kb genomic sequence in the centre of the mouse imprinting cluster on chromosome 7, which contained Tssc6, was compared with the orthologous region in the human [21]. As in the human, the Tssc6 locus was adjacent to Cd81. Again, comparison of the deduced amino acid sequence showed only weak homology with CD81. Ko et al. originally identified Tssc6 as an expressed sequence tag (EST) in extraembryonic tissues of the gastrula-stage embryo [22]. They subsequently designated it Phemx (Pan haematopoietic expression) [20] based on its expression pattern.

We have identified a novel 5′ exon of murine and human Tssc6/TSSC6 that encodes an in frame, upstream start codon, an N-terminal cytoplasmic domain and a transmembrane domain. Using computational methods we demonstrate that the predicted full length TSSC6 proteins have key features of the tetraspanin superfamily. In addition, we document the extensive alternative splicing that occurs at both the human and murine loci and examine the expression pattern of the murine Tssc6 gene.