The ADAMDEC1 (decysin) gene structure: evolution by duplication in a metalloprotease gene cluster on chromosome 8p12

Members of the ADAM superfamily of metalloprotease genes are involved in a number of biological processes, including fertilization, neurogenesis, muscle development, and the immune response. These proteins have been classified into several groups. The prototypic ADAM family is comprised of a pro-domain, a metalloprotease domain, a disintegrin domain, a cysteine-rich region, a transmembrane domain, and a variable cytoplasmic tail. We recently identified a novel member of this superfamily, ADAMDEC1 (decysin). Due to the partial lack of a disintegrin domain and the total lack of a cysteine-rich domain, this protein has been placed in a novel subclass of the ADAM gene family. We have investigated the gene structure of the human and mouse ADAMDEC1 and have revealed a metalloprotease gene cluster on human Chromosome 8p12 comprising ADAMDEC1, ADAM7, and ADAM28. Our results suggest that ADAMDEC1 has arisen by partial gene duplication from an ancestral gene at this locus and has acquired a novel function. ADAMDEC1 is expressed in the immune system, by dendritic cells and macrophages. The relatedness of ADAMDEC1, ADAM7, and ADAM28 suggests that these proteases share a similar function.     

Phylogenetic analysis of the cadherin superfamily.

Cadherins are a multigene family of proteins which mediate homophilic calcium-dependent cell adhesion and are thought to play an important role in morphogenesis by mediating specific intercellular adhesion. Different lines of experimental evidence have recently indicated that the site responsible for mediating adhesive interactions is localized to the first extracellular domain of cadherin. Based upon an analysis of the sequence of this domain, I show that cadherins can be classified into three groups with distinct structural features. Furthermore, using this sequence information a phylogenetic tree relating the known cadherins was assembled. This is the first such tree to be published for the cadherins. One cadherin subtype, neural cadherin (N-cadherin), shows very little sequence divergence between species, whereas all other cadherin subtypes show more substantial divergence, suggesting that selective pressure upon this domain may be greater for N-cadherin than for other cadherins. Phylogenetic analysis also suggests that the gene duplications which established the main branches leading to the different cadherin subtypes occurred very early in their history. These duplications set the stage for the diversified superfamily we now observe.     

Characterization of cadherin-24, a novel alternatively spliced type II cadherin

Cadherins comprise a superfamily of calcium-dependent cell-cell adhesion molecules. Within the superfamily are six subfamilies including type I and type II cadherins. Both type I and type II cadherins are composed of five extracellular repeat domains with conserved calcium-binding motifs, a single pass transmembrane domain, and a highly conserved cytoplasmic domain that interacts with beta-catenin and p120 catenin. In this study, we describe a novel cadherin, cadherin-24. It is a type II cadherin with a 781-codon open reading frame, which encodes a type II cadherin protein complete with five extracellular repeats containing calcium-binding motifs, a transmembrane domain, and a conserved cytoplasmic domain. Cadherin-24 has the unusual feature of being alternatively spliced in extracellular repeat 4. This alternative exon encodes 38 in-frame amino acids, resulting in an 819-amino-acid protein. Sequence analysis suggests the presence of beta-catenin and p120 catenin-binding sequences, and immunoprecipitation experiments confirm the ability of both forms of the novel cadherin to associate with alpha-catenin, beta-catenin, and p120 catenin. In addition, aggregation assays show that both forms of cadherin-24 mediate strong cell-cell adhesion.     

The quest for ligands and binding partners of atypical cadherin FAT1

A recent study in Scientific Reports identified glypican-3 (GPC3) as a novel extracellular interacting protein for FAT1 in hepato-cellular carcinoma (HCC) cells. FAT1 is a large transmembrane atypical cadherin with limited knowledge existing about its binding partners. While in Drosophila, dachsous (ds), another transmembrane member of the cadherin superfamily, is known to function as FAT1 ligand, no ligand is known in mammals so far. The revelation of GPC3 as a potential binding partner of FAT1 extracellular domain unfolds an opportunity to study potential triggers of FAT1 signaling in cancers. Available inhibitors of GPC3 in various phases of clinical trials also present an attractive option to curb GPC3-FAT1 signaling in tumors that overexpress these proteins.     

The fat-like gene of Drosophila is the true orthologue of vertebrate fat cadherins and is involved in the formation of tubular organs

Fat cadherins constitute a subclass of the large cadherin family characterized by the presence of 34 cadherin motifs. To date, three mammalian Fat cadherins have been described; however, only limited information is known about the function of these molecules. In this paper, we describe the second fat cadherin in Drosophila, fat-like (ftl). We show that ftl is the true orthologue of vertebrate fat-like genes, whereas the previously characterized tumor suppressor cadherin, fat, is more distantly related as compared with ftl. Ftl is a large molecule of 4705 amino acids. It is expressed apically in luminal tissues such as trachea, salivary glands, proventriculus, and hindgut. Silencing of ftl results in the collapse of tracheal epithelia giving rise to breaks, deletions, and sac-like structures. Other tubular organs such as proventriculus, salivary glands, and hindgut are also malformed or missing. These data suggest that Ftl is required for morphogenesis and maintenance of tubular structures of ectodermal origin and underline its similarity in function to a reported lethal mouse knock-out of fat1 where glomerular epithelial processes collapse. Based on our results, we propose a model where Ftl acts as a spacer to keep tubular epithelia apart rather than the previously described adhesive properties of the cadherin superfamily.     

Molecular cloning of a novel NF2/ERM/4.1 superfamily gene, ehm2, that is expressed in high-metastatic K1735 murine melanoma cells

We have cloned a novel gene, Ehm2, that is expressed in high-metastatic but not in low-metastatic K-1735 murine melanoma cells. The Ehm2 gene encodes a protein of 527 amino acid residues, showing up to 41% amino acid identity with the FERM domain of NF2/ERM/4.1 superfamily proteins, which have the function of connecting cell surface transmembrane proteins to cytoskeletal molecules. The Ehm2 gene was mapped to chromosome 4 and was expressed in the liver, lung, kidney, and testis and in 7- to 17-day embryos. The highest level of homology was observed with NBL4, which is a new subfamily protein of the NF2/ERM/4.1 superfamily. A human homologue of the mouse Ehm2 gene, showing significant homology (83% identity), was identified in the genomic DNA and EST databases. Furthermore, seven rat EST clones and one pig EST clone in the GenBank EST database were identified as having 83-92% sequence homology with the cDNA sequence of the mouse Ehm2 gene. Thus, Ehm2 is a highly conserved gene that encodes a novel member of the NF2/ERM/4.1 superfamily proteins.     

A comprehensive survey of cadherin superfamily gene expression patterns in Ciona intestinalis

We have carried out a comprehensive survey of the spatiotemporal expression of cadherin superfamily genes in the basal chordate Ciona intestinalis, as an example of a genome-wide expression study of a gene family directly regulating cellular processes in morphogenesis. We found 15 definitely expressed cadherin superfamily genes in the Ciona intestinalis genome. Up to the late gastrula stage, all identified delta-protocadherins and the type II classical cadherin, but not other subfamily members, were zygotically expressed. At later stages, however, all cadherin superfamily genes were expressed in the nervous system. These data are useful for understanding the role of these genes in Ciona development and the evolution of chordates.     

Molecular cloning of a novel immunoglobulin superfamily gene preferentially expressed by brain and testis

We have cloned and characterized a novel gene from both human and mouse that encodes a new member of the immunoglobulin superfamily. The gene is preferentially expressed in both brain and testis, and hence, termed BT-IgSF (brain- and testis-specific immunoglobulin superfamily). The predicted protein consists of V-type and C2-type immunoglobulin domains as well as a hydrophobic signal sequence, a single transmembrane region, and a cytoplasmic domain. Human BT-IgSF protein (431 amino acids) is 88% identical to the mouse protein (428 amino acids) and both show significant homology to coxsackie and adenovirus receptor (CAR) and endothelial cell-selective adhesion molecule (ESAM). We examined the expression of BT-IgSF with various cultured cells and found that the gene was expressed in both neurons and glial cells in vitro. Furthermore, the expression was preferentially detected in pyramidal cell layers of the dentate gyrus and hippocampus and in commissure fibers of the corpus callosum, in brain tissue sections examined. These findings suggest that BT-IgSF plays a role in the development or function of the central nervous system.     

Cloning and characterization of BS-cadherin, a novel cadherin from the colonial urochordate Botryllus schlosseri

The genomic DNA for a novel member of the cadherin family (BS-cadherin) was cloned and characterized from the colonial marine invertebrate, Botryllus schlosseri. Using a differential display of mRNA by means of PCR, a small cDNA fragment of 380 nucleotides was found to be specifically expressed in a colony undergoing allogeneic rejection processes, as compared with naïve parts of the same genotype. This cDNA fragment was used as a probe to screen a genomic library of Botryllus schlosseri. A genomic fragment containing an ORF of 2718 nucleotides, with no introns, was isolated. The encoded protein exhibits a typical structure of cadherins; an extracellular domain with conserved repeated sequences (cadherin signatures), a single transmembrane domain and a conserved cytoplasmic tail region. The BS-cadherin amino-acid sequence shows 32–35% identity to mature classical cadherins type I, e.g., N-, P- and E-cadherin as well as mature classical cadherins type II, e.g., human cadherin-6, -8 and OB-cadherin. This cadherin represents a new cadherin gene family, evolutionarily distant to all other known classical cadherins.     

Cloning and expression of cDNA encoding a neural calcium-dependent cell adhesion molecule: its identity in the cadherin gene family

The neural cadherin (N-cadherin) is a Ca2+-dependent cell-cell adhesion molecule detected in neural tissues as well as in non-neural tissues. We report here the nucleotide sequence of the chicken N-cadherin cDNA and the deduced amino acid sequence. The sequence data suggest that N-cadherin has one transmembrane domain which divides the molecule into an extracellular and a cytoplasmic domain; the extracellular domain contains internal repeats of characteristic sequences. When the N-cadherin cDNA connected with virus promoters was transfected into L cells which have no endogenous N-cadherin, the transformants acquired the N-cadherin-mediated aggregating property, indicating that the cloned cDNA contained all information necessary for the cell-cell binding action of this molecule. We then compared the primary structure of N-cadherin with that of other molecules defined as cadherin subclasses. The results showed that these molecules contain common amino acid sequences throughout their entire length, which confirms our hypothesis that cadherins make a gene family.     

Genetic and developmental characterization of Dmca1D,a calcium channel alpha1 subunit gene in Drosophila melanogaster.

To begin unraveling the functional significance of calcium channel diversity, we identified mutations in Dmca1D, a Drosophila calcium channel alpha1 subunit cDNA that we recently cloned. These mutations constitute the l(2)35Fa lethal locus, which we rename Dmca1D. A severe allele, Dmca1D(X10), truncates the channel after the IV-S4 transmembrane domain. These mutants die as late embryos because they lack vigorous hatching movements. In the weaker allele, Dmca1D(AR66), a cysteine in transmembrane domain I-S1 is changed to tyrosine. Dmca1D(AR66) embryos hatch but pharate adults have difficulty eclosing. Those that do eclose have difficulty in fluid-filling of the wings. These studies show that this member of the calcium channel alpha1 subunit gene family plays a nonredundant, vital role in larvae and adults.     

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.

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.     

Precise amounts of a novel member of a phosphotransferase superfamily are essential for growth and normal morphology in Caulobacter crescentus

The Caulobacter crescentus chromosomal clp locus contains the genes encoding the components of ClpXP, a multisubunit protease required for cell cycle progression in this organism. Here, we report the identification and characterization of cicA, a gene located between the clpX and clpP genes on the Caulobacter chromosome. cicA is a novel morphogene in C. crescentus and, like clpX and clpP, is essential for growth. A conditional cicA mutant stopped growth, but retained viability under restrictive conditions. In contrast, an increased concentration of CicA led to an immediate loss of the normal rod shape, an almost 10-fold increase of the cell's volume and a cell division block. In parallel with this drastic morphological change, cells rapidly lost viability. Primary sequence analysis suggested that the cicA gene encodes a member of a large superfamily of phosphotransferases, that include phosphoserine phosphatases, the ATPase domain of P-type ATPases and receiver domains of response regulators. Four conserved motifs of this protein family that have been implicated in the catalysis of phosphotransfer reactions were investigated by site-directed mutagenesis and were found to be critical for in vivo function of CicA. Based on our observations, we postulate that CicA is involved in essential phosphotransferase reactions in Caulobacter and that increased activity of CicA has a deleterious effect on cell wall biosynthesis, morphogenesis and cell division.     

Cloning and expression of nope, a new mouse gene of the immunoglobulin superfamily related to guidance receptors

The novel mouse gene Nope was identified due to its proximity to the Punc gene on chromosome 9. With a domain structure of four immunoglobulin domains, five fibronectin type III repeats, a single transmembrane domain, and a cytoplasmic domain, Nope encodes a new member of the immunoglobulin superfamily of cell surface proteins. It displays a high level of similarity to Punc, as well as to guidance receptors such as the Deleted in Colorectal Cancer protein and Neogenin. Nope is expressed during embryonic development in the notochord, in developing skeletal muscles, and later in the ventricular zone of the nervous system. In the adult brain, Nope can be detected in the hippocampus. Radiation hybrid mapping of Nope, Punc, and Neogenin placed all three genes in close vicinity on mouse chromosome 9.     

Cloning and expression of the large zebrafish protocadherin gene, Fat

The cadherin superfamily members play an important role in mediating cell-cell contact and adhesion (Takeichi, M., 1991. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251, 1451-1455). A distinct subfamily, neither belonging to the classical or protocadherins includes Fat, the largest member of the cadherin super-family. Fat was originally identified in Drosophila. Subsequently, orthologues of Fat have been described in man (Dunne, J., Hanby, A. M., Poulsom, R., Jones, T. A., Sheer, D., Chin, W. G., Da, S. M., Zhao, Q., Beverley, P. C., Owen, M. J., 1995. Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule. Genomics 30, 207-223), rat (Ponassi, M., Jacques, T. S., Ciani, L., ffrench, C. C., 1999. Expression of the rat homologue of the Drosophila fat tumour suppressor gene. Mech. Dev. 80, 207-212) and mouse (Cox, B., Hadjantonakis, A. K., Collins, J. E., Magee, A. I., 2000. Cloning and expression throughout mouse development of mfat1, a homologue of the Drosophila tumour suppressor gene fat [In Process Citation]. Dev. Dyn. 217, 233-240). In Drosophila, Fat has been shown to play an important role in both planar cell polarity and cell boundary formation during development. In this study we describe the characterization of zebrafish Fat, the first non-mammalian, vertebrate Fat homologue to be identified. The Fat protein has 64% amino acid identity and 80% similarity to human FAT and an identical domain structure to other vertebrate Fat proteins. During embryogenesis fat mRNA is expressed in the developing brain, specialised epithelial surfaces the notochord, ears, eyes and digestive tract, a pattern similar but distinct to that found in mammals.     

Expression of UNC-5 in the developing Xenopus visual system

Celsr, also called Flamingo (Fmi) genes encode proteins of the cadherin superfamily. Celsr cadherins are seven-pass transmembrane proteins with nine cadherin repeats in the extracellular domain, and an anonymous intracellular C-terminus. The Drosophila Fmi gene regulates epithelial planar cell polarity and dendritic field deployment. The three Flamingo gene orthologs in man and rodents are named, respectively, CELSR1-3 and Celsr1-3. Celsr1 and 2 are expressed during early development, in the brain and epithelia. In this report, we characterized further Celsr genes in the mouse, and examined their developmental pattern of expression. Each Celsr is expressed prominently in the developing brain following a specific pattern, suggesting that they serve distinct functions.