Amino acid sequence of mouse tenascin and differential expression of two tenascin isoforms during embryogenesis

We have isolated cDNA clones for mouse tenascin and analyzed expression of tenascin mRNAs during embryonic development of the kidney and gut. The deduced amino acid sequence of the mouse tenascin cDNAs shows a modular structure of repeats similar to chicken and human tenascin. In mouse there are 14.5 cysteine-rich repeats with similarity to the EGF repeat, followed by several repeats with similarity to the type III repeat of fibronectin. A longer variant contains 13 fibronectin type III repeats, whereas a shorter splice variant of mouse tenascin lacks the 5 type III repeats that occur directly after the fifth repeat in the longer variant. Contrary to the chicken and human sequences, mouse tenascin does not contain an RGD sequence in the third type III repeat implicated in cell attachment, or in any other positions. In Northern hybridizations to RNA from primary embryonic fibroblasts, the cDNA clone M 20/1 detects two mRNAs with sizes close to 6 and 8 kb. This, and the other data presented here suggest that the two major mouse tenascin polypeptides arise through an alternative RNA splicing. The two major mRNAs are differentially expressed during development. The 8-kb mRNA is more prominent than the 6-kb mRNA throughout prenatal kidney development, but during postnatal development the ratio of the two mRNAs changes. A different expression pattern is seen in the developing gut where the 6-kb mRNA predominates during embryogenesis with the 8-kb mRNA appearing later. The mRNA data of the developing gut correspond with previous protein data, which showed that the shorter Mr 210,000 polypeptide predominates during earlier developmental stages and the larger Mr 260,000 polypeptide appears later in the embryonic gut (Aufderheide, E., and P. Ekblom. 1988. J. Cell Biol. 107:2341-2349).     

A novel tenascin type III repeat is part of a complex of tenascin mRNA alternative splices.

Sequence analysis of two human tenascin encoding cDNA clones from a cDNA library of the U251 glioblastoma cell line revealed the presence of a novel 276 bp tenascin type III fibronectin like repeat. This alternatively spliced type III repeat designated AD1 is located between the previously identified repeats 10 and 11 and has sequence homology with human, chicken and mouse tenascin type III repeats. These results show that tenascin has at least 16 consecutive fibronectin like type III repeats. PCR amplification of random primed mRNA with specific type III repeat primers revealed a pattern of multiple alternative splices of AD1 and flanking type III repeats. The alternative splice variants were confirmed by direct sequencing. Differences were observed in the expression of the various alternative splices of tenascin mRNA between tumor and normal cells and may thus indicate differences in tenascin isoform expression and function in normal and tumor cells. PCR and Southern analysis of genomic DNA indicate that AD1 is coded by a single exon present in both human and mouse genome.     

Folding-unfolding of FN-III domains in tenascin: an elastically coupled two-state system

In a single-molecule atomic force microscopy (AFM) experiment, the tenascin molecule is stretched by an external force causing an elongation which is due to the unfolding of the FN-III modules. The features of the force-extension curves depend on the pulling speed and show a saw-tooth pattern (lower speeds) or a smooth pattern (higher speeds). In any case, the unfolded domains are elastically coupled to the unfolded modules, acting as transmitters of the external force. In this communication, the folding-unfolding process of the FN-III domains in tenascin is studied using reaction rate theory and a simple two-state model. The main hypothesis of the study is that, at microscopic level, the force needed to unfold a domain and the unfolding rate (unfolding velocity) can mimic the macroscopic process of measurement by AFM. As the external force is applied, the probability of unfolding increases as dictated by the reaction rate theory. Within this context, a relationship between the unfolding force and the unfolding velocity is obtained. The latter relation will describe microscopically the process in a phenomenological fashion. Moreover, while relating the results of this study with other experimental (AFM measurements) and theoretical (Monte Carlo simulations) data, we found that the graph of unfolding force-unfolding velocity is similar to that of external force-pulling velocity. The refolding process can also be studied within this model and the results show similar trends. The latter suggests a generic and universal behavior of such kind of molecular domains at least in the light of the proposed model.     

MPTP delta, a putative murine homolog of HPTP delta, is expressed in specialized regions of the brain and in the B-cell lineage.

Protein tyrosine phosphatases (PTPs), together with protein tyrosine kinases (PTKs), are involved in the regulation of cell activation, growth, and differentiation. To further elucidate the fine tuning of cell growth and differentiation through tyrosine phosphorylation, we tried to isolate mouse receptor-type PTP (RPTP) cDNA clones by screening mouse brain cDNA libraries with mouse CD45 PTP domain probes under reduced-stringency conditions. Characterization of isolated cDNA clones for RPTP showed that the cytoplasmic region contains two tandem repeats of PTP domain of about 230 amino acids with intrinsic phosphatase activity. The extracellular region was composed of immunoglobulin (Ig)-like domains and fibronectin type III (FN-III)-like domains. The gene was highly homologous to human PTP delta (HPTP delta) and thus was named MPTP delta (murine counterpart of HPTP delta). The MPTP delta gene appeared to generate at least three species of mRNA, which differ in the composition of the extracellular domain: type A, one Ig-like and four FN-III-like domains; type B, one Ig-like and eight FN-III-like domains; and type C, three Ig-like and eight FN-III-like domains. Interestingly, the 5' untranslated region and the leader peptide of types A and B were completely different from those of type C. Northern (RNA) blot analysis demonstrated that brain, kidney, and heart cells express three mRNA species of about 7 kb. Antibody directed against part of the extracellular domain of type A MPTP delta recognized a 210-kDa protein in brain and kidney lysates. In situ hybridization of brain samples revealed that MPTP delta mRNA is present in the hippocampus, thalamic reticular nucleus, and piriform cortex, where some Src family PTKs have been also demonstrated to exist. Although MPTP delta mRNA was not detected in lymphoid tissues, all of the pre-B-cell lines tested and one of three B-cell lines tested expressed MPTP delta mRNA, whereas antibody-producing B-cell hybridomas and T-cell and macrophage lines did not. Finally, the MPTP delta locus was tightly linked to the brown (b) locus on mouse chromosome 4.     

The expression of tenascin by neural crest cells and glia.

The extracellular matrix glycoprotein tenascin is concentrated in both the embryo and adult in regions where cell motility is taking place. For example, during avian neural crest morphogenesis tenascin is concentrated in the rostral half of the sclerotome, precisely where the neural crest cells themselves are found. Previous in vitro studies indicated that somite cells were the source of this tenascin, implying a role for tenascin in directing the ventral migration of neural crest cells and thus the establishment of the periodic arrangement of the PNS. In this study, we have used a cDNA probe to identify the source of tenascin found along the pathways of the neural crest using in situ hybridization. In tissue sections, individual cells found along the neural crest migratory pathways, both before entering the somites and within the somites, are strongly labelled by the tenascin cDNA. In vitro neural crest cells are more strongly labelled with the tenascin probe than somite cells. Finally, western blotting has been used to identify tenascin in culture medium conditioned by neural crest cells. This indicates that neural crest cells themselves are the source of much of the tenascin found lining their migratory pathways, and that interactions with somite cells may not be needed to induce the expression of tenascin. We have also studied the distribution of tenascin mRNA in the developing spinal cord and spinal ganglia. At embryonic days 7 and 10, tenascin cDNA hybridizes within cells that appear to be migrating from the ependymal layer to the white matter, as well as within cells in the dorsal roots.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the evolution of tenascin and fibronectin early in the chordate lineage

Fibronectin and tenascin are extracellular matrix glycoproteins that play important roles in cell adhesion and motility. In a previous study we provided evidence that tenascin first appeared early in the chordate lineage. As tenascin has been proposed to act, in part, through modulation of cell-fibronectin interactions, we sought here to identify fibronectin genes in non-vertebrate chordates and other invertebrates to determine if tenascin and fibronectin evolved separately or together, and to identify phylogenetically conserved features of both proteins. We found that the genome of the urochordate Ciona savignyi contains both a tenascin gene and a gene encoding a fibronectin-like protein with fibronectin type 1, 2 and 3 repeats. The genome of the cephalochordate Branchiostoma floridae (amphioxus) also has a tenascin gene. However, we could not identify a fibronectin-like gene in B. floridae, nor could we identify fibronectin or tenascin genes in echinoderms, protostomes or cnidarians. If urochordates are more closely related to vertebrates, tenascin may have evolved before fibronectin in an ancestor common to tunicates and amphioxus. Alternatively, tenascin and fibronectin may have evolved in an ancestor common to B. floridae and C. savignyi and the fibronectin gene was subsequently lost in the cephalochordate lineage. The fibronectin-like gene from C. savignyi does not encode the RGD motif for integrin binding found in all vertebrate fibronectins, and it lacks most of the fibronectin type 1 domains believed to be critical for fibrillogenesis. In contrast, the tenascin gene in B. floridae encodes multiple RGD motifs, suggesting that integrin binding is fundamental to tenascin function.     

Isolation and characterization of human fibroblast tenascin. An extracellular matrix glycoprotein of interest for developmental studies.

We have developed a biochemical method for purifying human tenascin from cultured fibroblasts or the culture medium. The method is a series of biochemical procedures including gel filtration, gelatin gel affinity chromatography and ion-exchange high performance liquid chromatography. The final preparation was identified as tenascin from its immunological cross-reactivity to antibody against chicken tenascin, strong hemagglutination activity which has been reported to be one of the biological functions of chicken tenascin, and from the electron microscopic study demonstrating a six-armed structure. Gel chromatography showed that intact human tenascin has an apparent molecular weight of over one million. Analysis of the purified tenascin with SDS-PAGE under reducing conditions demonstrated that tenascin consists of two kinds of subunits (250K and 190K). We established rat x mouse heterohybridoma cell lines which produce tenascin-specific antibodies. One monoclonal antibody (RCB1) was selected for immunohistochemical study and partially characterized. RCB1 bound native tenascin but not reduced and alkylated tenascin. Immunohistochemistry of normal and neoplastic tissues demonstrated that RCB1 bound the connective tissues surrounding the cancer nests and various normal tissues including interstitium of renal distal tubule, periosteum, endosteum, smooth muscles of digestive tract and media of arteries and arterioles.     

Chicken egg white cystatin. Molecular cloning, nucleotide sequence, and tissue distribution

A lambda gt11 chicken oviduct cDNA library was screened with a mixed synthetic oligonucleotide corresponding to amino acid residues 81-90 of chicken egg white cystatin, a cysteine proteinase inhibitor. Two initial cDNA clones of 367 and 431 bases were isolated. Both clones contained coding sequences for cystatin from amino acid residue 82 to the carboxyl end plus 3'-untranslated region and a poly(A)+ tail. The two clones utilized different polyadenylation signals located 55 nucleotides apart. Further screening of the library yielded a full-length cystatin cDNA. Sequence analysis indicated that cystatin contains an NH2-terminal extension of 23 amino acids which is probably a signal sequence. The cystatin cDNA hybridized to an mRNA of approximately 0.95 kilobase and was present in varying amounts in all chicken tissues examined. The highest concentration was found in the lung. Gizzard, brain, and heart contained lesser amounts of cystatin mRNA but considerably higher than oviduct. Among a limited number of embryonic tissues examined, significantly higher levels of the mRNA were found in liver and heart tissues when compared with the corresponding adult tissues. These results suggested that the expression of the chicken cystatin gene is tissue-dependent and under developmental control.     

Focal adhesion integrity is downregulated by the alternatively spliced domain of human tenascin [published erratum appears in J Cell Biol 1992 Feb;116(3):833]

Tenascin, together with thrombospondin and SPARC, form a family of matrix proteins that, when added to bovine aortic endothelial cells, caused a dose-dependent reduction in the number of focal adhesion- positive cells to approximately 50% of albumin-treated controls. For tenascin, a maximum response was obtained with 20-60 micrograms/ml of protein. The reduction in focal adhesions in tenascin-treated spread cells was observed 10 min after addition of the adhesion modulator, reached the maximum by 45 min, and persisted for at least 4 h in the continued presence of tenascin. This effect was fully reversible, was independent of de novo protein synthesis, and was neutralized by a polyclonal antibody to tenascin. Monoclonal antibodies to specific domains of tenascin (mAbs 81C6 and 127) were used to localize the active site to the alternatively spliced segment of tenascin. Furthermore, a recombinant protein corresponding to the alternatively spliced segment (fibronectin type III domains 6-12) was expressed in Escherichia coli and was active in causing loss of focal adhesions, whereas a recombinant form of a domain (domain 3) containing the RGD sequence had no activity. Chondroitin-6-sulfate effectively neutralized tenascin activity, whereas dermatan sulfate and chondroitin-4-sulfate were less active and heparan sulfate and heparin were essentially inactive. Studies suggest that galactosaminoglycans neutralize tenascin activity through interactions with cell surface molecules. Overall, our results demonstrate that tenascin, thrombospondin, and SPARC, acting as soluble ligands, are able to provoke the loss of focal adhesions in well-spread endothelial cells.     

Diverse forms of a receptor for acidic and basic fibroblast growth factors.

We recently reported the isolation of a chicken cDNA clone encoding a basic fibroblast growth factor (FGF) receptor that has three immunoglobulinlike domains in the extracellular region. We have now identified four unique human cDNA clones encoding previously unknown FGF receptor variants which contain only two immunoglobulinlike domains. Two of the human clones encode membrane-spanning receptors, and two encode putative secreted forms. Both the three- and two-immunoglobulinlike-domain forms mediate biological responsiveness to acidic and basic FGF. Thus, the first immunoglobulinlike domain of the three-domain form may have a function other than binding of acidic and basic FGF.     

Characterization of a Highly Conserved Human Homolog to the Chicken Neural Cell Surface Protein Bravo/Nr-CAM That Maps to Chromosome Band 7q31

The neuronal cell adhesion molecule Bravo/Nr-CAM is a cell surface protein of the immunoglobulin (Ig) superfamily and is closely related to the L1/NgCAM and neurofascin molecules, all of which contain six immunoglobulin domains, five fibronectin repeats, a transmembrane region, and an intracellular domain. Chicken Bravo/Nr-CAM has been shown to interact with other cell surface molecules of the Ig superfamily and has been implicated in specific pathfinding roles of axonal growth cones in the developing nervous system. We now report the characterization of cDNA clones encoding the human Bravo/Nr-CAM protein, which, like its chicken homolog, is composed of six V-like Ig domains and five fibronectin type III repeats. The human Bravo/Nr-CAM homolog also contains a transmembrane and intracellular domain, both of which are 100% conserved at the amino acid level compared to its chicken homolog. Overall, the human Bravo/Nr-CAM homolog is 82% identical to the chicken Bravo/Nr-CAM amino acid sequence. Independent cDNAs encoding four different isoforms were also identified, all of which contain alternatively spliced variants around the fifth fibronectin type III repeat, including one isoform that had been previously identified for chicken Bravo/Nr-CAM. Northern blot analysis reveals one mRNA species of approximately 7.0 kb in adult human brain tissue. Fluorescencein situhybridization maps the gene for human Bravo/Nr-CAM to human chromosome 7q31.1–q31.2. This chromosomal locus has been previously identified as containing a tumor suppressor candidate gene commonly deleted in certain human cancer tissues.     

Zebrafish tenascin-W,a new member of the tenascin family

A cDNA clone encoding tenascin-W, a novel member of the tenascin family, was isolated from a 20- to 28-h postfertilization (hpf) zebrafish cDNA library on the basis of the conserved epidermal growth factor-like domains represented in all tenascin molecules. An open reading frame of 2796 base pairs encodes a mature protein consisting of heptad repeats, a cysteine-rich amino terminal region, 3.5 epidermal growth factor-like repeats, five fibronectin type III homologous repeats, and a domain homologous to fibrinogen. These domains are the typical modular elements of molecules of the tenascin family. Sequence comparison demonstrated that TN-W shares homologies with the members of the tenascin family but is not a species homolog of any identified tenascin. The expression pattern of tn-w was analyzed by in situ hybridization in 1-day-old embryos, in 3-day-old larvae, and in juvenile zebrafish. At 24–25 hpf, tn-w mRNA was expressed in the lateral plate mesoderm, most conspicuously in the presumptive sclerotome. Migrating cells of sclerotomal and neural crest origins also showed high levels of expression. At 3 days, expression by sclerotomal and neural crest cells continued to be observed while expression in the somitic mesoderm was decreased. In juvenile fish, tn-w was expressed weakly by cells in the myosepta and, more strongly, by presumably nonneuronal cells in the dorsal root ganglia. In these tissues and at the same developmental stages, the expression of tn-w partially overlapped with the distribution of tn-c mRNA. In addition, tn-c was expressed in the central nervous system (CNS) and in the axial mesoderm, neither of which expressed tn-w at any of the age stages examined. The expression pattern of tn-w suggests an involvement in neural crest and sclerotome cell migration and in the formation of the skeleton. Similar and possibly overlapping functions could also be performed by tn-c, which appears to have additional functions during the development of the CNS. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 1–16, 1998