The Experts below are selected from a list of 96 Experts worldwide ranked by ideXlab platform
Rolf Kemler - One of the best experts on this subject based on the ideXlab platform.
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Characterization of recombinant Ecadherin (Uvomorulin) expressed in insect cells
2016Co-Authors: Kurt Herrenknecht, Rolf KemlerAbstract:Cadherins are Ca2+-dependent cell adhesion molecules that mediate cell adhesion by homophilic binding. Struc tural and functional analysis of the extracellular part of cadherins that mediates this binding has often been hampered by the availability of sufficient amount of protein. Therefore, we have expressed the extracellular region of E-cadherin (Uvomorulin) using the baculovirus expression vector system (BEVS). A recombinant bac ulovirus was generated that encodes the signal peptide, the precursor region and the extracellular part of the mature protein, under the control of the promotor for polyhedrin. Infection of insect cells with recombinant virus led to the expression of about 40 mg of the E-cad-herin fragment per 2xl09 infected cells. About half of the protein synthesized was secreted, either as mature protein or in its unprocessed form. The precursor pep-tide was removed by trypsin treatment in the presence of Ca2+ and recombinant protein was purified to homo geneity. Biochemical characterization of the recombi nant protein revealed a high degree of similarity with the mouse wild-type protein. Recombinant protein exhibited the known resistance to trypsin in the pres ence of Ca2+ and was recognized by two different con formation-sensitive monoclonal anti-E-cadherin anti bodies. Rabbit antibodies made against the recombinant protein recognized E-cadherin from different species. In spite of the high degree of structural resemblance recombinant E-cadherin was not able to inhibit E-cad-herin mediated cell-cell adhesion. Key words: cell adhesion molecule, recombinant E-cadheri
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conformational changes of the recombinant extracellular domain of e cadherin upon calcium binding
FEBS Journal, 1994Co-Authors: Sabine Pokutta, Rolf Kemler, Kurt Herrenknecht, Jurgen EngelAbstract:The cell-adhesion protein E-cadherin/Uvomorulin exhibits a calcium-dependent homoassociation. The effect of Ca2+ on the extracellular fragment of E-cadherin was studied using the recombinant protein expressed in the baculovirus expression system. The recombinant and native fragment of E-cadherin were found to be similar by many biochemical criteria [Herrenknecht, K. & Kemler, R. (1993) J. Cell Sci. 17, 147–154]. A large and reversible conformational transition was observed upon Ca2+ depletion. A change from a rod-like structure, 22 nm in length, to a more globular assembly of the five subdomains became evident by electron-microscopical analysis. In the presence of Ca2+, the circular dichroic spectra indicated predominantly β-structure but a more negative ellipticity was observed in the absence of Ca2+. The intrinsic tryptophan fluorescence decreased by 12% upon Ca2+ depletion. Both effects were used for calcium titrations which indicated calcium binding to several sites with average Kd values of 45–150 μM. Cleavage of the protein fragment by trypsin occurred only at low Ca2+ concentrations and from the calcium-dependence of cleavage rates, a Kd value of 24 μM was derived. The major site of cleavage was identified by partial sequencing to be located between the two putative calcium-binding sites in the third subdomain from the N-terminus. In agreement with earlier results with the native fragment, the recombinant protein did not associate in the presence or absence of Ca2+. We suggest the calcium-dependent homoassociation therefore depends on additional effects connected with the cell surface association of E-cadherin.
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characterization of recombinant e cadherin Uvomorulin expressed in insect cells
Journal of Cell Science, 1993Co-Authors: Kurt Herrenknecht, Rolf KemlerAbstract:SUMMARY Cadherins are Ca2+-dependent cell adhesion molecules that mediate cell adhesion by homophilic binding. Structural and functional analysis of the extracellular part of cadherins that mediates this binding has often been hampered by the availability of sufficient amount of protein. Therefore, we have expressed the extracellular region of E-cadherin (Uvomorulin) using the baculovirus expression vector system (BEVS). A recombinant baculovirus was generated that encodes the signal peptide, the precursor region and the extracellular part of the mature protein, under the control of the promotor for polyhedrin. Infection of insect cells with recombinant virus led to the expression of about 40 mg of the E-cadherin fragment per 2×109 infected cells. About half of the protein synthesized was secreted, either as mature protein or in its unprocessed form. The precursor peptide was removed by trypsin treatment in the presence of Ca2+ and recombinant protein was purified to homogeneity. Biochemical characterization of the recombinant protein revealed a high degree of similarity with the mouse wild-type protein. Recombinant protein exhibited the known resistance to trypsin in the presence of Ca2+ and was recognized by two different conformation-sensitive monoclonal anti-E-cadherin antibodies. Rabbit antibodies made against the recombinant protein recognized E-cadherin from different species. In spite of the high degree of structural resemblance recombinant E-cadherin was not able to inhibit E-cadherin mediated cell-cell adhesion.
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molecular organization of the Uvomorulin catenin complex
Journal of Cell Biology, 1992Co-Authors: Masayuki Ozawa, Rolf KemlerAbstract:The Ca(2+)-dependent cell adhesion molecule Uvomorulin is a member of the cadherin gene family. Its cytoplasmic region complexes with structurally defined proteins termed alpha-, beta-, and gamma-catenins. Here we show that A-CAM (N-cadherin), another member of this gene family, also associates with catenins suggesting that this complex formation may be a general property of the cadherins. For Uvomorulin it has been found that this association with catenins is of crucial importance for the adhesive function, but little is known about the molecular organization of the Uvomorulin-catenin complex. Using a combination of biochemical analyses we show that a single complex is composed of one molecule of Uvomorulin, one or two molecules of beta-catenin, and one molecule of alpha-catenin. Furthermore, beta-catenin seems to interact more directly with Uvomorulin. In pulse-chase experiments beta-catenin is already associated with the 135-kD Uvomorulin precursor molecule but the assembly of the newly synthesized alpha-catenin into the complex is only detected around the time of endoproteolytic processing.
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The structure of the gene coding for the mouse cell adhesion molecule Uvomorulin
Nucleic acids research, 1991Co-Authors: Martin Ringwald, H. Baribault, C. Schmidt, Rolf KemlerAbstract:We have recently shown that the Ca2+ dependent cell adhesion molecule Uvomorulin is encoded by a single gene, localized on mouse chromosome 8. Here we describe the organization of the Uvomorulin gene and give an initial characterization of the Uvomorulin promoter. Uvomorulin is encoded by 16 exons, which are distributed over a region of more than 40 kb genomic DNA. The exon structure of the genes for Uvomorulin and its chicken homologue L-CAM are nearly identical and thus highly conserved. The relationship between the exon structure and the structure of the Uvomorulin protein is analysed. The initiation site of transcription of the Uvomorulin gene is located 127 bp upstream of the translation start site in a GC-rich region with no TATA-box, but with a GC-box in position -48 and a CCAAT-box starting at position -65 with respect to the transcription start site. 1.6 kb of the Uvomorulin promoter (-1492 to + 92) confer cell type specific promoter activity to the CAT reporter gene. Homologies to known cis acting elements of other promoters are discussed.
W J Nelson - One of the best experts on this subject based on the ideXlab platform.
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alterations in the expression of Uvomorulin and na k adenosine triphosphatase during mouse skin tumor progression
American Journal of Pathology, 1992Co-Authors: B Ruggeri, W J Nelson, Jorge Caamano, Thomas J Slaga, Claudio J Conti, Andres J KleinszantoAbstract:Uvomorulin (E-cadherin), a cell adhesion molecule, and Na+,K(+)-adenosine triphosphatase (ATPase), a marker protein of the basal-lateral cell membrane domains of polarized epithelial cells, were investigated in a group of mouse skin tumors induced by a two-stage chemical carcinogenesis protocol and in cell lines derived from mouse skin papillomas and squamous cell carcinomas (SCC). Although these two markers were present in benign tumors and in nontumorigenic cell lines, the Na+,K(+)-ATPase showed an altered pattern of distribution that included the presence of enzyme not only in the basolateral domain but also on the apical domain of the cell membrane of basal and spinous cells in well-differentiated squamous cell carcinomas (SCC). In higher grade SCC, a loss of Na+,K(+)-ATPase immunoreactivity was simultaneously detected with a marginal or absent expression of Uvomorulin. The more differentiated SCC and papillomas expressed less Uvomorulin immunoreactivity than normal epidermal cells. Both markers were seen in tumor cell lines that produced well-differentiated SCC after subcutaneous inoculation into nude mice. Neither Na+,K(+)-ATPase nor Uvomorulin could be detected in cell lines that produced high grade, poorly differentiated SCC. Northern blots confirmed the absence of Uvomorulin mRNA in these highly malignant cell lines. These data indicate that progression from premalignant papilloma to low-grade SCC and subsequently to high-grade SCC is accompanied by loss of epithelial cell polarity as detected by changes in Na+,K(+)-ATPase and by decreased or absent expression of Uvomorulin in tumors and cell lines characterized by an advanced malignant phenotype.
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biosynthesis of the cell adhesion molecule Uvomorulin e cadherin in madin darby canine kidney epithelial cells
Journal of Biological Chemistry, 1991Co-Authors: Eileen M Shore, W J NelsonAbstract:The Ca(2+)-dependent cell adhesion molecule Uvomorulin is a transmembrane glycoprotein that functions at the cell surface to regulate epithelial cell recognition and adhesion. We have investigated the temporal and spatial regulation of Uvomorulin biosynthesis and cell surface expression in Madin-Darby canine kidney epithelial cells. We show that Uvomorulin is synthesized as a precursor polypeptide (Mr 135,000) that is core glycosylated in the endoplasmic reticulum. The precursor is processed to the mature polypeptide (Mr 120,000) shortly after addition of complex carbohydrate groups in the late Golgi complex, but prior to delivery of the polypeptide to the cell surface. However, glycosylation is not required for either efficient processing of the precursor or transport of Uvomorulin to the cell surface. At the cell surface, Uvomorulin is turned over rapidly (t1/2 approximately 5 h). Induction of Ca(2+)-dependent cell-cell contact results in rapid localization of cell surface Uvomorulin to regions of contact and an increase in the proportion of Uvomorulin that is insoluble in buffers containing Triton X-100. These results indicate several regulatory steps in the biosynthesis and cell surface expression of Uvomorulin in epithelial cells.
Eileen M Shore - One of the best experts on this subject based on the ideXlab platform.
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biosynthesis of the cell adhesion molecule Uvomorulin e cadherin in madin darby canine kidney epithelial cells
Journal of Biological Chemistry, 1991Co-Authors: Eileen M Shore, W J NelsonAbstract:The Ca(2+)-dependent cell adhesion molecule Uvomorulin is a transmembrane glycoprotein that functions at the cell surface to regulate epithelial cell recognition and adhesion. We have investigated the temporal and spatial regulation of Uvomorulin biosynthesis and cell surface expression in Madin-Darby canine kidney epithelial cells. We show that Uvomorulin is synthesized as a precursor polypeptide (Mr 135,000) that is core glycosylated in the endoplasmic reticulum. The precursor is processed to the mature polypeptide (Mr 120,000) shortly after addition of complex carbohydrate groups in the late Golgi complex, but prior to delivery of the polypeptide to the cell surface. However, glycosylation is not required for either efficient processing of the precursor or transport of Uvomorulin to the cell surface. At the cell surface, Uvomorulin is turned over rapidly (t1/2 approximately 5 h). Induction of Ca(2+)-dependent cell-cell contact results in rapid localization of cell surface Uvomorulin to regions of contact and an increase in the proportion of Uvomorulin that is insoluble in buffers containing Triton X-100. These results indicate several regulatory steps in the biosynthesis and cell surface expression of Uvomorulin in epithelial cells.
Stephen W Byers - One of the best experts on this subject based on the ideXlab platform.
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cell adhesion molecule Uvomorulin expression in human breast cancer cell lines relationship to morphology and invasive capacities
Cell Growth & Differentiation, 1991Co-Authors: Connie L Sommers, Erik W Thompson, J A Torri, R Kemler, Edward P Gelmann, Stephen W ByersAbstract:Loss of cell-cell adhesion in carcinoma cells may be an important step in the acquisition of an invasive, metastatic phenotype. We have examined the expression of the epithelial-specific cell adhesion molecule Uvomorulin (E-cadherin, cell-CAM 120/80, L-CAM) in human breast cancer cell lines. We find that fibroblastoid, highly invasive, vimentin-expressing breast cancer cell lines do not express Uvomorulin. Of the more epithelial-appearing, less invasive, keratin-expressing breast cancer cell lines, some express Uvomorulin, and some do not. We examined the morphologies of the cell lines in the reconstituted basement membrane matrix Matrigel and measured the ability of the cells to traverse a Matrigel-coated filter as in vitro models for detachment of carcinoma cells from neighboring cells and invasion through basement membrane into surrounding tissue. Colonies of Uvomorulin-positive cells have a characteristic fused appearance in Matrigel, whereas Uvomorulin-negative cells appear detached. Cells which are Uvomorulin negative and vimentin positive have a stellate morphology in Matrigel. We show that Uvomorulin is responsible for the fused colony morphology in Matrigel since treatment of Uvomorulin-positive MCF-7 cells with an antibody to Uvomorulin caused the cells to detach from one another but did not induce invasiveness in these cells, as measured by their ability to cross a Matrigel-coated polycarbonate filter in a modified Boyden chamber assay. Two Uvomorulin-negative, vimentin-negative cell lines are also not highly invasive as measured by this assay. We suggest that loss of Uvomorulin-mediated cell-cell adhesion may be one of many changes involved in the progression of a carcinoma cell to an invasive phenotype.
R Kemler - One of the best experts on this subject based on the ideXlab platform.
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cell adhesion molecule Uvomorulin expression in human breast cancer cell lines relationship to morphology and invasive capacities
Cell Growth & Differentiation, 1991Co-Authors: Connie L Sommers, Erik W Thompson, J A Torri, R Kemler, Edward P Gelmann, Stephen W ByersAbstract:Loss of cell-cell adhesion in carcinoma cells may be an important step in the acquisition of an invasive, metastatic phenotype. We have examined the expression of the epithelial-specific cell adhesion molecule Uvomorulin (E-cadherin, cell-CAM 120/80, L-CAM) in human breast cancer cell lines. We find that fibroblastoid, highly invasive, vimentin-expressing breast cancer cell lines do not express Uvomorulin. Of the more epithelial-appearing, less invasive, keratin-expressing breast cancer cell lines, some express Uvomorulin, and some do not. We examined the morphologies of the cell lines in the reconstituted basement membrane matrix Matrigel and measured the ability of the cells to traverse a Matrigel-coated filter as in vitro models for detachment of carcinoma cells from neighboring cells and invasion through basement membrane into surrounding tissue. Colonies of Uvomorulin-positive cells have a characteristic fused appearance in Matrigel, whereas Uvomorulin-negative cells appear detached. Cells which are Uvomorulin negative and vimentin positive have a stellate morphology in Matrigel. We show that Uvomorulin is responsible for the fused colony morphology in Matrigel since treatment of Uvomorulin-positive MCF-7 cells with an antibody to Uvomorulin caused the cells to detach from one another but did not induce invasiveness in these cells, as measured by their ability to cross a Matrigel-coated polycarbonate filter in a modified Boyden chamber assay. Two Uvomorulin-negative, vimentin-negative cell lines are also not highly invasive as measured by this assay. We suggest that loss of Uvomorulin-mediated cell-cell adhesion may be one of many changes involved in the progression of a carcinoma cell to an invasive phenotype.
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overexpression of Uvomorulin in a compaction negative f9 mutant cell line
Developmental Biology, 1991Co-Authors: Antonella Calogero, R Kemler, M Samuels, Tristan Darland, Steven A Edwards, Eileen D AdamsonAbstract:The mutant F9 cell line F9att-5.51 synthesizes reduced amounts of Uvomorulin (UM) protein and we hypothesized earlier (Adamson, Baribault, and Kemler, Dev. Biol. (1990), 138, 338) that this may account for its inability to compact into tightly aggregated balls of cells. Subsequently, when 5.51 cells are treated with retinoic acid to stimulate their differentiation, they are unable to form embryoid bodies as do wild-type cells which form an outer epithelial layer of visceral endoderm cells. We have now examined the possibility that the UM protein made in the mutant line is defective, but find that it is normal in structure and stability. The gene coding for UM appears to be normal as does the mRNA which is synthesized at a normal rate but is severely reduced in steady-state measurements of mutant cells. A rescue experiment was performed by increasing levels of UM in mutant cells by means of transfection with a UM expression vector. The resulting cells expressed abundant UM mRNA and protein but were still unable to form compacted aggregates and did not differentiate into embryoid bodies. Interestingly, the stability of endogenous UM mRNA was improved in the presence of exogenous UM; therefore, a positive feedback mechanism contributes to low mRNA levels in mutant cells. The accumulated data suggest that UM in 5.51 cells is unable to mount a compaction activity because a distal connecting link in the multicomponent process initiated by UM is missing or or aberrant. The missing component is likely to connect UM to actin and the cytoskeleton of the cell.
