Molecular cloning and characterization of CHM1L, a novel membrane molecule similar to chondromodulin-I

Chondromodulin-I (ChM-I) is a cartilage-specific glycoprotein that stimulates the growth of chondrocytes and inhibits the tube formation of endothelial cells. In the present study, we identified a novel ChM-I like molecule, designated ChM1L. Cloning of full length cDNAs of human, mouse, and rat ChM1L revealed that ChM1L encodes 317 amino acids novel type II transmembrane protein. ChM1L protein was expressed on the cell surface as N-glycosylated and non-N-glycosylated protein with molecular mass of 45 and 40 kDa, respectively. In adult mouse tissues, ChM1L mRNA was highly expressed in eye, skeletal muscle, and whole rib. The temporal pattern of ChM1L mRNA was examined using whole embryo at day 10 to 19 of gestation. After day 11, ChM1L mRNA was detected and its level was progressively elevated in association with development of mouse embryo. These data suggest that ChM1L is a novel membrane molecule which is similar to ChM-I that plays a regulatory role in eye, skeletal muscle, and development of embryo.     

Regulation of connexin 43-mediated gap junctional intercellular communication by Ca2+ in mouse epidermal cells is controlled by E- cadherin

Gap junctional intercellular communication (GJIC) of cultured mouse epidermal cells is mediated by a gap junction protein, connexin 43, and is dependent on the calcium concentration in the medium, with higher GJIC in a high-calcium (1.2 mM) medium. In several mouse epidermal cell lines, we found a good correlation between the level of GJIC and that of immunohistochemical staining of E-cadherin, a calcium-dependent cell adhesion molecule, at cell-cell contact areas. The variant cell line P3/22 showed both low GJIC and E-cadherin protein expression in low- and high-Ca2+ media. P3/22 cells showed very low E-cadherin mRNA expression. To test directly whether E-cadherin is involved in the Ca(2+)-dependent regulation of GJIC, we transfected the E-cadherin expression vector into P3/22 cells and obtained several stable clones which expressed high levels of E-cadherin mRNA. All transfectants expressed E-cadherin molecules at cell-cell contact areas in a calcium-dependent manner. GJIC was also observed in these transfectants and was calcium dependent. These results suggest that Ca(2+)-dependent regulation of GJIC in mouse epidermal cells is directly controlled by a calcium-dependent cell adhesion molecule, E-cadherin. Furthermore, several lines of evidence suggest that GJIC control by E-cadherin involves posttranslational regulation (assembly and/or function) of the gap junction protein connexin 43.     

Cell binding function of E-cadherin is regulated by the cytoplasmic domain.

Cadherins are a family of transmembrane glycoproteins responsible for Ca2+-dependent cell-cell adhesion. Their amino acid sequences are highly conserved in the cytoplasmic domain. To study the role of the cytoplasmic domain in the function of cadherins, we constructed expression vectors with cDNAs encoding the deletion mutants of E-cadherin polypeptides, in which the carboxy terminus was truncated at various lengths. These vectors were introduced into L cells by transfection, and cell lines expressing the mutant E-cadherin molecules were isolated. In all transfectants obtained, the extracellular domain of the mutant E-cadherins was exposed on the cell surface, and had normal Ca2+-sensitivity and molecular size. However, these cells did not show any Ca2+-dependent aggregation, indicating that the mutant molecules cannot mediate cell-cell binding. The mutant E-cadherin molecules could be released from cells by nonionic detergents, whereas a fraction of normal E-cadherin molecules could not be extracted with the detergent and appeared to be anchored to the cytoskeleton at cell-cell junctions. These results suggest that the cytoplasmic domain regulates the cell-cell binding function of the extracellular domain of E-cadherin, possibly through interaction with some cytoskeletal components.     

Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.     

SMMC 7721肝癌细胞蛋白激酶B活性增高可驱动有功能的上皮钙粘着蛋白到细胞表面(英)

为了研究蛋白激酶B（PKB）对上皮钙粘着蛋白（E-cadherin）的调节,使用了用胰岛素处理的野生型SMMC 7721细胞及稳定表达持续激活PKB的SMMC 7721细胞株(Gag-PKB/SMMC 7721).用RNA印迹法和蛋白质印迹法检测细胞E-cadherin 表达,发现通过胰岛素刺激或在细胞中表达持续激活PKB从而增加PKB活性,不影响E-cadherin的转录和蛋白质合成,但用流式细胞术和免疫荧光定位E-cadherin,则发现PKB活性增加能明显驱动E-cadherin到细胞表面,从而导致部分通过E-cadherin途径的细胞粘聚增加和细胞调亡的抑制.因此,我们提供新的证据表明,增加PKB活性可驱动有功能的E-cadherin分子到细胞表面.     

Ribosome-inactivating activity and cDNA cloning of antiviral protein isoforms of Chenopodium album

We have characterized a novel type I ribosome-inactivating protein (CAP30) from the leaves of Chenopodium album. Purified native CAP30 depurinated the ribosomes of Chenopodium, tomato, and tobacco leaves in vitro. To further characterize this protein, cDNA clones were isolated from a leaf cDNA library using a DNA probe derived from the N-terminal amino acid sequence. Two full-length cDNA clones, CAP30A and CAP30B, were isolated. The two clones were highly homologous (91.4% identity over 280 amino acids) at the deduced amino acid level. Both contain a putative signal peptide of 25 amino acid and a conserved domain commonly found in ribosome-inactivating proteins. This suggests that CAP30 is a single-chain ribosome-inactivating protein. Expression of CAP30 mRNA peaked twice, at 12 and 72 h, after tobacco mosaic virus (TMV) infection or wounding. Transformed Escherichia coli cells expressing pre- or mature CAP had greatly reduced growth rates. These results suggest that CAP30 functions as a broad-spectrum defense-related protein with both antiviral and anti-microbial activity.     

Expression in non-lymphoid cells of mouse recombinant immunoglobulin directed against the tumour marker human placental alkaline phosphatase

From a mouse hybridoma cell line secreting a monoclonal antibody directed against the tumour marker human placental alkaline phosphatase, mRNA coding for the H and L chains of this antibody was isolated and cloned as cDNA. Sequence analysis of the H and L chain cDNAs confirmed the IgG2b,kappa subtype previously established. Recloning the H and L chain cDNA information into SV40-based vectors enabled us to obtain expression of functional immunoglobulin upon cotransfection into COS or CHO dhfr- cells. This illustrates that non-lymphoid cells also have the capacity to assemble active immunoglobulins.     

A cloned chromogranin A (CgA) cDNA detects a 2.3Kb mRNA in diverse neuroendocrine tissues

CgA is a 72Kd protein of unknown function that is present in many neuroendocrine tissues and co-secreted with their resident hormones. We prepared a cDNA library to the mRNA from CgA-producing human medullary thyroid carcinoma (MTC) cells in the expression vector lambda gt11. The library was screened with a panel of one polyclonal and two monoclonal antibodies to CgA. The specificity of the antibodies for CgA was demonstrated by immunoassay, immunohistology, and immunoprecipitation of the in vitro translation products of mRNA from CgA-producing tissues. A chromogenic second antibody identified five immunoreactive clones. Their cDNA inserts were isolated after EcoRI digestion and agarose gel electrophoresis. These cDNAs were 32P-labelled and used as probes in Northern hybridization studies. An mRNA of approximately 2.3Kb was detected with the cDNA probes in human cell lines from MTC and lung cancers that were shown to produce CgA and in human pheochromocytoma and bovine adrenal medulla tissue. To confirm its identity, one of the putative CgA cDNAs was subcloned into a plasmid and was used to hybridization-arrest the in vitro translation of CgA mRNA. These studies demonstrate the cloning of cDNAs which hybridize with CgA mRNA from diverse neuroendocrine tissues.     

Vezatin, a protein associated to adherens junctions, is required for mouse blastocyst morphogenesis

Cell-cell interactions play a major role during preimplantation development of the mouse embryo. The formation of adherens junctions is a major feature of compaction, the first morphogenetic event that takes place at the 8-cell stage. Then, during the following two cell cycles, tight junctions form, and the outer layer of cells differentiate into a functional epithelium, leading to the formation of the blastocoel cavity. Until now, E-cadherin was the only transmembrane molecule localized in adherens junctions and required for early development. Vezatin is a transmembrane protein of adherens junctions, interacting with the E-cadherin-catenins complex. Here, we show that vezatin is expressed very early during mouse preimplantation development. It co-localizes with E-cadherin throughout development, being found all around the cell cortex before compaction and basolaterally in adherens junctions thereafter. In addition, vezatin is also detected in nuclei during most of the cell cycle. Finally, using a morpholino-oligonucleotide approach to inhibit vezatin function during preimplantation development, we observed that inhibition of vezatin synthesis leads to a cell cycle arrest with limited cell-cell interactions. This phenotype can be rescued when mRNAs coding for vezatin missing the 5'UTR are co-injected with the anti-vezatin morpholino-oligonucleotide. Cells derived from blastomeres injected with morpholino-oligonucleotide had a reduced amount of vezatin concomitantly with a decrease in the quantity of E-cadherin and beta-catenin localized in the areas of intercellular contact. Shift in E-cadherin cortical distribution was correlated with a strong decrease in E-cadherin mRNA and protein contents. Altogether, these observations demonstrate that vezatin is required for morphogenesis of the preimplantation mouse embryo.     

Biochemical processing of E-cadherin under cellular stress

The proteolytic cleavage pathways of E-cadherin endogenously expressed in MDCK (Madin-Darby canine kidney) cells were characterized in cells treated with antimycin A and deoxyglucose to examine transmembrane protein processing under cellular stress. E-cadherin is a type I transmembrane protein which operates as the cell adhesion molecule component of the adherens junction, a complex of proteins involved in epithelial tissue development and integrity. We now demonstrate that treatment of MDCK cells with antimycin A and deoxyglucose activates caspase mediated pathways that cleave E-cadherin. E-cadherin is cleaved into two major fragments, with the sizes predicted by the location of a caspase-3 cleavage consensus sequence. Cleavage of E-cadherin and deposition of the C-terminal fragment into the cytoplasm are inhibited by the caspase inhibitor DEVD-CHO. Thus, a major mechanism for E-cadherin cleavage and dissolution of the adherens junction under antimycin/deoxyglucose treatment is caspase mediated, initiated by activation of an apoptosis pathway.     

E-cadherin decreased human breast cancer cells sensitivity to staurosporine by up-regulating Bcl-2 expression

E-cadherin, a well-characterized cell-cell adhesion molecule, executes multifunction roles on cell behaviors. However, its effect on chemo-resistance remains controversial. In this study, we found that E-cadherin positive breast cell lines were less sensitive to staurosporine compared to E-cadherin negative ones. Next, we substantiated that the expression of E-cadherin in MDA-MB-435 cells could partly counteract the cytotoxic effect induced by staurosporine through a series of apoptosis assay. The resistance of E-cadherin over-expressing cells to staurosporine may due to the up-regulation of Bcl-2/Bax ratio. When E-cadherin interference plasmids were transfected into MCF-7 cells, Bcl-2 expression was down-regulated. Moreover, perturbation of E-cadherin function by blocking the cell-cell contact resulted in decreased cellular levels of Bcl-2 protein expression. All these results demonstrated the chemo-resistance function of E-cadherin in the condition of staurosporine treatment, therefore, might contribute effective therapeutic strategies in breast carcinoma.