Solution structures of the adhesion molecule DdCAD-1 reveal new insights into Ca(2+)-dependent cell-cell adhesion

DdCAD-1 is a novel Ca(2+)-dependent cell adhesion molecule that lacks a hydrophobic signal peptide and a transmembrane domain. DdCAD-1 is expressed by the social amoeba Dictyostelium discoideum at the onset of development. It is synthesized as a soluble protein and then transported to the plasma membrane by contractile vacuoles. Here we describe the novel features of the solution structures of Ca(2+)-free and Ca(2+)-bound monomeric DdCAD-1. DdCAD-1 contains two beta-sandwich domains, belonging to the betagamma-crystallin and immunoglobulin fold classes, respectively. Whereas the N-terminal domain has a major role in homophilic binding, the C-terminal domain tethers the protein to the cell membrane. From structural and mutational analyses, we propose a model for the Ca(2+)-bound DdCAD-1 dimer as a basis for understanding DdCAD-1-mediated cell-cell adhesion at the molecular level. Our results provide new insights into Ca(2+)-dependent mechanisms for cell-cell adhesion.     

Identification of a Ca2(+)-dependent cell-cell adhesion molecule in endothelial cells

Confluent cultures of aortic endothelial cells contain two different cell-cell adhesion mechanisms distinguished by their requirement for calcium during trypsinization and adhesion. A hybridoma clone was isolated producing a monoclonal antibody Ec6C10, which inhibits Ca2(+)-dependent adhesion of endothelial cells. There was no inhibition of Ca2(+)-independent adhesion of endothelial cells and only a minor effect on Ca2(+)-dependent adhesion of smooth muscle cells. Immunoblotting analysis shows that the antibody Ec6C10 recognizes a protein in endothelial but not epithelial cells with an apparent molecular weight of 135,000 in reducing conditions and 130,000 in non-reducing conditions. Monoclonal antibody Ec6C10 reacts with an antigen at the cell surface as shown by indirect immunofluorescence of confluent endothelial cells in a junctional pattern outlining the cobblestone morphology of the monolayer. Removal of extracellular calcium increased the susceptibility of the antigen recognized by antibody Ec6C10 to proteolysis by trypsin. The role of the Ca2(+)-dependent cell adhesion molecule in organization of the dense peripheral microfilament band in confluent endothelial cells was examined by adjusting the level of extracellular calcium to modulate cell-cell contact. Addition of the monoclonal antibody Ec6C10 at the time of the calcium switch inhibited the extent of formation of the peripheral F-actin band. These results suggest an association between cell-cell contact and the peripheral F-actin band potentially through the Ca2(+)-dependent CAM.     

EndoCAM: a novel endothelial cell-cell adhesion molecule

Cell-cell adhesion is controlled by many molecules found on the cell surface. In addition to the constituents of well-defined junctional structures, there are the molecules that are thought to play a role in the initial interactions of cells and that appear at precise times during development. These include the cadherins and cell adhesion molecules (CAMs). Representatives of these families of adhesion molecules have been isolated from most of the major tissues. The notable exception is the vascular endothelium. Here we report the identification of a cell surface molecule designated "endoCAM" (endothelial Cell Adhesion Molecule), which may function as an endothelial cell-cell adhesion molecule. EndoCAM is a 130-kD glycoprotein expressed on the surface of endothelial cells both in culture and in situ. It is localized to the borders of contiguous endothelial cells. It is also present on platelets and white blood cells. Antibodies against endoCAM prevent the initial formation of endothelial cell-cell contacts. Despite similarities in size and intercellular location, endoCAM does not appear to be a member of the cadherin family of adhesion receptors. The serologic and protease susceptibility characteristics of endoCAM are different from those of the known cadherins, including an endogenous endothelial cadherin. Although the precise biologic function of endoCAM has not been determined, it appears to be one of the molecules responsible for regulating endothelial cell-cell adhesion processes and may be involved in platelet and white blood cell interactions with the endothelium.     

Cadmium (Cd2+) disrupts Ca(2+)-dependent cell-cell junctions and alters the pattern of E-cadherin immunofluorescence in LLC-PK1 cells.

Recent findings from our laboratories have shown that Cd2+ has relatively specific damaging effects on the adhering and occluding junctions in the established porcine renal epithelial cell line, LLC-PK1. Results of the present studies show that the junction-perturbing effects of Cd2+ in LLC-PK1 cells are more pronounced when Cd2+ is applied to the basolateral cell surface than when it is applied to the apical surface, and that the severity of the effects is inversely related to the concentration of Ca2+ in the medium. Additional results show that exposure to sublethal concentrations of Cd2+ decreases the amount of E-cadherin that is associated with cell-cell contacts. These results suggest that Cd2+ damages Ca(2+)-dependent cell-cell junctions in LLC-PK1 cells by interacting with E-cadherin or a similar Ca(2+)-sensitive site that is oriented toward the basolateral cell surface.     

Development of a novel functional biosensor with a short Ca(2+)-dependent deoxyribozyme.

We develop a novel functional biosensor on a deoxyribozyme. A 5'-end-immobilized short Ca(2+)-dependent deoxyribozyme (dCGCTGGCAGGCTACAACGAGTCTTC) binds to a target RNA substrate (rGAAGACA decrease UGCCAGCG; decrease denotes an RNA cleavage site), and acts as an enzyme in the presence of Ca2+. It cleaves the target RNA substrate at one site of rAp decrease U in the asymmetric internal loop.     

Uvomorulin-catenin complex: cytoplasmic anchorage of a Ca2+-dependent cell adhesion molecule

The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins, named catenins, which are structurally related in different cell types of various species. This complex formation connects uvomorulin and cytoskeletal structures and might, moreover, be involved in other adhesion-dependent mechanisms.     

Involvement of blood-group-B-active trisaccharides in Ca2+-dependent cell-cell adhesion in the Xenopus blastula

 Despite their wide distribution in various organisms, no physiological roles have been proposed for the human blood-group-ABO (ABH)-active trisaccharides. Here we show that monoclonal antibodies against human blood-group-B-active trisaccharides (B-substance) completely block the Ca²⁺-dependent cell-cell adhesion system of frog (Xenopus laevis) embryonic cells. Synthetic B-substance or B-active glycopeptides also disrupt the Ca²⁺ -dependent cell-cell adhesion. These results suggest that blood-group-B-active substances play a role in cell-cell adhesion. Blood-group-B-active substances were found as glycoproteins and as glycosphingolipids. In order to identify B-active glycoproteins active in cell-cell adhesion, we purified B-active membrane glycoproteins by two-dimensional electrophoresis and found that they are 45- to 58-kDa proteins with pI(s) ranging from 4.0 to 5.3. They are glycosylphosphatidyl inositol (GPI) anchored. Amino acid sequence analysis showed that the purified B-active GPI-anchored proteins are homologues of soluble Xenopus cortical granule lectins (CGL). The results suggest that the B-active membrane glycoproteins are GPI-anchored forms of the lectin and are directly involved in frog Ca ²⁺-dependent cell-cell adhesion. Received: 16 September 1997 / Accepted 19 November 1997     

Molecular cloning of a human Ca2+-dependent cell-cell adhesion molecule homologous to mouse placental cadherin: its low expression in human placental tissues

P-cadherin is a subclass of Ca2+-dependent cell-cell adhesion molecules present in mouse placenta, where its localization suggests a function of connecting the embryo to the uterus (Nose, A., and M. Takeichi. 1986. J. Cell Biol. 103:2649-2658). We recently identified a human cadherin detected by an mAb capable of disrupting cell-cell adhesion of A-431 cells, and found that it was closely related immunochemically to mouse P-cadherin. Curiously, this cadherin was undetectable in human placenta by immunohistochemical examination (Shimoyama, Y., S. Hirohashi, S. Hirano, M. Noguchi, Y. Shimosato, M. Takeichi, and O. Abe. 1989. Cancer Res. 49:2128-2133). We here report the cloning and sequencing of cDNA clone encoding the human homologue of mouse P- cadherin. The deduced amino acid sequence of the human P-cadherin consists of 829 amino acid and shows striking homology with mouse P- cadherin. On Northern blot analysis, human P-cadherin was scarcely expressed in human placenta in contrast to mouse P-cadherin, which was abundantly expressed in mouse placenta throughout pregnancy, and it was shown that E-cadherin, but not P-cadherin, was the major cadherin molecule in human placenta. Moreover, NIH3T3 cells transfected with human P-cadherin cDNA expressed the functional cadherin molecule, which was identical to the cadherin we had previously identified using the mAb, showing that this molecule really does mediate cell-cell adhesion and that the cadherin we detected immunochemically is undoubtedly human P-cadherin. The results obtained in this study support the idea that P- cadherin plays little role, if any, in Ca2+-dependent cell-cell binding in human placental tissue at least after several weeks of pregnancy.     

Ca(2+)-dependent ubiquitination of calmodulin in yeast.

Recently we were able to show that calmodulin from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner by ubiquityl-calmodulin synthetase (uCaM-synthetase) from mammalian sources [R. Ziegenhagen and H.P. Jennissen (1990) FEBS Lett. 273, 253-256]. It was therefore of high interest to investigate whether this covalent modification of calmodulin also occurs in one of the simplest eukaryotes, the unicellular Saccharomyces cerevisiae. Yeast calmodulin was therefore purified from bakers yeast. In contrast to calmodulin from spinach and N. crassa it does not activate phosphorylase kinase. Crude yeast uCaM-synthetase conjugated ubiquitin Ca(2+)-dependently to yeast and mammalian (bovine) calmodulin. Yeast calmodulin was also a substrate for mammalian (reticulocyte) uCaM-synthetase. As estimated from autoradiograms the monoubiquitination product (first-order conjugate) of yeast calmodulin has an apparent molecular mass of ca. 23-26 kDa and the second-order conjugate an apparent molecular mass of ca. 28-32 kDa. Two to three ubiquitin molecules can be incorporated per yeast calmodulin. Experiments with methylated ubiquitin in the heterologous reticulocyte system indicate that, as with vertebrate calmodulins, only one lysine residue of yeast calmodulin reacts with ubiquitin so that the incorporation of multiple ubiquitin molecules will lead to a polyubiquitin chain. These results also indicate that the ability of coupling ubiquitin to calmodulin was acquired at a very early stage in evolution.     

Ca(2+)-dependent and Ca(2+)-independent mechanisms modulate whole-cell cationic currents in human neutrophils.

We used whole-cell, voltage-clamp methodology to study the activation and inhibition of cationic currents in neutrophil. Cationic channels involved were impermeable to N-methyl-D-glucamine and to choline, but permeable to Na+, K+, Cs+, tris(hydroxymethyl)amino-ethane, and tetraethylammonium. N-formyl-L-methionyl-L-leucyl-L-phenylalanine, the Ca(2+)-ionophore A23187, and phorbol myristate acetate activated the cationic current. Activated currents showed voltage dependence and outward rectification. The Ca(2+)-chelator 1,2 bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate markedly inhibited A23187-induced currents, but only partially decreased phorbol ester- or chemoattractant-induced currents. Dibutyryl cAMP diminished only the chemoattractant-induced currents. The adenosine analogs 5'N-ethylcarboxamidoadenosine and N6-cyclohexyladenosine blocked the currents induced by all agents. Thus, we conclude that activation and inhibition of cationic channels in human neutrophils involve both Ca(2+)-dependent and Ca(2+)-independent mechanisms.     

Ca(2+)-dependent interaction of triadin with histidine-rich Ca(2+)-binding protein carboxyl-terminal region.

A direct binding of HRC (histidine-rich Ca(2+)-binding protein) to triadin, the main transmembrane protein of the junctional sarcoplasmic reticulum (SR) of skeletal muscle, seems well supported. Opinions are still divided, however, concerning the triadin domain involved, either the cytoplasmic or the lumenal domain, and the exact role played by Ca(2+), in the protein-to-protein interaction. Further support for colocalization of HRC with triadin cytoplasmic domain is provided here by experiments of mild tryptic digestion of tightly sealed TC vesicles. Accordingly, we show that HRC is preferentially phosphorylated by endogenous CaM K II, anchored to SR membrane on the cytoplasmic side, and not by lumenally located casein kinase 2. We demonstrate that HRC can be isolated as a complex with triadin, following equilibrium sucrose-density centrifugation in the presence of mM Ca(2+). Here, we characterized the COOH-terminal portion of rabbit HRC, expressed and purified as a fusion protein (HRC(569-852)), with respect to Ca(2+)-binding properties, and to the interaction with triadin on blots, as a function of the concentration of Ca(2+). Our results identify the polyglutamic stretch near the COOH terminus, as the Ca(2+)-binding site responsible, both for the acceleration in mobility of HRC on SDS-PAGE in the presence of millimolar concentrations of Ca(2+), and for the enhancement by high Ca(2+) of the interaction between HRC and triadin cytoplasmic segment. (c)2001 Elsevier Science.     

Ca(2+)-dependent proteolysis in muscle wasting

Skeletal muscle wasting is a prominent feature of cachexia, a complex systemic syndrome that frequently complicates chronic diseases such as inflammatory and autoimmune disorders, cancer and AIDS. Muscle wasting may also develop as a manifestation of primary or neurogenic muscular disorders. It is now generally accepted that muscle depletion mainly arises from increased protein catabolism. The ubiquitin-proteasome system is believed to be the major proteolytic machinery in charge of such protein breakdown, yet there is evidence suggesting that Ca(2+)-dependent system, lysosomes and, in some conditions at least, even caspases are involved as well. The role of Ca(2+)-dependent proteolysis in skeletal muscle wasting is reviewed in the present paper. This system relies on the activity of calpains, a family of Ca(2+)-dependent cysteine proteases, whose regulation is complex and not completely elucidated. Modulations of Ca(2+)-dependent proteolysis have been associated with muscle protein depletion in various pathological contexts and particularly with muscle dystrophies. Calpains can only perform a limited proteolysis of their substrates, however they may play a critical role in initiating the breakdown of myofibrillar protein, by releasing molecules that become suitable for further degradation by proteasomes. Some evidence would also support a role for lysosomes and caspases in muscle wasting. Thus it cannot be excluded that different intracellular proteolytic systems may coordinately concur in shifting muscle protein turnover towards excess catabolism. Many different signals have been proposed as potentially involved in triggering the enhanced protein breakdown that underlies muscle wasting. How they are transduced to initiate the hypercatabolic response and to activate the proteolytic pathways remains largely unknown, however.     

Na(+)/Ca(2+) exchange facilitates Ca(2+)-dependent activation of endothelial nitric-oxide synthase.

Recent evidence suggests the expression of a Na(+)/Ca(2+) exchanger (NCX) in vascular endothelial cells. To elucidate the functional role of endothelial NCX, we studied Ca(2+) signaling and Ca(2+)-dependent activation of endothelial nitric-oxide synthase (eNOS) at normal, physiological Na(+) gradients and after loading of endothelial cells with Na(+) ions using the ionophore monensin. Monensin-induced Na(+) loading markedly reduced Ca(2+) entry and, thus, steady-state levels of intracellular free Ca(2+) ([Ca(2+)](i)) in thapsigargin-stimulated endothelial cells due to membrane depolarization. Despite this reduction of overall [Ca(2+)](i), Ca(2+)-dependent activation of eNOS was facilitated as indicated by a pronounced leftward shift of the Ca(2+) concentration response curve in monensin-treated cells. This facilitation of Ca(2+)-dependent activation of eNOS was strictly dependent on the presence of Na(+) ions during treatment of the cells with monensin. Na(+)-induced facilitation of eNOS activation was not due to a direct effect of Na(+) ions on the Ca(2+) sensitivity of the enzyme. Moreover, the effect of Na(+) was not related to Na(+) entry-induced membrane depolarization or suppression of Ca(2+) entry, since neither elevation of extracellular K(+) nor the Ca(2+) entry blocker 1-(beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazol e hydrochloride (SK&F 96365) mimicked the effects of Na(+) loading. The effects of monensin were completely blocked by 3', 4'-dichlorobenzamil, a potent and selective inhibitor of NCX, whereas the structural analog amiloride, which barely affects Na(+)/Ca(2+) exchange, was ineffective. Consistent with a pivotal role of Na(+)/Ca(2+) exchange in Ca(2+)-dependent activation of eNOS, an NCX protein was detected in caveolin-rich membrane fractions containing both eNOS and caveolin-1. These results demonstrate for the first time a crucial role of cellular Na(+) gradients in regulation of eNOS activity and suggest that a tight functional interaction between endothelial NCX and eNOS may take place in caveolae.     

Ca(2+)-dependent inactivation of a cloned cardiac Ca2+ channel alpha 1 subunit (alpha 1C) expressed in Xenopus oocytes.

The alpha 1 subunit of cardiac Ca2+ channel, expressed alone or coexpressed with the corresponding beta subunit in Xenopus laevis oocytes, elicits rapidly inactivating Ca2+ currents. The inactivation has the following properties: 1) It is practically absent in external Ba2+; 2) it increases with Ca2+ current amplitudes; 3) it is faster at more negative potentials for comparable Ca2+ current amplitudes; 4) it is independent of channel density; and 5) it does not require the beta subunit. These findings indicate that the Ca2+ binding site responsible for inactivation is encoded in the alpha 1 subunit and suggest that it is located near the inner channel mouth but outside the membrane electric field.     

Cell to cell adhesion systems in Xenopus laevis, the South African clawed toad. II: Monoclonal antibody against a novel Ca2+-dependent cell-cell adhesion glycoprotein on amphibian cells

We isolated a mouse monoclonal antibody that disrupts Ca2+-dependent cell-cell adhesion of amphibian (Xenopus laevis) cells. When added to culture medium, the monoclonal antibody completely disrupted cell-cell adhesion of amphibian cells in monolayer culture and specifically inhibited Ca2+-dependent cell-cell adhesion of dissociated cells in reaggregation experiments. The monoclonal antibody recognized a 140 kDa cell surface glycoprotein antigenically different from the previously reported Ca2+-dependent cell-cell adhesion molecules (cadherins).     

Immunologically unique and common domains within a family of proteins related to the retina Ca2+-dependent cell adhesion molecule, NcalCAM

Rabbit polyclonal antibodies raised to gp90, a fragment of the embryonic chick neural retina Ca2+-dependent adhesive molecule, gp130, recognize gp130 and inhibit Ca2+-dependent cell-cell adhesion. When tested against a panel of 10-day embryonic tissues, one of these antisera recognizes a component with a molecular weight identical to that of gp130 in embryonic chick cerebrum, optic lobe, hind brain, spinal cord and neural retina only; the second antiserum recognizes a similar component in all of the embryonic chick tissues tested. These data imply the existence of an extended family of closely related cell surface components with immunologically distinct subgroups each of which may mediate Ca2+-dependent cell-cell adhesion. As the term CAM, or cell adhesion molecule, has become common usage we propose to refer to these molecules as calCAMs, reflecting their calcium dependence. Analysis of fragments and endoglycosidase digests of NcalCAM have allowed a comparison of its structure with similar molecules from different tissues and species that have been implicated in Ca2+-dependent cell-cell adhesion.     

Ca(2+)-dependent protein kinase from the halotolerant green alga Dunaliella tertiolecta: partial purification and Ca(2+)-dependent association of the enzyme to the microsomes.

Ca(2+)-dependent protein kinase (CDPK) was purified 900-fold from the soluble fraction of Dunaliella tertiolecta cells by ammonium sulfate precipitation, DEAE-Toyopearl, phenyl-Sepharose, and hydroxylapatite column chromatography. The CDPK was activated by micromolar concentration of Ca2+ and required neither calmodulin nor phospholipids for its activation. The enzyme phosphorylated casein, myosin light chain, and histone type III-S (histone H-1), but did not phosphorylate protamine and phosvitin. The Km values for ATP and casein were 11 microM and 300 micrograms/ml, respectively. Phosphorylation of casein was inhibited by calmodulin antagonists, calmidazolium, trifluoperazine, and compound 48/80, but not affected by calmodulin. CDPK bound to phenyl-Sepharose in the presence of Ca2+ and was eluted by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). This suggests that hydrophobicity of the enzyme was increased by Ca2+. CDPK was also bound to the microsomes isolated from Dunaliella cells in the presence of micromolar concentration of Ca2+ and released in the presence of EGTA, suggesting the possibility of in vivo Ca(2+)-dependent association of the enzyme. The enzyme phosphorylated many proteins in the microsomes but few in the cytosol, if at all.