The cadherin-binding specificities of B-cadherin and LCAM

The cadherin family of calcium-dependent cell adhesion molecules plays an important part in the organization of cell adhesion and tissue segregation during development. The expression pattern and the binding specificity of each cadherin are of principal importance for its role in morphogenesis. B-Cadherin and LCAM, two chicken cadherins, have similar, but not identical, spatial and temporal patterns of expression. To examine the possibility that they might bind to one another in a heterophilic manner, we generated, by cDNA transfection, L- cell lines that express LCAM or B-cadherin. We then examined the abilities of these cells to coaggregate with each other and with other cadherin-expressing cells in short-term aggregation assays. The B- cadherin- and the LCAM-expressing cell lines segregate from P-, N-, or R-cadherin-expressing cells. B-cadherin- and LCAM-expressing cell lines, however, appear to be completely miscible, forming large mixed aggregates. Chick B-cadherin and murine E-cadherin also form mixed aggregates, indistinguishable from homophilic aggregates. Murine E- cadherin and chick LCAM coaggregate less completely, suggesting that the heterophilic interactions of these two cell lines are weak relative to homophilic interactions. These data suggest that heterophilic interactions between B-cadherin and LCAM are important during avian morphogenesis and help identify the amino acids in the binding domain that determine cadherin specificity.     

Identification and cloning of two species of cadherins in bovine endothelial cells.

By taking advantage of the extensive homology found in the cytoplasmic domains of several cloned cadherin molecules, we were able to identify two species of cadherins in bovine aortic endothelial cells using the polymerase chain reaction (PCR). The two species of PCR products were subsequently used as DNA probes to isolate the corresponding cDNA clones from bovine adrenal microvascular endothelial cells. Sequence comparison with other characterized cadherin molecules indicates that the major cDNA species encodes a cadherin molecule highly homologous to chicken and mouse N-cadherins, while the minor species is most homologous to mouse and human P-cadherins. Northern blot analysis with the corresponding cDNA probe showed a wide distribution of bovine N-cadherin among non-neuronal, as well as neuronal tissues, while P-cadherin was most abundant in kidney among all the bovine tissues tested, but was undetectable in placenta.     

Cloning of Five Human Cadherins Clarifies Characteristic Features of Cadherin Extracellular Domain and Provides Further Evidence for Two Structurally Different Types of Cadherin

The entire coding sequences for five possible human cadherins, named cadherin-4,-8,-11,-12 and-13, were determined. The deduced amino acid sequences of cadherin-4 and cadherin-13 showed high homology with those of chicken R-cadherin or chicken T-caciherin, suggesting that cadherin-4 and cadherin-13 are mammalian homologues of the chicken R-cadherin or T-cadherin. Comparison of the extracellular domain of these proteins with those of other cadherins and cadherin-related proteins clarifies characteristic structural features of this domain. The domain is subdivided into five subdomains, each of which contains a cadherin-specific motif characterized by well-conserved amino acid residues and short amino acid sequences. Moreover, each subdomain has unique features of its own. The comparison also provides additional evidence for two structurally different types of cadherins: the first type includes B-, E-, EP-, M, N-, P-and R-cadherins and cadherin-4; the second type includes cadherin-5 through cadherin-12. Cadherin-13 lacks the sequence corresponding to the cytoplasmic domain of typical cadherins, but the extracellular domain shares most of the features common to the extracellular domain of cadherins, especially those of the first type of cadherins, suggesting that cadherin-13 is a special type of cadherin. These results, and those of other recent cloning studies, indicate that many cadherins with different properties are expressed in various tissues of different organisms.     

Expression sequences and distribution of two primary cell adhesion molecules during embryonic development of Xenopus laevis

Studies of chicken embryos have demonstrated that cell adhesion molecules are important in embryonic induction and are expressed in defined sequences during embryogenesis and histogenesis. To extend these observations and to provide comparable evidence for heterochronic changes in such sequences during evolution, the local distributions of the neural cell adhesion molecule (N-CAM) and of the liver cell adhesion molecule (L-CAM) were examined in Xenopus laevis embryos by immunohistochemical and biochemical techniques. Because of the technical difficulties presented by the existence of multiple polypeptide forms of CAMs and by autofluorescence of yolk-containing cells, special care was taken in choosing and characterizing antibodies, fluorophores, and embedding procedures. Both N-CAM and L-CAM were found at low levels in pregastrulation embryos. During gastrulation, N-CAM levels increased in the presumptive neural epithelium and decreased in the endoderm, but L-CAM continued to be expressed in all cells including endodermal cells. During neurulation, the level of N-CAM expression in the neural ectoderm increased considerably, while remaining constant in non-neural ectoderm and diminishing in the somites; in the notochord, N-CAM was expressed transiently. Prevalence modulation was also seen at all sites of secondary induction: both CAMs increased in the sensory layer of the ectoderm during condensation of the placodes. During organogenesis, the expression of L-CAM gradually diminished in the nervous system while N-CAM expression remained high. In all other organs examined, the amount of one or the other CAM decreased, so that by stage 50 these two molecules were expressed in non-overlapping territories. Embryonic and adult tissues were compared to search for concordance of CAM expression at later stages. With few exceptions, the tissue distributions of N-CAM and L-CAM were similar in the frog and in the chicken from early times of development. In contrast to previous observations in the chicken and in the mouse, N-CAM expression was found to be high in the adult liver of Xenopus, whereas L-CAM expression was low. In the adult brain, N-CAM was expressed as three components of apparent molecular mass 180, 140, and 120 kD, respectively; in earlier stages of development only the 140-kD component could be detected. In the liver, a single N-CAM band appears at 160 kD, raising the possibility that this band represents an unusual N-CAM polypeptide. L-CAM appeared at all stages as a 124-kD molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cadherin-mediated cell adhesion and tissue segregation: qualitative and quantitative determinants

It is widely held that segregation of tissues expressing different cadherins results from cadherin-subtype-specific binding specificities. This belief is based largely upon assays in which cells expressing different cadherin subtypes aggregate separately when shaken in suspension. In various combinations of L cells expressing NCAM, E-, P-, N-, R-, or B-cadherin, coaggregation occurred when shear forces were low or absent but could be selectively inhibited by high shear forces. Cells expressing P- vs E-cadherin coaggregated and then demixed, one population enveloping the other completely. To distinguish whether this demixing was due to differences in cadherin affinities or expression levels, the latter were varied systematically. Cells expressing either cadherin at a lower level became the enveloping layer, as predicted by the Differential Adhesion Hypothesis. However, when cadherin expression levels were equalized, cells expressing P- vs E-cadherin remained intermixed. In this combination, "homocadherin" (E-E; P-P) and "heterocadherin" (E-P) adhesions must therefore be of similar strength. Cells expressing R- vs B-cadherin coaggregated but demixed to produce configurations of incomplete envelopment. This signifies that R- to B-cadherin adhesions must be weaker than either "homocadherin" adhesion. Together, cadherin quantity and affinity control tissue segregation and assembly through specification of the relative intensities of mature cell-cell adhesions.     

Cadherin cell adhesion molecules with distinct binding specificities share a common structure.

Ca2+-dependent cell--cell adhesion molecules, termed cadherins, are divided into subclasses with distinct tissue distributions and distinct cell-binding specificities. To elucidate the biochemical relationship of these subclasses, we compared the pattern of tryptic cleavage and the partial amino acid sequence of mouse liver E-cadherin with those of chicken brain N-cadherin. Although these two cadherins are distinct in their cell-binding and immunological specificities, they showed an identical mol. wt and a similar tryptic cleavage pattern. We isolated tryptic fragments of E- and N-cadherin, and determined the sequences of nine amino acid residues of their amino terminus. The results showed that sequences of amino acids from the amino terminus to the 7th residues are identical in these two cadherins. We thus suggest that cadherins with distinct specificities have a common genic origin.     

Cloning and expression of the chicken immunoglobulin joining (J)-chain cDNA

 The J chain is a component of polymeric immunoglobulin (Ig) molecules and may play an important role in their polymerization and the transport of polymeric Ig across epithelial cells. In this study, the primary structure of the chicken J chain was determined by sequencing cDNA clones. The cDNA had an open reading frame of 476 nucleotides encoding a putative protein of 158 amino acid residues including the signal sequence. The 3′ untranslated region consisted of 1216 nucleotides and a poly(A) tail. The deduced amino acid sequence of the chicken J chain had a high degree of homology to that of human, cow, rabbit, mouse, frog, and earthworm, with eight conserved Cys residues identical to the mammalian J chains. Northern blot hybridization performed with total RNA from various chicken tissues revealed high levels of J-chain mRNA expression in spleen, intestine, Harderian gland, and bursa of Fabricius, and low levels in the thymus. The J chain was expressed in the bursa as early as day 15 of embryogenesis. These data indicated that the chicken J-chain gene displays a high degree of homology with that of other species, and is expressed at an early stage of development of the chicken immune system. Received: 21 May 1999 / Revised: 30 August 1999     

The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy

We previously identified a 220-kD constitutive protein of the plasma membrane undercoat which colocalizes at the immunofluorescence microscopic level with cadherins and occurs not only in epithelial M., S. Yonemura, A. Nagafuchi, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). To clarify the nature and possible functions of this protein, we cloned its full-length cDNA and sequenced it. Unexpectedly, we found mouse 220-kD protein to be highly homologous to rat protein ZO-1, only a part of which had been already sequenced. This relationship was confirmed by immunoblotting with anti-ZO-1 antibody. As protein ZO-1 was originally identified as a component exclusively underlying tight junctions in epithelial cells, where cadherins are not believed to be localized, we analyzed the distribution of cadherins and the 220-kD protein by ultrathin cryosection immunoelectron microscopy. We found that in non-epithelial cells lacking tight junctions cadherins and the 220-kD protein colocalize, whereas in epithelial cells (e.g., intestinal epithelial cells) bearing well-developed tight junctions cadherins and the 220-kD protein are clearly segregated into adherens and tight junctions, respectively. Interestingly, in epithelial cells such as hepatocytes, which tight junctions are not so well developed, the 220-kD protein is detected not only in the tight junction zone but also at adherens junctions. Furthermore, we show in mouse L cells transfected with cDNAs encoding N-, P-, E-cadherins that cadherins interact directly or indirectly with the 220-kD protein. Possible functions of the 220-kD protein (ZO-1) are discussed with special reference to the molecular mechanism for adherens and tight junction formation.     

Interpretation of the cross sectional structure of skeletal muscle fibers. 2. Light and electron microscopic studies of m. rectus abdominis in Rana esculenta]

In both longitudinal and cross sections of rectus abdominis muscle of Rana esculenta three types of muscle fibres are identified by means of light and electron microscopy. A comparison is made between these fibre types in homologous muscles of frog and mammals (rat and mouse). In longitudinal sections of mammalian and frog muscle the Z-line can be used for discrimination of the fibre types A, B and C because that line is of different thickness in each type. The proportions of the thickness in frog and mammalian muscles are relatively the same, but the absolute values are different. In cross sections there are no differences between frog and mammalian muscle fibres concerning the typical form of myofibrils in type A- and B-fibres, whereas in type C-fibres the arrangement of the filaments in the Z- and H-layer is different in the members of both animal classes. The amount of mitochondria and lipid droplets is different as well. In the species examined the distribution of A-, B- and C-fibres changes within the whole muscle. In frog, this pattern depends on the level in which the muscle has been sectioned. This is not true for mammalian muscle. On the other hand both ends of the rectus abdominis muscle in frog, rat and mouse show an accumulation of B- and C-type fibres.     

Localization of HIV RNA in mitochondria of infected cells: potential role in cytopathogenicity

A novel member of the cadherin family of cell adhesion molecules has been characterized by cloning from rat liver, sequencing of the corresponding cDNA, and functional analysis after heterologous expression in nonadhesive S2 cells. cDNA clones were isolated using a polyclonal antibody inhibiting Ca(2+)-dependent intercellular adhesion of hepatoma cells. As inferred from the deduced amino acid sequence, the novel molecule has homologies with E-, P-, and N-cadherins, but differs from these classical cadherins in four characteristics. Its extracellular domain is composed of five homologous repeated domains instead of four characteristic for the classical cadherins. Four of the five domains are characterized by the sequence motifs DXNDN and DXD or modifications thereof representing putative Ca(2+)-binding sites of classical cadherins. In its NH2-terminal region, this cadherin lacks both the precursor segment and the endogenous protease cleavage site RXKR found in classical cadherins. In the extracellular EC1 domain, the novel cadherin contains an AAL sequence in place of the HAV sequence motif representing the common cell adhesion recognition sequence of E-, P-, and N-cadherin. In contrast to the conserved cytoplasmic domain of classical cadherins with a length of 150-160 amino acid residues, that of the novel cadherin has only 18 amino acids. Examination of transfected S2 cells showed that despite these structural differences, this cadherin mediates intercellular adhesion in a Ca(2+)-dependent manner. The novel cadherin is solely expressed in liver and intestine and was, hence, assigned the name LI-cadherin. In these tissues, LI- cadherin is localized to the basolateral domain of hepatocytes and enterocytes. These results suggest that LI-cadherin represents a new cadherin subtype and may have a role in the morphological organization of liver and intestine.     

Different features of the MHC class I heterodimer have evolved at different rates. Chicken B-F and beta 2-microglobulin sequences reveal invariant surface residues.

Chicken beta 2-microglobulin (beta 2m) and class I (B-F19 alpha chain) cDNA clones were isolated and the sequences compared to those of B-F Ag isolated from chicken E. These clones represent the major expressed class I molecules on E, with B-F alpha size variants evidently due to alternative use of small exons in the cytoplasmic region. The cDNA sequences were compared to turkey beta 2m, the apparent allele B-F12 alpha and other vertebrate homologs, using the 2.6 A structure of the human HLA-A2 molecule as a model. Both chicken alpha 1 and alpha 2 domains resemble mammalian classical class I molecules and the MHC-encoded nonclassical molecules more than CD1 or the class I-like FcR. In contrast, the chicken alpha 3 domain is equally homologous to all alpha 3 domains, to beta 2m and to class II beta 2 domains. For each pair of extracellular domains (alpha 1 vs alpha 2, alpha 3 vs beta 2m), the level of sequence homology between mammalian and avian molecules is quite different. This suggests that the structurally homologous domains have been under different selective pressures during evolution. There is a very strong G + C bias in alpha 3 and beta 2m, leading to an overall change in amino acid composition in B-F compared to class I molecules from other taxa. Many of the surface residues are quite diverged, particularly in alpha 3 and beta 2m. There are fewer changes in intra- and interdomain contact sites. Some residues with important functions are invariant, including seven residues that bind the ends of the peptide, two residues that bind CD8, and three residues that are phosphorylated. The positions of the allelic residues are conserved. There are other patches of invariant residues on alpha 1, alpha 2, and beta 2m; these might bind TCR or other molecules involved in class I function.     

cDNAs of cell adhesion molecules of different specificity induce changes in cell shape and border formation in cultured S180 cells

The liver cell adhesion molecule (L-CAM) and N-cadherin or adherens junction-specific CAM (A-CAM) are structurally related cell surface glycoproteins that mediate calcium-dependent adhesion in different tissues. We have isolated and characterized a full-length cDNA clone for chicken N-cadherin and used this clone to transfect S180 mouse sarcoma cells that do not normally express N-cadherin. The transfected cells (S180cadN cells) expressed N-cadherin on their surfaces and resembled S180 cells transfected with L-CAM (S180L cells) in that at confluence they formed an epithelioid sheet and displayed a large increase in the number of adherens and gap junctions. In addition, N-cadherin in S180cadN cells, like L-CAM in S180L cells, accumulated at cellular boundaries where it was colocalized with cortical actin. In S180L cells and S180cadN cells, L-CAM and N-cadherin were seen at sites of adherens junctions but were not restricted to these areas. Adhesion mediated by either CAM was inhibited by treatment with cytochalasin D that disrupted the actin network of the transfected cells. Despite their known structural similarities, there was no evidence of interaction between L-CAM and N-cadherin. Doubly transfected cells (S180L/cadN) also formed epithelioid sheets. In these cells, both N-cadherin and L-CAM colocalized at areas of cell contact and the presence of antibodies to both CAMs was required to disrupt the sheets of cells. Studies using divalent antibodies to localize each CAM at the cell surface or to perturb their distributions indicated that in the same cell there were no interactions between L-CAM and N-cadherin molecules. These data suggest that the Ca(++)-dependent CAMs are likely to play a critical role in the maintenance of epithelial structures and support a model for the segregation of CAM mediated binding. They also provide further support for the so-called precedence hypothesis that proposes that expression and homophilic binding of CAMs are necessary for formation of junctional structures in epithelia.     

Expression and localization of P-, K-, and OB-cadherin in the prepubertal rat ovary.

Classical and atypical cadherins mediate calcium-dependent cell adhesion and play an important role in morphogenetic processes. We have shown, previously, N- and E-cadherin expression in the rat ovary. This expression, however, was not associated with specific follicle-restructuring events such as antrum formation and segregation of mural from cumulus granulosa cells suggesting that other cadherins may serve this function. In this study, RT-PCR and immunostaining techniques showed that three other cadherins are expressed throughout prepubertal ovarian development in the rat: one classical (P-) cadherin, and two atypical (K- and OB-) cadherins. RT-PCR analysis of isolated ovarian tissue compartments (granulosa cells and the residual ovarian tissue) agreed with the immunostaining results. Immunostaining showed P- and K-cadherin expression by granulosa, as well as thecal/interstitial cells, and also in oocytes of primordial follicles. P-cadherin expression was absent in oocytes of follicles in later stages of development compared to K-cadherin, which was found in oocytes at all stages of folliculogenesis. P-, K-, and OB-cadherin were expressed by the ovarian surface epithelial cells of neonatal animals but only P- and OB-cadherin expression were maintained in these cells in 25 day-old animals. Cellular OB-cadherin staining was absent in follicles at all stages of development and its expression was restricted to the ovarian hilar region and portions of the stroma. In summary, cadherin expression and distribution profiles changed during ovarian growth and folliculogenesis suggesting a role for cadherins in organizational and morphogenetic processes within the developing rat ovary.     

Characterization of the putative avian CD2 homologue.

We describe two mouse mAb recognizing the putative chicken homologue of mammalian CD2 Ag and provide evidence for both structural and functional conservation between the avian and mammalian CD2 molecules. The antibodies were T cell-specific and immunoprecipitated a single diffuse band of Mr 40,000 from lysates of surface-labeled chicken thymocytes and peripheral T cells. Removal of N-linked carbohydrate with endo-beta-N-acetylglucosaminidase F revealed the core protein size of Mr 25,000. A rabbit antiserum raised against a synthetic peptide (CD2-300), composed of 18 amino acid residues of the conserved cytoplasmic domain of human, mouse, and rat CD2, precipitated an Ag similar to chicken CD2. Sequential precipitation with CD2-300 antiserum indicated the conservation of an avian and mammalian CD2 epitope. CD2 expression on thymocytes starts at day 11 of embryonic development, and, during subsequent development, thymic gamma delta cells are all CD2+, whereas most peripheral gamma delta-T cells lack CD2. Functional conservation between the chicken and mammalian CD2 molecules was demonstrated by the induction of DNA synthesis in chicken thymocytes and peripheral T cells with the combination of anti-CD2 mAb and PMA.     

Immunohistochemical localization of cadherin and catenin adhesion molecules in the murine growth plate.

Mouse tibial growth plates were examined for the presence of adhesion molecules using immunohistochemistry and RT-PCR. All of the components of the classical cadherin/catenin complex (cadherin, alpha-, beta-, and gamma-catenin), as well as a heavy presence of p120, were identified in the murine growth plate. All of the major cadherins (1-5, 11, 13, and 15) were, for the first time, identified and localized in the murine growth plate. We have demonstrated that most of the cadherins and catenins reside in the zone of hypertrophy. Only alpha-catenin and E-, P-, R-, and VE-cadherin were found in all regions of the growth plate. The results for T-cadherin were inconclusive.