Direct binding of cell polarity protein PAR-3 to cell-cell adhesion molecule nectin at neuroepithelial cells of developing mouse

PAR-3 is a cell polarity protein that localizes at tight junctions (TJs) by direct binding to an immunoglobulin (Ig)-like cell-cell adhesion molecule JAM-1 in mammalian epithelial cells. Another Ig-like cell-cell adhesion molecule nectin plays a role in the localization of JAM-1 at TJs in epithelial cells. Nectin furthermore plays a role in the organization of adherens junctions (AJs) and TJs. Nectin comprises a family of four members, nectin-1, -2, -3, and -4. Nectins are associated with the actin cytoskeleton through afadin, of which the PDZ domain binds to nectins through their C-terminal four amino acids. We show here that PAR-3 binds to nectin-1 and -3 in neuroepithelial cells of the embryonic telencephalon, which are equipped with AJs, but not with typical TJs. Nectin-1, -2, -3, and afadin, but not JAM-1, were concentrated at AJs in neuroepithelial cells of the embryonic telencephalon at E13.5 and PAR-3 co-localized with nectins. PAR-3 was co-immunoprecipitated with nectin-1 and -3, but not with nectin-2 or JAM-1, from the mouse whole brain at E13.5. Recombinant PAR-3 stoichiometrically bound to recombinant nectin-1 and -3. The first one of the three PDZ domains of PAR-3 bound to the C-terminal four amino acids of nectin-1 and -3. The affinities of PAR-3 and afadin for nectin-1 and -3 were similar. Cadherin-deficient L cells expressing nectin-1 and -3 formed nectin-1- and -3-based cell-cell junctions, respectively, where PAR-3 as well as afadin was recruited. These results indicate that nectin-1 and -3 are involved in the localization of PAR-3 at AJs in the neuroepithelial cells of the embryonic telencephalon.     

Implications of nectin-like molecule-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 in cell-cell adhesion and transmembrane protein localization in epithelial cells

Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules that play roles in organization of a variety of cell-cell junctions in cooperation with or independently of cadherins. Four nectins have been identified. Five nectin-like molecules, which have domain structures similar to those of nectins, have been identified, and we characterized here nectin-like molecule-2 (Necl-2)/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1. Necl-2 showed Ca2+-independent homophilic cell-cell adhesion activity. It furthermore showed Ca2+-independent heterophilic cell-cell adhesion activity with Necl-1/TSLL1/SynCAM3 and nectin-3. Necl-2 was widely expressed in rat tissues examined. Necl-2 localized at the basolateral plasma membrane in epithelial cells of the mouse gall bladder, but not at specialized cell-cell junctions, such as tight junctions, adherens junctions, and desmosomes. Nectins bind afadin, whereas Necl-2 did not bind afadin but bound Pals2, a membrane-associated guanylate kinase family member known to bind Lin-7, implicated in the proper localization of the Let-23 protein in Caenorhabditis elegans, the homologue of mammalian epidermal growth factor receptor. These results indicate the unique localization of Necl-2 and its possible involvement in localization of a transmembrane protein(s) through Pals2.     

Functional studies and distribution define a family of transmembrane AMPA receptor regulatory proteins

Functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in cerebellar granule cells requires stargazin, a member of a large family of four-pass transmembrane proteins. Here, we define a family of transmembrane AMPA receptor regulatory proteins (TARPs), which comprise stargazin, gamma-3, gamma-4, and gamma-8, but not related proteins, that mediate surface expression of AMPA receptors. TARPs exhibit discrete and complementary patterns of expression in both neurons and glia in the developing and mature central nervous system. In brain regions that express multiple isoforms, such as cerebral cortex, TARP-AMPA receptor complexes are strictly segregated, suggesting distinct roles for TARP isoforms. TARPs interact with AMPA receptors at the postsynaptic density, and surface expression of mature AMPA receptors requires a TARP. These studies indicate a general role for TARPs in controlling synaptic AMPA receptors throughout the central nervous system.     

Impact of two probiotic Lactobacillus strains feeding on fecal lactobacilli and weight gains in chicken

Two probiotic strains, Lactobacillus agilis JCM 1048 and L. salivarius subsp. salicinius JCM 1230 isolated from chicken intestine, exhibited probiotic characteristics that can be applied for chicken production. After 7 days of probiotic feeding (FD7), the count of intestinal lactobacilli in the probiotic group (group P, n=10) was significantly (p<0.05) higher than that in the control group (group C, n=9). After 40 days of probiotic feeding (FD40), the lactobacilli and enterococci counts were stable but the Enterobacteriaceae number was significantly reduced (p<0.05). A total of 163 isolated lactobacilli were identified as the L. acidophilus/gallinarum group (49.7%), L. agilis (30.7%), L. salivarius (9.2%), L. reuteri (9.2%), and Lactobacillus spp. (1.2%). The probiotic lactobacilli positively affected the Lactobacillus biota in chickens at FD7, with a significant increase in the number (p<0.05) of L. agilis and group P. The viable counts of each Lactobacillus species at FD40, however, showed no differences between two groups. An increasing incidence of L. agilis was also noted with probiotic feeding. The probiotic effect of two strains resulted in significantly increased weight gains (10.7%) of group P in comparison with group C at FD40 (p<0.01).     

Tage4/Nectin-like molecule-5 heterophilically trans-interacts with cell adhesion molecule Nectin-3 and enhances cell migration

Malignant transformation of cells causes disruption of cell-cell adhesion, enhancement of cell motility, and invasion into surrounding tissues. Nectins have both homophilic and heterophilic cell-cell adhesion activities and organize adherens junctions in cooperation with cadherins. We examined here whether Tage4, which was originally identified to be a gene overexpressed in colon carcinoma and has a domain structure similar to those of nectins, is involved in cell adhesion and/or migration. Tage4 heterophilically trans-interacted with nectin-3, but not homophilically with Tage4. Expression of Tage4 was markedly elevated in NIH3T3 cells transformed by an oncogenic Ki-Ras (V12Ras-NIH3T3 cells) as compared with that of wild-type NIH3T3 cells. trans-Interaction of Tage4 with nectin-3 enhanced motility of V12Ras-NIH3T3 cells. Tage4 did not bind afadin, a nectin- and actin filament-binding protein that connects nectins to the actin cytoskeleton and cadherins through catenins. Thus, Tage4 heterophilically trans-interacts with nectin-3 and regulates cell migration. Tage4 is tentatively re-named here nectin-like molecule-5 (necl-5) on the basis of its function and domain structure similar to those of nectins.     

Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190

S-Adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine to form adenosine and homocysteine. On the bases of crystal structures of the wild type enzyme and the D244E mutated enzyme complexed with 3'-keto-adenosine (D244E.Ado*), we have identified the important amino acid residues, Asp-130, Lys-185, Asp-189, and Asn-190, for the catalytic reaction and have proposed a catalytic mechanism (Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (2000) J. Biol. Chem. 275, 32147-32156). To confirm the proposed catalytic mechanism, we have made the D130N, K185N, D189N, and N190S mutated enzymes and measured the catalytic activities. The catalytic rates (k(cat)) of D130N, K185N, D189N, and N190S mutated enzymes are reduced to 0.7%, 0.5%, 0.1%, and 0.5%, respectively, in comparison with the wild type enzyme, indicating that Asp-130, Lys-185, Asp-189, and Asn-190 are involved in the catalytic reaction. K(m) values of the mutated enzymes are increased significantly, except for the N190S mutation, suggesting that Asp-130, Lys-185, and Asp-189 participate in the substrate binding. To interpret the kinetic data, the oxidation states of the bound NAD molecules of the wild type and mutated enzymes were measured during the catalytic reaction by monitoring the absorbance at 340 nm. The crystal structures of the WT and D244E.Ado*, containing four subunits in the crystallographic asymmetric unit, were re-refined to have the same subunit structures. A detailed catalytic mechanism of AdoHcyase has been revealed based on the oxidation states of the bound NAD and the re-refined crystal structures of WT and D244E.Ado*. Lys-185 and Asp-130 abstract hydrogen atoms from 3'-OH and 4'-CH, respectively. Asp-189 removes a proton from Lys-185 and produces the neutral N zeta (-NH(2)), and Asn-190 facilitates formation of the neutral Lys-185. His-54 and His-300 hold and polarize a water molecule, which nucleophilically attacks the C5'- of 3'-keto-4',5'-dehydroadenosine to produce 3'-keto-Ado.     

Inhibition of S-adenosylhomocysteine hydrolase by acyclic sugar adenosine analogue D-eritadenine. Crystal structure of S-adenosylhomocysteine hydrolase complexed with D-eritadenine

D-eritadenine (DEA) is a potent inhibitor (IC(50) = 7 nm) of S-adenosyl-l-homocysteine hydrolase (AdoHcyase). Unlike cyclic sugar Ado analogue inhibitors, including mechanism-based inhibitors, DEA is an acyclic sugar Ado analogue, and the C2' and C3' have opposite chirality to those of the cyclic sugar Ado inhibitors. Crystal structures of DEA alone and in complex with AdoHcyase have been determined to elucidate the DEA binding scheme to AdoHcyase. The DEA-complexed structure has been analyzed by comparing it with two structures of AdoHcyase complexed with cyclic sugar Ado analogues. The DEA-complexed structure has a closed conformation, and the DEA is located near the bound NAD(+). However, a UV absorption measurement shows that DEA is not oxidized by the bound NAD(+), indicating that the open-closed conformational change of AdoHcyase is due to the substrate/inhibitor binding, not the oxidation state of the bound NAD. The adenine ring of DEA is recognized by four essential hydrogen bonds as observed in the cyclic sugar Ado complexes. The hydrogen bond network around the acyclic sugar moiety indicates that DEA is more tightly connected to the protein than the cyclic sugar Ado analogues. The C3'-H of DEA is pointed toward C4 of the bound NAD(+) (C3'...C4 = 3.7 A), suggesting some interaction between DEA and NAD(+). By placing DEA into the active site of the open structure, the major forces to stabilize the closed conformation of AdoHcyase are identified as the hydrogen bonds between the backbone of His-352 and the adenine ring, and the C3'-H...C4 interaction. DEA has been believed to be an inactivator of AdoHcyase, but this study indicates that DEA is a reversible inhibitor. On the basis of the complexed structure, selective inhibitors of AdoHcyase have been designed.     

TGF-beta 1 as an enhancer of Fas-mediated apoptosis of lung epithelial cells

Transforming growth factor-beta 1 (TGF-beta 1) has important roles in lung fibrosis and the potential to induce apoptosis in several types of cells. We previously demonstrated that apoptosis of lung epithelial cells induced by Fas ligation may be involved in the development of pulmonary fibrosis. In this study, we show that TGF-beta1 induces apoptosis of primary cultured bronchiolar epithelial cells via caspase-3 activation and down-regulation of cyclin-dependent kinase inhibitor p21. Concentrations of TGF-beta 1 that were not sufficient to induce apoptosis alone could enhance agonistic anti-Fas Ab or rFas ligand-mediated apoptosis of cultured bronchiolar epithelial cells. Soluble Fas ligand in the bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF) also induced apoptosis of cultured bronchiolar epithelial cells that was significantly attenuated by anti-TGF-beta Ab. Otherwise, BALF from patients with hypersensitivity pneumonitis (HP) could not induce apoptosis on bronchiolar epithelial cells, despite its comparable amounts of soluble Fas ligand. The concentrations of TGF-beta 1 in BALF from patients with IPF were significantly higher compared with those in BALF from patients with HP or controls. Furthermore, coincubation with the low concentration of TGF-beta 1 and HP BALF created proapoptotic effects comparable with the IPF BALF. In vivo, the administration of TGF-beta 1 could enhance Fas-mediated epithelial cell apoptosis and lung injury via caspase-3 activation in mice. Our results demonstrate a novel role of TGF-beta 1 in the pathophysiology of pulmonary fibrosis as an enhancer of Fas-mediated apoptosis of lung epithelial cells.     

Phylogenetic analysis of cecal microbiota in chicken by the use of 16S rDNA clone libraries

The chicken cecum contains a great many bacteria, most of which are strict anaerobes. A strictly anaerobe culture-based method was used in the present study, in conjunction with the 16S rDNA clone library, to elucidate bacterial diversity and the phylogenetic relationship of cecal microbiota in the chicken. A comparative 16S rDNA sequence analysis of cultivated strains and retrieved clones from cecal contents was performed. Approximately 90% of the bacterial cells detected by microscopy did not form colonies on a medium 10 in plate-in-bottle. The 19 isolated strains yielded 11 distinct rDNA sequences, 58% of which were classified as low G + C gram-positive bacteria, 26% were related to Bacteroides spp., and 16% were classified as Proteobacteria. Based on the sequence analysis of 164 clones, 24% were identified to belong to 8 known species and 76% were considered to be 65 novel phylotypes. Approximately 94% of cloned sequences were classified into low G + C gram-positive bacteria, 4% were related to Bacteroides spp., and 2% were classified into Proteobacteria. Clostridium subcluster XIVa (38%), Clostridium cluster IV (13%), Lactobacillus spp. (24%), and Bacteroides spp. (4%) were the major groups constituting the cecal microbiota in chicken, in which the Clostridium subcluster XIVa was the most phylogenetically diverse group in chicken cecum. The 16S rDNA sequences of Lactobacillus acidophilus, L. crispatus, L. salivarius, and L. reuteri were the most frequently found in the Lactobacillus group in chicken cecum.     

The penta-EF-hand domain of ALG-2 interacts with amino-terminal domains of both annexin VII and annexin XI in a Ca2+-dependent manner

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand (PEF) protein family. ALG-2 forms a homodimer, a heterodimer with another PEF protein, peflin, and a complex with its interacting protein, named Alix or AIP1. We previously identified annexin XI as a novel ALG-2-binding partner. Both the N-terminal regulatory domain of annexin XI (Anx11N) and the ALG-2-binding domain of Alix/AIP1 are rich in Pro, Gly, Ala, Tyr and Gln. This PGAYQ-biased amino acid composition is also found in the N-terminal extension of annexin VII (Anx7N). Using recombinant ALG-2 proteins and the glutathione S-transferase (GST) fusion proteins of Anx7N and Anx11N, the direct Ca(2+)-dependent interaction was analyzed by a biotin-tagged ALG-2 overlay assay and by a real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. Both GST-Anx7N and GST-Anx11N showed similar binding kinetics against ALG-2 as well as ALG-2-DeltaN23, which lacked the hydrophobic N-terminal region. Two binding sites were predicted in both Anx7N and Anx11N, and the dissociation constants (K(d)) were estimated to be approximately 40-60 nM for the high-affinity site and 500-700 nM for the low-affinity site.