Akira Tohgo

Cyclic ADP-ribose Binds to FK506-binding Protein 12.6 to Release Ca2+ from Islet Microsomes

Cyclic ADP-ribose (cADPR) is a second messenger for Ca2+ mobilization via the ryanodine receptor (RyR) from islet microsomes for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373). In the present study, FK506, an immunosuppressant that prolongs allograft survival, as well as cADPR were found to induce the release of Ca2+ from islet microsomes. After islet microsomes were treated with FK506, the Ca2+ release by cADPR from microsomes was reduced. cADPR as well as FK506 bound to FK506-binding protein 12.6 (FKBP12.6), which we also found occurs naturally in islet microsomes. When islet microsomes were treated with cADPR, FKBP12.6 dissociated from the microsomes and moved to the supernatant, releasing Ca2+ from the intracellular stores. The microsomes that were then devoid of FKBP12.6 did not show Ca2+ release by cADPR. These results strongly suggest that cADPR may be the ligand for FKBP12.6 in islet RyR and that the binding of cADPR to FKBP12.6 frees the RyR from FKBP12.6, causing it to release Ca2+.

DNER acts as a neuron-specific Notch ligand during Bergmann glial development

Differentiation of CNS glia is regulated by Notch signaling through neuron-glia interaction. Here, we identified Delta/Notch-like EGF-related receptor (DNER), a neuron-specific transmembrane protein, as a previously unknown ligand of Notch during cellular morphogenesis of Bergmann glia in the mouse cerebellum. DNER binds to Notch1 at cell-cell contacts and activates Notch signaling in vitro. In the developing cerebellum, DNER is highly expressed in Purkinje cell dendrites, which are tightly associated with radial fibers of Bergmann glia expressing Notch. DNER specifically binds to Bergmann glia in culture and induces process extension by activating γ-secretase– and Deltex-dependent Notch signaling. Inhibition of Deltex-dependent, but not RBP-J–dependent, Notch signaling in Bergmann glia suppresses formation and maturation of radial fibers in organotypic slice cultures. Additionally, deficiency of DNER retards the formation of radial fibers and results in abnormal arrangement of Bergmann glia. Thus, DNER mediates neuron-glia interaction and promotes morphological differentiation of Bergmann glia through Deltex-dependent Notch signaling.

Cloning and characterization of cDNA encoding rat ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase (homologue to human CD38) from islets of Langerhans

We report the cloning and cDNA sequence of rat CD38, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase. Rat CD38 is composed of 303 amino acids and shares a high degree of homology with human and mouse CD38. Rat CD38 mRNA is expressed in various tissues including pancreatic islets but not in RINm5F cells.

Autoantibodies against CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) that impair glucose-induced insulin secretion in noninsulin- dependent diabetes patients.

Cyclic ADP-ribose (cADPR) has been shown to be a mediator for intracellular Ca2+ mobilization for insulin secretion by glucose in pancreatic beta cells, and CD38 shows both ADP-ribosyl cyclase to synthesize cADPR from NAD+ and cADPR hydrolase to hydrolyze cADPR to ADP-ribose. We show here that 13.8% of Japanese non-insulin-dependent diabetes (NIDDM) patients examined have autoantibodies against CD38 and that the sera containing anti-CD38 autoantibodies inhibit the ADP-ribosyl cyclase activity of CD38 (P </= 0.05). Insulin secretion from pancreatic islets by glucose is significantly inhibited by the addition of the NIDDM sera with anti-CD38 antibodies (P </= 0.04-0.0001), and the inhibition of insulin secretion is abolished by the addition of recombinant CD38 (P </= 0.02). The increase of cADPR levels in pancreatic islets by glucose was also inhibited by the addition of the sera (P </= 0.05). These results strongly suggest that the presence of anti-CD38 autoantibodies in NIDDM patients can be one of the major causes of impaired glucose-induced insulin secretion in NIDDM.

A missense mutation in the CD38 gene, a novel factor for insulin secretion: association with Type II diabetes mellitus in Japanese subjects and evidence of abnormal function when expressed in vitro

Summary Cyclic adenosine 5′diphosphate-ribose (cADPR) is thought to have a second messenger role in insulin secretion through mobilisation of Ca2 +. As human lymphocyte antigen CD38 has both ADP-ribosyl cyclase and cADPR hydrolase activity, it may be important in glucose-induced insulin secretion in islets. Thirty one randomly selected Japanese patients with Type II diabetes mellitus who had first-degree and/or second-degree relative(s) with Type II diabetes mellitus were screened for mutations of this gene using single-stranded conformation polymorphism. Two variant patterns in exon 3 and exon 4 of the CD38 gene were identified. The variant in exon 3 resulted in an amino acid substitution from Arg140 (CGG) to Trp (TGG). The Arg140Trp mutation was observed in 4 of 31 patients, and allele frequencies were significantly different in patients and the control subjects (p = 0.004). One patient with this mutation has two missense mutations on beta cell/liver glucose transporter (GLUT2) gene; her mother, who has impaired glucose tolerance, also has this mutation on the CD38 gene and one missense mutation on the GLUT2 gene. Enzyme activity studies using COS-7 cells expressing the Arg140Trp mutation showed a reduction in ADP-ribosyl cyclase and cADPR hydrolase activity of around 50 %. The Arg140Trp mutation on CD38 thus appears to contribute to the development of Type II diabetes mellitus via the impairment of glucose-induced insulin secretion in the presence of other genetic defects. [Diabetologia (1998) 41: 1024–1028]

Lysine 129 of CD38 (ADP-ribosyl Cyclase/Cyclic ADP-ribose Hydrolase) Participates in the Binding of ATP to Inhibit the Cyclic ADP-ribose Hydrolase

CD38 catalyzes not only the formation of cyclic ADP-ribose (cADPR) from NAD+ but also the hydrolysis of cADPR to ADP-ribose (ADPR), and ATP inhibits the hydrolysis (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). In the present study, using purified recombinant CD38, we showed that the cADPR hydrolase activity of CD38 was inhibited by ATP in a competitive manner with cADPR. To identify the binding site for ATP and/or cADPR, we labeled the purified CD38 with FSBA. Sequence analysis of the lysylendopeptidase-digested fragment of the labeled CD38 indicated that the FSBA-labeled residue was Lys-129. We introduced site-directed mutations to change the Lys-129 of CD38 to Ala and to Arg. Neither mutant was labeled with FSBA nor catalyzed the hydrolysis of cADPR to ADPR. Furthermore, the mutants did not bind cADPR, whereas they still used NAD+ as a substrate to form cADPR and ADPR. These results indicate that Lys-129 of CD38 participates in cADPR binding and that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38.

Assignment of CD38, the gene encoding human leukocyte antigen CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase), to chromosome 4p15

CD38 has been used as a phenotype marker of lymphocyte differentiation. Recently, we have demonstrated that cyclic ADP-ribose can be synthesized and hydrolyzed by CD38 and acts as a second messenger in insulin secretion from pancreatic beta-cells. We have mapped the CD38 gene to human chromosome 4p15 by fluorescence in situ hybridization.

Disturbance of cerebellar synaptic maturation in mutant mice lacking BSRPs, a novel brain-specific receptor-like protein family.

By DNA cloning, we have identified the BSRP (brain‐specific receptor‐like proteins) family of three members in mammalian genomes. BSRPs were predominantly expressed in the soma and dendrites of neurons and localized in the endoplasmic reticulum (ER). Expression levels of BSRPs seemed to fluctuate greatly during postnatal cerebellar maturation. Triple‐knockout mice lacking BSRP members exhibited motor discoordination, and Purkinje cells (PCs) were often innervated by multiple climbing fibers with different neuronal origins in the mutant cerebellum. Moreover, the phosphorylation levels of protein kinase Cα (PKCα) were significantly downregulated in the mutant cerebellum. Because cerebellar maturation and plasticity require metabotropic glutamate receptor signaling and resulting PKC activation, BSRPs are likely involved in ER functions supporting PKCα activation in PCs.

Impaired cerebellar functions in mutant mice lacking DNER.

DNER is a transmembrane protein carrying extracellular EGF repeats and is strongly expressed in Purkinje cells (PCs) in the cerebellum. Current study indicated that DNER functions as a new Notch ligand and mediates the functional communication via cell-cell interaction. By producing and analyzing knockout mice lacking DNER, we demonstrate its essential roles in functional and morphological maturation of the cerebellum. The knockout mice exhibited motor discoordination in the fixed bar and rota-rod tests. The cerebellum from the knockout mice showed significant retardation in morphogenesis and persistent abnormality in fissure organization. Histochemical and electrophysiological analyses detected that PCs retained multiple innervations from climbing fibers (CFs) in the mutant cerebellum. Synaptic transmission from parallel fibers (PFs) or CFs to PCs was apparently normal, while glutamate clearance at the PF-PC synapses was significantly impaired in the mutant mice. Moreover, the protein level of GLAST, the glutamate transporter predominantly expressed in Bergmann glia (BG), was reduced in the mutant cerebellum. Our results indicate that DNER takes part in stimulation of BG maturation via intercellular communication and is essential for precise cerebellar development.

Structural determination and characterization of a 40 kDa protein isolated from rat 40 S ribosomal subunit

We have purified a 40 kDa protein from the rat 40 S ribosomal subunit and determined its primary structure by amino acid and cDNA sequencing. The amino acid sequence of the 40 kDa protein shared 29–37% homology with prokaryotic ribosomal protein S2 of eubacteria and chloroplasts, indicating that the protein is a eukaryotic counterpart to prokaryotic S2. Moreover, the amino acid sequence shared 99% identity with those deduced from cDNAs for 68 kDa laminin binding proteins of human, murine and bovine origins. The cDNAs are capable of encoding polypeptides with predicted molecular mass of 33,000 which lacked typical signal sequences, N‐linked glycosylation sites and putative transmembrane domains. These results indicate that the eDNAs for 68 kDa laminin binding proteins actually code for the 40 kDa ribosomal protein.