Masaharu Noda

In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kDa tectal protein related to ligands for Eph receptor tyrosine kinases

The results of previous in vitro experiments indicate that a glycosylphosphatidylinositol (GPI)-anchored protein may play an important role in the guidance of temporal retinal axons during the formation of the topographically ordered retinotectal projection. We have purified and cloned a GPI-anchored, 25 kDa glycoprotein that is a good candidate for a molecule involved in this process. During the time of innervation by retinal ganglion cells, this protein is gradedly expressed in the posterior part of the developing tectum. In two different in vitro assay systems, the recombinant protein induces growth cone collapse and repulsion of retinal ganglion cell axons. These phenomena are observed for axons of temporal as well as nasal origin, indicating that an additional activity may be necessary to confer the nasotemporal specificity observed in previous assays. We named the protein RAGS (for repulsive axon guidance signal). The sequence of RAGS shows significant homology to recently identified ligands for receptor tyrosine kinases of the Eph subfamily.

Structural homology of Torpedo californica acetylcholine receptor subunits

The nicotinic acetylcholine receptor (AChR) from the electroplax of the ray Torpedo californica is composed of five subunits present in a molar stoichiometry of α2βγδ (refs 1–3) and contains both the binding site for the neurotransmitter and the cation gating unit (reviewed in refs 4–6). We have recently elucidated the complete primary structures of the α-, β- and δ-subunit precursors of the T. californica AChR by cloning and sequencing cDNAs for these polypeptides7,8. Here, we report the whole primary structure of the γ-subunit precursor of the AChR deduced from the nucleotide sequence of the cloned cDNA. Comparison of the amino acid sequences of the four subunits reveals marked homology among them. The close resemblance among the hydrophilicity profiles and predicted secondary structures of all the subunits suggests that these polypeptides are oriented in a pseudosymmetric fashion across the membrane. Each subunit contains four putative transmembrane segments that may be involved in the ionic channel. The transmembrane topology of the subunit molecules has also been inferred.

Isolation and structural organization of the human preproenkephalin B gene.

Recently, we have elucidated the primary structure of bovine adrenal preproenkephalin by determining the nucleotide sequence of cloned DNA complementary to its mRNA1. The structure of most of this precursor molecule has also been deduced by Gubler et al.2 using cDNA sequencing in conjunction with protein sequencing. Bovine preproenkephalin contains four copies of methionine-enkephalin3 (Met-enkephalin) and one copy each of leucine-enkephalin3 (Leu-enkephalin), Met-enkephalin-Arg6-Phe7 (ref. 4) and Met-enkephalin-Arg6-Gly7-Leu8 (refs 1, 2, 5). The region containing the repeated enkephalin and extended enkephalin sequences, which are each bounded by paired basic amino acid residues, is connected with a cysteine-containing amino-terminal sequence preceded by a signal peptide6. We have now studied the relationship between the repetitive structure of preproenkephalin and the structural organization of its gene by cloning a human genomic DNA segment containing the entire gene. We find that the general organization of the preproenkephalin gene is strikingly similar to that of the gene encoding the common precursor of corticotropin (ACTH) and β-lipotropin (β-LPH)7–9 (alternatively designated preproopiomelanocortin), another multi-hormone precursor. Furthermore, the complete mRNA and amino acid sequences of human preproenkephalin have been deduced from the corresponding gene sequence.

A missense mutation of the Na+ channel αII subunit gene Nav1.2 in a patient with febrile and afebrile seizures causes channel dysfunction

Generalized epilepsy with febrile seizures plus (GEFS+), a clinical subset of febrile seizures (FS), is characterized by frequent episodes beyond 6 years of age (FS+) and various types of subsequent epilepsy. Mutations in β1 and αI-subunit genes of voltage-gated Na+ channels have been associated with GEFS+1 and 2, respectively. Here, we report a mutation resulting in an amino acid exchange (R187W) in the gene encoding the α-subunit of neuronal voltage-gated Na+ channel type II (Nav1.2) in a patient with FS associated with afebrile seizures. The mutation R187W occurring on Arg187, a highly conserved residue among voltage-gated Na+ channels, was not found in 224 alleles of unaffected individuals. Whole-cell patch clamp recordings on human embryonic kidney (HEK) cells expressing a rat wild-type (rNav1.2) and the corresponding mutant channels showed that the mutant channel inactivated more slowly than wild-type whereas the Na+ channel conductance was not affected. Prolonged residence in the open state of the R187W mutant channel may augment Na+ influx and thereby underlie the neuronal hyperexcitability that induces seizure activity. Even though a small pedigree could not show clear cosegregation with the disease phenotype, these findings strongly suggest the involvement of Nav1.2 in a human disease and propose the R187W mutation as the genetic defect responsible for febrile seizures associated with afebrile seizures.

Differential regulation of three sodium channel messenger RNAs in the rat central nervous system during development.

The levels of the mRNAs encoding sodium channels I, II and III in various regions of the developing rat central nervous system (from embryonal day 10 to postnatal day 90) have been examined by blot hybridization analysis with specific probes. The three sodium channel mRNAs exhibit different temporal and regional expression patterns. The expression of sodium channel I mRNA rises after a lag phase to adult levels during the second and third postnatal weeks with stronger increases in caudal regions of the brain and in spinal cord. Sodium channel II mRNA increases steadily until the first postnatal week, keeping high adult levels in rostral regions of the brain or reaching low adult levels after the second postnatal week in most caudal regions of the brain and in spinal cord; cerebellum shows low levels during the first two postnatal weeks but high adult levels. In all regions, sodium channel III mRNA attains maximum levels around birth and decreases during the first and second postnatal weeks to reach variable low adult levels. These results suggest that sodium channel III is expressed predominantly at fetal and early postnatal stages and sodium channel I predominantly at late postnatal stages, whereas sodium channel II is expressed throughout the developmental stages studied with greater regional variability.

Primary structure of rat brain sodium channel III deduced from the cDNA sequence

The complete amino acid sequence of a third sodium channel (designated sodium channel III) from rat brain has been deduced by cloning and sequence analysis of the cDNA. This protein is homologous in amino acid sequence and shares characteristic structural features with other sodium channels.

A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II

A single point mutation of the rat sodium channel II reduces its sensitivity to tetrodotoxin and saxitoxin by more than three orders of magnitude. The mutation replaces glutamic acid 387 with a glutamine and has only slight effects on the macroscopic current properties, as measured under voltage‐clamp in Xenopus oocytes injected with the corresponding cDNA‐derived mRNA.

A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta.

Midkine is a 13-kDa heparin-binding growth factor with 45% sequence identity to pleiotrophin. Pleiotrophin has been demonstrated to bind to protein-tyrosine phosphatase ζ (PTPζ) with high affinity. In this study, we examined the binding of midkine to PTPζ by solid-phase binding assay. Midkine and pleiotrophin binding to PTPζ were equally inhibited by soluble pleiotrophin and also by some specific glycosaminoglycans. For both bindings, Scatchard analysis revealed low (3.0 nm) and high (0.58 nm) affinity binding sites. These results suggested that PTPζ is a common receptor for midkine and pleiotrophin. Midkine is structurally divided into the N- and C-terminal halves, and the latter exhibited full activity for PTPζ binding and neuronal migration induction. The C-terminal half contains two heparin-binding sites consisting of clusters of basic amino acids, Clusters I and II. A mutation at Arg78 in Cluster I resulted in loss of the high affinity binding and reduced neuronal migration-inducing activity, while mutations at Lys83 and Lys84 in Cluster II showed almost no effect on either activity. Chondroitinase ABC-treated PTPζ exhibited similar low affinity binding both to the native midkine and midkine mutants at Arg78. These results suggested that Arg78 in midkine plays an essential role in high affinity binding to PTPζ by interacting with the chondroitin sulfate portion of this receptor.

Mice deficient in protein tyrosine phosphatase receptor type Z are resistant to gastric ulcer induction by VacA of Helicobacter pylori

The vacuolating cytotoxin VacA produced by Helicobacter pylori causes massive cellular vacuolation in vitro and gastric tissue damage in vivo, leading to gastric ulcers, when administered intragastrically. Here we report that mice deficient in protein tyrosine phosphatase receptor type Z (Ptprz, also called PTP-ζ or RPTP-β, encoded by Ptprz) do not show mucosal damage by VacA, although VacA is incorporated into the gastric epithelial cells to the same extent as in wild-type mice. Primary cultures of gastric epithelial cells from Ptprz+/+ and Ptprz−/− mice also showed similar incorporation of VacA, cellular vacuolation and reduction in cellular proliferation, but only Ptprz+/+ cells showed marked detachment from a reconstituted basement membrane 24 h after treatment with VacA. VacA bound to Ptprz, and the levels of tyrosine phosphorylation of the G protein–coupled receptor kinase–interactor 1 (Git1), a Ptprz substrate, were higher after treatment with VacA, indicating that VacA behaves as a ligand for Ptprz. Furthermore, pleiotrophin (PTN), an endogenous ligand of Ptprz, also induced gastritis specifically in Ptprz+/+ mice when administered orally. Taken together, these data indicate that erroneous Ptprz signaling induces gastric ulcers.

Cloning, sequencing and expression of cDNA for a novel subunit of acetylcholine receptor from calf muscle

The nicotinic acetylcholine receptor (AChR) from fish electric organ has a subunit structure of α2βγδ, and this is thought to be also the case for the mammalian skeletal muscle AChR1–3. By cloning and sequencing the complementary or genomic DNAs, we have previously elucidated the primary structures of all four sub-units of the Torpedo californica electroplax4–6 and calf muscle AChR7–10 and of the α- and γ-subunits of the human muscle AChR7,11; the primary structures of the γ-subunit of the T. californien AChR12 and the α-subunit of the Torpedo marmorata AChR13,14 have also been deduced elsewhere. We have now cloned DNA complementary to the calf muscle messenger RNA encoding a novel polypeptide (the ε-subunit) whose deduced amino-acid sequence has features characteristic of the AChR subunits and which shows higher sequence homology with the γ-subunit than with the other subunits. cDNA expression studies indicate that the calf ε-subunit, as well as the calf γ-subunit, can replace the Torpedo γ-subunit to form the functional receptor in combination with the Torpedo α-, β- and δ-subunits.