Fumio Yoshikawa

Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand

In a variety of cells, the Ca2+ signalling process is mediated by the endoplasmic-reticulum-membrane-associated Ca2+ release channel, inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Being ubiquitous and present in organisms ranging from humans to Caenorhabditis elegans, InsP3R has a vital role in the control of cellular and physiological processes as diverse as cell division, cell proliferation, apoptosis, fertilization, development, behaviour, memory and learning. Mouse type I InsP3R (InsP3R1), found in high abundance in cerebellar Purkinje cells, is a polypeptide with three major functionally distinct regions: the amino-terminal InsP3-binding region, the central modulatory region and the carboxy-terminal channel region. Here we present a 2.2-Å crystal structure of the InsP3-binding core of mouse InsP3R1 in complex with InsP3. The asymmetric, boomerang-like structure consists of an N-terminal β-trefoil domain and a C-terminal α-helical domain containing an ‘armadillo repeat’-like fold. The cleft formed by the two domains exposes a cluster of arginine and lysine residues that coordinate the three phosphoryl groups of InsP3. Putative Ca2+-binding sites are identified in two separate locations within the InsP3-binding core.

Mutational Analysis of the Ligand Binding Site of the Inositol 1,4,5-Trisphosphate Receptor

To define the structural determinants for inositol 1,4,5-trisphosphate (IP3) binding of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), we developed a means of expressing the N-terminal 734 amino acids of IP3R1 (T734), which contain the IP3 binding region, in Escherichia coli. The T734 protein expressed in E. coli exhibited a similar binding specificity and affinity for IP3 as the native IP3R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduced IP3 binding activity. However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity was retrieved. Further N-terminal deletions over the first 228 amino acids completely abolished it again. C-terminal deletions up to residue 579 did not affect the binding activity, whereas those up to residue 568 completely abolished it. In addition, the expressed 356-amino acid polypeptide (residues 224-579) exhibited specific binding activity. Taken together, residues 226-578 were sufficient and close enough to the minimum region for the specific IP3 binding, and thus formed an IP3 binding “core.” Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734. We showed that single amino acid substitutions for 10 residues, which were widely distributed within the binding core and conserved among all members of the IP3R family, significantly reduced the binding activity. Among them, three (Arg-265, Lys-508, and Arg-511) were critical for the specific binding, and Arg-568 was implicated in the binding specificity for various inositol phosphates. We suggest that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of IP3.

Trypsinized Cerebellar Inositol 1,4,5-Trisphosphate Receptor STRUCTURAL AND FUNCTIONAL COUPLING OF CLEAVED LIGAND BINDING AND CHANNEL DOMAINS

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is a tetrameric intracellular inositol 1,4,5-trisphosphate (IP3)-gated Ca2+release channel (calculated molecular mass = ∼313 kDa/subunit). We studied structural and functional coupling in this protein complex by limited (controlled) trypsinization of membrane fractions from mouse cerebellum, the predominant site for IP3R1. Mouse IP3R1 (mIP3R1) was trypsinized into five major fragments (I–V) that were positioned on the entire mIP3R1 sequence by immuno-probing with 11 site-specific antibodies and by micro-sequencing of the N termini. Four fragments I–IV were derived from the N-terminal cytoplasmic region where the IP3-binding region extended over two fragments I (40/37 kDa) and II (64 kDa). The C-terminal fragment V (91 kDa) included the membrane-spanning channel region. All five fragments were pelleted by centrifugation as were membrane proteins. Furthermore, after solubilizing with 1% Triton X-100, all were co-immunoprecipitated with the C terminus-specific monoclonal antibody that recognized only the fragment V. These data suggested that the native mIP3R1-channel is an assembly of four subunits, each of which is constituted by non-covalent interactions of five major, well folded structural components I–V that are not susceptible to attack by mild trypsinolysis. Ca2+ release experiments further revealed that even the completely fragmented mIP3R1 retained significant IP3-induced Ca2+ release activity. These data suggest that structural coupling among five split components conducts functional coupling for IP3-induced Ca2+ release, despite the loss of peptide linkages. We propose structural-functional coupling in the mIP3R1, that is neighboring coupling between components I and II for IP3binding and long-distant coupling between the IP3 binding region and the channel region (component V) beyond trypsinized gaps for ligand gating.

Cooperative Formation of the Ligand-binding Site of the Inositol 1,4,5-Trisphosphate Receptor by Two Separable Domains

Limited trypsin digestion of mouse cerebellar membrane fractions leads to fragmentation of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) into five major components (Yoshikawa, F., Iwasaki, H., Michikawa, T., Furuichi, T., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 316–327). Here we report that trypsin-fragmented mouse IP3R1 (mIP3R1) retains significant inositol 1,4,5-trisphosphate (IP3) binding activity that is comparable to the intact receptor in affinity, capacity, and specificity. This is despite the fact that the IP3-binding core (residues 226–578), which is close to the minimum for high affinity binding, is completely split into two tryptic fragments at the Arg-343 and/or Arg-345, around the center of the core. Furthermore, we have examined whether binding activity could be complemented in vitro by mixing two distinct glutathioneS-transferase (GST) fusion proteins, which were respectively composed of residues 1–343 and 341–604, almost corresponding to two split binding components, and separately expressed in Escherichia coli. The GST-fused residues 1–343 (GN) showed no binding affinity for IP3, whereas the GST-fused residues 341–604 (GC) displayed weak but definite activity with an affinity >100-fold lower than that of the native receptor. Upon mixing of both GN and GC, a high affinity site comparable to the native site appeared. We suggest that the IP3-binding pocket consists of two non-covalently but tightly associated structural domains each of which has a discrete function: the C-terminal domain alone has low affinity for IP3, whereas the N-terminal one alone is incapable of binding but is capable of potentiating binding affinity.

Opalin, a Transmembrane Sialylglycoprotein Located in the Central Nervous System Myelin Paranodal Loop Membrane

In contrast to compact myelin, the series of paranodal loops located in the outermost lateral region of myelin is non-compact; the intracellular space is filled by a continuous channel of cytoplasm, the extracellular surfaces between neighboring loops keep a definite distance, but the loop membranes have junctional specializations. Although the proteins that form compact myelin have been well studied, the protein components of paranodal loop membranes are not fully understood. This report describes the biochemical characterization and expression of Opalin as a novel membrane protein in paranodal loops. Mouse Opalin is composed of a short N-terminal extracellular domain (amino acid residues 1–30), a transmembrane domain (residues 31–53), and a long C-terminal intracellular domain (residues 54–143). Opalin is enriched in myelin of the central nervous system, but not that of the peripheral nervous system of mice. Enzymatic deglycosylation showed that myelin Opalin contained N- and O-glycans, and that the O-glycans, at least, had negatively charged sialic acids. We identified two N-glycan sites at Asn-6 and Asn-12 and an O-glycan site at Thr-14 in the extracellular domain. Site-directed mutations at the glycan sites impaired the cell surface localization of Opalin. In addition to the somata and processes of oligodendrocytes, Opalin immunoreactivity was observed in myelinated axons in a spiral fashion, and was concentrated in the paranodal loop region. Immunogold electron microscopy demonstrated that Opalin was localized at particular sites in the paranodal loop membrane. These results suggest a role for highly sialylglycosylated Opalin in an intermembranous function of the myelin paranodal loops in the central nervous system.

Phospholipase D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia.

Background Phospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)4-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4. Methodology/Principal Findings PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity. Conclusions/Significance Results showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells.

Systematizing and Cloning of Genes Involved in the Cerebellar Cortex Circuit Development

The cerebellar cortical circuit of mammals develops via a series of magnificent cellular events in the postnatal stage of development to accomplish the formation of functional circuit architectures. The contribution of genetic factors is thought to be crucial to cerebellar development. Therefore, it is essential to analyze the underlying transcriptome during development to understand the genetic blueprint of the cerebellar cortical circuit. In this review, we introduce the profiling of large numbers of spatiotemporal gene expression data obtained by developmental time-series microarray analyses and in situ hybridization cellular mRNA mapping, and the creation of a neuroinformatics database called the Cerebellar Development Transcriptome Database. Using this database, we have identified thousands of genes that are classified into various functional categories and are expressed coincidently with related cellular developmental stages. We have also suggested the molecular mechanisms of cerebellar development by functional characterization of several identified genes (Cupidin, p130Cas, very-KIND, CAPS2) responsible for distinct cellular events of developing cerebellar granule cells. Taken together, the gene expression profiling during the cerebellar development demonstrates that the development of cerebellar cortical circuit is attributed to the complex but orchestrated transcriptome.

Deciphering the genetic blueprint of cerebellar development by the gene expression profiling informatics

The brain is the ultimate genetic system to which a large number of genes are devoted. To extract and visualize biological information from such large data sets accumulated in the post-sequencing era, the use of bioinformatics would be a very powerful means. To understand the genetic basis of mouse cerebellar postnatal development, we have analyzed the whole transcription or gene expression (transcriptome) during the developmental stages on a genome-wide basis, and have systematized the spatio-temporal gene expression profile information in a comprehensive database (Cerebellar Development Transcriptome [CDT] database) from a bioinformatics point of view. This CDT database would open up a new field for deciphering the genetic blueprint for cerebellar development.

Age-dependent redistribution and hypersialylation of the central myelin paranodal loop membrane protein Opalin in the mouse brain

Opalin/Tmem10 is a myelin-associated sialylglycoprotein that is specific to only the mammalian central nervous system. However, little is known about the properties or function of this protein. Here, we analyzed the expression and glycosylation patterns of Opalin in the postnatal mouse brain. Immunolocalization patterns of Opalin were similar to those of myelin basic protein in juvenile and adolescent mice. On the other hand, in the adult mouse brain, decreasing immunoreactivity for Opalin was observed in the hindbrain region especially in the cerebellar white matter compared with the corpus callosum in the forebrain. In addition, Opalin showed increasing molecular size with mouse aging. This age-dependent increase in Opalin molecular weight was mainly owing to hypersialylation of O-glycans. These results indicate that the regional redistribution and the degree of sialylation of Opalin protein are age-dependently regulated in mouse brains.

Mammalian-Specific Central Myelin Protein Opalin Is Redundant for Normal Myelination: Structural and Behavioral Assessments.

Opalin, a central nervous system-specific myelin protein phylogenetically unique to mammals, has been suggested to play a role in mammalian-specific myelin. To elucidate the role of Opalin in mammalian myelin, we disrupted the Opalin gene in mice and analyzed the impacts on myelination and behavior. Opalin-knockout (Opalin−/−) mice were born at a Mendelian ratio and had a normal body shape and weight. Interestingly, Opalin−/− mice had no obvious abnormalities in major myelin protein compositions, expression of oligodendrocyte lineage markers, or domain organization of myelinated axons compared with WT mice (Opalin+/+) mice. Electron microscopic observation of the optic nerves did not reveal obvious differences between Opalin+/+ and Opalin−/− mice in terms of fine structures of paranodal loops, transverse bands, and multi-lamellae of myelinated axons. Moreover, sensory reflex, circadian rhythm, and locomotor activity in the home cage, as well as depression-like behavior, in the Opalin−/− mice were indistinguishable from the Opalin+/+ mice. Nevertheless, a subtle but significant impact on exploratory activity became apparent in Opalin−/− mice exposed to a novel environment. These results suggest that Opalin is not critical for central nervous system myelination or basic sensory and motor activities under conventional breeding conditions, although it might be required for fine-tuning of exploratory behavior.