Identification and characterization of Veph, a novel gene encoding a PH domain-containing protein expressed in the developing central nervous system of vertebrates

We isolated Veph, a novel gene encoding a pleckstrin homology (PH) domain-containing protein from a mouse. Veph was strongly expressed in the embryonic brain, and its expression level gradually decreased in later stages. In situ hybridization analysis of sectioned embryo brains revealed that Veph was expressed exclusively in the ventricular zone. We then isolated a zebrafish orthologue of Veph (zVeph). As observed in the mouse gene, zVeph was expressed in the ventricular zone of developing brain and spinal cord. Blockage of zVeph expression by injection of zVeph-specific morpholino antisense oligo into zebrafish fertilized eggs resulted in a defect in the midbrain-hindbrain boundary and otic vesicle formation, suggesting the important function of zVeph in central nervous system (CNS) development. On the other hand, homozygous knockout mice of Veph showed no significant defect in the CNS, pointing to possible different functions of Veph between the zebrafish and mouse.     

Identification and molecular characterization of BP75, a novel bromodomain-containing protein.

We here describe the identification and characterization of a novel bromodomain-containing protein, the bromodomain protein of 75 kDa (BP75). Initially, we identified BP75 in a two-hybrid screening for proteins that interact with the first PDZ (acronym for post-synaptic density protein PSD-95, Drosophila discs large tumor suppressor DlgA and the tight junction protein ZO-1) domain in protein tyrosine phosphatase-BAS-like (PTP-BL). We found that BP75 is expressed ubiquitously and show that both BP75 and a PTP-BL deletion mutant consisting of the first PDZ domain are located mainly in the nucleus, although cytoplasmic localization is also evident. Full-length PTP-BL, on the contrary, is predominantly localized in the cytoplasm, although some basal nuclear staining is observed. The described molecular interaction may reflect a mechanism of coupling submembraneous signalling events and nuclear events.     

Identification, cloning and characterization of a novel nuclear protein, HA95, homologous to A-kinase anchoring protein 95

Previously, we have identified and characterized nuclear AKAP95 from man which targets cyclic AMP (cAMP)-dependent protein kinase (PKA)-type II to the condensed chromatin/spindle region at mitosis. Here we report the cloning of a novel nuclear protein with an apparent molecular mass of 95 kDa that is similar to AKAP95 and is designated HA95 (homologous to AKAP95). HA95 cDNA sequence encodes a protein of 646 amino acids that shows 61% homology to the deduced amino acid sequence of AKAP95. The HA95 gene is located on chromosome 19p13.1 immediately upstream of the AKAP95 gene. Both HA95 and AKAP95 genes contain 14 exons encoding similar regions of the respective proteins, indicating a previous gene duplication event as the origin of the two tandem genes. Despite their apparent similarity, HA95 does not bind RII in vitro. HA95 contains a putative nuclear localization signal in its N-terminal domain. It is localized exclusively into the nucleus as demonstrated in cells transfected with HA95 fused to either green fluorescence protein or the c-myc epitope. In the nucleus, the HA95 protein is found as complexes directly associated with each other or indirectly associated via other nuclear proteins. In interphase, HA95 is co-localized with AKAP95, but the two proteins are not biochemically associated. At metaphase, both proteins co-localize with condensed chromosomes. The similarity in sequence and localization of HA95 and AKAP95 suggests that the two molecules constitute a novel family of nuclear proteins that may exhibit related functions.     

Identification and characterization of a novel angiostatin-binding protein by the display cloning method

Angiostatin is a potent anti-angiogenic protein. To examine the angiostatin-interacting proteins, we used the display-cloning method with a T7 phage library presenting human cDNAs. The specific T7 phage clone that bound to the immobilized angiostatin was isolated, and a novel gene encoding the displayed polypeptide on the isolated T7 phage was identified. The displayed angiostatin-binding sequence was expressed in E. coli as a soluble protein and purified to homogeneity. This novel angiostatin-binding region interacted specifically to angiostatin with a dissociation constant of 3.4 x 10(-7) M. A sequence analysis showed that the identified sequence was a part of the large ORF of 1,998 amino acids, whose function has not yet been characterized. A Northern analysis indicated that the gene containing the angiostatin-binding sequence was expressed differentially in the developmental stages or cell types.     

Full sequence of neurocalcin, a novel calcium-binding protein abundant in central nervous system.

We determined the cDNA sequence for neurocalcin, a novel calcium-binding protein in bovine brain. This clone (pCalN) has 582 nucleotides in the open reading frame including the termination codon TGA, 11 nucleotides of the 5' leader and 1251 nucleotides of the 3' noncoding region. The deduced amino acid sequence revealed that neurocalcin is composed of 193 amino acids, has a molecular mass of 22,284 daltons, and contains three putative calcium-binding sites (EF-hand motifs). By Northern blot analysis, 3.8kbp mRNA was detected in brain. The deduced amino acid sequence had a strong homology to visinin (46.5%) and recoverin (51.6%) in retina, suggesting that neurocalcin may play a visinin- or recoverin-like role in brain.     

cDNA cloning and characterization of a novel nucleolar protein.

In an initial study of anti-nuclear antibodies in the chronic inflammatory bladder disease interstitial cystitis, we reported that 7% of interstitial cystitis patients studied had autoantibodies to the nucleolus. We now report that, using an autoimmune serum from a patient with interstitial cystitis, we have identified and partially characterized a novel protein with an M(r) of approximately 55 kDa (hereafter referred to as No55) localized to the granular component of the nucleolus. No55 was initially characterized by diffuse nucleolar immunofluorescence staining in interphase cells and by Western blotting as a 55-kDa doublet on whole-cell extracts. During mitosis, No55 was associated with chromosomes and appeared in prenucleolar bodies during telophase, but it did not colocalize with p80-coilin in coiled bodies. Immunoelectron microscopy revealed that No55 was localized uniformly throughout the granular component of the nucleolus compared with a more peripheral localization of nucleolar granular component protein B23. On segregation of the nucleolus with actinomycin D, No55 remained with the granular component of the segregated nucleolus, whereas protein B23 was found predominantly in the nucleoplasm. Finally, a cDNA expression library was screened with the human autoantibody against No55, and a 2.4-kb insert was isolated, subcloned to homogeneity, and then sequenced. Analysis of this sequence showed an open reading frame of approximately 1.3 kb coding for 437 amino acids with a predicted molecular weight of 50 kDa. A search of the gene sequence database indicated homology with SC65, a rat synaptonemal complex protein. Therefore, on the basis of molecular weight, nucleolar sublocalization, response to actinomycin D, and cDNA sequence determination, No55 is a novel protein of the interphase nucleolus.     

Purification, characterization and molecular cloning of TGP1, a novel G-DNA binding protein from Tetrahymena thermophila.

G-DNA, a polymorphic family of four-stranded DNA structures, has been proposed to play roles in a variety of biological processes including telomere function, meiotic recombination and gene regulation. Here we report the purification and cloning of TGP1, a G-DNA specific binding protein from Tetrahymena thermophila. TGP1 was purified by three-column chromatographies, including a G-DNA affinity column. Two major proteins (approximately 80 and approximately 40 kDa) were present in the most highly purified column fraction. Renaturation experiments showed that the approximately 80 kDa protein contains TGP1 activity. Biochemical characterization showed that TGP1 is a G-DNA specific binding protein with a preference for parallel G-DNAs. The TGP1/DNA complex has a dissociation constant (Kd) of approximately 2.2 x 10(-8) M and TGP1 can form supershift in gel mobility shift assays. The cDNA coding TGP1 was cloned and sequenced based upon an internal peptide sequence obtained from the approximately 80 kDa protein. Sequence analyses showed that TGP1 is a basic protein with a pI of 10.58, and contains two extensively hydrophilic and basic domains. Homology searches revealed that TGP1 is a novel protein sharing weak similarities with a number of proteins.     

Isolation and characterization of a basic encephalitogenic protein from the central nervous system of sheep.

A basic protein has been purified from sheep brain. The purified protein sedimented in the analytical centrifuge at 56,000 r.p.m. as an homogenous product. This protein induced an allergic encephalitis when injected into guinea pigs. Some physiochemical properties of the protein were studied: the sedimentation coefficient was 1.52 and the molecular weight was 20,000 +/- 2,000, as estimated by electrophoresis in acrylamide gels containing SDS and urea; the specific extinction coefficient (see article) was 6.01 +/- 0.20. The aminoacid composition of the molecule was determined and its most prominent aspects are a high content of arginine and lysine, the presence of a single tryptophan, the total absence of cysteine and cystine and a blocked N-terminal residue. All these properties are very close to those of human and bovine encephalitogenic proteins.     

Identification of a novel glucose transporter-like protein-GLUT-12

Facilitative glucose transporters exhibit variable hexose affinity and tissue-specific expression. These characteristics contribute to specialized metabolic properties of cells. Here we describe the characterization of a novel glucose transporter-like molecule, GLUT-12. GLUT-12 was identified in MCF-7 breast cancer cells by homology to the insulin-regulatable glucose transporter GLUT-4. The GLUT-12 cDNA encodes 617 amino acids, which possess features essential for sugar transport. Di-leucine motifs are present in NH(2) and COOH termini at positions similar to the GLUT-4 FQQI and LL targeting motifs. GLUT-12 exhibits 29% amino acid identity with GLUT-4 and 40% to the recently described GLUT-10. Like GLUT-10, a large extracellular domain is predicted between transmembrane domains 9 and 10. Genomic organization of GLUT-12 is highly conserved with GLUT-10 but distinct from GLUTs 1-5. Immunofluorescence showed that, in the absence of insulin, GLUT-12 is localized to the perinuclear region in MCF-7 cells. Immunoblotting demonstrated GLUT-12 expression in skeletal muscle, adipose tissue, and small intestine. Thus GLUT-12 is potentially part of a second insulin-responsive glucose transport system.     

Identification of a novel human adenylate kinase. cDNA cloning, expression analysis, chromosome localization and characterization of the recombinant protein.

Adenylate kinases have an important role in the synthesis of adenine nucleotides that are required for cellular metabolism. We report the cDNA cloning of a novel 22-kDa human enzyme that is sequence related to the human adenylate kinases and to UMP/CMP kinase of several species. The enzyme was expressed in Escherichia coli and shown to catalyse phosphorylation of AMP and dAMP with ATP as phosphate donor. When GTP was used as phosphate donor, the enzyme phosphorylated AMP, CMP, and to a small extent dCMP. Expression as a fusion protein with the green fluorescent protein showed that the enzyme is located in the cytosol. Northern blot analysis with mRNA from eight different human tissues demonstrated that the enzyme was expressed exclusively in brain, with two mRNA isoforms of 2.4 and 4.0 kb. The gene that encoded the enzyme was localized to chromosome 1p31. Based on the substrate specificity and the sequence similarity with the previously identified human adenylate kinases, we have named this novel enzyme adenylate kinase 5.     

Identification of a novel neuromedin U receptor subtype expressed in the central nervous system

Neuromedin U is a neuropeptide prominently expressed in the upper gastrointestinal tract and central nervous system. Recently, GPR66/FM-3 (NmU-R1) was identified as a specific receptor for neuromedin U. A BLAST search of the GenBank(TM) genomic database using the NmU-R1 cDNA sequence revealed a human genomic fragment encoding a G protein-coupled receptor that we designated NmU-R2 based on its homology to NmU-R1. The full-length NmU-R2 cDNA was subsequently cloned, stably expressed in 293 cells, and shown to mobilize intracellular calcium in response to neuromedin U. This response was dose-dependent (EC(50) = 5 nm) and specific in that other neuromedins did not induce a calcium flux in receptor-transfected cells. Expression analysis of human NmU-R2 demonstrated its mRNA to be most highly expressed in central nervous system tissues. Based on these data, we conclude that NmU-R2 is a novel neuromedin U receptor subtype that is likely to mediate central nervous system-specific neuromedin U effects.     

Identification of a novel estrogen-sulfating cytosolic SULT from zebrafish: molecular cloning, expression, characterization, and ontogeny study

By searching the expressed sequence tag database, a zebrafish cDNA encoding a putative cytosolic sulfotransferase (SULT) was identified. Sequence analysis indicated that this zebrafish SULT belongs to the SULT1 cytosolic SULT gene family. The recombinant form of this novel zebrafish SULT, expressed using the pGEX-2TK expression system and purified from transformed BL21 (DE3) Escherichia coli cells, displayed sulfating activities specifically for estrone and 17beta-estradiol among various endogenous compounds tested as substrates. The enzyme also exhibited sulfating activities toward some xenobiotic phenolic compounds. This new zebrafish SULT showed dual pH optima, at 6.5 and 10-10.5, with estrone or n-propyl gallate as substrate. Kinetic constants of the sulfation of estrone, 17beta-estradiol, and n-propyl gallate were determined. Developmental stage-dependent expression experiments revealed a significant level of expression of this novel zebrafish estrogen-sulfating SULT at the beginning of the hatching period during embryogenesis, which continued throughout the larval stage onto maturity.     

Expression of a novel human ornithine decarboxylase-like protein in the central nervous system and testes

Ornithine decarboxylase (ODC) is the key enzyme of polyamine synthesis. The physiological activity of ODC is associated with cell proliferation, and high ODC activities are encountered in rapidly growing cancer cells. We have cloned a cDNA for a novel human protein that is 54% identical to ODC and 45% identical to antizyme inhibitor (AZI). mRNA for ODC-paralogue (ODC-p) was found only in the central nervous system and testes, suggesting a role in terminal differentiation rather than cell proliferation. ODC-p occurs at least in eight alternatively spliced forms. In vitro translated ODC-p did not decarboxylate ornithine, whereas, in vivo, one splice variant exerted modest ODC-like activity upon expression in COS-7 cells. ODC-p has a unique mutation in cysteine 360, where this ornithine decarboxylase reaction-directing residue is substituted by a valine. This substitution might lead to an enzymatic reaction that differs from typical ODC activity. ODC-p might also function as a brain- and testis-specific AZI.