Altering the substrate chain-length specificity of an alpha-glucosidase

Dextran glucosidases show high sequence identity (50%) to Bacillus sp. SAM1606 alpha-glucosidase, which is more specific for short-chain substrates. Sequence comparison of these enzymes as well as molecular modeling studies predicted that the extension of loop 4 of the (beta/alpha)(8)-barrel fold may be responsible for the narrower specificity of SAM1606 alpha-glucosidase with respect to substrate chain length. Indeed, deletion mutants of SAM1606 alpha-glucosidase that lack this extension showed higher relative activities toward dextran and long-chain isomaltooligosaccharides. Kinetic and thermodynamic analyses of oligosaccharide hydrolysis catalyzed by SAM1606 alpha-glucosidase and its deletion mutants suggested that the loss of such extension(s) in loop 4 should energetically destabilize the Michaelis complexes with long-chain substrates to result in smaller differences between the activation free energies for the enzymatic hydrolyses of isomaltoheptaose and isomaltose than those observed for the wild-type enzyme. This is the reason that dextran glucosidase, whose loop 4 is shorter in length, shows broader substrate chain-length specificity than does SAM1606 alpha-glucosidase.     

EXT gene family member rib-2 is essential for embryonic development and heparan sulfate biosynthesis in Caenorhabditis elegans

EXT gene family members including EXT1, EXT2, and EXTL2 are glycosyltransferases required for heparan sulfate biosynthesis. To examine the biological functions of rib-2, a member of the Caenorhabditis elegans EXT gene family, we generated a mutant worm lacking the rib-2 gene using the UV-TMP method followed by sib-selection. Inactivation of rib-2 alleles induced developmental abnormalities in F2 and F3 homozygous worms, while F1 heterozygotes showed a normal morphology. The F2 homozygous progeny generated from the F1 heterozygous hermaphrodites somehow developed to adult stage but exhibited abnormal characteristics such as developmental delay and egg-laying defects. The F3 homozygous progeny from the F2 homozygous hermaphrodites showed early developmental defects and most of the F3 worms stopped developing during the gastrulation stage. Whole-mount staining analysis for heparan sulfate using Toluidine blue (pH 2.5) revealed a defect of heparan sulfate biosynthesis in the F2 homozygotes. The analysis using fluorometric post-column high-performance liquid chromatography also uncovered reduced production of heparan sulfate in the rib-2 mutant. These results indicate that rib-2 is essential for embryonic development and heparan sulfate biosynthesis in C. elegans.     

Hippocampal cytochrome P450s synthesize brain neurosteroids which are paracrine neuromodulators of synaptic signal transduction

Hippocampal pyramidal neurons and granule neurons of adult male rats are equipped with a complete machinery for the synthesis of pregnenolone, dehydroepiandrosterone, 17beta-estradiol and testosterone as well as their sulfate esters. These brain neurosteroids are synthesized by cytochrome P450s (P450scc, P45017alpha and P450arom) from endogenous cholesterol. Synthesis is acutely dependent on the Ca(2+) influx attendant upon neuron-neuron communication via N-methyl-D-aspartate (NMDA) receptors. Pregnenolone sulfate, estradiol and corticosterone rapidly modulate neuronal signal transduction and the induction of long-term potentiation via NMDA receptors and putative membrane steroid receptors. Brain neurosteroids are therefore promising neuromodulators that may either activate or inactivate neuron-neuron communication, thereby mediating learning and memory in the hippocampus.     

Parkin cleaves intracellular alpha-synuclein inclusions via the activation of calpain

Mutations in the alpha-synuclein and parkin genes cause heritable forms of Parkinson's disease. In the present study, we examined the possible functional relationship between the parkin and alpha-synuclein genes in a conditionally immortalized embryonic hippocampal cell (H19-7) line. Whereas transient transfection of alpha-synuclein into neuronal H19-7 cells caused the formation of its intracytoplasmic inclusions and a significant cell death, the combined overexpression of parkin restored the alpha-synuclein-induced decrease in cell viability to control levels. In addition, the overexpression of parkin was found to generate selective cleavage of alpha-synuclein. Furthermore, the cytoprotective effect of parkin on alpha-synuclein-induced cell death was not inhibited in the presence of a proteasome inhibitor. Interestingly, the overexpression of parkin induced the activation of an intracellular cysteine protease, calpain, but not caspase, and the cytoprotective effect of parkin on alpha-synuclein cytotoxicity was significantly inhibited by the presence of calpain-specific inhibitors. In conclusion, our results suggest that parkin accelerates the degradation of alpha-synuclein via the activation of the nonproteasomal protease, calpain, leading to the prevention of alpha-synuclein-induced cell death in embryonic hippocampal progenitor cells.     

Cloning and characterization of a new human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase,designated pp-GalNAc-T13, that is specifically expressed in neurons and synthesizes GalNAc alpha-serine/threonine antigen

To date, 10 members of the UDP-N-acetyl-alpha-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (pp-GalNAc-T) family have been cloned and analyzed in human. In this study, we cloned and analyzed a novel human pp-GalNAc-T from an NT2 cell cDNA library, and we named it pp-GalNAc-T13. In amino acid sequences, pp-GalNAc-T13 was highly homologous, showing 84.3% identity, to pp-GalNAc-T1. Real time PCR analysis revealed pp-GalNAc-T13 to be highly and restrictively expressed in the brain and present at very low or undetectable levels in other tissues, in contrast to the ubiquitous expression of pp-GalNAc-T1. pp-GalNAc-T13 was abundantly expressed in all neuroblastoma cells examined and primary cultured neurons but not in glioblastoma cells and primary cultured astrocytes. pp-GalNAc-T13 exhibited much stronger activity to transfer GalNAc to mucin peptides, such as Muc5Ac and MUC7, than did pp-GalNAc-T1. In addition, pp-GalNAc-T13 differed in substrate specificity to pp-GalNAc-T1. pp-GalNAc-T13 was able to form a triplet Tn epitope, three consecutive GalNAc-Ser/Thr structures, on peptides encoded in syndecan-3, a proteoglycan expressed in neurons. pp-GalNAc-T13-deficient mice have been established in a previous work. Immunohistochemical study showed a remarkable decrease in Tn antigen expression in the cerebellum of the pp-GalNAc-T13 knockout mouse. pp-GalNAc-T13 would be a major enzyme responsible for the synthesis of O-glycan and specifically the Tn antigen epitope in neurons.     

Siah-1 facilitates ubiquitination and degradation of synphilin-1

Parkinson's disease is a common neurodegenerative disorder characterized by loss of dopaminergic neurons and appearance of Lewy bodies, cytoplasmic inclusions that are highly enriched with ubiquitin. Synphilin-1, alpha-synuclein, and Parkin represent the major components of Lewy bodies and are involved in the pathogenesis of Parkinson's disease. Synphilin-1 is an alpha-synuclein-binding protein that is ubiquitinated by Parkin. Recently, a mutation in the synphilin-1 gene has been reported in patients with sporadic Parkinson's disease. Although synphilin-1 localizes close to synaptic vesicles, its function remains unknown. To investigate the proteins that interact with synphilin-1, the present study performed a yeast two-hybrid screening and identified a novel interacting protein, Siah-1 ubiquitin ligase. Synphilin-1 and Siah-1 proteins were endogenously expressed in the central nervous system and were found to coimmunoprecipitate each other in rat brain homogenate. Confocal microscopic analysis revealed colocalization of both proteins in cells. Siah-1 was found to interact with the N terminus of synphilin-1 through its substrate-binding domain and to specifically ubiquitinate synphilin-1 via its RING finger domain. Siah-1 facilitated synphilin-1 degradation via the ubiquitin-proteasome pathway more efficiently than Parkin. Siah-1 was found to not facilitate ubiquitination and degradation of wild type or mutant alpha-synuclein. Synphilin-1 inhibited high K+-induced dopamine release from PC12 cells. Siah-1 was found to abrogate the inhibitory effects of synphilin-1 on dopamine release. Such findings suggest that Siah-1 might play a role in regulation of synphilin-1 function.     

Protein 4.1N is required for translocation of inositol 1,4,5-trisphosphate receptor type 1 to the basolateral membrane domain in polarized Madin-Darby canine kidney cells

Protein 4.1N was identified as a binding molecule for the C-terminal cytoplasmic tail of inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) using a yeast two-hybrid system. 4.1N and IP(3)R1 associate in both subconfluent and confluent Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell line. In subconfluent MDCK cells, 4.1N is distributed in the cytoplasm and the nucleus; IP(3)R1 is localized in the cytoplasm. In confluent MDCK cells, both 4.1N and IP(3)R1 are predominantly translocated to the basolateral membrane domain, whereas 4.1R, the prototypical homologue of 4.1N, is localized at the tight junctions (Mattagajasingh, S. N., Huang, S. C., Hartenstein, J. S., and Benz, E. J., Jr. (2000) J. Biol. Chem. 275, 30573-30585), and other endoplasmic reticulum marker proteins are still present in the cytoplasm. Moreover, the 4.1N-binding region of IP(3)R1 is necessary and sufficient for the localization of IP(3)R1 at the basolateral membrane domain. A fragment of the IP(3)R1-binding region of 4.1N blocks the localization of co-expressed IP(3)R1 at the basolateral membrane domain. These data indicate that 4.1N is required for IP(3)R1 translocation to the basolateral membrane domain in polarized MDCK cells.     

Rat brain pericyte cell lines expressing beta2-adrenergic receptor,angiotensin II receptor type 1A,klotho, and CXCR4 mRNAs despite having endothelial cell markers

Pericytes are an integral component of blood capillaries, but their involvement in a variety of conditions and diseases, including hypertension and multiple sclerosis, is poorly understood. In order to analyze the mRNA expression of markers related to hypertension and multiple sclerosis in rat brain pericytes, we have established brain capillary pericyte cell lines from temperature-sensitive SV40 large T antigen transgenic rats. The newly established clones showed similar biochemical and morphological properties to primary pericytes. The expression of endothelial cell-related markers Flt-1, Flk-1, Tie-1, and Tie-2 was evaluated by RT-PCR analysis. beta2-Adrenergic receptor (beta2-AR), angiotensin II receptor type1A (AT1A), and klotho were also evaluated as markers related to hypertension and multiple sclerosis. All of the isolated clones expressed beta2-AR, AT1A and klotho genes. They also stably expressed Flt-1 and Tie-2, while Flk-1, Tie-1 and CXCR4 were expressed only at low levels in some of the clones. The expressions of AT1 in TR-PCT1 were determined by Western blotting. Angiotensin II stimulated migration of pericytes. This effect was blocked by an AT1 antagonist. The pericyte cell lines established here are pluripotent, and should be useful for analysis of the reactivity and biological roles of pericytes.     

Oligodeoxynucleotides without CpG motifs work as adjuvant for the induction of Th2 differentiation in a sequence-independent manner

The outcomes of immune responses are regulated by various parameters including how Ags are handled by APCs. In this study, we describe the intrinsic immunomodulatory characteristics of oligodeoxynucleotides (ODNs) that improve the Ag presentation by APCs. ODNs (20-mer) containing CpG motifs induced strong Th1-skewed responses. In contrast, those without CpG motifs enhanced cytokine production by effector Th cells without particular skewing toward Th1 responses or induced the differentiation of unprimed CD4(+) T cells toward Th2 cells. These functional features were prominently envisaged when ODNs were conjugated to the Ag, and were underlain by the facilitated binding of ODN-conjugated Ag to Ia(+) cells. Despite the functional differences between ODNs with CpG motifs and those without CpG motifs, both ODNs bound to Ia(+) cells with similar affinity and kinetics. Immunoenhancing activities of the ODNs were not sequence-dependent; the characteristics, including the facilitation of Ag capture, enhancement of effector Th cell responses, and induction of Th2 cells, were shared by randomly synthesized ODNs conjugated to Ag. This is the first study suggesting that ODNs, independent of the sequences, enhance immune responses through the promoted capture of ODN-conjugated Ag by APCs.     

The relative contribution of IL-4 receptor signaling and IL-10 to susceptibility to Leishmania major

The roles of IL-10 and IL-4 receptor signaling were evaluated in a murine model of Leishmania major infection. In previous studies the L. major substrain LV39 caused progressive, nonhealing lesions in BALB/c mice deficient for IL-4R alpha-chain (IL-4R alpha), while substrain IR173 was highly controlled. To explore whether IL-10 is responsible for inducing susceptibility to LV39, wild-type and IL-4R alpha(-/-) mice were treated with anti-IL-10R mAb, and in a genetic approach, the IL-4R alpha(-/-) mice were crossed with BALB/c IL-10(-/-) mice. In contrast to the lack of resistance conferred by IL-4R alpha gene deletion, partial resistance to LV39 was conferred by IL-10 gene deletion or treatment of BALB/c mice with anti-IL-10R mAb. Lesion sizes and LV39 parasite numbers were further and dramatically reduced in both anti-IL-10R-treated IL-4R alpha(-/-) mice and IL-4R alpha x IL-10 double knockouts. Anti-IL-10R mAb treatment further suppressed parasite growth in IL-4R alpha(-/-) mice infected with L. major IR173. Production of IFN-gamma was only increased relative to wild-type or littermate controls in IL-4R alpha(-/-) mice with complementary defects in IL-10. Comparisons of IFN-gamma-treated infected macrophages in vitro indicated that LV39 required 25- to 500-fold greater concentrations of IFN-gamma than IR173-infected macrophages to achieve a similar efficiency of parasite killing. These studies suggest that regardless of parasite substrain, IL-10 is as important as IL-4/IL-13 in promoting susceptibility to L. major and even more so for those substrains that are relatively resistant to IFN-gamma mediated killing.