Expression of the β-Galactoside α1,2-Fucosyltransferase Gene Suppresses Axonal Outgrowth of Neuro2a Neuroblastoma Cells

Abstract: The axonal outgrowth of cells of Neuro2a, a mouse neuroblastoma cell line, was suppressed on expression of the β-galactoside α1,2-fucosyltransferase (α1,2-FT) gene. We recently cloned two types of rabbit α1,2-FT, RFT-I and RFT-II. RFT-I exhibits comparable kinetic properties and structural homology with human H gene α1,2-FT, and RFT-II shows comparable kinetic parameters with human Se gene α1,2-FT. Neuro2a cells expressing RFT-I (N2A-RFT-I) contained a large amount of fucosyl GM1 instead of GM1 and GD1a, major gangliosides in the parent Neuro2a cells, whereas Neuro2a cells expressing RFT-II (N2A-RFT-II) showed a subtle change in the ganglioside pattern. N2A-RFT-II and parent Neuro2a cells showed axonal outgrowth in serum-free medium on the exogenous addition of GM1, whereas N2A-RFT-I cells exhibited multiple neurite sprouts but not axonal outgrowth. This phenotype was fully recovered by N2A-RFT-I cells on the addition of d - threo -1-phenyl-2-decanoylamino-3-morpholino-1-propanol and α- l -fucosidase to the culture medium, which resulted in pronounced reduction of fucosyl GM1 expression. These results suggested that expression of H-type α1,2-FT, and subsequent incorporation of fucose into glycolipids and glycoproteins, especially the formation of fucosyl GM1, modifies the response of neuronal cells to stimuli that induce axonal extension.     

Presence of α7 nicotinic acetylcholine receptors on dorsal root ganglion neurons proved using knockout mice and selective α-neurotoxins in histochemistry

In complex tissues where multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed, immunohistochemistry has been the most popular tool for investigation of nAChR subunit distribution. However, recent studies with nAChR subunit knockout mice demonstrated that a large panel of antibodies is unsuitable. Thus, we aimed to develop a histochemical method for selective labeling of α7 nAChR with neurotoxins, utilizing α7 nAChR-transfected cells, dorsal root ganglia (DRG) and spinal cord from wild-type and knockout mouse. The specificity of Alexa Fluor 488-conjugated α-bungarotoxin (Alexa-αBgt) was demonstrated in binding to α7-transfected cells inhibited by long-chain α-cobratoxin (CTX), but not short-chain α-neurotoxin II (NTII). In contrast, binding to Torpedo muscle-type nAChRs and to motor end plates in mouse tongue sections was prevented by both CTX and NTII. In tissue sections of DRG, expressing all neuronal nAChR subunits, only CTX precluded Alexa-αBgt labeling of neurons, with no staining for α7 nAChR knockout tissue. It proved that α7 nAChRs are the major αBgt-binding sites in mouse DRG. Corresponding results were obtained for terminals in the spinal cord. Thus, we present a protocol utilizing Alexa-αBgt and non-labeled CTX/NTII that allows specific histochemical detection of α7 nAChR with a spatial resolution at the level of single axon terminals.     

Gene regulation of α4β2 nicotinic receptors: microarray analysis of nicotine-induced receptor up-regulation and anti-inflammatory effects

α4β2 Nicotinic acetylcholine receptors play an important role in the reward pathways for nicotine. We investigated whether receptor up-regulation of α4β2 nicotinic acetylcholine receptors involves expression changes for non-receptor genes. In a microarray analysis, 10 μM nicotine altered expression of 41 genes at 0.25, 1, 8 and 24 h in hα4β2 SH-EP1 cells. The maximum number of gene changes occurred at 8 h, around the initial increase in ³[H]-cytisine binding. Quantitative RT-PCR corroborated gene induction of endoplasmic reticulum proteins CRELD2, PDIA6, and HERPUD1, and suppression of the pro-inflammatory cytokines IL-1β and IL-6. Nicotine suppresses IL-1β and IL-6 expression at least in part by inhibiting NFκB activation. Antagonists dihydro-β-erythroidine and mecamylamine blocked these nicotine-induced changes showing that receptor activation is required. Antagonists alone or in combination with nicotine suppressed CRELD2 message while increasing α4β2 binding. Additionally, small interfering RNA knockdown of CRELD2 increased basal α4β2 receptor expression, and antagonists decreased CRELD2 expression even in the absence of α4β2 receptors. These data suggest that endoplasmic reticulum proteins such as CRELD2 can regulate α4β2 expression, and may explain antagonist actions in nicotine-induced receptor up-regulation. Further, the unexpected finding that nicotine suppresses inflammatory cytokines suggests that nicotinic α4β2 receptor activation promotes anti-inflammatory effects similar to α7 receptor activation.     

Diverse Expression of β1,4-N-Acetylgalactosaminyltransferase Gene in the Adult Mouse Brain

Abstract: Among various tissues of mouse, β1,4- N -acetylgalactosaminyltransferase (GM2/GD2 synthase) gene is expressed predominantly in the brain. Further analysis of the gene expression in the mouse CNS was performed by northern blotting and by enzyme assays using extracts from various parts of the CNS. In situ hybridization was also done to investigate the distribution of cells generating GM2/GD2 synthase. In northern blots, diverse levels of the gene expression were observed, depending on the regions examined. By in situ hybridization, pyramidal cells in the hippocampus, granular cells in dentate gyrus and cerebral cortex, Purkinje cells in cerebellum, and mitral cells in the olfactory bulb expressed high levels of the mRNA; these results corresponded to the results obtained by northern blot. Enzyme levels in these sites were accordingly high. However, enzyme levels in certain areas with low mRNA intensities, such as thalamus and pons medulla, were higher than expected from the results of northern blotting. The significance of the high gene expression in certain areas for brain function and the reason for the discrepancy between mRNA level and enzyme activity in some regions are discussed.     

N-Glycosylation in the Moss Physcomitrella patens is Organized Similarly to that in Higher Plants

Abstract: Allergenicity of plant glycoproteins in humans may prevent the use of plants as production factories for pharmaceutically important proteins. The major difference between plant and mammalian N-glycans is the presence of xylosyl and α1,3-fucosyl residues in the former. In a first step towards "humanization" of the N-glycosylation pathway in the moss Physcomitrella patens, which could be an excellent system for industrial production of therapeutic proteins, we isolated the cDNAs and genes for N-acetylglucosaminyltransferase I (GNTI), α1,3-fucosyltransferase, and β1,2-xylosyltransferase. Sequence analysis revealed that all three proteins are homologous to their counterparts from higher plants, however, the conservation of the primary structure was only 35 - 45 %. The gene encoding the key enzyme of the pathway, gntI, was disrupted in P. patens by homologous recombination. Although the mutation of this gene in mouse or A. thaliana led to a significantly altered pattern of N-glycans, the glycosylation pattern in the gntI knockouts did not differ from that in wild-type moss and was identical to that in higher plants. Protein secretion, analysed in assays with recombinant human VEGF₁₂₁ protein, was not affected in the knockouts. We conclude from our findings that the N-glycosylation pathway in P. patens is identically organized to that in higher plants. However, P. patens probably possesses more than one isoform of GNTI which complicates a straightforward knockout. Therefore, and since complex type structures appear more desirable than oligomannosidic N-glycans, future modifications of the pathway should target α1,3-fucosyltransferase and/or β1,2-xylosyltransferase.     

The α5(H105R) mutation impairs α5 selective binding properties by altered positioning of the α5 subunit in GABAA receptors containing two distinct types of α subunits

GABA_A receptors are pentameric ligand-gated ion channels that are major mediators of fast inhibitory neurotransmission. Clinically relevant GABA_A receptor subtypes are assembled from α5(1-3, 5), β1-3 and the γ2 subunit. They exhibit a stoichiometry of two α, two β and one γ subunit, with two GABA binding sites located at the α/β and one benzodiazepine binding site located at the α/γ subunit interface. Introduction of the H105R point mutation into the α5 subunit, to render α5 subunit-containing receptors insensitive to the clinically important benzodiazepine site agonist diazepam, unexpectedly resulted in a reduced level of α5 subunit protein in α5(H105R) mice. In this study, we show that the α5(H105R) mutation did not affect cell surface expression and targeting of the receptors or their assembly into macromolecular receptor complexes but resulted in a severe reduction of α5-selective ligand binding. Immunoprecipitation studies suggest that the diminished α5-selective binding is presumably due to a repositioning of the α5(H105R) subunit in GABA_A receptor complexes containing two different α subunits. These findings imply an important role of histidine 105 in determining the position of the α5 subunit within the receptor complex by determining the affinity for assembly with the γ2 subunit.     

The significance and effect of tandem repeats within the Mycobacterium tuberculosis leuA gene on α-isopropylmalate synthase

The 57-bp tandem repeats located in the Mycobacterium tuberculosis leuA gene code for the α-isopropylmalate synthase (α-IPMS). It is unique to this pathogen. It was previously demonstrated that the leuA -coding sequence Rv3710, containing the tandem repeats, can be translated to an active α-IPMS. The objective of the present study was to investigate the significance and effect of the two 57-bp tandem repeats upon gene expression and the general properties of α-IPMS. The putative M. tuberculosis H37Rv leuA gene with and without the tandem repeats was cloned by PCR and expressed in an Escherichia coli host. The enzyme product was studied for general properties, comparing that from a native leuA gene containing two repeats and that from the 57-bp tandem repeats deletion mutant. Upon deletion of the two 57-bp tandem repeats, the expression level of leuA from M. tuberculosis H37Rv was comparable with that of the native form. The general properties of the two types of enzymes were similar. They were both functional with the same range of optimal temperature and optimal pH for activity and with similar enzyme stability. Deletion of the repeats had no detectable effect on leuA expression level or the general properties of the enzyme product.     

The loop between β-strands β2 and β3 and its interaction with the N-terminal α-helix is essential for biogenesis of α7 nicotinic receptors

Recently, we have shown that the α-helix present at the N-termini of α7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking β-strands β2 and β3 (loop 3). We studied the role of this loop as well as its interaction with the helix in membrane receptor expression. Residues from Asp62 to Val75 in loop 3 were mutated. Mutations of conserved amino acids, such as Asp62, Leu65 and Trp67 abolished membrane receptor expression in Xenopus oocytes. Others mutations, at residues Asn68, Ala69, Ser70, Tyr72, Gly74, and Val 75 were less harmful although still produced significant expression decreases. Steady state levels of wild-type and mutant α7 receptors (L65A, W67A, and Y72A) were similar but the formation of pentameric receptors was impaired in the latter (W67A). Mutation of critical residues in subunits of heteromeric nicotinic acetylcholine receptors (α3β4) also abolished their membrane expression. Complementarity between the helix and loop 3 was evidenced by studying the expression of chimeric α7 receptors in which these domains were substituted by homologous sequences from other subunits. We conclude that loop 3 and its docking to the α-helix is an important requirement for receptor assembly.     

Role of the N-terminal α-helix in biogenesis of α7 nicotinic receptors

We studied the role of the α-helix present at the N-terminus of nicotinic acetylcholine receptor (nAChR) subunits in the expression of functional channels. Deletion of this motif in α7 subunits abolished expression of nAChRs at the membrane of Xenopus oocytes. The same effect was observed upon substitution by homologous motifs of other ligand-gated receptors. When residues from Gln4 to Tyr15 were individually mutated to proline, receptor expression strongly decreased or was totally abolished. Equivalent substitutions to alanine were less harmful, suggesting that proline-induced break of the α-helix is responsible for the low expression. Steady-state levels of wild-type and mutant subunits were similar but the formation of pentameric receptors was impaired in the latter. In addition, those mutants that reached the membrane showed a slightly increased internalization rate. Expression of α7 nAChRs in neuroblastoma cells confirmed that mutant subunits, although stable, were unable to reach the cell membrane. Analogous mutations in heteromeric nAChRs (α3β4 and α4β2) and 5-HT_3A receptors also abolished their expression at the membrane. We conclude that the N-terminal α-helix of nAChRs is an important requirement for receptor assembly and, therefore, for membrane expression.     

alpha(1,2)-fucosylation prevents sialyl Lewis x expression and E-selectin-mediated adhesion of fucosyltransferase VII-transfected cells.

E-selectin is a cytokine-inducible, calcium-dependent endothelial cell adhesion molecule that plays a critical role in the leucocyte-endothelium interaction during inflammation and is thought to contribute to the metastatic dissemination of tumour cells. Like the other selectins, E-selectin binds to ligands carrying the tetrasaccharide sialyl-Lewis x (NeuAcalpha2,3Galbeta1,4[Fucalpha1, 3]GlcNAc)1 or its isomer sialyl-Lewis a (NeuAcalpha2, 3Galbeta1, 3[Fucalpha1,4]GlcNAc). We examined the effect of expressing the H-type alpha(1,2)-fucosyltransferase or the alpha(2, 6)-sialyltransferase on the synthesis of sialyl-Lewis x by alpha(1, 3)fucosyltransferase. We found that H-type alpha(1, 2)-fucosyltransferase but not alpha(2,6)-sialyltransferase, strongly inhibited sialyl-Lewis x expression and E-selectin adhesion. We assume that H-type alpha(1,2)-fucosyltransferase competes with the endogenous alpha(2,3)-sialyltransferase for the N-acetyllactosamine structures assigned to further serve as acceptors for alpha(1, 3)fucosyltransferase.     

α2-Macroglobulin Attenuates β-Amyloid Peptide 1–40 Fibril Formation and Associated Neurotoxicity of Cultured Fetal Rat Cortical Neurons

Abstract: β-Amyloid peptides (Aβ) are deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease. The neurotoxicity of Aβ in cultured neurons is dependent on its aggregation state, but the factors contributing to aggregation and fibril formation are poorly understood. In the present study, we investigated whether α₂-macroglobulin (α₂M), a protein present in neuritic plaques and elevated in Alzheimer's disease brain, is a potential regulatory factor for Aβ fibril formation. Previous studies in our laboratory have shown that α₂M is an Aβ binding protein. We now report that, in contrast to another plaque-associated protein, α₁-antichymotrypsin, α₂M coincubated with Aβ significantly reduces aggregation and fibril formation in vitro. Additionally, cultured fetal rat cortical neurons are less vulnerable to the toxic actions of aged Aβ following pretreatment with α₂M. We postulate that α₂M is able to maintain Aβ in a soluble state, preventing fibril formation and associated neurotoxicity.     

αγ-Enolase in the Rat: Ontogeny and Tissue Distribution

Abstract: The rat brain enolases are dimers composed of α and γ subunits. At pH 8.6 αγ-enolase seemed to be stable, and no evidence was found for the possible formation of αγ-enolase from αα-enolase and γγ-enolase in the course of rat brain homogenization. During ontogeny of the rat forebrain, αγ-enolase was formed before γγ-enolase. The half-maximal specific concentrations were reached at postnatal days 14 and 23, respectively. The distribution of αγ- and γγ-enolase in various rat brain areas was also investigated. In all areas both forms were present. In neuroendocrine tissues αγ-enolase was present at a much higher concentration than γγ-enolase. The ratio between γγ-enolase and αγ-enolase may be indicative of the degree of neuronal maturation, a conclusion further substantiated by the high ratio observed in cerebellum and the low ratio observed in olfactory bulbs, both compared with the ratio in forebrain.     

β-Synuclein occurs in vivo in lipid-associated oligomers and forms hetero-oligomers with α-synuclein

α-synuclein (αS) and β-synuclein (βS) are homologous proteins implicated in Parkinson's disease and related synucleinopathies. While αS is neurotoxic and its aggregation and deposition in Lewy bodies is related to neurodegeneration, βS is considered as a potent inhibitor of αS aggregation and toxicity. No mechanism for the neuroprotective role of βS has been described before. Here, we report that similar to αS, βS normally occurs in lipid-associated, soluble oligomers in wild-type (WT) mouse brains. We partially purified βS and αS proteins from whole mouse brain by size exclusion followed by ion exchange chromatography and found highly similar elution profiles. Using this technique, we were able to partially separate βS from αS and further separate βS monomer from its own oligomers. Importantly, we show that although αS and βS share high degree of similarities, βS oligomerization is not affected by increasing cellular levels of polyunsaturated fatty acids (PUFAs), while αS oligomerization is dramatically enhanced by PUFA. We show the in vivo occurrence of hetero-oligomers of αS and βS and suggest that βS expression inhibits PUFA-enhanced αS oligomerization by forming hetero-oligomers up to a quatramer that do not further propagate.     

Identification of a new alpha1,2-fucosyltransferase involved in O-antigen biosynthesis of Escherichia coli O86:B7 and formation of H-type 3 blood group antigen

Escherichia coli O86 possesses high human blood group B activity because of its O-antigen structure, sharing the human blood group B epitope. In this study, the wbwK gene of E. coli O86:B7 was expressed and purified as the GST fusion protein. Thereafter, the wbwK gene was biochemically identified to encode an alpha1,2-fucosyltransferase through radioactivity assays, as well as mass spectrometry and NMR spectroscopy. WbwK shows strict substrate specificity and only recognizes Gal beta1,3GalNAc alpha-OR (T-antigen and derivatives) as the acceptor to generate the H-type 3 blood group antigen. In contrast to other alpha1,2-fucosyltransferases, WbwK does not display activity toward the simple substrate Gal beta-OMe. Comparison with another recently characterized alpha1,2-fucosyltransferase (WbsJ) of E. coli O128:B12 indicates a low level of amino acid identity between them; however, they share a common acceptor substrate, Gal beta1,3GalNAc alpha-OR. Domain swapping between WbwK and WbsJ revealed that the smaller variable domains located in the C-terminus determine substrate specificity, whereas the larger variable domain in the N-terminus might play a role in forming the correct conformation for substrate binding or for localization of the alpha1,2-fucosyltransferase involved in O-antigen biosynthesis. In addition, milligram scale biosynthesis of the H-type 3 blood group antigen was explored using purified recombinant WbwK. WbwK may have potential applications in masking T-antigen, the tumor antigen, in vivo.     

Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µ M nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration-dependent manner with IC₅₀ values in the 10 n M range following incubation for 24 h and in the 1 n M range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 n M nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first-order process for both subtypes; half-lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first-order for both subtypes; half-lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor-mediated neurotransmitter release.