Glutamate-Dependent Translational Control of Glutamine Synthetase in Bergmann Glia Cells.

Glutamate is the major excitatory transmitter of the vertebrate brain. It exerts its actions through the activation of specific plasma membrane receptors expressed both in neurons and in glial cells. Recent evidence has shown that glutamate uptake systems, particularly enriched in glia cells, trigger biochemical cascades in a similar fashion as receptors. A tight regulation of glutamate extracellular levels prevents neuronal overstimulation and cell death, and it is critically involved in glutamate turnover. Glial glutamate transporters are responsible of the majority of the brain glutamate uptake activity. Once internalized, this excitatory amino acid is rapidly metabolized to glutamine via the astrocyte-enriched enzyme glutamine synthetase. A coupling between glutamate uptake and glutamine synthesis and release has been commonly known as the glutamate/glutamine shuttle. Taking advantage of the established model of cultured Bergmann glia cells, in this contribution, we explored the gene expression regulation of glutamine synthetase. A time- and dose-dependent regulation of glutamine synthetase protein and activity levels was found. Moreover, glutamate exposure resulted in the transient shift of glutamine synthetase mRNA from the monosomal to the polysomal fraction. These results demonstrate a novel mode of glutamate-dependent glutamine synthetase regulation and strengthen the notion of an exquisite glia neuronal interaction in glutamatergic synapses.

Glutamate regulates eEF1A phosphorylation and ribosomal transit time in Bergmann glial cells.

Glutamate, the major excitatory transmitter in the vertebrate brain, is involved in neuronal development and synaptic plasticity. Glutamatergic stimulation leads to differential gene expression patterns in neuronal and glial cells. A glutamate-dependent transcriptional control has been established for several genes. However, much less is known about the molecular events that modify the translational machinery upon exposure to this neurotransmitter. In a glial model of cerebellar cultured Bergmann cells, glutamate induces a biphasic effect on [(35)S]-methionine incorporation into proteins that suggests that the elongation phase of protein biosynthesis is the target for regulation. Indeed, after a 15 min exposure to glutamate a transient increase in elongation factor 2 phosphorylation has been reported, an effect mediated through the activation of the elongation factor 2 kinase. In this contribution, we sought to characterize the phosphorylation status of the eukaryotic elongation factor 1A (eEF1A) and the ribosomal transit time under glutamate exposure. A dose-dependent increase in eEF1A phosphorylation was found after a 60 min glutamate treatment; this phenomenon is Ca(2+)/CaM dependent, blocked with Src and phosphatidyl-inositol 3-kinase inhibitors and with rapamicyn. Concomitantly, the ribosomal transit time was increased with a 15 min glutamate exposure. After 60 more minutes, the average time used by the ribosomes to complete a polypeptide chain had almost returned to its initial level. These results strongly suggest that glutamate exerts an exquisite time-dependent translational control in glial cells, a process that might be critical for glia-neuron interactions.

Heterologous expression and biochemical characterization of an alpha1,2-mannosidase encoded by the Candida albicans MNS1 gene.

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with alpha1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an alpha1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble alpha1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise alpha1,2-mannosidases in other biological systems as well.

Kex2 protease converts the endoplasmic reticulum alpha1,2-mannosidase of Candida albicans into a soluble cytosolic form.

Cytosolic alpha-mannosidases are glycosyl hydrolases that participate in the catabolism of cytosolic free N-oligosaccharides. Two soluble alpha-mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in Candida albicans. We demonstrate that addition of pepstatin A during the preparation of cell homogenates enriched alpha-mannosidase E-I at the expense of E-II, indicating that the latter is generated by proteolysis during cell disruption. E-I corresponded to a polypeptide of 52 kDa that was associated with mannosidase activity and was recognized by an anti-alpha1,2-mannosidase antibody. The N-mannan core trimming properties of the purified enzyme E-I were consistent with its classification as a family 47 alpha1,2-mannosidase. Differential density-gradient centrifugation of homogenates revealed that alpha1,2-mannosidase E-I was localized to the cytosolic fraction and Golgi-derived vesicles, and that a 65 kDa membrane-bound alpha1,2-mannosidase was present in endoplasmic reticulum and Golgi-derived vesicles. Distribution of alpha-mannosidase activity in a kex2Delta null mutant or in wild-type protoplasts treated with monensin demonstrated that the membrane-bound alpha1,2-mannosidase is processed by Kex2 protease into E-I, recognizing an atypical cleavage site of the precursor. Analysis of cytosolic free N-oligosaccharides revealed that cytosolic alpha1,2-mannosidase E-I trims free Man8GlcNAc2 isomer B into Man7GlcNAc2 isomer B. This is believed to be the first report demonstrating the presence of soluble alpha1,2-mannosidase from the glycosyl hydrolases family 47 in a cytosolic compartment of the cell.

Heterologous expression and biochemical characterization of an alpha 1, 2-mannosidase encoded by the Candida albicans MNS1 gene

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with 1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an ±1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble ±1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise ±1,2-mannosidases in other biological systems as well.

Conversion of alpha1,2-mannosidase E-I from Candida albicans to alpha1,2-mannosidase E-II by limited proteolysis.

Previous studies demonstrated the presence in Candida albicans ATCC 26555 of two soluble alpha1,2-mannosidases: E-I and E-II. In contrast, in the C. albicans CAI-4 mutant only E-I was detected and it could be processed by a membrane-bound proteolytic activity from the ATCC 26555 strain, generating an active 43 kDa polypeptide. Here, alpha1,2-mannosidase E-I from strain ATCC 26555 was purified by conventional methods of protein isolation and affinity chromatography in Concanavalin A-Sepharose 4B. Analytical electrophoresis of the purified enzyme revealed two polypeptides of 52 and 23 kDa, the former being responsible for enzyme activity as revealed by zymogram analysis. Time course proteolysis with an aspartyl protease from Aspergillus saitoi, converted alpha1,2-mannosidase E-I into an active polypeptide of 43 kDa which trimmed Man(9)GlcNAc(2), generating Man(8)GlcNAc(2) isomer B and mannose. Trimming was inhibited preferentially by 1-deoxymannojirimycin. Both, the molecular mass and the enzyme properties of the proteolytic product were identical to those described for alpha1,2-mannosidase E-II therefore supporting the notion that E-I is the precursor of E-II.

Circadian oscillations of RPCH gene expression in the eyestalk of the crayfish Cherax quadricarinatus.

The RPCH and beta-actin cDNAs from the crayfish Cherax quadricarinatus were amplified, cloned and sequenced. The primary structure sequences of these cDNAs were compared to other members of the AKH/RPCH family. Fluctuations in the amount of the C. quadricarinatus RPCH and beta-actin mRNAs, as cDNAs, were quantified every 3h by RT-PCR. Single cosinor analysis supports the notion of beta-actin and RPCH mRNA circadian behavior in animals subjected to 12h:12h light/dark regimes. In constant darkness RPCH mRNA concentration changes to ultradian cycles.

Hydrolysis of Man9GlcNAc2 and Man8GlcNAc2 oligosaccharides by a purified alpha-mannosidase from Candida albicans.

A soluble alpha-mannosidase from Candida albicans was purified to homogeneity by sequential size exclusion, ion exchange, and affinity chromatographies in columns of Sepharose CL6B, DEAE Bio-Gel A, and Concanavalin A Sepharose 4B, respectively. Analytical electrophoresis of the purified preparation in 10% SDS-polyacrylamide gels stained with Coomassie blue revealed a single polypeptide of 43 kDa that was responsible for enzyme activity. The purified enzyme primarily trimmed Man(9)GlcNAc(2) to produce Man(8)GlcNAc(2) isomer B and mannose as a function of time of incubation up to 12 h at 37 degrees C. Prolonged incubation with the enzyme resulted in the accumulation after 24 h of other oligosaccharides corresponding to Man(7)GlcNAc(2) and probably Man(6)GlcNAc(2). These two products were also observed when Man(8)GlcNAc(2) isomer B instead of Man(9)GlcNAc(2) was used as substrate. Other oligosaccharides, such as Man(6)GlcNAc(2)-Asn, Man(5)GlcNAc(2)-Asn, and the alpha1,3- and alpha1,6-linked mannobiosides, were not hydrolyzed at all. These properties are consistent with an alpha1,2-mannosidase that may represent a new member of the glycosylhydrolase family 47.

The genomic organization of the open reading frame of the red pigment concentrating hormone gene in the blue crab Callinectes sapidus.

The open reading frame (ORF) of the gene for the precursor of the octapeptide Red Pigment Concentrating Hormone (RPCH) from the blue crab Callinectes sapidus was cloned by PCR with oligonucleotides targeted to the initiation and the end of the translation coding sequences. A 272 bp intron was characterized between nucleotides 343 and 344 of the reported cDNA, present in the region coding for the last amino acids of the precursor related peptide of RPCH. The intron genomic structure here described is similar to that reported for the gene coding for the Adipokinetic Hormone (AKH) of the grasshopper Schistocerca nitans.