Expression of human GLUD2 glutamate dehydrogenase in human tissues: Functional implications

Neurology Laboratory, Medical School, University of Crete, Heraklion, Crete, Greece.
Neurochemistry International (Impact Factor: 2.65). 06/2012; 61(4):455-62. DOI: 10.1016/j.neuint.2012.06.007
Source: PubMed

ABSTRACT Glutamate dehydrogenase (GDH), a mitochondrial enzyme with a key metabolic role, exists in the human in hGDH1 and hGDH2 isoforms encoded by the GLUD1 and GLUD2 genes, respectively. It seems that GLUD1 was retroposed to the X chromosome where it gave rise to GLUD2 via random mutations and natural selection. Of these, evolutionary Gly456Ala substitution dissociated hGDH2 from GTP control, while replacement of Arg443 by Ser drastically modified basal activity, heat stability, optimal pH, allosteric regulation and migration pattern in SDS-PAGE, thus suggesting an effect on enzyme's conformation. While GLUD2-specific transcripts have been detected in human brain, retina and testis, data on the endogenous hGDH2 protein are lacking. Given the housekeeping nature of hGDH1 and its high homology to hGDH2, the specific detection of hGDH2 in tissues presents a challenge. To develop an antibody specific for hGDH2, we considered that an epitope containing the Arg443Ser change was an attractive target. We accordingly used a peptide that corresponds to residues 436-447, with Ser at position 443, to immunize rabbits and succeeded in raising a polyclonal antibody specific for hGDH2. Western blots showed that human testis contained equal amounts of hGDH2 and hGDH1 and that both isoproteins localized to the mitochondrial fraction. In human brain, however, hGDH2 expression was lower than that of hGDH1. Immuno-histochemical studies on human testis and cerebral cortex, showed punctuate, organelle-like hGDH2 immuno-labeling in sertoli cells and in astrocytes, respectively, consistent with the mitochondrial localization of the enzyme. Similar studies in kidney revealed that hGDH2 is expressed in epithelial cells of the proximal convoluted tubule. As hGDH2 can metabolize glutamate at relatively low pH without the GTP constrain, it may function efficiently under conditions of relative acidification that prevail in astrocytes following glutamate uptake. Similarly, in the kidney, hGDH2 could contribute to enhanced excretion of ammonia under acidosis.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Mammalian glutamate dehydrogenase (GDH) is an evolutionarily conserved enzyme central to the metabolism of glutamate, the main excitatory transmitter in mammalian CNS. Its activity is allosterically regulated and thought to be controlled by the need of the cell for ATP. While in most mammals, GDH is encoded by a single GLUD1 gene that is widely expressed (housekeeping; hGDH1 in the human), humans and other primates have acquired via retroposition a GLUD2 gene encoding an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. Whereas hGDH1 shows high levels of expression in the liver, hGDH2 is expressed in human testis, brain and kidney. Recent studies have provided significant insight into the functional adaptation of hGDH2. This includes resistance to GTP control, enhanced sensitivity to inhibition by estrogens and other endogenous allosteric effectors, and ability to function in a relatively acidic environment. While inhibition of hGDH1 by GTP, derived from Krebs cycle, represents the main mechanism by which the flux of glutamate through this pathway is regulated, dissociation of hGDH2 from GTP control may provide a biological advantage by permitting enzyme function independently of this energy switch. Also, the relatively low optimal pH for hGDH2 is suited for transmitter glutamate metabolism, as glutamate uptake by astrocytes leads to significant mitochondrial acidification. Although mammalian GDH is a housekeeping enzyme, its levels of expression vary markedly among the various tissues and among the different types of cells that constitute the same organ. In this paper, we will review existing evidence on the cellular and subcellular distribution of GDH in neural and non-neural tissues of experimental animals and humans, and consider the implications of these findings in biology of these tissues. Special attention is given to accumulating evidence that glutamate flux through the GDH pathway is linked to cell signaling mechanisms that may be tissue-specific.
    Neurochemical Research 01/2014; 39(3). DOI:10.1007/s11064-013-1235-5 · 2.55 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In vitro and in vivo studies have shown that glutamate can be oxidized for energy by brain astrocytes. The ability to harvest the energy from glutamate provides astrocytes with a mechanism to offset the high ATP cost of the uptake of glutamate from the synaptic cleft. This brief review focuses on oxidative metabolism of glutamate by astrocytes, the specific pathways involved in the complete oxidation of glutamate and the energy provided by each reaction.
    Frontiers in Endocrinology 01/2013; 4:191. DOI:10.3389/fendo.2013.00191
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mammalian glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, interconnecting carbon skeleton and nitrogen metabolism. In addition, it functions as an energy switch by its ability to fuel the Krebs cycle depending on the energy status of the cell. As GDH lies at the intersection of several metabolic pathways, its activity is tightly regulated by several allosteric compounds that are metabolic intermediates. In contrast to other mammals that have a single GDH-encoding gene, humans and great apes possess two isoforms of GDH (hGDH1 and hGDH2, encoded by the GLUD1 and GLUD2 genes, respectively) with distinct regulation pattern, but remarkable sequence similarity (they differ, in their mature form, in only 15 of their 505 amino-acids). The GLUD2 gene is considered a very young gene, emerging from the GLUD1 gene through retro-position only recently (<23 million years ago). The new hGDH2 iso-enzyme, through random mutations and natural selection, is thought to have conferred an evolutionary advantage that helped its persistence through primate evolution. The properties of the two highly homologous human GDHs have been studied using purified recombinant hGDH1 and hGDH2 proteins obtained by expression of the corresponding cDNAs in Sf21 cells. According to these studies, in contrast to hGDH1 that maintains basal activity at 35-40 % of its maximal, hGDH2 displays low basal activity that is highly responsive to activation by rising levels of ADP and/or L-leucine which can also act synergistically. While hGDH1 is inhibited potently by GTP, hGDH2 shows remarkable GTP resistance. Furthermore, the two iso-enzymes are differentially inhibited by estrogens, polyamines and neuroleptics, and also differ in heat-lability. To elucidate the molecular mechanisms that underlie these different regulation patterns of the two iso-enzymes (and consequently the evolutionary adaptation of hGDH2 to a new functional role), we have performed mutagenesis at sites of difference in their amino acid sequence. Results showed that the low basal activity, heat-lability and estrogen sensitivity of hGDH2 could be, at least partially, ascribed to the Arg443Ser evolutionary change, whereas resistance to GTP inhibition has been attributed to the Gly456Ala change. Other amino acid substitutions studied thus far cannot explain all the remaining functional differences between the two iso-enzymes. Also, the Arg443Ser/Gly456Ala double mutation in hGDH1 approached the properties of wild-type hGDH2, without being identical to it. The insights into the structural mechanism of enzymatic regulation and the implications in cell biology provided by these findings are discussed.
    Neurochemical Research 02/2014; 39(3). DOI:10.1007/s11064-014-1251-0 · 2.55 Impact Factor