Abstract |
Background: Glutamate dehydrogenase (GDH) is an abundant mitochondrial enzyme
which catalyzes the interconversion of glutamate to α-ketoglutarate. Humans possess
two functional genes which encode for GDH, the ubiquitously expressed GLUD1
(coding for hGDH1) and the tissue specific GLUD2 (coding for hGDH2). The two isoenzymes,
although sharing all but 15 out of their 505 amino acids in their mature form,
differ significantly in their regulation properties. hGDH2 is mainly detected on brain,
kidney and testis, where it is found to co-localize with hGDH1 in the mitochondrial
matrix. Both iso-enzymes operate by forming homo-hexamers, but it is currently
unknown if they form hetero-hexamers at the areas where they co-localize. Besides
their physiological role in metabolism, genetic alterations in the GLUD1 and GLUD2
genes have been associated with pathophysiological conditions. Specifically,
mutations of the GLUD1 gene lead to the hyperinsulinism / hyperammonemia
syndrome, whereas a polymorphism in GLUD2 has been associated with accelerated
Parkinson’s disease age of onset. Furthermore, there is evidence that hGDH plays a
role in Alzheimer’s disease associated neurodegeneration (as its overexpression leads
to age associated degenerative changes in the mouse hippocampus), even though
proof for this in human patients suffering from dementia is lacking.
Aims: Aim of this study is to further analyze hGDH1 and hGDH2 properties by
employing functional, structural as well as genetic methods. Specifically, we
investigated whether hGDH1 and hGDH2 are capable of forming hetero-hexamers
when they are co-expressed, and what particular properties these complexes may
have. Secondly, we aimed to obtain the so far unsolved crystal structure of hGDH2
which will enable us to perform a valid and accurate comparison with hGDH1 (the
structure of which is already known). Lastly, we analyzed the genetic variations’
spectrum of GLUD1 and GLUD2 genes in the well characterized Cretan Aging Cohort
of 201 subjects, comprising of patients suffering from dementia and mild cognitive
impairment (MCI), as well as of cognitive normal controls.
Methods: To accomplish the first part of our aims, we created hetero-hexameric hGDH
complexes in Sf21 cells, using the Baculovirus expression system. We employed coimmunoprecipitation
and affinity chromatography to discriminate between in vitro
mixtures of separately expressed hGDHs and in vivo 1:1 co-expressed hGDHs. Then
we performed a series of kinetic studies to characterize the possible hetero-hexamers’
properties. For the second part, we produced hGDH2 in large-scale Sf21 cultures and we purified it to homogeneity, before crystallization by repeated trials using different
methods and reagents. For the genetic studies, we obtained Whole Exome
Sequencing data from 201 Cretan elderly individuals, from which 100 suffered from
dementia, 20 were classified as MCI and 81 were cognitive normal controls. After
characterizing their dementia related genetic background, we focused on GLUD1 and
GLUD2 genes in respect to their variations.
Results: In the present study we show that hGDHs can form, apart from homohexamers,
hetero-hexameric complexes, when they are co-expressed. These heterohexamers
possess unique enzymatic properties, which under specific conditions
(absence of ADP, L-leucine activation, heat stability) are intermediate between the
properties of pure hGDH1 and hGDH2 (and thus comparable to the 1:1 in vitro mixture
of the two homo-hexamers), whereas they tend to resemble hGDH2’s behavior in
respect with GTP and DES inhibition. In the second part of the study, we managed for
the first time to produce high-quality hGDH2 crystals of 3.2Å resolution for structure
characterization. In the third part of the study (genetic analysis of GLUD1 and GLUD2
genes), we found the Ser498Ala GLUD2 variant to be more common in controls
(16.05%, n=81) than in patients with dementia (3.0%, n=100; p=0.003), and these
results were verified by including in our analyses an additional local cohort.
Conclusion: Our study raises the possibility that hGDH1 and hGDH2 form heterohexamers
in the cells where they are co-expressed. As the functional properties of
these complexes are distinct from those of homo-hexamers, this association might
provide versatility to human cells by equipping them with more than two iso-enzymes.
Structural studies on hGDH2 are expected, not only to explore its particularities, but
also to decipher the structural basis of its enzymatic differences from its highly
homologous hGDH1 iso-enzyme. Finally, in the culturally and genetically
homogeneous cohort of aged adults of the island of Crete, we found that the Ser498Ala
GLUD2 variant was associated with lower risk for dementia in our population.
|