Cellular studies of the two main isoforms of human d-aspartate oxidase.

Human d-aspartate oxidase (hDASPO) is a FAD-dependent enzyme responsible for the degradation of d-aspartate (d-Asp). In the mammalian central nervous system, d-Asp behaves as a classical neurotransmitter, it is thought to be involved in neural development, brain morphology and behavior, and appears to be involved in several pathological states, such as schizophrenia and Alzheimer's disease. Apparently, the human DDO gene produces alternative transcripts encoding for three putative hDASPO isoforms, constituted by 341 (the 'canonical' form), 369, and 282 amino acids. Despite the increasing interest in hDASPO and its physiological role, little is known about these different isoforms. Here, the additional N-terminal peptide present in the hDASPO_369 isoform only has been identified in hippocampus of Alzheimer's disease female patients, while peptides corresponding to the remaining part of the protein were present in samples from male and female healthy controls and Alzheimer's disease patients. The hDASPO_369 isoform was largely expressed in E. coli as insoluble protein, hampering with its biochemical characterization. Furthermore, we generated U87 human glioblastoma cell clones stably expressing hDASPO_341 and, for the first time, hDASPO_369 isoforms; the latter protein showed a lower expression compared with the canonical isoform. Both protein isoforms are active (showing similar kinetic properties), localize to the peroxisomes, are very stable (a half-life of approximately 100 h has been estimated), and are primarily degraded through the ubiquitin-proteasome system. These studies shed light on the properties of hDASPO isoforms with the final aim to clarify the mechanisms controlling brain levels of the neuromodulator d-Asp.

Changes in D-aspartic acid and D-glutamic acid levels in the tissues and physiological fluids of mice with various D-aspartate oxidase activities.

D-Aspartic acid (D-Asp) and D-glutamic acid (D-Glu) are currently paid attention as modulators of neuronal transmission and hormonal secretion. These two D-amino acids are metabolized only by D-aspartate oxidase (DDO) in mammals. Therefore, in order to design and develop new drugs controlling the D-Asp and D-Glu amounts via regulation of the DDO activities, changes in these acidic D-amino acid amounts in various tissues are expected to be clarified in model animals having various DDO activities. In the present study, the amounts of Asp and Glu enantiomers in 6 brain tissues, 11 peripheral tissues and 2 physiological fluids of DDO(+/+), DDO(+/-) and DDO(-/-) mice were determined using a sensitive and selective two-dimensional HPLC system. As a result, the amounts of D-Asp were drastically increased with the decrease in the DDO activity in all the tested tissues and physiological fluids. On the other hand, the amounts of D-Glu were almost the same among the 3 strains of mice. The present results are useful for designing new drug candidates, such as DDO inhibitors, and further studies are expected.

Thyroid hormones and D-aspartic acid, D-aspartate oxidase, D-aspartate racemase, H2O2, and ROS in rats and mice.

Total concentrations of thyroid hormones T(3) and T(4), and of their free forms, FT(3) and FT(4), D-aspartic acid (D-Asp), D-aspartate oxidase (D-AspO), D-aspartate racemase, H(2)O(2), and ROS (reactive oxygen species) were determined in rats and mice. T(3) and T(4) were 1 and 50 ng/ml, respectively, in serum, and 750 and 40000 ng/g, respectively, in thyroid. Concentrations of the free forms FT(3) and FT(4) were ca. 250 times lower than their respective total concentrations. The endogenous content of D-Asp in thyroid gland was ca. 100 nmol/g tissue, whereas the activity of D-AspO was ca. 80 units/mg thyroid, and that of D-aspartate racemase was ca. 15 units/mg thyroid. H(2)O(2) Concentration in rat and mouse thyroid gland was ca. 290 pmol/g thyroid, and the concentration of ROS was ca. 10 pmol/DCF/min/mg protein. H(2)O(2) is essential for the iodination of the tyrosyl residues to produce mono- and diiodotyrosine that are the precursors for the synthesis of T(3) and T(4). Production of H(2)O(2) in thyroid glands occurs by oxidation of endogenous D-Asp by D-AspO (D-Asp+O(2)+H(2)O-->alpha-oxaloacetate+NH(3)+H(2)O(2)). D-Aspartate racemase catalyzes the in vivo production of D-Asp from L-Asp. Thus, interaction of endogenous D-Asp, D-AspO, and D-aspartate racemase in thyroid gland constitutes an additional biochemical pathway for the production of H(2)O(2) and consequently for the synthesis of thyroid hormones.