Abstract
d-Aspartate is an endogenous free amino acid in the brain, endocrine tissues, and exocrine tissues in mammals, and it plays several physiological roles. In the testis, d-aspartate is detected in elongate spermatids, Leydig cells, and Sertoli cells, and implicated in the synthesis and release of testosterone. In the hippocampus, d-aspartate strongly enhances N-methyl-d-aspartate receptor-dependent long-term potentiation and is involved in learning and memory. The existence of aspartate racemase, a candidate enzyme for d-aspartate production, has been suggested. Recently, mouse glutamic-oxaloacetic transaminase 1-like 1 (Got1l1) has been reported to synthesize substantially d-aspartate from l-aspartate and to be involved in adult neurogenesis. In this study, we investigated the function of Got1l1 in vivo by generating and analyzing Got1l1 knockout (KO) mice. We also examined the enzymatic activity of recombinant Got1l1 in vitro. We found that Got1l1 mRNA is highly expressed in the testis, but it is not detected in the brain and submandibular gland, where d-aspartate is abundant. The d-aspartate contents of wild-type and Got1l1 KO mice were not significantly different in the testis and hippocampus. The recombinant Got1l1 expressed in mammalian cells showed l-aspartate aminotransferase activity, but lacked aspartate racemase activity. These findings suggest that Got1l1 is not the major aspartate racemase and there might be an as yet unknown d-aspartate-synthesizing enzyme.
Highlights
Some free d-amino acids exist at high concentrations in several tissues in mammals. d-Aspartate is present in some tissues such as those in the hippocampus, pineal body, and pituitary in the brain, the submandibular gland (SMG), and the testis (Furuchi and Homma 2005; Hashimoto and Oka 1997; Masuda et al 2003)
For the construction of the glutamic-oxaloacetic transaminase 1-like 1 (Got1l1)-targeting vector, the 5′ homology arm of 5.2 kb (−3,648 to +1,533; the nucleotide residues of the mouse bacterial artificial chromosome (BAC) clone are numbered in the 5′–3′ direction, beginning with the A of ATG, the initiation site of translation in Got1l1, which refers to position +1, and the preceding residues are indicated by negative numbers) and 3′ homology arm of 6.9 kb (+1,800 to +8,730) from the BAC clone were subcloned into the pDONR P4-P1R and pDONER P2R-P3 vectors (Invitrogen, Carlsbad, CA), respectively, using a counter-selection BAC modification kit (Gene Bridges, Dresden, Germany)
Unlike in the previous study, the recombinant Got1l1 was not obtained in a soluble fraction of E. coli when the E. coli pET-vector system was used
Summary
Some free d-amino acids exist at high concentrations in several tissues in mammals. d-Aspartate is present in some tissues such as those in the hippocampus, pineal body, and pituitary in the brain, the submandibular gland (SMG), and the testis (Furuchi and Homma 2005; Hashimoto and Oka 1997; Masuda et al 2003). D-Aspartate is present in some tissues such as those in the hippocampus, pineal body, and pituitary in the brain, the submandibular gland (SMG), and the testis (Furuchi and Homma 2005; Hashimoto and Oka 1997; Masuda et al 2003). D-Aspartate is involved in the synthesis and release of the luteinizing hormone in the pituitary and melatonin release in the pineal body (D’Aniello et al 2000; Ishio et al 1998; Takigawa et al 1998). Several studies have shown the functions of d-aspartate, the enzyme producing d-aspartate was not identified in mammals. A recombinant Got1l1 protein is a pyridoxal 5′-phosphate (PLP)-dependent enzyme and synthesizes substantial d-aspartate from l-aspartate and only one-fifth as much l-glutamate, with very little d-glutamate in vitro. The function and contribution of Got1l1 as an aspartate racemase has not been clarified in vivo
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