Abstract
Dysfunction of D-amino acid oxidase (DAO) and DAO activator (DAOA)/G72 genes have been linked to neuropsychiatric disorders. The glutamate hypothesis of schizophrenia has proposed that increased DAO activity leads to decreased D-serine, which subsequently may lead to N-methyl-D-aspartate (NMDA) receptor hypofunction. It has been shown that DAOA binds to DAO and increases its activity. However, there are also studies showing DAOA decreases DAO activity. Thus, the effect of DAOA on DAO is controversial. We aimed to understand the effect of DAOA on DAO activity in neuron-like (SH-SY5Y), astrocyte-like (1321N1) and kidney-like (HEK293) human cell lines. DAO activity was measured based on the release of hydrogen peroxide and its interaction with Amplex Red reagent. We found that DAOA increases DAO activity only in HEK293 cells, but has no effect on DAO activity in SH-SY5Y and 1321N1 cells. This might be because of different signaling pathways, or due to lower DAO and DAOA expression in SH-SY5Y and 1321N1 cells compared to HEK293 cells, but also due to different compartmentalization of the proteins. The lower DAO and DAOA expression in neuron-like SH-SY5Y and astrocyte-like 1321N1 cells might be due to tightly regulated expression, as previously reported in the human post-mortem brain. Our simulation experiments to demonstrate the interaction between DAOA and human DAO (hDAO) showed that hDAO holoenzyme [hDAO with flavine adenine dinucleotide (FAD)] becomes more flexible and misfolded in the presence of DAOA, whereas DAOA had no effect on hDAO apoprotein (hDAO without FAD), which indicate that DAOA inactivates hDAO holoenzyme. Furthermore, patch-clamp analysis demonstrated no effect of DAOA on NMDA receptor activity in NR1/NR2A HEK293 cells. In summary, the interaction between DAO and DAOA seems to be cell type and its biochemical characteristics dependent which still needs to be elucidated.
Highlights
The human D-amino acid oxidase (DAO/DAAO) gene is located at chromosome 12q24, and encodes for a ∼39 kDa protein of 347 amino acids (Verrall et al, 2010)
We found that the endogenous DAO activator (DAOA) mRNA levels were significantly lower in DAO, pEGFPN1+pCMV3-c-Myc, pCMV3-c-Myc transfected than G72+DAO transfected SH-SY5Y (F = 3656.23, df = 3, p < 0.0001), 1321N1 (F = 112.39, df = 3, p < 0.0001) and HEK293 cells (F = 858.86, df = 3, p < 0.0001; Figure 5B)
DAO and DAOA genes are alleged to be involved in pathophysiology of neuropsychiatric disorders such as schizophrenia and bipolar disorder (Detera-Wadleigh and McMahon, 2006; Allen et al, 2008; Prata et al, 2008; Gatt et al, 2015), but the interactions between these genes remains unclear to date
Summary
The human D-amino acid oxidase (DAO/DAAO) gene is located at chromosome 12q24, and encodes for a ∼39 kDa protein of 347 amino acids (Verrall et al, 2010). The human DAO activator (DAOA)/G72 gene is a primate specific gene located at chromosome 13q33, and encodes for a ∼20 kDa protein of 153 amino acids (Benzel et al, 2008). It catalyzes the oxidation of D-amino acids through concomitant reduction of flavine adenine dinucleotide (FAD), producing corresponding imino acid, which is hydrolyzed to yield ammonia and corresponding α-keto acid. DAO can regulate the function of NMDA receptors via D-serine breakdown
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