pLG72 Modulates Intracellular D-Serine Levels through Its Interaction with D-Amino Acid Oxidase

Abstract

Human genes coding for pLG72 and D-amino acid oxidase have recently been linked to the onset of schizophrenia. pLG72 was proposed as an activator of the human FAD-containing flavoprotein D-amino acid oxidase (hDAAO). In the brain this oxidizes D-serine, a potent activator of N-methyl-D-aspartate receptor. We have investigated the mechanistic regulation of hDAAO by pLG72. Immunohistochemical analyses revealed that hDAAO and pLG72 are both expressed in astrocytes of the human cortex, where they most likely interact, considering their partial overlapping subcellular distribution and their coimmunoprecipitation. We demonstrated that the specific in vitro interaction of the two proteins yields a complex composed of 2 hDAAO homodimers and 2 pLG72 molecules. Binding of pLG72 did not affect the kinetic properties and FAD binding ability of hDAAO; instead, a time-dependent loss of hDAAO activity in the presence of an excess of pLG72 was found. The binding affects the tertiary structure of hDAAO, altering the amount of the active form. We finally demonstrated that overexpression of hDAAO in glioblastoma cells decreases the levels of D-serine, an effect that is null when pLG72 is coexpressed. These data indicate that pLG72 acts as a negative effector of hDAAO. Therefore, a decrease in the synaptic concentration of D-serine as the result of an anomalous increase in hDAAO activity related to hypoexpression of pLG72 may represent a molecular mechanism by which hDAAO and pLG72 are involved in schizophrenia susceptibility.Schizophrenia is one of the most widely spread psychiatric disorders; it is a complex disease or, more likely, a group of related illnesses to which an individual has a strong genetic predisposition (1). Among the identified schizophrenia susceptibility genes (2), the gene G72 encodes for several splicing isoforms; pLG72 represents the longest open reading frame (153 amino acids), which is mainly expressed in brain (3). G72 is present only in primates: there are no homologues of this gene in databases nor has sequence analysis of the putative open reading frame revealed any likely function (2, 3). Yeast twohybrid experiments using pLG72 as bait identified D-amino acid oxidase (EC 1.4.3.3, DAAO 4 ) on 12q24 as a putative interacting partner, and preliminary functional measurements showed that pLG72 should function as an in vitro activator of pig kidney DAAO (pkDAAO) (3). DAAO is a FAD-containing flavoenzyme that catalyzes the oxidative deamination of D-amino acids to the corresponding ␣-keto acids, hydrogen peroxide and ammonia (4, 5).Based on current findings we can hypothesize that in brain, the physiological role of DAAO is to modulate the levels of D-serine, an important glial-derived messenger that acts as the endogenous allosteric modulator of the glutamatergic NMDA receptor subtype (6 -8). D-and L-serine can be reversibly isomerized in astrocytic glia, which unsheathes synapses, by serine racemase. Compelling evidence has indicated that glutamate neurotransmission hypofunction is associated with symptoms...

Effect of ligand binding on human D‐amino acid oxidase: Implications for the development of new drugs for schizophrenia treatment

“…We also demonstrated that CPZ binds to the apoprotein form of hDAAO with an affinity similar to that estimated for FAD. 9 Furthermore, and differently to that observed for FAD binding, the presence of benzoate does not modify the K d for CPZ binding to hDAAO apoprotein.…”

“…The aliphatic phenothiazine CPZ is a dopamine D2 receptor antagonist that was introduced in the treatment of schizophrenia in the early 1950s; it also acts as a FAD-competitive inhibitor of hDAAO (K d ¼ 5 lM). 9,17 We here demonstrate that the different ligands affect the conformation and stability of the holoenzyme and apoprotein forms of the human flavoenzyme as well as the cellular concentration of hDAAO differently. We also provide evidence that these ligands do not alter the interaction between hDAAO and pLG72 and that affect the time course of cellular D-serine concentration similarly.…”

“…6 We recently demonstrated that in vitro hDAAO specifically interacts with pLG72, yielding a % 200-kDa complex constituted by 2 hDAAO homodimers and 2 pLG72 monomers. [7][8][9] This interaction results in a time-dependent loss of hDAAO activity which is mainly due to alteration of the tertiary structure of hDAAO. Furthermore, we confirmed in vivo the hDAAO-pLG72 interaction and demonstrated that the cellular concentration of D-serine decreases in U87 glioblastoma cells transfected with a plasmid encoding for hDAAO but is not modified in those simultaneously transfected with cDNAs encoding both pLG72 and hDAAO.…”

“…Furthermore, we confirmed in vivo the hDAAO-pLG72 interaction and demonstrated that the cellular concentration of D-serine decreases in U87 glioblastoma cells transfected with a plasmid encoding for hDAAO but is not modified in those simultaneously transfected with cDNAs encoding both pLG72 and hDAAO. 9 Therefore, we proposed a molecular mechanism by which hDAAO and pLG72 are involved in schizophrenia susceptibility: an anomalous increase in hDAAO activity (e.g., related to pLG72 hypoexpression) will result in a decrease in the synaptic concentration of D-serine, thus causing hypofunctionality of NMDA receptormediated neurotransmission. Importantly, and in agreement with the aforementioned model, hDAAO is now considered the target for a new class of drugs (hDAAO inhibitors) to treat schizophrenia.…”

“…15 In this work we analyze the consequences of ligand binding on conformation and stability of hDAAO alone and in complex with pLG72. To reach this goal, the substrate D-serine or the pseudo-substrate trifluoro-D-alanine (CF 3 -D-Ala), benzoate (the most studied substrate-competitive inhibitor of hDAAO, K d ¼ 7 lM), 1,9,16 and the drug chlorpromazine (CPZ) were employed. The aliphatic phenothiazine CPZ is a dopamine D2 receptor antagonist that was introduced in the treatment of schizophrenia in the early 1950s; it also acts as a FAD-competitive inhibitor of hDAAO (K d ¼ 5 lM).…”