The degradation (by distinct pathways) of human d‐amino acid oxidase and its interacting partner pLG72 – two key proteins in d‐serine catabolism in the brain

Abstract

Human D-amino acid oxidase (EC 1.4.3.3; hDAAO) is a peroxisomal flavoenzyme significantly enriched in the mammalian brain. hDAAO has been proposed to play (with serine racemase; EC 5.1.1.18) an essential role in the catabolism of D-serine, an 'atypical' key signalling molecule that acts as allosteric activator of the N-methyl-D-aspartate-type glutamate receptor (NMDAr). hDAAO and its interacting partner pLG72 have been related to schizophrenia, a highly disabling psychiatric disorder in which a dysfunction of NMDA-mediated neurotransmission is widely assumed to occur. We previously demonstrated that the D-serine cellular concentration depends on hDAAO and pLG72 expression levels and that newly-synthesized hDAAO interacts with its modulator in the cytosol, being progressively destabilized and inactivated. To obtain insight into the mechanisms regulating cellular D-serine levels, we investigated the degradation pathways of hDAAO and pLG72 in U87 glioblastoma cells stably expressing enhanced yellow fluorescent protein-hDAAO (peroxisomal), hDAAO-enhanced yellow fluorescent protein (cytosolic) or pLG72-enhanced cyan fluorescent protein (mitochondrial) proteins. hDAAO is a long-lived protein: the peroxisomal fraction of this flavoprotein is degraded via the lysosomal/endosomal pathway (and blocking this pathway increases the cellular hDAAO activity and decreases D-serine levels), whereas the cytosolic portion is ubiquitinated and targeted to the proteasome. By contrast, pLG72 shows a rapid turnover (t 1/2 % 25-40 min) and is degraded via the proteasome system, albeit not ubiquitinated. Overexpression of pLG72 increases the turnover of hDAAO, in turn playing a protective role against excessive D-serine depletion.

Novel insights into renal d-amino acid oxidase accumulation: propiverine changes DAAO localization and peroxisomal size in vivo

“…Co-staining with antibodies against mono-and polyubiquitylated conjugates indicated that cytosolic and nuclear protein accumulations are positive for ubiquitin (data not shown). This is in line with the fact that hDAAO was shown to be ubiquitylated in glioblastoma cells (Cappelletti et al 2013). Concurrent manipulative in vitro experiments also demonstrated that proteasomal inhibition resulted in a comparable cytosolic and nuclear accumulation of DAAO (Luks et al, unpublished data).…”

“…However, both DAAO and α2u-globulin inherently have a long half-life (hDAAO: approx. 60 h (Cappelletti et al 2013);α2u-globulin: approx. 6 h (Lehman-McKeeman et al 1990)), whereby binding of chemicals to α2u-globulin extends this half-life markedly and exacerbates the accumulation of α2u-globulin in the lysosomes of exposed male rats (Swenberg et al 1989;Lehman-McKeeman et al 1990;Frazier et al 2012).…”

Novel biosensors based on optimized glycine oxidase

“…the enzyme that degrades the neuromodulator D-serine in vivo [18]. The standard assay for glycine requires the derivatization of the amino acid followed by separation (and quantification) by means of HPLC on a C8 column, each run requiring 60 min.…”

“…Cellular amino acids were extracted as described previously [18]: 2.5 9 10 5 U87 cells were resuspended in 1 mL of ice-cold 5% trichloroacetic acid, sonicated and centrifuged for 45 min at 16 000 g. For the HPLC assay, the soluble fraction was extracted with water-saturated ether, neutralized and derivatized with o-phthaldialdehyde/N-acetyl-L-cysteine. Glycine was resolved by HPLC chromatography on a 5-lm Waters C8 reversed-phase column (Waters Corp., Milford, MA, USA) eluted under isocratic conditions (100 mM sodium acetate buffer, pH 6.2, 1% tetrahydrofuran at 1 mLÁmin À1 ; 60 min per run).…”

“…The novel sensing assay shows a detection limit ≤ 0.5 lM (slightly lower than commercial assay kits) and was used to assay glycine and sarcosine in biological fluid, i.e. in a U87 glioblastoma cell line used to study the modulation of D-serine level by the catabolic enzyme D-amino acid oxidase [18,25]; D-serine and glycine are alternative ligands required, together with glutamate, to activate NMDARs [21,26]. The glycine concentration detected by the H244K GO-based assay was in good agreement with the value obtained by using the reference HPLC method (7.5 versus 6.7 lM, respectively) but in 1 s, compared with the 60 min required by chromatographic separation.…”