Elucidating the role of the pLG72 R30K substitution in schizophrenia susceptibility

Abstract

In the human brain, pLG72 interacts with the flavoenzyme d-amino acid oxidase (hDAAO), which is involved in catabolism of d-serine, a co-agonist of N-methyl-d-aspartate receptors (NMDAR). Here, we investigated the wild-type pLG72, the R30K variant associated with schizophrenia susceptibility, and the K62E variant. The protein conformation, oligomeric state, ligand-, and hDAAO-binding properties are only slightly modified by the substitutions. All pLG72 variants inhibit hDAAO and lead to an increase in cellular (d/d+l)-serine. However, the R30K pLG72 is significantly more prone to degradation than the R30 and the K62E variants in a cell system, thus possessing a lower ability to interact/inhibit hDAAO. This links R30K pLG72 with the hyperactivity of hDAAO, the decreased d-serine level, and NMDAR hypofunction observed in schizophrenia-affected patients.

Biochemical Properties of Human D-Amino Acid Oxidase

“…These ligands do not alter the far-UV CD spectrum of the flavoenzyme (not shown). Notably, we previously demonstrated that pLG72, the specific hDAAO modulator (Sacchi et al, 2008 ), also interacts with nucleotide triphosphates although no ATPase/GTPase activity was assayed (Sacchi et al, 2017 ). Indeed, hDAAO activity on D-alanine (at 2 or 28 mM final concentration) is not modified by the simultaneous presence of 10 mM magnesium ions and ATP or GTP (10 μM).…”

D-Amino Acid Oxidase-pLG72 Interaction and D-Serine Modulation

“…In past years, we produced recombinant wild-type, R30K (the SNP associated with schizophrenia susceptibility), and the K62E variant of pLG72 (SNP rs9558562) in E. coli cells as inclusion bodies, reaching ≈100 mg/L of fermentation broth (Molla et al, 2006a ; Sacchi et al, 2017 ). In all cases, the recombinant pLG72 proteins were refolded using the anionic detergent N-lauroylsarcosine, a reducing agent and at a basic pH-value.…”

“…Correctly folded pLG72 showed an absorbance maximum centered at 267 nm. The detergent affects the oligomeric state of pLG72: the protein was monodispersed at a detergent concentration ≥0.025%; under optimal conditions, all the pLG72 variants were dimeric (Molla et al, 2006a ; Sacchi et al, 2017 ). The substitutions R30K and K62E only slightly modified the protein conformation, yielding a moderately more rigid conformation than for the previous one (T m was 3–8°C higher than that of wild-type pLG72).…”

“…pLG72 binds aromatic compounds, i.e., the cofactors FAD, FMN, and riboflavin, the antipsychotic drug chlorpromazine (CPZ), the nucleotides GDP/GTPγS, and the sugar β-D-galactopyranoside, see Table 1 (Chumakov et al, 2002 ; Sacchi et al, 2008 , 2017 ). It was previously proposed that the activity of FMN-containing oxidoreductase of complex I in mitochondria is regulated by pLG72 binding (Otte et al, 2011 ): as the K d values for FMN/FAD were similar for all pLG72 variants (Sacchi et al, 2017 ), we exclude that the R30K substitution favors such an interaction. Notwithstanding the ability to bind nucleotides, the pLG72 variants did not show GTPase or ATPase activity (Sacchi et al, 2017 ).…”

Competitive Inhibitors Unveil Structure/Function Relationships in Human D-Amino Acid Oxidase

“…Recently, by applying low-resolution techniques (i.e., limited proteolysis couple to mass spectroscopy and cross-linking experiments) structural determinants of the proteins involved in the formation of the interface surface in the complex could be mapped. This study proposed a model of the hDAAO-pLG72 complex in which the N-terminal region of the protein plays an important role in forming the oligomerization interface (Birolo et al, 2016 ; Sacchi et al, 2017 ). Binding of pLG72 to hDAAO in vitro does not alter the kinetic parameters on D-Ser and the affinity for the FAD cofactor of hDAAO but rather increases the rate of hDAAO inactivation.…”