title: Structure-function relationships in human d-amino acid oxidase variants corresponding to known SNPs.
abstract: In the brain, d-amino acid oxidase plays a key role in modulating the N-methyl-d-aspartate receptor (NMDAR) activation state, catalyzing the stereospecific degradation of the coagonist d-serine. A relationship between d-serine signaling deregulation, NMDAR dysfunction, and CNS diseases is presumed. Notably, the R199W substitution in human DAAO (hDAAO) was associated with familial amyotrophic lateral sclerosis (ALS), and further coding substitutions, i.e., R199Q and W209R, were also deposited in the single nucleotide polymorphism database. Here, we investigated the biochemical properties of these different hDAAO variants. The W209R hDAAO variant shows an improved d-serine degradation ability (higher activity and affinity for the cofactor FAD) and produces a greater decrease in cellular d/(d+l) serine ratio than the wild-type counterpart when expressed in U87 cells. The production of H2O2 as result of excessive d-serine degradation by this hDAAO variant may represent the factor affecting cell viability after stable transfection. The R199W/Q substitution in hDAAO altered the protein conformation and enzymatic activity was lost under conditions resembling the cellular ones: this resulted in an abnormal increase in cellular d-serine levels. Altogether, these results indicate that substitutions that affect hDAAO functionality directly impact on d-serine cellular levels (at least in the model cell system used). The pathological effect of the expression of the R199W hDAAO, as observed in familial ALS, originates from both protein instability and a decrease in kinetic efficiency: the increase in synaptic d-serine may be mainly responsible for the neurotoxic effect. This information is expected to drive future targeted treatments.
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, via J. H. Dunant 3, 21100 Varese, Italy; The Protein Factory, Politecnico di Milano, ICMR-CNR, Università degli Studi dell'Insubria, via Mancinelli 7, 20131 Milano, Italy.
authors.forename: Pamela
authors.initials: P
authors.lastname: Cappelletti
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, via J. H. Dunant 3, 21100 Varese, Italy; The Protein Factory, Politecnico di Milano, ICMR-CNR, Università degli Studi dell'Insubria, via Mancinelli 7, 20131 Milano, Italy.
authors.forename: Luciano
authors.initials: L
authors.lastname: Piubelli
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, via J. H. Dunant 3, 21100 Varese, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Murtas
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, via J. H. Dunant 3, 21100 Varese, Italy; The Protein Factory, Politecnico di Milano, ICMR-CNR, Università degli Studi dell'Insubria, via Mancinelli 7, 20131 Milano, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Caldinelli
authors.affiliation: The Protein Factory, Politecnico di Milano, ICMR-CNR, Università degli Studi dell'Insubria, via Mancinelli 7, 20131 Milano, Italy; CNR, Istituto di Chimica del Riconoscimento Molecolare, Sezione Adolfo Quilico, via M. Bianchi 9, 20131 Milano, Italy.
authors.forename: Mattia
authors.initials: M
authors.lastname: Valentino
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, via J. H. Dunant 3, 21100 Varese, Italy; The Protein Factory, Politecnico di Milano, ICMR-CNR, Università degli Studi dell'Insubria, via Mancinelli 7, 20131 Milano, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Molla
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, via J. H. Dunant 3, 21100 Varese, Italy; The Protein Factory, Politecnico di Milano, ICMR-CNR, Università degli Studi dell'Insubria, via Mancinelli 7, 20131 Milano, Italy.
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, via J. H. Dunant 3, 21100 Varese, Italy; The Protein Factory, Politecnico di Milano, ICMR-CNR, Università degli Studi dell'Insubria, via Mancinelli 7, 20131 Milano, Italy. Electronic address: silvia.sacchi@uninsubria.it.
authors.forename: Silvia
authors.initials: S
authors.lastname: Sacchi
mesh_terms: D045744:Cell Line, Tumor; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D020641:Polymorphism, Single Nucleotide; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D008024:Ligands; D005182:Flavin-Adenine Dinucleotide; D003605:D-Amino-Acid Oxidase
keywords: Amyotrophic lateral sclerosis; Enzymatic activity; Flavoprotein; NMDAR-mediated neurotransmission; d-Amino acid oxidase; d-Serine metabolism