title: Characterization of human DAAO variants potentially related to an increased risk of schizophrenia.
abstract: Considering the key role of d-serine in N-methyl-d-aspartate receptor-mediated neurotransmission, it is highly relevant to define the role that enzymes play in d-serine synthesis and degradation. In particular, the details of regulation of the d-serine catabolic human enzyme d-amino acid oxidase (hDAAO) are unknown although different lines of evidence have shown it to be involved in schizophrenia susceptibility. Here we investigated the effect of three single nucleotide polymorphisms and known mutations in hDAAO, i.e., D31H, R279A, and G331V. A very low amount of soluble G331V hDAAO is produced in E. coli cells: the recombinant variant enzyme is fully active. Human U87 glioblastoma cells transiently transfected for G331V hDAAO show a low viability, a significant amount of protein aggregates, and augmented apoptosis. The recombinant D31H and R279A hDAAO variants do not show alterations in tertiary and quaternary structures, thermal stability, binding affinity for inhibitors, and the modulator pLG72, whereas the kinetic efficiency and the affinity for d-serine and for FAD were higher than for the wild-type enzyme. While these effects for the substitution at position 31 cannot be structurally explained, the R279A mutation might affect the hDAAO FAD-binding affinity by altering the "structurally ambivalent" peptide V47-L51. In agreement with the observed increased activity, expression of D31H and R279A hDAAO variants in U87 cells produces a higher decrease in cellular d/(d+l) serine ratio than the wild-type counterpart. In vivo, these substitutions could affect cellular d-serine concentration and its release at synapsis and thus might be relevant for schizophrenia susceptibility.
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, Varese, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Caldinelli
authors.forename: Silvia
authors.initials: S
authors.lastname: Sacchi
authors.forename: Gianluca
authors.initials: G
authors.lastname: Molla
authors.forename: Marco
authors.initials: M
authors.lastname: Nardini
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
mesh_terms: D017209:Apoptosis / Q000235:genetics; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D002746:Chlorpromazine / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D003605:D-Amino-Acid Oxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D020022:Genetic Predisposition to Disease / Q000235:genetics*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D018613:Microscopy, Confocal; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D020125:Mutation, Missense; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012307:Risk Factors; D012559:Schizophrenia / Q000201:enzymology / Q000235:genetics*; D012694:Serine / Q000378:metabolism; D013696:Temperature
chemical_list: D002352:Carrier Proteins; C475071:DAOA protein, human; D047908:Intracellular Signaling Peptides and Proteins; D050505:Mutant Proteins; D005182:Flavin-Adenine Dinucleotide; D012694:Serine; D003605:D-Amino-Acid Oxidase; D020169:Caspases; D002746:Chlorpromazine