D-amino acid oxidase activity is inhibited by an interaction with bassoon protein at the presynaptic active zone - PubMed (original) (raw)
D-amino acid oxidase activity is inhibited by an interaction with bassoon protein at the presynaptic active zone
Michael Popiolek et al. J Biol Chem. 2011.
Abstract
Schizophrenia is a highly heritable neuropsychiatric disorder affecting ∼1% of the world's population. Linkage and association studies have identified multiple candidate schizophrenia susceptibility genes whose functions converge on the glutamatergic neurotransmitter system. One such susceptibility gene encoding D-amino acid oxidase (DAO), an enzyme that metabolizes the NMDA receptor (NMDAR) co-agonist D-serine, has the potential to modulate NMDAR function in the context of schizophrenia. To further investigate its cellular regulation, we sought to identify DAO-interacting proteins that participate in its functional regulation in rat cerebellum, where DAO expression is especially high. Immunoprecipitation with DAO-specific antibodies and subsequent mass spectrometric analysis of co-precipitated proteins yielded 24 putative DAO-interacting proteins. The most robust interactions occurred with known components of the presynaptic active zone, such as bassoon (BSN) and piccolo (PCLO). The interaction of DAO with BSN was confirmed through co-immunoprecipitation assays using DAO- and BSN-specific antibodies. Moreover, DAO and BSN colocalized with one another in cultured cerebellar granule cells and in synaptic junction membrane protein fractions derived from rat cerebellum. The functional consequences of this interaction were studied through enzyme assay experiments, where DAO enzymatic activity was significantly inhibited as a result of its interaction with BSN. Taking these results together, we hypothesize that synaptic D-serine concentrations may be under tight regulation by a BSN-DAO complex. We therefore predict that this mechanism plays a role in the modulation of glutamatergic signaling through NMDARs. It also furthers our understanding of the biology underlying this potential therapeutic entry point for schizophrenia and other psychiatric disorders.
Figures
FIGURE 1.
Characterization of a DAO-specific polyclonal antibody. A, an anti-DAO antibody revealed two immunoreactive bands at ∼37 and 40 kDa in immunoblots of lysates from a stable HEK293 cell line, expressing hDAO (lane 1), but not of control HEK293 lysates (lane 2). The anti-DAO antibody detected a single immunoreactive band at ∼39 kDa in immunoblots of rat cerebellar lysates (lane 3) but not of lysates from rat spleen (lane 4), the latter of which is not expected to express DAO. B, when the anti-DAO antibody was preincubated with the immunizing peptide, no immunoreactive bands were detected in lysates of either the hDAO stable line or rat cerebellum (lanes 1 and 3). C, anti-DAO, covalently coupled to Dynal magnetic beads, immunoprecipitated (IP) DAO protein from rat cerebellar lysates, as determined by Western blotting (IB) with anti-DAO antibody (lane 1). In contrast, DAO was not immunoprecipitated when anti-DAO was preincubated with the immunogenic peptide (lane 2) or when beads were covalently coupled to nonspecific rabbit IgG in place of anti-DAO (lane 3). Rat cerebellar lysate (Input; lane 4) was used at one-tenth the volume to verify that DAO was present in starting material. D, DAO and BSN coimmunoprecipitate with one another from rat cerebellar lysates. BSN was immunoprecipitated from rat cerebellar lysates by both anti-BSN (lane 1) and anti-DAO (lane 3) antibodies but not by nonspecific mouse (lane 2) or rabbit (lane 4) IgG controls. DAO was immunoprecipitated from rat cerebellar lysates by both anti-BSN (lane 1) and anti-DAO (lane 3) antibodies but not by nonspecific mouse (lane 2) or rabbit (lane 4) IgG controls.
FIGURE 2.
Determination of the DAO-binding domain within BSN. Shown is a schematic BSN diagram depicting the functional domains: double zinc finger domains (Zn1 and Zn2) and predicted coiled-coil regions (cc1, cc2, and cc3). A stable HEK293 cell line expressing DAO was transfected with either BSN 95–3938 (A), BSN 95–609 (B), BSN 95–3263 (C), BSN 1692–3263 (D), or BSN 2715–3263 (E), each fused to GFP at its N terminus. Lysates were subjected to immunoprecipitation (IP) with a mouse anti-GFP antibody (lane 3) or nonspecific mouse IgG (lane 2) and immunoblotted with an anti-GFP antibody (top) or an anti-DAO antibody (bottom). The combined data suggest that the domain required for the interaction with DAO resides between amino acids 2715 and 3263 of the BSN polypeptide.
FIGURE 3.
DAO and BSN, but not catalase, cofractionate in a cerebellar synaptic junction membrane protein fraction. A, schematic diagram of the fractionation protocol employed to determine the subcellular localization of DAO in rat cerebellum. B, DAO is present in a fraction containing synaptic junction membrane proteins (Sjmp), including PSD-95 and BSN but not catalase. Catalase, a peroxisomal marker, was not detected in the Sjmp fraction even when the immunoblot was overexposed, confirming that the DAO detected in this fraction is not derived from peroxisomal membranes. Due to a low yield, the Sjmp sample was loaded at half the amount found in all of the other lanes. SnpF, synaptosomal fraction.
FIGURE 4.
DAO partially colocalizes with BSN in cultured cerebellar granule neurons. CGNs were cultured for 2 weeks, fixed with either paraformaldehyde (A) or methanol (B), and subjected to double-label immunofluorescence microscopy with rabbit anti-DAO (red) and mouse anti-BSN (green) antibodies. Images of neuronal processes were collected with a 100× oil immersion objective. Arrows, anti-DAO immunofluorescent puncta that colocalize with that of anti-BSN; arrowheads, anti-DAO immunofluorescent puncta that do not colocalize with that of anti-BSN. Crossed arrows, anti-BSN immunofluorescent puncta that do not colocalize with that of anti-DAO. Colocalizing puncta appear yellow in the merged images (right panels). Size bar, 2 μm.
FIGURE 5.
BSN inhibition of DAO enzymatic activity. A stable HEK293 cell line expressing DAO was transfected with cDNA encoding GFP-BSN 2715–3263 at a concentration of 20, 100, or 500 ng/30,000 cells. A, BSN 2715–3263 protein expression increased with increasing cDNA concentrations, as measured by GFP immunoreactivity on Western blots. Neither DAO nor GAPDH protein expression was altered by the BSN 2713–3263 expression. B, DAO enzymatic activity decreased in concert with increasing BSN 2713–3263 expression relative to GFP-transfected DAO HEK293 cells. *, p < 0.05; ***, p < 0.001 by one-way analysis of variance followed by Dunnett's multiple comparison against GFP-transfected cells.
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