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. 2011 Jun 17;286(24):21535-45.
doi: 10.1074/jbc.M111.221143. Epub 2011 Apr 4.

Structural and functional impairments of polysialic acid by a mutated polysialyltransferase found in schizophrenia

Ryo Isomura  1 , Ken Kitajima, Chihiro Sato
Affiliations

Structural and functional impairments of polysialic acid by a mutated polysialyltransferase found in schizophrenia

Ryo Isomura et al. J Biol Chem. .

Abstract

Polysialic acid (polySia), a unique acidic glycan modifying neural cell adhesion molecule (NCAM), is known to regulate embryonic neural development and adult brain functions. Polysialyltransferase STX is responsible for the synthesis of polySia, and two single nucleotide polymorphisms (SNPs) of the coding region of STX are reported from schizophrenic patients: SNP7 and SNP9, respectively, giving STX(G421A) with E141K and STX(C621G) with silent mutations. In this study, we focused on these mutations and a binding activity of polySia to neural materials, such as brain-derived neurotrophic factor (BDNF). Here we describe three new findings. First, STX(G421A) shows a dramatic decrease in polySia synthetic activity on NCAM, whereas STX(C621G) does not. The STX(G421A)-derived polySia-NCAM contains a lower amount of polySia with a shorter chain length. Second, polySia shows a dopamine (DA) binding activity, which is a new function of polySia as revealed by frontal affinity chromatography for measuring the polySia-neurotransmitter interactions. Interestingly, the STX(G421A)-derived polySia-NCAM completely loses the DA binding activity, whereas it greatly diminishes but does not lose the BDNF binding activity. Third, an impairment of the polySia structure with an endosialidase modulates the DA-mediated Akt signaling. Taken together, impairment of the amount and quality of polySia may be involved in psychiatric disorders through impaired binding to BDNF and DA, which are deeply involved in schizophrenia and other psychiatric disorders, such as depression and bipolar disorder.

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Figures

FIGURE 1.
FIGURE 1.
In vitro enzyme activity of polysialyltransferases. A, in vitro enzyme activity of STX(WT), STX(G421A), STX(C621G), and PST(WT) toward NCAM-Fc. The enzyme reaction was carried out at 37 °C for 3 h in 10-μl reaction mixtures containing each enzyme, 50 mm MES buffer (pH 6.0), 10 mm MnCl2, 0.5% Triton CF-54, 10 μm CMP-[14C]Neu5Ac, and NCAM-Fc. A 5-μl aliquot was spotted on Whatman 3 MM paper and developed in ethanol, 1 m ammonium acetate (7:3, v/v), pH 7.5. After air-drying, the amount of incorporated [14C]Neu5Ac remaining at the origin was determined using a BAS 2000 imaging analyzer (Fuji Film, Tokyo, Japan). The data are expressed as the mean ± S.D. (error bars), with at least 3 replicates/experiment. The relative activity of each polysialyltransferase was determined by comparing with the activity of STX(WT), which was set to 100%. ***, p = 0.00022 (<0.001). B, in vitro enzyme activity of STX(WT), STX(G421A), and STX(C621G), in the presence of PST(WT), toward NCAM-Fc. The data are expressed as the mean ± S.D., with at least 3 replicates/experiment. The relative activity of each pair of polysialyltransferases was determined by comparing to the activity of STX(WT)/PST(WT), which was set to 100%. ***, significance at p = 0.00012 (<0.001). +, presence of enzyme. ++, presence of enzyme in a double amount. −, absence of enzyme.
FIGURE 2.
FIGURE 2.
PolySia formation on NCAM by STX(WT), STX(G421A), and STX(C621G) in the absence and presence of PST(WT) in the transfected cells. A, polySia-NCAM-Fc secreted from CHO cells stably expressing NCAM-Fc and each of STX(WT), STX(G421A), or STX(C621G) was purified from the culture medium. The purified polySia-NCAM-Fc was subjected to Western blotting with anti-polySia (12E3) and anti-NCAM (H-300). The relative intensity of polySia-NCAM immunostainings was densitometrically determined. The data are expressed as the mean ± S.D. (error bars), with 3 replicates/experiment. The relative intensity for STX(WT) was set to 100%. ***, significance at p = 0.00075 (<0.001). B, polySia-NCAM-Fc secreted from CHO cells stably expressing NCAM-Fc and each pair of PST(WT) and STX(WT), STX(G421A), or STX(C621G) was purified from culture medium and subjected to Western blotting as described in A. The relative intensity of the STX(WT)/PST(WT) pair was set to 100%. **, significance at p = 0.0011 (<0.01). +, presence of enzyme. −, absence of enzyme.
FIGURE 3.
FIGURE 3.
Mono Q anion exchange chromatography of polySia-NCAM-Fc synthesized by each STX/PST pair. polySia-NCAM-Fc secreted from CHO cells stably expressing NCAM-Fc and each of STX(WT), STX(G421A), or STX(C621G) in the presence of the PST(WT) was collected from culture medium using a protein G-Sepharose column and subjected to Mono Q anion exchange chromatography. The NaCl gradient is indicated in the graph by the dotted line. The eluate was collected and dot-blotted onto PVDF membrane, followed by immunostaining with anti-polySia (12E3) antibody. The polySia staining exhibited by each sample prior to anion exchange chromatography was set equal to 1.0. The 12E3-relative activity shows the amounts of polySia in each fraction. ■, polySia-NCAM-Fc derived from STX(WT); ●, polySia-NCAM-Fc derived from STX(G421A); ▴, polySia-NCAM-Fc derived from STX(C621G). The relationship between NC and the NaCl concentration at which polySia-NCAM-Fc was eluted is indicated in the figure. The relationship with log(NC) and NaCl concentration was already confirmed (28).
FIGURE 4.
FIGURE 4.
Interaction between polySia-NCAM and BDNF. Top, titration of BDNF with STX(WT)-derived polySia-NCAM (7–900 ng as BSA); middle, titration of BDNF with STX(G421A)-derived polySia-NCAM (7–900 ng as BSA); bottom, titration of BDNF with STX(C621G)-derived polySia-NCAM (7–900 ng as BSA). The principle of this method was well described (28). The calculated EC50 values are indicated below each gel. +, anode region; −, cathode region; >, loaded position. The BDNF-polySia-NCAM complex is boxed by a dotted line.
FIGURE 5.
FIGURE 5.
Interaction between polySia and selected neurotransmitters as measured by frontal affinity chromatography. A, elution profiles of various catecholamines after passing through columns with immobilized polySia. B, histamines. C, serotonins. The neurotransmitters were dissolved in PBS at concentrations of 10 μm (blue), 20 μm (pink), and 30 μm (yellow). Two ml of each solution was applied to the column (126 μl) through a 2-ml loop at a flow rate of 0.125 ml/min at 25 °C. Each elution pattern is superimposed on that of acetylcholine (black). The Kd values obtained by three independent experiments are summarized in Table 2.
FIGURE 6.
FIGURE 6.
Interaction between polySia-NCAM and DA measured by frontal affinity chromatography. Elution profiles of DA after application to a column of immobilized STX(WT)-derived polySia-NCAM (A), STX(G421A)-derived polySia-NCAM (B), and STX(C621G)-derived polySia-NCAM (C). DA was dissolved in PBS at concentrations of 10 μm (blue), 20 μm (pink), and 30 μm (yellow), and 2 ml of each solution was applied to the column (126 μl) through a 2-ml loop at a flow rate of 0.125 ml/min at 25 °C. Each elution pattern is superimposed on that of acetylcholine (black). The Kd values of STX(WT)- and STX(C621G)-derived polySia-NCAM were 8.6 and 19 μm, respectively. The Kd value of STX(G421A)-derived polySia-NCAM could not be calculated.
FIGURE 7.
FIGURE 7.
Effects of polySia expression on DA-induced Akt signaling. A, RT-PCR of STX, PST, NCAM, and D2DR. The cDNA derived from SK-N-SH cells was used as template for PCR that was performed with specific primers as described in supplemental Table S1. The PCR products were analyzed with 1% agarose gels and visualized with ethidium bromide. B, phosphorylation of Akt after the DA stimulation. SK-N-SH cells were treated with or without Endo-N (Endo-N(+) or Endo-N(−), respectively) or with boiled EndoN (bEndo-N(+)). The polysialylation state was analyzed by the immunostaining of polySia (top, polySia). The cells were then treated with DA for 0, 30, 60, and 120 min, and the homogenates were analyzed by Western blotting with anti-phosphorylated Akt antibody and anti-Akt antibody (middle and bottom, pAkt and Akt). The immunostainings were densitometrically measured, and the relative ratio of pAkt/Akt was plotted for each treatment (right): Endo-N(−) (filled squares), Endo-N(+) (filled circles), and bEndo-N(+) (filled triangles). The pAkt/Akt value at 0 min was set to 1.0. The data are expressed as the mean ± S.D. (error bars), with at least 3 replicates/experiment. ** and *, p < 0.01 and p < 0.1, respectively, compared with the case with Endo-N(−).
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