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Figure 1. Comparison of brain weight, protein concentration, and Sia concentration for the five different vertebrate brains. Brains from five different vertebrates (mouse, chicken, turtle, xenopus, goldfish, (n = 3)) were used. (A) Comparison of the brain weights of the five vertebrate brains. (B) Protein amount (g) per brain weight (g). Brains were homogenized using lysis buffer, and a bicinchoninic acid (BCA) assay was performed to determine the protein concentration. (C) Sia amount (mg) per protein amount (g). Brain homogenate was treated with a strong acid to achieve hydrolysis of sialylglycoconjugates. All Sia residues were completely released and labeled with 1,2-dimethylenedioxybenzen (DMB). Sia–DMB was separated using a Wako Handy octadecylsilyl (ODS) column (250 mm × 4.6 mm, Wako). The absolute amount of Sia was calculated based on the authentic Neu5Ac. * indicates p < 0.05. ** indicates p < 0.01. |
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Figure 2. SDS–PAGE/Western blotting of polySia–NCAM derived from five vertebrate brains. Brains from five vertebrates (mouse, chicken, turtle, xenopus, goldfish, n = 3) were used. PolySia expression was analyzed using SDS-PAGE/Western blotting. The brain homogenates were separated by SDS-PAGE (7% polyacrylamide gel) and blotted onto polyvinylidene difluoride (PVDF) membrane (10 µg as protein/lane). The polySia-NCAM was visualized using anti-polySia antibodies, (A) 12E3, (B) 735, and (C) 12F8. The left panel represents the immunoblots. The right panel shows the relative intensity of the anti-polySia antibody. Mouse staining was set to 1.0. * indicates p < 0.05. ** indicates p < 0.01. |
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Figure 3. Native-PAGE/Western blotting of polySia-NCAM in brains from five different vertebrates. Brains from five vertebrates (mouse, chicken, turtle, xenopus, goldfish, n = 3) were used. PolySia expression was analyzed using native-PAGE/Western blotting. The brain homogenates of five vertebrate brains were separated by native-PAGE (7% polyacrylamide gel) and blotted onto PVDF membranes (10 µg as protein/lane). The polySia-NCAM was visualized using anti-polySia antibodies, (A) 12E3, (B) 735, and (C) 12F8. The left panel represents the immunoblots. The right panel shows the relative intensity of the polySia antibody. Mouse staining was set to 1.0. * indicates p < 0.05. ** indicates p < 0.01. |
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Figure 4. SDS-PAGE/native-PAGE MAP. The S/N MAP of brains from five different vertebrates is described based on the results of blots with each antibody. The results of SDS-PAGE (Figure 2) and native-PAGE (Figure 3) were used. (A) The typical S/N map of 12E3, 735, and 12F8 staining. (B) Relative slope value, K. The value for mouse was set to 1.0. The left, middle, and right panels stand for 12E3, 735, and 12F8, respectively. The values were obtained from the S/N maps from five different vertebrate brains (n = 3). * indicates p < 0.05. ** indicates p < 0.01. |
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Figure 5. ELISA analysis of polySia-NCAM in brains from five different vertebrates. Brain homogenates (250 ng as protein) from five vertebrates (mouse, chicken, turtle, xenopus, goldfish, n = 3) were immobilized onto a 96-well plate and blocked with 2% BSA. The wells were then incubated with 12E3, 735, or 12F8 antibody before or after the Endo-N treatments. After color development, ELISA values of the Endo-N-treated wells were subtracted from those of the Endo-N-untreated wells. The left, middle, and right panels show the relative binding intensity of the 12E3, 735, and 12F8 antibodies, respectively. Triplicate analyses are shown with error bars. * indicates p < 0.05. ** indicates p < 0.01. |
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Figure 6. MH–FAEC analysis (Oligo-analysis) of brain homogenates from five vertebrate brains. Brains from five vertebrates (mouse, chicken, turtle, xenopus, goldfish, n = 1) were used. Brain homogenates (100 µg as protein) were subjected to mild acid hydrolysis followed by DMB derivatization. DMB-labeled oligo/polySia chains were applied to an anion exchange chromatography-HPLC analysis. |
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Figure 7. Fluorometric C7/C9 analysis of brain homogenates of five different vertebrate brains. Brains from five vertebrates (mouse, chicken, turtle, xenopus, and goldfish, n = 3) were used. Brain homogenates (20 µg as protein) were subjected to the fluorometric C7/C9 method to measure the average degree of polymerization (DP). (A) Typical chromatograms of C7/C9 analysis of each brain. (B) Relative (C7+C9)/C7 index in each brain. * indicates p < 0.05. ** indicates p < 0.01. Mouse value was set to 1.0. |
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Figure 8. Net negative charge analysis of polySia-NCAM and endo-N-treated NCAM from brain homogenates of five vertebrate brains. Brains from five vertebrates (mouse, chicken, turtle, xenopus and goldfish, n = 1) were analyzed using DEAE-Sephadex A-25 anion-exchange chromatography. (A) Elution profiles of polySia-NCAM and endo-N-treated NCAM. Crude samples or endo-N-treated samples (500 μg as protein) were applied to the column, and eluted in a step-wise manner using 1.5 mL of 2 mM Tris-HCl (pH 8.0) buffer containing 0.1% Triton and NaCl (0.2, 0.4, 0.6, 0.8, 1.0, and 3.0 M). Then, 10 μL of the sample from each fraction was analyzed by SDS-PAGE/Western blotting. Crude samples were analyzed for the majority of the polySia on NCAM using the anti-polySia antibody 12E3 (solid line), while the Endo-N-treated samples, i.e., oligoSia-NCAM, were analyzed using the anti-NCAM antibody (broken line). Flow-through (FT) and wash fractions comprised only 0.1% Triton and 2 mM Tris-HCl (pH 8.0) with 0.15 M NaCl. The sum of all fraction intensities detected by Western blotting was set to 1.0. (B) Pie charts of the distribution of negative charges represented as NaCl concentration for each animal. Based on the chromatograms (left panels) of the crude sample, the intensity of 12E3 staining from each of the indicated NaCl concentrations was summed, and expressed as its proportions (%) to the summation of the intensities for all NaCl concentrations used. Blue, pink, grey, yellow, and sky blue represent 0.4 M, 0.6 M, 0.8 M, 1.0 M, and 3.0 M, respectively. |
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Figure 9. Sephacryl S-500 chromatograms of polySia-NCAM of brain homogenates from five vertebrate brains. Brains from five different vertebrates (mouse, chicken, turtle, xenopus, and goldfish, n = 1) were analyzed by the gel filtration. The elution profiles were monitored by native-PAGE/Western blotting of each fraction using 12E3 (Figure S3A). The left, middle, and right panels for each animal brain show the elution profiles in the regions of HMW (above 720 kDa), MMW (480–720 kDa), and LMW (less than 480 kDa), respectively. The sum of the intensity of the immunostaining of the fractions was set to 1.0. At the bottom panels, merged images of the profiles for five different vertebrate brains are shown. |
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Figure 10. The polySia/Sia ratio and EQ value. The polySia/Sia ratio was calculated according to the intensity of anti-polySia-antibody per protein divided by the Sia amount per protein. (A) polySia/Sia ratio evaluated by SDS-PAGE/Western blotting. (B) PolySia/Sia ratio evaluated by native-PAGE/Western blotting. (C) The EQ value as a standard for evaluating the intelligence of vertebrates was calculated using an equation: EQ = K × [brain weight]/[body weight ]3/4 (K = 10, K was set to 10 for the convenience of calculation, although there were deviations among 8~16. * indicates p < 0.05. ** indicates p < 0.01. |
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Figure 11. The quantity and quality of polySia and the features of polySia-NCAM in five different vertebrate brains. PolySia-NCAM is highly regulated in both quantity and quality (DP, net negative charge, and size) among the five different vertebrate brains. |
