1 Supplementary Material
Characterization of Ac1GT1b and Ac2GT1b
The reaction with GT1b ganglioside yielded two products (Fig. 1A). The bands were named starting from the product with the lower Rf as Ac1GT1b and Ac2GT1b respectively. After their isolation using an Iatrobeads silica column, the number of transferred O-acetyl groups was determined with MALDI-TOF/MS. The spectrum of Ac1GT1b showed three peaks (m/z) of 2213.1, 2235.1 and 2241.1. The first two peaks (2213.1 and 2235.1) correspond to mono-O-acetylated GT1b, containing a ceramide with a long chain base (LCB) of 18:1 and fatty acid (F.A) of 18:0 in a monoanionic form with two sodiums (Na+) [M-2Na+-H]- and three sodiums [M-3Na+-H]-, respectively. The peak of 2241.1 corresponds to mono-O-acetylated GT1b, containing a ceramide with a LCB of 18:1 and F.A. of 20:0 in monoanionic form with two sodiums (Na+) [M-2Na+-H]-.
The position of the O-acetyl group in Ac1GT1b was characterized by negative electrospray-MS (Fig.S1A and S1B). Two species with three negative charges [M/3-H]=722.9 (Fig.S1A) and [M/3-H]=732.4 (Fig.S1B) corresponding to the species of mono-O-acetylated GT1b m=2168.7 that contains a ceramide with a LCB of 18:1 and F.A. of 18:0; and m=2197.2 that contains a ceramide with a LCB of 18:1 and F.A. of 20:0, respectively were fragmented. There are two low molecular weight peaks (m/z) of B0=290.2 and B1=332.2, corresponding to the sialic acid and the O-acetylated sialic
acid, respectively. The absence of O-acetylated forms of the fragments Y0
(Glcβ1-1’Cer), Y1 (Galβ1-4Glcβ1-1’Cer), Y2 (GalNAcβ1-4Galβ1-4Glcβ1-1’Cer) and Y3
(Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1’Cer) confirmed that the O-acetylation is in the sialic acid. The peaks of B2=581.28 and B3=623.28 were present in both spectra
2 fragment of two bound sialic acid with a single O-acetylation in one of them (O-acetyl-NeuAcα2-8NeuAc), respectively. These results suggest that Ac1GT1b might be a mixture of two isomers, one with the O-acetylation in one of the two bound sialic acids (O-acetyl-NeuAcα2-8NeuAc), and the second isomer with the O-acetylation in the sialic acid bound to galactose (O-acetyl-Neu5Acα2-3 Gal). The fragments Y4 and Y5.
correspond to gangliosides GM1a (Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1’Cer) or GM1b (Neu5Acα2-3Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-(Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1’Cer) and their O-acetylated forms respectively. The fragments Y6 and Y7 correspond to the gangliosides
GD1a (Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1’Cer) or GD1b Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1’Cer and their O-acetylated forms respectively. The fragments Y4, Y5. Y6 and Y7 confirm that Ac1GT1b
is a mix of two isomers. The digestion of Ac1GT1b with neuraminidase from Clostridium perfringens showed one band with the same Rf as GM1a (Fig.S1C), suggesting that the inner sialic acid bound to the other sialic acid (NeuAcα2-8NeuAc) was not O-acetylated. In conclusion, the two isomers of Ac1GT1b might be [Ac-O-Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1’Cer] and [Neu5Acα2-3Galβ1-3GalNAcβ1-4(Ac-O-Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1’Cer].
The spectrum of Ac2GT1b showed three peaks (m/z) of 2255.0, 2261.1 and 2283.1. The first peak of 2255.0 corresponds to di-O-acetylated GT1b, containing a ceramide with a long chain base (LCB) of 18:1 and fatty acid (F.A) of 18:0 in a monoanionic form with two sodiums (Na+) [M-2Na+-H]-. The last two peaks of 2261.1 and 2284.1 correspond to di-O-acetylated GT1b that contains a ceramide with a LCB of 18:1 and F.A. of 20:0 in monoanionic form with one sodium [M-Na+-H]- and two
3 The characterization of the position of the two O-acetyl groups in Ac2GT1b by negative electrospray-MS (Fig.S1D and S1E) showed that the O-acetylated sialic acids were the same as the isomers of Ac1GT1b [Ac-O-Neu5Acα2-3 Galβ1-3GalNAcβ1-4(Ac-O-Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1’Cer].
Characterization of PrGD1b
We tested whether SOAT could O-propionylate the GD1b ganglioside using Propionyl Coenzyme A as substrate. The reaction rendered a new band with a different Rf from GD1b (Fig.1C) with a yield of about 15%. After the isolation of this band with an Iatrobeads silica column, the number of transferred O-propionyl groups was determined with MALDI-TOF/MS. The spectrum of O-propionylGD1b (PrGD1b) showed two peaks (m/z) of 1942.4 and 1914.4, corresponding to mono-O-propionylated GD1b, containing a ceramide with a long chain base (LCB) of 18:1 and fatty acid (F.A) of 20:0 in a monoanionic form with one sodium (Na+) [M-Na+-H]- and a
mono-O-propionylated GD1b, containing a ceramide with a long chain base (LCB) of 18:1 and fatty acid (F.A) of 18:0 in a monoanionic form with one sodium (Na+) [M-Na+-H]-, respectively.
The position of the O-propionyl group in PrGD1b was characterized by negative electrospray-MS (Fig.S2A and S2B). Two species with two negative charges [M/2-H]=960.6 (Fig.S2A) and [M/2-H]=946.2 (Fig.S2B) corresponding both to the mono-O-propionylated GD1b m=1921.2, containing a ceramide with a LCB of 18:1 and F.A of 20:0; and m=1893.2, containing a ceramide with a LCB of 18:1 and F.A of 18:0, respectively were fragmented. There are two low molecular peaks (m/z) of B0=290.2
and B1=346.2 that correspond to the sialic acid and the O-propionylated sialic acid,
4 Y1 (Galβ1-4Glcβ1-1’Cer), Y2 (GalNAcβ1-4Galβ1-4Glcβ1-1’Cer) and Y3
(Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1’Cer) confirms that the O-propionylation is only in the sialic acid. The peak of B2=637.3 present in both spectra corresponds to a fragment of
two bound sialic acids with a single O-propionylation in one of them (O-propionyl-NeuAcα2-8NeuAc). The fragment Y4 that corresponded to the ganglioside GM1a
(Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1’Cer), confirmed that the inner sialic acid was not O-propionylated. In conclusion, the PrGD1b is the GD1b ganglioside with one O-propionylation in the outer sialic acid [Galβ1-3GalNAcβ1-4(Pr-O-Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1’Cer].
Figure S1: Negative electrospray characterization of the products obtained in the O-acetylation of GT1b ganglioside with SOAT. Negative electrospray spectra of (A) mono-O-acetyl-GT1b ganglioside (Ac1GT1b) that contains a ceramide with a LCB of 18:1 and F.A. of 18:0 (d18:1/18:0) and (B) mono-O-acetyl-GT1b ganglioside (Ac1GT1b) that contains a ceramide with a LCB of 18:1 and F.A. of 20:0 8 (d18:1/20:0). Inset, the fragmentation schemes of the two mono-O-acetyl-GT1b species obtained in the reaction with SOAT. (C) TLC of GT1b and Ac1GT1b after neuraminidase and alkaline treatment compared to standard gangliosides (GM3, GM2, GM1a, GD1a, GD1b and GT1b), Stds. We used alkaline hydrolysis with sodium hydroxide and digestions with neuraminidase to determine the position of the O-acetylation in Ac1GT1b. Alkaline hydrolysis of Ac1GT1b reduced its Rf in TLC to the
same Rf as the GT1b ganglioside and confirmed that Ac1GT1b was O-acetylated
(compare lanes 5 and 6). Neuraminidase from Clostridium perfringens could hydrolyze only both outer sialic acids of ganglioside GT1b and not the sialic acid bound to the galactose close to the ceramide (Rauvala, 1979), increasing the Rf of GT1b to the same
5 neuraminidase digestion plus alkaline hydrolysis (lane 8) increased the Rf value of
Ac1GT1b to the same Rf as the GM1a ganglioside, demonstrating that Ac1GT1b was
not O-acetylated in the inner sialic acid bound to the galactose close to the ceramide. In conclusion, the enzymatic reaction produced a mixture of two isomers, corresponding to AcGT1b with a single O-acetylation 4(Ac-O-Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1’Cer] and [Neu5Acα2-3Galβ1-3GalNAcβ1-4(Ac-O-Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1’Cer]. Neuraminidase digestion of both GT1b (lane 3) and Ac1GT1b (lane 7) or neuraminidase digestion plus alkaline hydrolysis of both GT1b (lane 4) and Ac1GT1b (lane 8) gives only GM1a as product, demonstrating that Ac1GT1b is not O-acetylated in the inner alpha-2,3 bound sialic acid to the galactose of lactosyl-ceramide. Negative electrospray spectra of (D) di-O-acetylated-GT1b ganglioside (Ac2GT1b) that contains a ceramide with a LCB of 18:1 and F.A. of 18:0 (d18:1/18:0) and (E) di-O-acetylated-GT1b ganglioside (Ac2GT1b) that contains a ceramide with a LCB of 18:1 and F.A. of 20:0 8 (d18:1/20:0). Inset, the fragmentation schemes of di-O-acetyl-GT1b.
Figure S2: Negative electrospray characterization of the products obtained in the O-propionylation of GD1b ganglioside with SOAT. Negative electrospray spectra of (A) mono-O-propionyl-GD1b ganglioside (PrGD1b) that contains a ceramide with a LCB of 18:1 and F.A. of 18:0 (d18:1/18:0) and (B) mono-O-propionyl-GD1b ganglioside (PrGD1b) that contains a ceramide with a LCB of 18:1 and F.A. of 20:0 8 (d18:1/20:0). Inset, the fragmentation scheme of mono-O-propionyl-GD1b.
Figure S3: MTT assay of (A) BV-2 cells and (B) rat microglia. Cells were pretreated with different concentrations of neurostatin for 30 minutes and then treated with LPS (400 ng/ml for BV-2 cells and 200 ng/ml for rat microglia) for 24 hours. The results are
6 (A) the mean ± SEM of three experiments in triplicates for BV-2 cells and (B) the mean ± SEM of four experiments in duplicates for rat microglia.
References
Rauvala, H., 1979. Monomer-micelle transition of the ganglioside GM1 and the hydrolysis by Clostridium perfringens neuraminidase. Eur J Biochem 97, 555-564.
7
B A
8 C Figure S1
Stds 1 2 3 4 5 6 7 8
NaOH
- + - + - + - +
Neuram
. - - + + - - + +
GT1b Ac1GT1b
GM3 GT1b GM2 GM1a GD1a GD1b9 D
Figure S1
10 REFERENCES
Rauvala, H., 1979. Monomer-micelle transition of the ganglioside GM1 and the hydrolysis by Clostridium perfringens neuraminidase. Eur J Biochem 97, 555-564.
B A
11 Figure S3 A B
