Biochemical studies on sphingolipids of Artemia franciscana: novel neutral glycosphingolipids

Abstract

Neutral glycosphingolipids containing one to six sugars in their oligosaccharide chains have been isolated from cysts of the brine shrimp Artemia franciscana. The structures of these glycolipids were identified by methylation analysis, partial acid hydrolysis, gas-liquid chromatography, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and proton nuclear magnetic resonance spectroscopy to be Glcβ1-Cer, Manβ1-4Glcβ1-Cer, Fucα1-3Manβ1-4Glcβ1-Cer, GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GlcNAcα1-2Fucα1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer (CPS), and GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer (CHS). Two glycosphingolipids, CPS and CHS, were characterized as novel structures. Because Artemia contains a certain series of glycosphingolipids (-Fucα3Manβ4GlcβCer), which differ from the core sugar sequences reported thus far, we tentatively designated the glycosphingolipids characterized as nonarthro-series ones. Furthermore, CHS exhibited a hybrid structure of arthro-series and nonarthro-series sugar chain. Two novel glycosphingolipids were characterized from the brine shrimp Artemia franciscana; one was composed of arthrotetraose and a branching fucose attached to N-acetylglucosamine residue, and the other was composed of CPS with an additional N-acetylglucosamine residue attached to the branching fucose.

Fig. 1.

Thin-layer chromatogram of neutral glycosphingolipids isolated from the brine shrimp Artemia franciscana. Lane 1, total neutral glycosphingolipid fraction obtained by QAE-Sephadex A-25 and Florisil column chromatographies; lanes 2 to 9, isolated CMS, CDS, nAtCTS, AtCTS, nAtCTeS, AtCTeS, CPS, and CHS, respectively. The HPTLC plate was developed in chloroform-methanol-water (60:40:10, v/v/v). The spots were visualized by orcinol-sulfuric acid reagent.

Fig. 2.

Gas chromatograms of partially methylated alditol acetates derived from the isolated GSLs. A: CMS; B: CDS; C: nAtCTS; D: AtCTS; E: nAtCTeS; F: AtCTeS; G: CPS; H: CHS; a: 1Glc; b: 1Man; c: 1,4Glc; d: 1Fuc; e: 1,4Glc and 1,3Man; f: 1GlcNAc; g: 1,2Fuc; h: 1GalNAc; i: 1,4GlcNAc; j: 1,3,4GlcNAc.

Fig. 3.

Thin-layer chromatogram of hydrolyzates of CHS with HCl. 1, intact CHS; 2, hydrolyzates before separation; 3, product with mono-saccharide residue (CHS-M); 4, product with di-saccharide residue (CHS-D); 5, product with tri-saccharide residue (CHS-T); 6, product with tetra-saccharide residue (CHS-Q); 7, remaining CHS. The plate was developed with chloroform-methanol-water (60:40:10, v/v/v). Spots were visualized with orcinol-sulfuric acid reagent.

Fig. 4.

Positive-ion reflector mode MALDI-TOF MS spectra of the isolated GSLs. A: CMS; a: [M+Na]⁺ ion at m/z 722.78; b: [M+Na]⁺ ion at m/z 738.77; c: [M+Na]⁺ ion at m/z 778.86; d: [M+Na]⁺ ion at m/z 792.87; B: CDS; a: [M+Na]⁺ ion at m/z 884.62; b: [M+Na]⁺ ion at m/z 898.64; c: [M+Na]⁺ ion at m/z 940.69; d: [M+Na]⁺ ion at m/z 954.70; C: nAtCTS; a: [M+Na]⁺ ion at m/z 1030.67; b: [M+Na]⁺ ion at m/z 1044.68; c: [M+Na]⁺ ion at m/z 1086.73; d: [M+Na]⁺ ion at m/z 1100.74; D: AtCTS; a: [M+Na]⁺ ion at m/z 1143.69; b: [M+Na]⁺ ion at m/z 1157.69; E: nAtCTeS; a: [M+Na]⁺ ion at m/z 1233.88; b: [M+Na]⁺ ion at m/z 1247.89; c: [M+Na]⁺ ion at m/z 1289.95; d: [M+Na]⁺ ion at m/z 1303.96; F: AtCTeS; a: [M+Na]⁺ ion at m/z 1290.78; b: [M+Na]⁺ ion at m/z 1304.80; c: [M+Na]⁺ ion at m/z 1346.84; d: [M+Na]⁺ ion at m/z 1360.85; G: CPS; a: [M+Na]⁺ ion at m/z 1493.18; b: [M+Na]⁺ ion at m/z 1507.16; H: CHS; a: [M+Na]⁺ ion at m/z 1695.54; b: [M+Na]⁺ ion at m/z 1709.55.

Fig. 5.

Anomeric proton regions of the ¹H-NMR spectra of the isolated GSLs. A: CMS; B: CDS; C: nAtCTS; D: AtCTS; E: nAtCTeS; F: AtCTeS; G: CPS; H: CHS; I: Glcβ; II: Manβ; III: Fucα; IV: GlcNAcβ; V: GlcNAcα; VI: GalNAcβ; *, Fuc5H.