Biochemical studies on sphingolipids of Artemia franciscana: complex neutral glycosphingolipids

Abstract

Brine shrimp are primitive crustacean arthropodal model organisms, second to daphnia, which can survive in high-salinity environments. Their oviposited cysts, cuticle-covered diapausing eggs, are highly resistant to dryness. To elucidate specialties of brine shrimp, this study characterized glycosphingolipids, which are signal transduction-associated material. A group of novel and complex fucosyl glycosphingolipids were separated and identified from cysts of the brine shrimp Artemia franciscana by repeated lipid extraction, alkaline methanolysis, acid treatment, successive column chromatography, and post-source decay measurements by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Structures of the glycosphingolipids were elucidated by conventional structural characterization and mass spectrometry, and the compounds were identified as GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, and GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer. These compounds also contained a branching, non-arthro-series disaccharide with an α-GlcNAc terminus, similar to that found in a previously reported ceramide hexasaccharide (III(3)(GlcNAcα2Fucα)-At4Cer). The glycans within these complex GSLs are longer than reported glycans of the animal kingdom containing α-GlcNAc terminus. These complex GSLs as well as the longest GSL with ten sugar residues, ceramide decasaccharide (CDeS), contain the fucosylated LacdiNAc sequence reported to associate with parasitism/immunosuppression and the α-GlcNAc terminus reported to show a certain antibacterial effect in other reports. CDeS, the longest GSL of this species, was found in the highest amount, which indicates that CDeS may be functionally important.

Fig. 1

A thin-layer chromatogram shows the separation of neutral glycosphingolipids separated from the brine shrimp A. franciscana. a Fractionation by linear gradient elution. Lane 1, total neutral glycosphingolipid fraction; lanes 2–9, previously reported CMS, CDS, nAtCTS, AtCTS, nAtCTeS, AtCTeS, CPS, and CHS, respectively; lane 10, CHpS₁; lane 11, CHpS₂; lane 12, COS₁; lane 13, mixture of COS₂, CNS, and CDeS; lane 14, CDeS. b Fractionation by stepwise elution. Lane 1, total neutral glycosphingolipid fraction; lanes 2 and 3, nonpolar material fraction; lane 4, CMS; lane 5, CMS and CDS; lane 6, nAtCTS, AtCTS, and nAtCTeS; lane 7, AtCTeS; lane 8, CPS and CHS; lane 9, CHpS₁, CHpS₂, and COS₁; lane 10, mixture of COS₂, CNS, and CDeS; lanes 11 and 12, non-GSL fractions. c Purification of the fraction containing COS₂, CNS, and CDeS by Iatrobeads column chromatography using ammoniacal propanol. Lane 1, total neutral glycosphingolipid fraction; lane 2, before fractionation; lane 3, non-GSL fraction; lane 4, CDeS; lane 5, CNS; lane 6, COS₂. The HPTLC (a and c) and TLC (b) plates were developed in (a and b) C/M/W (60:40:10, v/v/v), (c) 1-propanol/water/ammonium hydroxide (70:30:5, v/v/v) or C/M/W (55:45:10, v/v/v). The spots were visualized by orcinol- H₂SO₄ reagent

Fig. 2

Gas chromatograms of partially methylated alditol acetates derived from the separated GSLs. (a) CHpS₁; (b) CHpS₂; (c) COS₁; (d) COS₂; (e) CNS; (f) CDeS; A, 1,2Fuc; B, 1,4Glc and 1,3Man; C, 1GlcNAc; D, 1GalNAc; E, 1,3GlcNAc; F, 1,3,4GlcNAc; G, 1,3GalNAc; H, 1,4GlcNAc; I, 1Fuc, *, contaminant of phthalic acid-like material

Fig. 3

Positive-ion reflector mode MALDI-TOF MS spectra of the separated GSLs. a CHpS₁; A, [M+Na]⁺ ion at m/z 1899.47; B, [M+Na]⁺ ion at m/z 1913.42; b CHpS₂; A, [M+Na]⁺ ion at m/z 1898.82; B, [M+Na]⁺ ion at m/z 1912.78; c COS₁; A, [M+Na]⁺ ion at m/z 2101.84; B, [M+Na]⁺ ion at m/z 2115.81; d COS₂; A, [M+Na]⁺ ion at m/z 2044.96; B, [M+Na]⁺ ion at m/z 2058.95; e CNS; A, [M+Na]⁺ ion at m/z 2248.38; B, [M+Na]⁺ ion at m/z 2262.31; f CDeS; A, [M+Na]⁺ ion at m/z 2451.45; B, [M+Na]⁺ ion at m/z 2465.42

Fig. 4

Representative positive-ion PSD spectra in MALDI-TOF MS of the separated GSLs. a CHpS₁; b CNS. Black arrows indicate mass differences between fragments with a ceramide molecular group. Gray arrows indicate mass differences between fragments without a ceramide molecular group. All fragments were detected as sodium adducts. The spectrum of b was expanded by a factor of 5 in the low-molecular-weight region

Fig. 5

Anomeric proton regions of the ¹H-NMR spectra of the separated GSLs. a CHpS₁; b CHpS₂; c COS₂; d CNS; e CDeS; I: Glcβ; II: GalNAcβ; III: Manβ; IV: GlcNAcβ; V: Fucα and GlcNAcα; VI: Fucα; *: FucH5

Fig. 6

Summary of the putative biosynthetic pathway of non-arthro and arthro-series neutral GSLs in the cyst of the brine shrimp