Novel myelin penta- and hexa-acetyl-galactosyl-ceramides: structural characterization and immunoreactivity in cerebrospinal fluid

Abstract

Fast migrating cerebrosides (FMC) are derivatives of galactosylceramide (GalCer). The structures of the most hydrophobic FMC-5, FMC-6, and FMC-7 were determined by electrospray ionization linear ion-trap mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy complementing previous NMR spectroscopy and gas chromatography-mass spectrometry to be 3-O-acetyl-sphingosine-GalCer derivatives with galactose O-acetyl modifications. FMC-5 and FMC-6 are 3-O-acetyl-sphingosine-2,3,4,6-tetra-O-acetyl-GalCer with nonhydroxy and hydroxy-N-fatty-acids, while FMC-7 has an additional O-acetylation of the 2-hydroxy-fatty acid. The immuno-reactivity in human cerebrospinal fluid (CSF) to these acetylated glycolipids was examined in central nervous system (CNS) infectious disease, noninflammatory disorders, and multiple sclerosis (MS). Screening for lipid binding in MS and other neurological disease groups revealed that the greatest anti-hydrophobic FMC reactivity was observed in the inflammatory CNS diseases (meningitis, meningo-encephalitis, and subacute sclerosing panencephalitis). Some MS patients had increased reactivity with the hydrophobic FMCs and with glycoglycerophospholipid MfGL-II from Mycoplasma fermentans. The cross-reactivity of highly acetylated GalCer with microbial acyl-glycolipid raises the possibility that myelin-O-acetyl-cerebrosides, bacterial infection, and neurological disease are linked.

Fig. 1.

Primary structure of different glycolipids examined in this study. A: Diagram represents endogenous antigens, such as GalCer (R1 = H, R2 = H, R3−R6 = H) (1); FMC-7 (R1 = OAc, R2−R6 = Ac) (structure described here); and sulfatide (R1-R4 = H, R5 = HSO⁻₄, R6 = H) (33). R1 and R2 represent hydroxy and nonhydroxy fatty acyl groups, respectively. B: Mycoplasma fermentans glycolipid MfGL-II (19). C: LPS from Escherichia coli J5 (34). Abbreviations: FMC, fast migrating cerebroside; GalCer, galactosylceramide; GlcNAc, N-acetylglucosamine; Hep, L-glycero-D-manno-heptopyranose; Kdo, 3-deoxy-D-manno-octulopyranosonic acid; LPS, lipopolisaccharide.

Fig. 2.

Thin layer chromatogram of the purified fast migrating cerebrosides FMC-5, FMC-6, and FMC-7. Plates (5 × 20 cm) were resolved in A: chloroform:hexane:methanol 75:18:1.2 (v/v/v); B: chloroform:hexane:methanol:glacial acetic acid 75:18:1.2:0.6 (v/v/v/v) and the bands were visualized with DPA-aniline spray. Lane 1 is FMC-7; lane 2, FMC-6; lane 3, FMC-5; and lane 4, a mixture of FMC-5, -6, and -7. Abbreviations: DPA, diphenylamine-aniline; FMC, fast migrating cerebroside.

Fig. 3.

⁺ESI-LTQ-MS¹ (ThermoFisher LTQ) molecular adduct profiles acquired for [M + Li]⁺ adducts of A: rat brain FMC-5, -6, -7; B: chemically acetylated mixed GalCer fraction from bovine brain; and C: chromatographically purified rat brain FMC-7. Abbreviations: ESI-LTQ-MS, electrospray ionization-linear ion trap-mass spectrometry; FMC, fast migrating cerebroside; GalCer, galactosylceramide.

Fig. 4.

⁺ESI-LTQ-MS¹ (ThermoFisher LTQ) [M + Li]⁺ adduct profiles acquired for three fractions (Fr.) isolated by silicic acid chromatography from the same chemically acetylated bovine brain GalCer that yielded the profile in Figure 3B. A: Fr. 225-246 (FMC-5); B: Fr. 168-186; and C: Fr. 56-86 (FMC-7). Abbreviations: ESI-LTQ-MS, electrospray ionization-linear ion trap-mass spectrometry; FMC, fast migrating cerebroside; GalCer, galactosylceramide.

Fig. 5.

One-dimensional proton NMR spectrum of FMC-7 in pure DMSO-d₆ at 27°C. A: Downfield region (5.8–3.4 ppm). B: Upfield region (2.8–0.4 ppm). “Sph” and “Fa” refer to protons of the sphingosine and fatty-N-acyl chains, respectively; “cis” refers to vinyl and allyl protons of unsaturated fatty-N-acyl groups; “H” refers to protons of the ring and exocyclic hydroxymethyl groups of the hexose residue; “imp” refers to a signal from a uncharacterized but commonly occurring impurity; “DMSO” refers to the residual proton signal from the deuterated solvent which characteristically appears at 2.50 ppm. Abbreviations: FMC, fast migrating cerebroside; GalCer, galactosylceramide; NMR, nuclear magnetic resonance spectroscopy.

Fig. 6.

Immuno-TLC of A: anti-FMC-7 antibody with LPS from E. coli J5 strain (lane 1), glycoglycerolipid MfGL-II from Mycoplasma fermentans (lane 2), and FMC-7 (lane 3); B: MS CSF IgG with: LPS from E. coli J5 strain (lane 1), glycoglycerolipid MfGL-II from Mycoplasma fermentans (lane 2), and FMC-7 (lane 3); C: anti-FMC-5/-6/-7 antibody with: EBs Chlamydia pneumoniae (lane 1), MfGL-II (lane 2), mixture FMC-5/-6/-7 (lane 3), and GalCer (lane 4); D: MS CSF IgG with FMC-5/-6/-7 mixture (lane 1), FMC-1 (lane 2), EBs Chlamydia pneumoniae (lane 3), and MfGL-II (lane 4). The bands were resolved on the plate using for 6A and 6B→ n-propanol:methanol:water (50:30:20; v/v/v) and for 6C and 6D→ n-propanol:methanol:water (60:20:15; v/v/v), respectively, and then visualized with DAB staining. Abbreviations: CSF, cerebrospinal fluid; FMC, fast migrating cerebroside; GalCer, galactosylceramide; LPS, lipopolisaccharide; MS, multiple sclerosis.

Fig. 7.

XY scatter graphs of reactivity of antigens A: FMC-7, B: GalCer, C: sulfatide, D: MfGL-II, and E: LPS from E. coli J5 in MS patients. RRMS in relapse (n = 5); RRMS in remission (n = 11); SPMS (n = 10); PPMS (n = 3); controls NI-OND (n = 4); I-OND (n = 5). The patients with CNS disease (I-OND) had the following diagnoses: meningitis (3), meningo-encephalitis (1), and subacute sclerosing panencephalitis (SSPE) (1). The group of patients with noninflammatory or NI-OND encompasses neuropathy (3) and cerebellar degeneration (1). Horizontal bars indicate median values. Abbreviations: CNS, central nervous system; FMC, fast migrating cerebroside; GalCer, galactosylceramide; LPS, lipopolisaccharide; MS, multiple sclerosis; OND, other neurological diseases; PPMS, primary progressive MS; RRMS, relapsing remitting MS; SPMS, secondary progressive MS.