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A novel assay method for glycosphingolipid deacylase by enzyme-linked immunochemical detection of lysoglycosphingolipid

  • Published:
Lipids

Abstract

Lysoglycosphingolipids consist of a sphingoid long-chain base and monosaccharide or complex sugar, and they lack the fatty acyl group present in native glycosphingolipids. Less than 1 pmol of lyso-Forssman glycolipid and lysoganglioside GM1 were detected on a thin-layer chromatogram by an enzyme-linked immunochemical coloration method with anti-Forssman glycolipid antibody (FOM-1) and cholera toxin B subunit, respectively. Each spot between 1 and 100 pmol lyso-Forssman glycolipid was immunostained as densely as that of the same amount of native Forssman glycolipid. The density of the lyso-Forssman glycolipid spots increased proportionally with increment in the amount of lysoglycolipid. The density of spots of 0.2–100 pmol lysoganglioside GM1 was also proportional to the amount of each lyso-GM1 spot. These results indicated that less than 1 to 100 pmol of deacylated glycosphingolipid was quantifiable by the immunochemical coloration method with sugar chain-specific antibodies. Glycosphingolipid deacylase, which cleaved an amide bond between the sphingoid long-chain base and fatty acyl chain in ceramide of glycosphingolipid, was assayed by detecting the lyso-Forssman glycolipid produced. Lipophilic compounds, recovered from an aliquot of the reaction mixture of Forssman glycolipid and crude enzyme at appropriate times, were analyzed by thin-layer chromatography. It was found that lyso-Forssman glycolipid was produced in the first 1–2 h by the enzyme and production increased with incubation time. This coloration method is more sensitive and specific than the visualization method with a non-specific reagent such as orcinol-sulfuric acid reagent.

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Abbreviations

FOM-1:

anti-Forssman glycolipid antibody, manufactured by Biomedicals AG (August, Switzerland)

Gb4 Cei:

globoside

HPTLC:

high-performance thin-layer chromatography

HRP:

horseradish peroxidase

PBS:

phosphate-buffered saline

PVP:

polyvinylpyrrolidone

TLC:

thin-layer chromatography

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Authors and Affiliations

  1. Hokkaido National Industrial Research Institute. AIST. MITI, 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, 062-8517, Sapporo, Japan

    Kazuo Izumi & Michiko Takagi Sawada

Authors
  1. Kazuo Izumi
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  2. Michiko Takagi Sawada
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Correspondence to Kazuo Izumi.

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Izumi, K., Sawada, M.T. A novel assay method for glycosphingolipid deacylase by enzyme-linked immunochemical detection of lysoglycosphingolipid. Lipids 36, 97–101 (2001). https://doi.org/10.1007/s11745-001-0674-z

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  • DOI: https://doi.org/10.1007/s11745-001-0674-z

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