Evidence for a glycolipid anchor of gp64, a putative cell‐cell adhesion protein of Polysphondylium pallidum

Abstract

The membrane-bound glycoprotein (gp64) of the cellular slime mold Polysphondylium pallidum, is a putative cell-cell adhesion protein identified by adhesion-blocking antibody fragments. Since gp64 can be purified in a few days and in substantial yields, it is a good candidate for clarifying the structure of a cell-cell adhesion protein. This study reveals that gp64 possesses a glycolipid anchor which is sensitive to deamination but resistant to phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. Although the anchor resistance to phosphatidylinositol-specific phospholipase C can be ascribed to the presence of an additional acyl chain on the inositol ring in the glycosyl phosphatidylinositol anchors, this was not the case. After a mild-base treatment that released an additional acyl chain on the inositol ring, only a trace amount of fatty acid was detected but, after strong acid hydrolysis, we detected both amide-linked fatty acids and a long-chain base. The long-chain base was further analysed by gas-chromatography/mass spectrometry and was found to be phytosphingosine. Both fatty acids and myo-inositol were also analysed by gas-chromatography/mass spectrometry. These data suggest that gp64 possesses a glycolipid anchor which contains ceramide and myo-inositol.

Glycosylphosphatidylinositol (GPI)-Anchored Proteins.

“…* Also, GP64, a cell-surface protein of Polysphondylium pallidum (a slime mold), has ceramide in its GPI structure. 29) GPI precursors in their carboxyl-terminals are transported to Golgi complex where the proteins are subjected to further modification in the GPI moiety, and finally inserted into the plasma membrane as mature GPI-anchored proteins.…”

Fatty acid composition of the cellular slime mold Polysphondylium pallidum

“…; Quadrex, New Haven, CT). The separated FAME were identified by comparing their retention times with those of authentic standards and by GC-MS, and were quantitated with a data analyzer (Chromato-Integrator D-2500; Hitachi) as previously described (10). Hydrogenation was carried out in glacial acetic acid with PtO 2 as a catalyst at room temperature for 6 h (11).…”