Structural elucidation of novel phosphocholine-containing glycosylinositol-phosphoceramides in filamentous fungi and their induction of cell death of cultured rice cells

Abstract

Novel ZGLs (zwitterionic glycosphingolipids) have been found in and extracted from the mycelia of filamentous fungi ( Acremonium sp.) isolated from soil. Five ZGLs (ZGL1-ZGL5) were structurally elucidated by sugar compositional analysis, methylation analysis, periodate oxidation, matrix-assisted laser-desorption ionization-time-of-flight MS, (1)H-NMR spectroscopy and fast-atom bombardment MS. Their chemical structures were as follows: GlcN(alpha1-2)Ins1-P-1Cer (ZGL1), Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL2), Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL3), PC-->6Man(alpha1-6)GlcN(alpha1-2)Ins1- P -1Cer (ZGL4), and PC-->6Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL5) (where Cer is ceramide and PC is phosphocholine). In addition, one acidic glycosphingolipid, which was the precursor of ZGLs, was also characterized as inositol-phosphoceramide. The core structure of the ZGLs, GlcN(alpha1-2)Ins1- P, is rather different from those found in other fungi, such as Man(alpha1-2)Ins1- P and Man(alpha1-6)Ins1- P. Interestingly, the terminal mannose residue of ZGL4 and ZGL5 was modified further with a PC group. The presence of PC-containing glycosylinositol-phosphoceramides has not been reported previously in any organism. The ceramide constituents of both ZGLs and acidic glycosphingolipid were essentially the same, and consisted of a 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid base and 2-hydroxytetracosanoic acid (>90%) as the major fatty acid. ZGLs were found to cause cell death in suspensions of cultured rice cells. The cell death-inducing activity of ZGLs is probably due to the characteristic glycan moiety of Man(alpha1-6)GlcN, and PC-containing ZGLs had high activity. This study is the first to demonstrate that fungal glycosylinositol-phosphoceramides induce cell death in cultured rice cells.

Newly Discovered Neutral Glycosphingolipids in Aureobasidin A-resistant Zygomycetes

“…After methanolysis, the fatty acid methyl esters were extracted three times with 400 l of n-hexane and then analyzed by capillary gas-liquid chromatography (GLC)/MS (14,15). The remaining methanolic phase was evaporated to dryness for deacidification under a nitrogen stream.…”

“…Carbohydrate and Fatty Acid Composition Analyses-For determination of the compositions of the fatty acids and sugars in GSLs, 100 -200 g of GSLs were methanolyzed in thick glass test tubes with 200 l of freshly prepared 1 M anhydrous methanolic HCl using a microwave oven (14,15). After methanolysis, the fatty acid methyl esters were extracted three times with 400 l of n-hexane and then analyzed by capillary gas-liquid chromatography (GLC)/MS (14,15).…”

“…Extraction and Purification of Sphingolipids-Sphingolipids were prepared from mycelia by consecutive extractions, as described elsewhere (14). Lipid extracts were saponified with 0.5 M KOH in methanolwater (95:5, v/v) at 37°C for 6 h. The hydrolysate was acidified to pH 1.0 with concentrated HCl and then dialyzed against tap water for 2 days followed by concentration and precipitation with acetone.…”

“…Materials-QAE-Sephadex A-25, DEAE Sephadex A-25, and D-[U- 14 C]glucose were purchased from Amersham Biosciences. Iatrobeads 6RS-8060 was obtained from Iatron Laboratories Inc. Silica gel 60 precoated plates were from Merck, magnesium silicate (Florisil) was from Nacalai Tesque, green coffee bean ␣-galactosidase was from Sigma, and jack bean ␤-galactosidase was from Seikagaku Co. Aureobasidin A was obtained from Takara Bio Inc. All other reagents used were of best grade available commercially.…”

“…The neutral fraction was further purified by silica gel chromatography (column, 15 ϫ 600 mm) with a linear gradient elution system of chloroform-methanol-water (400 ml of 90:10:0.5 by volume to 420 ml of 40:60:10 by volume). The polar fraction was then applied to a column of DEAE-Sephadex A-25 (20 ϫ 200 mm, acetate form), as described elsewhere (14).…”