Abstract
A glycophosphoceramide concentrate prepared from tobacco leaves was shown to contain a mixture of related lipids (Kaul, K., and Lester, R. L. (1975) Plant Physiol. 55, 120-129; Kaul, K., and Lester, R. L. (1978) Biochemistry 17, 3569-3575) with the simplest and most abundant components having the structure GlcN(+/- Ac)(alpha 1 leads to 4)GlcUA(alpha 1 leads to 2)myoinositol-1-O-phosphorylceramide (Hsieh, T. C.-Y., Kaul, K., Laine, R. A., and Lester, R. L. (1978) Biochemistry 17, 3575-3579). To determine the structure of the more complex members of this series, a mixture of oligosaccharides was prepared from a carboxyl-reduced glycophosphoceramide concentrate by alkali-catalyzed hydrolysis and alkaline phosphatase treatment. A combination of reverse-phase high pressure liquid chromatography (Wells, G. B., and Lester, R. L. (1979) Anal. Biochem. 97, 184-190), normal-phase high pressure liquid chromatography, and thin layer chromatography were used to resolve several oligosaccharides as acetylated derivatives. Products of methylation analysis, CrO3 oxidation, and deacetylation-deamination were identified using chemical ionization mass spectrometry to give the following novel structures. A major tetrasaccharide was completely characterized as Gal(alpha 1 leads to 4)GlcNAc(alpha 1 leads to 4)GlcUA(alpha 1 leads to 2)myoinositol. An additional structure of a minor tetrasaccharide was partially characterized as GlcNAc(alpha 1 leads to 4)GlcUA(alpha 1 leads to ?)myoinositol(O leads from 1 alpha)Man. These are representatives of a class of acidic glycolipids from plants, possibly analogous to the acidic gangliosides found in animal cell membranes.
Highlights
A glycophosphoceramide concentrate prepared fromcomplete structure of a ceramide-phosphotrisaccharidewhich tobacco leaves was shown to contain a mixtureof re- comprises 20% of the glycophosphoceramide concentrate prelatedlipids(Kaul, K., andLester, R.L. (1975) Plunt pared from tobacco leaves [5].This structure, GlcNAc(cul4
184-190), normal-phase high pressure liquid chroma- phasechromatography.Fractions were further purified by tography, and thin layer chromatography were utsoed thin layer chromatography to yield a trisaccharide and two resolve several oligosaccharides as acetylated deriva- tetrasaccharide fractions in pure form
Hi-FlosilLiquid Chromatography of thePeracetylated Oligosaccharide Mixture-The peracetylated oligosaccharide mixture obtained from the carboxyl-reduced glycophosphosphingolipid concentrate was chromatographed in a HiFlosil silica gel column as shown in Fig. 1.The major fraction, Fraction 3 (Fig. l),contained a mixture of oligosaccharides further resolved by reverse-phase HPLC asdescribed below
Summary
PURIFICATION AND CHARACTERIZATION OF A NOVEL TETRASACCHARIDE DERIVED FROM TOBACCO LEAF GLYCOLIPIDS*. A glycophosphoceramide concentrate prepared fromcomplete structure of a ceramide-phosphotrisaccharidewhich tobacco leaves was shown to contain a mixtureof re- comprises 20% of the glycophosphoceramide concentrate prelatedlipids(Kaul, K., andLester, R.L. (1975) Plunt pared from tobacco leaves [5].This structure, GlcNAc(cul. (1978) 4)GlcUA(al+ 2)inositol-1-0-phosphorylceramidem, ay be a Biochemistry 17,3569-3575) with the simpleasntd most basic core structure of the tobacco glycophosphoceramides. GlcN(kAc)(al + 4)GlcUA(al + 2)myoinositol-l-O- contain galactose and arabinose in addition to the trisacchapRmwca3en5h.aer7doAmsa9samp.)pb,.lrhkaeTiedronaposerdlcaiydonoLrlfeceneetedtecsrphrteaefhminrsmroto,irssnimaedpeRrtehe.iata(ehHLtscae,.bsasaiy(rsee1bthm9raot,7urlxi8kexcTy)atat.luut-BlCmirrr-ieeeco.ed-acYnoouhtffta.ce,.leoKmtydAhlziiaegsegutocdrllmosyh,yaKmcyoco1dr.cb,p7erhLi,honaac3olaryoi5sntimsp7dieio5hose,pn-fosl-exrptctihoohdeWnoecccpheceehaonroranntasmroscpawttitihedetouredfice.rzeenoTesrtmachscmreoiw(bi5mcdheo)eip.nctlhcehcetoewenmletyprphraoteathenpveeaewnrmaatptsfairoorejoonbpmrtaatroeietfnthdreaaeaddsncaebacqyorcublfhiaoigatsxoretyisqdrla-ueecrmacenhdoptauvihrtcioiineedsdgsereverse-phase high pressureliquidchromatography glycophosphoceramide mixture and acetylating the re-. 184-190), normal-phase high pressure liquid chroma- phasechromatography.Fractions were further purified by tography, and thin layer chromatography were utsoed thin layer chromatography to yield a trisaccharide and two resolve several oligosaccharides as acetylated deriva- tetrasaccharide fractions in pure form.
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