Glycosphingolipids structure, determination

Structural studies of glycosphingolipids involves determination of the structure of the oligosaccharide chain and of the lipid moiety. For the oligosaccharide chain, it is necessary to determine the composition, molar ratio, and sequence of the monosaccharides, their pyranose or furanose nature, and the position of glycosidic bonds and their configuration for the lipid moiety, the composition of the fatty acids and sphingosine bases must be determined. Used for these purposes are the classical, chemical methods, conventionally accepted in the chemistry of carbohydrates and lipids and based on the degradation of compounds, enzymic, and physicochemical methods, primarily mass spectrometry and n.m.r. spectroscopy. 

The studies discussed above have all dealt with glycosphingolipids isolated from neutrophils that were derived from normal whole blood. Wherrett (16) has presented detailed structural analysis of a tetraglycosylceramide isolated from polymorphonuclear leukocytes obtained from the urine of a patient with a urinary tract infection. This glycosphingolipid was determined to be lactoneotetraosylceramide. 

New ceramide digalactosides were isolated by Hayashi s group from the marine sponge Halichondria japonica.24 To the major glycosphingolipid was assigned the structure 86, except for the stereochemistry, using FAB/MS, IR and NMR spectroscopy. The first synthesis of the ceramide 87 and therefore the structure determination were described by us.25... 

Dell A, Oates JE, Morris HR, Egge H. Structure determination of carbohydrates and glycosphingolipids by fast atom bombardment mass-spectrometry. Int J Mass Spectrom Ion Processes 1983 46 415-418. 

Ann and Adams (1993) have explored the use of alkali metal ions as adducts to improve structural determination of other types of sphingolipids. CID spectra of [M + Cat]" ions (Caf = alkali metal ions) of ceramides and neutral glycosphingolipids provide immediate information about the lengths of the sphingolipid base and A -acyl chains and, most importantly, the locations of substituents on the A -acyl chain. 

Hsu, F.F., Turk, J. (2001) Structural determination of glycosphingolipids as lithiated adducts by electrospray ionization mass spectrometry using low-energy coUisional-activated dissociation on a triple stage quadrupole instrument. Journal of the American Society for Mass Spectrometry, 12, 61-79. 

Ann Q, Adams J. Structure determination of ceramides and neutral glycosphingolipids by collisional activation of [M +Li] ions. J Am Soc Mass Spectrom. 1992 3 260-3. 

Mass spectrometry of intact glycosphingolipids by direct probe insertion and soft ionisation procedures has become one of the major means of structure determination for these compounds [460,896,974]. information is thereby obtained on the nature of both the iipid and carbohydrate moieties, aithough the presence of different moiecuiar species hampers the interpretation of the spectra greatiy. Detailed discussion of this aspect is beyond the scope of this book. 

Measurements of the quantities of glycolipids inserted into the membrane have also been reported by a technique based on the use of C-labeled lipid anchors. In this method, the carbohydrate (a-o-Man) was covalently coupled to the anchor at the surface of a pre-formed vesicle. Indeed, the liposome structure was shown to remain intact in the treatment. Nevertheless, the measurement of the incorporated mannose was performed after separation of bound and unbound material by centrifugation. The yields of coupling were shown to increase with the increase of the initial mannose/ C-anchor ratio, but non covalent insertions were displayed at high initial mannose concentrations. Therefore, the aforementioned method was not as accurate as could have been expected for the use of radioactive materials [142]. Radiolabeled phospholipids were also used for such determinations thus the amounts of glycosphingolipids incorporated into liposomes were quantified by the use of H-phospholipids whereas the amounts of glycolipids were determined by a sphingosine assay [143]. 

This information was obtained from samples of less than 5 pg by electron impact/desorption direct probe mass spectrometry. On the basis of complete structural analyles, to be presented elsewhere, we have been able to determine that the four fractions isolated thus far actually contain six different glycosphingolipids with the following structures ... 

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