Lactosylceramide molecular species specificity of rat

Six naturally occurring and three synthetic molecubetween the molecular species composition of those glylar species of lactosylceramide (Laccer) were used to examine cosphingolipids with more complex carbohydrate chains the molecular species specificity of CMP-N-acetylneuramiand their simpler (shorter carbohydrate chain) glyconate:lactosylceramide a2,3-sialyltransferase in a Golgi-rich fracsphingolipid precursors (7). Moreover, several investigattion of rat liver. The enzyme molecular species specificity was determined either in the presence of nonspecific lipid transfer ors have reported that there is no precursor-product protein or in the presence of detergents. Assays performed in the relationship between the simple and more complex glycopresence of transfer protein showed that for those lactosylcerasphingolipid classes (8, 9). This has led to suggestions that mide molecular species with either d18:l or d18:O long chain base each glycosphingolipid class is synthesized by its own multhe enzyme activity decreased linearly as the effective carbon number of the long chain base decreased the activity of the ensimple glYcosPhingoliPids represents only a small fraction zyme twice as much as a corresponding increase in the carbon of the total cellular pool of these glycolipids (7, 8). number of the fatty acid. On the other hand, when the enzyme Kannagi, Nudelman, and Hakomori (11) have suggested activity was assayed in the presence of detergents, there was no that, at least for glycosphingolipids with complex carb&ysignificant difference in activity among the various molecular species of lactosylceramide based upon the carbon number of drate *ains9 the Observed variation in mo1ecular ‘pecies the fatty acid or on the presence of a double bond in the long composition ofthe glYcosPhindiPid chses could be due to chain base. However, the decrease in enzyme activity with an inthe fatty acid specificity of the glycosyltransferases that syncrease in the carbon number of the long chain base persisted. thesize the various glycosphhgolipid classes. However, no I These results demonstrate that sialyltransferase has bindstudy has yet been reported examming the molecular sFcies ing specificity with respect to the long chain base, but not the fatspecificity of any of the glycosyltransferases using defined ty acid. ’I’he apparent molecular species towards the fatty acid is related to the aqueous solubility of the various LacCer molenaturaUY occuning molecular species Of gl~osphin@’lipid cular species. -Kadowaki, H., L. A. Symanski, K. E. Rye-Sias substrate. we, therefore, examined the molecular species kora, and R. S. Koff. Lactosylceramide molecular species specificity of sialyltransferaw (CMP-N-acetylneuramispecificity of rat liver CMP-N-acety1neuraminate:lactosylceranate:lactosylceramide cr2,3-sidyltransferase (EC 2.4.99.9)) mide sialyltransferase. J. Lipid Res. 1989. 30: 1789-1797. which catalyzes the transfer of N-acetylneuraminic acid Supplementary key words rat liver Golgi apparatus (Ne&) from CMP-NeuAc to lactosylceramide (LacCer) to form the simplest ganglioside, GM3. Sialyltransferase was chosen for this study because LacCer is the precursor not number of the fatty acid increased, An increase in the carbon tienzyme comp1ex (lo) Or that the actua1 precursor pool Of Glycosphingolipids are synthesized in the Golgi membrane and endoplasmic reticulum (l), and are subse~ s&ia@i‘ ; GgOseFer, GaINWl-PGal l91-4-GIc~ quently transported to the plasma membrane, where they Abb-&, ns: mer, G@l-alc-ceramide; G a C e r , GdNW1participate in a variety of cell surface-related events (2-6). Glycosphingolipid biosynthesis proceeds by the sequential (&D=+Cer, GaZel -~*Ml*@1*lCdk GM3, NeuAm23Gal~1-4Glc-ceramide; CMP-NeuAc, CMP-N-acetylneuraminic acid; quentlY to an increasingly complex carbohydrate chain. HPLC, high performance liquid chromatography. The long chain base Although each tissue has a characteristic glycosphingolipabbreviations as suggested by Breimer, Karlsson, and Samuelsson (39) id class and ceramide molecular species pattern, it is a are used throughout. For example, in the notation d18:1-18:0, the d18:l represents the long chain base sphingosine (1,3-dihydroxy-2-aminooctageneral Phenomenon that there is no Obvious relationship decene) and 18:O represents the fatty acid (octadecanoic acid). addition Of a carbohydrate, first to ceramide and subseRRT, relative retention time; GLC, gas-liquid chromatography; Journal of Lipid Research Volume 30, 1989 1789 by on A ril 4, 2008 w w w .j.org D ow nladed fom only of GM3 and the higher gangliosides, but also of several other glycosphingolipid classes including the lacto, lactoneo, and glob0 series, each of which has its own characteristic molecular species pattern. It is therefore likely that the enzymes that metabolize LacCer will exhibit some specificity towards the various LacCer molecular species. EXPERIMENTAL PROCEDURES