Characterization of Crithidia fasciculata oligosaccharide-lipid and its elongation by bovine mammary microsomes.
It was recently shown that a Man7(GlcNAc)2-lipid species serves as the precursor for the biosynthesis of asparagine-linked glycoproteins in the trypanosomatid Crithidia fasciculata. Preliminary results indicated it to be similar to the intermediate in the major pathway for the biosynthesis of lipid-linked Glc3Man9(GlcNAc)2 in animal systems. To explore the potential of this glycolipid as an acceptor for studying the biosynthesis of mammary glycoproteins, we conducted a detailed structural analysis of the labelled Man7(GlcNAc)2-lipid isolated from exponentially growing cells of C. fasciculata. The results showed its structure to be Man alpha 1----2Man alpha 1----2Man alpha 1----3(Man alpha 1----2Man alpha 1----3Man alpha 1----6)Man beta----GlcNAc beta----GlcNAc, identical to the nonasaccharide synthesized by the animal systems. Incubation of the Man7(GlcNAc)2-lipid with bovine mammary microsomes along with GDP-mannose or mannosyl-phosphodolichol elongates it to give Man9(GlcNAc)2-lipid having the same structure as the corresponding intermediate in animal systems. Inhibition of the elongation reaction by EDTA or amphomycin indicates that the intermediary formation of mannosyl-phosphodolichol is required for the incorporation of mannose residues into the nonasaccharide-lipid. Mannosyl.phosphoretinol failed to serve as a mannosyl donor in this reaction.
D000117:Acetylglucosamine / Q000302:isolation & purification*; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography / Q000379:methods; D003421:Crithidia / Q000032:analysis*; D005260:Female; D005944:Glucosamine / Q000031:analogs & derivatives*; D005959:Glucosidases; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000378:metabolism*; D008358:Mannose / Q000302:isolation & purification* / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism*; D009844:Oligosaccharides / Q000302:isolation & purification*
D009844:Oligosaccharides; D005959:Glucosidases; D005944:Glucosamine; D008358:Mannose; D000117:Acetylglucosamine