Characterization of Crithidia fasciculata oligosaccharide-lipid and its elongation by bovine mammary microsomes

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Abstract
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 Manα1 → 2Manα1 → 2Manα1 → 3(Manα1 → 2Manα1 → 3Manα1 → 6)Manβ→ GlcNAcβ→ 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.