Letter to the Glycoforum: Improved Protocols For Preparing Lipid-Linked And Related Saccharides For Fluorophore-Assisted Carbohydrate Electrophoresis (FACE).
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ABSTRACT: The abundant cell-surface lipophosphoglycan (LPG) of Leishmania parasites plays a central role throughout the eukaryote's life cycle. A number of LPG-defective mutants and their complementing genes have been isolated and have proven invaluable in assessing the importance of LPG and related glycoconjugates in parasite virulence. While ricin agglutination selection protocols frequently result in lpg− mutants, one Leishmania donovani variant we isolated, named JABBA, was found to be lpg+. Procyclic (logarithmic) JABBA expresses significant amounts of a large-sized LPG, larger than observed from procyclic wild type but similar in size to LPG from wild type from metacyclic (stationary) phase. Structural analysis of the LPG from logarithmically grown JABBA by capillary electrophoresis protocols revealed that it averaged 30 repeat units composed of the unsubstituted Gal(β1,4)Man(α1)-PO4 typical of wild-type L. donovani. Analysis of JABBA LPG caps indicated that 20% is branched trisaccharide Gal(β1,4)[Glc(β1,2)]Man and tetrasaccharide Gal(β1,4)[Glc(β1,2)Man(α1,2)]Man instead of the usual Gal(β1,4)Man and Man(α1,2)Man terminating caps. Consistent with these structural observations, analyses of the relevant glycosyltransferases in JABBA microsomes involved in LPG biosynthesis showed a 2-fold increase in elongating mannosylphosphoryltransferase activity and up-regulation of a β-glucosyltransferase activity. Furthermore, the caps of JABBA LPG are cryptic in presentation as shown by the loss of binding by the lectins, ricin, peanut agglutinin and concanavalin A and reduced accessibility of the terminal galactose residues to oxidation by galactose oxidase. These results indicate that LPG from JABBA is intriguingly similar to the larger LPG in wild-type parasites that arises following the differentiation of the non-infectious procyclic promastigotes to infectious, metacyclic forms.Glycobiology 12/2014; 25(4). DOI:10.1093/glycob/cwu130 · 3.75 Impact Factor