title: Glucosylceramide transferase in Giardia preferentially catalyzes the synthesis of galactosylceramide during encystation.
abstract: The stage differentiation from trophozoite to cyst (i.e., encystation) is an essential step for Giardia to survive outside its human host and spread the infection via the fecal-oral route. We have previously shown that Giardia expresses glucosylceramide transferase 1 (GlcT1) enzyme, the activity of which is elevated during encystation. We have also reported that blocking the activity of gGlcT1 interferes with the biogenesis of encystation-specific vesicles (ESVs) and cyst viability in Giardia. To further understand the role of this enzyme and how it regulates encystation, we overexpressed, knocked down, and rescued the giardial GlcT1 (gGlcT1) gene and measured its enzymatic activity in live parasites as well as in isolated membrane fractions using NBD-ceramide and UDP-glucose or UDP-galactose. We observed that gGlcT1 is able to catalyze the synthesis of both glucosylceramide (GlcCer) and galactosylceramide (GalCer), however the synthesis of GalCer is 2-3 fold higher than of GlcCer. Although both activities follow Michaelis-Menten kinetics, the bindings of UDP-glucose and UDP-galactose with the enzyme appear to be non-competitive and independent of each other. The modulation of gGlcT1 synthesis concomitantly influenced the expression cyst-wall protein (CWP) and overall encystation. We propose that gGlcT1 is a unique enzyme and that Giardia uses this enzyme to synthesize both GlcCer and GalCer to facilitate the process of encystation/cyst production.
authors.affiliation: Infectious Disease and Immunology Cluster, Border Biomedical Research Center (BBRC), University of Texas at El Paso, El Paso, TX 79968-0519, USA; Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519/USA, Tel: (915) 747-6896∥.|Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968-0519, USA; Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519/USA, Tel: (915) 747-6896∥.
authors.forename: Leobarda
authors.initials: L
authors.lastname: Robles-Martinez
authors.affiliation: Infectious Disease and Immunology Cluster, Border Biomedical Research Center (BBRC), University of Texas at El Paso, El Paso, TX 79968-0519, USA; Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519/USA, Tel: (915) 747-6896∥.|Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968-0519, USA; Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519/USA, Tel: (915) 747-6896∥.
authors.forename: Tavis L
authors.initials: TL
authors.lastname: Mendez
authors.affiliation: Infectious Disease and Immunology Cluster, Border Biomedical Research Center (BBRC), University of Texas at El Paso, El Paso, TX 79968-0519, USA; Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519/USA, Tel: (915) 747-6896∥.|Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968-0519, USA; Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519/USA, Tel: (915) 747-6896∥.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Apodaca
authors.affiliation: Infectious Disease and Immunology Cluster, Border Biomedical Research Center (BBRC), University of Texas at El Paso, El Paso, TX 79968-0519, USA; Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519/USA, Tel: (915) 747-6896∥.|Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968-0519, USA; Department of Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968-0519/USA, Tel: (915) 747-6896∥.
authors.forename: Siddhartha
authors.initials: S
authors.lastname: Das
mesh_terms: D000595:Amino Acid Sequence; D004789:Enzyme Activation; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D005872:Giardia / Q000502:physiology*; D005873:Giardiasis / Q000469:parasitology; D005963:Glucosylceramides / Q000096:biosynthesis*; D006031:Glycosylation; D007700:Kinetics; D000072417:Protein Domains; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005963:Glucosylceramides; D015800:Protozoan Proteins; D014166:Transferases
keywords: Anti-sense morpholino oligonucleotide; Encystation; Galactosylceramide; Giardia lamblia; Glucosylceramide; Glucosylceramide transferase; Sphingolipid