ABSTRACT
Giardia intestinalis trophozoites encyst when they are exposed to bile. During encystment, events related to the inducible synthesis of a novel N-acetyl-D-galactosamine (GalNAc) homopolymer, occur. Within the first 6 h of encystment, mRNA for glucosamine 6-P isomerase (GPI), the first inducible enzyme unique to this pathway appears, oxygen uptake rates double from non-encysting levels, and metronidazole (MTZ) inhibits oxygen uptake. Within 12 h, GPI and its activity are detectable and OU decreases 50% from non-encysting levels; glucose's stimulation and MTZ's inhibition of oxygen uptake cease. In contrast, aspartate uptake remained constant throughout the 40 h monitored. Two genes, gpi 1 and 2 encode for GPI, but only gpi1 is expressed during encystment. Glucosamine 6-P (GlcN6P), the synthetic product of GPI, activates UDP-N-acetylglucosamine (UDP-GlcNAc) pyrophosphorylase, a downstream enzyme, 3 to 5-fold in the direction of UDP-GlcNAc synthesis. UDP-GlcNAc is epimerized to UDP-GalNAc and UDP-GalNAc is polymerized by "cyst wall synthase" (beta 1 --> 3 GalNAc transferase) into a highly insoluble beta 1,3-linked homopolymer. This GalNAc polysaccharide, the major component of cyst wall filaments, forms, in conjunction with polypeptides, the outer cyst wall of Giardia.
Subject(s)
Acetylgalactosamine/metabolism , Aldose-Ketose Isomerases/genetics , Gene Expression Regulation, Enzymologic , Giardia lamblia/growth & development , Polysaccharides/metabolism , Acetylgalactosamine/genetics , Aldose-Ketose Isomerases/metabolism , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Genes, Protozoan , Giardia lamblia/genetics , Giardia lamblia/metabolism , Giardiasis/parasitology , Humans , Molecular Sequence DataABSTRACT
sqv (squashed vulva) genes comprise a set of eight independent loci in Caenorhabditis elegans required zygotically for the invagination of vulval epithelial cells and maternally for normal oocyte formation and embryogenesis. Sequencing of sqv-3, sqv-7, and sqv-8 suggested a role for the encoded proteins in glycolipid or glycoprotein biosynthesis. Using a combination of in vitro analysis of SQV enzymatic activities, sqv(+)-mediated rescue of vertebrate cell lines, and biochemical characterization of sqv mutants, we show that sqv-3, -7, and -8 all affect the biosynthesis of glycosaminoglycans and therefore compromise the function of one specific class of glycoconjugates, proteoglycans. These findings establish the importance of proteoglycans and their associated glycosaminoglycans in epithelial morphogenesis and patterning during C. elegans development.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans/genetics , Carrier Proteins/genetics , Glycosyltransferases/genetics , Hexosyltransferases/genetics , Monosaccharide Transport Proteins , Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins , Animals , COS Cells , Caenorhabditis elegans/embryology , Gene Expression Regulation, Developmental , Helminth Proteins/genetics , Morphogenesis/geneticsABSTRACT
Giardia synthesizes UDP-GalNAc during cyst wall formation (encystment) via a pathway of inducible enzymes similar to that used to synthesize chitin or peptidoglycan and that includes the UTP-requiring UDP-N-acetylglucosamine pyrophosphorylase. Although it has never been reported as a regulatory enzyme in any system studied to date, kinetic data including Hill plots demonstrate clearly that UDP-N-acetylglucosamine pyrophosphorylase activity, purified from encysting Giardia, is allosterically activated anabolically by physiological levels of glucosamine 6-phosphate (3 microm). Capillary electrophoresis demonstrates that within 24 h after trophozoites are induced to encyst, the level of glucosamine 6-phosphate increases 3-fold over that of non-encysting cells and that by 48 h into encystment the level of glucosamine 6-phosphate has decreased to non-encysting levels or below. UDP-N-acetylglucosamine pyrophosphorylase protein is present constitutively in encysting as well as non-encysting cells. UDP-N-acetylglucosamine pyrophosphorylase immunoaffinity purified from encysting and non-encysting cells exhibited the same molecular weight, amino acid composition, and circular dichroism spectra. Moreover, regardless of whether the enzyme came from encysting or non-encysting cells, the change in its circular dichroism spectra and up to a 6-fold increase in its specific activity anabolically were due to its activation with glucosamine 6-phosphate. Thus, the data support the idea that UDP-N-acetylglucosamine pyrophosphorylase is a major regulatory point in amino sugar synthesis in encysting Giardia and that its allosteric anabolic activation may shift the equilibrium of this pathway toward UDP-GalNAc synthesis.
Subject(s)
Giardia lamblia/enzymology , Nucleotidyltransferases/metabolism , Allosteric Regulation , Amino Acids/analysis , Animals , Blotting, Western , Chromatography, Affinity , Circular Dichroism , Enzyme Activation , Kinetics , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/isolation & purification , Substrate SpecificityABSTRACT
The biosynthesis of the carbohydrate component of the cyst wall of the protozoan parasite Giardia lamblia, a polymer of N-acetylgalactosamine (GalNac), is by a pathway that is initiated with the conversion of fructose 6-phosphate to glucosamine 6-phosphate by an aminating isomerase, glucose 6-phosphate isomerase. This enzyme appears only after Giardia trophozoites are induced to start the production of cyst wall components after bile is added. To investigate whether induction of glucosamine 6-phosphate isomerase is by protein modification or by transcription activation, its gene was cloned and sequenced. Two genes, gpi1 and gpi2, encoding putative glucosamine 6-phosphate isomerases were identified but one, gpi1 was expressed. The transcript for gpi1 appeared not earlier than 6 h after cells were induced with bile salts. These results show that the first enzyme in the pathway leading to GalNac synthesis in encysting Giardia cyst wall biosynthesis is under transcriptional control.
