Cyst wall synthase: N-acetylgalactosaminyltransferase activity is induced to form the novel N-acetylgalactosamine polysaccharide in the Giardia cyst wall
Uridine-5′-diphospho-N-acetylgalactosamine (UDP-GalNAc) is required in the formation of the outer filamentous wall of Giardia and is synthesized by inducible enzymes in the cytosol of encysting trophozoites. In this study, an inducible enzyme activity that is associated with a particle population isolated from encysting Giardia is reported, and this activity exclusively incorporates [1-14C]GalNAc (from UDP-[14C]GalNAc) into an ethanol precipitate with the same properties as the filamentous cyst wall of Giardia. This ethanol precipitate exhibits characteristics of Giardia cyst wall filaments in that both contain GalNAc as the only sugar moieties and are SDS-insoluble, proteinase- and alkali-resistant and acid-hydrolysable. However, since the precise chemical nature of the ethanol precipitate remains unknown, this enzyme activity is referred to tentatively as cyst wall synthase (CWS). CWS activity peaks in cells between 24 and 36 h of encystment and exhibits a high affinity and marked specificity for UDP-GalNAc as its substrate. UDP-N-acetylglucosamine, UDP-glucose, UDP-galactose, d-glucosamine and d-galactosamine were not incorporated into the ethanol precipitate. Partially purified CWS activity exhibits an apparent Km of 0·048 mM for UDP-GalNAc, a Vmax of 0·70 nmol min−1 (mg protein)−1 and a requirement for divalent cations in the following order of preference: Ca2+, Mg2+>Co2+>>>Mn2+, Zn2+. EDTA inhibits CWS activity.
DuranA., CabibE.1978; Solubilization and partial purification of yeast chitin synthase. Confirmation of the zymogenic nature of the enzyme. J Biol Chem 253:4419–4425
ErlandsenS. L., BemrickW. J., PauleyJ.1989; High-resolution electron microscopic evidence for the filamentous structure of the cyst wall in Giardia muris and Giardia duodenalis. J Parasitol 75:787–797[CrossRef]
FaubertG., ReinerD. S., GillinF. D.1991; Giardia lamblia: regulation of secretary vesicle formation and loss of ability to reattach during encystation in vitro. Exp Parasitol 72:345–354[CrossRef]
FeelyD. E., ErlandsenS. L., ChaseD. G.1984; Structure of the trophozoite and cyst. In Giardia and Giardiasis pp. 3–31Edited byErlandsenS., MeyerE. New York: Plenum;
GerwigG. J., van KuikJ. A., LeeflangB. R., KamerlingJ. P., VliegenthartJ. F., KarrC. D., JarrollE. L.2002; The Giardia intestinalis filamentous cyst wall contains a novel β(1-3)-N-acetyl-d-galactosamine polymer: a structural and conformational study. Glycobiology 12:499–505[CrossRef]
LujanH. D., MarottaA., MowattM. R., SciakyN., Lippincott-ScwartzJ., NashT. E.1995a; Developmental induction of Golgi structure and function in the primitive eukaryote Giardia lamblia. J Biol Chem 270:4612–4618[CrossRef]
LujanH. D., MowattM. R., ConradJ. T., BowersB., NashT. E.1995b; Identification of a novel Giardia lamblia cyst wall protein with leucine-rich repeats. Implications for secretory granule formation and protein assembly into the cyst wall. J Biol Chem 270:29307–29313[CrossRef]
MowattM. R., LujanH. D., CottenD. B., BowersB., YeeJ., NashT. E., StibbsH. H.1995; Developmentally regulated expression of a Giardia lamblia cyst wall protein gene. Mol Microbiol 15:955–963[CrossRef]
Cyst wall synthase: N-acetylgalactosaminyltransferase activity is induced to form the novel N-acetylgalactosamine polysaccharide in the Giardia cyst wall