During the life cycle of the pathogenic protistan parasite, Giardia must differentiate into a metabolically-reduced infective form known as the cyst prior to exiting the host continuing the fecal-oral route of transmission. The mature cyst is surrounded by a thick filamentous outer cyst wall, once suggested to be composed of chitin (a β1-4-linked N-acetylglucosamine homopolymer) as in some fungal and other more closely related protozoan wall filaments. Because of the near cosmopolitan nature of chitin among eukaryotic microbes, crustaceans, insects, and some algae, and because of early lectin-binding data, this view was casually accepted for Giardia wall filaments. However, no direct biochemical evidence was provided in support of the claim.
To address the nature of the cyst wall filaments, our direct biochemical data demonstrated the presence of a large amount of the unique cyst-specific sugar N-acetylgalactosamine (GalNAc) in isolated cyst walls with only trace amounts of the GalNAc precursor N-acetylglucosamine (GlcNAc) found in mature cysts, and the presence of an inducible UDP-GalNAc synthesizing pathway including an inducible UDP-N-acetylgalactosamine 4'-epimerase responsible for the synthesis of the UDP-GalNAc precursor to the Giardia cyst wall filaments.
Now we demonstrate the presence of a novel β1,3-N-acetylgalactosaminyl transferase activity (β3GaN-T) responsible for the synthesis of the unique cyst wall homopolymer of (1,3)-β-
-acetylgalactopyranosamine that comprises ca. 63% of the dry weight and essentially 100% of the carbohydrate of isolated filamentous cyst walls.
This newly described transferase activity catalyzes the following reaction: UDP-GalNAc + (GalNAc-β1,3-GalNAc)n → (GalNAc-β1,3-GalNAc) n+1 + UDP and has been given the common name cyst wall synthase (CWS) for its unique action in synthesizing the cyst wall carbohydrate homopolymer. The IUPAC-UMB systematic nomenclature proposed for CWS is UDP-N-acetylgalactosamine : (1→3)-β-
-acetylgalactopyranosamine β(1→3)- N
-acetylgalactosaminyltransferase (UDP-GalNAc : (GalNAc-β1-3-GalNAc) n
-acetylgalactosaminyltransferase). This enzyme activity may be abbreviated as β3GaN-T following current literature convention. The β3GaN-T activity was induced during encystment more than 600-fold from less than 0.001 nmol min-1
protein (limit of detection) with peak activity at 24-36 hours post-induction and was purified 155-fold from microsomal encystment specific vesicles (ESV) by differential and isopycnic centrifugation, followed by detergent extraction. β3GaN-T co-localized with cyst wall specific proteins CWP1 and CWP2 to the microsomal ESV sub-population distinct from the lysosome-like peripheral vacuoles. The vesicle-associated Ca2+
-dependent activity was specific for the incorporation of [ 14
C]-GalNAc from UDP-[1-14
C]GalNAc into an ethanol- or TCA-precipitate or filtrate with a Kmapp
and a Vmaxapp
for UDP-GalNAc of 49.3 μM and 0.701 nmol min-1
protein, respectively. An endogenous or exogenous acceptor was not identified, which is consistent with other processive glycan synthases. β3GaN-T did not incorporate radiolabel from UDP-[1- 14
C]GlcNAc or other typical UDP-sugar substrates regardless of cofactor tested. Substrate analogs such as UDP-GlcNAc, Nikkomycin Z, Polyoxin D, and uridine 5'- tri, di, and monophosphate inhibited enzyme activity in a concentration-dependent manner.
The β3GaN-T reaction product was chemically-resistant in a manner similar to that of isolated cyst wall filaments, which were characterized with respect to composition, linkage, conformation, branching, substitution/modification, and chain length. Analysis revealed that the isolated filamentous cyst walls are composed of 63% dry weight N-acetylgalactopyranosamine in a β1→3 unbranched, unsubstituted, and nearly completely N-acetylated homopolymer of at least 23 residues in length, after partial acid hydrolysis.