Biology of Giardia lamblia. Detection of N-acetyl-D-glucosamine as the only surface saccharide moiety and identification of two distinct subsets of trophozoites by lectin binding.

Abstract

Lectins and glycosidases of known sugar specificity were used as probes to analyze the surface carbohydrate moieties of G. lamblia trophozoites and in particular to determine whether chitin or oligomeric D-GlcNAc is present in the trophozoite form of the parasite as well as on the cyst. Of 13 lectins with varying sugar specificity, only D-GlcNAc-specific lectins bound specifically to the trophozoite surface as determined by light microscopy and EM. A striking finding was the identification of two distinct subsets of trophozoites, distinguished by reactivity with WGA and detected by light microscopy and EM as well as by flow cytometry. Unlike the cyst wall, the trophozoite D-GlcNAc residues were resistant to chitinase treatment. In contrast N-acetyl-beta-D-glucosaminidase abolished WGA binding suggesting that the lectin binds to terminal beta-linked D-GlcNAc residues. These residues were identified as being present on surface glycoproteins by Western blotting of parasite membrane proteins using WGA as a probe. This study identifies D-GlcNAc as the only saccharide moiety detectable by lectin binding on the surface of G. lamblia trophozoites and demonstrates that in contrast to the cyst, chitin is not present in the trophozoite. In addition two distinct subsets of trophozoites were identified based on reactivity with WGA and may represent varying stages of differentiation from trophozoite to cyst.

Conserved Processes and Lineage-Specific Proteins in Fungal Cell Wall Evolution

“…6). This high degree of conservation is consistent with an ancestral and continuing role of chitin in cell walls of many eukaryotes (4,7,43,44,55,57).…”

“…Cavalier-Smith has argued that fungal cell walls are derived from ancestral chitinous walls of desiccation-resistant cysts in many protist clades (7,8). Certainly, chitinous cysts and spores are present in other eukaryote kingdoms, including Amoebozoa (such as Entamoeba [11]), Chromalveolata (Phytophthora [43,44]), and Excavata (Giardia [57]). In addition, S. cerevisiae walls share extracellular disulfide cross-links and GPI-anchored cell adhesion molecules with Amoebozoa, perhaps the earliest-diverged eukaryotes (Fig.…”

Growth inhibition of the intestinal parasite Giardia lamblia by a dietary lectin is associated with arrest of the cell cycle.

“…However, at the concentration of WGA used in this study, i.e., 100 ttg/ml we found that only 10.6% of trophozoites were agglutinated by the lectin (data not shown) even though it binds to trophozoite glycoproteins as assayed by fluorescence microscopy (9), light and electron microscopy (10), and FACSO analysis (11). The basis for this lack of cell agglutination despite the presence of numerous WGA-binding sites on the trophozoite surface glycoconjugates is not clear.…”

“…Organisms were harvested, washed three times in PBS, and lysed with 1% Triton X-100 in PBS containing 2 mM PMSF overnight at 4°C. Lysates were centrifuged at 175 g for 5 min to pellet nuclei and the supematant boiled with sample buffer before electrophoresis on a 7% SDS-polyacrylamide gel (10). Separated proteins were electrotransferred to nitrocellulose and probed with 1 ,ug/ml of biotinylated WGA, using the avidin-biotin alkaline phosphatase technique as described earlier (11).…”

“…The nature and properties of carbohydrate residues of glycoproteins on the plasma membrane of diverse cell types have been assessed using lectins, a class of sugar binding proteins of nonimmune origin (8). The presence of such lectin receptors on the surface membrane of Giardia lamblia has been shown previously (9,10). Of a variety of lectins tested, only wheat germ agglutinin (WGA),1 tomato lectin, and succinylated wheat germ agglutinin (S-WGA), bound specifically to carbohydrate determinants on the surface of Giardia trophozoites.…”