Previously, on the basis of lectin binding and glycosidase digestion assays, we have suggested that N-acetyl-d-glucosamine residues (GlcNAc) are major structural components of both trophozoites and in vivo cysts of the intestinal parasite Giardia lamblia. In this report we confirm that GlcNAc is present both in trophozoites and in vitro cysts as assessed by lectin binding and glycosidase digestion assays, galactosyltransferase labeling, immunochemical analysis using antibodies specific for GlcNAc and its β1-4 oligomers, and by gas chromatography/mass spectrometry (GC/MS). The results show that wheatgerm agglutinin (WGA) binds specifically to intact trophozoites and in vitro cysts as well as to SDS-PAGE separated proteins. WGA binding to the separated proteins was markedly reduced after their digestion with N-acetyl-β-d-glucosaminidase, supporting the conclusion that WGA is reacting with terminal β-linked GlcNAc residues. Labeling of trophozoites and cysts by 3H-exogalactosylation with galactosyltransferase further confirmed the presence of terminal GlcNAc in both surface and intracellular glycoproteins. The presence of GlcNAc is also supported by microfluorometric analysis using antibodies to (GlcNAc)1, (GlcNAc)2, and (GlcNAc)3, which revealed a sugar-inhibitable binding of the antibody to live trophozoites. Finally, the presence of GlcNAc in both cysts and trophozoites was unequivocally confirmed by GC/MS analysis of detergent-extracted membranes and glycoproteins isolated by affinity chromatography on WGA-agarose. GC/MS analysis also revealed mannose (Man), N-acetyl-d-galactosamine (GalNAc), fucose (Fuc), galactose (GaI), glucose (Glc) and N-acetylneuraminic acid (NANA) to be present in cysts. All these sugars were also present in trophozoites, except for GalNAc. The glycoproteins isolated by WGA affinity chromatography were 5- to 40-fold enriched in GlcNAc, further supporting the conclusion that WGA reacts with GlcNAc in Giardia. In summary, the data presented here provide biological and chemical evidence for GlcNAc in both cysts and trophozoites of G. lamblia and are consistent with previously published results from this and other laboratories. © 1990.
