Abstract
Although the existence of O-linked oligosaccharide residues in glycoproteins of Plasmodium falciparum has been shown, the existence of N-linked glycoproteins is still a matter of controversy and skepticism. This report demonstrates the unequivocal presence of N-linked glycoproteins in P. falciparum, principally in the ring and young trophozoite stages of the intraerythrocytic cycle. These glycoproteins lose their capacity to bind to concanavalin A-Sepharose after treatment of cultures with tunicamycin under conditions that do not affect protein synthesis. When the glycoproteins were treated with N-Glycanase(R), oligosaccharides were released. It was possible to identify an N-linked glycoprotein of >200 kDa in the ring stage and also N-linked glycoproteins in the range of 200-30 kDa in the trophozoite stage. Treatment of trophozoites with 12 microM tunicamycin inhibited differentiation to the schizont stage. To our knowledge, this is the first report in the literature unequivocally showing N-linked glycoproteins in trophozoites of P. falciparum as well as their importance for the differentiation of the intraerythrocytic stages of this parasite.
Highlights
This report demonstrates the unequivocal presence of N-linked glycoproteins in P. falciparum, principally in the ring and young trophozoite stages of the intraerythrocytic cycle
Inhibition of Parasite Development in Cultures of P. falciparum Treated with Antibiotics—Treatment of P. falciparum with 12 M TUNYC for 96 h caused a 90% inhibition in development of the young trophozoite to the schizont stage of the second cycle, without pigment formation
Parasites at the old trophozoite stage (35– 40-h culture, 83– 88-h TUNYC exposure) of the second cycle were interrupted in their development and died (Fig. 1, photo 6Ј)
Summary
Reagents were obtained from the following sources. RPMI 1640 medium, RPMI 1640 medium without glucose and methionine, Hepes, hypoxanthine, glucose, gentamycin, TUNYC, CHX, Tris, EDTA, SDS, 2-mercaptoethanol, Nonidet P-40, Triton X-100, phenylmethylsulfonyl. Fluoride, iodoacetamide, N␣-p-tosyl-lysine chloromethyl ketone, leupeptin, methyl ␣-D-mannopyranoside, methyl ␣-D-glucopyranoside, N-acetylglucosamine, N,NЈ-diacetylchitobiose, trichloroacetic acid, trifluoroacetic acid, n-butyl alcohol, pyridine, dextran blue, and maltooligosaccharides were purchased from Sigma. Percoll® and concanavalin A-Sepharose were purchased from Pharmacia Chemicals (Uppsala, Sweden). Plasmagel® was purchased from Laboratoire Roger Bellon (Nevilly-sur-Seine, France). L-[35S]Methionine, D-[U-14C]glucose, D-[U14C]mannose, and Amplify® were obtained from Amersham International (Buckinghamshire, United Kingdom). N-Glycanase® and O-Glycanase® were purchased from Genzyme (Cambridge, MA). Bio-Gel P-4 was acquired from Bio-Rad. Standard of N-glycans: Man3GlcNAc2 and Man9GlcNAc2 were purchased from Oxford Glycosystem (Abingdon, UK)
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