Structural Characterization of NETNES, a Novel Glycoconjugate in Trypanosoma cruzi Epimastigotes

Abstract

The unicellular stercorarian protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas' disease. The epimastigote form of the parasite is covered in a dense coat of glycoinositol phospholipids and short glycosylphosphatidylinositol (GPI)-anchored mucinlike molecules. Here, we describe the purification and structural characterization of NETNES, a relatively minor but unusually complex glycoprotein that coexists with these major surface components. The mature glycoprotein is only 13 amino acids in length, with the sequence AQENETNESGSID, and exists in two forms with either four or five post-translational modifications. These are either one or two asparagine-linked oligomannose glycans, two linear ␣-mannose glycans linked to serine residues via phosphodiester linkages, and a GPI membrane anchor attached to the C-terminal aspartic acid residue. The variety and density of post-translational modifications on an unusually small peptide core make NETNES a unique type of glycoprotein. The N-glycans are predominantly Man␣1-6(Man␣1-3) Man␣1-6(Man␣1-3)Man␤1-4GlcNAc␤1-4GlcNAc␤1-Asn; the phosphate-linked glycans are a mixture of (Man␣1-2) 0 -3 Man1-P-Ser; and the GPI anchor has the structure Man␣1-2(ethanolamine phosphate)Man␣1-2Man␣1-6Man␣1-4(2-aminoethylphosphonate-6)GlcN␣1-6-myoinositol-1-P-3(sn-1-O-(C 16:0 )alkyl-2-O-(C 16:0 )acylglycerol). Four putative NETNES genes were found in the T. cruzi genome data base. These genes are predicted to encode 65-amino acid proteins with cleavable 26-amino acid Nterminal signal peptides and 26-amino acid C-terminal GPI addition signal peptides.

Fate of Glycosylphosphatidylinositol (GPI)-Less Procyclin and Characterization of Sialylated Non-GPI-Anchored Surface Coat Molecules of Procyclic-Form Trypanosoma brucei

“…This could be due to difficulties in the enzyme reaching the glycosylation sites because of an unusual cross-linked/aggregated physical state and/or the presence of ␣1-to ␣3-linked fucose attached to the chitobiosyl core of the glycans, which is known to render N-glycans resistant to PNGase F (49). In addition, it is possible that the material contains O-linked oligosaccharides, as found in T. cruzi mucins (35,44), and/or phosphatelinked oligosaccharides, as found in leishmania (13), T. cruzi (12,23), and Trypanosoma congolense (48). A detailed analysis of the carbohydrate moieties in the high-MW glycoconjugate fraction will be undertaken but is beyond the scope of this report.…”

Sugar Nucleotide Pools of Trypanosoma brucei , Trypanosoma cruzi , and Leishmania major

“…The phosphosaccharide repeat chains of leishmania PPGs are attached to protein via Man␣1-P-Ser linkages (49). Although considerably less abundant, P-linked glycans have also been found in T. cruzi, in gp72 (40) and the NETNES glycoprotein (61), and in T. congolense GARP (113). They may also exist in the T. brucei gp72 homologue Fla-1 (77).…”

Trypanosoma congolense Procyclins: Unmasking Cryptic Major Surface Glycoproteins in Procyclic Forms

“…The sample (recovered in the lower phase of the butanol-water partition) was then dried and submitted to deamination (27). The released phosphatidylinositol molecules were recovered after partition with 1-butanol and analyzed, in negative-ion mode, with a Finnigan-Thermoquest LCQ-Duo ion trap electrospray mass spectrometer.…”