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

Abstract

A Trypanosoma brucei TbGPI12 null mutant that is unable to express cell surface procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB 3 H 4 labeling, that the cell surface of the TbGPI12 null parasites contains glycoconjugates that terminate in sialic acid linked to galactose. Following desialylation, a high-apparent-molecularweight glycoconjugate fraction was purified by ricin affinity chromatography and gel filtration and shown to contain mannose, galactose, N-acetylglucosamine, and fucose. The latter has not been previously reported in T. brucei glycoproteins. A proteomic analysis of this fraction revealed a mixture of polytopic transmembrane proteins, including P-type ATPase and vacuolar proton-translocating pyrophosphatase. Immunolocalization studies showed that both could be labeled on the surfaces of wild-type and TbGPI12 null cells. Neither galactose oxidase-NaB 3 H 4 labeling of the non-GPI-anchored surface glycoconjugates nor immunogold labeling of the P-type ATPase was affected by the presence of procyclins in the wild-type cells, suggesting that the procyclins do not, by themselves, form a macromolecular barrier.

A Cell-surface Phylome for African Trypanosomes

“…Experimental studies of the cell-surface demonstrate that non-protein glycoconjugates could play an equal role in regulating host-parasite interactions, for example, a protease-resistant surface molecule (PRS) is known to dominate the surface of procyclic-stage T. conglolense [79]. T. brucei expresses various glycoconjugates on their surfaces that only become apparent in null mutants that cannot express the major surface glycoprotein [103]–[104]. Even considering the protein component, low abundance genes not considered in the CSP may still perform a vital role; for example, the haptoglobin-hemoglobin receptor (Tb927.6.440; [105]) responsible for resistance to trypanolytic factor by T. brucei is single-copy.…”

O -fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii

“…Proteins were pulled down from total cell lysate by incubation with biotinylated AAL (Vector Labs) and Dynabeads MyOne Streptavidin T1 (Thermo Fisher) and were eluted by incubation with αMeFuc (Carbosynth). See SI Methods for details (47).…”

Identification of clinically approved small molecules that inhibit growth and affect transcript levels of developmentally regulated genes in the African trypanosome

“…In our study, we failed to identify any compounds that produced procyclin protein on the surface of parasites, underscoring the already established paradigm that additional developmental steps may be required to remodel the parasite surface following transcription of the relevant RNAs. In addition, defects in glycosylation of the procyclin protein and/or defects in the addition of the GPI anchor could also explain the lack of procyclin protein observed on the surface of the parasites [65][66][67][68]. Given that the bromodomain inhibitor I-BET151 has previously been shown to increase both RNA and protein levels of procyclin [10], a more targeted screen using a library of epigenetic inhibitors might uncover more compounds that lead to this phenotype in the EP1/GFP reporter line.…”