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Updated: Aug 19, 2026
High-throughput Gene Tagging in Trypanosoma brucei
Published on: August 12, 2016
The procyclic acidic repetitive proteins of Trypanosoma brucei. Purification and post-translational modification
1Rockefeller University, New York 10021.
Abstract:
The procyclic acidic repetitive protein (PARP) of Trypanosoma brucei was purified by cell fractionation followed by ion-exchange and concanavalin A-Sepharose affinity chromatography. PARP is membrane-bound and comprises about 1% of the total procyclic trypanosome protein or 6 x 10(6) molecules per parasite. The results of NH2-terminal sequencing and amino acid analysis indicate that PARP is processed by removal of an N-terminal signal sequence and the hydrophobic COOH terminus. Metabolic labeling of PARP with [3H] ethanolamine is consistent with attachment of the protein to the membrane via a glycosylphosphatidylinositol anchor. The glycolipid can be removed by base hydrolysis or nitrous acid deamination but is not susceptible to bacterial phosphatidylinositol-specific phospholipase C.
Insights
The procyclic acidic repetitive protein (PARP) from Trypanosoma brucei is membrane-bound and attached via a glycosylphosphatidylinositol anchor. This protein is processed by removing signal and hydrophobic sequences.
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Trypanosoma brucei is a protozoan parasite causing African trypanosomiasis.
- The procyclic acidic repetitive protein (PARP) is a major surface protein in procyclic forms of T. brucei.
- Understanding PARP's structure and membrane attachment is crucial for studying parasite biology.
Purpose of the Study:
- To purify and characterize the procyclic acidic repetitive protein (PARP) from Trypanosoma brucei.
- To investigate the membrane-binding mechanism and processing of PARP.
- To determine the nature of the linkage between PARP and the parasite membrane.
Main Methods:
- Cell fractionation to isolate membrane-bound proteins.
- Ion-exchange chromatography for protein purification.
- Concanavalin A-Sepharose affinity chromatography.
- NH2-terminal sequencing and amino acid analysis.
- Metabolic labeling with [3H] ethanolamine.
- Chemical treatments (base hydrolysis, nitrous acid deamination) to cleave anchors.
Main Results:
- PARP was successfully purified from procyclic Trypanosoma brucei.
- PARP is a membrane-bound protein, constituting approximately 1% of total procyclic trypanosome protein.
- NH2-terminal sequencing and amino acid analysis revealed processing, including removal of an N-terminal signal sequence and a hydrophobic C-terminus.
- Metabolic labeling indicated that PARP is attached to the membrane via a glycosylphosphatidylinositol (GPI) anchor.
- The GPI anchor was sensitive to base hydrolysis and nitrous acid deamination but resistant to bacterial phosphatidylinositol-specific phospholipase C (PI-PLC).
Conclusions:
- PARP is a processed, membrane-bound protein in Trypanosoma brucei.
- The protein is anchored to the membrane by a GPI anchor, suggesting a conserved mechanism for membrane association.
- The resistance to bacterial PI-PLC indicates a potential difference in the anchor structure compared to other organisms or a specific modification.
- These findings contribute to understanding the molecular composition and membrane association of key trypanosome proteins.
