Cell-free synthesis and functional characterization of sphingolipid synthases from parasitic trypanosomatid protozoa

Technology

Annotation & Function

Summary

The Trypanosoma brucei genome has four highly similar genes encoding sphingolipid synthases (TbSLS1-4).

Description

The Trypanosoma brucei genome has four highly similar genes encoding sphingolipid synthases (TbSLS1-4). TbSLSs are polytopic membrane proteins that are essential for viability of the pathogenic bloodstream stage of this human protozoan parasite and, consequently, can be considered as potential drug targets. TbSLS4 was shown previously to be a bifunctional sphingomyelin/ethanolamine phosphorylceramide synthase, whereas functions of the others were not characterized. Using a recently described liposome-supplemented cell-free synthesis system, which eliminates complications from background cellular activities, we now unambiguously define the enzymatic specificity of the entire gene family. TbSLS1 produces inositol phosphorylceramide, TbSLS2 produces ethanolamine phosphorylceramide, and TbSLS3 is bifunctional, like TbSLS4. These findings indicate that TbSLS1 is uniquely responsible for synthesis of inositol phosphorylceramide in insect stage parasites, in agreement with published expression array data (17). This approach also revealed that the Trypanosoma cruzi ortholog (TcSLS1) is a dedicated inositol phosphorylceramide synthase. The cell-free synthesis system allowed rapid optimization of the reaction conditions for these enzymes and site-specific mutagenesis to alter end product specificity. A single residue at position 252 (TbSLS1, Ser(252); TbSLS3, Phe(252)) strongly influences enzymatic specificity. We also have used this system to demonstrate that aureobasidin A, a potent inhibitor of fungal inositol phosphorylceramide synthases, does not significantly affect any of the TbSLS activities, consistent with the phylogenetic distance of these two clades of sphingolipid synthases. These results represent the first application of cell-free synthesis for the rapid preparation and functional annotation of integral membrane proteins and thus illustrate its utility in studying otherwise intractable enzyme systems.

Figure


uploads/sphingolipid_synthesis_pathway.gif

Sphingolipid synthesis pathway. 3-KDS, 3-keto-dihydrosphingosine; PL, phospholipid; DAG, diacylglycerol. Homologs of all indicated enzymes (italics) are found in the T. brucei and L. major genomes: serine palmitoyl transferase, Tb927.4.1020 (subunit 1) and Tb10.70.3220 (subunit 2); 3-ketosphinganine reductase, Tb927.10.4040; ceramide synthase, Tb927.8.7730; dihydroceramide desaturase, Tb927.6.3000; sphingolipid synthases, Tb09.211.1000-1030. The proposed site of action of aureobasidin A (AbA?) is indicated.

Publication

Sevova ES, Goren MA, Schwartz KJ, Hsu FF, Turk J, Fox BG, Bangs JD. Cell-free synthesis and functional characterization of sphingolipid synthases from parasitic trypanosomatid protozoa. J Biol Chem 2010, 285(27):20580-7. PubMed ID: 20457606 | Search SBKB Publications portal | PMC Link

Contact

Brian Fox (bgfox@biochem.wisc.edu)

Transmembrane Protein Center

Link




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Last edited:Tue 08 Jan 2013 - 4 years, 5 months ago