NDLI: Cell-free Synthesis and Functional Characterization of Sphingolipid Synthases from Parasitic Trypanosomatid Protozoa*

Content Provider	PubMed Central
Author	Sevova, Elitza S. Goren, Michael A. Schwartz, Kevin J. Hsu, Fong-fu Turk, John Fox, Brian G. Bangs, James D.
Abstract	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, Ser252; TbSLS3, Phe252) 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.
Related Links	http://dx.doi.org/10.1074/jbc.m110.127662
Ending Page	20587
Page Count	8
Starting Page	20580
File Format	PDF
ISSN	00219258
e-ISSN	1083351X
Journal	The Journal of Biological Chemistry
Issue Number	27
Volume Number	285
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2010-07-02
Access Restriction	Open
Rights Holder	American Society for Biochemistry and Molecular Biology
Subject Keyword	Cell Biology Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Cell-free Synthesis and Functional Characterization of Sphingolipid Synthases from Parasitic Trypanosomatid Protozoa

Ceramide synthases at the centre of sphingolipid metabolism and biology

Characterization of dolichol diphosphate oligosaccharide: protein oligosaccharyltransferase and glycoprotein-processing glucosidases occurring in trypanosomatid protozoa.

The three cortical membranes of the gregarines (parasitic protozoa). Characterization of the membrane proteins of Gregarina blaberae *

Characterization of cell-free protein-synthesis systems from undeveloped and developing Artemia embryos.

Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism[S]

The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes.

Subunit Movements in Single Membrane-bound H+-ATP Synthases from Chloroplasts during ATP Synthesis

Sphingolipids in parasitic protozoa

Cell-free Synthesis and Functional Characterization of Sphingolipid Synthases from Parasitic Trypanosomatid Protozoa*

Similar Documents

Cell-free Synthesis and Functional Characterization of Sphingolipid Synthases from Parasitic Trypanosomatid Protozoa

Ceramide synthases at the centre of sphingolipid metabolism and biology

Characterization of dolichol diphosphate oligosaccharide: protein oligosaccharyltransferase and glycoprotein-processing glucosidases occurring in trypanosomatid protozoa.

The three cortical membranes of the gregarines (parasitic protozoa). Characterization of the membrane proteins of Gregarina blaberae *

Characterization of cell-free protein-synthesis systems from undeveloped and developing Artemia embryos.

Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism[S]

The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes.

Subunit Movements in Single Membrane-bound H+-ATP Synthases from Chloroplasts during ATP Synthesis

Sphingolipids in parasitic protozoa

Cell-free Synthesis and Functional Characterization of Sphingolipid Synthases from Parasitic Trypanosomatid Protozoa*