Cell-free synthesis and functional characterization of sphingolipid synthases from parasitic trypanosomatid protozoa.
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.
Sevova
Elitza S
ES
Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.
Goren
Michael A
MA
Schwartz
Kevin J
KJ
Hsu
Fong-Fu
FF
Turk
John
J
Fox
Brian G
BG
Bangs
James D
JD
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D002474:Cell-Free System; D002518:Ceramides / Q000378:metabolism; D003001:Cloning, Molecular; D016678:Genome; D006801:Humans; D008081:Liposomes; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013104:Spheroplasts / Q000235:genetics; D013107:Sphingolipids / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014345:Trypanosoma / Q000235:genetics* / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism; D014352:Trypanosomiasis / Q000235:genetics* / Q000378:metabolism
D020413:3' Untranslated Regions; D002518:Ceramides; D008081:Liposomes; D013107:Sphingolipids
Exp Parasitol. 2004 Mar-Apr;106(3-4):135-49
15172221
J Biol Chem. 2006 Oct 6;281(40):29421-5
16905542
J Biol Chem. 2004 Apr 30;279(18):18688-93
14976195
Nucleic Acids Symp Ser. 2000;(44):9-10
12903243
Mol Microbiol. 2008 Oct;70(2):281-96
18699867
EMBO J. 2007 Feb 21;26(4):1094-104
17290222
J Biol Chem. 1997 Apr 11;272(15):9809-17
9092515
J Lipid Res. 1986 Dec;27(12):1294-303
3559392
Clin Chim Acta. 1982 May 6;121(1):111-6
6282500
J Biol Chem. 2006 Sep 22;281(38):28200-9
16861742
Cell Mol Life Sci. 2007 Sep;64(17):2270-84
17558466
EMBO J. 2003 Nov 17;22(22):6016-26
14609948
Protein Expr Purif. 2008 Dec;62(2):171-8
18765284
J Antibiot (Tokyo). 1991 Sep;44(9):919-24
1938613
J Lipid Res. 2009 Nov;50(11):2270-7
19454763
Biochim Biophys Acta. 1981 Jun 22;644(2):341-50
7020760
Biochim Biophys Acta. 1996 May 20;1300(3):233-9
8679689
Comp Biochem Physiol B. 1977;58(2):195-9
400952
Mol Biochem Parasitol. 2009 Nov;168(1):16-23
19545591
Protein Expr Purif. 2006 Jun;47(2):562-70
16377204
EMBO J. 2004 Jan 14;23(1):33-44
14685263
BMC Genomics. 2009 Sep 11;10:427
19747379
J Cell Biol. 1994 Sep;126(5):1133-48
8063853
Curr Opin Microbiol. 2006 Aug;9(4):352-8
16798065
Cell. 1989 Mar 10;56(5):793-800
2924349
J Cell Biol. 2009 Jun 15;185(6):1013-27
19506037
Mol Biol Cell. 2009 Oct;20(20):4444-57
19726565
Biochem J. 1995 Aug 15;310 ( Pt 1):255-61
7646454
J Cell Sci. 2002 Aug 15;115(Pt 16):3253-63
12140257
Biochem J. 2005 Apr 15;387(Pt 2):519-29
15569002
Methods Enzymol. 2009;463:647-73
19892197
Mol Microbiol. 2005 Mar;55(5):1566-78
15720561
J Cell Sci. 2004 Dec 1;117(Pt 25):5955-64
15564373
Mol Biol Cell. 2009 Nov;20(22):4739-50
19759175