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• W2015386056 abstract "Fungal glucosylceramides play an important role in plant-pathogen interactions enabling plants to recognize the fungal attack and initiate specific defense responses. A prime structural feature distinguishing fungal glucosylceramides from those of plants and animals is a methyl group at the C9-position of the sphingoid base, the biosynthesis of which has never been investigated. Using information on the presence or absence of C9-methylated glucosylceramides in different fungal species, we developed a bioinformatics strategy to identify the gene responsible for the biosynthesis of this C9-methyl group. This phylogenetic profiling allowed the selection of a single candidate out of 24–71 methyltransferase sequences present in each of the fungal species with C9-methylated glucosylceramides. A Pichia pastoris knock-out strain lacking the candidate sphingolipid C9-methyltransferase was generated, and indeed, this strain contained only non-methylated glucosylceramides. In a complementary approach, a Saccharomyces cerevisiae strain was engineered to produce glucosylceramides suitable as a substrate for C9-methylation. C9-methylated sphingolipids were detected in this strain expressing the candidate from P. pastoris, demonstrating its function as a sphingolipid C9-methyltransferase. The enzyme belongs to the superfamily of S-adenosylmethionine-(SAM)-dependent methyltransferases and shows highest sequence similarity to plant and bacterial cyclopropane fatty acid synthases. An in vitro assay showed that sphingolipid C9-methylation is membrane-bound and requires SAM and Δ4,8-desaturated ceramide as substrates. Fungal glucosylceramides play an important role in plant-pathogen interactions enabling plants to recognize the fungal attack and initiate specific defense responses. A prime structural feature distinguishing fungal glucosylceramides from those of plants and animals is a methyl group at the C9-position of the sphingoid base, the biosynthesis of which has never been investigated. Using information on the presence or absence of C9-methylated glucosylceramides in different fungal species, we developed a bioinformatics strategy to identify the gene responsible for the biosynthesis of this C9-methyl group. This phylogenetic profiling allowed the selection of a single candidate out of 24–71 methyltransferase sequences present in each of the fungal species with C9-methylated glucosylceramides. A Pichia pastoris knock-out strain lacking the candidate sphingolipid C9-methyltransferase was generated, and indeed, this strain contained only non-methylated glucosylceramides. In a complementary approach, a Saccharomyces cerevisiae strain was engineered to produce glucosylceramides suitable as a substrate for C9-methylation. C9-methylated sphingolipids were detected in this strain expressing the candidate from P. pastoris, demonstrating its function as a sphingolipid C9-methyltransferase. The enzyme belongs to the superfamily of S-adenosylmethionine-(SAM)-dependent methyltransferases and shows highest sequence similarity to plant and bacterial cyclopropane fatty acid synthases. An in vitro assay showed that sphingolipid C9-methylation is membrane-bound and requires SAM and Δ4,8-desaturated ceramide as substrates. Sphingolipids of fungi and plants can be grouped into different classes based on the nature of their polar headgroups. In the case of GIPC, 2The abbreviations used are: GIPC, glycosylinositolphosphorylceramide; IPC, inositolphosphorylceramide; CDS, coding sequence; contig, group of overlapping clones; DNP, 2,4-dinitrophenyl; ESI, electrospray ionization; HPLC, high performance liquid chromatography; MCS, multiple cloning site; MS, mass spectrometry; SAM, S-adenosylmethionine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 2The abbreviations used are: GIPC, glycosylinositolphosphorylceramide; IPC, inositolphosphorylceramide; CDS, coding sequence; contig, group of overlapping clones; DNP, 2,4-dinitrophenyl; ESI, electrospray ionization; HPLC, high performance liquid chromatography; MCS, multiple cloning site; MS, mass spectrometry; SAM, S-adenosylmethionine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. an inositol residue is phosphodiester-linked to the hydrophobic part of the molecule, the ceramide backbone. In the case of cerebrosides, the ceramide is directly linked to a glucosyl or galactosyl residue that can form the base for further glycosylations. The ceramide backbone usually carries several functional groups such as hydroxy groups, (E)- or (Z)-double bonds, methyl branches at aliphatic or olefinic carbon atoms, or even cyclopropane rings. Different patterns of functional groups are characteristic for GIPC versus cerebrosides as well as for different organisms (see Fig. 1) (1Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 1999; 1426: 347-357Crossref PubMed Scopus (168) Google Scholar, 2Toledo M.S. Levery S.B. Straus A.H. Suzuki E. Momany M. Glushka J. Moulton J.M. Takahashi H.K. Biochemistry. 1999; 38: 7294-7306Crossref PubMed Scopus (101) Google Scholar, 3Toledo M.S. Levery S.B. Straus A.H. Takahashi H.K. J. Lipid Res. 2000; 41: 797-806Abstract Full Text Full Text PDF PubMed Google Scholar, 4Warnecke D. Heinz E. Cell. Mol. Life Sci. 2003; 60: 919-941Crossref PubMed Scopus (133) Google Scholar, 5Tan R.X. Chen J.H. Nat. Prod. Rep. 2003; 20: 509-534Crossref PubMed Scopus (90) Google Scholar). The functional significance of these differences has yet to be investigated.The genes responsible for most functional groups have been cloned (reviewed in Refs. 4Warnecke D. Heinz E. Cell. Mol. Life Sci. 2003; 60: 919-941Crossref PubMed Scopus (133) Google Scholar, 6Daum G. Lees N.D. Bard M. Dickson R. Yeast. 1998; 14: 1471-1510Crossref PubMed Scopus (514) Google Scholar, 7Sperling P. Warnecke D. Heinz E. Top. Curr. Genet. 2004; 6: 337-381Crossref Google Scholar, 8Dunn T.M. Lynch D.V. Michaelson L.V. Napier J.A. Ann. Bot. 2004; 93: 483-497Crossref PubMed Scopus (131) Google Scholar), and now their biological functions will be characterized by reverse genetic, molecular, and cell biological approaches. A structural feature hardly studied so far is the C9-methyl branch at the (E)-Δ8-double bond of fungal cerebrosides. Many fungi share a canonical glucosylceramide structure with (E,E)-9-methylsphinga-4,8-dienine as sphingoid base, carrying (E)-double bonds at the Δ4- and Δ8-positions and a methyl branch at the C9-position (4Warnecke D. Heinz E. Cell. Mol. Life Sci. 2003; 60: 919-941Crossref PubMed Scopus (133) Google Scholar). Besides in fungi, a glucosylceramide with the same structure has been found in the sea anemone Metridium senile (9Karlsson K.-A. Leffler H. Samuelsson B.E. Biochim. Biophys. Acta. 1979; 574: 79-93Crossref PubMed Scopus (50) Google Scholar), the first organism described to contain C9-methylated sphingolipids, as well as in the protist Thraustochytrium globosum (10Jenkins K.M. Jensen P.R. Fenical W. Tetrahedron Lett. 1999; 40: 7637-7640Crossref Scopus (31) Google Scholar). Structurally related glucosylceramides containing an additional Δ10-double bond have been found in T. globosum, in several species of starfish, in the marine sponge Agelas mauritianus, and in the sea squirt Phallusia fumigata (10Jenkins K.M. Jensen P.R. Fenical W. Tetrahedron Lett. 1999; 40: 7637-7640Crossref Scopus (31) Google Scholar, 11Irie A. Kubo H. Hoshi M. J. Biochem. (Tokyo). 1990; 107: 578-586Crossref PubMed Scopus (42) Google Scholar, 12Higuchi R. Harano Y. Mitsuyuki M. Isobe R. Yamada K. Miyamoto T. Komori T. Liebigs Ann. 1996; 1996: 593-599Crossref Scopus (30) Google Scholar, 13Jin W. Rinehart K.L. Jares-Erijman E.A. J. Org. Chem. 1994; 59: 144-147Crossref Scopus (131) Google Scholar, 14Natori T. Morita M. Akimoto K. Koezuka Y. Tetrahedron. 1994; 50: 2771-2784Crossref Scopus (398) Google Scholar, 15Durán R. Zubía E. Ortega M.J. Naranjo S. Salvá J. Tetrahedron. 1998; 54: 14597-14602Crossref Scopus (46) Google Scholar).There are several studies pointing to important biological functions of fungal glucosylceramides. Fungal glucosylceramides carrying a C9-methyl group as well as certain plant glucosylceramides induce fruiting body formation in the fungus Schizophyllum commune (16Kawai G. Ohnishi M. Fujino Y. Ikeda Y. J. Biol. Chem. 1986; 261: 779-784Abstract Full Text PDF PubMed Google Scholar). Human patients with a Cryptococcus neoformans infection as well as rabbits infected with Pseudallescheria boydii develop antibodies binding to C9-methylated fungal glucosylceramides. The purified antibodies inhibit fungal growth in culture (17Rodrigues M.L. Travassos L.R. Miranda K.R. Franzen A.J. Rozental S. de Souza W. Alviano C.S. Barreto-Bergter E. Infect. Immun. 2000; 68: 7049-7060Crossref PubMed Scopus (175) Google Scholar, 18Pinto M.R. Rodrigues M.L. Travassos L.R. Haido R.M.T. Wait R. Barreto-Bergter E. Glycobiology. 2002; 12: 251-260Crossref PubMed Scopus (80) Google Scholar). Fungal glucosylceramides can elicit defense responses in rice plants and cell suspension cultures of rice, and treatment with these fungal glucosylceramides protects rice plants against fungal infection (19Koga J. Yamauchi T. Shimura M. Ogawa N. Oshima K. Umemura K. Kikuchi M. Ogasawara N. J. Biol. Chem. 1998; 273: 31985-31991Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 20Umemura K. Ogawa N. Yamauchi T. Iwata M. Shimura M. Koga J. Plant Cell Physiol. 2000; 41: 676-683Crossref PubMed Scopus (68) Google Scholar, 21Umemura K. Ogawa N. Koga J. Iwata M. Usami H. Plant Cell Physiol. 2002; 43: 778-784Crossref PubMed Scopus (65) Google Scholar). The plant defensin RsAFP2 from radish seeds interacts specifically with fungal glucosylceramides (22Thevissen K. Warnecke D.C. François I.E.J.A. Leipelt M. Heinz E. Ott C. Zähringer U. Thomma B.P.H.J. Ferket K.K.A. Cammue B.P.A. J. Biol. Chem. 2004; 279: 3900-3905Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Pichia pastoris and Candida albicans strains deficient in the gene responsible for glucosylceramide synthesis are resistant to RsAFP2 (22Thevissen K. Warnecke D.C. François I.E.J.A. Leipelt M. Heinz E. Ott C. Zähringer U. Thomma B.P.H.J. Ferket K.K.A. Cammue B.P.A. J. Biol. Chem. 2004; 279: 3900-3905Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Because the C9-methyl group is the prime structural feature distinguishing fungal glucosylceramides from those of plants and mammals (Fig. 1), it is likely that C9-methylation of fungal glucosylceramides plays an important role in the interactions between fungi and other organisms.To enable the investigation of these proposed functions with molecular techniques, we were interested in identifying the gene encoding the methyltransferase that introduces the C9-methyl branch into fungal sphingolipids, as well as in directly demonstrating its biochemical function. We developed a bioinformatics strategy based on phylogenetic profiling that exploits the pattern of presence or absence of C9-methylated glucosylceramides in several fungi with completely sequenced genomes. After identifying candidate genes, we tested their involvement in sphingolipid C9-methylation by generating a knock-out mutant of the yeast P. pastoris. In a complementary experiment, a Saccharomyces cerevisiae strain was engineered to directly demonstrate the formation of C9-methylated sphingolipids. Finally, membrane association and substrate specificity of sphingolipid C9-methylation in P. pastoris were investigated by an in vitro assay.EXPERIMENTAL PROCEDURESPhylogenetic ProfilingData Base Construction and Sequence Retrieval—The complete sets of protein sequences from several fungi, plants, and animals were downloaded from the web pages of the sequencing projects listed in Supplemental Table S1 and compiled into a single data base. Swiss-Prot species identification codes (www.expasy.org/cgi-bin/speclist) were included in the sequence titles as species identifiers.Protein sequences belonging to the superfamily of SAM-dependent methyltransferases were retrieved using PSI-Blast (23Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59167) Google Scholar) with the S. cerevisiae sterol methyltransferase Erg6p (YML008C) as query sequence (10 iterations, inclusion threshold e ≤ 10–4).Clustering of SAM-dependent Methyltransferase Sequences—SAM-dependent methyltransferase sequences were compared pairwise using blastp (23Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59167) Google Scholar) (e-value e ≤ 10–4). The resulting score matrix was used as input for the TRIBE-MCL protocol (Ref. 24Enright A.J. Van Dongen S. Ouzounis C.A. Nucleic Acids Res. 2002; 30: 1575-1584Crossref PubMed Scopus (2471) Google Scholar, available at micans.org/mcl) with parameter I = 1.1. For each cluster containing more than four sequences, a phylogenetic tree was constructed with ClustalX (25Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35206) Google Scholar).Groups of Orthologous Sequences—Within the phylogenetic tree for each cluster, groups of orthologous sequences were identified by comparing the branching pattern of the tree with the phylogeny of the species from which the sequences originate. A group of orthologous sequences is defined as a subtree of the phylogenetic tree in which the branches originate only from speciation events but not from gene duplications within the same species. Genes duplicated within a single species were counted as one gene if the duplicated sequences were more similar to each other than to any other sequence.Groups of orthologous sequences as defined above may be nested, i.e. when sequences A, B, and C form a group of orthologous sequences with branching pattern ((A,B), C), then the sequences A and B form themselves a smaller group that also fits the definition. In such cases, only the enclosing group was considered for phylogenetic profiling.Phylogenetic Profiles—The phylogenetic profile of a group of sequences is defined as the set of species from which the sequences originate (26Pellegrini M. Marcotte E.M. Thompson M.J. Eisenberg D. Yeates T.O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4285-4288Crossref PubMed Scopus (1450) Google Scholar). The difference between two phylogenetic profiles was quantified by counting how many sequences were present in only one of the two profiles. The number of differences was counted as the number of sequences present only in the first profile plus the number of sequences present only in the second profile.Data Handling—Scripts for the handling of sequence data and phylogenetic trees were written in the programming language Python (www.python.org) with the Biopython libraries (www.biopython.org).Knock-out of the Sphingolipid C9-Methyltransferase in P. pastorisA knock-out cassette for an exact deletion of the candidate C9-methyltransferase CDS was constructed by triple fusion PCR as described in Ref. 27Shevchuk N.A. Bryksin A.V. Nusinovich Y.A. Cabello F.C. Sutherland M. Ladisch S. Nucleic Acids Res. 2004; 32: e19Crossref PubMed Scopus (250) Google Scholar. All reactions were performed with PfuTurbo DNA polymerase (Stratagene). The primers used are listed in Supplemental Table S2. The underlined parts of the chimeric primers Up-R-Zeo-F and Down-F-Zeo-R are the reverse complement to the primers Zeo-F and Zeo-R, respectively. The Zeocin resistance cassette from the pGAPZ-B vector (Invitrogen) was amplified with primers Zeo-F and Zeo-R. 3099 bp of the genomic locus containing the candidate C9-methyltransferase CDS was amplified from genomic DNA of the P. pastoris strain GS115 (28Cregg J.M. Barringer K.J. Hessler A.Y. Madden K.R. Mol. Cell. Biol. 1985; 5: 3376-3385Crossref PubMed Scopus (450) Google Scholar) with primers Up-F and Down-R. From this PCR product, upstream and downstream sequences directly flanking the CDS were amplified with the nested primers Up-nested-F/Up-R-Zeo-F (530 bp) and Down-nested-R/Down-F-Zeo-R (516 bp), respectively. The upstream and downstream sequences and the Zeocin resistance cassette were fused in a single PCR for 12 rounds without primers. From this, the full-length fusion product was amplified in a separate reaction with primers Up-nested-F and Down-nested-R. The knock-out cassette was ligated into the SmaI site of the pBluescript II KS(–)-vector (Stratagene) and checked by sequencing.The knock-out cassette was excised from the plasmid with PstI/XbaI and used to transform electrocompetent cells of the strain GS115 (his4). Transformants were selected on YPD plates (34Treco D.A. Lundblad V. Current Protocols in Molecular Biology.in: Asubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Albright L.M. Coen D.M. Varki A. John Wiley & Sons, New York1993Google Scholar) containing 100 mg/liter Zeocin. Correct integration of the knock-out cassette was confirmed by PCR with the primer pairs Up-F/Zeo-int-R and Down-R/Zeo-int-F as positive and Up-F/Int-R and Down-R/Int-F as negative controls.Knock-out of SUR2 in S. cerevisiaeA knock-out cassette for an exact deletion of the SUR2 CDS (YDR297W) was constructed by PCR with long primers as described in Ref. 29Lundblad V. Hartzog G. Moqtaderi Z. Current Protocols in Molecular Biology.in: Asubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Albright L.M. Coen D.M. Varki A. John Wiley & Sons, New York1997Google Scholar. The primers used are listed in Supplemental Table S3. The kanamycin resistance cassette of the plasmid pUG6 (30Güldener U. Heck S. Fielder T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1341) Google Scholar) was amplified with primers SUR2-kan-F and SUR2-kan-R. These primers contain ≈80 bp complementary to the sequences directly flanking the SUR2 CDS (underlined). The PCR reaction was performed with Taq DNA polymerase (Invitrogen). The knock-out cassette was ligated into the pGEM-T vector (Promega).The knock-out cassette was excised from the plasmid with NotI/SacII and used to transform chemically competent cells of the strain UTL-7A (MATa ura3–52 trp1 leu2–3/112). Transformants were selected on YPD plates containing 200 mg/liter G-418. Correct integration of the knock-out cassette was confirmed by PCR with primer pairs SUR2-up-F/SUR2-up-R and SUR2-down-F/SUR2-Down-R as positive controls and primer pair SUR2-up-F/SUR2-Down-R as combined positive and negative control.An S. cerevisiae Expression SystemIdentification of the Δ4- and Δ8-Desaturases from P. pastoris—For PCR-based cloning of the P. pastoris Δ4- and Δ8-desaturases, a genomic DNA library of the P. pastoris strain GS115 (28Cregg J.M. Barringer K.J. Hessler A.Y. Madden K.R. Mol. Cell. Biol. 1985; 5: 3376-3385Crossref PubMed Scopus (450) Google Scholar) was used as template. The primers used are listed in Supplemental Table S4.To amplify the Δ4-desaturase, degenerate primers were deduced from a multiple alignment of amino acid sequences from C. albicans, Neurospora crassa, Schizosaccharomyces pombe, Arabidopsis thaliana, Lycopersicon esculentum, Drosophila melanogaster, and Homo sapiens belonging to the DES family (31Ternes P. Franke S. Zähringer U. Sperling P. Heinz E. J. Biol. Chem. 2002; 277: 25512-25518Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). PCR-amplification was carried out with the degenerate primers Delta4-deg-F and Delta4-deg-R at an annealing temperature of 48 °C.To amplify the Δ8-desaturase, degenerate primers were deduced from a multiple alignment of amino acid sequences from Δ5- and Δ6-fatty acid desaturases and Δ8-sphingolipid desaturases described in Ref. 32Sperling P. Lee M. Girke T. Zähringer U. Stymne S. Heinz E. Eur. J. Biochem. 2000; 267: 3801-3811Crossref PubMed Scopus (72) Google Scholar. PCR amplification was carried out using the degenerate primers Delta8-deg-F and Delta8-deg-R at an annealing temperature of 40 °C and resulted in PCR products of 177 bp for the Δ4-desaturase and 564 bp for the Δ8-desaturase.For the identification of full-length Δ4- and Δ8-desaturase sequences, the P. pastoris genomic DNA library was screened with these PCR products using the DIG System (Roche Molecular Biochemicals). The inserts of positive clones for each desaturase were sequenced, with CDS of 1083 bp (Δ4-desaturase) and 1629 bp (Δ8-desaturase) encoding polypeptides of 360 and 542 amino acids, respectively. 3The nucleotide sequences of the Δ4- and Δ8-desaturases from P. pastoris have been deposited in the GenBank™ database with accession numbers AY700778 and AY700777. The GenPept accession numbers of the deduced amino acid sequences are AAU10085 and AAU10084, respectively. Cloning of the P. pastoris Δ4- and Δ8-Desaturases—The complete CDSs of the P. pastoris Δ4- and Δ8-desaturases were amplified from genomic clones by PCR with PfuTurbo DNA polymerase (Stratagene, Δ4-desaturase) or Herculase DNA Polymerase (Stratagene, Δ8-desaturase) with the primers Delta4-F and Delta4-R (Δ4-desaturase), and Delta8-F and Delta8-R (Δ8-desaturase) (Supplemental Table S4). The primers included adapter sequences containing the indicated restriction sites (underlined). The PCR products were ligated into the pESC-URA vector (Stratagene), with the CDS of the Δ4-desaturase in the NotI and BglII sites of MCS1 and the CDS of the Δ8-desaturase in the BamHI and XhoI sites of MCS2. In this way, three different plasmids were generated: One plasmid carrying the Δ4-desaturase, one carrying the Δ8-desaturase, and one carrying both desaturases. The cloned CDSs were checked by sequencing.Cloning of a Candidate Sphingolipid C9-Methyltransferase—The complete CDS of a candidate sphingolipid C9-methyltransferase from P. pastoris was amplified from genomic DNA of the P. pastoris strain GS115 with the primers C9-F and C9-R (Supplemental Table S4). The primers included adapter sequences containing the indicated restriction sites (underlined). The PCR products were ligated into the BamHI and EcoRI sites of the pYES3-CT vector (Invitrogen). The resulting plasmid was checked by sequencing.Transformation of S. cerevisiae—For the experiment shown in Fig. 3, chemically competent cells of the S. cerevisiae SUR2 knock-out strain were transformed with the empty vector pESC-URA (Fig. 3A) or with either the Δ4-desaturase (Fig. 3B), the Δ8-desaturase (Fig. 3C), or both desaturases (Fig. 3D) ligated into pESC-URA.FIGURE 3Expression of the P. pastoris Δ4- and Δ8-desaturases in an S. cerevisiae strain lacking the sphingolipid C4-hydroxylase SUR2. A, S. cerevisiae cells transformed with the empty vector contained the sphingoid bases C18-sphinganine (d18:0) and C20-sphinganine (d20:0). B, S. cerevisiae cells expressing the P. pastoris Δ4-desaturase contained the additional sphingoid bases C18-(E)-sphing-4-enine (d18:1Δ4) and C20-(E)-sphing-4-enine (d20:1Δ4). C, S. cerevisiae cells expressing the P. pastoris Δ8-desaturase contained the additional sphingoid base C18-(E)-sphing-8-enine (d18:1Δ8). D, S. cerevisiae cells expressing both the P. pastoris Δ4- and Δ8-desaturases contained the three additional sphingoid bases C18-(E,E)-sphinga-4,8-dienine (d18:2Δ4,8), C20-(E,E)-sphinga-4,8-dienine (d20:2Δ4,8), and C20-(E)-sphing-4-enine (d20:1Δ4). The sphingoid bases from whole cells were analyzed as DNP derivatives by HPLC.View Large Image Figure ViewerDownload Hi-res image Download (PPT)For the experiment shown in Fig. 4, chemically competent cells of the S. cerevisiae SUR2 knock-out strain were transformed simultaneously with the empty vector pYES3-CT (Fig. 4A) or the candidate C9-methyltransferase ligated in pYES3-CT (Fig. 4B), with the Δ4-desaturase and the Δ8-desaturase ligated into pESC-URA, and with the human glucosylceramide synthase (33Leipelt M. Warnecke D. Zähringer U. Ott C. Müller F. Hube B. Heinz E. J. Biol. Chem. 2001; 276: 33621-33629Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) ligated into both MCS of the pESC-LEU vector (Invitrogen). The plasmid containing the glucosylceramide synthase was kindly provided by M. Leipelt, Universität Hamburg.FIGURE 4Expression of the candidate sphingolipid C9-methyltransferase from P. pastoris in S. cerevisiae. A, S. cerevisiae cells lacking the sphingolipid C4-hydroxylase SUR2 while expressing the P. pastoris Δ4- and Δ8-desaturases, the human glucosylceramide synthase, and the candidate sphingolipid C9-methyltransferase from P. pastoris contained the C9-methylated sphingoid base C18-(E,E)-9-methylsphinga-4,8-dienine (d18:2–9m) as well as the non-methylated sphingoid bases shown in Fig. 3D. B, enlarged section of the chromatogram shown in A. The methylated sphingoid base C18-(E,E)-9-methylsphinga-4,8-dienine (d18:2–9m) is only present in the S. cerevisiae cells expressing the candidate C9-methyltransferase (top), but not in the control (bottom). C, comparing the ion trace of the ion with m/z = 476 (bottom) to the chromatogram (top) confirms that the peak at 20 min is indeed C18-(E,E)-9-methylsphinga-4,8-dienine (d18:2–9m). D, the ESI-MS spectrum of the peak at 20 min shows that this peak contains only the ion with m/z = 476 corresponding to C18-(E,E)-9-methylsphinga-4,8-dienine and its heavier isotope with m/z = 477. The sphingoid bases from whole cells were analyzed as DNP derivatives by HPLC (A and B) and by HPLC/MS in negative ion mode (C and D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Lipid AnalysisCulture of P. pastoris—A preculture was grown in liquid YPD medium for 24 h at 30 °C. 400 ml of YPD medium was inoculated 1:100 from the preculture and incubated again for 24 h at 30 °C. The cells were harvested by centrifugation.Culture of S. cerevisiae—Precultures were grown in complete minimal medium (34Treco D.A. Lundblad V. Current Protocols in Molecular Biology.in: Asubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Albright L.M. Coen D.M. Varki A. John Wiley & Sons, New York1993Google Scholar) containing 2% (w/v) glucose at 30 °C for 3 days. To induce expression under the control of the GAL1 and GAL10 promotors, the cells were washed, and 100 ml of complete minimal medium containing 2% (w/v) raffinose and 2% (w/v) galactose was inoculated to give a starting optical density of A600 = 0.02. The cultures were incubated at 30 °C for 48 h, and the cells were harvested by centrifugation.Isolation of Glucosylceramides from P. pastoris—Approximately 10 g (fresh weight) of P. pastoris cells was resuspended in 20 ml of 0.45% (w/v) NaCl and boiled in a water bath for 15 min. The cells were sedimented by centrifugation, and the lipids were extracted by shaking in 30 ml of CHCl3/MeOH, 1:2 for 24 h at 8 °C followed by 30 ml of CHCl3/MeOH, 2:1. The lipid extract was washed by phase partitioning with CHCl3/MeOH/0.45% (w/v) NaCl (8:4:3) and subsequently evaporated. The lipids were redissolved in CHCl3 and applied to a 500 mg/6 ml Strata SI-2 column (Phenomenex). The column was flushed with 30 ml of CHCl3 before the glycolipids were eluted with 30 ml of acetone/2-propanol, 9:1. The glucosylceramides were purified from the acetone/2-propanol fraction by preparative TLC on Silica Gel 60 plates (Merck) in CHCl3/MeOH, 85:15.Analysis of the Sphingoid Base Composition—The sphingoid base composition of P. pastoris glucosylceramides and of total S. cerevisiae cells was analyzed as described in Ref. 31Ternes P. Franke S. Zähringer U. Sperling P. Heinz E. J. Biol. Chem. 2002; 277: 25512-25518Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar. Briefly, sphingolipids were hydrolyzed with 10% (w/v) Ba(OH)2 in 1,4-dioxane/H2O, 1:1 (v/v) (20 h at 110 °C) (35Morrison W.R. Hay J.D. Biochim. Biophys. Acta. 1970; 202: 460-467Crossref PubMed Scopus (79) Google Scholar). The free sphingoid bases were extracted, washed, and converted to their DNP derivatives with one part of 0.5% (v/v) methanolic 1-fluoro-2,4-dinitrobenzene and four parts of 2 m boric acid/KOH, pH 10.5 (30 min at 60 °C) (36Karlsson K.-A. Chem. Phys. Lipids. 1970; 5: 6-43Crossref PubMed Scopus (243) Google Scholar). The DNP-derivatized sphingoid bases were extracted and purified by TLC in CHCl3/MeOH, 90:10.Analysis by reverse-phase HPLC was performed on a Multospher RP18-5 250 × 4 mm column (CS-Chromatographie) with a flow rate of 0.8 ml/min and a concave gradient from 84 to 100% methanol/acetonitrile/2-propanol, 10:3:1, against H2O in 55 min. The elution was monitored with a UV detector (ThermoQuest) at 350 nm.Reversed-phase HPLC/MS with electrospray ionization (ESI) of DNP-derivatized sphingoid bases was performed on a MAT 95XL-Trap instrument (ThermoQuest) in negative ion mode. The eluate of the HPLC was split using a low dead volume T-piece with 5% entering the ESI-sprayer and 95% a UV detector (Hitachi).In Vitro Methyltransferase AssayPreparation of Sphingolipid Substrates—Glucosylceramides from P. pastoris wild-type cells, from the P. pastoris sphingolipid C9-methyltransferase knock-out strain, and from cucumber leaves were isolated as described above. Ceramides were prepared from the purified glucosylceramides by periodic acid oxidation followed by β-elimination with 1,1-dimethylhydrazine (37Heinze F.J. Linscheid M. Heinz E. Anal. Biochem. 1984; 139: 126-133Crossref PubMed Scopus (13) Google Scholar). Briefly, purified glucosylceramides were incubated in 0.2 m methanolic HIO4 (90 min at room temperature). The" @default.
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