Identification of Fungal Sphingolipid C9-methyltransferases by Phylogenetic Profiling

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 membranebound and requires SAM and ⌬4,8-desaturated ceramide as substrates.

Functional Characterization of a Higher Plant Sphingolipid Δ4-Desaturase: Defining the Role of Sphingosine and Sphingosine-1-Phosphate in Arabidopsis

“…In common with many fungi, the methylotrophic yeast P. pastoris synthesizes GlcCers that contain D4-unsaturated LCBs, predominantly in the form of 9-methyl-sphinga-4,8-dienine. The three P. pastoris enzymes (sphingolipid C9-methyltransferase and sphingolipid D4-and D8-desaturases) responsible for the synthesis of this LCB were recently identified and characterized by heterologous expression in S. cerevisiae (Ternes et al, 2006). In this study, we deleted the intrinsic P. pastoris sphingolipid D4-desaturase gene to establish a mutant strain suitable as an expression host to characterize heterologously expressed sphingolipid D4-desaturase candidate genes.…”

“…The LCB composition of P. pastoris glucosylceramides was analyzed as described by Ternes et al (2006). Similar methods were used for the analyses of Arabidopsis and Commelina communis sphingolipid LCBs, according to the methods of Borner et al (2005) and Tonon et al (2005).…”

Sphingolipids from the human fungal pathogen Aspergillus fumigatus

“…The absence of filipin staining in GlcCer deficient strain or mutant with altered ceramide structure showed that GlcCer is essential for the polarisome organisation at the apex [26,27]. The function the methyl group at the C-9, which is absent in plant and mammalian GlcCer structures [17], has been studied in yeast and filamentous fungi. The deletion of the specific methyl-transferase involved in GlcCer synthesis leads to defects in hyphal growth and in virulence of pathogenic fungal strains [18,24,28,29].…”

“…Then, three enzymatic reactions occur sequentially that lead to ∆4-and ∆8-desaturation and addition of a methyl group at the C-9 position on the LCB (Fig. 2) [16][17][18]. Finally, a β-glucosyltransferase, Gcs1 for glucosylceramide synthase, uses UDP-Glc as sugar donor to transfer a glucose unit onto the primary hydroxyl group to form β-glucosylceramide [19].…”

Identification and Functional Characterization of the 2-Hydroxy Fatty N-Acyl-Δ3(E)-desaturase from Fusarium graminearum

“…Many enzymes necessary for modifications of the ceramide backbone (glycosyltransferases, desaturases, hydroxylases, C9-methyltransferase) have already been identified and functionally characterized in plants and fungi (13)(14)(15)(16)(17)(18)(19)(20)(21). However, a gene corresponding to a desaturase with ⌬3(E)-regioselectivity has not yet been cloned.…”