In Silico Analysis of the Fucosylation-Associated Genome of the Human Blood Fluke Schistosoma mansoni: Cloning and Characterization of the Fucosyltransferase Multigene Family

Abstract

Fucosylated glycans of the parasitic flatworm Schistosoma mansoni play key roles in its development and immunobiology. In the present study we used a genome-wide homology-based bioinformatics approach to search for genes that contribute to fucosylated glycan expression in S. mansoni, specifically the α2-, α3-, and α6-fucosyltransferases (FucTs), which transfer L-fucose from a GDP-L-fucose donor to an oligosaccharide acceptor. We identified and in silico characterized several novel schistosome FucT homologs, including six α3-FucTs and six α6-FucTs, as well as two protein O-FucTs that catalyze the unrelated transfer of L-fucose to serine and threonine residues of epidermal growth factor- and thrombospondin-type repeats. No α2-FucTs were observed. Primary sequence analyses identified key conserved FucT motifs as well as characteristic transmembrane domains, consistent with their putative roles as fucosyltransferases. Most genes exhibit alternative splicing, with multiple transcript variants generated. A phylogenetic analysis demonstrated that schistosome α3- and α6-FucTs form monophyletic clades within their respective gene families, suggesting multiple gene duplications following the separation of the schistosome lineage from the main evolutionary tree. Quantitative decreases in steady-state transcript levels of some FucTs during early larval development suggest a possible mechanism for differential expression of fucosylated glycans in schistosomes. This study systematically identifies the complete repertoire of FucT homologs in S. mansoni and provides fundamental information regarding their genomic organization, genetic variation, developmental expression, and evolutionary history.

Functional characterization of Schistosoma mansoni fucosyltransferases in Nicotiana benthamiana plants

“…After sequencing of the S. mansoni genome in 2009 various putative SmFucT coding sequences were found, of which 14 full-length genes could be amplified from cDNA 20 , 25 . Currently, these SmFucTs are characterized based on sequence analysis 20 and SmFucTF is the only functionally characterized enzyme based on chemoenzymatic assays 21 . However, characterization based on sequences or chemoenzymatic assays can differ from the in vivo biological function.…”

“…Fucosylated glycan motifs are synthesized by specific fucosyltransferases (FucTs), however, most of the S. mansoni FucTs (SmFucTs) are not yet functionally characterized. Via a homology-based genome-wide bioinformatics approach, 14 full-length SmFucT sequences were characterized in silico and were previously amplified from cDNA 20 . Of these 14 sequences, two have been classified as O-SmFucTs involved in transfer of fucose to a serine or threonine residue.…”

In silico analysis of the fucosylation-associated genome of the human blood fluke Schistosoma mansoni: cloning and characterization of the enzymes involved in GDP-L-fucose synthesis and Golgi import

“…In addition to known Golgi-associated GFTs, search queries included the ER-resident transporter Efr , which imports GDP-L-fucose donor substrates for consumption by ER-associated protein O-FucTs in Drosophila . These searches failed to identify a homologous ER-type GFT in S. mansoni despite the previous finding that schistosomes express two putative ER-resident protein O-fucosyltransferases [3]. Notably, Ishikawa et al .…”

“…Primers used for reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE) are provided (see Additional file 1: Table S1A-C). RT-PCR and RACE protocols were performed as detailed in [3] and are summarized as follows: Miracidia, 2-day in vitro -cultivated primary sporocysts and mixed-sex adults (i.e., pooled male and female worms) were washed with artificial pond water (miracidia), CBSS (sporocysts) or mammalian phosphate-buffered saline (8.41 mM Na 2 HPO 4 , 1.65 mM NaH 2 PO 4 ·H 2 O, 146.4 mM NaCl, pH 7.4; adults), and total (“raw”) RNA was extracted using TRIzol® Reagent (Invitrogen). Genomic contamination was removed with TURBO™ DNase (Applied Biosystems, Foster City, CA, USA), and the resultant DNA-free RNA was converted to RT-PCR-ready cDNA using the SuperScript® III First-Strand Synthesis System (Invitrogen).…”

“…Recent studies by Fitzpatrick et al . [2] and Peterson et al [3] inventoried the schistosome α3- and α6-fucosyltransferases (FucTs), which transfer L-fucose from a GDP-L-fucose nucleotide-sugar donor to an oligosaccharide acceptor to create α3 and α6 linkages, respectively. These studies also demonstrated stage- and gender-specific variations in FucT gene transcription, which may contribute to differential fucosyl-glycotope expression that has been reported among stages of S. mansoni [4-7].…”