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

Nathan A Peterson Tavis K Anderson Xiao-Jun Wu 0 Timothy P Yoshino 0 0 Current address: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin , 2115 Observatory Drive, Madison, WI 53706 , USA Background: Carbohydrate structures of surface-expressed and secreted/excreted glycoconjugates of the human blood fluke Schistosoma mansoni are key determinants that mediate host-parasite interactions in both snail and mammalian hosts. Fucose is a major constituent of these immunologically important glycans, and recent studies have sought to characterize fucosylation-associated enzymes, including the Golgi-localized fucosyltransferases that catalyze the transfer of L-fucose from a GDP-L-fucose donor to an oligosaccharide acceptor. Importantly, GDP-L-fucose is the only nucleotide-sugar donor used by fucosyltransferases and its availability represents a bottleneck in fucosyl-glycotope expression. Methods: A homology-based genome-wide bioinformatics approach was used to identify and molecularly characterize the enzymes that contribute to GDP-L-fucose synthesis and Golgi import in S. mansoni. Putative functions were further investigated through molecular phylogenetic and immunocytochemical analyses. Results: We identified homologs of GDP-D-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose3,5-epimerase-4-reductase (GMER), which constitute a de novo pathway for GDP-L-fucose synthesis, in addition to a GDP-L-fucose transporter (GFT) that putatively imports cytosolic GDP-L-fucose into the Golgi. In silico primary sequence analyses identified characteristic Rossman loop and short-chain dehydrogenase/reductase motifs in GMD and GMER as well as 10 transmembrane domains in GFT. All genes are alternatively spliced, generating variants of unknown function. Observed quantitative differences in steady-state transcript levels between miracidia and primary sporocysts may contribute to differential glycotope expression in early larval development. Additionally, analyses of protein expression suggest the occurrence of cytosolic GMD and GMER in the ciliated epidermal plates and tegument of miracidia and primary sporocysts, respectively, which is consistent with previous localization of highly fucosylated glycotopes. Conclusions: This study is the first to identify and characterize three key genes that are putatively involved in the synthesis and Golgi import of GDP-L-fucose in S. mansoni and provides fundamental information regarding their genomic organization, genetic variation, molecular phylogenetics, and developmental expression in intramolluscan larval stages. - Background The deoxyhexose sugar L-fucose is a major constituent of an array of immunologically important carbohydrates that are presented on surface-expressed and secreted/ excreted glycoconjugates of the human blood fluke Schistosoma mansoni (reviewed by [1]). Although the schistosome glycome is perhaps the most extensively characterized among invertebrates, relatively little is known about the enzymatic machinery responsible for its expression. Recent studies by Fitzpatrick et al. [2] and Peterson et al. [3] inventoried the schistosome 3- and 6-fucosyltransferases (FucTs), which transfer L-fucose from a GDP-L-fucose nucleotide-sugar donor to an oligosaccharide acceptor to create 3 and 6 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]. While the population composition and cellular organization of the expressed glycosyltransferases are key determinants affecting carbohydrate structural diversity, other factors are also important, including nucleotide-sugar donor availability, Golgi membrane dynamics, intralumenal pH, and competition for donor/acceptor substrates [8]. In S. mansoni, this means that GDP-L-fucose synthesis and Golgi import, which dictate fucose donor availability in the Golgi, likely contribute to differential fucosyl-glycotope expression. However, to date, no studies have examined these aspects of fucosylation in schistosomes. In general, GDP-L-fucose synthesis is localized in the cytosol and can occur by two possible metabolic pathways, the de novo and salvage pathways (reviewed by [9]), which constitute approximately 90% and 10%, respectively, of total GDP-L-fucose synthesis in mammalian cells [10]. In de novo synthesis, GDP-D-mannose is converted to GDP-L-fucose in three steps by GDP-D-mannose-4,6dehydratase (GMD, EC 4.2.1.47) and the bifunctional enzyme GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (GMER, EC 1.1.1.271; also called GDP-L-fucose synthase). Alternatively, the salvage pathway generates GDP-L-fucose from free cytosolic L-fucose in two steps, which are generally catalyzed by L-fucokinase (Fuk) and L-fucose-1-phosphate guanylyltransferase (FPGT; also called GDP-L-fucose pyrophosphorylase). Both pathways are summarized in Figure 1. GMD and GMER are well conserved across prokaryotic and eukaryotic taxa in terms of both structure and function [11], but the salvage pathway exhibits some variation. While homologs of Fuk and FPGT have been described in several mammalian species [12-15], the salvage pathway in Bacteroides and Arabidopsis comprises a single bifunctional enzyme (Fkp in Bacteroides; FKGP in Arabidopsis) that exhibits both Fuk and FPGT activities [16,17]. Elements of a salvage pathway do not exist in Drosophila [18] and only a Fuk homolog has been identified in C. elegans [11]. How GDP-L-fucose is synthesized in S. mansoni is unknown. In eukaryotes, fucosylation occurs primarily in the Golgi. Consequently, following GDP-L-fucose synthesis in the cytosol, the activated fucose is imported into the Golgi lumen where it can be utilized by Golgi-localized FucTs. This translocation is driven by a GDP-L-fucose transporter (GFT), which couples GDP-L-fucose entry with equimolar exit (i.e., antiportation) of GMP, a downstream byproduct of fucosylation (reviewed by [19]). Previous studies indicate that GDP-L-fucose synthesis and transport are essential processes in the production of fucosylated glycans. For example, increased expression of GMD, GMER and GFT was linked to higher levels of fucosylation in human hepatocellular carcinoma [20,21] and elevated expression of sialyl Lewis X during inflammation and tumorigenesis [22]. Additionally, Omasa et al. [23] observed decreased fucosylation of recombinant human antithrombin III following RNAi-mediated knockdown of GFT in transfected Chinese hamster ovary cells. The essential role of GFT in proper fucosylation is further evidenced in humans by the rare autosomal recessive syndrome leukocyte adhesion deficiency type II (LADII), which is characterized by severe psychomotor and growth retardation, facial malformation, and persistent and recurrent infections with marked neutrophilia [24]. Red blood cells of (...truncated)