An essential, kinetoplastid-specific GDP-Fuc: β-D-Gal α-1,2-fucosyltransferase is located in the mitochondrion of Trypanosoma brucei

Abstract

The biosynthesis of guanosine 5′-diphospho-β-L-fucose (GDP-Fuc), the activated donor for fucose, has been shown to be essential in the parasite Trypanosoma brucei. Fucose is a common constituent of eukaryotic glycan structures, but it has been rarely found in trypanosomatid glycoconjugates. A single putative T. brucei fucosyltransferase (TbFUT1) gene was identified in the trypanosome genome. The encoded TbFUT1 protein was enzymatically active when expressed in Escherichia coli. Structural characterization of its reaction products identified it as a GDP-Fuc: β-D-galactose α-1,2-fucosyltransferase, with a preference for a Galβ1,3GlcNAcβ1-O-R acceptor motif among the substrates tested. Conditional null mutants of the TbFUT1 gene demonstrated that it is essential for growth of the mammalian-infective bloodstream form and insect vector dwelling procyclic form of the parasite. Unexpectedly, TbFUT1 was localized in the mitochondrion of T. brucei and found to be essential for mitochondrial function in bloodstream form trypanosomes, suggesting this kinetoplastid parasite possesses an unprecedented and essential mitochondrial fucosyltransferase activity.SIGNIFICANCEThe sugar fucose is a well-known component of cell-surface glycoproteins and glycolipids and typically plays roles in cell-cell adhesion. Fucose is generally incorporated into glycoproteins and glycolipids by fucosyltransferase enzymes that reside in the Golgi apparatus. Here we show that the single fucosyltransferase of the protozoan parasite Trypanosoma brucei, causative agent of human and animal African trypanosomiasis, resides in the mitochondrion and not the Golgi apparatus. While the exact role of fucosylation in the parasite mitochondrion remains to be determined, it is essential for mitochondrial function and for parasite growth and survival. The unusual nature of this parasite enzyme, and its orthologues in related parasite pathogens, suggests that selective inhibitors may have therapeutic potential across a family of parasites.

A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and catalytic activity is essential in the parasitic protozoan Leishmania

“…5B). Overall the activity seen with the substrates tested mirrored those observed elsewhere for TbFUT1, where a wider range was tested but with LNB similarly being most active (1).…”

“…A 16 aa truncation (MSKAR) was fully functional and mitochondrially targeted, a 17 aa truncation (MKAR) was partially functional and mitochondrially targeted, and a 18 aa truncation (MAR) was not functional but partially mitochondrially targeted (Fig S4). These data suggest first that LmjFUT1 may possess a cryptic mitochondrial targeting sequence, resembling the N terminus of TbFUT1, which we show elsewhere is also mitochondrially targeted (1). These data further suggest that in trypanosomatids critical residues of the Motif IV region may extend functionally further towards the N terminus than in other species.…”

“…A variety of fucose modifications have been described in prokaryotes, and the internal fucose within the SKP1 pentasaccharide D-Galα1-6-D-Galα1-L-Fucα1-2-D-Galα1-3GlcNAc of several single-celled eukaryotes is not recognized by lectins or antibodies tested here (64). Studies searching for the cellular FUT1-dependent products in T. brucei have been similarly unrequited (1). Future studies will be necessary to resolve the nature of the GDP-Fucose and FUT1dependent product(s) in trypanosomatids, which genetic and biochemical data strongly predict must nonetheless exist.…”

“…Assays and resolution of products by HPTLC were performed as described (1). Briefly, Leishmania lysates were incubated with Anti-HA-beads in the presence of in the presence of 1 μCi GDP[ 3 H]Fuc (American Radiochemicals) and 1 mM LNB in 50 mM TrisHCl, 25 mM KCl, pH 7.2 in a final volume of 25 μl for 2 h at 37°C.…”

Viral-eukaryotic gene exchange drives infection mode and cellular evolution

“…4e). A recent report also suggests an essential role for the fucosyltransferase in kinetoplast function in T. brucei 43 , altogether indicating that these viralderived glycosyltransferases were co-opted for use in the kinetoplast at the same time as it underwent massive evolutionary change. These data, along with the tendency for viruses to modify cell surfaces, suggest that viral-derived genes may have played various roles in the evolution of cellular morphology across the eukaryotic tree of life, possibly affecting the diversi cation of eukaryotic forms.…”