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

Abstract

Fucose is a common component of eukaryotic cell-surface glycoconjugates, generally added by Golgi-resident fucosyltransferases. Whereas fucosylated glycoconjugates are rare in kinetoplastids, the biosynthesis of the nucleotide sugar GDP-Fuc has been shown to be essential in Trypanosoma brucei. Here we show that the single identifiable T. brucei fucosyltransferase (TbFUT1) is a GDP-Fuc: β-D-galactose α-1,2-fucosyltransferase with an apparent preference for a Galβ1,3GlcNAcβ1-O-R acceptor motif. Conditional null mutants of TbFUT1 demonstrated that it is essential for both the mammalian-infective bloodstream form and the insect vector-dwelling procyclic form. Unexpectedly, TbFUT1 was localized in the mitochondrion of T. brucei and found to be required for mitochondrial function in bloodstream form trypanosomes. Finally, the TbFUT1 gene was able to complement a Leishmania major mutant lacking the homologous fucosyltransferase gene (Guo et al., 2021). Together these results suggest that kinetoplastids possess an unusual, conserved and essential mitochondrial fucosyltransferase activity that may have therapeutic potential across trypanosomatids.

Common and unique features of glycosylation and glycosyltransferases in African trypanosomes

“…The discovery that the biosynthesis of the nucleotide sugar GDP-fucose was required for parasite growth in T. brucei [76] and in Leishmania major [77], suggested that these organisms contain one or more essential FUT genes. A single GT11 family FUT, TbFUT1, was identified in the T. brucei genome and shown to be essential in BSF and PCF cells [78]. Similarly, the L. major orthologue (LmjFUT1) is essential for cell growth [79].…”

“…Similarly, the L. major orthologue (LmjFUT1) is essential for cell growth [79]. The most curious feature of TbFUT1 and LmjFUT1 is their localisation to the parasite mitochondria [78,79]. Recombinant TbFUT1 has GDP-Fuc : βGal α1-2-fucosyltransferase activity, common amongst GT11 family enzymes, with an apparent preference for acceptor substrates containing a terminal LNB (Galβ1-3GlcNAc) motif [78].…”

“…The most curious feature of TbFUT1 and LmjFUT1 is their localisation to the parasite mitochondria [78,79]. Recombinant TbFUT1 has GDP-Fuc : βGal α1-2-fucosyltransferase activity, common amongst GT11 family enzymes, with an apparent preference for acceptor substrates containing a terminal LNB (Galβ1-3GlcNAc) motif [78]. The origin of FUT1 is unlike that of the GT67 family which expanded from a common eukaryotic β3GT ancestor.…”

“…Whereas recombinant TbFUT1 and LmjFUT1 have been shown to fucosylate exogenous peptide and/or glycan substrates, their native mitochondrial substrates have not yet been identified. Phenotypic analysis of a TbFUT1 conditional null mutant in BSF cells reveals that depleting TbFUT1 causes a loss of mitochondrial membrane potential, linked to the activity of the F o F 1 -ATP synthase [78]. BSF T. brucei lack the proton pumping respiratory chain complexes normally expressed in mitochondria; instead the F o F 1 -ATP synthase works in reverse mode such that catalytic rotation of the F 1 moiety via ATP hydrolysis actively pumps protons out of the mitochondrial matrix via the membrane multimeric F o c-ring pore.…”

“…The effect of TbFUT1 depletion in insect stage PCF appears to be different. Here, cell death upon TbFUT1 conditional depletion takes longer to manifest than in BSF cells [78]. However, the PCF form of T. brucei uses F o F 1 -ATP more conventionally, generating ATP from the mitochondrial proton gradient, in conjunction with fully expressed and functional respiratory chain composed of complexes I-IV [81].…”