TbGT8 is a bifunctional glycosyltransferase that elaborates N-linked glycans on a protein phosphatase AcP115 and a GPI-anchor modifying glycan in Trypanosoma brucei

Abstract

The procyclic form of Trypanosoma brucei expresses procyclin surface glycoproteins with unusual glycosylphosphatidylinositol-anchor side chain structures that contain branched N-acetyllactosamine and lacto-N-biose units. The glycosyltransferase TbGT8 is involved in the synthesis of the branched side chain through its UDP-GlcNAc: βGal β1-3N-acetylglucosaminyltransferase activity. Here, we explored the role of TbGT8 in the mammalian bloodstream form of the parasite with a tetracycline-inducible conditional null T. brucei mutant for TbGT8. Under non-permissive conditions, the mutant showed significantly reduced binding to tomato lectin, which recognizes poly-N-acetyllactosamine-containing glycans. Lectin pull-down assays revealed differences between the wild type and TbGT8 null-mutant T. brucei, notably the absence of a broad protein band with an approximate molecular weight of 110 kDa in the mutant lysate. Proteomic analysis revealed that the band contained several glycoproteins, including the acidic ecto-protein phosphatase AcP115, a stage-specific glycoprotein in the bloodstream form of T. brucei. Western blotting with an anti-AcP115 antibody revealed that AcP115 was approximately 10 kDa smaller in the mutant. Enzymatic de-N-glycosylation demonstrated that the underlying protein cores were the same, suggesting that the 10-kDa difference was due to differences in N-linked glycans. Immunofluorescence microscopy revealed the colocalization of hemagglutinin epitope-tagged TbGT8 and the Golgi-associated protein GRASP. These data suggest that TbGT8 is involved in the construction of complex poly-N-acetyllactosamine-containing type N-linked and GPI-linked glycans in the Golgi of the bloodstream and procyclic parasite forms, respectively.

Identification of a glycosylphosphatidylinositol anchor-modifying β1-3 galactosyltransferase in Trypanosoma brucei

“…Using a well-established workflow for the functional and biochemical characterization of T. brucei GTs ( Izquierdo, Nakanishi, et al 2009 ; Izquierdo et al 2013 ), we designate TbGT3 as primarily a GPI side chain modifying UDP-Gal : βGlcNAc β1-3 Gal-transferase. However, like TbGT8, which is both a GPI side-chain modifying UDP-GlcNAc:βGal β1-3 GlcNAc-transferase ( Izquierdo, Nakanishi, et al 2009 ) and involved in large complex N -glycan synthesis in bloodstream form parasites ( Nakanishi et al 2014 ), TbGT3 is likely also to be involved in some way in the synthesis of large complex N -glycans in the bloodstream form of the parasite ( Atrih et al 2005 ; Izquierdo, Nakanishi, et al 2009 ).…”

“… Model of GPI anchor side-chain biosynthesis initiation in procyclic form trypanosomes. The first branching point in the GPI anchor side-chain is produced by TbGT8, a GlcNAc-transferase that acts on the terminal digalactose moiety of the GPI anchor ( Izquierdo, Nakanishi, et al 2009 ; Nakanishi et al 2014 ). The resulting GPI structure can be further substituted either by (i) TbGT3 Gal-transferase, described in this paper, that adds a Gal residue to the non-reducing–t-GlcNAc or (ii) an unknown Gal-transferase, labelled as “?…”

“…The first of these genes to be studied were TbGT8 , a UDP-GlcNAc:βGal β1-3 GlcNAc-transferase that modifies the complex GPI anchor side chains of the procyclins ( Izquierdo, Nakanishi, et al 2009 ) and also elaborates complex N -glycans in bloodstream from parasites ( Nakanishi et al 2014 ), and TbGT11 , a UDP-GlcNAc:αMan β1-2 GlcNAc-transferase equivalent to GnTI and involved in the synthesis of complex N -glycan structures ( Damerow et al 2014 ).…”

A UDP-GlcNAc : βGal β1-6 GlcNAc transferase involved in bloodstream formN-glycan and procyclic form GPI anchor elaboration inTrypanosoma brucei

“…However, neither enzyme is essential for growth in vitro or for the establishment of tsetse fly infections. Analysis of N-linked glycosylation in BSF mutants revealed that TbGT8 deficient mutants also have pronounced changes in N-glycosylation, as manifested by reduced wheat germ agglutinin (WGA) and ricin binding (13,32). The presence of GlcNAcβ1-3Gal inter-LacNAc linkages in both BSF Nlinked glycans and PCF GPI anchors presumably explains the dual function of TbGT8 and it is possible that other functional dualities may exist for other Trypanosoma GTs.…”

A Gene of the β3-Glycosyltransferase Family Encodes N-Acetylglucosaminyltransferase II Function in Trypanosoma brucei

“…The comparable motifs in the T. brucei genes are slightly different, WG, Y(I,V,F) X K X DDD, and ED(A/V/I/L/M)(M/L) X (G/A), but nevertheless, they identify the parasite genes as belonging to the β3GT superfamily ( 26 ). One of these genes ( TbGT8 ) encodes a β1–3 GlcNAc transferase and another ( TbGT3 ) a β1–3 Gal transferase that modifies the complex GPI anchor side chains of the procyclins (the major surface glycoproteins of the procyclic life cycle stage) ( 26 – 28 ). However, we recently reported that another gene ( TbGT11 ) encodes a β1–2 GlcNAc transferase that performs a similar role to members of the N -acetylglucosaminyltransferase I family, in that it transfers GlcNAc in β1–2 linkage to the 3-arm of Manα1–6(Manα1–3)Manβ1-4GlcNAcβ1–4GlcNAc ( 1 ).…”