Deletion of the TbALG3 gene demonstrates site-specific N-glycosylation and N-glycan processing in Trypanosoma brucei

Sujatha Manthri 1 M Lucia S G uther 1 Luis Izquierdo 1 Alvaro Acosta-Serrano 0 Michael A J Ferguson 1 0 The Wellcome Centre for Molecular Parasitology, Glasgow Biomedical Research Centre, University of Glasgow , Glasgow G12 8TA, Scotland, UK 1 The Division of Biological Chemistry and Drug Discovery, The Wellcome Trust Biocentre, College of Life Sciences, University of Dundee , Dundee DD1 5EH We recently suggested a novel site-specific N-glycosylation mechanism in Trypanosoma brucei whereby some protein N-glycosylation sites selectively receive Man9GlcNAc2 from Man9GlcNAc2-PP-Dol while others receive Man5GlcNAc2 from Man5GlcNAc2-PP-Dol. In this paper, we test this model by creating procyclic and bloodstream form null mutants of TbALG3, the gene that encodes the -mannosyltransferase that converts Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PPDol. The procyclic and bloodstream form TbALG3 null mutants grow with normal kinetics, remain infectious to mice and tsetse flies, respectively, and have normal morphology. However, both forms display aberrant N-glycosylation of their major surface glycoproteins, procylcin, and variant surface glycoprotein, respectively. Specifically, procyclin and variant surface glycoprotein N-glycosylation sites that are modified with Man9GlcNAc2 and processed no further than Man5GlcNAc2 in the wild type are glycosylated less efficiently but processed to complex structures in the mutant. These data confirm our model and refine it by demonstrating that the biantennary glycan transferred from Man5GlcNAc2-PP-Dol is the only route to complex N-glycans in T. brucei and that Man9GlcNAc2-PP-Dol is strictly a precursor for oligomannose structures. The origins of site-specific Man5GlcNAc2 or Man9GlcNAc2 transfer are discussed and an updated model of N-glycosylation in T. brucei is presented. - The trypanosmatid Trypanosoma brucei is a parasitic protozoan organism that causes nagana in cattle and human African sleeping sickness. The organism undergoes a complex life cycle, involving major biochemical and morphological changes, be1To whom correspondence should be addressed: Tel. +44-1382-384219; Fax +44-1382-348896; e-mail: tween its mammalian host and tsetse fly vector. These changes include complete remodeling of the major cell surface coat molecules. The tsetse midgut-dwelling procyclic form of T. brucei has a surface coat of 3 106 polyanionic, rod-like, procyclin glycoproteins (Mowatt and Clayton 1987; Roditi et al. 1987, 1989; Richardson et al. 1988; Treumann et al. 1997) as well as other unidentified glycoproteins (Guther et al. 2006). In T. brucei D strain 427, used in this study, the parasites contain (per diploid o w genome) two copies of the GPEET1 gene, encoding a procy- lon clin with 6 Gly-Pro-Glu-Glu-Thr repeats; one copy each of ad the EP1-1 and EP1-2 genes, encoding EP1 procyclins with fed 30 and 25 Glu-Pro repeats, respectively; two copies of the rom EP2-1 gene, encoding EP2 procyclin with 25 Glu-Pro repeats; th and two copies of the EP3-1 gene, encoding EP3 procyclin /t:p with 22 Glu-Pro repeats (Acosta-Serrano et al. 1999; Roditi /lgy and Clayton 1999). The EP1 and EP3 procyclins contain a sin- cob gle N-glycosylation site, at the N-terminal side of the Glu- .ox PErnodor-eHpe-saetndsiotimveaintr,iaonctecnunpaierdy Mexacnlu5sGivlceNlyAbc2y oaligcoosnavcecnhtiaornidael jfruodo (Treumann et al. 1997). Neither EP2 nor GPEET procyclin is ran N-glycosylated but GPEET1 procyclin is phosphorylated on six .lso out of seven Thr residues (Butikofer et al. 1999; Mehlert et al. /rg 1999). GPEET and EP procyclins contain similar glycosylphos- by phatidylinositol (GPI) membrane anchors, based on the ubiqui- eug tous ethanolamine-P-6Man1-2Man1-6Man1-4GlcN1-6PI tso core (Ferguson 1999), where in this case, the phosphatidylinos- nO itol (PI) lipid is a 2-O-acyl-lyso-PI structure, substituted with tco large branched poly-N-acetyllactosamine structures that can ter- reb minate with 2-3-linked sialic acid residues (Ferguson et al. 20 1993; Treumann et al. 1997). ,20 The bloodstream form of T. brucei has a surface coat of 5 14 106 variant surface glycoprotein (VSG) homodimers (Mehlert, Richardson, et al. 1998). The VSG coat serves as a physical barrier to components of the host complement system and undergoes antigenic variation (Pays et al. 2004; Taylor and Rudenko 2006). There are many VSG genes and each encodes a GPIanchored glycoprotein with one to three N-glycosylation sites (Mehlert, Richardson, et al. 1998). The cell line used in this study expresses VSG variant 221 (also known as MiTat1.2). VSG221 has a GPI anchor with the same core as the procyclins but with a dimyristoyl-PI component and carbohydrate side chains of between two and six Gal residues (Mehlert, Zitzmann, et al. 1998). VSG221 has two N-glycosylation sites: the Asn428 site, five residues from the GPI attachment site, is occupied mostly by Endo-H-sensitive oligomannose structures (Man59GlcNAc2), whilst the Asn263 site is occupied by small Endo-H-resistant biantennary structures ranging from Man34GlcNAc2 to GalGlcNAcMan3GlcNAc2 (Zamze et al. 1991). Protein N-glycosylation in eukaryotes serves a wide variety of functions including signaling through interaction with lectins, protein stabilization, protease resistance, endocytic sorting functions, and protein folding (Varki 1993; Rudd and Dwek 1997; Helenius and Aebi 2004). In eukaryotes, a precursor for N-glycosylation is built up in the endoplasmic reticulum (ER) on the lipid carrier dolichol pyrophosphate (Dol-PP) (Burda and Aebi 1999). The glycan portion of this Dol-PP-oligosaccharide is transferred en bloc by the action of oligosaccharyltransferase (OST), generally during protein translation and sequestration into the lumen of the ER, to Asn residues within Asn-X-Ser/Thr sequons (Helenius and Aebi 2004). Processing of the precursor structure by glycosidase and glycosyltransferase enzymes within the ER and Golgi apparatus generates the final set of mature structures (Kornfeld R and Kornfeld S 1985; Schachter 2000). In most eukaryotes, the mature precursor used by OST is Glc3Man9GlcNAc2-PP-Dol. However, genomic and experimental comparisons have shown that some lower eukaryotes do not possess all the ALG genes needed to make Glc3Man9GlcNAc2PP-Dol and that they transfer smaller glycans to protein (Parodi 1993; Samuelson et al. 2005). Differences in the compositions and donor specificities of eukaryotic OST complexes, which usually contain eight different subunits, have also been noted (Kelleher and Gilmore 2006; Kelleher et al. 2007). Seminal work by Parodi and colleagues on several trypanosomatid parasites (excluding T. brucei) showed that protein N-glycosylation in these organisms is aberrant (reviewed in Parodi 1993). None of these organisms make Dol-P-Glc and so fail to make glucosylated Dol-PP-oligosaccharide precursors. The mature Dol-PP-oligosaccharide species used for transfer to protein vary according to species. For example, Trypanos (...truncated)