Apicomplexan C-Mannosyltransferases Modify Thrombospondin Type I-containing Adhesins of the TRAP Family

Abstract

In many metazoan species, an unusual type of protein glycosylation, called C-mannosylation, occurs on adhesive thrombospondin type 1 repeats (TSRs) and type I cytokine receptors. This modification has been shown to be catalyzed by the Caenorhabditis elegans DPY-19 protein and orthologues of the encoding gene were found in the genome of apicomplexan parasites. Lately, the micronemal adhesin thrombospondin-related anonymous protein (TRAP) was shown to be C-hexosylated in Plasmodium falciparum sporozoites. Here, we demonstrate that also the micronemal protein MIC2 secreted by Toxoplasma gondii tachyzoites is C-hexosylated. When expressed in a mammalian cell line deficient in C-mannosylation, P. falciparum and T. gondii Dpy19 homologs were able to modify TSR domains of the micronemal adhesins TRAP/MIC2 family involved in parasite motility and invasion. In vitro, the apicomplexan enzymes can transfer mannose to a WXXWXXC peptide but, in contrast to C. elegans or mammalian C-mannosyltransferases, are inactive on a short WXXW peptide. Since TSR domains are commonly found in apicomplexan surface proteins, C-mannosylation may be a common modification in this phylum.

O-fucosylation of thrombospondin-like repeats is required for processing of MIC2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites

“…1A). The targeted HCD and ETD experiments allowed localization of a dHexHex disaccharide on the predicted POFUT2 acceptor residues on MIC2 TSR1 and TSR4 and a dHex +/-Hex on TSR5 (25). TSR3 is also modified with a dHexHex, although the modification site could not be assigned ( Table 1, Figs.…”

“…TSR6 lacks a consensus site for POFUT2 and is not modified by C-Man; TSR2 lacks an Ofucosylation consensus site and was not sampled. TSR3 and TSR4, in addition to the previously described TSR5 (25), are modified with C-Man. Lastly, while MIC2 contains four potential Nglycosylation sites, we observed peptides containing unmodified Asn at residues 463, 470, and 680, suggesting that these N-glycan sites are unoccupied ( Fig.…”

“…Four TSRs of MIC2 are modified by a dHex with or without Hex. Glycosylation on tachyzoite secreted proteins was analyzed as described in (25). Briefly, secretion was induced with 1% ethanol (7), and the peptides resulting from reduction, alkylation, and trypsin digestion of the secreted protein fraction were analyzed by nanoLC-MS/MS using a Thermo Q Exactive quadrupole Orbitrap MS with low energy CID.…”

“…In metazoans, fucose is transferred on already folded TSR domains (23) and can be elongated by a β1,3-glucose by action of the glucosyltransferase B3GLCT (24). Mass spectrometry analyses of P. falciparum TRAP and circumsporozoite protein (CSP) indicate that a hexose (Hex) residue elongates the fucose on Apicomplexa TSRs; this sugar may be glucose, but has not yet been unequivocally identified (21,25).…”

POFUT2-mediated O-glycosylation of MIC2 is dispensable for Toxoplasma gondii tachyzoites

“…Endogenous TSR O-glycosylation has yet to be observed in T. gondii, however it does encode a putative POFUT2 (TGGT1_273550) and the recombinant expression of T. gondii proteins in CHO cells does results in modification of parasite protein with the GlcFuc and C-Man glycans (21). Here, we generate a T. gondii line to facilitate bulk purification of micronemal protein 2 (MIC2) directly from tachyzoites in order to map endogenous glycosylation sites on this motilityassociated adhesin that possesses six TSR domains.…”