Characterisation of unusual glycosyltransferases from lower eukaryotes

Background.

The differences in glycosylation between protists, insects, nematodes and mammals present challenges in terms of their immunogenicity and the understanding of the molecular basis of their production. At the same time, non-mammalian systems are also a potentially useful resource in terms of altered glycoprotein properties, e.g. in terms of the immunomodulatory potential of their glycans, or of enzymes with unusual substrate specificities. In our previous glycomic studies we have uncovered unusual modifications of glycans from a variety of sources (trichomonads, amoebae, molluscs, nematodes and insects); examples include trifucosylated N-glycan cores, xylosylation, galactosylation, blood group A, phosphorylcholine, methylation, sulphate and methylphosphate (Schiller et al., 2012a).

Aims and methods.

In the proposed work, cDNAs predicted to encode glycosyltransferases from protist or nematode sources will be cloned and expressed in either yeast, insect or mammalian cells (MATTANOVICH, GRABHERR). Previous studies in my group on cloning invertebrate glycosyltransferases, especially fucosyltransferases, have been based on identifying genes by homology, also in combination with the use of mutant lines and/or glycan arrays for substrate determination. A similar approach will also be used for the target genes in this PhD thesis showcase. Forms of relevant enzymes will be prepared by soluble expression (i.e., with a secretion signal rather than a transmembrane domain), and then be assayed in vitro using HPLC or MS-based tests as to their enzymatic properties. The effect of both soluble and full-length forms on expression of carbohydrate epitopes will be assessed by Western blotting and flow cytometry (GRILLARI).

In this thesis, the planned focus will be on homologues of N-glycan-modifying xylosyltransferases in Trichomonas and Acanthamoeba. Relevant xylosylated N-glycans have been detected in both species (Paschinger et al., 2012; Schiller et al., 2012b). Some trichomonad homologues have already been cloned in my laboratory, but activity is yet to be determined. In theory, one or more xylosyltransferase homologues from both species should be responsible for the expression of anti-horseradish peroxidase epitopes, and thus this antibody will be used to screen for the presence of this epitope in the transfectedcells. It is expected that these xylosyltransferases will modify pauci- and oligomannosidic N-glycans and such substrates for in vitro assays will be generated ‘in house’, purchased if available, or obtained from international collaborators. If available within the next four years, relevant glycan arrays will also be used in in vitro ‘gain-of-epitope’ assays. Selected xylosyltransferases will be characterised in detail using a range of biochemical/biophysical methods, including steady-state kinetics, site-directed mutagenesis, differential scanning calorymetry, or X-ray crystallography.
The outcome will be the identification and characterization of novel glycosyltransferases whose definition will aid elucidation of glycan-biosynthesis pathways in one obligate parasite (Trichomonas; the most widespread sexually-transmitted non-viral pathogen in the human population) and one opportunistic parasite (Acanthamoeba; causing keratitis in some contact lens wearers or encephalitis in immunocompromised patients).

Paschinger, K., Hykollari, A., Razzazi-Fazeli, E., Greenwell, P., Leitsch, D., Walochnik, J., Wilson, I.B.H. (2012) The N-glycans of Trichomonas vaginalis contain variable core and antennal modifications. Glycobiology 22, 300-313
Schiller, B., Hykollari, A., Yan, S., Paschinger, K., Wilson, I.B.H. (2012a) Complicated N-linked glycans in simple organisms. Biol. Chem. (Hoppe-Seyler) 393, 661–673
Schiller, B., Makrypidi, G., Razzazi-Fazeli, E., Paschinger, K., Walochnik, J., Wilson, I.B.H. (2012b) Exploring the unique N-glycome of the opportunistic human pathogen Acanthamoeba. J. Biol. Chem. 287, 43191-43204