Cloning, complementation and expression of UDP Glucose 4′ Epimerase from Trypanosoma brucei and T. cruzi

Abstract

The protozoan parasites Trypanosoma brucei and 7: cruzi are the causative agents of African Sleeping Sickness and Chagas' Disease, respectively. Their surface molecules play a crucial role in protection and infectivity. Galactose residues are thought to play an important role in parasite survival: 7: brucei bloodstream forms are covered by a layer of variant surface glycoprotein (VSG) attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor which is modified by a branched a-Gal side chain. In 7: cruzi p-galactopyranoside residues of m u c k are the acceptor sites for the trans-sialylation reaction which is essential for parasite survival. The oxidoreductase UDP-glucose 4' epimerase interconverts glucose and galactose. In mammals this enzyme is important for converting Gal to Glc because excess Gal can be toxic. In trypanosomes, the reverse reaction (Glc to Gal) is probably more important since Glc is freely available from blood whereas they cannot transport Gal. We have cloned the epimerase from both 7: brucei and 7: cruzi using partial sequences found by database searches. The parasite epimerase genes were cloned into pUCl8; these plasmids successfully rescued an E. coliepimerase mutant (galE-) for growth on McConkey agar with galactose as the sole carbon source. Rescued cells had the epimerase gene in reading frame +1 and grew as large red colonies, whereas their mutant counterparts formed smaller, paler colonies. We have also expressed the parasite epimerases from pET15b and pQE-30. Overexpressed protein will be used for enzyme assays and crystallisation trials. We will also attempt to make epimerase knockouts in I: brucei and 7: cruzi to assess the role of Gal in these parasites. MFIS: a high throughput technology for studying the ligandreceptor interactionThe Multi-parameter Fluorescence Immunosensor System (MFIS) is designed to study the specificity and cross-reactivity of the ligand-receptor interaction on a large scale. Using a micro-spotting device, many thousands of ligands or receptors can be patterned on a micro-glass slide. A solution containing specific ligands or receptors with distinct fluorescent tags can then be applied on the slide and their binding monitored by fluorescence signals. To investigate the feasibility of this method, we have taken advantage of a wellestablished antigen-antibody system, the dextrans and anti-dextran antibodies. A panel of purified dextran preparations with different linkage compositions and ratios of terminal/internal epitopes were immobilized on a surface-treated glass slide and than incubated with anti-dextran antibodies of defined specificities, either cavitytype or groove-type. The former is specific for the terminal nonreducing end structure of alpha(l,6)dextran; the latter recognizes the internal linear chain of the polysaccharide. When a cavity-type mAb, 16.4.12E, was applied on the glass slide, it bound to the immobilized alpha( 1,6)dextran preparations having branches but not those with an internal linear chain structure. By contrast, a...