Specificity in signal transduction among glycosylphosphatidylinositols of Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp.

Glycosylphosphatidylinositols (GPIs) and related glycoconjugates of parasite origin have been shown to regulate both the innate and acquired immune systems of the host. This is achieved through the activation of novel GPI-dependent signalling pathways in macrophages, lymphocytes and other cell types. Parasite GPIs impart at least two distinct signals to host cells through the structurally distinct inositolphosphoglycan (IPG) and fatty acid domains. Binding of IPG to as yet uncharacterized cell surface receptor(s) leads to activation of src-family protein tyrosine kinases: depending upon structure, GPI-derived fatty acids can either activate or antagonize protein kinase C, and may enter the sphingomyelinase pathway. The degree of fatty acid saturation may also contribute to signalling activity. Thus, variation in structure of parasite GPIs imparts different properties of signal transduction upon this class of glycolipid. The divergent activities of GPIs from various protozoal taxa reflect global aspects of the host/parasite relationship, suggesting that GPI signalling is a central determinant of disease in malaria, leishmaniasis and both American and African trypanosomiases.

Chromosomal promoter replacement in Saccharomyces cerevisiae: construction of conditional lethal strains for the cloning of glycosyltransferases from various organisms.

Heterologous complementation in yeast has been a successful tool for cloning and characterisation of genes from various organisms. Therefore we constructed conditionally lethal Saccharomyces cerevisiae strains by replacing the endogenous promoter from the genes of interest (glycosyltransferases) by the stringently regulated GAL1-promoter, by a technique called chromosomal promoter replacement. Such yeast strains were constructed for the genes Alg 1, Alg7, Sec59, Wbp1 involved in N-Glycosylation, the genes Gpi2, Gpi3/Spt14, Gaal, Pis1, involved in GPI-anchor biosynthesis and Dpm involved in both pathways. All strains show the expected conditionally lethal phenotype on glucose-containing medium when expression of the respective gene is turned off.

Cloning and functional expression of the human GlcNAc-1-P transferase, the enzyme for the committed step of the dolichol cycle, by heterologous complementation in Saccharomyces cerevisiae.

The gene for the human dolichol cycle GlcNAc-1-P transferase (ALG7/GPT) was cloned by screening a human lung fibroblast cDNA library. The library was constructed in a Saccharomyces cerevisiae expression vector, and the positive clone was identified by complementation of the conditional lethal S.cerevisiae strain YPH-A7-GAL. This strain was constructed by replacing the endogenous promoter of the GPT-gene by the stringently regulated GAL1-promoter. This construct allows to specifically suppress the endogenous enzyme activity. The insert of the positive clone displayed an open reading frame of 1200 nucleotides, coding for a putative protein of 400 amino acids with a calculated molecular weight of 44.7 kDa. The deduced protein sequence shows a homology of over 90% when compared with other mammalian GPT sequences, thus resembling the close phylogenetic relationship between mammalian species. This homology however decreases to 40-50% when compared to more distantly related organisms such as S.cerevisiae , Schizosaccharomyces pombe , or Leishmania amazonensis . Biochemical characterization of the recombinant protein showed that it is functionally expressed in the S.cerevisiae strain YPH-A7-GAL. GlcNAc- and GlcNAc2-PP-Dolichol biosynthesis could be shown with isolated S.cerevisiae membranes from cells harboring the recombinant plasmid and grown on glucose thus suppressing transcription of the endogenous gene. Synthesis could be stimulated by dolicholphosphate and was inhibited by tunicamycin. These results show that we have cloned the human GlcNAc-1-P transferase by heterologous complementation in S. cerevisiae, a strategy that may be useful for the cloning and characterization of glycosyltransferases from a variety of organisms.

Cloning and functional expression of glycosyltransferases from parasitic protozoans by heterologous complementation in yeast: the dolichol phosphate mannose synthase from Trypanosoma brucei brucei.

The gene for the enzyme dolichol phosphate mannose (Dol-P-Man) synthase from the parasitic protozoan Trypanosoma brucei brucei (T. brucei) was cloned by screening a T. brucei cDNA library and then sequenced. The library was constructed in a yeast expression vector and the positive clone was identified by complementation of a temperature-sensitive defect in the yeast strain DPM 1-6 [Orlean, Albright and Robbins (1988) J. Biol. Chem. 263, 17499-17507]. The insert of this clone displayed an open reading frame of 801 nucleotides coding for a putative protein of 267 amino acids. The deduced protein sequence showed an identity of 49% and a similarity of 69% with the published yeast sequence. Additional features of the T. brucei sequence are the presence of a putative signal sequence, a C-terminal transmembrane domain, a consensus sequence for phosphorylation by cAMP-dependent protein kinase and a stretch of five nucleotides immediately upstream from the putative initiation codon that could function as a prokaryotic ribosome binding site. A consensus sequence for dolichol binding (FI/VXF/YXXIPFXF/Y) found in the yeast protein could not be detected in the putative transmembrane domain of the T. brucei sequence. Biochemical characterization of the recombinant protein showed that it is functionally expressed in the yeast strain DPM 1-6 and Escherichia coli. In both constructs Dol-P-Man synthesis was shown in a cell-free system. Synthesis was stimulated by exogenous dolichol phosphate and inhibited by amphomycin. These results confirm that we have cloned the T. brucei Dol-P-Man synthase by heterologous complementation in yeast, an approach that might be applicable for other glycosyltransferases from various sources.