Abstract
Glucosylceramides are membrane lipids in most eukaryotic organisms and in a few bacteria. The physiological functions of these glycolipids have only been documented in mammalian cells, whereas very little information is available of their roles in plants, fungi, and bacteria. In an attempt to establish appropriate experimental systems to study glucosylceramide functions in these organisms, we performed a systematic functional analysis of a glycosyltransferase gene family with members of animal, plant, fungal, and bacterial origin. Deletion of such putative glycosyltransferase genes in Candida albicans and Pichia pastoris resulted in the complete loss of glucosylceramides. When the corresponding knock-out strains were used as host cells for homologous or heterologous expression of candidate glycosyltransferase genes, five novel glucosylceramide synthase (UDP-glucose:ceramide glucosyltransferase) genes were identified from the plant Gossypium arboreum (cotton), the nematode Caenorhabditis elegans, and the fungi Magnaporthe grisea, Candida albicans, and P. pastoris. The glycosyltransferase gene expressions led to the biosynthesis of different molecular species of glucosylceramides that contained either C18 or very long chain fatty acids. The latter are usually channeled exclusively into inositol-containing sphingolipids known from Saccharomyces cerevisiae and other yeasts. Implications for the biosynthesis, transport, and function of sphingolipids will be discussed.
Highlights
Glycosylceramides are present in almost all eukaryotic organisms and in a few bacteria
When the corresponding knock-out strains were used as host cells for homologous or heterologous expression of candidate glycosyltransferase genes, five novel glucosylceramide synthase (UDP-glucose:ceramide glucosyltransferase) genes were identified from the plant Gossypium arboreum, the nematode Caenorhabditis elegans, and the fungi Magnaporthe grisea, Candida albicans, and P. pastoris
The first cDNA coding for a human GCS has been cloned [11]. This success has opened new possibilities for analyzing GlcCer functions [12, 13], by studying the phenotypes resulting from gene deletions in knock-out mice [14]. These studies address different aspects of GlcCer functions: (i) GlcCer are membrane lipids and contribute to the physical properties and physiological functions of membranes; (ii) GlcCer serves as basic precursor for over 300 species of glycosphingolipids found in different mammalian cell types; and (iii) GlcCer synthesis and degradation are believed to contribute to the control of the level of ceramide, which is regarded as a second messenger involved in many biological processes such as heat stress response and apoptosis [15,16,17]
Summary
Glycosylceramides are present in almost all eukaryotic organisms and in a few bacteria. Very little is known about the functions and intracellular location of GlcCer in nonanimal organisms such as plants, fungi, and bacteria Progress in this field is hampered by the lack of a genetic approach, since no genes or cDNAs coding for GCS have been cloned or identified from these organisms so far (except for our preliminary communication on a putative GCS from Candida albicans [19]). The aim of the present work was the cloning of GCS from nonanimal organisms and establishment of novel model systems suitable for alterations in GlcCer content These genetically modified organisms will enable investigations on various aspects of GlcCer synthesis, transport, and functions, which should extend and complement the studies limited so far to mammalian cells. The mechanism of this separation of two sphingolipid pools is not under-
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