Glucosylceramide synthases, a gene family responsible for the biosynthesis of glucosphingolipids in animals, plants, and fungi.

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.

DsbA and DsbC affect extracellular enzyme formation in Pseudomonas aeruginosa.

DsbA and DsbC proteins involved in the periplasmic formation of disulfide bonds in Pseudomonas aeruginosa were identified and shown to play an important role for the formation of extracellular enzymes. Mutants deficient in either dsbA or dsbC or both genes were constructed, and extracellular elastase, alkaline phosphatase, and lipase activities were determined. The dsbA mutant no longer produced these enzymes, whereas the lipase activity was doubled in the dsbC mutant. Also, extracellar lipase production was severely reduced in a P. aeruginosa dsbA mutant in which an inactive DsbA variant carrying the mutation C34S was expressed. Even when the lipase gene lipA was constitutively expressed in trans in a lipA dsbA double mutant, lipase activity in cell extracts and culture supernatants was still reduced to about 25%. Interestingly, the presence of dithiothreitol in the growth medium completely inhibited the formation of extracellular lipase whereas the addition of dithiothreitol to a cell-free culture supernatant did not affect lipase activity. We conclude that the correct formation of the disulfide bond catalyzed in vivo by DsbA is necessary to stabilize periplasmic lipase. Such a stabilization is the prerequisite for efficient secretion using the type II pathway.

Characterization of UDP-glucose:ceramide glucosyltransferases from different organisms.

Cerebrosides are typical membrane lipids of many organisms. They occur in plants, fungi, animals, humans and some prokaryotes. Almost all of our knowledge on the physiological functions of cerebrosides results from experimental data obtained with mammalian cells. However, very little is known about the roles played by these lipids in plants and fungi. To initiate such investigations we have cloned and characterized a ceramide glucosyltransferase from the yeast Candida albicans. Functional expression of this gene in Saccharomyces cerevisiae led to the accumulation of new glycolipids which were not present in wild-type baker's yeast. They were identified by MS and NMR spectroscopy as beta-D-glucopyranosyl ceramides. The ceramide moieties of these cerebrosides comprised phytosphinganine and mainly long-chain (C(26)) alpha-hydroxy fatty acids in amide linkage. We also generated a ceramide glucosyltransferase-knock-out strain of C. albicans which was devoid of cerebrosides. The viability of this mutant showed that for this organism glucosyl ceramides are not essential for vegetative growth on complete or minimal media. In addition, we have cloned and functionally expressed one of the three putative glucosylceramide synthases from Caenorhabditis elegans, as well as a corresponding enzyme from Pichia pastoris.