Katharina vom Dorp

Fatty acid phytyl ester synthesis in chloroplasts of Arabidopsis.

This work identifies two phytyl ester synthases that act in fatty acid phytyl ester synthesis in chloroplasts during senescence and nitrogen deprivation to maintain the integrity of the photosynthetic membrane during abiotic stress and senescence. During stress or senescence, thylakoid membranes in chloroplasts are disintegrated, and chlorophyll and galactolipid are broken down, resulting in the accumulation of toxic intermediates, i.e., tetrapyrroles, free phytol, and free fatty acids. Chlorophyll degradation has been studied in detail, but the catabolic pathways for phytol and fatty acids remain unclear. A large proportion of phytol and fatty acids is converted into fatty acid phytyl esters and triacylglycerol during stress or senescence in chloroplasts. We isolated two genes (PHYTYL ESTER SYNTHASE1 [PES1] and PES2) of the esterase/lipase/thioesterase family of acyltransferases from Arabidopsis thaliana that are involved in fatty acid phytyl ester synthesis in chloroplasts. The two proteins are highly expressed during senescence and nitrogen deprivation. Heterologous expression in yeast revealed that PES1 and PES2 have phytyl ester synthesis and diacylglycerol acyltransferase activities. The enzymes show broad substrate specificities and can employ acyl-CoAs, acyl carrier proteins, and galactolipids as acyl donors. Double mutant plants (pes1 pes2) grow normally but show reduced phytyl ester and triacylglycerol accumulation. These results demonstrate that PES1 and PES2 are involved in the deposition of free phytol and free fatty acids in the form of phytyl esters in chloroplasts, a process involved in maintaining the integrity of the photosynthetic membrane during abiotic stress and senescence.

Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.

Ablation of Neuronal Ceramide Synthase 1 in Mice Decreases Ganglioside Levels and Expression of Myelin-associated Glycoprotein in Oligodendrocytes

Background: Ceramide synthase 1 catalyzes the synthesis of C18 ceramide and is mainly expressed in neurons of the brain. Results: Ablation of ceramide synthase 1 decreases ganglioside levels and expression of oligodendrocytic myelin-associated glycoprotein in motor-impaired mice. Conclusion: CerS1-derived C18 gangliosides are essential for cerebellar development and neurodevelopmentally regulated behavior in mice. Significance: Neuronal gangliosides regulate expression of myelin-associated glycoprotein in oligodendrocytes. Ceramide synthase 1 (CerS1) catalyzes the synthesis of C18 ceramide and is mainly expressed in the brain. Custom-made antibodies to a peptide from the C-terminal region of the mouse CerS1 protein yielded specific immunosignals in neurons but no other cell types of wild type brain, but the CerS1 protein was not detected in CerS1-deficient mouse brains. To elucidate the biological function of CerS1-derived sphingolipids in the brain, we generated CerS1-deficient mice by introducing a targeted mutation into the coding region of the cers1 gene. General deficiency of CerS1 in mice caused a foliation defect, progressive shrinkage, and neuronal apoptosis in the cerebellum. Mass spectrometric analyses revealed up to 60% decreased levels of gangliosides in cerebellum and forebrain. Expression of myelin-associated glycoprotein was also decreased by about 60% in cerebellum and forebrain, suggesting that interaction and stabilization of oligodendrocytic myelin-associated glycoprotein by neuronal gangliosides is due to the C18 acyl membrane anchor of CerS1-derived precursor ceramides. A behavioral analysis of CerS1-deficient mice yielded functional deficits including impaired exploration of novel objects, locomotion, and motor coordination. Our results reveal an essential function of CerS1-derived ceramide in the regulation of cerebellar development and neurodevelopmentally regulated behavior.

Remobilization of Phytol from Chlorophyll Degradation Is Essential for Tocopherol Synthesis and Growth of Arabidopsis

Tocopherol synthesis and growth of Arabidopsis require the activity of the phytyl-phosphate kinase VTE6 in the phosphorylation cascade of the phytol salvage pathway. Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for the phytyl-phosphate kinase from Arabidopsis thaliana (At1g78620) was identified via a phylogeny-based approach. This gene was designated VITAMIN E DEFICIENT6 (VTE6) because the leaves of the Arabidopsis vte6 mutants are tocopherol deficient. The vte6 mutant plants are incapable of photoautotrophic growth. Phytol and phytyl-phosphate accumulate, and the phytyl-diphosphate content is strongly decreased in vte6 leaves. Phytol feeding and enzyme assays with Arabidopsis and recombinant Escherichia coli cells demonstrated that VTE6 has phytyl-P kinase activity. Overexpression of VTE6 resulted in increased phytyl-diphosphate and tocopherol contents in seeds, indicating that VTE6 encodes phytyl-phosphate kinase. The severe growth retardation of vte6 mutants was partially rescued by introducing the phytol kinase mutation vte5. Double mutant plants (vte5 vte6) are tocopherol deficient and contain more chlorophyll, but reduced amounts of phytol and phytyl-phosphate compared with vte6 mutants, suggesting that phytol or phytyl-phosphate are detrimental to plant growth. Therefore, VTE6 represents the missing phytyl-phosphate kinase, linking phytol release from chlorophyll with tocopherol synthesis. Moreover, tocopherol synthesis in leaves depends on phytol derived from chlorophyll, not on de novo synthesis of phytyl-diphosphate from geranylgeranyl-diphosphate.

Inactivation of Ceramide Synthase 6 in Mice Results in an Altered Sphingolipid Metabolism and Behavioral Abnormalities

Background: Ceramide synthases N-acylate (dihydro-)sphingosine to (dihydro-)ceramide in mammals. Results: Enzymatically inactive ceramide synthase 6 in mice (CerS6KO) results in an altered sphingolipid metabolism and behavioral abnormalities. Conclusion: Catalytically active CerS6 is necessary to maintain sphingolipid homeostasis in mice. Significance: The CerS6KO mouse reveals for the first time the metabolic and physiological consequences of CerS6 inactivation. The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for β-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is ∼35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain.

Ceramide synthase 4 deficiency in mice causes lipid alterations in sebum and results in alopecia.

Five ceramide synthases (CerS2-CerS6) are expressed in mouse skin. Although CerS3 has been shown to fulfill an essential function during skin development, neither CerS6- nor CerS2-deficient mice show an obvious skin phenotype. In order to study the role of CerS4, we generated CerS4-deficient mice (Cers4-/-) and CerS4-specific antibodies. With these biological tools we analysed the tissue distribution and determined the cell-type specific expression of CerS4 in suprabasal epidermal layers of footpads as well as in sebaceous glands of the dorsal skin. Loss of CerS4 protein leads to an altered lipid composition of the sebum, which is more solidified and therefore might cause progressive hair loss due to physical blocking of the hair canal. We also noticed a strong decrease in C20 1,2-alkane diols consistent with the decrease of wax diesters in the sebum of Cers4-/- mice. Cers4-/- mice at 12 months old display additional epidermal tissue destruction due to dilated and obstructed pilary canals. Mass spectrometric analyses additionally show a strong decrease in C20-containing sphingolipids.

Ceramide Synthase 5 Is Essential to Maintain C16:0-Ceramide Pools and Contributes to the Development of Diet-induced Obesity

Ceramides are bioactive sphingolipids, which are composed of sphingoid bases carrying acyl chains of various lengths. Ceramides are synthesized by a family of six ceramide synthases (CerS) in mammals, which produce ceramides with different N-linked acyl chains. Increased ceramide levels are known to contribute to the development of obesity and insulin resistance. Recently, it has been demonstrated that the ceramide acylation pattern is of particular importance for an organism to maintain energy homeostasis. However, which of the CerS family members are involved in this process is not yet completely known. Using newly developed CerS5 knock-out mice, we show here that CerS5 is essential to maintain cellular C16:0 sphingolipid pools in lung, spleen, muscle, liver, and white adipose tissue. Glycerophospholipid levels in CerS5-deficient mice were not altered. We found a strong impact of CerS5-dependent ceramide synthesis in white adipose tissue after high fat diet feeding. In skeletal muscle, liver, and spleen, C16:0-ceramide levels were altered independent of feeding conditions. The loss of CerS5 is associated with reduced weight gain and improved systemic health, including maintenance of glucose homeostasis and reduced white adipose tissue inflammation after high fat diet challenge. Our findings indicate that reduction of endogenous C16:0-ceramide by genetic inhibition of CerS5 is sufficient to ameliorate obesity and its comorbidities.

Accumulation of glycolipids and other non-phosphorous lipids in Agrobacterium tumefaciens grown under phosphate deprivation

Phosphate deficiency is characteristic for many natural habitats, resulting in different physiological responses in plants and bacteria including the replacement of phospholipids by glycolipids and other phosphorous-free lipids. The plant pathogenic bacterium Agrobacterium tumefaciens, which is free of glycolipids under full nutrition, harbors an open reading frame (ORF) coding for a processive glycosyltransferase (named as Pgt). This glycosyltransferase was previously shown to synthesize glucosylgalactosyldiacylglycerol (GGD) and digalactosyldiacylglycerol (DGD) after heterologous expression. The native function of this enzyme and the conditions for its activation remained unknown. We show here that Pgt is active under phosphate deprivation synthesizing GGD and DGD in Agrobacterium. A corresponding deletion mutant (Δpgt) is free of these two glycolipids. Glycolipid accumulation is mainly regulated by substrate (diacylglycerol) availability. Diacylglycerol and the total fatty acid pool are characterized by an altered acyl composition in dependence of the phosphate status with a strong decrease of 18:1 and concomitant increase of 19:0 cyclo during phosphate deprivation. Furthermore, Agrobacterium accumulates two additional unknown glycolipids and diacylglycerol trimethylhomoserine (DGTS) during phosphate deprivation. Accumulation of all these lipids is accompanied by a reduction in phospholipids from 75 to 45% in the wild type. A further non-phosphorous lipid, ornithine lipid, was not increased but its degree of hydroxylation was elevated under phosphate deprivation. The lack of GGD and DGD in the Δpgt mutant has no effect on growth and virulence of Agrobacterium, suggesting that these two lipids are functionally replaced by DGTS and the two unknown glycolipids under phosphate deprivation.

Functional characterization of enzymes catalyzing ceramide phosphoethanolamine biosynthesis in mice

Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS)2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.

Altered epidermal lipid processing and calcium distribution in the KID syndrome mouse model Cx26S17F

The keratitis–ichthyosis–deafness (KID) syndrome is caused by mutations in the gap junctional channel protein connexin 26 (Cx26), among them the mutation Cx26S17F. Heterozygous Cx26S17F mice resemble the human KID syndrome, i.e. exhibiting epidermal hyperplasia and hearing impairments. Newborn Cx26S17F mice show a defective epidermal water barrier as well as altered epidermal lipid secretion and location. Linoleoyl ω‐esterified ceramides are strongly decreased on the skin surface of Cx26S17F mice. Moreover, the epidermal calcium gradient is altered in the mutant mice. These alterations may be caused by an abnormal Cx26S17F channel function that leads to a defective epidermal water barrier, which in turn may trigger the hyperproliferation seen in the KID syndrome.