[Show abstract][Hide abstract] ABSTRACT: Owing to its capability of discriminating subtle mass-altering structural differences such as double bonds or elongated acyl chains, matrix-assisted laser desorption/ionization (MALDI)-based imaging mass spectrometry (IMS) has emerged as a powerful technique for analysis of lipid distribution in tissue at moderate spatial resolution of about 50 micrometer. However, it is still unknown if MS1-signals and ion intensity images correlate with the corresponding apparent lipid concentrations. Analyzing renal sulfated glycosphingolipids, sulfatides, we validate for the first time IMS-signal identities using corresponding sulfatide-deficient kidneys. To evaluate the extent of signal quenching effects interfering with lipid quantification, we surgically dissected the three major renal regions (papillae, medulla and cortex) and systematically compared MALDI IMS of renal sulfatides with quantitative analyses of corresponding lipid extracts by on target MALDI TOF-MS and by UPLC-ESI-(QqQ)MS2. Our results demonstrate a generally strong correlation (R2 > 0.9) between the local relative sulfatide signal intensity in MALDI IMS and absolute sulfatide quantities determined by the other two methods. However, high concentrations of sulfatides in papillae and medulla result in up to 4-fold signal suppression. In conclusion, our study suggests that MALDI IMS is useful for semi-quantitative dissection of relative local changes of sulfatides and possibly other lipids in tissue.
The Journal of Lipid Research 10/2014; · 4.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mammalian kidneys are rich in sulfatides. Especially papillary sulfatides contribute to renal adaptation to chronic metabolic acidosis. Due to differences in their ceramide anchors, the structural diversity of renal sulfatides is large. However, the underling biological function of this complexity is not understood. As a compound's function and its tissue location are intimately connected, we analyzed individual renal sulfatide distributions of control and ceramide synthase 2-deficient mice by imaging mass spectrometry and by LC-MS2, (in controls for cortex, medulla, and papillae separately). To explain locally different structures, we compared our lipid data with regional mRNA levels of corresponding anabolic enzymes. The combination of IMS and in source decay-LC-MS2 analyses revealed exclusive expression of C20-sphingosine-containing sulfatides within renal papillae, whereas conventional C18-sphingosine-containing compounds were predominant in medulla, and sulfatides with a C18-phytosphingosine were restricted to special cortical structures. Ceramide synthase 2 deletion resulted in bulk loss of sulfatides with C23/C24-acyl chains, but did not lead to decreased urinary pH as previously observed in sulfatide-depleted kidneys. Reasons may be the almost unchanged C22-sulfatid levels and constant total renal sulfatide levels due to compensation with C16- to C20-acyl chain-containing compounds. Intriguingly, CerS2-deficient kidneys were completely depleted of phytosphingosine-containing cortical sulfatides without any compensation.
The Journal of Lipid Research 09/2014; · 4.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The lipid-rich stratum corneum functions as a barrier against pathogens and desiccation inter alia by an unbroken meshwork of extracellular lipid lamellae. These lamellae are composed of cholesterol, fatty acids and ceramides in an equimolar ratio. The huge class of skin ceramides consists of 3 groups, I) "classical" long and very long chain ceramides, II) ultra-long chain ceramides, and III) ω-esterified ultra-long chain ceramides, which are esterified either with linoleic acid or with cornified envelope proteins and are required for the water permeability barrier. Here, we describe 1-O-acyl ceramides as a new class of epidermal ceramides in humans and mice. These ceramides contain in both, N- and 1-O-position long to very long acyl chains. They derive from the group I of classical ceramides and make up 5% of all esterified ceramides. Considering their chemical structure and hydrophobicity, we presume 1-O-acylceramides to contribute to the water barrier homeostasis. Biosynthesis of 1-O-acylceramides is not dependent on lysosomal phospholipase A2. However, glucosylceramide synthase deficiency was followed by a 7-fold increase of 1-O-acylceramides, which then contributed 30% to all esterified ceramides. Furthermore, loss of neutral glucosylceramidase resulted in decreased levels of a 1-O-acylceramide subgroup. Therefore, we propose 1-O-acylceramides to be synthesized at ER-related sites.
The Journal of Lipid Research 09/2013; · 4.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gaucher disease (GD) is the most common inherited lysosomal storage disorder in humans, caused by mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GBA1). GD is clinically heterogeneous and although the type of GBA1 mutation plays a role in determining the type of GD, it does not explain the clinical variability seen among patients. Cumulative evidence from recent studies suggests that GBA2 could play a role in the pathogenesis of GD and potentially interacts with GBA1.
We used a framework of functional and genetic approaches in order to further characterize a potential role of GBA2 in GD. Glucosylceramide (GlcCer) levels in spleen, liver and brain of GBA2-deficient mice and mRNA and protein expression of GBA2 in GBA1-deficient murine fibroblasts were analyzed. Furthermore we crossed GBA2-deficient mice with conditional Gba1 knockout mice in order to quantify the interaction between GBA1 and GBA2. Finally, a genetic approach was used to test whether genetic variation in GBA2 is associated with GD and/ or acts as a modifier in Gaucher patients. We tested 22 SNPs in the GBA2 and GBA1 genes in 98 type 1 and 60 type 2/3 Gaucher patients for single- and multi-marker association with GD.
We found a significant accumulation of GlcCer compared to wild-type controls in all three organs studied. In addition, a significant increase of Gba2-protein and Gba2-mRNA levels in GBA1-deficient murine fibroblasts was observed. GlcCer levels in the spleen from Gba1/Gba2 knockout mice were much higher than the sum of the single knockouts, indicating a cross-talk between the two glucosylceramidases and suggesting a partially compensation of the loss of one enzyme by the other. In the genetic approach, no significant association with severity of GD was found for SNPs at the GBA2 locus. However, in the multi-marker analyses a significant result was detected for p.L444P (GBA1) and rs4878628 (GBA2), using a model that does not take marginal effects into account.
All together our observations make GBA2 a likely candidate to be involved in GD etiology. Furthermore, they point to GBA2 as a plausible modifier for GBA1 in patients with GD.
[Show abstract][Hide abstract] ABSTRACT: Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) has become a method of choice in lipid analysis, as it provides localization information for defined lipids that is not readily accessible with non-mass spectrometric methods. Most current MALDI matrices have been found empirically. Nevertheless, preferential matrix properties for many analyte classes are poorly understood and may differ between lipid classes. We used rational matrix design and semi-automated screening for the discovery of new matrices suitable for MALDI-IMS of lipids. Utilizing Smartbeam- and nitrogen lasers for MALDI, we systematically compared doubly substituted α Cyanocinnamic acid derivatives (R1 CCA-R2) with respect to their ability to serve as negative ion matrix for various brain lipids. We identified 4 Phenyl-α-cyanocinnamic acid amide (Ph-CCA-NH2) as a novel negative-ion matrix that enables analysis and imaging of various lipid classes by MALDI-MS. We demonstrate that Ph-CCA-NH2 displays superior sensitivity and reproducibility compared to matrices commonly employed for lipids. A relatively small number of background peaks and good matrix suppression effect could make Ph CCA NH2 a widely applicable tool for lipid analysis.
[Show abstract][Hide abstract] ABSTRACT: The epidermis and in particular its outermost layer the stratum corneum provides terrestrial vertebrates with a pivotal defensive barrier against water loss, xenobiotics and harmful pathogens. A vital demand for this epidermal permeability barrier is the lipid-enriched lamellar matrix that embeds the enucleated corneocytes. Ceramides are the major components of these highly ordered intercellular lamellar structures, in which linoleic acid- and protein-esterified ceramides are crucial for structuring and maintaining skin barrier integrity. In this review, we describe the fascinating diversity of epidermal ceramides including 1-O-acylceramides. We focus on epidermal ceramide biosynthesis emphasizing its metabolic and topological requirements and discuss enzymes that may be involved in α- and ω-hydroxylation. Finally, we turn to epidermal ceramide regulation, highlighting transcription factors and liposensors recently described to play crucial roles in modulating skin lipid metabolism and epidermal barrier homeostasis. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier.
Biochimica et Biophysica Acta 08/2013; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Skin barrier function is primarily assigned to the outer epidermal layer, the stratum corneum (SC), mainly composed of corneocytes and lipid-enriched extracellular matrix. Epidermal ceramides (Cers) are essential barrier lipids, containing ultra-long-chain (ULC) fatty acids (FAs) with a unique ω-hydroxy group, which is necessary for binding to corneocyte proteins. In the SC, Cers are believed to derive from glucosylated intermediates, namely glucosylceramides (GlcCers), as surmised from human Gaucher's disease and related mouse models. Tamoxifen (TAM)-induced deletion of the endogenous GlcCer-synthesizing enzyme UDP-glucose:ceramide glucosyltransferase (UGCG) in keratin K14-positive cells resulted in epidermal GlcCer depletion. Although free extractable Cers were elevated in total epidermis and as well in SC, protein-bound Cers decreased significantly in Ugcg(f/fK14CreERT2) mice, indicating glucosylation to be required for regular Cer processing as well as arrangement and extrusion of lipid lamellae. The almost complete loss of protein-bound Cers led to a disruption of the water permeability barrier (WPB). UGCG-deficient mice developed an ichthyosis-like skin phenotype marked by impaired keratinocyte differentiation associated with delayed wound healing. Gene expression profiling of Ugcg-mutant skin revealed a subset of differentially expressed genes involved in lipid signaling and epidermal differentiation/proliferation, correlating to human skin diseases such as psoriasis and atopic dermatitis. Peroxisome proliferator-activated receptor beta/delta (PPARβ/δ), a Cer-sensitive transcription factor was identified as potential mediator of the altered gene sets.
Human Molecular Genetics 06/2013; · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Urinary ammonium excretion by the kidney is essential for renal excretion of sufficient amounts of protons and to maintain stable blood pH. Ammonium secretion by the collecting duct epithelia accounts for the majority of urinary ammonium; it is driven by an interstitium-to-lumen NH3 gradient due to the accumulation of ammonium in the medullary and papillary interstitium. Here, we demonstrate that sulfatides, highly charged anionic glycosphingolipids, are important for maintaining high papillary ammonium concentration and increased urinary acid elimination during metabolic acidosis. We disrupted sulfatide synthesis by a genetic approach along the entire renal tubule. Renal sulfatide-deficient mice had lower urinary pH accompanied by lower ammonium excretion. Upon acid diet, they showed impaired ammonuria, decreased ammonium accumulation in the papilla, and chronic hyperchloremic metabolic acidosis. Expression levels of ammoniagenic enzymes and Na(+)-K(+)/NH4(+)-2Cl(-) cotransporter 2 were higher, and transepithelial NH3 transport, examined by in vitro microperfusion of cortical and outer medullary collecting ducts, was unaffected in mutant mice. We therefore suggest that sulfatides act as counterions for interstitial ammonium facilitating its retention in the papilla. This study points to a seminal role of sulfatides in renal ammonium handling, urinary acidification, and acid-base homeostasis.
Proceedings of the National Academy of Sciences 05/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glycosphingolipids (GSL) have been associated with a variety of diseases including cancer and autosomal dominant polycystic kidney disease (ADPKD). In contrast to glucosylceramide and gangliosides, alterations in complex neutral GSLs such as globotetraosylceramide (Gb4Cer) have not been investigated in ADPKD yet, and mass spectrometry analysis of Gb4Cer from tissue extracts remains challenging. To this end, we introduce PrimaDrop as an improved and widely applicable sample preparation method for automated MALDI-MS analysis of lipid extracts, which promotes homogeneous co-crystallization and enables relative quantification by indirect TLC-MALDI-TOF MS against an internal bradykinin standard. Application of the method for detailed investigation of Gb4Cer isoforms in kidneys of an ADPKD rat model revealed increased levels of sphingoid-base containing isoforms in cystic kidneys, whereas changes were subtle for Gb4Cer-containing phytosphingoid-bases. We furthermore established an absolute LC-ESI-MS/MS quantification method and demonstrate that absolute quantities of Gb4Cer correlate well with relative quantities obtained by indirect TLC-MALDI-TOF MS. Taken together, our study proposes an effective sample preparation method for automated analysis of lipid extracts and TLC eluates and suggests that indirect HPTLC-MALDI-TOF MS with automated data acquisition is a viable option for analysis of neutral glycosphingolipids, and that Gb4Cer may play a role in the pathogenesis of ADPKD.
[Show abstract][Hide abstract] ABSTRACT: 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 immuno-signals in neurons and no other cell types of wild type brain, but did not detect the CerS1 protein 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 causes a foliation defect, progressive shrinkage and neuronal apoptosis in the cerebellum. Mass spectrometric analyses reveal up to 60% decreased levels of gangliosides in cerebellum and forebrain. Expression of Myelin Associated Glycoprotein (MAG) is also decreased by about 60% in cerebellum and forebrain, suggesting that interaction and stabilization of oligodendrocytic MAG protein 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 regulation of cerebellar development and neurodevelopmentally-regulated behavior.
Journal of Biological Chemistry 10/2012; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glycosphingolipids (GSLs) constitute major components of enterocytes and were hypothesized to be potentially important for intestinal epithelial polarization. The enzyme UDP-glucose ceramide glucosyltransferase (Ugcg) catalyzes the initial step of GSL biosynthesis. Newborn and adult mice with enterocyte-specific genetic deletion of the gene Ugcg were generated. In newborn mutants lacking GSLs at day P0, intestinal epithelia were indistinguishable from those in control littermates displaying an intact polarization with regular brush border. However, those mice were not consistently able to absorb nutritional lipids from milk. Between postnatal days 5 and 7, severe defects in intestinal epithelial differentiation occurred accompanied by impaired intestinal uptake of nutrients. Villi of mutant mice became stunted, and enterocytes lacked brush border. The defects observed in mutant mice caused diarrhea, malabsorption, and early death. In this study, we show that GSLs are essential for enterocyte resorptive function but are primarily not for polarization; GSLs are required for intracellular vesicular transport in resorption-active intestine.
Journal of Biological Chemistry 07/2012; 287(39):32598-616. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Myristoyl-CoA (CoA):protein N-myristoyltransferase (NMT) catalyzes protein modification through covalent attachment of a C14 fatty acid (myristic acid) to the N-terminal glycine of proteins, thus promoting protein-protein and protein-membrane interactions. NMT is essential for the viability of numerous human pathogens and is also up-regulated in several tumors. Here we describe a new, nonradioactive, ELISA-based method for measuring NMT activity. After the NMT-catalyzed reaction between a FLAG-tagged peptide and azido-dodecanoyl-CoA (analog of myristoyl-CoA), the resulting azido-dodecanoyl-peptide-FLAG was coupled to phosphine-biotin by Staudinger ligation, captured by plate-bound anti-FLAG antibodies and detected by streptavidin-peroxidase. The assay was validated with negative controls (including inhibitors), corroborated by HPLC analysis, and demonstrated to function with fresh or frozen tissues. Recombinant murine NMT1 and NMT2 were characterized using this new method. This versatile assay is applicable for exploring recombinant NMTs with regard to their activity, substrate specificity, and possible inhibitors as well as for measuring NMT-activity in tissues.
The Journal of Lipid Research 07/2012; 53(11):2459-68. · 4.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glycolipids have been shown to serve specialized functions in cell signalling, proliferation and differentiation processes, which are all important during liver regeneration. We previously generated beta-glucosidase 2 (GBA2) knockout mice that accumulate the glycolipid glucosylceramide in various tissues, including the liver. The present study addressed the role of GBA2-deficiency and subsequent glucosylceramide accumulation in liver regeneration.
Gba2 knockout and wild-type mice were subjected to two-third partial hepatectomy. Mice were sacrificed at different time points, blood was collected, and the remnant liver was removed. Glucosylceramide and ceramide were quantified using mass spectrometry from whole liver and isolated hepatocytes. Serum and hepatocytic supernatant of IL-6, TNF-α and TGF-β levels were measured using ELISA. Cell signalling proteins were analysed using immunoblots.
Regenerating liver after partial hepatectomy showed a significant increase of hepatic glucosylceramide in GBA2-deficient mice compared to controls. Accumulation of glucosylceramide was associated with a delay in liver regeneration and reduced serum levels of IL-6 and TNF-α. Furthermore, reduced IL-6 led to decreased expression of the phosphorylated form of the signal transducer and activator of transcription 3 (P-STAT3).
We conclude that increased glucosylceramide affects cytokine- and growth factor-mediated signalling pathways during liver regeneration. Thus, the repression of IL-6/STAT3 signalling pathway seems to be one of the mechanisms for the delay of liver regeneration in GBA2-deficient mice.
Liver international: official journal of the International Association for the Study of the Liver 07/2012; 32(9):1354-62. · 4.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The stratum corneum as the outermost epidermal layer protects against exsiccation and infection. Both the underlying cornified envelope (CE) and the intercellular lipid matrix contribute essentially to these two main protective barriers. Epidermis-unique ceramides with ultra-long-chain acyl moities (ULC-Cers) are key components of extracellular lipid lamellae (ELL) and are bound to CE proteins, thereby contributing to the cornified lipid envelope (CLE). Here, we identified human and mouse ceramide synthase 3 (CerS3), among CerS1-6, to be exclusively required for the ULC-Cer synthesis in vitro and of mouse CerS3 in vivo. Deficiency of CerS3 in mice results in complete loss of ULC-Cers (≥C26), lack of continuous ELL and a non-functional CLE. Consequently, newborn mutant mice die shortly after birth from transepidermal water loss. Mutant skin is prone to Candida albicans infection highlighting ULC-Cers to be pivotal for both barrier functions. Persistent periderm, hyperkeratosis and deficient cornification are hallmarks of mutant skin demonstrating loss of Cers to trigger a keratinocyte maturation arrest at an embryonic pre-barrier stage.
Human Molecular Genetics 02/2012; 21(3):586-608. · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 2-Hydroxylated fatty acid (HFA)-containing sphingolipids are abundant in mammalian skin and are believed to play a role in the formation of the epidermal barrier. Fatty acid 2-hydroxylase (FA2H), required for the synthesis of 2-hydroxylated sphingolipids in various organs, is highly expressed in skin, and previous in vitro studies demonstrated its role in the synthesis of HFA sphingolipids in human keratinocytes. Unexpectedly, however, mice deficient in FA2H did not show significant changes in their epidermal HFA sphingolipids. Expression of FA2H in murine skin was restricted to the sebaceous glands, where it was required for synthesis of 2-hydroxylated glucosylceramide and a fraction of type II wax diesters. Absence of FA2H resulted in hyperproliferation of sebocytes and enlarged sebaceous glands during hair follicle morphogenesis and anagen (active growth phase) in adult mice. This was accompanied by a significant up-regulation of the epidermal growth factor receptor ligand epigen in sebocytes. Loss of FA2H significantly altered the composition and physicochemical properties of sebum, which often blocked the hair canal, apparently causing a delay in the hair fiber exit. Furthermore, mice lacking FA2H displayed a cycling alopecia with hair loss in telogen. These results underline the importance of the sebaceous glands and suggest a role of specific sebaceous gland or sebum lipids, synthesized by FA2H, in the hair follicle homeostasis.
Journal of Biological Chemistry 05/2011; 286(29):25922-34. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 2-Hydroxylated fatty acid (HFA)-containing sphingolipids are abundant in mammalian skin and are believed to play a role in
the formation of the epidermal barrier. Fatty acid 2-hydroxylase (FA2H), required for the synthesis of 2-hydroxylated sphingolipids
in various organs, is highly expressed in skin, and previous in vitro studies demonstrated its role in synthesis of HFA-sphingolipids
in human keratinocytes. Unexpectedly, however, mice deficient in FA2H did not show significant changes in their epidermal
HFA-sphingolipids. Expression of FA2H in murine skin was restricted to the sebaceous glands, where it was required for synthesis
of 2-hydroxylated glucosylceramide and a fraction of type II wax diesters. Absence of FA2H resulted in hyperproliferation
of sebocytes and enlarged sebaceous glands during hair follicle morphogenesis and anagen (active growth phase) in adult mice.
This was accompanied by a significant up-regulation of the epidermal growth factor receptor ligand Epigen expression in sebocytes.
Loss of FA2H significantly altered the composition and physicochemical properties of sebum, which often blocked the hair canal,
apparently causing a delay in the hair fiber exit. Furthermore, mice lacking FA2H displayed a cycling alopecia with hair loss
in telogen. These results underline the importance of the sebaceous glands and suggest a role of specific sebaceous gland
or sebum lipids, synthesized by FA2H, in the hair follicle homeostasis.
Journal of Biological Chemistry 05/2011; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sulfatides, a class of acidic glycosphingolipids, are highly expressed in mammalian myelin and in kidney, where they are thought to stabilize neuronal structures and signaling and to influence osmotic stability of renal cells, respectively. Recently, 9-aminoacridine (9-AA) has been introduced as a negative ion matrix that displays high selectivity for low complexity galactosylceramid-I(3)-sulfate sulfatides and that is suitable for quantitative analysis by matrix-assisted desorption/ionization (MALDI) mass spectrometry (MS). Analyzing acidic fractions of lipid extracts and cryosections from kidneys of wild type and arylsulfatase A-deficient (ASA -/-) mice, we demonstrate that 9-AA also enables sensitive on-target analysis as well as imaging of complex lactosylceramide-II(3)-sulfate and gangliotetraosylceramide-II(3), IV(3) bis-sulfate sulfatides by MALDI-TOF/TOF MS. Utilizing the MALDI imaging MS technique, we show differential localization in mouse kidney of (1) sulfatides with identical ceramide anchors, but different glycan-sulfate head groups but also of (2) sulfatides with identical head groups but with different acyl- or sphingoid base moieties. A comparison of MALDI images of renal sulfatides from control and sulfatide storing arylsulfatase A-deficient (ASA -/-) mice revealed relative expression differences, very likely reflecting differences in sulfatide turnover of the various renal cell types. These results establish MALDI imaging MS with 9-AA matrix as a label-free method for spatially resolved ex vivo investigation of the relative turnover of sulfatides in animal models of human glycosphingolipid storage disease.
Analytical and Bioanalytical Chemistry 02/2011; 401(1):53-64. · 3.66 Impact Factor