Publications by authors named "Maria J Ferraz"

25 Publications

  • Page 1 of 1

Correction of pathology in mice displaying Gaucher disease type 1 by a clinically-applicable lentiviral vector.

Mol Ther Methods Clin Dev 2021 Mar 3;20:312-323. Epub 2020 Dec 3.

Department of Molecular Medicine and Gene Therapy, Lund University, Lund, Sweden.

Gaucher disease type 1 (GD1) is an inherited lysosomal disorder with multisystemic effects in patients. Hallmark symptoms include hepatosplenomegaly, cytopenias, and bone disease with varying degrees of severity. Mutations in a single gene, glucosidase beta acid 1 (), are the underlying cause for the disorder, resulting in insufficient activity of the enzyme glucocerebrosidase, which in turn leads to a progressive accumulation of the lipid component glucocerebroside. In this study, we treat mice with signs consistent with GD1, with hematopoietic stem/progenitor cells transduced with a lentiviral vector containing an RNA transcript that, after reverse transcription, results in codon-optimized cDNA that, upon its integration into the genome encodes for functional human glucocerebrosidase. Five months after gene transfer, a highly significant reduction in glucocerebroside accumulation with subsequent reversal of hepatosplenomegaly, restoration of blood parameters, and a tendency of increased bone mass and density was evident in vector-treated mice compared to non-treated controls. Furthermore, histopathology revealed a prominent reduction of Gaucher cell infiltration after gene therapy. The vector displayed an oligoclonal distribution pattern but with no sign of vector-induced clonal dominance and a typical lentiviral vector integration profile. Cumulatively, our findings support the initiation of the first clinical trial for GD1 using the lentiviral vector described here.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omtm.2020.11.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806948PMC
March 2021

Human glucocerebrosidase mediates formation of xylosyl-cholesterol by β-xylosidase and transxylosidase reactions.

J Lipid Res 2021 Jan 6;62:100018. Epub 2021 Jan 6.

Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, The Netherlands. Electronic address:

Deficiency of glucocerebrosidase (GBA), a lysosomal β-glucosidase, causes Gaucher disease. The enzyme hydrolyzes β-glucosidic substrates and transglucosylates cholesterol to cholesterol-β-glucoside. Here we show that recombinant human GBA also cleaves β-xylosides and transxylosylates cholesterol. The xylosyl-cholesterol formed acts as an acceptor for the subsequent formation of di-xylosyl-cholesterol. Common mutant forms of GBA from patients with Gaucher disease with reduced β-glucosidase activity were similarly impaired in β-xylosidase, transglucosidase, and transxylosidase activities, except for a slightly reduced xylosidase/glucosidase activity ratio of N370S GBA and a slightly reduced transglucosylation/glucosidase activity ratio of D409H GBA. XylChol was found to be reduced in spleen from patients with Gaucher disease. The origin of newly identified XylChol in mouse and human tissues was investigated. Cultured human cells exposed to exogenous β-xylosides generated XylChol in a manner dependent on active lysosomal GBA but not the cytosol-facing β-glucosidase GBA2. We later sought an endogenous β-xyloside acting as donor in transxylosylation reactions, identifying xylosylated ceramide (XylCer) in cells and tissues that serve as donor in the formation of XylChol. UDP-glucosylceramide synthase (GCS) was unable to synthesize XylChol but could catalyze the formation of XylCer. Thus, food-derived β-D-xyloside and XylCer are potential donors for the GBA-mediated formation of XylChol in cells. The enzyme GCS produces XylCer at a low rate. Our findings point to further catalytic versatility of GBA and prompt a systematic exploration of the distribution and role of xylosylated lipids.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1194/jlr.RA120001043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903134PMC
January 2021

Glucosylated cholesterol in skin: Synthetic role of extracellular glucocerebrosidase.

Clin Chim Acta 2020 Nov 16;510:707-710. Epub 2020 Sep 16.

Medical Biochemistry Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands. Electronic address:

The existence of glucosylated cholesterol (GlcChol) in tissue has recently been recognized. GlcChol is generated from glucosylceramide (GlcCer) and cholesterol through transglucosylation by two retaining β-glucosidases, GBA and GBA2. Given the abundance of GBA, GlcCer and cholesterol in the skin's stratum corneum (SC), we studied the occurrence of GlcChol. A significant amount of GlcChol was detected in SC (6 pmol/mg weight). The ratio GlcChol/GlcCer is higher in SC than epidermis, 0.083 and 0.011, respectively. Examination of GlcChol in patients with Netherton syndrome revealed comparable levels (11 pmol/mg). Concluding, GlcChol was identified as a novel component in SC and is likely locally metabolized by GBA. The physiological function of GlcChol in the SC warrants future investigation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cca.2020.09.017DOI Listing
November 2020

Lyso-glycosphingolipids: presence and consequences.

Essays Biochem 2020 09;64(3):565-578

Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA, Leiden, The Netherlands.

Lyso-glycosphingolipids are generated in excess in glycosphingolipid storage disorders. In the course of these pathologies glycosylated sphingolipid species accumulate within lysosomes due to flaws in the respective lipid degrading machinery. Deacylation of accumulating glycosphingolipids drives the formation of lyso-glycosphingolipids. In lysosomal storage diseases such as Gaucher Disease, Fabry Disease, Krabbe disease, GM1 -and GM2 gangliosidosis, Niemann Pick type C and Metachromatic leukodystrophy massive intra-lysosomal glycosphingolipid accumulation occurs. The lysosomal enzyme acid ceramidase generates the deacylated lyso-glycosphingolipid species. This review discusses how the various lyso-glycosphingolipids are synthesized, how they may contribute to abnormal immunity in glycosphingolipid storing lysosomal diseases and what therapeutic opportunities exist.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/EBC20190090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7517347PMC
September 2020

Role of β-glucosidase 2 in aberrant glycosphingolipid metabolism: model of glucocerebrosidase deficiency in zebrafish.

J Lipid Res 2019 11 27;60(11):1851-1867. Epub 2019 Sep 27.

Department of Medical Biochemistry Leiden Institute of Chemistry, Leiden, The Netherlands

β-glucosidases [GBA1 (glucocerebrosidase) and GBA2] are ubiquitous essential enzymes. Lysosomal GBA1 and cytosol-facing GBA2 degrade glucosylceramide (GlcCer); GBA1 deficiency causes Gaucher disease, a lysosomal storage disorder characterized by lysosomal accumulation of GlcCer, which is partly converted to glucosylsphingosine (GlcSph). GBA1 and GBA2 also may transfer glucose from GlcCer to cholesterol, yielding glucosylated cholesterol (GlcChol). Here, we aimed to clarify the role of zebrafish Gba2 in glycosphingolipid metabolism during Gba1 deficiency in zebrafish (), which are able to survive total Gba1 deficiency. We developed Gba1 (), Gba2 (), and double () zebrafish knockouts using CRISPR/Cas9 and explored the effects of both genetic and pharmacological interventions on GlcCer metabolism in individual larvae. Activity-based probes and quantification of relevant glycolipid metabolites confirmed enzyme deficiency. GlcSph increased in larvae (0.09 pmol/fish) but did not increase more in larvae. GlcCer was comparable in and WT larvae but increased in and larvae. Independent of Gba1 status, GlcChol was low in all larvae (0.05 vs. 0.18 pmol/fish in WT). Pharmacologic inactivation of zebrafish Gba1 comparably increased GlcSph. Inhibition of GlcCer synthase (GCS) in Gba1-deficient larvae reduced GlcCer and GlcSph, and concomitant inhibition of GCS and Gba2 with iminosugars also reduced excessive GlcChol. Finally, overexpression of human GBA1 and injection of recombinant GBA1 both decreased GlcSph. We determined that zebrafish larvae offer an attractive model to study glucosidase actions in glycosphingolipid metabolism in vivo, and we identified distinguishing characteristics of zebrafish Gba2 deficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1194/jlr.RA119000154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824494PMC
November 2019

Adipocytes harbor a glucosylceramide biosynthesis pathway involved in iNKT cell activation.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 08 30;1864(8):1157-1167. Epub 2019 Apr 30.

Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, the Netherlands. Electronic address:

Background: Natural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis.

Results: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell activity.

Conclusion: Glucosylceramide biosynthesis pathway is important for endogenous lipid antigen activation of iNKT cells in adipocytes.

Significance: Unraveling adipocyte-iNKT cell communication may help to fight obesity-induced AT dysfunction. Overproduction and/or accumulation of ceramide and ceramide metabolites, including glucosylceramides, can lead to insulin resistance. However, glucosylceramides also fulfill important physiological functions. They are presented by antigen presenting cells (APC) as endogenous lipid antigens via CD1d to activate a unique lymphocyte subspecies, the CD1d-restricted invariant (i) natural killer T (NKT) cells. Recently, adipocytes have emerged as lipid APC that can activate adipose tissue-resident iNKT cells and thereby contribute to whole body energy homeostasis. Here we investigate the role of the glucosylceramide biosynthesis pathway in the activation of iNKT cells by adipocytes. UDP-glucose ceramide glucosyltransferase (Ugcg), the first rate limiting step in the glucosylceramide biosynthesis pathway, was inhibited via chemical compounds and shRNA knockdown in vivo and in vitro. β-1,4-Galactosyltransferase (B4Galt) 5 and 6, enzymes that convert glucosylceramides into potentially inactive lactosylceramides, were subjected to shRNA knock down. Subsequently, (pre)adipocyte cell lines were tested in co-culture experiments with iNKT cells (IFNγ and IL4 secretion). Inhibition of Ugcg activity shows that it regulates presentation of a considerable fraction of lipid self-antigens in adipocytes. Furthermore, reduced expression levels of either B4Galt5 or -6, indicate that B4Galt5 is dominant in the production of cellular lactosylceramides, but that inhibition of either enzyme results in increased iNKT cell activation. Additionally, in vivo inhibition of Ugcg by the aminosugar AMP-DNM results in decreased iNKT cell effector function in adipose tissue. Inhibition of endogenous glucosylceramide production results in decreased iNKT cells activity and cytokine production, underscoring the role of this biosynthetic pathway in lipid self-antigen presentation by adipocytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbalip.2019.04.016DOI Listing
August 2019

Cytosolic glucosylceramide regulates endolysosomal function in Niemann-Pick type C disease.

Neurobiol Dis 2019 07 12;127:242-252. Epub 2019 Mar 12.

School of Pharmacy, De Montfort University, The Gateway, Leicester LE1 9BH, UK. Electronic address:

Niemann-Pick type C disease (NPCD) is a neurodegenerative disease associated with increases in cellular cholesterol and glycolipids and most commonly caused by defective NPC1, a late endosomal protein. Using ratiometric probes we find that NPCD cells show increased endolysosomal pH. In addition U18666A, an inhibitor of NPC1, was found to increase endolysosomal pH, and the number, size and heterogeneity of endolysosomal vesicles. NPCD fibroblasts and cells treated with U18666A also show disrupted targeting of fluorescent lipid BODIPY-LacCer to high pH vesicles. Inhibiting non-lysosomal glucocerebrosidase (GBA2) reversed increases in endolysosomal pH and restored disrupted BODIPY-LacCer trafficking in NPCD fibroblasts. GBA2 KO cells also show decreased endolysosomal pH. NPCD fibroblasts also show increased expression of a key subunit of the lysosomal proton pump vATPase on GBA2 inhibition. The results are consistent with a model where both endolysosomal pH and Golgi targeting of BODIPY-LacCer are dependent on adequate levels of cytosolic-facing GlcCer, which are reduced in NPC disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nbd.2019.03.005DOI Listing
July 2019

Dissociation of globotriaosylceramide and impaired endothelial function in α-galactosidase-A deficient EA.hy926 cells.

Mol Genet Metab 2018 12 2;125(4):338-344. Epub 2018 Nov 2.

Department of Internal Medicine, University of Michigan Medical School, University of Michigan, Ann Arbor, Michigan, USA. Electronic address:

Fabry disease, a rare, X-linked lysosomal storage disease, arises from deficiency of the lysosomal hydrolase, α-galactosidase A (GLA) which disrupts the catabolism of globo- series glycosphingolipids (GSLs). One potential link between GLA deficiency and vascular dysfunction may be changes in endothelial nitric oxide synthase (eNOS) function. GLA-deficient EA.hy926 cells were obtained by siRNA knockdown of GLA expression and by mutation of GLA with CRISPR/Cas9 gene editing to investigate the effects of GLA deficiency on eNOS. As previously observed with siRNA knockdown of GLA, globotriaosylceramide (Gb3) accumulated in EA.hy926 cells. In contrast, Gb3 did not accumulate in CRISPR/Cas9 gene edited GLA-deficient cells, but instead, globotetraosylceramide (Gb4). However, in both the siRNA and CRISPR/Cas9 models globotriaosylsphingosine (lyso-Gb3) was elevated. As was previously observed with siRNA knockdown of GLA expression, CRISPR/Cas9 GLA-deficient cells had lower eNOS activity. Restoring GLA activity in GLA-deficient cells with exogenous GLA treatment improved eNOS activity. In contrast, treating cells with the glucosylceramide synthase inhibitor, eliglustat, decreased NOS activity. These results suggest that eNOS uncoupling is due to GLA deficiency, and not necessarily due to elevated Gb3 per se. It was observed that lyso-Gb3 inhibits eNOS activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymgme.2018.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6554739PMC
December 2018

α-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease.

J Biol Chem 2018 06 19;293(26):10042-10058. Epub 2018 Apr 19.

From the Departments of Medical Biochemistry,

α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in leaf extracts, enabling the identification and biochemical characterization of an α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking -glycans and was active toward 4-methylumbelliferyl-α-d-galactopyranoside substrate ( = 0.17 mm) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.001774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028973PMC
June 2018

Gluco-1 H-imidazole: A New Class of Azole-Type β-Glucosidase Inhibitor.

J Am Chem Soc 2018 04 4;140(15):5045-5048. Epub 2018 Apr 4.

Department of Chemistry, York Structural Biology Laboratory , University of York , Heslington, York YO10 5DD , United Kingdom.

Gluco-azoles competitively inhibit glucosidases by transition-state mimicry and their ability to interact with catalytic acid residues in glucosidase active sites. We noted that no azole-type inhibitors described, to date, possess a protic nitrogen characteristic for 1 H-imidazoles. Here, we present gluco-1 H-imidazole, a gluco-azole bearing a 1 H-imidazole fused to a glucopyranose-configured cyclitol core, and three close analogues as new glucosidase inhibitors. All compounds inhibit human retaining β-glucosidase, GBA1, with the most potent ones inhibiting this enzyme (deficient in Gaucher disease) on a par with glucoimidazole. None inhibit glucosylceramide synthase, cytosolic β-glucosidase GBA2 or α-glucosidase GAA. Structural, physical and computational studies provide first insights into the binding mode of this conceptually new class of retaining β-glucosidase inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.8b02399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942873PMC
April 2018

A Fluorescence Polarization Activity-Based Protein Profiling Assay in the Discovery of Potent, Selective Inhibitors for Human Nonlysosomal Glucosylceramidase.

J Am Chem Soc 2017 10 29;139(40):14192-14197. Epub 2017 Sep 29.

Structural Biology Laboratory, Department of Chemistry, The University of York , York YO10 5DD, United Kingdom.

Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chemical tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining β-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell extracts containing recombinant, overexpressed glycosidase as the easily accessible enzyme source.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.7b07352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5677758PMC
October 2017

1,6-Cyclophellitol Cyclosulfates: A New Class of Irreversible Glycosidase Inhibitor.

ACS Cent Sci 2017 Jul 13;3(7):784-793. Epub 2017 Jul 13.

Department of Bio-organic Synthesis and Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.

The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here we present the synthesis, conformational analysis, and application of novel 1,6-cyclophellitol cyclosulfates. We show that 1,6--cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose C chair conformation matches that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacts more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acscentsci.7b00214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5532717PMC
July 2017

Human Alpha Galactosidases Transiently Produced in Leaves: New Insights in Substrate Specificities with Relevance for Fabry Disease.

Front Plant Sci 2017 21;8:1026. Epub 2017 Jun 21.

Department of Medical Biochemistry, Leiden Institute of ChemistryLeiden, Netherlands.

Deficiency of α-galactosidase A (α-GAL) causes Fabry disease (FD), an X-linked storage disease of the glycosphingolipid globtriaosylcerammide (Gb3) in lysosomes of various cells and elevated plasma globotriaosylsphingosine (Lyso-Gb3) toxic for podocytes and nociceptive neurons. Enzyme replacement therapy is used to treat the disease, but clinical efficacy is limited in many male FD patients due to development of neutralizing antibodies (Ab). Therapeutic use of modified lysosomal α--acetyl-galactosaminidase (α-NAGAL) with increased α-galactosidase activity (α-NAGAL) has therefore been suggested. We transiently produced in leaves functional α-GAL, α-NAGAL, and α-NAGAL enzymes for research purposes. All enzymes could be visualized with activity-based probes covalently binding in their catalytic pocket. Characterization of purified proteins indicated that α-NAGAL is improved in activity toward artificial 4MU-α-galactopyranoside. Recombinant α-NAGAL and α-NAGAL are not neutralized by Ab-positive FD serum tested and are more stable in human plasma than α-GAL. Both enzymes hydrolyze the lipid substrates Gb3 and Lyso-Gb3 accumulating in Fabry patients. The addition to FD sera of α-NAGAL, and to a lesser extent that of α-NAGAL, results in a reduction of the toxic Lyso-Gb3. In conclusion, our study suggests that modified α-NAGAL might reduce excessive Lyso-Gb3 in FD serum. This neo-enzyme can be produced in and might be further developed for the treatment of FD aiming at reduction of circulating Lyso-Gb3.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2017.01026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478728PMC
June 2017

Carba-cyclophellitols Are Neutral Retaining-Glucosidase Inhibitors.

J Am Chem Soc 2017 05 5;139(19):6534-6537. Epub 2017 May 5.

Department of Chemistry, University of York , Heslington, York, YO10 5DD, U.K.

The conformational analysis of glycosidases affords a route to their specific inhibition through transition-state mimicry. Inspired by the rapid reaction rates of cyclophellitol and cyclophellitol aziridine-both covalent retaining β-glucosidase inhibitors-we postulated that the corresponding carba "cyclopropyl" analogue would be a potent retaining β-glucosidase inhibitor for those enzymes reacting through the H transition-state conformation. Ab initio metadynamics simulations of the conformational free energy landscape for the cyclopropyl inhibitors show a strong bias for the H conformation, and carba-cyclophellitol, with an N-(4-azidobutyl)carboxamide moiety, proved to be a potent inhibitor (K = 8.2 nM) of the Thermotoga maritima TmGH1 β-glucosidase. 3-D structural analysis and comparison with unreacted epoxides show that this compound indeed binds in the H conformation, suggesting that conformational strain induced through a cyclopropyl unit may add to the armory of tight-binding inhibitor designs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.7b01773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437670PMC
May 2017

Accurate quantification of sphingosine-1-phosphate in normal and Fabry disease plasma, cells and tissues by LC-MS/MS with (13)C-encoded natural S1P as internal standard.

Clin Chim Acta 2016 Aug 21;459:36-44. Epub 2016 May 21.

Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, The Netherlands. Electronic address:

We developed a mass spectrometric procedure to quantify sphingosine-1-phosphate (S1P) in biological materials. The use of newly synthesized (13)C5 C18-S1P and commercial C17-S1P as internal standards rendered very similar results with respect to linearity, limit of detection and limit of quantitation. Caution is warranted with determination of plasma S1P levels. Earlier it was reported that S1P is elevated in plasma of Fabry disease patients. We investigated this with the improved quantification. No clear conclusion could be drawn for patient plasma samples given the lack of uniformity of blood collection and plasma preparation. To still obtain insight, plasma and tissues were identically collected from α-galactosidase A deficient Fabry mice and matched control animals. No significant difference was observed in plasma S1P levels. A significant 2.3 fold increase was observed in kidney of Fabry mice, but not in liver and heart. Comparative analysis of S1P in cultured fibroblasts from normal subjects and classically affected Fabry disease males revealed no significant difference. In conclusion, accurate quantification of S1P in biological materials is feasible by mass spectrometry using the internal standards (13)C5 C18-S1P or C17-S1P. Significant local increases of S1P in the kidney might occur in Fabry disease as suggested by the mouse model.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cca.2016.05.017DOI Listing
August 2016

Biochemical response to substrate reduction therapy versus enzyme replacement therapy in Gaucher disease type 1 patients.

Orphanet J Rare Dis 2016 Mar 24;11:28. Epub 2016 Mar 24.

Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.

Background: We retrospectively compared biochemical responses in type 1 Gaucher disease patients to treatment with glycosphingolipid synthesis inhibitors miglustat and eliglustat and ERT.

Methods: Seventeen GD1 patients were included (n = 6 eliglustat, (two switched from ERT), n = 9 miglustat (seven switchers), n = 4 ERT (median dose 60U/kg/m). Plasma protein markers reflecting disease burden (chitotriosidase, CCL18) and lipids reflecting substrate accumulation (glucosylsphingosine, glucosylceramide) were determined. Also, liver and spleen volumes, hemoglobin, platelets, and fat fraction were measured.

Results: In patients naïve to treatment, chitotriosidase, CCL18 and glucosylsphingosine decreased comparably upon eliglustat and ERT treatment, while the response to miglustat was less. After 2 years, median decrease of chitotriosidase was 89% (range 77-98), 88% (78-92) and 37% (29-46) for eliglustat, ERT and miglustat naïve patients respectively; decrease of CCL18 was 73% (63-78), 54% (43-86), and 10% (3-18); decrease of glucosylsphingosine was 86% (78-93), 78% (65-91), 48% (46-50). Plasma glucosylceramide in eliglustat treated patients (n = 4) reached values below the normal range (n = 20 healthy controls). Biochemical markers decreased or stabilized in switchers from ERT to eliglustat (n = 2), but less in miglustat switchers (n = 7). Clinical parameters responded comparably upon eliglustat and ERT treatment.

Conclusions: Our explorative study provides evidence that biochemical markers respond comparably in patients receiving eliglustat treatment and ERT, while the corresponding response to miglustat treatment is less.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13023-016-0413-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806476PMC
March 2016

Lysosomal glycosphingolipid catabolism by acid ceramidase: formation of glycosphingoid bases during deficiency of glycosidases.

FEBS Lett 2016 Mar 3;590(6):716-25. Epub 2016 Mar 3.

Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands.

Glycosphingoid bases are elevated in inherited lysosomal storage disorders with deficient activity of glycosphingolipid catabolizing glycosidases. We investigated the molecular basis of the formation of glucosylsphingosine and globotriaosylsphingosine during deficiency of glucocerebrosidase (Gaucher disease) and α-galactosidase A (Fabry disease). Independent genetic and pharmacological evidence is presented pointing to an active role of acid ceramidase in both processes through deacylation of lysosomal glycosphingolipids. The potential pathophysiological relevance of elevated glycosphingoid bases generated through this alternative metabolism in patients suffering from lysosomal glycosidase defects is discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.12104DOI Listing
March 2016

Lyso-glycosphingolipid abnormalities in different murine models of lysosomal storage disorders.

Mol Genet Metab 2016 Feb 23;117(2):186-93. Epub 2015 Dec 23.

Department of Medical Biochemistry, Academic Medical Center, 1105, AZ, Amsterdam, The Netherlands; Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, 2333, CC, Leiden, The Netherlands. Electronic address:

In lysosomal glycosphingolipid storage disorders, marked elevations in corresponding glycosphingoid bases (lyso-glycosphingolipids) have been reported, such as galactosylsphingosine in Krabbe disease, glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease. Using LC–MS/MS, we comparatively investigated the occurrence of abnormal lyso-glycosphingolipids in tissues and plasma of mice with deficiencies in lysosomal α-galactosidase A, glucocerebrosidase and galactocerebrosidase. The nature and specificity of lyso-glycosphingolipid abnormalities are reported and compared to that in correspondingly more abundant N-acylated glycosphingolipids. Specific elevations in tissue and plasma globotriaosylsphingosine were detected in α-galactosidase A-deficient mice; glucosylsphingosine in glucocerebrosidase-deficient mice and galactosylsphingosine in galactocerebrosidase-deficient animals. A similar investigation was conducted for two mouse models of Niemann Pick type C (Npc1nih and Npc1nmf164), revealing significant tissue elevation of several neutral glycosphingolipids and concomitant increased plasma glucosylsphingosine. This latter finding was recapitulated by analysis of plasma of NPC patients. The value of plasma glucosylsphingosine in biochemical confirmation of the diagnosis of NPC is discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymgme.2015.12.006DOI Listing
February 2016

Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular β-glucosidases.

J Lipid Res 2016 Mar 2;57(3):451-63. Epub 2016 Jan 2.

Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands Departments of Medical Biochemistry Leiden Institute of Chemistry, Leiden, The Netherlands

The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading β-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa. Furthermore, the natural occurrence of 1-O-cholesteryl-β-D-glucopyranoside (GlcChol) in mouse tissues and human plasma is demonstrated using LC-MS/MS and (13)C6-labeled GlcChol as internal standard. In cells, the inhibition of GBA increases GlcChol, whereas inhibition of GBA2 decreases glucosylated sterol. Similarly, in GBA2-deficient mice, GlcChol is reduced. Depletion of GlcCer by inhibition of GlcCer synthase decreases GlcChol in cells and likewise in plasma of inhibitor-treated Gaucher disease patients. In tissues of mice with Niemann-Pick type C disease, a condition characterized by intralysosomal accumulation of cholesterol, marked elevations in GlcChol occur as well. When lysosomal accumulation of cholesterol is induced in cultured cells, GlcChol is formed via lysosomal GBA. This illustrates that reversible transglucosylation reactions are highly dependent on local availability of suitable acceptors. In conclusion, mammalian tissues contain GlcChol formed by transglucosylation through β-glucosidases using GlcCer as donor. Our findings reveal a novel metabolic function for GlcCer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1194/jlr.M064923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4766994PMC
March 2016

Mass spectrometric quantification of glucosylsphingosine in plasma and urine of type 1 Gaucher patients using an isotope standard.

Blood Cells Mol Dis 2015 Apr 17;54(4):307-14. Epub 2015 Jan 17.

Department of Medical Biochemistry, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands. Electronic address:

Deficiency of glucocerebrosidase (GBA) leads to Gaucher disease (GD), an inherited disorder characterised by storage of glucosylceramide (GlcCer) in lysosomes of tissue macrophages. Recently, we reported marked increases of deacylated GlcCer, named glucosylsphingosine (GlcSph), in plasma of GD patients. To improve quantification, [5-9] (13)C5-GlcSph was synthesised for use as internal standard with quantitative LC-ESI-MS/MS. The method was validated using plasma of 55 GD patients and 20 controls. Intra-assay variation was 1.8% and inter-assay variation was 4.9% for GlcSph (m/z 462.3). Plasma GlcSph levels with the old and new methods closely correlate (r=0.968, slope=1.038). Next, we analysed GlcSph in 24h urine samples of 30 GD patients prior to therapy. GlcSph was detected in the patient samples (median 1.20nM, range 0.11-8.92nM), but was below the limit of quantification in normal urine. Enzyme replacement therapy led to a decrease of urinary GlcSph of GD patients, coinciding with reductions in plasma GlcSph and markers of Gaucher cells (chitotriosidase and CCL18). In analogy to globotriaosylsphingsone in urine of Fabry disease patients, additional isoforms of GlcSph differing in structure of the sphingosine moiety were identified in GD urine samples. In conclusion, GlcSph can be sensitively detected by LC-ESI-MS/MS with an internal isotope standard. Abnormalities in urinary GlcSph are a hallmark of Gaucher disease allowing biochemical confirmation of diagnosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bcmd.2015.01.006DOI Listing
April 2015

Development of an acid ceramidase activity-based probe.

Chem Commun (Camb) 2015 Apr;51(28):6161-3

Leiden Institute of Chemistry, Leiden University, P. O. Box 9502, 2300 RA Leiden, The Netherlands.

Acid ceramidase is responsible for the ultimate step in the catabolism of (glyco)sphingolipids by hydrolysis of ceramide into sphingosine and free fatty acid. Deficiency in acid ceramidase is the molecular basis of Farber disease. Here we report the synthesis and characterization of an activity-based acid ceramidase probe.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c5cc00356cDOI Listing
April 2015

Potent and selective activity-based probes for GH27 human retaining α-galactosidases.

J Am Chem Soc 2014 Aug 11;136(33):11622-5. Epub 2014 Aug 11.

Leiden Institute of Chemistry and The Netherlands Proteomics Centre, Leiden University , P.O. Box 9502, 2300 RA Leiden, The Netherlands.

Lysosomal degradation of glycosphingolipids is mediated by the consecutive action of several glycosidases. Malfunctioning of one of these hydrolases can lead to a lysosomal storage disorder such as Fabry disease, which is caused by a deficiency in α-galactosidase A. Herein we describe the development of potent and selective activity-based probes that target retaining α-galactosidases. The fluorescently labeled aziridine-based probes 3 and 4 inhibit the two human retaining α-galactosidases αGal A and αGal B covalently and with high affinity. Moreover, they enable the visualization of the endogenous activity of both α-galactosidases in cell extracts, thereby providing a means to study the presence and location of active enzyme levels in different cell types, such as healthy cells versus those derived from Fabry patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja507040nDOI Listing
August 2014

Gaucher disease and Fabry disease: new markers and insights in pathophysiology for two distinct glycosphingolipidoses.

Biochim Biophys Acta 2014 May 13;1841(5):811-25. Epub 2013 Nov 13.

Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands; Department of Bio-organic Synthesis, Leiden Institute of Chemistry, The Netherlands. Electronic address:

Gaucher disease (GD) and Fabry disease (FD) are two relatively common inherited glycosphingolipidoses caused by deficiencies in the lysosomal glycosidases glucocerebrosidase and alpha-galactosidase A, respectively. For both diseases enzyme supplementation is presently used as therapy. Cells and tissues of GD and FD patients are uniformly deficient in enzyme activity, but the two diseases markedly differ in cell types showing lysosomal accumulation of the glycosphingolipid substrates glucosylceramide and globotriaosylceramide, respectively. The clinical manifestation of Gaucher disease and Fabry disease is consequently entirely different and the response to enzyme therapy is only impressive in the case of GD patients. This review compares both glycosphingolipid storage disorders with respect to similarities and differences. Presented is an update on insights regarding pathophysiological mechanisms as well as recently available biochemical markers and diagnostic tools for both disorders. Special attention is paid to sphingoid bases of the primary storage lipids in both diseases. The value of elevated glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease for diagnosis and monitoring of disease is discussed as well as the possible contribution of the sphingoid bases to (patho)physiology. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbalip.2013.11.004DOI Listing
May 2014

Fetal asphyxia induces acute and persisting changes in the ceramide metabolism in rat brain.

J Lipid Res 2013 Jul 26;54(7):1825-33. Epub 2013 Apr 26.

Department of Neuroscience, Maastricht University, School of Mental Health and Neuroscience (MHeNS), Maastricht, The Netherlands.

Fetal asphyctic preconditioning, induced by a brief episode of experimental hypoxia-ischemia, offers neuroprotection to a subsequent more severe asphyctic insult at birth. Extensive cell stress and apoptosis are important contributing factors of damage in the asphyctic neonatal brain. Because ceramide acts as a second messenger for multiple apoptotic stimuli, including hypoxia/ischemia, we sought to investigate the possible involvement of the ceramide pathway in endogenous neuroprotection induced by fetal asphyctic preconditioning. Global fetal asphyxia was induced in rats by clamping both uterine and ovarian vasculature for 30 min. Fetal asphyxia resulted in acute changes in brain ceramide/sphingomyelin metabolic enzymes, ceramide synthase 1, 2, and 5, acid sphingomyelinase, sphingosine-1-phosphate phosphatase, and the ceramide transporter. This observation correlated with an increase in neuronal apoptosis and in astrocyte number. After birth, ceramide and sphingomyelin levels remained high in fetal asphyxia brains, suggesting that a long-term regulation of the ceramide pathway may be involved in the mechanism of tolerance to a subsequent, otherwise lethal, asphyctic event.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1194/jlr.M034447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3679385PMC
July 2013

Elevated plasma glucosylsphingosine in Gaucher disease: relation to phenotype, storage cell markers, and therapeutic response.

Blood 2011 Oct 25;118(16):e118-27. Epub 2011 Aug 25.

Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands.

Gaucher disease, caused by a deficiency of the lysosomal enzyme glucocerebrosidase, leads to prominent glucosylceramide accumulation in lysosomes of tissue macrophages (Gaucher cells). Here we show glucosylsphingosine, the deacylated form of glucosylceramide, to be markedly increased in plasma of symptomatic nonneuronopathic (type 1) Gaucher patients (n = 64, median = 230.7 nM, range 15.6-1035.2 nM; normal (n = 28): median 1.3 nM, range 0.8-2.7 nM). The method developed for mass spectrometric quantification of plasma glucosylsphingosine is sensitive and robust. Plasma glucosylsphingosine levels correlate with established plasma markers of Gaucher cells, chitotriosidase (ρ = 0.66) and CCL18 (ρ = 0.40). Treatment of Gaucher disease patients by supplementing macrophages with mannose-receptor targeted recombinant glucocerebrosidase results in glucosylsphingosine reduction, similar to protein markers of Gaucher cells. Since macrophages prominently accumulate the lysoglycosphingolipid on glucocerebrosidase inactivation, Gaucher cells seem a major source of the elevated plasma glucosylsphingosine. Our findings show that plasma glucosylsphingosine can qualify as a biomarker for type 1 Gaucher disease, but that further investigations are warranted regarding its relationship with clinical manifestations of Gaucher disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2011-05-352971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3685900PMC
October 2011