Cloning and Characterization of a Novel Human Alkaline Ceramidase

Mao, Cungui; Xu, Ruijuan; Szulc, Zdzisław M.; Bielawska, Alicja; Galadari, Sehamuddin; Obeid, Lina M.

doi:10.1074/jbc.m102818200

Cited by 155 publications

(175 citation statements)

References 43 publications

(61 reference statements)

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“…Overexpression of YPC1 or YDC1 in lag1⌬lac1⌬ cells increased the amount of M(IP) 2 C to at least wild-type levels ( Figure 8A), and different forms of this sphingolipid appeared depending on the gene overexpressed. This is consistent with differences in the specificity of the two ceramidases for phytoceramide and dihydroceramide (Mao et al, 1997(Mao et al, , 2000b with the upper band being M(IP) 2 C with dihydroceramide and the lower band with phytoceramide. Similar observations came from [ 3 H]DHS long-term labeling ( Figure 8B).…”

Section: Overproduction Of the Ceramidases Ypc1p And Ydc1p In The Lagsupporting

confidence: 59%

“…Even though YPL087w was not identified in the high-copy suppressor screen, the cloning and overexpression of this gene with the use of the plasmid pWB824 in the lag1⌬lac1⌬ deletion strain restored growth to ϳ65% of the wild-type rate (Figure 7, row 4).YBR183w has been found to encode an alkaline phytoceramidase with reverse action and was named YPC1 (Mao et al, 2000a). YPL087w was reported to encode a dihydroceramidase named Ydc1p (Mao et al, 2000b). Like Ypc1p, Ydc1p exhibits a reverse ceramidase action but to a lower extent than Ypc1p, which explains why we did not detect it in our screening.…”

Section: Overproduction Of the Ceramidases Ypc1p And Ydc1p In The Lagcontrasting

confidence: 49%

“…Recently, Mao and coworkers (Mao et al, 2000a(Mao et al, , 2000b) have identified two genes, YPC1 and YDC1, involved in the FB1-resistant and acyl-CoA-independent ceramide synthase. These data prompted us to test the CoA dependence of the LAG1/LAC1-dependent ceramide synthase activity.…”

Section: Localization Of the Sphingolipid Synthesis Defect In Double mentioning

confidence: 99%

“…Ceramide is synthesized mainly from fatty acyl-CoA and PHS or DHS by a CoA-dependent ceramide synthase. Recently, two genes, YPC1 and YDC1, have been shown to have a minor ceramide synthase activity due to their reverse ceramidase action (Mao et al, 2000a(Mao et al, , 2000b. This ceramide synthase activity is independent of CoA and is not inhibited by fumonisin B1 (FB1).…”

Section: Introductionmentioning

confidence: 99%

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Lag1p and Lac1p Are Essential for the Acyl-CoA–dependent Ceramide Synthase Reaction inSaccharomyces cerevisae

Schorling

Vallée

Barz

et al. 2001

MBoC

261

268

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Lag1p and Lac1p are two homologous transmembrane proteins of the endoplasmic reticulum in Saccharomyces cerevisiae. Homologous genes have been found in a wide variety of eukaryotes. In yeast, both genes, LAC1 and LAG1, are required for efficient endoplasmic reticulum-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins. In this study, we show that lag1⌬lac1⌬ cells have reduced sphingolipid levels due to a block of the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction. The sphingolipid synthesis defect in lag1⌬lac1⌬ cells can be partially corrected by overexpression of YPC1 or YDC1, encoding ceramidases that have been reported to have acyl-CoA-independent ceramide synthesis activity. Quadruple mutant cells (lag1⌬lac1⌬ypc1⌬ydc1⌬) do not make any sphingolipids, but are still viable probably because they produce novel lipids. Moreover, lag1⌬lac1⌬ cells are resistant to aureobasidin A, an inhibitor of the inositolphosphorylceramide synthase, suggesting that aureobasidin A may be toxic because it leads to increased ceramide levels. Based on these data, LAG1 and LAC1 are the first genes to be identified that are required for the fumonisin B1-sensitive and acyl-CoAdependent ceramide synthase reaction.

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Section: Overproduction Of the Ceramidases Ypc1p And Ydc1p In The Lagsupporting

confidence: 59%

Section: Overproduction Of the Ceramidases Ypc1p And Ydc1p In The Lagcontrasting

confidence: 49%

Section: Localization Of the Sphingolipid Synthesis Defect In Double mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Lag1p and Lac1p Are Essential for the Acyl-CoA–dependent Ceramide Synthase Reaction inSaccharomyces cerevisae

Schorling

Vallée

Barz

et al. 2001

MBoC

261

268

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“…9 and have predicted molecular masses of 36.4 and 37.2 kDa respectively. A human homologue of YPC1p has been cloned and characterized [7]. It has a pH optimum of 9.5 and a substrate preference for phytoceramide.…”

Section: Introductionmentioning

confidence: 99%

Identification of a novel amidase motif in neutral ceramidase

Galadari

Mao

et al. 2006

Biochemical Journal

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Neutral CDases (ceramidases) are newly identified enzymes with important roles in cell regulation, but little is known about their catalytic mechanisms. In the present study the full-length human neutral CDase was cloned and expressed in the yeast doubleknockout strain ∆ypc1∆ydc1, which lacks the yeast CDases YPC1p and YDC1p. Biochemical characterization of the human neutral CDase showed that the enzyme exhibited classical Michaelis-Menten kinetics, with an optimum activity at pH 7.5. Activity was enhanced by Na + and Ca 2+ . Mg 2+ and Mn 2+ were somewhat stimulatory, but Zn 2+ , Cu 2+ and Fe 2+ inhibited the enzyme. Dithiothreitol and 2-mercaptoethanol dose-dependently inhibited neutral CDase. In order to identify which amino acids were involved in the catalytic action of neutral CDase, the purified enzyme was subjected to chemical modifications. It was observed that the serine residue modifier di-isopropyl fluorophosphate dose-dependently inhibited activity, implicating a serine residue in the catalytic action. From an alignment of the sequences of the neutral CDases from different species, all conserved serine residues were selected for site-directed mutagenesis. Of the six aligned serine residues that were mutated to alanine, only the S354A mutant lost its activity totally. Ser 354 falls within a very highly conserved hexapeptide sequence GDVSPN, which itself was in the middle of a larger conserved sequence, namely NXGDVSPNXXG P / X XC. Moreover, mutations of Asp 352 and Cys 362 in the consensus sequence to alanine resulted in loss of activity of neutral CDase. Hence the present study identified a novel amidase sequence containing a critical serine residue that may function as a nucleophile in the hydrolytic attack on the amide bond present in ceramide.

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Glycosphingolipide – Natur, Funktion und pharmakologische Modulierung

Wennekes

Berg

Boot³

et al. 2009

Angewandte Chemie

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Die Entdeckung der Glycosphingolipide wird im Allgemeinen Johan L. W. Thudichum zugeschrieben, der 1884 über die chemische Zusammensetzung des Gehirns publizierte. In seinen Studien isolierte er aus ethanolischen Hirnextrakten mehrere Verbindungen, die er als Cerebroside bezeichnete. Eine dieser Verbindungen, Phrenosin (heute als Galactosylceramid bekannt), unterwarf er einer sauren Hydrolyse, aus der drei Komponenten hervorgingen. Eine Komponente identifizierte er als Fettsäure, eine andere als Isomer zur D‐Glucose, heute als D‐Galactose bekannt. Die dritte Komponente mit “alkaloidaler Natur” gab Thudichum “viele Rätsel” auf, weshalb er sie Sphingosin nannte, nach dem mythologischen Rätsel der Sphinx. Derzeit sind Sphingolipide und ihre glycosidierten Derivate Gegenstand intensiver Forschung mit dem Ziel, ihre Funktion für die strukturelle Integrität der Zellmembran, ihre Teilnahme in Erkennungs‐ und Signalübertragungsvorgängen und besonders ihre Beteiligung an pathologischen Prozessen, die als Ursache menschlicher Krankheiten (z. B. von Sphingolipidosen und Typ‐2‐Diabetes) gelten, aufzuklären. Dieser Aufsatz wird auf neuere Befunde zur Biosynthese, Funktion und zum Abbau von Glycosphingolipiden im Menschen eingehen, mit Fokus auf das Glycosphingolipidglucosylceramid. Besondere Aufmerksamkeit richtet sich auf die klinische Bedeutung von Verbindungen, die mit den Faktoren des Glycosphingolipid‐Metabolismus wechselwirken.

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Cloning and Characterization of a Novel Human Alkaline Ceramidase

Cited by 155 publications

References 43 publications

Lag1p and Lac1p Are Essential for the Acyl-CoA–dependent Ceramide Synthase Reaction inSaccharomyces cerevisae

Lag1p and Lac1p Are Essential for the Acyl-CoA–dependent Ceramide Synthase Reaction inSaccharomyces cerevisae

Identification of a novel amidase motif in neutral ceramidase

Glycosphingolipide – Natur, Funktion und pharmakologische Modulierung

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