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PubMed1999

A vital role for glycosphingolipid synthesis during development and differentiation.

Glycosphingolipids (GSLs) are believed to be integral for the dynamics of many cell membrane events, including cellular interactions, signaling, and trafficking. We have investigated their roles in development and differentiation by eliminating the major synthesis pathway of GSLs through targeted disruption of the Ugcg gene encoding glucosylceramide synthase. In the absence of GSL synthesis, embryogenesis proceeded well into gastrulation with differentiation into primitive germ layers and patterning of the embryo but was abruptly halted by a major apoptotic process. In vivo, embryonic stem cells deficient in GSL synthesis were again able to differentiate into endodermal, mesodermal, and ectodermal derivatives but were strikingly deficient in their ability to form well differentiated tissues. In vitro, however, hematopoietic and neuronal differentiation could be induced. The results demonstrate that the synthesis of GSL structures is essential for embryonic development and for the differentiation of some tissues and support the concept that GSLs are involved in crucial cell interactions mediating these processes.

Animals↗
PubMed1995

Purification, characterization, and biosynthesis of human acid ceramidase.

Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) is the lysosomal enzyme catalyzing the hydrolysis of ceramide to sphingosine and free fatty acid. Its inherited deficiency causes ceramide accumulation in Farber's disease. The enzyme was purified to apparent homogeneity from human urine by sequential chromatography on octyl-Sepharose, concanavalin A-Sepharose, blue-Sepharose, and DEAE-cellulose. The final preparation, which was enriched approximately 4450-fold over the starting material, resulted in a polypeptide of approximately 50 kDa and could be reduced into two subunits of approximately 13 (alpha) and approximately 40 (beta) kDa. Treatment of the purified enzyme with endoglycosidase H or peptido-N-glycanase F reduced the molecular mass of the beta subunit to approximately 30-35 and approximately 27 kDa, respectively. In contrast, the molecular mass of the alpha subunit was unchanged. The purified enzyme had an apparent Km of 149 microM and a Vmax of 136 nmol/mg/h using N-lauroylsphingosine as substrate. Polyclonal antibodies were raised against the purified urinary enzyme and used to investigate the biosynthesis of acid ceramidase. Immunoprecipitation studies on metabolically labeled skin fibroblasts indicated that both subunits arose from a single precursor of approximately 55 kDa. A minor portion of newly synthesized acid ceramidase was secreted into the medium as a monomeric 47-kDa protein, indicating that generation of the mature heterodimeric enzyme occurred in endosomal and/or lysosomal compartments.

Acid Ceramidase↗
PubMed1995

Photoaffinity labeling of human lysosomal beta-hexosaminidase B. Identification of Glu-355 at the substrate binding site.

The carbene precursor 3-azi-1-[([6-3H]-2-acetamido-2-deoxy-1-beta-D-galactopyranosyl)thi o -butane (also designated [3H]-1-ATB-GalNAc) has been used as a photoaffinity label for human lysosomal beta-hexosaminidase B (Hex B, EC 3.2.1.52) purified to apparent homogeneity from postmortal liver. [3H]-1-ATB-GalNAc behaved as an active site-directed inhibitor, which bound covalently to Hex B upon photolysis at 350 nm and resulted in 15% inactivation of enzyme activity. Up to 75% of the inactivation of Hex B was prevented by including the competitive inhibitor 2-acetamido-2-deoxy-D-glucono-1,5-lactone in the photoaffinity experiment. Incubation of [3H]-1-ATB-GalNAc with the enzyme followed by irradiation and subsequent separation of the three polypeptides composing the beta-subunit led mainly to labeling of the beta a-polypeptide. Subsequent proteolysis of beta a with trypsin and separation of the resulting peptides by high pressure liquid chromatography yielded one prominently labeled peptide fraction. Edman degradation resulted in the sequence E339ISEVFPDQFIHLGGD-EVEFK359. However, no modified amino acid was detected, indicating that the photoaffinity label was presumably bound to the peptide by a labile ester linkage. This was proven when the radiolabel was almost completely released from the peptide by treatment with aqueous ammonium hydroxide. Simultaneously, Glu-355 was converted into Gln-355, which is located within a region of Hex B that shows considerable homology with the alpha-subunit of human hexosaminidase A and other hexosaminidases from various species.

Affinity Labels↗
PubMed1996

Expression of the three alternative forms of the sphingolipid activator protein precursor in baby hamster kidney cells and functional assays in a cell culture system.

Sphingolipid activator proteins (SAPs) are non-enzymatic glycoproteins required for lysosomal degradation of various sphingolipids with short oligosaccharide chains by their respective exohydrolases. Four of these (SAP-A to SAP-D or saposins A to D) are derived from a common precursor by proteolytic processing. Alternative splicing of the SAP-precursor gene results in insertion of additional 6 or 9 bases of exon 8' or 8, respectively, into the SAP-B coding region of the transcribed mRNAs. To examine the features of the three different SAP-precursor proteins (prosaposins), the respective cDNAs were stably expressed in baby hamster kidney cells. Pulse-chase experiments with transfected cells and endocytosis studies on human fibroblasts showed that synthesis, transport, and maturation of all SAP-precursor led to formation of the four mature SAPs (SAP-A to SAP-D). In order to determine the biological function of the three different SAP-B isoforms, SAP-precursor-deficient human fibroblasts were loaded with recombinant SAP-precursor proteins with or without 2- and 3-amino acid insertions, respectively, purified from the medium of the baby hamster kidney cells. They were found to stimulate at nanomolar concentrations the turnover of biosynthetically labeled ceramide, glucosylceramide, and lactosylceramide. Since the physiological function of SAP-B is to stimulate the degradation of sulfatide by arylsulfatase A (EC 3.1.6.1) and globotriaosylceramide by beta-galactosidase (EC 3.2.1.23) loading studies with the respective exogenously labeled lipids on SAP-precursor-deficient fibroblasts were performed. Addition of different purified SAP-precursors to the medium of the lipid-loaded fibroblasts showed positive stimulation of the lipid degradation by all three SAP-B isoforms derived from the SAP-precursors. These findings establish that all three forms of the SAP-B can function as sulfatide/globotriaosylceramide activator.

Alternative Splicing↗
PubMed1996

Identification of domains in human beta-hexosaminidase that determine substrate specificity.

The lysosomal beta-hexosaminidases are dimers composed of alpha and beta subunits. beta-Hexosaminidase A (alphabeta) is a heterodimer, whereas hexosaminidase B (betabeta) and S (alphaalpha) are homodimers. Although containing a high degree of amino acid identity, each subunit expresses a unique active site that can be distinguished by a differential ability to hydrolyze charged substrates. The site on the beta-subunit primarily degrades neutral substrates, whereas the alpha-subunit site is, in addition, active against sulfated substrates. Isozyme specificity is also exhibited with glycolipid substrates. Among human isozymes, only beta-hexosaminidase A together with the GM2 activator protein can degrade the natural substrate, GM2 ganglioside, at physiologically significant rates. To identify the domains of the human beta-hexosaminidase subunits that determine substrate specificity, we have generated chimeric subunits containing both alpha- and beta-subunit sequences. The chimeric constructs were expressed in HeLa cells to screen for activity and then selected constructs were produced in the baculovirus expression system to assess their ability to degrade GM2 ganglioside in the presence of GM2 activator protein. Generation of activity against the sulfated substrate required the substitution of two noncontinuous alpha-subunit sequences (amino acids 1-191 and 403-529) into analogous positions of the beta-subunit. Chimeric constructs containing only one of these regions linked to the beta-subunit sequence showed either neutral substrate activity only (amino acids 1-191) or lacked enzyme activity entirely (amino acids 403-529). Neither the chimeras nor the wild-type subunits displayed activator-dependent GM2-hydrolyzing activity when expressed alone. However, one chimeric subunit containing alpha amino acids 1-191 fused with beta amino acids 225 to 556, when co-expressed with the wild-type alpha-subunit, showed activity comparable with that of recombinant beta-hexosaminidase A formed by the co-expression of the alpha- and beta-subunits. This result indicates that the beta-subunit amino acids 225-556 contribute an essential function in the GM2-hydrolyzing activity of beta-hexosaminidase A.

Amino Acid Sequence↗
PubMed1996

Biosynthesis, processing, and targeting of sphingolipid activator protein (SAP )precursor in cultured human fibroblasts. Mannose 6-phosphate receptor-independent endocytosis of SAP precursor.

Sphingolipid activator proteins (SAPs) are essential cofactors for the lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acidic exohydrolases. SAP-A, -B, -C, and -D derive from proteolysis of a 73-kDa glycoprotein, the SAP precursor. In the present publication, we studied the intracellular transport and the endocytosis of SAP precursor in human skin fibroblasts. Our data indicate that SAP precursor bears phosphate residues on noncomplex carbohydrate chains linked to the SAP-C and the SAP-D domain and sulfate residues on complex carbohydrate chains located within the SAP-A, -C, and possibly the SAP-D domain. Treatment of fibroblasts with either bafilomycin A1 or 3-methyladenine indicates that proteolytic cleavage of SAP precursor begins as early as in the late endosomes. To determine whether targeting of SAP precursor depends on mannose 6-phosphate residues, we analyzed the processing of SAP precursor in I-cell disease fibroblasts. In these cells nearly normal amounts of newly synthesized SAP-C were found, although secretion of SAP precursor was enhanced 2-3-fold. Moreover, SAP-C could be localized to lysosomal structures by indirect immunofluorescence in normal and in I-cell disease fibroblasts. Mannose 6-phosphate was not found to interfere significantly with endocytosis of SAP precursor. Normal fibroblasts internalized SAP precursor secreted from I-cells nearly as efficiently as the protein secreted from normal cells. To our surprise, deglycosylated SAP precursor was taken up by mannose 6-phosphate receptor double knock out mouse fibroblasts more efficiently than the glycosylated protein. We propose that intracellular targeting of SAP precursor to lysosomes is only partially dependent on mannose 6-phosphate residues, whereas its endocytosis occurs in a carbohydrate-independent manner.

Animals↗
PubMed1996

Molecular cloning and characterization of a full-length complementary DNA encoding human acid ceramidase. Identification Of the first molecular lesion causing Farber disease.

Human acid ceramidase ((AC) N-acylsphingosine amidohydrolase, EC 3.5. 1.23) hydrolyzes the sphingolipid ceramide into sphingosine and free fatty acid. Ceramide is an essential component of all sphingolipids and an important cell-signaling molecule. Moreover, an inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease. Human AC was purified from urine, and 117 amino acid residues were determined by microsequencing. Degenerative oligonucleotide probes were then constructed and used to screen for human fibroblast and pituitary cDNA libraries. Several partial cDNA clones were obtained, and two of these were combined to construct a full-length cDNA containing a 17-base pair (bp) 5'-untranslated sequence, a 1185-bp open reading frame encoding 395 amino acids, a 1110-bp 3'-untranslated sequence, and an 18-bp poly(A) tail. Transient expression of the full-length cDNA in COS-1 cells led to a 10-fold increase in AC activity. In addition, biosynthetic studies carried out in the transfected cells demonstrated that 13-kDa (alpha) and 40-kDa (beta) AC subunits were derived from a common 55-kDa precursor encoded by the full-length cDNA. This protein pattern was identical to that seen in normal human skin fibroblasts. A homoallelic point mutation (T222K) was also identified in the AC gene of a patient suffering from Farber disease, further confirming the authenticity of the full-length cDNA.

Acid Ceramidase↗
PubMed1997

cis-4-Methylsphingosine decreases sphingolipid biosynthesis by specifically interfering with serine palmitoyltransferase activity in primary cultured neurons.

The effect of six different structurally modified sphingosine analogues on biosynthesis of sphingolipids was studied in primary cultured murine cerebellar neurons. Treatment of cells with cis-4-methylsphingosine at micromolar levels resulted in a markedly decreased sphingolipid biosynthesis, whereas the other compounds examined, trans-4-methylsphingosine, cis-5-methylsphingosine, trans-5-methylsphingosine, cis-sphingosine, and 1-deoxysphingosine, inhibited sphingolipid biosynthesis less efficiently. The inhibition of sphingolipid biosynthesis by the various compounds was paralleled by a decrease of serine palmitoyltransferase activity in situ. For cis-4-methylsphingosine the inhibitory effect on serine palmitoyltransferase activity was shown to be concentration- and time-dependent. Half-maximal reduction of enzyme activity occurred after 24 h of treatment with 10 microM of the compound. The activity of other enzymes of sphingolipid biosynthesis as well as phospholipid and protein biosynthesis was not affected. Analysis of the sphingoid moiety of cellular sphingolipids suggests that the sphingosine analogues listed above were subject to degradation rather than being utilized as precursors for sphingolipid biosynthesis by cultured neurons. Except of 1-deoxysphingosine, the other five sphingosine analogues were shown to be substrates for sphingosine kinase in vitro. After 24 h of treatment of primary cerebellar neurons with the various sphingosine analogues the relative percentage of the respective intracellular 1-phosphate derivatives paralleled exactly the inhibitory effect on serine palmitoyltransferase activity observed when cells were treated with the unphosphorylated compounds. In contrast to the respective 1-phosphate derivatives of the other methyl-branched sphingosine analogues examined, cis-4-methylsphingosine 1-phosphate showed an intracellular accumulation suggesting a delayed turnover rate in cultured murine neurons for this compound. These results suggest that the inhibitory effect of the sphingosine analogues on serine palmitoyltransferase is mediated by their respective 1-phosphate derivatives and that the pronounced effect of cis-4-methylsphingosine is caused by a high intracellular concentration of cis-4-methylsphingosine 1-phosphate. cis-4-Methylsphingosine, in addition, caused drastic changes in cell morphology of primary cerebellar neurons, which were not observed when these cells were treated with one of the other sphingosine analogues examined.

Acyltransferases↗
PubMed1997

Characterization of ceramide synthesis. A dihydroceramide desaturase introduces the 4,5-trans-double bond of sphingosine at the level of dihydroceramide.

Ceramide (N-acylsphingosine) biosynthesis has been proposed to involve introduction of the 4,5-trans-double bond of sphingosine after synthesis of dihydroceramide (i.e. N-acylsphinganine). For the first time, the in vitro conversion of dihydroceramide to ceramide has been demonstrated using rat liver microsomes and N-[1-14C]octanoyl-D-erythro-sphinganine (st-H2Cer) and either NADH or NADPH as co-substrate; the apparent Km values for st-H2Cer and NADH were 340 and 120 microM, respectively. Molecular oxygen is required for enzymatic activity, and cyanide, divalent copper, as well as antibodies raised against cytochrome b5 are inhibitory, which suggests that this enzyme should be named dihydroceramide desaturase based on these similarities with the mechanism of delta9-desaturase (stearoyl-CoA desaturase). Factors that influenced the activity of dihydroceramide desaturase include the alkyl chain length of the sphingoid base (in the order C18 > C12 > C8) and fatty acid (C8 > C18); the stereochemistry of the sphingoid base (D-erythro- > L-threo-dihydroceramides); the nature of the headgroup, with the highest activity with dihydroceramide, but some (approximately 20%) activity with dihydroglucosylceramide, however); and the ability to utilize alternative reductants (ascorbic acid could substitute for a reduced pyridine nucleotide, but was inhibitory at higher concentrations). Dihydroceramide desaturase was inhibited by dithiothreitol, which suggests that it might be possible to alter ceramide synthesis by varying the thiol status of hepatocytes. Consistent with this hypothesis, when rat hepatocytes were cultured in varying concentrations of N-acetylcysteine (5 and 10 mM), there was a decrease in the relative incorporation of [14C]serine into [14C]ceramide. These studies have conclusively established the pathway of ceramide synthesis via desaturation of dihydroceramide and have uncovered several properties of this reaction that warrant further consideration for their relevance to both sphingolipid metabolism and signaling.

Animals↗
PubMed1997

Biosynthesis, processing, and intracellular transport of GM2 activator protein in human epidermal keratinocytes. The lysosomal targeting of the GM2 activator is independent of a mannose-6-phosphate signal.

The processing, intracellular transport, and endocytosis of the GM2 activator protein (GM2AP), an essential cofactor of beta-hexosaminidase A for the degradation of ganglioside GM2, was investigated in human epidermal keratinocytes. The GM2AP precursor is synthesized as an 18-kDa peptide, which is singly glycosylated, resulting in 22-kDa high mannose and 24-27-kDa complex glycoforms. A small portion of the 22-kDa form bears phosphomannosyl residues. About 30% of the GM2AP precursor is secreted during 12 h after synthesis, consisting almost exclusively of complex glycoforms. In a post-Golgi compartment, the intracellular remainder is converted to a 20-kDa mature form within 24 h, bearing a heavily trimmed N-glycan on a 17-kDa backbone. Interestingly, even nonglycosylated GM2AP is delivered to the lysosome, as shown by tunicamycin treatment and subcellular fractionation. Also, its endocytosis is independent of carbohydrate-linked signals and is even more effective for nonglycosylated GM2AP. We conclude that a mannose-6-phosphate-independent pathway for the lysosomal delivery of GM2AP exists in cultured human keratinocytes.

Biological Transport↗
PubMed1998

1-Methylthiodihydroceramide, a novel analog of dihydroceramide, stimulates sphinganine degradation resulting in decreased de novo sphingolipid biosynthesis.

1-Methylthiodihydroceramide (10 microM) decreased de novo ceramide biosynthesis by about 90% in primary cultured cerebellar neurons. Accordingly, de novo formation of sphingomyelin and of glycosphingolipids, all of which contain ceramide in their backbone, was reduced in a time- and concentration-dependent manner by up to 80%. Complex sphingolipid synthesis was restored upon addition of dihydroceramide or ceramide, in micromolar concentrations, to the culture medium, suggesting that none of the glycosyltransferases involved in glycosphingolipid biosynthesis is inhibited by this analog. Assays of the enzymes catalyzing sphinganine biosynthesis, as well as its N-acylation to form dihydroceramide, revealed that they were also not affected. In contrast, there was a 2.5-fold increase in the activity of sphinganine kinase. Reduction of de novo sphingolipid biosynthesis by 1-methylthiodihydroceramide is therefore due to its ability to deplete cells of newly formed free sphinganine. As a consequence of depletion of sphinganine levels, 1-methylthiodihydroceramide disrupted axonal growth in cultured hippocampal neurons in a manner similar to that reported for direct inhibitors of sphingolipid synthesis; thus, there was essentially no axon growth after incubation with 1-methylthiodihydroceramide between days 2 and 3, and co-incubation with short acyl chain analogs of ceramide (5 microM) antagonized the inhibition of growth. Interestingly, the D-erythro and the L-threo isomere were equally effective, but the corresponding free base as well as other structurally related compounds did not affect either sphingolipid biosynthesis or neuronal growth.

Animals↗
PubMed1999

Sphingolipid activator proteins are required for epidermal permeability barrier formation.

The epidermal permeability barrier is maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the major components of these multilayered membranes, derive in large part from hydrolysis of glucosylceramides mediated by stratum corneum beta-glucocerebrosidase (beta-GlcCerase). Prosaposin (pSAP) is a large precursor protein that is proteolytically cleaved to form four distinct sphingolipid activator proteins, which stimulate enzymatic hydrolysis of sphingolipids, including glucosylceramide. Recently, pSAP has been eliminated in a mouse model using targeted deletion and homologous recombination. In addition to the extracutaneous findings noted previously, our present data indicate that pSAP deficiency in the epidermis has significant consequences including: 1) an accumulation of epidermal glucosylceramides together with below normal levels of ceramides; 2) alterations in lipids that are bound by ester linkages to proteins of the cornified cell envelope; 3) a thickened stratum lucidum with evidence of scaling; and 4) a striking abnormality in lamellar membrane maturation within the interstices of the stratum corneum. Together, these results demonstrate that the production of pSAP, and presumably mature sphingolipid activator protein generation, is required for normal epidermal barrier formation and function. Moreover, detection of significant amounts of covalently bound omega-OH-GlcCer in pSAP-deficient epidermis suggests that deglucosylation to omega-OH-Cer is not a requisite step prior to covalent attachment of lipid to cornified envelope proteins.

Animals↗
PubMed2000

Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain.

The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.

Amides↗
PubMed2000

Mice expressing only monosialoganglioside GM3 exhibit lethal audiogenic seizures.

Gangliosides are a family of glycosphingolipids that contain sialic acid. Although they are abundant on neuronal cell membranes, their precise functions and importance in the central nervous system (CNS) remain largely undefined. We have disrupted the gene encoding GD3 synthase (GD3S), a sialyltransferase expressed in the CNS that is responsible for the synthesis of b-series gangliosides. GD3S-/- mice, even with an absence of b-series gangliosides, appear to undergo normal development and have a normal life span. To further restrict the expression of gangliosides, the GD3S mutant mice were crossbred with mice carrying a disrupted GalNAcT gene encoding beta1,4-N-acetylgalactosaminyltransferase. These double mutant mice expressed GM3 as their major ganglioside. In contrast to the single mutant mice, the double mutants displayed a sudden death phenotype and were extremely susceptible to induction of lethal seizures by sound stimulus. These results demonstrate unequivocally that gangliosides play an essential role in the proper functioning of the CNS.

Age Factors↗
PubMed2000

Interfacial regulation of acid ceramidase activity. Stimulation of ceramide degradation by lysosomal lipids and sphingolipid activator proteins.

The lysosomal degradation of ceramide is catalyzed by acid ceramidase and requires sphingolipid activator proteins (SAP) as cofactors in vivo. The aim of this study was to investigate how ceramide is hydrolyzed by acid ceramidase at the water-membrane interface in the presence of sphingolipid activator proteins in a liposomal assay system. The degradation of membrane-bound ceramide was significantly increased both in the absence and presence of SAP-D when anionic lysosomal phospholipids such as bis(monoacylglycero)phosphate, phosphatidylinositol, and dolichol phosphate were incorporated into substrate-bearing liposomes. Higher ceramide degradation rates were observed in vesicles with increased membrane curvature. Dilution assays indicated that acid ceramidase remained bound to the liposomal surface during catalysis. Not only SAP-D, but also SAP-C and SAP-A, were found to be stimulators of ceramide hydrolysis in the presence of anionic phospholipids. This finding was confirmed by cell culture studies, in which SAP-A, -C, and -D reduced the amount of ceramide storage observed in fibroblasts of a patient suffering from prosaposin deficiency. Strong protein-lipid interactions were observed for both SAP-D and acid ceramidase in surface plasmon resonance experiments. Maximum binding of SAP-D and acid ceramidase to lipid bilayers occurred at pH 4.0. Our results demonstrate that anionic, lysosomal lipids are required for efficient hydrolysis of ceramide by acid ceramidase.

Anions↗
PubMed2001

Degradation of membrane-bound ganglioside GM2 by beta -hexosaminidase A. Stimulation by GM2 activator protein and lysosomal lipids.

According to a recent hypothesis, glycosphingolipids originating from the plasma membrane are degraded in the acidic compartments of the cell as components of intraendosomal and intralysosomal vesicles and structures. Since most previous in vitro investigations used micellar ganglioside GM2 as substrate, we studied the degradation of membrane-bound ganglioside GM2 by water-soluble beta-hexosaminidase A in the presence of the GM2 activator protein in a detergent-free, liposomal assay system. Our results show that anionic lipids such as the lysosomal components bis(monoacylglycero)phosphate or phosphatidylinositol stimulate the degradation of GM2 by beta-hexosaminidase A up to 180-fold in the presence of GM2 activator protein. In contrast, the degradation rate of GM2 incorporated into liposomes composed of neutral lysosomal lipids such as dolichol, cholesterol, or phosphatidylcholine was significantly lower than in negatively charged liposomes. This demonstrates that both, the GM2 activator protein and anionic lysosomal phospholipids, are needed to achieve a significant degradation of membrane-bound GM2 under physiological conditions. The interaction of GM2 activator protein with immobilized membranes was studied with surface plasmon resonance spectroscopy at an acidic pH value as it occurs in the lysosomes. Increasing the concentration of bis(monoacylglycero)phosphate in immobilized liposomes led to a significant drop of the resonance signal in the presence of GM2 activator protein. This suggests that in the presence of bis(monoacylglycero)phosphate, which has been shown to occur in inner membranes of the acidic compartment, GM2 activator protein is able to solubilize lipids from the surface of immobilized membrane structures.

G(M2) Activator Protein↗
PubMed2001

CD95 signaling via ceramide-rich membrane rafts.

Clustering seems to be employed by many receptors for transmembrane signaling. Here, we show that acid sphingomyelinase (ASM)-released ceramide is essential for clustering of CD95. In vitro and in vivo, extracellularly orientated ceramide, released upon CD95-triggered translocation of ASM to the plasma membrane outer surface, enabled clustering of CD95 in sphingolipid-rich membrane rafts and apoptosis induction. Whereas ASM deficiency, destruction of rafts, or neutralization of surface ceramide prevented CD95 clustering and apoptosis, natural ceramide only rescued ASM-deficient cells. The data suggest CD95-mediated clustering by ceramide is prerequisite for signaling and death.

Apoptosis↗
PubMed2001

Human acid ceramidase: processing, glycosylation, and lysosomal targeting.

The biosynthesis of human acid ceramidase (hAC) starts with the expression of a single precursor polypeptide of approximately 53-55 kDa, which is subsequently processed to the mature, heterodimeric enzyme (40 + 13 kDa) in the endosomes/lysosomes. Secretion of hAC by either fibroblasts or acid ceramidase cDNA-transfected COS cells is extraordinarily low. Both lysosomal targeting and endocytosis critically depend on a functional mannose 6-phosphate receptor as judged by the following criteria: (i) hAC-precursor secretion by NH(4)Cl-treated fibroblasts and I-cell disease fibroblasts, (ii) inhibition of the formation of mature heterodimeric hAC in NH(4)Cl-treated fibroblasts or in I-cell disease fibroblasts, and (iii) blocked endocytosis of hAC precursor by mannose 6-phosphate receptor-deficient fibroblasts or the addition of mannose 6-phosphate. The influence of the six individual potential N-glycosylation sites of human acid ceramidase on targeting, processing, and catalytic activity was determined by site-directed mutagenesis. Five glycosylation sites (sites 1-5 from the N terminus) are used. The elimination of sites 2, 4, and 6 has no influence on lysosomal processing or enzymatic activity of recombinant ceramidase. The removal of sites 1, 3, and 5 inhibits the formation of the heterodimeric enzyme form. None of the mutant ceramidases gave rise to an increased rate of secretion, suggesting that lysosomal targeting does not depend on one single carbohydrate chain.

Ammonium Chloride↗