Plasma membrane production of ceramide fromganglioside GM3 in human fibroblasts
Rea Valaperta,* Vanna Chigorno,* Luisa Basso,* Alessandro Prinetti,*Roberto Bresciani,† Augusto Preti,† Taeko Miyagi,‡ and Sandro Sonnino*,1
Department of Medical Chemistry, Biochemistry and Biotechnology and Center of Excellence onNeurodegenerative Diseases, University of Milan, Segrate, Italy; †Department of Biomedical Sciencesand Biotechnology, University of Brescia, Brescia, Italy; and ‡Division of Biochemistry, ResearchInstitute, Miyagi Prefectural Cancer Center, Natori, Miyagi, Japan
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5077fje
SPECIFIC AIMS
Ceramide is a key lipid molecule necessary to regulatesome cellular processes, including apoptosis and celldifferentiation, and its production has been shown tobe due to sphingomyelin hydrolysis or acylation ofsphingosine. We investigated whether bioactive plasmamembrane ceramide can be produced as well fromgangliosides by detachment of sugar units due to theaction of membrane-bound glycosylhydrolases.
PRINCIPAL FINDINGS
1. Ceramide is produced from GM3 at the plasmamembrane in normal human fibroblasts
Figure 1, lane 1, shows the radioactive sphingolipidpattern of cells fed with GM3-containing radioactivesphingosine. After TLC separation, the catabolic prod-ucts lactosylceramide and ceramide were identified,together with SM obtained by recycling of sphingosine.To verify whether a part of the radioactive ceramideformed from GM3 administered to cells was producedin the plasma membrane, we fed cells with GM3 afterblocking lysosomal activity or endocytosis. Figure 1,lanes 2–4, shows that GM3-derived ceramide was pro-duced by cells, even after blocking lysosomal activity orendocytosis. These results suggest that the observedceramide is produced in the membrane concentration.
2. Overexpression of plasma membrane sialidaseNeu3 resulted in the increased ceramide productionfrom GM3 in the plasma membrane
The above described set of experiments were repeatedon cells overexpressing the plasma membrane sialidaseNeu3. Stable transfectants were established, expressinga C-terminally hemagglutinin tagged form of mousesialidase Neu3. In the transfected cells, the protein wasmainly localized on the plasma membrane, and siali-
dase activity was determined on natural substrates ac-tivities on GM3 and GD1a were 2 times higher thanmock in cells. Figure 1, lane 1a, shows that transfectedcells fed with GM3, produced lactosylceramide, cer-amide, and SM. Transfected cells produced about 3times more ceramide than in mock cells, as well as asimilar quantity of lactosylceramide. However, cer-amide but not lactosylceramide was formed by blockingthe cell lysosomal activity or endocytosis by transfectedcells fed with GM3 (Fig. 1, lanes 2a to 4a).
3. Overexpression of plasma membrane sialidaseNeu3 resulted in profound alteration in sphingolipidmetabolism and composition
The sphingolipid pattern of human fibroblasts andNeu3-transfected cells, as determined after feedingtritiated sphingosine to cells, lipid extraction, TLCseparation of the total lipid mixture and radioimagingare reported in Fig. 2. In agreement with previousfindings, GM3 and Gb3Cer are the main components ofthe sphingolipid mixture together with sphingomyelin,while ceramide is hardly detectable.
In Neu3 cells with comparison to normal fibroblasts,we observed a minor, but statistically significant, reduc-tion in the GM3 content, a minor increase of LacCer,and a dramatic decrease of Gb3Cer. In addition to this,ceramide was clearly present at higher levels in Neu3overexpressing cells, and we calculated a sixfold in-crease compared to mock cells. Despite the enzymeoverexpression and increase of activity, only a very smalldecrease in GM3 content was observed in stably trans-fected fibroblasts. Then we investigated the activity ofthe GM3 synthase SAT1. In stably transfected cells, the
1 Correspondence: Department of Medical Chemistry, Bio-chemistry and Biotechnology and Center of Excellence onNeurodegenerative Diseases, University of Milan, Via FratelliCervi 93, Segrate 20090 (Milan, Italy). E-mail: [email protected]
activity of SAT1 increased �150% compared to normalcells. The increase of enzyme activity was parallel to anincrease of SAT1 mRNA. This suggests that SAT1maximum activity did not change, but that more en-zyme was available. The higher availability of SAT1 couldexplain why the Neu3 overexpression in transfectedcells does not produce any change in GM3 content.
SAT1 was not the only sphingolipid metabolic en-zyme altered by Neu3 overexpression. A marked in-crease in the activity of �-galactosidase (9,655�888pmol/h/mg cell protein in Neu3-transfected cells vs.1,125�145 pmol/h/mg cell protein in mock cells) and�-glucosidase (64,045�4,589 pmol/h/mg cell proteinin Neu3-transfected cells vs. 16,334�1,234 pmol/h/mgcell protein in mock cells) was observed. A modestincrease in the activity of neutral sphingomyelinase(32.1�3.7 pmol/h/mg cell protein in Neu3-transfectedcells vs. 21.1�1.5 pmol/h/mg cell protein in mockcells) was measured. On the other hand, no statisticallysignificant differences were observed between Neu3-transfected cells and mock cells in the enzyme activitiesof Neu1 lysosomal sialidase, �-galactosidase, acidic, basic,and neutral ceramidases and acidic sphingomyelinase.
4. Overexpression of plasma membrane sialidase Neu3resulted a reduced cell growth and increased apoptosis
The growth rate of Neu3-transfected cells was highlyreduced. Neu3 overexpression caused a marked dimi-nution of [3H]thymidine incorporation, indicating in-hibition of cell proliferation. The effect of Neu3 over-expression on programmed cell death was alsoevaluated. In Neu3 stable transfected human fibro-blasts, the expression of the apoptosis-suppressing pro-
tein Bcl-2 was markedly reduced compared to mocktransfected cells, cytosolic cytocrome c was detectable,and caspase-3 was cleaved to its active fragment, indicatingthat an apoptotic pathway is executing in these cells.
CONCLUSIONS AND SIGNIFICANCE
Glycosphingolipids are components of the cell plasmamembranes where they participate in the organizationof lipid domains and modulate several aspects of thesignal transduction processes. Plasma membranes gly-cosylhydrolases could be the natural candidate formodifications of the cell surface glycolipids. Within themembrane-associated glycosylhydrolases, the mem-brane-bound sialidase Neu3 has been characterized.Neu3 has been cloned and then shown to be associatedwith sphingolipid-enriched membrane domains.
In this paper, we show the involvement of Neu3 andother membrane-associated glycosylhydrolases in pro-cessing gangliosides belonging to the plasma mem-branes of human fibroblasts in culture, namely GM3(Fig. 3).
The production at the cell surface of normal fibro-blasts of a minor amount of ceramide was clearly shownby feeding cells with GM3 ganglioside-containing radio-active sphingosine. GM3 was administered to cellsunder experimental conditions that allow gangliosidesto enter into the cell plasma membranes and to be-come indistinguishable from the endogenous com-pounds. A part of the radioactive GM3 entered in themetabolic pathway, and we observed the production ofsome LacCer, Cer, and SM (Fig. 1). Radioactive SM canbe formed only by recycling of sphingosine, and thisconfirms that GM3 reaches lysosomes where sphin-gosine is produced. To understand whether a part of
Figure 1. Radioactive lipids from mock Neu3 and cells fedwith radioactive GM3. Mock and Neu3-transfected cells (lane1) were treated with ammonium chloride 10 mM (lane 2) orchloroquine 50 �M (lane 3) or kept at 4°C (lane 4). Then thecells were fed with [3H]GM3. Lipids from mock and Neu3-transfected cells were extracted and separated by HPTLC.
Figure 2. Sphingolipids in Neu3 cells. Mock and Neu3-transfected cells were labeled with [3-3H]sphingosine, andthe total lipids were separated by HPTLC. Lanes 1 and 2,mock cells; lanes 3 and 4, Neu3-transfected cells.
1228 Vol. 20 June 2006 VALAPERTA ET AL.The FASEB Journal
catabolic process occurred also at the cell surface, wemaintained cells under conditions that do not allowlysosomal activity or block endocytosis. Figure 1 showsthat under these experimental conditions, no lactosyl-ceramide and SM could be observed, while ceramidewas produced. Neu3 overexpressing fibroblasts withdouble Neu3 activity, subjected to the above experi-ments gave similar qualitative results but producedceramide in much higher quantities, about 3 timesmore under all the experimental conditions. Theseresults demonstrate that: 1) the experimental condi-tions used to block lysosomal activity and endocytosisare effective to prevent GM3 from reaching lysosomes;2) the LacCer observed under normal experimentalconditions is produced in the lysosomes; 3) blockinglysosomal activity and endocytosis, ceramide is pro-duced at the plasma membranes; and 4) a part ofceramide produced under normal conditions is pro-duced at the cell surface.
The production of ceramide at the plasma mem-branes of mammal cells has been always associated withthe activity of sphingomyelinase on sphingomyelin. Forthe first time, we show that ceramide is produced at theplasma membrane starting from glycosphingolipids.
We did not observe the production of LacCer andGlcCer, blocking cell lysosomal activity and endocytosis,after administration to cells of radioactive GM3. Thissuggests that both galactosidase and glucosidase areavailable at the plasma membrane, and the rates ofdetachment of neutral sugars are higher than that ofsialic acid. Surprisingly, in Neu3 cells, in which theproduction of Cer is very high after administration ofGM3, no radioactive neutral glycolipids could be ob-served blocking cell lysosomal activity and endocytosis.This could suggest that, together with the increase ofsialidase activity, an increase of the activity of mem-brane-associated galactosidase and glucosidase couldoccur. Indeed, we found that the total cell galactosidase
and glucosidase activities were highly increased inNeu3-overexpressing cells.
Together with the increased production of ceramide,many biochemical events were strongly modified inNeu3 cells. The increased sialidase activity was notfollowed by a parallel decrease of the cell GM3 content.As shown in Fig. 2, the GM3 cellular contents in controland Neu3 cells are only slightly different, which appearsin contrast with the high content of ceramide in Neu3cells. Surprisingly, we found that the increase of siali-dase content and activity occurred together with acorresponding increase of the content and activity ofSAT1 (the sialyltransferase known also as GM3 syn-thase). Thus, some plasma membrane GM3 disappearsas it becomes a substrate for the cell surface hydrolyticprocess GM33 LacCer3 GlcCer3 Cer, and a similarquantity is substituted by neosynthesized GM3. Thisexplains the loss of Gb3Cer in Neu3-overexpressingcells. Gb3Cer is formed by addition of an �-galactose toLacCer. Probably, LacCer is not available to the �-galac-tosyltransferase, being used to produce GM3 by SAT1.
A direct correlation between the increase of mem-brane-bound sialidase Neu3 and the increase of otherenzymes of the glycosphingolipid metabolism is not soevident. At least as far as it concerns SAT1, we foundthat both the protein and the activity increased. Glyco-sphingolipids, with cholesterol, are components ofplasma membrane lipid domains that are believed tocontain the switch of several functional events. There issolid information to suggest that changes of the com-position and organization of these domains can modu-late the functional events. We had dramatic changes ofthe glycosphingolipid pattern after Neu3 cell overex-pression (Fig. 2). This could be responsible of signalsthat are able to modify the contents of the enzymes forthe glycosphingolipid catabolism. In addition to this,the new glycosphingolipid pattern could be responsiblefor the activation of the membrane-associated galacto-sidase and glucosidase throughout direct glycolipid-protein interactions. Glycosphingolipid ability to mod-ulate membrane-bound enzymes has been well studiedand detailed in the past.
The biochemical events occurring with the increaseof Neu3 activity lead to cell death. This is not surpris-ing, due to the increase of ceramide. Ceramide hasbeen shown to participate in some way in the activationof the apoptotic process and to be released fromplasma membrane sphingomyelin by a plasma mem-brane-associated sphingomyelinase.
Twenty-five years ago, it was suggested that polysialo-gangliosides, after biosynthesis in the Golgi and trans-port to the plasma membranes, could be substrates fora membrane-associated sialidase that could regulate thecorrect ratio between gangliosides at different sialicacid content and neutral glycolipids. Our results sug-gest that the role of Neu3, the membrane-associatedsialidase, is to correctly maintain in the plasma mem-branes the ganglioside pattern and content necessaryfor the cell communications.
Figure 3. Representation of the plasma membrane-associatedsialidase Neu3 overexpression effects in human fibroblasts.The Neu3 overexpression in these cells determined severalcell signals that caused up-regulation of enzymes, such as theplasma membrane-associated �-galactosidase and �-glucosi-dase and the intracellular GM3 synthase. The �-galactosidaseand �-glucosidase activities produced ceramide from gangli-oside GM3 in the plasma membrane. The high concentrationof ceramide acts on two important cells mechanisms: reduc-tion of cell proliferation and induction of apoptosis. Theincreased GM3 synthase expression maintains a constantconcentration of GM3.
1229CERAMIDE FROM GM3 AT THE CELL SURFACE
The FASEB Journal • FJ Express Full-Length Article
Plasma membrane production of ceramide fromganglioside GM3 in human fibroblasts
Rea Valaperta,* Vanna Chigorno,* Luisa Basso,* Alessandro Prinetti,*Roberto Bresciani,† Augusto Preti,† Taeko Miyagi,‡ and Sandro Sonnino*,1
Department of Medical Chemistry, Biochemistry and Biotechnology and Center of Excellence onNeurodegenerative Diseases, University of Milan, Segrate, Italy; †Department of Biomedical Sciencesand Biotechnology, University of Brescia, Brescia, Italy; and ‡Division of Biochemistry, ResearchInstitute, Miyagi Prefectural Cancer Center, Natori, Miyagi, Japan
ABSTRACT Ceramide is a key lipid molecule neces-sary to regulate some cellular processes, includingapoptosis and cell differentiation. In this context, itsproduction has been shown to occur via sphingomyelinhydrolysis or sphingosine acylation. Here, we show thatin human fibroblasts, plasma membrane ceramide isalso produced from ganglioside GM3 by detachment ofsugar units. Membrane-bound glycosylhydrolases havea role in this process. In fact, the production ofceramide from GM3 has been observed even underexperimental conditions able to block endocytosis orlysosomal activity, and the overexpression of theplasma membrane ganglioside sialidase Neu3 corre-sponded to a higher production of ceramide in theplasma membrane. The increased activity of Neu3 wasparalleled by an increase of GM3 synthase mRNA andGM3 synthase activity. Neu3-overexpressing fibroblastswere characterized by a reduced proliferation rate andhigher basal number of apoptotic cells in comparisonwith wild-type cells. A similar behavior was observedwhen normal fibroblasts were treated with exogenousC2-ceramide.—Valaperta, R., Chigorno, V., Basso, L.,Prinetti, A., Bresciani, R., Preti, A., Miyagi, T., andSonnino, S. Plasma membrane production of ceramidefrom ganglioside GM3 in human fibroblasts. FASEB J.20, E450–E461 (2006)
Plasma membrane sphingolipids are a class of lipidmediators. Following various stimuli, the production oftheir catabolic fragments ceramide, sphingosine, andsphingosine-1-phosphate, leads to cell proliferation,cell differentiation, or apoptotic cell death, cell con-traction, retraction, and migration (2). Sphingomyelinis considered the precursor of the sphingoid frag-ments (3). Ceramide glycanase, the enzyme thatcatalyzes the one-step release of the oligosaccharidechain from glycosphingolipids with the liberation offree ceramide, has been described in bacteria andlow eukaryotes (4).
Gangliosides, sialic acid containing glycosphingolip-ids (1), have been described to participate to the
cell-to-cell signaling process and to the process of signaltransduction through the membrane (5). Sialidasesremove sialic acid residues from sialocompounds (6).The membrane-associated sialidase Neu3 (7–9) triggersselective ganglioside desialylation in neuroblastomacells, thus modulating cell growth, differentiation, andneuritogenesis (10–11). Neu3 has been shown to re-lease sialic acid from sialoglycolipids of neighboringcells through cell-to-cell interactions in COS-7 cells (9).A specific role of Neu3 has been proposed in cancer(12–13), in which high concentrations of the enzymewould maintain high levels of lactosylceramide consid-ered an antiapoptotic compound, thus allowing cellproliferation.
Here, we report the first direct evidence that theplasma membrane-associated sialidase Neu3, in coordi-nation with other cell surface glycosylhydrolases, mod-ulates cell proliferation and cell apoptosis, by regulat-ing the production of plasma membrane ceramidefrom gangliosides in cultured human fibroblasts.
MATERIALS AND METHODS
High-performance silica gel thin-layer plates (HPTLC Kiesel-gel 60, 10 �10 cm) were purchased from Merck GmbH(Milan, Italy). Vibrio cholerae sialidase was from Sigma (Milan,Italy). The methylumbelliferyl derivatives of �-Gal and �-glu-cose were from Sigma. [6-3H]-acetyl-D-mannosamine (20Ci/mmole) was from American Radiolabeled Chemicals (St.Louis, MO) and [3H]thymidine (12 Ci/mmole) and[3-3H]sphingosine (23.1 Ci/mmole) were from Perkin Elmer(Norwalk, CT).
Sphingosine was prepared from cerebroside (14); C2-cer-amide was prepared by acetylation of sphingosine using aceticanhydride in methanol; [1-3H]sphingosine (2.0 Ci/mmole) wasprepared from sphingosine (15). Ganglioside GM3 was pre-pared from bovine brain GM1 (16). The preparation ofisotopically labeled [3-3H(sphingosine)]GM3 (specific radioac-
1 Correspondence: Department of Medical Chemistry, Bio-chemistry and Biotechnology and Center of Excellence onNeurodegenerative Diseases, University of Milan, Via FratelliCervi 93, Segrate 20090 (Milan, Italy). E-mail: [email protected]
tivity, 2.3 Ci/mmole) and [3-3H(sphingosine)]GD1a (specificradioactivity, 1.3 Ci/mmole) have been described in detail(17). Bovine brain SM (Avanti Polar Lipids, Alabaster, AL)was labeled as for GM3. LacCer was prepared by mild acidichydrolysis of GM1 (18). [3-3H(sphingosine)]LacCer was pre-pared from [3-3H(sphingosine)]GM3 (specific radioactivity,2.3 Ci/mmole) by treatment with vibrio cholerae sialidase (18).[1-3H(sphingosine)]Cer (specific radioactivity, 2.2 Ci/mmole)was prepared by direct acylation of [1-3H(sphingosine)] (spe-cific radioactivity, 2.0 Ci/mmole) with palmitic anhydride.Radioactive sphingolipids were extracted from cells fed with[1-3H]sphingosine, purified, characterized as described pre-viously and used as chromatographic standards (19).
Normal human skin fibroblasts were cultured and propa-gated as described previously (20) in 100-mm dishes(0.42�0.10 mg cell protein/dish), using 10% FCS-EMEM andused for the experiments when confluent. Mock and Neu3cells were cultured in 100-mm plastic dishes containing 15%FCS-EMEM.
Expression of mouse ganglioside sialidase cDNA in normalhuman fibroblasts
The plasmid construct (PC) DNA-Neu3HA (21) was digestedwith EcorI restriction enzyme. The product of the digestionwas cloned into pIRES-neo eukaryotic expression vector. Thepresence, orientation and fidelity of the cDNA in the vectorwere confirmed by DNA sequencing. Human fibroblasts weretransfected by nucleofection with Nucleofector System (In-strumentation Laboratory, Barcelona, Spain), with the emptyexpression vector or the expression vector containing themouse ganglioside sialidase cDNA by the following proce-dures. Cells that were washed twice with PBS were resus-pended in Nucleofector solution, according to manufactur-er’s instructions, at a final concentration of 5 � 105 cells/100�l. The suspension was added to sterile cuvettes with 5 �g ofplasmid DNA. The cuvette was placed on ice for 10 min andelectrophorated using specific program by InstrumentationLaboratory. The cuvette was placed on ice for an additional10 min and cells were cultured in 4 ml of medium in 35-mMdishes. 24 h after nucleofection, selection started in growthmedium containing 200 �g/ml neomycin. Following 5 wk ofselection, few colonies were subcloned and expanded.
Total RNA was isolated from mock and Neu3 transfected cellsusing TRIZOL reagent, followed by RNase free DNase Itreatment according to the manufacturer’s instructions.cDNA was prepared from 1 �g total RNA using cDNAsynthesis kit for real-time polymerase chain reaction (RT-PCR; Invitrogen, Carlsbad, CA), according to the manufac-turer’s protocol. Fifty nanograms of each cDNA were dilutedto a volume of 25 �l PCR mix (Premix Taq, Ex TaqTM
R-polymerase chain reaction Version, Takara Bio Inc., Shiga,Japan) containing primers and probe.
The analysis of human endogenous Neu3 in mock and inNeu3-transfected cells was performed using Smart CyclerSystem (CEPHED).
The sequences and final concentrations of primers in thereaction mixture for human sialidase Neu3 were: 0.3 �Mforward 5�-CCTGAAGCCACTGATGGAA -3�, 0.3 �M reverse5�-TTCCTGCCTGACACAATCTG-3� and 0.2 �M probe FAM-5�-CCACACTACCGGGGCATCGG-3�-tamra (GenBankTM ac-cession no. NM_006656). For amplification, the initial dena-turation at 95°C for 10 s (1 cycle) was followed by a secondstep at 95°C for 10 s, 60°C for 15 s (40 cycles). To normalize
data obtained, the �-actin expression was used as internalcontrol. The sequences of forward primer was 5�-CGACAG-GATGCAGAAGGAG-3� and reverse primer was 5�-ACATCT-GCTGGAAGGTGGA-3�.
The relative expression of endogenous Neu3 in Neu3transfected cells compared to mock cells was normalized tothe expression of human �-actin and was calculated byequation 2-��Ct where Ct is the cycle threshold and ��Ct �(Ct
Ct�-actin)mock cells. To demonstrate that amplification effi-
ciency of Neu3 and �-actin were approximately equal, �Ctwere determined using 5, 10, 25, 50 and 100 ng of totalRNA. After the amplification by real-time PCR the productswere loaded on gel electrophoresis.
RNA isolation and RT-PCR analysis
Total RNA was isolated by the single-step acid-guanidineisothiocyanate-phenol-chloroform extraction method, ac-cording to the manufacturer’s instructions. One microgramof RNA was treated with 1 U of RNase-free DNase (Invitro-gen) for 15 min at room temperature to remove any possibleDNA contamination. Complementary DNA (cDNA) was syn-thesized with 4 �g of total RNA using SuperScriptTM IIIReverse Transcriptase (RT; Invitrogen) in a final 20-�l reac-tion vol.
For PCR amplification of mouse Neu3-hemagglutinincDNA we used a forward primer 5�-GACTTGGTGGCGTGTT-TGTT-3� and reverse primer 5�-TTAAGCGTAATCCGGAA-CATC-3� (GenBankTM accession no. NM_016720). Reversesequence primer is localized on tag hemagglutinin (HA); forthe GM3 synthase cDNA, we used a forward primer 5�-AATGGCGCTGTTATTTGAGC-3� and reverse primer 5�-CT-GGCAAGAGTTCCAAGAGG-3� (GenBankTM accession no.AY152815) and for the Gb3Cer synthase cDNA we used aforward primer 5�-TTCTCAAGAACCTGCGGAAC-3� and re-verse primer 5�-GATCCAGCCGTTGTAGTGGT-3� (GenBankaccession no. NM_017436). As in control, housekeeping gene�-actin cDNA was measured at the same time. Thirty-fivecycles were performed at 94°C (denaturation), 58°C (anneal-ing) and 72°C (elongation). The PCR products were sub-jected to electrophoresis in 1,2% (w/v) agarose gel and werevisualized by UV after ethidium bromide staining.
Indirect immunofluorescence
Mock and Neu3-transfected cells, grown in slide culturechamber, were briefly washed with PBS and fixed for 10 minwith 4% paraformaldehyde in PBS. For permeabilization,cells were incubated for 15 min in the presence of 0.5%Triton X-100 in PBS for 2 min. Cells were then washed twicefor 5 min with PBS and incubated for 1 h with mouseanti-hemagglutinin (1: 200, Sigma) or with a commercialmonoclonal antibody for �6�1-integrin (1:200, Serotec, Ra-leigh, NC), in PBS containing 1% BSA. Samples were thenwashed twice with PBS for 10 min and incubated withCy2-conjugated donkey antimouse IgG (1:200, Jackson Immu-noResearch Laboratories, West Grove, PA) for detection ofthe Neu3-hemagglutinin and Cy3-conjugated donkey anti-mouse IgG (1:200, Jackson ImmunoResearch Laboratories)for detection of the �6�1-integrin, in PBS containing 1% BSA.After two washes with PBS samples were embedded withfluorescence mounting medium (DAKO, Glostrup, Den-mark). Laser confocal analysis was performed with Bio-Rad1024 system (Bio-Rad Laboratories, Hercules, CA) and im-ages were processed with Adobe Photoshop software.
E451CERAMIDE FROM GM3 AT THE CELL SURFACE
Cell proliferation assay
5 � 105 cells were grown in 35-mm Falcon dishes andincubated with 1 �l medium containing 0.5 �Ci of [3H]thy-midine. After 1.5, 6, 20, and 48 h at 37°C, cells were harvestedwith 500 �l PBS, treated with 500 �l trichloroacetic acid 20%,shaken for 30 min at 4°C and centrifuged at 13,000 rpm for15 min. After the removal of the supernatant, 500 �l of 20%trichloroacetic acid were added to the pellets, and mixturestreated as before. After 1 h at 37°C, 50 �l of 0.4 M HCl wereadded to the reaction mixtures that were analyzed for radio-activity content.
Determination of cell apoptosis markers
Cell homogenates from mock and stable transfected cells (35�g of cell protein) were analyzed by SDS/PAGE gel electro-phoresis, proteins were transferred to PVDF membranes, andthe presence of Bcl-2 and caspase-3 were assessed by immu-noblotting with specific antibodies, followed by reaction withperoxidase-conjugated secondary antibody (Ab) and en-hanced chemiluminescence detection.
Expression of cytosolic cytochrome c was determined asdescribed previously (22).
Cell treatments with C2-ceramide and DNA fragmentationassay
Mock and Neu3-transfected cells were plated in 96-well plasticplates (104 cells/well) and cultured for 48 h. Then the cellswere incubated for 6, 12, and 24 h with 25 � 106 MC2-ceramide. After incubation, DNA fragmentation was inves-tigated using a cell death detection ELISAPLUS kit (RocheDiagnostics, Munich, Germany), according to the manufac-turer’s protocol. Each measurement was done in triplicateand the apoptotic index defined by the quantification ofmono- and oligonucleosomes. The optical density405 is nor-malized to the total milligrams protein content of the sampleused in the assay.
Metabolic tritium labeling of cell sphingolipids
Mock and transfected cells (48 h after seeding) were fed 3 �108 M [3-3H]sphingosine (2 ml/dish). After a 2-h pulse, themedium was removed and replaced with fresh mediumwithout radioactive sphingosine, and cells were incubated for48 h (chase), allowing metabolic radiolabeling of all sphin-golipids (including ceramide, SM, neutral glycolipids, andgangliosides) (19).
Metabolic tritium labeling of cell sialoglycoproteins
Mock and transfected cells (48 h after seeding) were fed2.25 � 107 M [6-3H]acetyl-D-mannosamine (5 ml/dish) inculture medium for 24 h and then chased for 24 and 48 h.After the chase, the cells were washed twice with PBS andresuspended in sterile water. 30 �g of cell proteins wereseparated by 10% SDS-PAGE and blotted to a PVDF mem-brane. Radioactive proteins were visualized by radioimaging.
Lipid extraction and analysis
Cells were harvested in ice-cold water (2 ml) by scraping witha rubber policeman, and lyophilized. Lipids were extractedtwice with chloroform/methanol/water 2:1:01 by volume(first extraction, 1.5 ml; second extraction, 0.25 ml). Totallipid extracts were analyzed by HPTLC performed with the
solvent system chloroform/methanol/0.2% aqueous CaCl2,55:45:10 (v/v), followed by radioactivity imaging and radio-activity quantification. The identity of radioactive lipids wasassessed by comparison with standard lipids.
Enzyme activities
Activity of GM3 synthase, ceramidase, �-galactosidase, siali-dase and sphingomyelinase were determined in cell homog-enates. Negative controls were performed using heat-inacti-vated cell homogenates (100°C for 3 min). The enzymaticreactions were stopped by adding chloroform/methanol(2:1) and analyzed by HPTLC using the solvent systemchloroform/methanol/water, 55:20:3 by volume or 55:45:10chloroform/methanol/0.2% aqueous CaCl2. Separated ra-dioactive lipids were detected and quantified by radioactivityimaging.
GM3 synthase activity was assayed as described previously ina cell-free assay using [3-3H(sphingosine)]LacCer as a substrate(23).
To perform ceramide assay, cell homogenates were pre-pared as described for the GM3 synthase assay (23). Theactivity of ceramidases was assayed as described previouslyusing [1-3H(sphingosine)]Cer as a substrate (23–24).
�-Galactosidase, �-glucosidase and �-galactosidase assayswere performed on cells. Then cells washed twice with PBS,suspended in sterile water, and pelleted. Activities on artificialsubstrates were determined as reported (25).
�-Galactosidase activity was determined on the naturalsubstrate. In each reaction tube containing 0.1 ml of sodiumtaurocholate (1 mg/ml in C/M 2/1) and 50 �g of thesubstrate [3-3H(sphingosine)]LacCer (corresponding to 1,35
Figure 1. Radioactive lipids from Neu3 cells fed with radioac-tive GM3. Mock (lanes 1 to 4) and Neu3-transfected (lanes 1ato 4a) cells were treated with 10 mM ammonium chloride(lanes 2 and 2a) or 50 �M chloroquine (lanes 3 and 3a) or keptat 4°C (lanes 4 and 4a) for 30 min in complete EMEM. Lanes1 and 1a are control cells (cells not submitted to ammoniumchloride, chloquine or 4°C treatments). Then the cells werefed with 4.5 � 106 M [3H]GM3 (in the presence of 10 mMammonium chloride, 50 �M chloroquine, or maintained at4°C) for 12 h. Lipids from mock and Neu3-transfected cellswere extracted and separated by HPTLC using the solventsystems: chloroform/methanol/0.2% aqueous CaCl2 55:45:10by vol.
E452 Vol. 20 June 2006 VALAPERTA ET AL.The FASEB Journal
nCi), from a stock solution in chloroform/methanol 2/1(v/v), were dried under nitrogen flow. To this mixture, 0.1 mlof sterile water was added and the tubes were sonicated.�-Galactosidase activity was assayed by adding 0.2 ml of 0.2 Msodium citrate buffer (pH 4.8) containing 20 mM NaCl, 0.1ml of enzyme source (containing 350 �g cellular protein) ina total reaction volume of 400 �l. Incubation was carried outat 37°C for 2 h.
Sialidase assays were performed on mock and stable Neu3fibroblasts. Then, they were washed twice with PBS, sus-pended in sterile water, and pelleted. The membrane boundNeu3 and the lysosomal Neu1 were assayed as describedpreviously (26), using both GD3 and GD1a gangliosides.
For the sphingomyelinase assays, cell homogenates wereprepared in 150 mM sodium cacodylate-HCl buffer, pH 6.6(20 mg of cell protein/ml) with protease inhibitors (2 mM4-(2-aminoethyl)benzenesulfonyl fluoride, 0.0016 mM aproti-nin, 0.044 mM leupeptin, 0.08 mM bestatin, 0.03 mM pepsta-tin A, 0.028 mM E-64) (Sigma) and 0.2% Triton-X 100. Theactivity of acid and neutral sphingomyelinase was assayed asfollows. In each reaction tube, 25 �l of 0.2% Triton-X100(v/v) in chloroform/ methanol (2:1) was mixed with 0.74 �Mof [3H]sphingomyelin (corresponding to 13.5 nCi) from a
stock solution in chloroform/ methanol (2:1) and driedunder N2. To this mixture, 25 �l of 250 mM sodium acetatebuffer, pH 5.2 (for the assay of the acidic enzyme) or 25 �l of40 mM HEPES, 5 mM MgCl2, pH 7.4 (for the assay of neutralenzyme) and 25 �l of cell homogenate (containing 100 �g ofprotein for the acidic sphingomyelinase and 10 �g for theneutral enzyme) were added in a total reaction volume of 50�l. The incubation was performed at 37°C for 2 h withcontinuous shaking.
Activity of Neu3 and membrane-associatedsphingomyelinase on [3-3H(sphingosine)]GM3 and of [3-3H(sphingosine)]SM-administered to cells
[3-3H(sphingosine)]GM3 and [3-3H(sphingosine)]SM were ad-ministered to cells, and their fate was determined underconditions that prevent lysosomal catabolism. To do this, cellswere kept at 36°C with 50 �M chloroquine or 10 mMammonium chloride or kept at 4°C for 30 min in EMEM.After removal of the medium and rapid washing of cells withEMEM, 2 ml of the medium containing the radioactive lipidwere added to each dish, and the cells were incubated at 36°Cin the presence of 50 �M chloroquine or 10 mM ammoniumchloride, or at 4°C for 12 h. Medium containing tritium-labeled GM3 or SM was prepared as follows. The tritiatedsphingolipid, dissolved in propan-1-ol/water, 7:3 (v/v), waspipetted into a sterile tube and dried under a nitrogenstream. The residue of [3-3H(sphingosine)]GM3 was solubi-lized in Eagle’s minimum essential medium (EMEM) contain-ing 10% FCS, 1% glutamine, 1% penicillin/streptomycin, toobtain a GM3 concentration of 4.5 � 106 M. The residue of[3-3H(sphingosine)]SM was solubilized in the above mediumwithout FCS to obtain a final concentration of 6 � 106 M.
At the end of incubation, cells were washed four times withcomplete EMEM, twice with PBS and scraped off with waterby mean of a rubber policeman. Samples were lyophilizedand submitted to lipid extraction resulting in a delipidizedpellet and a total lipid mixture. Radioactive lipids wereseparated by HPTLC (1000 dpm/lane), and analyzed byradioimaging (48 h acquisition) using a Beta-Imager 2000Instrument (Biospace, Paris, France).
Other analytical methods
The radioactivity associated with lipids, with total lipid ex-tracts and with DNA was determined by liquid scintillationcounting.
The protein assays were carried out according to Lowry(27), using BSA as the reference standard or using thebicinchoninic acid protein assay kit.
Figure 2. mRNA expressions of mouse Neu3-hemagglutininand of endogenous Neu3 in mock and Neu3-transfected cells.A) Expression levels of mRNA of mouse Neu3-hemagglutininby RT-PCR in mock (lane 1) and Neu3 cells (lane 2). �-actinmRNA expression was measured as internal control in mock(lane 3) and in Neu3 cells (lane 4). B) Gel electrophoresisexpression of endogenous Neu3 mRNA in mock (lane 1) andNeu3-hemagglutinin-transfected cells (lane 2) after semiquan-titative real-time PCR. �-actin mRNA expression was mea-sured as an internal control in mock (lane 3) and in Neu3cells (lane 4).
TABLE 1. Semi-quantitative Real-Time PCR data of endogenous Neu3 in Neu3-HA transfected and mock cells
The relative expression of endogenous Neu3 in Neu3-HA transfected cells was compared to that in mock cells. The expression wascalculated after normalization to the internal control �-actin by the equation 2-� � Ct, where � � Ct � (Ctendogenous Neu3 Ct�-actin)Neu3 cells (Ctendogenous Neu3 Ct�-actin)mock cells. Ct, cycle threshold. The absolute values of the slope of log RNA amount versus �Ct were 0.033 and 0.045,for Neu3-HA and Mock cells, respectively, as determined using 5, 10, 25, 50, and 100 ng total RNA.
E453CERAMIDE FROM GM3 AT THE CELL SURFACE
RESULTS
Figure 1, lane 1, shows the radioactive sphingolipidpattern of cells fed with radioactive GM3. After TLCseparation, the catabolic products lactosylceramide andceramide were identified, together with some SM. Thepresence of radioactive SM has been shown to beassociated with the very rapid recycling of lysosomalsphingosine produced in the catabolism of adminis-tered ganglioside (19).
To verify whether a part of the radioactive ceramideformed from GM3 administered to cells was producedat the plasma membrane, we fed cells with GM3 also inthe presence of chloroquine and ammonium chloride,which are known to block lysosomal activity (28–30), orat 4°C, a condition known to block endocytosis (31–33). Figure 1, lanes 2 to 4, shows that ceramide andtraces of lactosylceramide, but not SM, are producedfrom GM3 in fibroblasts under experimental condi-tions which are able to block lysosomal activity orendocytosis. The lack of SM production is good proofthat GM3 in the presence of chloroquine or ammo-nium chloride does not actually reach lysosomes or is inany way metabolized in the lysosomal compartment. Inabsence of lysosomal catabolism of GM3, sphingosinecannot be formed and recycled for the biosynthesis ofSM. These results suggest that the observed ceramide isproduced in the membrane.
The above described set of experiments was repeatedon cells overexpressing the plasma membrane sialidaseNeu3 to examine the possible increase in production ofceramide, parallel to the increase of membrane boundsialidase activity.
A full-length cDNA encoding mouse sialidase Neu3cDNA, having the HA tag at the C-terminal was clonedand inserted into a mammalian expression vector.Transfected fibroblasts did not show any morphologicalchange with respect to control cells. Positive cloneswere determined on the basis of Neu3-hemagglutininmRNA levels by RT-PCR. Figure 2A shows the expres-sion levels of mRNA of mouse Neu3-hemagglutinin inNeu3-hemagglutinin-transfected cells. Of course, as ex-pected, in mock cells, we did not find any Neu3-hemagglutinin product. To analyze hypotheticalchanges of endogenous Neu3 after cell transfectionwith mouse Neu3-hemagglutinin mRNA, we performedsemiquantitative real-time PCR. Figure 2B and Table 1shows that any change of the Neu3 content occurred inNeu3-hemagglutinin transfected cells with respect tomock cells.
Figure 3 shows that the protein was mainly localizedon plasma membrane in transfected cells. These cellshad sialidase activity 2 times higher than mock cells, asdetermined on natural substrates GM3 and GD1a (Ta-ble 2).
Figure 1, lane 1a, shows that transfected cells fed withGM3, produced lactosylceramide, ceramide, and SM.Transfected cells produced a high quantity of cer-amide, about 3 times that produced by mock cells, anda similar quantity of lactosylceramide (Table 3). In-
stead, ceramide, but not lactosylceramide, was formedblocking the cell lysosomal activity with chloroquineand ammonium chloride, or maintaining cells at 4°C toblock endocytosis by transfected cells fed with GM3(Fig. 1, lanes 2a to 4a). These results obtained feedingNeu3 cells with GM3 confirm that some ceramide isproduced at the plasma membrane, by involvement ofNeu3, whereas the observed lactosylceramide is largelyproduced in lysosomes.
Neutral glycosphingolipids could not be observed
Figure 3. Immunofluorescence detection of Neu3. A) Stain-ing of nonpermeabilized Neu3 cells with anti-hemagglutinin.B) Staining of permeabilized Neu3 cells with anti-hemagglu-tinin showing a predominant plasma-membrane distribution.C) Staining of permeabilized Neu3 cells with a monoclonalmouse anti �6�1-integrin Ab. D) Merged image of B and C.Specimens were analyzed by using MRC-1024 confocal lasersystem (Bio-Rad), and images were processed with AdobePhotoshop software.
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blocking the lysosomal ganglioside catabolism. Thissuggests that in the membrane, detachment of neutralsugars is faster than that of sialic acid. In addition,intermediate catabolytes could not even be observed inNeu3 cells in which the sialidase activity is quite high.This would suggest that in Neu3 cells the plasmamembrane galactosidase and glucosidase activities arealso increased. There are no methods to determineplasma membrane activities of these two enzymes, butTable 2 shows that the total cell enzyme activities on theneutral sugars dramatically increased in transfectedcells.
The activity of the three known ceramidases, theneutral, the acidic and the basic, were comparable intransfected and normal cells. Thus, ceramide accumu-lates in transfected cells.
The sphingolipid pattern of human fibroblasts andNeu3-transfected cells, as determined after feedingtritiated sphingosine to cells, lipid extraction, TLCseparation of the total lipid mixture and radioimagingare reported in Figure 4A. The glycosphingolipid pat-tern of fibroblasts is in agreement with previous infor-mation (34), suggesting that GM3 and Gb3Cer are themain components of the glycosphingolipid mixture.However, Fig. 4 shows also that sphingomyelin is one ofthe main sphingolipids, while ceramide is hardly detect-able.
In Neu3 cells with comparison to normal fibroblasts,we observed a minor, but statistically significant, reduc-tion in the GM3 content, a minor increase of LacCer,and a dramatic decrease of Gb3Cer. In addition to this,ceramide was clearly present at higher levels in Neu3overexpressing cells, and we calculated a 6-fold increasewith respect to mock cells. The sphingolipid percentdistributions in normal and Neu3-transfected cells arereported in Table 4.
Figure 4B shows the radioactive sialoprotein patternin normal and Neu3 fibroblasts fed for two days [6-3H]-acetyl-D-mannosamine, a specific precursor of sialicacid. Cells incorporated the same radioactivity, but it isclear that in Neu3-transduced cells, the radioactivityassociated with proteins was lower than in normal cells,suggesting that Neu3 is capable of acting on sialopro-teins.
Despite the enzyme overexpression and increase ofactivity, only a very minor decrease in GM3 content wasobserved in stably transfected fibroblasts. Then weinvestigated the activity of the GM3 synthase SAT1.Figure 5A shows that in stably transfected cells theactivity of SAT1 increased by 150% with respect tonormal cells, as determined in an in vitro assay per-formed with [3-3H(sphingosine)]LacCer and cold CMP-Neu5Ac. The increase of enzyme activity was parallel toan increase of SAT1 mRNA (Fig. 5B, C). This suggests
TABLE 2. In vitro enzyme activities on natural and artificial substrates, pmol/hour/mg cell protein
Ceramide, lactosylceramide, and SM are produced by recycling of the radioactive sphingoid fragment from GM3 taken up by Mock andNeu3-transfected fibroblasts after administration of the GM3 to cells under conditions that prevent the lysosomal activity. The total radioactivityassociated with cells was very similar � 10%. Data are mean of 3 experiments with sd of � 15%.
E455CERAMIDE FROM GM3 AT THE CELL SURFACE
that SAT1 maximum activity did not change, but thatmore enzyme was available. The higher availability ofSAT1 could explain why the Neu3 overexpression intransfected cells does not produce any change in GM3content. Thus, in Neu3 cells, the high turnover of GM3largely consumes LacCer, which is no more available asa substrate for the synthesis of Gb3Cer. In confirmationof this, we found that the Gb3Cer synthase mRNAcontent did not change in Neu3 cells (Fig. 5D, E).
Cell surface SM is precursor of ceramide. Tritium-labeled SM was administered to cells cultured in thepresence of chloroquine. We did not find any statisticaldifference in ceramide contents between control andNeu3 cells, suggesting that the sphingomyelinase activ-ity is not responsible, or it is only to a small extent, forthe increase of ceramide in Neu3-transfected cells. Onthe other hand, in both control cells and cells main-tained in the presence of chloroquine, ceramide isproduced in a similar amount, suggesting that a con-stant quantity of ceramide is produced out of thelysosomes from SM (Fig. 6 and Table 5).
The rate of growth of Neu3-transfected cells wasreduced. This was determined by measuring DNAsynthesis through the incorporation of [3H]thymidinein cells. As shown in Fig. 7, Neu3 overexpression causeda marked diminution of [3H]thymidine incorporation,indicating inhibition of DNA synthesis.
The effect of Neu3 overexpression on programmedcell death was also evaluated. In Neu3 stable transfectedhuman fibroblasts, the expression of the apoptosis-suppressing protein Bcl-2 was markedly reduced re-spect to mock transfected cells (Fig. 8A), cytosoliccytocrome c was detectable (Fig. 8B) and caspase-3 wascleaved into its active fragment (Figure 8C), suggestingthat the apoptotic pathway is executing in these cells.Quantitative evaluation of the apoptotic cell death (Fig.9) indicated that the entity of the process was 8 timeshigher in Neu3 than in mock cells.
To correlate a specific role of high concentration ofceramide in Neu3 cells with the apoptotic process, weadministered C2-ceramide, a synthetic cell-permeableceramide analog (35), to cells. Figure 9 shows that after12 h-incubation with ceramide, the rate of cell death inmock cells was very similar to that of nontreated Neu3cells. In both mock and Neu3 cells, C2-treatmentprogressively induced cell death by apoptosis. Never-theless, data shown in Fig. 9 suggest that the highervalue of apoptotic index in Neu3 cells with respect tomock cells after C2-ceramide treatment, is probably dueto the higher basal concentration of intracellular apo-ptotic ceramide in the transfected fibroblasts.
DISCUSSION
Glycosphingolipids are components of the cell plasmamembranes where, through interactions with differentproteins, they can play an important role in modulatingseveral aspects of the signal transduction processes (5).Plasma membranes glycosylhydrolases (9, 36–38) could
Figure 4. Sphingolipids and sialoglycoproteins in Neu3 cells.A) Mock and Neu3 cells were pulsed with [3-3H]Sphingosine,3 � 108 M final concentration. After a 48 h chase, cells wereharvested and treated for lipid analysis (see Material andMethods). Total lipids were separated by HPTLC using thesolvent system chloroform/methanol/0.2% CaCl2 55:45:10 byvolume. Radioactive lipids were detected by digital autora-diography using a Beta-Imager 2000 Instrument; 1000 dpm/lane, 48 h acquisition. Lanes 1 and 2, mock cells; lanes 3 and4, Neu3 transfected cells. B) Mock and Neu3-transfected cellswere treated with 10 � 106 dpm/ml of [3H]N-acetylman-nosamine for 24-h pulse followed by 24 or 48-h chase. At theend of the incubation, the cells were harvested, and the cellhomogenates (30 �g) were separated by electrophoresis on a10% SDS-PAGE gel and the proteins were transferred toPVDF membranes. Radioactive proteins were detected bydigital autoradiography; 1000 dpm/lane, 65 h acquisition.Lanes 1 and 3, Neu3 cells incubated for 24 h and 48 chaserespectively; lanes 2 and 4, mock cells incubated for 24 h and48 h chase respectively. �-tubulin and �-actin were used asinternal controls.
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be the natural candidate for modifications of the cellsurface glycolipids, participating to the modulation ofthe signal transduction processes. Within the mem-brane associated glycosylhydrolases, the membrane-bound sialidase Neu3 has been characterized (7–9).
In this paper, we show the involvement of Neu3 andother membrane-associated glycosylhydrolases in pro-cessing gangliosides belonging to the plasma mem-branes of human fibroblasts in culture, namely GM3(34).
The production at the cell surface of normal fibro-blasts of a small amount of ceramide was clearly shownby feeding cells with GM3 ganglioside-containing triti-ated sphingosine. GM3 was administered to cells underexperimental conditions that allowed gangliosides toenter into the cell plasma membranes and to becomeindistinguishable from the endogenous compounds(39–41). A part of the radioactive GM3 entered in themetabolic pathway, and we observed the production ofsome LacCer, Cer and SM (Fig. 1). Radioactive SM canbe formed only by recycling of sphingosine, and thisconfirms that GM3 reaches lysosomes where sphin-gosine is produced. To understand whether a part ofcatabolic process occurred out of the lysosomes, wetreated cells with ammonium chloride or chloroquine.Both ammonium chloride and chloroquine have beenwidely used to inhibit lysosomal activity and in studiesaimed to clarify the glycolipid intracellular traffickingand metabolism (28–30). Figure 1 shows that, underthese two experimental conditions, no lactosylceramideand SM could be observed, while ceramide was pro-duced. Neu3 overexpressing fibroblasts with doubleNeu3 activity subjected to the above treatments gavesimilar qualitative results but produced ceramide levelsalmost 3 times more(Table 3). These results demon-strate that the experimental conditions used to blocklysosomal activity are effective, as indicated by thecomplete lack of products deriving by the recycling of[3H]sphingosine that under our experimental condi-tions is generated in the active lysosomes from GM3(19). Thus the large portion of cell ceramide observedis produced out of lysosomes.
To understand whether GM3 is converted to cer-amide at the plasma membrane, experimental condi-tions known to block internalization of plasma mem-brane components via endocytosis were also used.
Incubation of cells at low temperature has been widelyused in the past for this purpose (31–33). When humanskin fibroblasts were incubated with fluorescent sphin-golipid analogs for 30 min at 10°C and observed byfluorescence microscopy, only plasma membrane label-ing was observed (31). The use of temperatures be-tween 2 and 10°C is a well-established method todistinguish endocytosis from other mechanisms of in-ternalization of membrane components, as endocytosisdoes not occur in mammalian cells to any significantextent at temperatures below 11°C (32, 42). As shownin Fig. 1, when GM3 was administered to cells at 4°C, nolactosylceramide and SM could be observed, but stillceramide was produced (Table 3). These results, takentogether, indicate that ceramide is produced in anextralysosomal compartment and strongly suggest thatthe site of ceramide production is the plasma mem-brane.
The production of ceramide, at the plasma mem-branes of mammal cells, has been always associated tothe activity of sphingomyelinase on sphingomyelin. Forthe first time, we show that ceramide is produced at theplasma membrane starting from glycosphingolipids.
We did not observe the production of LacCer andGlcCer, blocking cell lysosomal activity and endocytosis,after administration of cells of radioactive GM3. Thissuggests that both galactosidase and glucosidase areavailable at the plasma membrane, and the rates ofdetachment of neutral sugars are higher than that ofsialic acid. Surprisingly, in Neu3 cells, where the pro-duction of Cer is very high after administration of GM3,no radioactive neutral glycolipids could be observedblocking cell lysosomal activity and endocytosis. This,together with the increase of sialidase activity, couldsuggest that an increase of the activity of membrane-associated galactosidase and glucosidase could occur.Unfortunately, no assays are available to discriminateplasma membrane galactosidase and glucosidase activ-ities from the lysosomal ones. Nevertheless, we foundthat the total cell galactosidase and glucosidase activi-ties were highly increased in Neu3 overexpressing cells(Table 1).
Together with the increased production of ceramide,many biochemical events were strongly modified inNeu3 cells. The increased sialidase activity was notfollowed by a parallel decrease of the cell GM3 content.
TABLE 4. Sphingolipid distribution in normal and Neu3 stably transfected human fibroblasts in culture
Neu3 cells Mock cells
nCi/mg protein % nCi/mg protein % nCi/mg protein % nCi/mg protein %
nCi/mg protein and % distribution from 2 separate experiments.
E457CERAMIDE FROM GM3 AT THE CELL SURFACE
As shown in Fig. 4, the GM3 cellular contents in controland Neu3 cells are only slightly different. This appearsin contrast with the high content of ceramide in Neu3cells. Surprisingly, we found that the increase of siali-dase content and activity occurred together with acorrespondent increase of the content and activity of
Figure 5. Expression of GM3 synthase and Gb3Cer synthase inNeu3 cells. A–C) Sialyltransferase (SAT1, GM3 synthase). Dand E) �-Galactosyltransferase (Gb3Cer synthase). A) Sialyl-transferase activity in Mock and Neu3 cells was assayed usingdifferent concentration of ganglioside as substrate (2�M, 5�Mor 10�M [1-3H(Sphingosine)]LacCer). Data are expressed aspicomoles of formed GM3/hr/mg of cell proteins and are themeans � sd of 3 independent experiments. B) RT-PCRs were
performed with the sialyltransferase-specific primers by usingcDNA from Mock (lane 1) and Neu3 cells (lane 2). �-actinmRNA expression was measured as internal control in mock(lane 3) and in Neu3 cells (lane 4). In the original gel, weanalyzed �-actin mRNA expression in duplicate. We consid-ered this redundant and for this reason, rather than prepar-ing a figure with 6 lanes, the duplicate lanes for the �-actinexpression are not shown. The two lanes regarding SAT1from another portion of the same gel are superimposed overthe duplicate lanes. C) Quantification of mRNA expressionlevels of SAT1 in Neu3-transfected cells with comparison tomock cells. The quantitative analysis of each spot was per-formed by Gel Doc 2000 Software Analysis. D) RT-PCRs wereperformed with the �-galactosyltransferase-specific primers byusing cDNA from mock (lane 1) and Neu3-transfected cells(lane 2). �-actin mRNA expression was measured as internalcontrol in mock (lane 3) and in Neu3-transfected cells (lane4). E) Quantification, as above, of mRNA expression levels of�-galactosyltransferase in Neu3-transfected cells with compar-ison to mock cells.
Figure 6. Radioactive lipids from Neu3 cells fed with radioactiveSM. Mock and Neu3-transfected cells were treated with 50 �Mchloroquine for 30 min in EMEM in the absence of FCS. Thenthe cells were fed with 6.0 � 106M [3H]SM and maintained inthe presence of 50 �M chloroquine for 12 h. Lipids from mockand Neu3-transfected cells were extracted and separated byHPTLC using the solvent systems: chloroform/methanol/0.2%aqueous CaCl2 55:45:10 by vol. Radioactive lipids were de-tected by digital autoradiography using a Beta-Imager 2000Instrument; 1000 dpm/lane, 48 h acquisition. Lane 1, mockcells; lane 2, Neu3 transfected cells; lane 3, mock cells withchloroquine; lane 4, Neu3-transfected cells with chloroquine.
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SAT1 (the sialyltransferase known also as GM3 syn-thase) (see Fig. 5). Thus, some plasma membrane GM3disappears, as it becomes a substrate for the cell surfacehydrolytic process GM33 LacCer3 GlcCer3 Cer, anda similar quantity is substituted by neosynthesized GM3.Thus, to support the GM3 turnover, a large amount ofLacCer is necessary. LacCer is substrate for both SAT1and Gb3Cer synthase (Gb3Cer is formed by addition ofan �-galactose to LacCer). But we did not find anyincrease in Gb3Cer synthase (�-galactosyltransferase)expression. Thus, LacCer is not available to the �-galac-tosyltransferase, as it is used to produce GM3 by SAT1,and this explains the loss of Gb3Cer in Neu3 overex-pressing cells.
A direct correlation between the increase of mem-brane bound sialidase Neu3 and the increase of otherenzymes of the glycosphingolipid metabolism is not soevident. At least as far as it concerns SAT1, we foundthat both the protein and the activity increased (Fig. 5).Thus a signal was necessary to synthesize more mRNASAT1. Glycosphingolipids, together with cholesterol,are components of plasma membrane lipid domains,which are believed to contain the switch of severalfunctional events. There is solid information that sug-gests that changes of the composition and organization
of these domains can modulate the functional events.We had dramatic changes of the glycosphingolipidpattern after Neu3 cell overexpression (Fig. 4). Thiscould be responsible for signals that can modify thecontents of the enzymes for the glycosphingolipidcatabolism. In addition to this, the new glycosphingo-lipid pattern could be responsible for the activation ofthe membrane-associated galactosidase and glucosidasethroughout direct glycolipid-protein interactions. Gly-cosphingolipid ability to modulate membrane-boundenzymes has been well studied and described in detailin the past (43–46).
The biochemical events occurring with the increaseof Neu3 activity lead to cell death. This is not surpris-ing, because of the increase of ceramide. Ceramide,released from plasma membrane sphingomyelin by aplasma membrane-associated sphingomyelinase, withneutral or acidic optimal pH, has been shown to
Production of ceramide from SM taken up by Mock and Neu3-transfected fibroblasts after administration of SM to cells underconditions that prevent lysosomal activity. The total radioactivityassociated with cells was very similar � 10%. Data are the mean of 3experiments.
Figure 7. [3H]Thymidine incorporation in Neu3. Mock (solidbars) and Neu3-transfected cells (gray bars) were grown in35-mm dishes in 10% FCS-EMEM. 24 h after plating cells, themedium was removed and replaced with 1 ml of EMEMcontaining 0.5 �Ci of [3H]thymidine. After 1.5, 6, 20, and48 h at 37°C, cells were harvested with phosphate-bufferedsaline and treated with 10% trichloroacetic acid. The amountof radioactivity associated with labeled DNA was determinedby liquid scintillation counting. Data are presented as mean �sd of 3 experiments and are expressed as dpm.
Figure 8. Bcl-2, cytochrome c and caspase-3 expression inNeu3 cells. The levels of Bcl-2 (A), cytochrome c (B) andcaspase-3 (C) were assessed by Western blot analysis in mockand Neu3-transfected cells. Stable transfectant cells showeddecreased Bcl-2 protein and increased cytochrome c proteinlevels, whereas the amount of cleavage fragment of caspase-3was found to be higher in Neu3 cells compared with controlcells. �-tubulin was determined as an internal control.
E459CERAMIDE FROM GM3 AT THE CELL SURFACE
participate in some way in the activation of the apopto-tic process (47). We have effectively shown that cer-amide promotes apoptosis when added to normal fibro-blasts and that it increases the existing apoptoticprocess when added to Neu3 cells (Fig. 9). This sup-ports a correlation between the high content of cer-amide produced by the high expression of Neu3 andthe high rate of death by apoptosis in Neu3 cells. Ofcourse, it is now necessary to look for physiologicalligands able to increase directly or indirectly Neu3activity/quantity. Some experiments in this directionare now in progress. In addition to this, we would liketo stress that our results have been obtained usingnontransformed cells and that for the first time, theyassociate Neu3 overexpression to increase apoptosis.The role of Neu3 was previously studied by overexpress-ing the enzyme in tumor cells. Results from thesestudies were very different from what we report here. Infact, Neu3 overexpression in tumor cells led to anincrease of the LacCer cell content and enhanced anantiapoptotic effect (12). The different results could berelated to the fact that in tumor cells the glycosylationprocess is aberrant.
Figure 6 shows that feeding of sphingomyelin, iso-topically tritium labeled at position 3 of sphingosine, tohuman fibroblasts leads to the formation of someceramide, as expected. Nevertheless, in Neu3 overex-pressing cells, a similar amount of ceramide was pro-duced. This would confirm that ceramide produced inNeu3 cells comes mainly from the degradation of GM3but do not exclude that the minor increase of neutralsphingomyelinase activity can produce a minor quan-tity of it from sphingomyelin, in vivo.
Twenty-five years ago, it was suggested (48) thatpolysialogangliosides, after biosynthesis in the Golgiand transport to the plasma membranes, could besubstrates for a membrane-associated sialidase able toregulate the correct ratio between gangliosides at dif-ferent sialic acid content and neutral glycolipids. Ourresults suggest that the role of Neu3, the membrane-associated sialidase, is to correctly maintain in the
plasma membranes the ganglioside pattern and thecontent necessary for the cell communications.
This work was supported by COFIN-PRIN (2002), Con-siglio Nazionale delle Ricerche (PF Biotechnology) and FIRB;Italy.
REFERENCES
1. IUPAC-IUBMB Joint Commission on Biochemical Nomencla-ture (1997) Pure Appl. Chem. 69, 2475–2487. (1998). Carbohydr.Res. 312, 167–175
2. Hannun, Y. A., Luberto, C., and Argraves, K. M. (2001) Enzymesof sphingolipid metabolism: from modular to integrative signal-ing. Biochemistry. 40, 4893–4903
3. Alessenko, A. V. (2000) The role of sphingomyelin cycle metab-olites in transduction of signals of cell proliferation, differenti-ation and death. Membrane Cell Biol. 13, 303–320
4. Ito, M., and Yamagata, T. (1986) A novel glycosphingolipid-degrading enzyme cleaves the linkage between the oligosaccha-ride and ceramide of neutral and acidic glycosphingolipids.J. Biol. Chem. 261, 14278–14282
5. Hakomori, S., and Igarashi, Y. (1995) Functional role of glyco-sphingolipids in cell recognition and signaling. J. Biochem. 118,1091–1093
6. Monti, E., Preti, A., Venerando, B., and Borsani, G. (2002)Recent development in mammalian sialidase molecular biology.Neurochem. Res. 27, 649–663
7. Miyagi, T., Sagawa, J., Konno, K., Handa, S., and Tsuiki, S.(1990) Biochemical and immunological studies on two distinctganglioside-hydrolyzing sialidases from the particulate fractionof rat brain. J. Biochem. (Tokyo). 107, 787–793
8. Monti, E., Bassi, M. T., Papini, N., Riboni, M., Manzoni, M.,Venerando, B., Croci, G., Preti, A., Ballabio, A., Tettamanti, G.,and Borsani, G. (2000) Identification and expression of NEU3,a novel human sialidase associated to the plasma membrane.Biochem. J. 349, 343–351
9. Papini, N., Anastasia, L., Tringali, C., Croci, G., Bresciani, R.,Yamaguchi, K., Miyagi, T. Preti, A., Prinetti, A., Prioni, S.,Sonnino, S., Tettamanti, G., Venerando, B., and Monti, E.(2004) The plasma membrane-associated sialidase MmNEU3modifies the ganglioside pattern of adjacent cells supporting itsinvolvement in cell-to-cell interactions. J. Biol. Chem. 279,16989–16995
10. Kopitz, J., von Reitzenstein, C., Muhl, C., and Cantz, M. (1994)Role of plasma membrane ganglioside sialidase of humanneuroblastoma cells in growth control and differentiation.Biochem. Biophys. Res. Commun. 199, 1188–1193
11. Kopitz, J., Muhl, C., Ehemann, V., Lehmann, C., and Cantz, M.(1997) Effects of cell surface ganglioside sialidase inhibition ongrowth control and differentiation of human neuroblastomacells. Eur. J. Cell Biol. 73, 1–9
12. Kakugawa, Y., Wada, T., Yamaguchi, K., Yamanami, H., Ouchi,K., Sato, I., and Miyagi, T. (2002) Up-regulation of plasmamembrane-associated ganglioside sialidase (Neu3) in humancolon cancer and its involvement in apoptosis suppression. Proc.Natl. Acad. Sci. U. S. A. 99, 10718–10723
13. Hakomori, S. (2002) Glycosylation defining cancer malignancy:new wine in an old bottle. Proc. Natl. Acad. Sc.i U. S. A. 99,10231–10233
14. Carter, H. E., Rothfus, J. A., and Gigg, R. (1961) Biochemistry ofthe sphingolipids: XII. conversion of cerebrosides to ceramidesand sphingosine; structure of Gaucher cerebroside. J. Lipid Res.2, 228–234
15. Toyokuni, T., Nisar, M., Dean, B., and Hakomori, S. (1991) Afacile and regiospecific titration of sphingosine: synthesis of (2S,3R, 4E)-2-amino-4-octadecene-1,3-diol-1-3H. J. Labeled Compd.Radiopharm. 29, 567–574
16. Mauri, L., Casellato, R., Kirschner, G., and Sonnino, S. (1999) Aprocedure for the preparation of GM3 ganglioside from GM1-lactone. Glycoconjugate J. 16, 197–203
17. Sonnino, S., Chigorno, V., and Tettamanti, G. (2000) Prepara-tion of radioactive gangliosides, [3H] or [14C] isotopically
Figure 9. Apoptotic index in mock and Neu3 cells. Quantita-tive apoptosis in mock (solid bars) and Neu3 cells (gray bars)treated with 25 � 106 M C2-ceramide for 6, 12 and 24 h. Thelevel of DNA fragmentation was determined by ELISA assay.The DNA fragments in cell lysates were quantified at 450 nM.Data are presented as mean � sd of 3 experiments and areexpressed as absorbance on total milligrams of protein.
E460 Vol. 20 June 2006 VALAPERTA ET AL.The FASEB Journal
labeled at the oligosaccharide or ceramide moieties. MethodEnzymol. 311, 639–656
18. Sonnino, S., Ghidoni, R., Galli, G., and Tettamanti, G. (1978)On the structure of a new, fucose containing ganglioside frompig cerebellum. J. Neurochem. 31, 947–956
19. Chigorno, V., Riva, C., Valsecchi, M., Nicolini, M., Brocca, P.,and Sonnino, S. (1997) Metabolic processing of gangliosides byhuman fibroblasts in culture. Formation and re-cycling ofseparate pools of sphingosine. Eur. J. Biochem. 250, 661–669
20. Leroy, J. G., Ho, W. M., MacBrinn, M. C., Zielke, K., Jacob, J.,and O’Brien, J. S. (1972) I-cell disease: biochemical studies.Pediatr. Res. 6, 752–757
21. Hasegawa, T., Yamaguchi, K., Wada, T., Takeda, A., Itoyama, Y.,and Miyagi, T. (2000) Molecular cloning of mouse gangliosidesialidase and its increased expression in neuro2a cell differen-tiation. J. Biol. Chem. 275, 8007–8015. Erratum in: J. Biol. Chem.(2000). 275, 14778
22. Bossy-Wetzel, E., Newmeyer, D. D., and Green, D. R. (1998)Mitochondrial cytochrome c release in apoptosis occurs up-stream of DEVD-specific caspase activation and independentlyof mitochondrial transmembrane depolarisation. EMBO J. 17,37–49
23. Prinetti, A., Basso, L., Appierto, V., Villani, M.G., Valsecchi, M.,Loberto, N., Prioni, S., Chigorno, V., Cavadini, E., Formelli, F.,and Sonnino, S. (2003) Altered sphingolipid metabolism inN-(4-hydroxyphenyl)-retinamide-resistant A2780 human ovar-ian carcinoma cells. J. Biol. Chem. 278, 5574–5583
24. Bielawska, A., Greenberg, M. S., Perry, D., Jayadev, S., Shayman,J. A., McKay, C., and Hannun, Y. A. (1996) (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol as an inhibitor of ce-ramidase. J. Biol. Chem. 271, 12646–12654
25. Tettamanti, G., Lombardo, A., Marchesini, S., Giuliani, A.,Berra, B., Di Donato, S., Rimoldi, M., and Bertagnoglio, B.(1977) Lipid storage diseases: biochemical diagnosis. Biochem.Exp. Biol. 13, 45–60
26. Chigorno, V., Cardace, G., Pitto, M., Sonnino, S., Ghidoni, R.,and Tettamanti, G. (1986) A radiometric assay for gangliosidesialidase applied to the determination of the enzyme subcellularlocation in cultured human fibroblasts. Anal. Biochem. 153,283–294
27. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.(1951) Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193, 265–275
28. Marques, C., Pereira, P., Taylor, A., Liang, J. N., Reddy, V. N.,Szweda, L. I., Shang, F. Ubiquitin-dependent lysosomal degra-dation of the HNE-modified proteins in lens epithelial cells.FASEB J. 18, 1424–1426, 2004
29. Fang, D., and Kerppola, T. K. (2004) Ubiquitin-mediated fluo-rescence complementation reveals that Jun ubiquitinated byItch/AIP4 is localized to lysosomes. Proc. Natl. Acad. Sci. U. S. A.101, 14782–14787
30. Koh, Y.H., von Armin, C.A., Hyman, B.T., Tanzi, R.E., Tesco, G.(2005) BACE is degraded via the lysosomal pathway. J. Biol.Chem. 280, 32499–32504
31. Puri, V., Watanabe, R., Singh, R.D., Dominguez, M., Brown, J.C.,Wheatley, C.L., Marks, D.L., Pagano, R. E. (2001) Clathrin-dependent and -independent internalization of plasma mem-brane sphingolipids initiates two Golgi targeting pathways. J. CellBiol. 154, 535–547
32. Marks, D.L., Singh, R.D., Choudhury, A., Wheatley, C.L., Pa-gano, R. E. (2005) Use of fluorescent sphingolipid analogs tostudy lipid transport along the endocytic pathway. Methods 36,186–195
33. Furlaneto, R. W. (1988) Receptor-mediated endocytosis andlysosomal processing of insulin-like growth factor I by mitogeni-cally responsive cells. Endocrinology 122, 2044–2053
34. Dawson, G., Matalon, R., and Dorfman, A. (1972) Glycosphin-golipids in cultured human skin fibroblasts. I. Characterizationand metabolism in normal fibroblasts. J. Biol. Chem. 247, 5944–5950
35. Obeid L.M., Linardic C.M., Karolak L.A., Hannun, Y. A. (1993)Programmed cell death induced by ceramide. Science 259,1769–1771
36. Lukong, K. E., Seyrantepe, V., Landry, K., Trudel, S., Ahmad, A.,Gahl, W. A., Lefrancois, S., Morales, C. R., and Pshezhetsky,A. V. (2001) Intracellular distribution of lysosomal sialidase iscontrolled by the internalization signal in its cytoplasmic tail.J. Biol. Chem. 276, 46172–46181
37. Cordero, O. J., Merino, A., Paez de la Cadena, M., Bugia, B.,Nogueira, M., Vinuela, J. E., Martinez-Zorzano, V. S., de Carlos,A., and Rodriguez-Berrocal, F. J. (2001) Cell surface humanalpha-L-fucosidase. Eur. J. Biochem. 268, 3321–3331
38. Mencarelli S., Cavalieri S., Magini A., Tancini B., Basso L.,Lemansky P., Hasilik A., Li Y-T, Chigorno V., Orlacchio A.,Emiliani, C., and Sonnino, S. (2005) Direct immunological andbiochemical identification of plasma membrane associated ma-ture �-hexosaminidase a in human cells. FEBS Lett. 579, 5501–5506
39. Chigorno V., PittoM., Cardace G., Acquatti D., Kirschner G.,Sonnino S., Guidoni, R., and Tettamanti, G. (1985) Associationof gangliosides to fibroblasts in culture: A Study performed withGM1(14C)-labelled at the sialic acid acetyl group. GlycoconjugateJ. 2, 279–291
40. Radsak, K., Schwarzmann, G., and Wiegandt, H. (1982) Studieson the cell association of exogenously added sialo-glycolipids.Hoppe Seylers Z Physiol. Chem. 363, 263–272
41. Schwarzmann, G., Hoffmann-Bleihauer, P., Schubert, J., Sand-hoff, K., and Marsh, D. (1983) Incorporation of gangliosideanalogues into fibroblast cell membranes. A spin-label study.Biochemistry 22, 5041–5048
42. Martin, O. C., and Pagano, R. E. (1994) Internalization andsorting of a fluorescent analogue of glucosylceramide to theGolgi apparatus of human skin fibroblasts: utilization of endo-cytic and nonendocytic transport mechanisms. J. Cell Biol. 125,769–781
43. Partington, C. R., and Daly, J. W. (1979) Effect of gangliosideson adenylate cyclase activity in rat cerebral cortical membranes.Mol. Pharmacol. 15, 484–491
44. Leon, A., Facci, L., Toffano, G., Sonnino, S., and Tettamanti, G.(1981) Activation of (Na�, K�)-ATPase by nanomolar concen-trations of GM1 ganglioside. J. Neurochem. 37, 350–357
45. Kreutter, D., Kim, J. Y. H., Goldenring, J. R., Rasmussen, H.,Ukomadu, C., DeLorenzo, J., and Yu, R. K. (1987) Regulation ofprotein kinase C activity by gangliosides. J. Biol. Chem. 262,1633–1637
46. Yates, A. J., Walters, J. D., Wood, C. L., and Johnson, D. (1989)Ganglioside modulation of cyclic AMP-dependent protein ki-nase and cyclic nucleotide phosphodiesterase in vitro. J. Neuro-chem. 53, 162–167
47. Andrieu-Abadie, N., and Levade, T. (2002) Sphingomyelinhydrolysis during apoptosis. Biochim. Biophys. Acta 1585, 126–134
48. Scheel, G., Acevedo, E., Conzelmann, E., Nehrkorn, H., andSandhoff, K. (1982) Model for the interaction of membrane-bound substrates and enzymes. Hydrolysis of ganglioside GD1aby sialidase of neuronal membranes isolated from calf brain. EurJ Biochem. 127, 245–253
Received for publication September 13, 2005.Accepted for publication January 3, 2006.
