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48. Department of Medical Chemistry, Biochemistry and Biotechnology and Center of Excellence on Neurodegenerative Diseases, University of Milan, Segrate, Italy;
Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia, Italy; and
Division of Biochemistry, Research Institute, Miyagi Prefectural Cancer Center, Natori, Miyagi, Japan
1Correspondence: Department of Medical Chemistry, Biochemistry and Biotechnology and Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Fratelli Cervi 93, Segrate 20090 (Milan, Italy). E-mail: sandro.sonnino{at}unimi.it
SPECIFIC AIMS
Ceramide is a key lipid molecule necessary to regulate some cellular processes, including apoptosis and cell differentiation, and its production has been shown to be due to sphingomyelin hydrolysis or acylation of sphingosine. We investigated whether bioactive plasma membrane ceramide can be produced as well from gangliosides by detachment of sugar units due to the action of membrane-bound glycosylhydrolases.
PRINCIPAL FINDINGS
1. Ceramide is produced from GM3 at the plasma membrane in normal human fibroblasts
Figure 1
, lane 1, shows the radioactive sphingolipid pattern of cells fed with GM3-containing radioactive sphingosine. After TLC separation, the catabolic products lactosylceramide and ceramide were identified, together with SM obtained by recycling of sphingosine. To verify whether a part of the radioactive ceramide formed from GM3 administered to cells was produced in the plasma membrane, we fed cells with GM3 after blocking lysosomal activity or endocytosis. Figure 1
, lanes 2–4, shows that GM3-derived ceramide was produced by cells, even after blocking lysosomal activity or endocytosis. These results suggest that the observed ceramide is produced in the membrane concentration.
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2. Overexpression of plasma membrane sialidase Neu3 resulted in the increased ceramide production from GM3 in the plasma membrane
The above described set of experiments were repeated on cells overexpressing the plasma membrane sialidase Neu3. Stable transfectants were established, expressing a C-terminally hemagglutinin tagged form of mouse sialidase Neu3. In the transfected cells, the protein was mainly localized on the plasma membrane, and sialidase activity was determined on natural substrates activities on GM3 and GD1a were 2 times higher than mock in cells. Figure 1
, lane 1a, shows that transfected cells fed with GM3, produced lactosylceramide, ceramide, and SM. Transfected cells produced about 3 times more ceramide than in mock cells, as well as a similar quantity of lactosylceramide. However, ceramide but not lactosylceramide was formed by blocking the cell lysosomal activity or endocytosis by transfected cells fed with GM3 (Fig. 1
, lanes 2a to 4a).
3. Overexpression of plasma membrane sialidase Neu3 resulted in profound alteration in sphingolipid metabolism and composition
The sphingolipid pattern of human fibroblasts and Neu3-transfected cells, as determined after feeding tritiated sphingosine to cells, lipid extraction, TLC separation of the total lipid mixture and radioimaging are reported in Fig. 2
. In agreement with previous findings, GM3 and Gb3Cer are the main components of the sphingolipid mixture together with sphingomyelin, while ceramide is hardly detectable.
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In Neu3 cells with comparison to normal fibroblasts, we observed a minor, but statistically significant, reduction 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 Neu3 overexpressing cells, and we calculated a sixfold increase compared to mock cells. Despite the enzyme overexpression and increase of activity, only a very small decrease in GM3 content was observed in stably transfected fibroblasts. Then we investigated the activity of the GM3 synthase SAT1. In stably transfected cells, the activity of SAT1 increased 
150% compared to normal cells. The increase of enzyme activity was parallel to an increase of SAT1 mRNA. This suggests that SAT1 maximum activity did not change, but that more enzyme was available. The higher availability of SAT1 could explain why the Neu3 overexpression in transfected cells does not produce any change in GM3 content.
SAT1 was not the only sphingolipid metabolic enzyme altered by Neu3 overexpression. A marked increase in the activity of ßbeta;-galactosidase (9,655±888 pmol/h/mg cell protein in Neu3-transfected cells vs. 1,125±145 pmol/h/mg cell protein in mock cells) and ßbeta;-glucosidase (64,045±4,589 pmol/h/mg cell protein in Neu3-transfected cells vs. 16,334±1,234 pmol/h/mg cell protein in mock cells) was observed. A modest increase in the activity of neutral sphingomyelinase (32.1±3.7 pmol/h/mg cell protein in Neu3-transfected cells vs. 21.1±1.5 pmol/h/mg cell protein in mock cells) was measured. On the other hand, no statistically significant differences were observed between Neu3-transfected cells and mock cells in the enzyme activities of Neu1 lysosomal sialidase, 
-galactosidase, acidic, basic, and neutral ceramidases and acidic sphingomyelinase.
4. Overexpression of plasma membrane sialidase Neu3 resulted a reduced cell growth and increased apoptosis
The growth rate of Neu3-transfected cells was highly reduced. Neu3 overexpression caused a marked diminution of [3H]thymidine incorporation, indicating inhibition of cell proliferation. The effect of Neu3 overexpression on programmed cell death was also evaluated. In Neu3 stable transfected human fibroblasts, the expression of the apoptosis-suppressing protein Bcl-2 was markedly reduced compared to mock transfected cells, cytosolic cytocrome c was detectable, and caspase-3 was cleaved to its active fragment, indicating that an apoptotic pathway is executing in these cells.
CONCLUSIONS AND SIGNIFICANCE
Glycosphingolipids are components of the cell plasma membranes where they participate in the organization of lipid domains and modulate several aspects of the signal transduction processes. Plasma membranes glycosylhydrolases could be the natural candidate for modifications of the cell surface glycolipids. Within the membrane-associated glycosylhydrolases, the membrane-bound sialidase Neu3 has been characterized. Neu3 has been cloned and then shown to be associated with sphingolipid-enriched membrane domains.
In this paper, we show the involvement of Neu3 and other membrane-associated glycosylhydrolases in processing gangliosides belonging to the plasma membranes of human fibroblasts in culture, namely GM3 (Fig. 3
).
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The production at the cell surface of normal fibroblasts of a minor amount of ceramide was clearly shown by feeding cells with GM3 ganglioside-containing radioactive sphingosine. GM3 was administered to cells under experimental conditions that allow gangliosides to enter into the cell plasma membranes and to become indistinguishable from the endogenous compounds. A part of the radioactive GM3 entered in the metabolic pathway, and we observed the production of some LacCer, Cer, and SM (Fig. 1)
. Radioactive SM can be formed only by recycling of sphingosine, and this confirms that GM3 reaches lysosomes where sphingosine is produced. To understand whether a part of catabolic process occurred also at the cell surface, we maintained cells under conditions that do not allow lysosomal activity or block endocytosis. Figure 1
shows that under these experimental conditions, no lactosylceramide and SM could be observed, while ceramide was produced. Neu3 overexpressing fibroblasts with double Neu3 activity, subjected to the above experiments gave similar qualitative results but produced ceramide in much higher quantities, about 3 times more under all the experimental conditions. These results demonstrate that: 1) the experimental conditions used to block lysosomal activity and endocytosis are effective to prevent GM3 from reaching lysosomes; 2) the LacCer observed under normal experimental conditions is produced in the lysosomes; 3) blocking lysosomal activity and endocytosis, ceramide is produced at the plasma membranes; and 4) a part of ceramide produced under normal conditions is produced at the cell surface.
The production of ceramide at the plasma membranes of mammal cells has been always associated with the activity of sphingomyelinase on sphingomyelin. For the first time, we show that ceramide is produced at the plasma membrane starting from glycosphingolipids.
We did not observe the production of LacCer and GlcCer, blocking cell lysosomal activity and endocytosis, after administration to cells of radioactive GM3. This suggests that both galactosidase and glucosidase are available at the plasma membrane, and the rates of detachment of neutral sugars are higher than that of sialic acid. Surprisingly, in Neu3 cells, in which the production of Cer is very high after administration of GM3, no radioactive neutral glycolipids could be observed blocking cell lysosomal activity and endocytosis. This could suggest that, together with the increase of sialidase activity, an increase of the activity of membrane-associated galactosidase and glucosidase could occur. Indeed, we found that the total cell galactosidase and glucosidase activities were highly increased in Neu3-overexpressing cells.
Together with the increased production of ceramide, many biochemical events were strongly modified in Neu3 cells. The increased sialidase activity was not followed by a parallel decrease of the cell GM3 content. As shown in Fig. 2
, the GM3 cellular contents in control and Neu3 cells are only slightly different, which appears in contrast with the high content of ceramide in Neu3 cells. Surprisingly, we found that the increase of sialidase content and activity occurred together with a corresponding increase of the content and activity of SAT1 (the sialyltransferase known also as GM3 synthase). Thus, some plasma membrane GM3 disappears as it becomes a substrate for the cell surface hydrolytic process GM3
LacCer GlcCer Cer, and a similar quantity is substituted by neosynthesized GM3. This explains the loss of Gb3Cer in Neu3-overexpressing cells. Gb3Cer is formed by addition of an -galactose to LacCer. Probably, LacCer is not available to the -galactosyltransferase, being used to produce GM3 by SAT1.
A direct correlation between the increase of membrane-bound sialidase Neu3 and the increase of other enzymes of the glycosphingolipid metabolism is not so evident. At least as far as it concerns SAT1, we found that both the protein and the activity increased. Glycosphingolipids, with cholesterol, are components of plasma membrane lipid domains that are believed to contain the switch of several functional events. There is solid information to suggest that changes of the composition and organization of these domains can modulate the functional events. We had dramatic changes of the glycosphingolipid pattern after Neu3 cell overexpression (Fig. 2)
. This could be responsible of signals that are able to modify the contents of the enzymes for the glycosphingolipid catabolism. In addition to this, the new glycosphingolipid pattern could be responsible for the activation of the membrane-associated galactosidase and glucosidase throughout direct glycolipid-protein interactions. Glycosphingolipid ability to modulate membrane-bound enzymes has been well studied and detailed in the past.
The biochemical events occurring with the increase of Neu3 activity lead to cell death. This is not surprising, due to the increase of ceramide. Ceramide has been shown to participate in some way in the activation of the apoptotic process and to be released from plasma membrane sphingomyelin by a plasma membrane-associated sphingomyelinase.
Twenty-five years ago, it was suggested that polysialogangliosides, after biosynthesis in the Golgi and transport to the plasma membranes, could be substrates for a membrane-associated sialidase that could regulate the correct ratio between gangliosides at different sialic acid content and neutral glycolipids. Our results suggest that the role of Neu3, the membrane-associated sialidase, is to correctly maintain in the plasma membranes the ganglioside pattern and content necessary for the cell communications.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5077fje
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  M. Valsecchi, L. Mauri, R. Casellato, S. Prioni, N. Loberto, A. Prinetti, V. Chigorno, and S. Sonnino Ceramide and sphingomyelin species of fibroblasts and neurons in culture J. Lipid Res., February 1, 2007; 48(2): 417 - 424. [Abstract] [Full Text] [PDF]
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