The monoclonal antibody M-DC8 defines a major subset of human blood dendritic cells (DCs). Here we identify the M-DC8 structure as 6-sulfo LacNAc, a novel carbohydrate modification of the P selectin glycoprotein ligand 1 (PSGL-1). In contrast to previously described blood DCs, M-DC8+ DCs lack the cutaneous lymphocyte antigen (CLA) on PSGL-1 and fail to bind P and E selectin. Yet they express anaphylatoxin receptors (C5aR and C3aR) and the Fcgamma receptor III (CD16), which recruit cells to inflammatory sites. While sharing with DC1 the expression of myeloid markers and a potent capacity to prime T cells in vitro, M-DC8+ DCs produce far more TNF-alpha in response to the bacterial endotoxin lipopolysaccharide (LPS). Thus, 6-sulfo LacNAc-expressing DCs appear as a novel proinflammatory DC subset.

GD3 synthase is rapidly activated in different cell types after specific apoptotic stimuli. De novo synthesized GD3 accumulates and contributes to the apoptotic program by relocating to mitochondrial membranes and inducing the release of apoptogenic factors. We found that sialic acid acetylation suppresses the proapoptotic activity of GD3. In fact, unlike GD3, 9-O-acetyl-GD3 is completely ineffective in inducing cytochrome c release and caspase-9 activation on isolated mitochondria and fails to induce the collapse of mitochondrial transmembrane potential and cellular apoptosis. Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3. The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis. Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3. Thus, sialic acid acetylation critically controls the proapoptotic activity of GD3.

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F1C fimbriae are correlated with uropathogenic Escherichia coli strains. Although F1C fimbriae mediate binding to kidney tubular cells, their receptor is not known. In this paper, we demonstrate for the first time specific carbohydrate residues as receptor structure for F1C-fimbria-expressing E. coli. The binding of the F1C fimbriated recombinant E. coli strain HB101(pPIL110-54) and purified F1C fimbriae to reference glycolipids of different carbohydrate compositions was evaluated by using thin-layer chromatography (TLC) overlay and solid-phase binding assays. TLC fimbrial overlay analysis revealed the binding ability of purified F1C fimbriae only to glucosylceramide (GlcCer), ␤1-linked galactosylceramide 2 (GalCer2) with nonhydroxy fatty acids, lactosylceramide, globotriaosylceramide, paragloboside (nLc 4 Cer), lactotriaosylceramide, gangliotriaosylceramide (asialo-GM 2 [GgO 3 Cer]) and gangliotetraosylceramide (asialo-GM 1 [GgO 4 Cer]). The binding of purified F1C fimbriae as well as F1C fimbriated recombinant E. coli strain HB101(pPIL110-54) was optimal to microtiter plates coated with asialo-GM 2 (GgO 3 Cer). The bacterial interaction with asialo-GM 1 (GgO 4 Cer) and asialo-GM 2 (GgO 3 Cer) was strongly inhibited only by disaccharide GalNAc␤1-4Gal␤ linked to bovine serum albumin. We observed no binding to globotetraosylceramide or Forssman antigen (Gb 5 Cer) glycosphingolipids or to sialic-acid-containing gangliosides. It was demonstrated that the presence of a GalCer or GlcCer residue alone is not sufficient for optimal binding, and additional carbohydrate residues are required for high-affinity adherence. Indeed, the binding efficiency of F1C fimbriated recombinant bacteria increased by 19-fold when disaccharide sequence GalNAc␤1-4Gal␤ is linked to glucosylceramide as in asialo-GM 2 (GgO 3 Cer). Thus, it is suggested that the disaccharide sequence GalNAc␤1-4Gal␤ of asialo-GM 2 (GgO 3 Cer) which is positioned internally in asialo-GM 1 (GgO 4 Cer) is the high-affinity binding epitope for the F1C fimbriae of uropathogenic E. coli.

We have previously demonstrated induction of O-acetylated sialoglycoproteins on lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). These molecules promote survival of lymphoblasts by preventing apoptosis. Although O-acetylated sialoglycoproteins are over expressed, the status of O-acetylation of gangliosides and their role in lymphoblasts survival remains to be explored in ALL patients. Here, we have observed enhanced levels of 9-O-acetylated GD3 (9-O-AcGD3) in the lymphoblasts of patients and leukaemic cell line versus disialoganglioside GD3 in comparison to the normal cells. Localization of GD3 and 9-O-AcGD3 on mitochondria of patient's lymphoblasts has been demonstrated by immuno-electron microscopy. The exogenous administration of GD3-induced apoptosis in lymphoblasts as evident from the nuclear fragmentation and sub G0/G1 apoptotic peak. In contrast, 9-O-AcGD3 failed to induce such apoptosis. We further explored the mitochondria-dependent pathway triggered during GD3-induced apoptosis in lymphoblasts. GD3 caused a time-dependent depolarization of mitochondrial membrane potential, release of cytochrome c and 7.4- and 8-fold increased in caspase 9 and caspase 3 activity respectively. However, under identical conditions, an equimolar concentration of 9-O-AcGD3 failed to induce similar effects. Interestingly, 9-O-AcGD3 protected the lymphoblasts from GD3-induced apoptosis when administered in equimolar concentrations simultaneously. In situ de-O-acetylation of 9-O-AcGD3 with sodium salicylate restores the GD3-responsiveness to apoptotic signals. Although both GD3 and 9-O-acetyl GD3 localize to mitochondria, these two structurally related molecules may play different roles in ALL-disease biology. Taken together, our results suggest that O-acetylation of GD3, like that of O-acetylated sialoglycoproteins, might be a general strategy adopted by leukaemic blasts towards survival in ALL.

Gesellschaft fur Biotechnologische Forschung mbH BraunschweigMouse spleen cells were prepared from CBA/J mice, and T lymphocytes were selectively stimulated with the T cell mitogen concanavalin A and further propagated in the presence of the T cell growth factor interleukin-2. The T cells were metabolically labeled with ~-[l-'~C]galactose and D[1-'4C]glUcOSamine, and the gangliosides were extracted and purified by DEAE-Sepharose column chromatography. Carbohydrate backbone structures of the asialogangliosides, prepared by mild acid hydrolysis, were determined by (1) high-performance liquid chromatography, (2) treatment with exoglycosidases and (3) immunostaining. Monosialylated gangliosides were isolated by gradient elution from DEAE-Sepharose and further separated by preparative high-performance thin-layer chromatography in two solvent systems. Isolated fractions were characterized by (1) preparation of asialogangliosides by mild acid hydrolysis, (2) the action of Vibrio cholerae neuraminidase, and (3) fast-atombombardment mass spectrometry. The following structures were identified : IVNeuAc-GgOse4Cer; IVNeuGcGgOse4Cer; IVNeuAc-GgOse5Cer; and 1VNeu-Gc-GgOse5Cer. The latter two gangliosides were not detected on B lymphoblasts and may be T-cell-specific structures. All gangliosides were heterogeneous in their ceramide moieties, being substituted with C16: o, CZ4: o, and CZ4: fatty acids. A preliminary study of several other mouse strains showed n o strain-specific genetic variations in the T cell gangliosides. The possible role of these gangliosides is discussed.

Among 52 Saccharomyces cerevisiae fatty acid synthetase (fas)mutants screened for their ability to incorporate 14C‐labeled pantothenic acid into the fatty acid synthetase multienzyme complex, especially those of fas‐complementation group VII had lost this ability. The purified fatty acid synthetase complexes of all the 19 fas‐mutants available from this group were shown to be free of 14C‐labeled pantothenic acid. The amount of pantothenate incorporated into the enzymes of several other fas‐mutants of the non‐pleiotropic complementation groups II, V, VI and VIII, however, was similar to that observed with wild‐type fatty acid synthetase. The purified pantothenate‐free fatty acid synthetases of five different group VII fas‐mutants have been tested for the seven known component enzymes of the complex. In all mutants, only the β‐ketoacyl synthetase was inactive whereas in vitro all the other partial activities were unimpaired. By sodium dodecylsulfate‐polyacrylamide‐gel electrophoresis, no differences could be observed between the protein structure of the pantothenate‐free mutant fatty acid synthetase and that of the wild‐type comp ex. Both were separated into three different components A, B and C with molecular weights of 185000, 180000 and 177000, respectively. However, some fas‐mutants consist only of the components A and B, others only of B and C. The study of various [14C]‐pantothenate‐labeled mutant fatty acid synthetases suggests that component C originates from A presumably by limited proteolysis. In the undegraded complex AB, [14C]pantothenate is only associated with the component A. Since in one of the mutants studied A is completely converted to C, it is concluded that A is one distinct component rather than a group of components with identical molecular weights. It is tentatively suggested that the gene product of fas 2, one of the two known fatty‐acid‐synthetase gene clusters in yeast, is only one, single and multifunctional polypeptide chain. Therefore, it appears that in yeast, the acyl carrier protein is not an individual protein component of the fatty acid synthetase complex, but only a distinct region of the multifunctional polypeptide chain encoded by fas 2.

The ganglioside GD3 (Neu5Aca8Neu5Aca3Galb4GlcCer) is an intracellular lipid messenger that induces apoptosis by targeting mitochondria in various cell types. GD3 can also promote apoptosis when externally added to cells. Previous studies showed that the proapoptotic effects of GD3 can be counteracted by 9-O-acetylation. To determine whether 9-O-acetyl GD3 (acGD3) has a general antiapoptotic potential, the apoptosis-sensitive Jurkat cell line and an apoptosis-sensitive variant of the cell line Molt-4 were preincubated with micromolar concentrations of acGD3 and then treated with inducers of apoptosis. A reduced apoptotic index and an increased cell viability were observed. On the other hand, when the Jurkat cells were treated with GD3 for extended periods of time, a population was selected that was resistant to apoptosis induction by N-acetyl sphingosine as well as by the anti-leukemic drug daunorubicin. Comparative analysis of gangliosides revealed the formation of acGD3 in the resistant Jurkat cells that was not found in the apoptosis-sensitive cells. Conversely, exposing the acGD3 positive and apoptosis-resistant cell line Molt-4 to the Odeacetylating activity of salicylate resulted in a complete disappearance of acGD3 and an enhanced sensitivity to N-acetyl sphingosine-mediated apoptosis. Formation of acGD3 might thus represent a new mechanism how tumor cells can escape apoptosis. ' 2006 Wiley-Liss, Inc.Key words: apoptosis; 9-O-acetyl GD3; gangliosideThe intracellular ganglioside GD3 (Neu5Aca8Neu5Aca3Galb 4GlcCer) has been shown to serve as a second lipid messenger in the apoptotic pathway induced by FAS/FASL, TNF-a or b-amyloid in diverse lymphoid and myeloid cell types.1-3 Various effects have been ascribed to GD3, such as production of reactive oxygen species (ROS), 4 opening of the mitochondrial permeability transition pore (PTP), 4 release of cytochrome C, 1,4,5 activation of caspases 6 and inhibition of the translocation of NFjB to the nucleus. 7 The importance of ROS and PTP in the GD3-mediated apoptotic pathway is stressed by the finding that GD3-induced mitochondrial changes are prevented by antioxidants such as butylated hydroxytoluene (BHT) 2 and by preincubation of the cells with cyclosporin A, an inhibitor of the PTP.8 Despite the obvious involvement of mitochondria, the exact mechanism how GD3 promotes apoptosis still remains elusive.Accumulation of GD3 has been reported in a variety of tumors.9-11 The concomitant presence of an acetylated modification of GD3, 9-O-acetyl GD3 (acGD3), was observed in some tumors such as melanoma 12,13 and breast cancer, 14,15 as well as in tumor cell lines like MOLT 4 16 and SKMel28. 17In a previous report, we have shown that the proapoptotic activity of GD3 is suppressed by acetylation. 18 However, it remained unclear whether the observed resistance to apoptosis is due to inactivation of GD3 by acetylation or, rather, to an antiapoptotic potential of acGD3 on its own. To approach this question, more information is needed about the effects of acGD3 when applied ...