ABSTRACT
GPx8 is a glutathione peroxidase homolog inserted in the membranes of endoplasmic reticulum (ER), where it seemingly plays a role in controlling redox status by preventing the spill of H2O2. We addressed the impact of GPx8 silencing on the lipidome of microsomal membranes, using stably GPx8-silenced HeLa cells. The two cell lines were clearly separated by Principal Component Analysis (PCA) and Partial Least Square Discriminant analysis (PLS-DA) of lipidome. Considering in detail the individual lipid classes, we observed that unsaturated glycerophospholipids (GPL) decreased, while only in phosphatidylinositols (PI) a substitution of monounsaturated fatty acids (MUFA) for polyunsaturated fatty acids (PUFA) was observed. Among sphingolipids (SL), ceramides (CER) decreased while sphingomyelins (SM) and neutral glycophingolipids (nGSL) increased. Here, in addition, longer chains than in controls in the amide fatty acid were present. The increase up to four folds of the CER (d18:1; c24:0) containing three hexose units, was the most remarkable species increasing in the differential lipidome of siGPx8 cells. Quantitative RT-PCR complied with lipidomic analysis specifically showing an increased expression of: i) acyl-CoA synthetase 5 (ACSL5); ii) CER synthase 2 and 4; iii) CER transporter (CERT); iv) UDP-glucosyl transferase (UDP-GlcT), associated to a decreased expression of UDP-galactosyl transferase (UDP-GalT). A role of the unfolded protein response (UPR) and the spliced form of the transcription factor XBP1 on the transcriptional changes of GPx8 silenced cells was ruled-out. Similarly, also the involvement of Nrf2 and NF-κB. Altogether our results indicate that GPx8-silencing of HeLa yields a membrane depleted by about 24% of polyunsaturated GPL and a corresponding increase of saturated or monounsaturated SM and specific nGSL. This is tentatively interpreted as an adaptive mechanism leading to an increased resistance to radical oxidations. Moreover, the marked shift of fatty acid composition of PI emerges as a possibly relevant issue in respect to the impact of GPx8 on signaling pathways.
Subject(s)
Endoplasmic Reticulum , Hydrogen Peroxide , Ceramides , Glutathione Peroxidase/genetics , HeLa Cells , Humans , PeroxidasesABSTRACT
Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed 11 single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched lacto-N-neohexaose (Galß4GlcNAcß6[Galß4GlcNAcß3]Galß4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity.
Subject(s)
Enterotoxigenic Escherichia coli/genetics , Enterotoxins/chemistry , Enterotoxins/genetics , Models, Molecular , Mutagenesis, Site-Directed , Binding Sites , Carbohydrates/chemistry , Crystallography, X-Ray , Humans , Molecular Conformation , Protein Binding , Structure-Activity Relationship , Surface Plasmon ResonanceABSTRACT
Glucosylceramides (GlcCer) are the main neutral glycosphingolipids expressed in fungal cells. In this work, glucosylceramides (GlcCer) were extracted from three strains of Scedosporium (Pseudallescheria) boydii, one strain of Pseudallescheria ellipsoidea and one strain of Pseudallescheria angusta and purified by several chromatographic steps. Using high-performance thin layer chromatography (HPTLC), we found a similarity between GlcCer obtained from all of the analysed strains. A detailed structural analysis of the P. ellipsoidea GlcCer was performed via electrospray ionization mass spectrometry (ESI-MS) and confirmed in 1- and 2-D heteronuclear NMR experiments ((1)H-(13) C HSQC). GlcCer species produced by mycelial forms of these strains displayed the same structure previously demonstrated by our group for P. boydii, Cryptococcus neoformans, Pseudallescheria minustipora, Fusarium solani, and Colletotrichum gloesporioides. A monoclonal antibody (mAb) against GlcCer was used for immunofluorescence experiments. Our results revealed that GlcCer is present on the surface of these fungi, and no difference was observed in the GlcCer structure of the present set of strains in terms of geographic or clinical origin, suggesting a conserved GlcCer structure similar to those previously described for Scedosporium apiospermum, Scedosporium aurantiacum, and P. minutispora. The surface distribution of GlcCer in these fungi is suggestive of the involvement of this molecule in fungal growth.
Subject(s)
Glucosylceramides/chemistry , Mycoses/microbiology , Pseudallescheria/metabolism , Scedosporium/metabolism , Glucosylceramides/metabolism , Humans , Molecular Structure , Pseudallescheria/chemistry , Pseudallescheria/isolation & purification , Scedosporium/chemistry , Scedosporium/isolation & purification , Spectrometry, Mass, Electrospray IonizationABSTRACT
In order to evaluate the relationship between MDR of ovarian cancer and expression of neutral glycosphingolipids(N-GSLs) in ovarian cancer cell lines, the effects of TAM and VRP on the growth of COC1/DDP were assayed by MTT method. N-GSLs of the cells were isolated and purified with the modified Hakamoris method and analysed by HPTLC. The results showed that the expression of N-GSLs was different between parent cell lines and resistant cell sublines, the level of CMH was higher in COC1/DDP than in COC1. TAM and VRP could render multidrug-resistant cells sensitive to chemotherapy, while the level of CMH concomitantly was sharply decreased. It suggested that the expression of N-GSLs is associated with MDR of ovarian cancer, and CMH may be a kind of MDR related glycolipids in ovarian cancer.
ABSTRACT
To investigate the role of tumor multidrug resistance (MDR) associated with CMH in the immunological evasion of MDR tumor cells, CMH was separated from neutral glycolipids of KBv 200by column chromatography, and the expression of B7 in DC was detected by flow cytometry. The results showed that the expression of B7 in DC was inhibited by CMH, it suggested the tumor MDR associated with glycolipids may inhibit human immune response by its effects on DC.
ABSTRACT
In order to study the relationship between the expression of neutral glycosphingolipids(N-GSLs) and MDR, N-GSLs in MDR cell line KBv 200 and its parental cell line KB were studied. The N-GSLs were extracted and purified from the KB and KBv 200 cells according to the modified method of Hakomori, then analysed by High Performance Thin Layer Chromatography (HPTLC). The effects of PPMP on the expression of N-GSLs in KBv 200 cell line were also studied by the above method, its reversion on the KBv 200 cells to Vincristine (VCR) was examined by MTT method. The results showed that the levels of CMH and CDH in KBv 200 cells were higher than in KB cells, CMH was much more markedly increased, PPMP could reverse MDR by inhibiting CMH synthesis. It suggested that CMH is MDR associated N-GSLs in KBv 200 cell line, and inhibition of CMH expression in tumor cells maybe a new way to reverse MDR.
ABSTRACT
To investigate a simple method visualizing neutral glycosphingolipid (N-GSLs) with iodine vapor, N-GSLs were isolated from the normal brain tissue of human by a modificated method of Hakomori, and then analyzed by HPTLC and visualized respectively by spraying aniline-diphenylamine-H 3PO 4 reagent and immersing into a jar full of iodine vapor. The plate should be kept 110℃ for 5 min until the coloration emerged by using the first method,and the N-GSLs were slowly made visible with a blue coloration. The N-GSLs were immediately made visible with a red-brown coloration by using the second method, and the color visible on the plates could emerge again after vaporized. Moreover, the second method was more sensitive than the first. The location of individual N-GSLs was the same by using the two methods. It suggested that a new method for visualizing N-GSLs has been established, it is simple, economic, rapid, innocuity and more sensitive, and the results are reproducible.
