Iris Meisen

Facing glycosphingolipid–Shiga toxin interaction: dire straits for endothelial cells of the human vasculature

The two major Shiga toxin (Stx) types, Stx1 and Stx2, produced by enterohemorrhagic Escherichia coli (EHEC) in particular injure renal and cerebral microvascular endothelial cells after transfer from the human intestine into the circulation. Stxs are AB5 toxins composed of an enzymatically active A subunit and the pentameric B subunit, which preferentially binds to the glycosphingolipid globotriaosylceramide (Gb3Cer/CD77). This review summarizes the current knowledge on Stx-caused cellular injury and the structural diversity of Stx receptors as well as the initial molecular interaction of Stxs with the human endothelium of different vascular beds. The varying lipoforms of Stx receptors and their spatial organization in lipid rafts suggest a central role in different modes of receptor-mediated endocytosis and intracellular destiny of the toxins. The design and development of tailored Stx neutralizers targeting the oligosaccharide–toxin recognition event has become a very real prospect to ameliorate or prevent life-threatening renal and neurological complications.

Thin-layer chromatography, overlay technique and mass spectrometry: a versatile triad advancing glycosphingolipidomics.

Much effort is currently invested in the development of mass spectrometry-based strategies for investigating the entirety of glycosphingolipids (GSLs) of a certain cell type, tissue, organ or body encompassing the respective glycosphingolipidome. As part of the investigation of the vertebrate glycosphingolipidome, GSL analysis is undergoing rapid expansion owing to the application of novel mass spectrometry techniques acting as the linchpin in the network of collaborations challenged to unravel structural and functional aspects of GSLs. Difficulties may arise in the determination of the exact structures of GSLs due to the heterogeneity of the sugar moiety varying in the number and sequence of monosaccharides, and their anomeric configuration and linkage type, which make up the principal items of the glyco code of biologically active carbohydrate chains. The ceramide variability caused by the diversity of the long-chain amino alcohol and the fatty acid, which both may vary in chain length, degree of unsaturation, and type and number of substituents, further contributes to the increasing number of possible GSL species. In view of this heterogeneity, a single-method analytical mass spectrometry (MS) technique without auxiliary tools yields limited data, providing only partial structural information of individual GSLs in complex mixtures. Approaching this challenge, current advances on a triad system matching three complementary methods are described in this review: (i) silica gel based TLC separation of GSLs, (ii) their overlay detection on the TLC plate (mostly based on antibody-mediated recognition), and (iii) direct and indirect MS based structural characterization, i.e. directly on the TLC plate or in lipid extracts from silica gel. We will focus on recent improvements by employing antibodies, AB(5) toxins and bacteria for direct IR-MALDI-o-TOF MS and indirect ESI-QTOF MS analysis of GSLs. We believe that the combinatorial approach using conventional TLC and modern mass spectrometry provides a developmental advance in exploring the glycosphingolipidome of biological material.

Promiscuous Shiga toxin 2e and its intimate relationship to Forssman

Shiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) represents the major virulence factor responsible for the pig edema disease which is characterized by hemorrhagic lesions, neurological disorders and often fatal outcomes. Stx2e-producing strains from the intestine of slaughtered pigs (n = 3), feces of piglets with postweaning diarrhea or edema disease (n = 12) and feces of humans with asymptomatic infections or mild diarrhea (n = 13) were comparatively analyzed for the binding specificities of Stx2e to glycosphingolipids (GSLs) of the globo-series. Besides equivalent binding towards globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), we could demonstrate specific interaction of Stx2e preparations from human and porcine STEC isolates with Forssman GSL. Notably, Forssman GSL was recognized neither by structurally closely related Stx2 nor by Stx1 derived from human STEC isolates conferring Stx2e a unique recognition feature. Noteworthy, 7 (54%) of the 13 human and 8 (53%) of the 15 pig Stx2e samples exhibited cytotoxic action towards human brain microvascular endothelial cells. Our findings provide a basis for further exploring the functional role of the promiscuous receptor repertoire of Stx2e and the exact nature of the mechanisms that underlie different pathological outcomes of Stx2e-producing STEC in humans and pigs.

Direct coupling of high-performance thin-layer chromatography with UV spectroscopy and IR-MALDI orthogonal TOF MS for the analysis of cyanobacterial toxins.

Cyanobacteria are pathogenic prokaryotes and known for producing a high variety of cyclic hepatotoxic peptides in fresh and brackish water. Prominent members of these toxins are microcystin LR (MC LR) and nodularin (Nod), which are under suspicion to cause cancer. Various analytical methods have been reported for the detection of these cyclopeptides, and these are mainly based on liquid chromatography combined with mass spectrometric techniques. Here, we introduce a new approach based on the direct coupling of high-performance thin-layer chromatography (HPTLC) with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF MS) using the liquid matrix glycerol. The analysis of the cyclopeptides involves the application of three complementary methods: (i) HPTLC separation of MC LR and Nod, (ii) their detection and quantification by UV spectroscopy at lambda = 232 nm, and (iii) direct identification of separated analytes on the HPTLC plate by IR-MALDI-o-TOF MS. Calibration curves exhibited a linear relationship of amount of analyte applied for HPTLC and UV absorption (R(2) > 0.99). The limits of detection were 5 ng for UV spectroscopy and 3 ng for mass spectrometric analysis of individual peptides. This novel protocol greatly improves the sensitive determination of toxins from pathogenic cyanobacteria in complex water samples. It was successfully applied to the detection and quantification of MC LR and Nod in a spiked, processed environmental water sample.

Identification of Neuropeptides from the Sinus Gland of the Crayfish Orconectes limosus Using Nanoscale On-line Liquid Chromatography Tandem Mass Spectrometry

In this article, a novel and sensitive analytical strategy for direct characterization of neuropeptides from the X-Organ-sinus gland neurosecretory system of the crayfish Orconectes limosus is presented. A desalted extract corresponding to 0.5 sinus gland equivalents was analyzed in a nanoflow liquid chromatography system coupled to quadrupole time-of-flight tandem mass spectrometry (nanoLC-QTOF MS/MS). The existence and structural identity of four crustacean hyperglycemic hormone precursor-related peptide variants and two new genetic variants of the pigment-dispersing hormone, not detected by conventional chromatographic systems, molecular cloning, or immunochemical methods before, was revealed. The here-presented approach of the combined LC-QTOF MS/MS technique is a powerful tool to discover new peptide hormones in biological systems, due to its sensitivity, accuracy, and speed.

Two genetic variants of the crustacean hyperglycemic hormone (CHH) from the Australian crayfish, Cherax destructor: detection of chiral isoforms due to posttranslational modification

From sinus glands of the Australian crayfish Cherax destructor, two genetic variants of the crustacean hyperglycemic hormone (CHH) were isolated by HPLC and fully characterized by mass spectrometry and Edman sequencing. Both CHH A (8350.38 Da) and CHH B (8370.34 Da) consist of 72 amino acid residues, with pyroGlu as N-terminus and an amidated (Val-NH2) C-terminus. They differ in 14 residues (81% identity). Both sequences are significantly different from those of the hitherto known three CHHs of Astacoidea species (Northern hemisphere crayfish), which among themselves are extremely conserved. This may reflect the long, separate evolution of the Astacoidea lineage and the Parastacoidea (Southern hemisphere crayfish) lineage, to which Cherax belongs. CHH A and CHH B genes are expressed at comparable levels, as indicated by the similar amounts of mature peptides in the sinus gland. In addition to each of the major peptides, which share the identical N-terminal tripeptide pyroGlu-Val-L-Phe, one chiral isoform containing pyroGlu-Val-D-Phe was identified. Compared to the main peptides, the amounts of the D-isoforms are lower, but significant, amounting to 30-40% of L-isoforms. These results demonstrate that two genes can give rise to a total of four different peptides in the secretory terminals of the sinus gland. All peptides gave a highly significant hyperglycemic in vivo response in C. destructor.

Expression of Shiga toxin 2e glycosphingolipid receptors of primary porcine brain endothelial cells and toxin-mediated breakdown of the blood–brain barrier

Shiga toxin (Stx) 2e, released by certain Stx-producing Escherichia coli, is presently the best characterized virulence factor responsible for pig edema disease, which is characterized by hemorrhagic lesions, neurological disorders and often fatal outcomes. Although Stx2e-mediated brain vascular injury is the key event in development of neurologic signs, the glycosphingolipid (GSL) receptors of Stx2e and toxin-mediated impairment of pig brain endothelial cells have not been investigated so far. Here, we report on the detailed structural characterization of Stx2e receptors globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), which make up the major neutral GSLs in primary porcine brain capillary endothelial cells (PBCECs). Various Gb3Cer and Gb4Cer lipoforms harboring sphingenine (d18:1) or sphinganine (d18:0) and mostly a long-chain fatty acid (C20-C24) were detected. A notable batch-to-batch heterogeneity of primary endothelial cells was observed regarding the extent of ceramide hydroxylation of Gb3Cer or Gb4Cer species. Gb3Cer, Gb4Cer and sphingomyelin preferentially distribute to detergent-resistant membrane fractions and can be considered lipid raft markers in PBCECs. Moreover, we employed an in vitro model of the blood-brain barrier (BBB), which exhibited strong cytotoxic effects of Stx2e on the endothelial monolayer and a rapid collapse of the BBB. These data strongly suggest the involvement of Stx2e in cerebral vascular damage with resultant neurological disturbance characteristic of edema disease.

Tumor-associated CD75s gangliosides and CD75s-bearing glycoproteins with Neu5Acα2-6Galβ1-4GlcNAc-residues are receptors for the anticancer drug rViscumin

The anticancer drug rViscumin, currently under clinical development, has been shown in previous studies to be a sialic acid specific ribosome inactivating protein (RIP). Comparative binding assays with the CD75s‐specific monoclonal antibodies HB6 and J3‐89 revealed rViscumin to be a CD75s‐specific RIP due to identical binding characteristics toward CD75s gangliosides. The receptor gangliosides are IV6nLc4Cer, VI6nLc6Cer, and the newly characterized ganglioside VIII6nLc8Cer, all three carrying the Neu5Acα2‐6Galβ1‐4GlcNAc motif. To elucidate the clinical potential of the rViscumin targets, CD75s gangliosides were determined in several randomly collected gastrointestinal tumors. The majority of the tumors showed an enhanced expression of CD75s gangliosides compared with the unaffected tissues. The rViscumin binding specificity was further investigated with reference glycoproteins carrying sialylated and desialylated type II N‐glycans. Comparative Western blots of rViscumin and ricin, an rViscumin homologous but galactoside‐specific RIP, revealed specific recognition of type II N‐glycans with CD75s determinants by rViscumin, whereas ricin failed to react with terminally sialylated oligosaccharides such as CD75s motifs and others. This strict binding specificity of rViscumin and the increased expression of CD75s gangliosides in various tumors suggest this anticancer drug as a promising candidate for an individualised adjuvant therapy of human tumors.

Shiga toxin glycosphingolipid receptor expression and toxin susceptibility of human pancreatic ductal adenocarcinomas of differing origin and differentiation

Abstract Shiga toxins (Stxs) are composed of an enzymatically active A subunit (StxA) and a pentameric B subunit (StxB) that preferentially binds to the glycosphingolipid (GSL) globo\xadtriaosylceramide (Gb3Cer/CD77) and to a reduced extent to globotetraosylceramide (Gb4Cer). The identification of Gb3Cer as a tumor-associated GSL in human pancreatic cancer prompted us to investigate the expression of Gb3Cer and Gb4Cer in 15 human pancreatic ductal adenocarcinoma cell lines derived from primary tumors and liver, ascites, and lymph node metastases. Thin-layer chromatography overlay assays revealed the occurrence of Gb3Cer in all and of Gb4Cer in the majority of cell lines, which largely correlated with transcriptional expression analysis of Gb3Cer and Gb4Cer synthases. Prominent Gb3Cer and Gb4Cer lipoform heterogeneity was based on ceramides carrying predominantly C16:0 and C24:0/C24:1 fatty acids. Stx2-mediated cell injury ranged from extremely high sensitivity (CD50 of 0.94 pg/ml) to high refractiveness (CD50 of 5.8 μg/ml) and to virtual resistance portrayed by non-determinable CD50 values even at the highest Stx2 concentration (10 μg/ml) applied. Importantly, Stx2-mediated cytotoxicity did not correlate with Gb3Cer expression (the preferential Stx receptor), suggesting that the GSL receptor content does not primarily determine cell sensitivity and that other, yet to be delineated, cellular factors might influence the responsiveness of cancer cells.

Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes

Haemolytic anaemia is one of the characteristics of life‐threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes. We employed an ex vivo erythropoiesis model using mobilized CD34+ haematopoietic stem/progenitor cells from human blood and monitored expression of Stx receptors and Stx2a‐mediated cellular injury of developing erythrocytes. CD34+ haematopoietic stem/progenitor cells were negative for Stx2a receptors and resistant towards the toxin. Expression of Stx2a‐binding glycosphingolipids and toxin sensitivity was apparent immediately after initiation of erythropoietic differentiation, peaked for basophilic and polychromatic erythroblast stages and declined during maturation into orthochromatic erythroblasts and reticulocytes, which became highly refractory to Stx2a. The observed Stx‐mediated toxicity towards erythroblasts during the course of erythropoiesis might contribute, although speculative at this stage of research, to the anaemia caused by Stx‐producing pathogens.