A Topographical Atlas of Shiga Toxin 2e Receptor Distribution in the Tissues of Weaned Piglets.

Shiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) is the primary virulence factor in the development of pig edema disease shortly after weaning. Stx2e binds to the globo-series glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galß1-4Glcß1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer), the latter acting as the preferential Stx2e receptor. We determined Stx receptor profiles of 25 different tissues of a male and a female weaned piglet using immunochemical solid phase binding assays combined with mass spectrometry. All probed tissues harbored GSL receptors, ranging from high (category I) over moderate (category II) to low content (category III). Examples of Gb4Cer expression in category I tissues are small intestinal ileum, kidney pelvis and whole blood, followed by colon, small intestinal duodenum and jejunum belonging to category II, and kidney cortex, cerebrum and cerebellum as members of category III organs holding true for both genders. Dominant Gb3Cer and Gb4Cer lipoforms were those with ceramides carrying constant sphingosine (d18:1) and a variable C16:0, C22:0 or C24:1/C24:0 fatty acid. From the mapping data, we created a topographical atlas for Stx2e receptors in piglet tissues and organs, which might be helpful to further investigations on the molecular and cellular mechanisms that underlie infections of Stx2e-producing STEC in pigs and their zoonotic potential for humans.

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.

Characterization of urinary tract infection-associated Shiga toxin-producing Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC), a subgroup of Shiga toxin (Stx)-producing E. coli (STEC), is a leading cause of diarrhea and hemolytic-uremic syndrome (HUS) in humans. However, urinary tract infections (UTIs) caused by this microorganism but not associated with diarrhea have occasionally been reported. We geno- and phenotypically characterized three EHEC isolates obtained from the urine of hospitalized patients suffering from UTIs. These isolates carried typical EHEC virulence markers and belonged to HUS-associated E. coli (HUSEC) clones, but they lacked virulence markers typical of uropathogenic E. coli. One isolate exhibited a localized adherence (LA)-like pattern on T24 urinary bladder epithelial cells. Since the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) are well-known receptors for Stx but also for P fimbriae, a major virulence factor of extraintestinal pathogenic E. coli (ExPEC), the expression of Gb3Cer and Gb4Cer by T24 cells and in murine urinary bladder tissue was examined by thin-layer chromatography and mass spectrometry. We provide data indicating that Stxs released by the EHEC isolates bind to Gb3Cer and Gb4Cer isolated from T24 cells, which were susceptible to Stx. All three EHEC isolates expressed stx genes upon growth in urine. Two strains were able to cause UTI in a murine infection model and could not be outcompeted in urine in vitro by typical uropathogenic E. coli isolates. Our results indicate that despite the lack of ExPEC virulence markers, EHEC variants may exhibit in certain suitable hosts, e.g., in hospital patients, a uropathogenic potential. The contribution of EHEC virulence factors to uropathogenesis remains to be further investigated.

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.

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 AB(5) 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.

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

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 (CD(50) of 0.94 pg/ml) to high refractiveness (CD(50) of 5.8 µg/ml) and to virtual resistance portrayed by non-determinable CD(50) 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.

The human H3N2 influenza viruses A/Victoria/3/75 and A/Hiroshima/52/2005 preferentially bind to α2-3-sialylated monosialogangliosides with fucosylated poly-N-acetyllactosaminyl chains.

Among influenza A viruses, subtype H3N2 is the major cause of human influenza morbidity and is associated with seasonal epidemics causing annually half million deaths worldwide. Influenza A virus infection is initiated via hemagglutinin that binds to terminally sialylated glycoconjugates exposed on the surface of target cells. Gangliosides from human granulocytes were probed using thin-layer chromatography overlay assays for their binding potential to H3N2 virus strains A/Victoria/3/75 and A/Hiroshima/52/2005. Highly polar gangliosides with poly-N-acetyllactosaminyl chains showing low chromatographic mobility exhibited strong virus adhesion which was entirely abolished by sialidase treatment. Auxiliary overlay assays using anti-sialyl Lewis(x) (sLe(x)) monoclonal antibodies showed identical binding patterns compared with those performed with the viruses. A comprehensive structural analysis of fractionated gangliosides by electrospray ionization quadrupole time-of-flight mass spectrometry revealed sLe(x) gangliosides with terminal Neu5Acα2-3Galß1-4(Fucα1-3)GlcNAc epitope and extended neolacto (nLc)-series core structures as the preferential virus binding gangliosides. More precisely, sLe(x) gangliosides with nLc8, nLc10 and nLc12Cer cores, carrying sphingosine (d18:1) and a fatty acid with variable chain length (mostly C24:0, C24:1 or C16:0) in the ceramide moiety and one or two additional internal fucose residues in the oligosaccharide portion, were identified as the preferred receptors recognized by H3N2 virus strains A/Victoria/3/75 and A/Hiroshima/52/2005. This study describes glycan-binding requirements of hemagglutinin beyond binding to glycans with a specific sialic acid linkage of as yet undefined neutrophil receptors acting as ligands for H3N2 viruses. In addition, our results pose new questions on the biological and clinical relevance of this unexpected specificity of a subtype of influenza A viruses.

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.

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.

Normal silica gel and reversed phase thin-layer chromatography coupled with UV spectroscopy and IR-MALDI-o-TOF-MS for the detection of tetracycline antibiotics.

Tetracyclines (TCs) form a group of bacteriostatic antibiotics with closely related structures and similar chemical and physicochemical properties. They are widely employed as therapeutics in human and veterinary medicine. Here, we introduce the combination of UV spectroscopic detection of high-performance thin-layer chromatography (HPTLC)-separated TCs with direct analysis on solid phase using infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF-MS). Normal silica gel phase- and water-wettable hybrid C(18) reversed phase layers (RP-18W) both allowed HPTLC separation and sensitive UV spectroscopic detection followed by MS analysis of distinct TCs bands using the liquid matrix glycerol. The novel approach of direct IR-MALDI-o-TOF-MS analysis resulted in the unequivocal identification of four structurally different TCs employed in this study, and linear calibration curves were produced for analyte amounts from 20 ng to 1 µg. MS analysis of TCs from RP-18W HPTLC plates was found to be superior when compared to the spectra acquired from the silica gel layer. Ionic analytes obtained from the RP-18W surface are mainly detected as protonated species of high abundance accompanied by a reduced formation of adducts as well as background ions arising from the matrix or the stationary phase. This results in decreased complexity of the spectra and enhanced sensitivity of the combinatorial method. An approximate detection limit of 5 ng of individual TCs in mixtures by combining RP-18W HPTLC with IR-MALDI-o-TOF-MS offers a novel timely and cost-efficient method for tracing TCs.

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.

Application of combined high-performance thin-layer chromatography immunostaining and nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometry to the structural characterization of high- and low-affinity binding ligands of Shiga toxin 1.

Shiga toxin 1 (Stx1) represents an AB5 toxin produced by enterohemorrhagic Escherichia coli, which cause gastrointestinal diseases in humans that are often followed by potentially fatal systemic complications, such as acute encephalopathy and hemolytic uremic syndrome. The expression of the preferential Stx1 receptor, Gb3Cer/CD77 (Gal alpha1-4Gal beta1-4Glc beta1-1Cer), is one of the primary determinants of susceptibility to tissue injury. Due to the clinical importance of this life-threatening toxin, a combined strategy of preparative high-performance thin-layer chromatography (HPTLC) overlay assay and mass spectrometry was developed for the detection and structural characterization of Stx1-binding glycosphingolipids (GSLs). A preparation of neutral GSLs from human erythrocytes, comprising 21.4% and 59.1% of the high- and low-affinity Stx1-binding ligands Gb3Cer/CD77 and Gb4Cer, respectively, was separated on silica gel precoated HPTLC plates and probed for the presence of Stx1 receptors. Stx1 positive on the one hand and anti-Gb3Cer/CD77 and anti-Gb4Cer antibody positive bands from parallel reference runs on the other hand were extracted with chloroform/methanol/water (30/60/8, v/v/v). These crude extracts were used without any further purification for a detailed structural analysis by nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) in the negative ion mode. In all extracts investigated, neutral GSLs were detected as singly charged deprotonated molecular ions, [M-H]-, and neither buffer-derived salt adducts nor coextracted contaminants from the overlay assay procedure or the silica gel layer were observed. For the structural characterization of Stx1- and antibody-binding GSLs low-energy collision-induced dissociation (CID) was applied to high and low abundant receptor species of the crude extracts. All MS/MS spectra obtained contained full series of Y-type ions, B-type ions and additional ions generated by ring cleavages of the sugar moiety. Only analytical quantities in the microgram scale of a single GSL species within the complex GSL mixture were required for the structural MS characterization of Stx1 ligands as Gb3Cer/CD77 and Gb4Cer. This effective combined HPTLC/MS procedure offers a broad range of applications, not only for toxins of bacterial origin, but also for any GSL-binding agents such as plant-derived lectins or human proteins with yet unknown binding specificities.

Analysis of gangliosides directly from thin-layer chromatography plates by infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry with a glycerol matrix.

A novel method is presented for direct coupling of high-performance thin-layer chromatography (HPTLC) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of biomolecules. A first key feature is the use of a liquid matrix (glycerol), which provides a homogeneous wetting of the silica gel and a simple and fast MALDI preparation protocol. A second is the use of an Er:YAG infrared laser, which ablates layers of approximately 10-microm thickness of analyte-loaded silica gel and provides a soft desorption/ionization of even very labile analyte molecules. The orthogonal time-of-flight mass spectrometer employed in this study, finally provides a high accuracy of the mass determination, which is independent of any irregularity of the silica gel surface. The analytical potential of the method is demonstrated by the compositional mapping of a native GM3 (II(3)-alpha-Neu5Ac-LacCer) ganglioside mixture from cultured Chinese hamster ovary cells. The analysis is characterized by a high relative sensitivity, allowing the simultaneous detection of various major and minor GM3 species directly from individual HPTLC analyte bands. The lateral resolution of the direct HPTLC-MALDI-MS analysis is defined by the laser focus diameter of currently approximately 200 microm. This allows one to determine mobility profiles of individual species with a higher resolution than by reading off the chromatogram by optical absorption. The fluorescent dye primuline was, furthermore, successfully tested as a nondestructive, MALDI-compatible staining agent.

Oxygenation alters ganglioside expression in rat liver following partial hepatectomy.

Gangliosides from livers of weanling rats were analyzed after 15% partial hepatectomy (PH) and different pre- and post-operative hyberbaric oxygenation (pre- and postHBO). Neu5Ac was the predominant ganglioside-derived sialic acid (>85%) compared to Neu5Gc. Almost identical low total sialic acid content (Neu5Ac+Neu5Gc) of the control and operated nonHBO animals opposed a 6.4- to 7.6-fold increase in pre- and postHBO animals (69.26 and 81.64pmol/mg wet weight, respectively). NanoESI-QTOF mass spectrometry combined with HPTLC immunostaining revealed GM3(Neu5Ac) and GM3(Neu5Gc) as major gangliosides, correlating with the respective sialic acid concentrations. Minor neolacto-series gangliosides were enhanced in preHBO and postHBO, but GM1-core gangliosides only in preHBO rats. GM2 and GalNAc-GM1b were clearly detectable in oxygenated rats compared to traces in the control and nonHBO animals. These results point at a functional role of gangliosides in liver growth regulation and reconstitution after PH combined with pre- and post-operative HBO treatment.

Tumor-associated CD75s gangliosides and CD75s-bearing glycoproteins with Neu5Acalpha2-6Galbeta1-4GlcNAc-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 Neu5Acalpha2-6Galbeta1-4GlcNAc 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.

Direct analysis of silica gel extracts from immunostained glycosphingolipids by nanoelectrospray ionization quadrupole time-of-flight mass spectrometry.

A combined strategy of preparative high-performance thin-layer chromatography overlay assay and mass spectrometry was established for the structural characterization of immunostained glycosphingolipids (GSLs) in silica gel extracts. Crude chloroform/methanol/water (30/60/8, v/v/v) extracts of immunostained TLC bands were analyzed by nanoelectrospray low-energy CID mass spectrometry without further purification. The GSL species investigated were isomeric monosialogangliosides of the neolacto series from a ganglioside preparation of human granulocytes, the disialoganglioside GD3 from a human melanoma lipid extract, and ganglio series Gg3Cer of a neutral GSL preparation from murine lymphoreticular MDAY-D2 cells. For the specific detection of lipid-bound oligosaccharides, polyclonal chicken IgY, murine monoclonal IgG3, and IgM antibodies were used. The resulting mass spectra show that only analytical quantities of approximately 1 microg of a single GSL within a complex mixture are required for the structure determination of immunostained GSLs by MS and MS/MS. All species investigated were detected as singly charged deprotonated molecular ions, and neither buffer-derived salt adducts nor coextracted contaminants from the immunostaining procedure or the silica gel layer were observed. This effective HPTLC-MS-joined procedure offers a wide range of applications for any carbohydrate binding agents such as bacterial toxins, plant lectins, and others.

Mistletoe lectin I is a sialic acid-specific lectin with strict preference to gangliosides and glycoproteins with terminal Neu5Ac alpha 2-6Gal beta 1-4GlcNAc residues.

Mistletoe lectin I (ML-I) is a type II ribosome-inactivating protein, which inhibits the protein biosynthesis at the ribosomal level. ML-I is composed of a catalytically active A-chain with rRNA N-glycosidase activity and a B-chain with carbohydrate binding specificities. Using comparative solid-phase binding assays along with electrospray ionization tandem mass spectrometry, ML-I was shown to preferentially bind to terminally alpha2-6-sialylated neolacto series gangliosides from human granulocytes. IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer were identified as ML-I receptors, whereas the isomeric alpha2-3-sialylated neolacto series gangliosides were not recognized. Only marginal binding of ML-I to terminal galactose residues of neutral glycosphingolipids with a Galbeta1-4Glc or Galbeta1-4GlcNAc sequence was determined, whereas a distal Galalpha1-4Gal, GalNAcbeta1-3Gal, or GalNAcbeta1-4Gal disaccharide did not bind at all. Among the glycoproteins investigated in Western blot and microwell adsorption assays, only those carrying Neu5Acalpha2-6Galbeta1-4GlcNAc residues, exclusively, predominantly, or even as less abundant constituents in an assembly with Neu5Acalpha2-3Galbeta1-4GlcNAc-terminated glycans, displayed high ML-I binding capacity. From our data we conclude that (i) ML-I has to be considered as a sialic acid- and not a galactose-specific lectin and (ii) neolacto series gangliosides and sialoglycoproteins with type II glycans, which share the Neu5Acalpha2-6Galbeta1-4GlcNAc terminus, are true ML-I receptors. This strict preference might help to explain the immunostimulatory potential of ML-I toward certain leukocyte subpopulations and its therapeutic success as a cytotoxic anticancer drug.

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.

Discrimination of neolacto-series gangliosides with alpha2-3- and alpha2-6-linked N-acetylneuraminic acid by nanoelectrospray ionization low-energy collision-induced dissociation tandem quadrupole TOF MS.

A combined strategy of thin-layer chromatography immunostaining and negative ion nanoelectrospray low-energy CID mass spectrometry was established for the differentiation of isomeric alpha2-3 and alpha2-6 sialylated neolacto-series monosialogangliosides from human granulocytes. The gangliosides investigated differed in the ceramide moiety by substitution with C16:0 or C24:1 fatty acid and in their oligosaccharide chains due to nLc4 and nLc6 core structures. With respect to the type of sialylation, the homogeneity of the HPLC-purified ganglioside fractions was verified by use of specific anti-Neu5Acalpha2-3Galbeta1-4GlcNAc-R and anti-Neu5Acalpha2-6Galbeta1-4GlcNAc-R antibodies. A clear-cut series of fragment ions for both types of isomeric gangliosides, carrying alpha2-3- and alpha2-6-linked neuraminic acid, respectively, was obtained by low-energy CID. Additionally, a characteristic ring cleavage was detected exclusively in all species with Neu5Acalpha2-6Galbeta1-4GlcNAc terminus, regardless of ceramide fatty acid and oligosaccharide chain lengths. The diagnostic (0,2)X(4/6) ions, generated by ring cleavage of an alpha2-6-linked neuraminic acid are accompanied by a simultaneous decrease of the corresponding Y(4)/Y(6) ions. These results suggest the unequivocal discrimination of individual alpha2-3- and alpha2-6-sialylated neolacto-series monosialogangliosides by distinct fragmentation patterns in low-energy CID tandem MS.

Influence of targeting ligand flexibility on receptor binding of particulate drug delivery systems.

Receptor-mediated drug targeting via nanoengineered particulate delivery systems is an emerging field. However, little is known about how such magic bullets should be assembled to yield optimal targeting efficiency. Here we investigated the influence of targeting ligand flexibility on binding of ligand-coated microparticles to cell surface receptors. Using the ganglioside G(M1)-binding B subunit of cholera toxin as ligand and fluorescent microparticles as a model delivery system, conjugates with different numbers of linkages between ligand and particle were prepared and tested for their efficiency to bind to live fibroblast monolayers. Our results show that multiple bonds between ligand and particle reduce the targeting rate by up to 50% compared to constructs where ligands are attached via single aliphatic chains. Thus, for maximum performance, targeted particulate drug delivery systems should be assembled such that ligands are attached via single sigma bonds only, allowing the ligand molecules to adopt an optimal binding conformation.