Globoside Is an Essential Intracellular Factor Required for Parvovirus B19 Endosomal Escape.

Human parvovirus B19 (B19V), like most parvoviruses, possesses phospholipase A2 (PLA2) activity, which is thought to mediate endosomal escape by membrane disruption. Here, we challenge this model and find evidence for a mechanism of B19V entry mediated by the glycosphingolipid globoside without endosome disruption and retrograde transport to the Golgi. We show that B19V PLA2 activity requires specific calcium levels and pH conditions that are not optimal in endosomes. Accordingly, endosomal membrane integrity was maintained during B19V entry. Furthermore, endosomes remained intact when loaded with MS2 bacteriophage particles pseudotyped with multiple B19V PLA2 subunits, providing superior enzymatic potential compared to native B19V. In globoside knockout cells, incoming viruses are arrested in the endosomal compartment and the infection is blocked. Infection can be rescued by promoting endosomal leakage with polyethyleneimine (PEI), demonstrating the essential role of globoside in facilitating endosomal escape. Incoming virus colocalizes with Golgi markers and interfering with Golgi function blocks infection, suggesting that globoside-mediated entry involves the Golgi compartment, which provides conditions favorable for the lipolytic PLA2. Our study challenges the current model of B19V entry and identifies globoside as an essential intracellular receptor required for endosomal escape.

Globoside and the mucosal pH mediate parvovirus B19 entry through the epithelial barrier.

Parvovirus B19 (B19V) is transmitted primarily via the respiratory route, however, the mechanism involved remains unknown. B19V targets a restricted receptor expressed in erythroid progenitor cells in the bone marrow. However, B19V shifts the receptor under acidic conditions and targets the widely expressed globoside. The pH-dependent interaction with globoside may allow virus entry through the naturally acidic nasal mucosa. To test this hypothesis, MDCK II cells and well-differentiated human airway epithelial cell (hAEC) cultures were grown on porous membranes and used as models to study the interaction of B19V with the epithelial barrier. Globoside expression was detected in polarized MDCK II cells and the ciliated cell population of well-differentiated hAEC cultures. Under the acidic conditions of the nasal mucosa, virus attachment and transcytosis occurred without productive infection. Neither virus attachment nor transcytosis was observed under neutral pH conditions or in globoside knockout cells, demonstrating the concerted role of globoside and acidic pH in the transcellular transport of B19V. Globoside-dependent virus uptake involved VP2 and occurred by a clathrin-independent pathway that is cholesterol and dynamin-dependent. This study provides mechanistic insight into the transmission of B19V through the respiratory route and reveals novel vulnerability factors of the epithelial barrier to viruses.

CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors.

The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the ß-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the ß-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay-Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the ß-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the ß-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.

Glycosphingolipids are mediators of cancer plasticity through independent signaling pathways.

The molecular repertoire promoting cancer cell plasticity is not fully elucidated. Here, we propose that glycosphingolipids (GSLs), specifically the globo and ganglio series, correlate and promote the transition between epithelial and mesenchymal cells. The epithelial character of ovarian cancer remains stable throughout disease progression, and spatial glycosphingolipidomics reveals elevated globosides in the tumor compartment compared with the ganglioside-rich stroma. CRISPR-Cas9 knockin mediated truncation of endogenous E-cadherin induces epithelial-to-mesenchymal transition (EMT) and decreases globosides. The transcriptomics analysis identifies the ganglioside-synthesizing enzyme ST8SIA1 to be consistently elevated in mesenchymal-like samples, predicting poor outcome. Subsequent deletion of ST8SIA1 induces epithelial cell features through mTORS2448 phosphorylation, whereas loss of globosides in ΔA4GALT cells, resulting in EMT, is accompanied by increased ERKY202/T204 and AKTS124. The GSL composition dynamics corroborate cancer cell plasticity, and further evidence suggests that mesenchymal cells are maintained through ganglioside-dependent, calcium-mediated mechanisms.

Primary Human Colon Epithelial Cells (pHCoEpiCs) Do Express the Shiga Toxin (Stx) Receptor Glycosphingolipids Gb3Cer and Gb4Cer and Are Largely Refractory but Not Resistant towards Stx.

Shiga toxin (Stx) is released by enterohemorrhagic Escherichia coli (EHEC) into the human intestinal lumen and transferred across the colon epithelium to the circulation. Stx-mediated damage of human kidney and brain endothelial cells and renal epithelial cells is a renowned feature, while the sensitivity of the human colon epithelium towards Stx and the decoration with the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galß1-4Glcß1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) is a matter of debate. Structural analysis of the globo-series GSLs of serum-free cultivated primary human colon epithelial cells (pHCoEpiCs) revealed Gb4Cer as the major neutral GSL with Cer (d18:1, C16:0), Cer (d18:1, C22:1/C22:0) and Cer (d18:1, C24:2/C24:1) accompanied by minor Gb3Cer with Cer (d18:1, C16:0) and Cer (d18:1, C24:1) as the dominant lipoforms. Gb3Cer and Gb4Cer co-distributed with cholesterol and sphingomyelin to detergent-resistant membranes (DRMs) used as microdomain analogs. Exposure to increasing Stx concentrations indicated only a slight cell-damaging effect at the highest toxin concentration of 1 µg/mL for Stx1a and Stx2a, whereas a significant effect was detected for Stx2e. Considerable Stx refractiveness of pHCoEpiCs that correlated with the rather low cellular content of the high-affinity Stx-receptor Gb3Cer renders the human colon epithelium questionable as a major target of Stx1a and Stx2a.

Human parvovirus B19 interacts with globoside under acidic conditions as an essential step in endocytic trafficking.

The glycosphingolipid (GSL) globoside (Gb4) is essential for parvovirus B19 (B19V) infection. Historically considered the cellular receptor of B19V, the role of Gb4 and its interaction with B19V are controversial. In this study, we applied artificial viral particles, genetically modified cells, and specific competitors to address the interplay between the virus and the GSL. Our findings demonstrate that Gb4 is not involved in the binding or internalization process of the virus into permissive erythroid cells, a function that corresponds to the VP1u cognate receptor. However, Gb4 is essential at a post-internalization step before the delivery of the single-stranded viral DNA into the nucleus. In susceptible erythroid Gb4 knockout cells, incoming viruses were arrested in the endosomal compartment, showing no cytoplasmic spreading of capsids as observed in Gb4-expressing cells. Hemagglutination and binding assays revealed that pH acts as a switch to modulate the affinity between the virus and the GSL. Capsids interact with Gb4 exclusively under acidic conditions and dissociate at neutral pH. Inducing a specific Gb4-mediated attachment to permissive erythroid cells by acidification of the extracellular environment led to a non-infectious uptake of the virus, indicating that low pH-mediated binding to the GSL initiates active membrane processes resulting in vesicle formation. In summary, this study provides mechanistic insight into the interaction of B19V with Gb4. The strict pH-dependent binding to the ubiquitously expressed GSL prevents the redirection of the virus to nonpermissive tissues while promoting the interaction in acidic intracellular compartments as an essential step in infectious endocytic trafficking.

Shiga Toxin (Stx)-Binding Glycosphingolipids of Primary Human Renal Cortical Epithelial Cells (pHRCEpiCs) and Stx-Mediated Cytotoxicity.

Human kidney epithelial cells are supposed to be directly involved in the pathogenesis of the hemolytic-uremic syndrome (HUS) caused by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC). The characterization of the major and minor Stx-binding glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), respectively, of primary human renal cortical epithelial cells (pHRCEpiCs) revealed GSLs with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Using detergent-resistant membranes (DRMs) and non-DRMs, Gb3Cer and Gb4Cer prevailed in the DRM fractions, suggesting their association with microdomains in the liquid-ordered membrane phase. A preference of Gb3Cer and Gb4Cer endowed with C24:0 fatty acid accompanied by minor monounsaturated C24:1-harboring counterparts was observed in DRMs, whereas the C24:1 fatty acid increased in relation to the saturated equivalents in non-DRMs. A shift of the dominant phospholipid phosphatidylcholine with saturated fatty acids in the DRM to unsaturated species in the non-DRM fractions correlated with the GSL distribution. Cytotoxicity assays gave a moderate susceptibility of pHRCEpiCs to the Stx1a and Stx2a subtypes when compared to highly sensitive Vero-B4 cells. The results indicate that presence of Stx-binding GSLs per se and preferred occurrence in microdomains do not necessarily lead to a high cellular susceptibility towards Stx.

Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry.

The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, Pk) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid substitution (p.Q211E) ramps up the enzyme's promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galß1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.

The binding mechanism of the virulence factor Streptococcus suis adhesin P subtype to globotetraosylceramide is associated with systemic disease.

Streptococcus suis is part of the pig commensal microbiome but strains can also be pathogenic, causing pneumonia and meningitis in pigs as well as zoonotic meningitis. According to genomic analysis, S. suis is divided into asymptomatic carriage, respiratory and systemic strains with distinct genomic signatures. Because the strategies to target pathogenic S. suis are limited, new therapeutic approaches are needed. The virulence factor S. suis adhesin P (SadP) recognizes the galabiose Galα1-4Gal-oligosaccharide. Based on its oligosaccharide fine specificity, SadP can be divided into subtypes PN and PO We show here that subtype PN is distributed in the systemic strains causing meningitis, whereas type PO is found in asymptomatic carriage and respiratory strains. Both types of SadP are shown to predominantly bind to pig lung globotriaosylceramide (Gb3). However, SadP adhesin from systemic subtype PN strains also binds to globotetraosylceramide (Gb4). Mutagenesis studies of the galabiose-binding domain of type PN SadP adhesin showed that the amino acid asparagine 285, which is replaced by an aspartate residue in type PO SadP, was required for binding to Gb4 and, strikingly, was also required for interaction with the glycomimetic inhibitor phenylurea-galabiose. Molecular dynamics simulations provided insight into the role of Asn-285 for Gb4 and phenylurea-galabiose binding, suggesting additional hydrogen bonding to terminal GalNAc of Gb4 and the urea group. Thus, the Asn-285-mediated molecular mechanism of type PN SadP binding to Gb4 could be used to selectively target S. suis in systemic disease without interfering with commensal strains, opening up new avenues for interventional strategies against this pathogen.

Expression of Ganglioside Disialosyl Globopentaosyl Ceramide in Prostate Biopsy Specimens as a Predictive Marker for Recurrence after Radical Prostatectomy.

Carbohydrate antigens are associated with carcinogenesis, cancer invasion, and metastasis and their expression reflect biological activities of various cancers. We previously reported that expression of disialosyl globopentaosyl ceramide (DSGb5), one of carbohydrate antigens, in radical prostatectomy specimens independently predicted biochemical recurrence (i.e., elevating serum prostate specific antigen without recurrent lesions in the image) after radical prostatectomy. However, it is important to evaluate the prognosis at the diagnosis. In this study we investigated DSGb5 expression in prostate biopsy specimens to develop a novel biomarker for providing appropriate management. Between 2005 and 2011, patients who underwent both prostate biopsy and radical prostatectomy in our institution were included. The median follow-up period was 88 months. DSGb5 expression was assessed by immunohistochemical staining and defined 116 patients as high DSGb5 expression (42 patients) or low DSGb5 expression (74 patients). High DSGb5 expression was significantly associated with lymphovascular invasion in radical prostatectomy specimens on both univariate and multivariable analyses (p = 0.028, 0.027). On multivariable analysis, Gleason Score in prostatectomy specimen, positive resection margin, and DSGb5 expression in the biopsy specimen were independently associated with biochemical recurrence-free survival following radical prostatectomy (p = 0.004, 0.008, 0.024). When targeting only patients with negative resection margin, DSGb5 expression was significantly associated with biochemical recurrence-free survival on both univariate and multivariable analyses (p = 0.006, 0.007). DSGb5 expression in prostate biopsy specimens is predictive of lymphovascular invasion and biochemical recurrence-free survival following radical prostatectomy. DSGb5 is a potential biomarker for preoperatively predicting oncological outcomes of prostate cancer.

Blood group P1 antigen-bearing glycoproteins are functional but less efficient receptors of Shiga toxin than conventional glycolipid-based receptors.

Shiga toxin (STx) is a virulence factor produced by enterohemorrhagic Escherichia coli. STx is taken up by mammalian host cells by binding to the glycosphingolipid (GSL) globotriaosylceramide (Gb3; Galα1-4Galß1-4Glc-ceramide) and causes cell death after its retrograde membrane transport. However, the contribution of the hydrophobic portion of Gb3 (ceramide) to STx transport remains unclear. In pigeons, blood group P1 glycan antigens (Galα1-4Galß1-4GlcNAc-) are expressed on glycoproteins that are synthesized by α1,4-galactosyltransferase 2 (pA4GalT2). To examine whether these glycoproteins can also function as STx receptors, here we constructed glycan-remodeled HeLa cell variants lacking Gb3 expression but instead expressing pA4GalT2-synthesized P1 glycan antigens on glycoproteins. We compared STx binding and sensitivity of these variants with those of the parental, Gb3-expressing HeLa cells. The glycan-remodeled cells bound STx1 via N-glycans of glycoproteins and were sensitive to STx1 even without Gb3 expression, indicating that P1-containing glycoproteins also function as STx receptors. However, these variants were significantly less sensitive to STx than the parent cells. Fluorescence microscopy and correlative light EM revealed that the STx1 B subunit accumulates to lower levels in the Golgi apparatus after glycoprotein-mediated than after Gb3-mediated uptake but instead accumulates in vacuole-like structures probably derived from early endosomes. Furthermore, coexpression of Galα1-4Gal on both glycoproteins and GSLs reduced the sensitivity of cells to STx1 compared with those expressing Galα1-4Gal only on GSLs, probably because of competition for STx binding or internalization. We conclude that lipid-based receptors are much more effective in STx retrograde transport and mediate greater STx cytotoxicity than protein-based receptors.

LecA (PA-IL): A Galactose-Binding Lectin from Pseudomonas aeruginosa.

LecA/PA-IL (Pfam PF07828) is a soluble galactose-binding lectin from bacterium Pseudomonas aeruginosa. The lectin is specific for α-galactose present on glycosphingolipids of the globoside family and has therefore been proposed to play a role in cell adhesion and in internalization of bacteria in epithelial cells. The lectin has also direct toxic activity. Search for high-affinity inhibitors can be performed on the recombinant lectin, with use of surface plasmon resonance assays and structural studies.

Globoside Is Dispensable for Parvovirus B19 Entry but Essential at a Postentry Step for Productive Infection.

Globoside (Gb4) is considered the primary receptor of parvovirus B19 (B19V); however, its expression does not correlate well with the attachment and restricted tropism of the virus. The N terminus of VP1 (VP1u) of B19V interacts with an as-yet-unknown receptor required for virus internalization. In contrast to Gb4, the VP1u cognate receptor is expressed exclusively in cells that B19V can internalize. With the aim of clarifying the role of Gb4 as a B19V receptor, we knocked out the gene B3GalNT1 coding for the enzyme globoside synthase in UT7/Epo cells. Consequently, B3GalNT1 transcripts and Gb4 became undetectable in the knockout (KO) cells without affecting cell viability and proliferation. Unexpectedly, virus attachment, internalization, and nuclear targeting were not disturbed in the KO cells. However, NS1 transcription failed, and consequently, genome replication and capsid protein expression were abrogated. The block could be circumvented by transfection with a B19V infectious clone, indicating that Gb4 is not required after the generation of viral double-stranded DNA with resolved inverted terminal repeats. While in wild-type (WT) cells, occupation of the VP1u cognate receptor with recombinant VP1u disturbed virus binding and blocked the infection, antibodies against Gb4 had no significant effect. In a mixed population of WT and KO cells, B19V selectively infected WT cells. This study demonstrates that Gb4 does not have the expected receptor function, as it is dispensable for virus entry; however, it is essential for productive infection, explaining the resistance of the rare individuals lacking Gb4 to B19V infection.IMPORTANCE Globoside has long been considered the primary receptor of B19V. However, its expression does not correlate well with B19V binding and uptake and cannot explain the pathogenesis or the remarkable narrow tissue tropism of the virus. By using a knockout cell line, we demonstrate that globoside does not have the expected function as a cell surface receptor required for B19V entry, but it has an essential role at a postentry step for productive infection. This finding explains the natural resistance to infection associated with individuals lacking globoside, contributes to a better understanding of B19V restricted tropism, and offers novel strategies for the development of antiviral therapies.

[Current status and future prospect of enzyme replacement therapy for Fabry disease].

Fabry disease is characterized by deficient activity of α-galactosidase A, which results in accumulation of glycolipids, such as globotriaosylceremide, in various tissue. Clinical symptoms are varied. In childhood, pain in extremities, hypohidrosis, and angiokeratoma are main symptoms, In adulthood, renal, cardiac and cerebrovascular symptoms are occurred In past, only symptomatic treatments were available. In early 2000th, enzyme replacement therapy was developed after positive results of clinical trials. Ten years after approval, the data of long term safety and efficacy of enzyme replacement.

The P1 histo-blood group antigen is present on human red blood cell glycoproteins.

BACKGROUND: The P1 antigen was first described in 1927 and belongs to the P1PK histo-blood group system, together with Pk and NOR. The A4GALT-encoded 4-α-galactosyltransferase synthesizes these antigens and has been considered to extend glycolipids exclusively. However, contradicting studies have been published regarding the presence of P1 on human glycoproteins. In other species, P1 occurs on glycoproteins. Furthermore, human ABH antigens occur on both glycolipids and glycoproteins and are biochemically related to P1. Thus, we hypothesized that P1 is present on RBC glycoproteins in humans. STUDY DESIGN AND METHODS: RBCs of known P1 /P2 status (phenotype and rs8138197 genotype) were used. The RBC surface glycans were modified with α-galactosidases, papain, and/or peptide-N-glycosidase F. RBC membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblot. A new P 1 /P 2 -allelic discrimination assay based on rs5751348 was validated. RESULTS: P1 occurs on various glycoproteins, seen as smearlike patterns in anti-P1-stained immunoblots with RBC membranes of P1 but not P2 or p phenotype. There was a significant difference between the staining of P 1 -homozygous and P 1 -heterozygous RBCs (P 1 P 1 > P 1 P 2 ), as well as intragenotypic variation. Immunoblotting banding patterns show major carriers at approximately 50 and 100 kDa. P1 staining was lost after treatment of RBCs with α-galactosidase of broad Galα-1,3/4/6-specificity. Peptide-N-glycosidase F treatment reduced the P1 signal, while papain or α-1,3-specific galactosidase did not. P 1 /P 2 status was confirmed by a new rs5751348 assay. CONCLUSION: Our data indicate that the P1 antigen can reside on human RBC glycoproteins. Glycosidase studies suggest that at least part of the epitopes occur on N-glycans.

Identification of lysosomal and extralysosomal globotriaosylceramide (Gb3) accumulations before the occurrence of typical pathological changes in the endomyocardial biopsies of Fabry disease patients.

PURPOSE: Evaluation standards and treatment initiation timing have been debated for a long time, particularly for late-onset Fabry disease (FD), because of its slow progression. However, early initiation of enzyme replacement therapy (ERT) for FD could be effective in stabilizing the disease progression and potentially preventing irreversible organ damage. We aimed to examine globotriaosylceramide (Gb3) deposits in patients' endomyocardial biopsies to understand the early pathogenesis of FD cardiomyopathy. METHODS: Immunofluorescent (IF) staining of Gb3 and lysosomal-associated membrane protein 1 (LAMP-1) was performed on endomyocardial biopsies of patients suspected of Fabry cardiomyopathy who had negative or only slight Gb3 accumulation determined by toluidine blue staining and electron microscopic examination. RESULTS: The IF staining results revealed that all patients examined had abundant Gb3 accumulation in their cardiomyocytes, including the ones who are negative for inclusion bodies. Furthermore, we found that early Gb3 deposits were mostly confined within lysosomes, while they appeared extralysosomally at a later stage. CONCLUSION: A significant amount of lysosomal Gb3 deposits could be detected by IF staining in cardiac tissue before the formation of inclusion bodies, suggesting the cardiomyocytes might have been experiencing cellular stress and damage early on, before the appearance of typical pathological changes of FD during the disease progression.

Glycolipid iGb3 feedback amplifies innate immune responses via CD1d reverse signaling.

The cross-talk between cellular lipid metabolism and the innate immune responses remains obscure. In addition to presenting lipid antigens to Natural Killer T-cells (NKT cells), the Cluster of Differentiation 1D Glycoprotein (CD1d) might mediate reverse signaling in antigen-presenting cells (APCs). Here we found CD1d deficiency attenuated Toll-like receptor (TLR)-triggered inflammatory innate responses in macrophages and dendritic cells, protecting mice from endotoxin shock. TLR activation in macrophages induced metabolic changes of glycosphingolipids (GSLs), among which glycolipid isoglobotrihexosylceramide (iGb3) was rapidly produced. The endogenously generated iGb3 bound CD1d in endosomal compartments and then synergized with the initially activated TLR signal to induce Tyr332 phosphorylation of CD1d intracellular domain. This led to the recruitment and activation of proline-rich tyrosine kinase 2 (Pyk2). Pyk2 interacted with IκB kinase ß (IKKß) and TANK-binding kinase 1 (TBK1), and enhanced tyrosine phosphorylation of Tyr188/199 of IKKß and Tyr179 of TBK1 and thus, their activation to promote full activation of TLR signaling. Thus, intracellular CD1d reverse signaling, triggered by endogenous iGb3, amplifies inflammatory innate responses in APCs. Our findings identify a non-canonical function of CD1d reverse signaling activated by lipid metabolite in the innate immune response.

Shiga toxin-glycosphingolipid interaction: Status quo of research with focus on primary human brain and kidney endothelial cells.

Shiga toxin (Stx)-mediated injury of the kidneys and the brain represent the major extraintestinal complications in humans upon infection by enterohemorrhagic Escherichia coli (EHEC). Damage of renal and cerebral endothelial cells is the key event in the pathogenesis of the life-threatening hemolytic uremic syndrome (HUS). Stxs are AB5 toxins and the B-pentamers of the two clinically important Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid globotriaosylceramide (Gb3Cer, Galα4Galß4Glcß1Cer) and to less extent to globotetraosylceramide (Gb4Cer, GalNAcß3Galα4Galß4Glcß1), which are expected to reside in lipid rafts in the plasma membrane of the human endothelium. This review summarizes the current knowledge on the Stx glycosphingolipid receptors and their lipid membrane ensemble in primary human brain microvascular endothelial cells (pHBMECs) and primary human renal glomerular endothelial cells (pHRGECs). Increasing knowledge on the precise initial molecular mechanisms by which Stxs interact with cellular targets will help to develop specific therapeutics and/or preventive measures to combat EHEC-caused diseases.

Protocols for Glycosyltransferase Assays: Ganglioside Globoside and Lewis-X Intermediate-Lactosylceramide Biosyntheses in Eukaryotic Systems.

Protocols for assay of 24 different Glycolipid-Glycosyltransferases (GSL-GLTs) of the eukaryotic systems are described. Problems of quantitating the activities in crude membranes are also described. Different separation methods (for separation of substrate, donors, and the product of the reaction) have been described based on the paper chromatography or high voltage paper electrophoresis in 1.0% Na2B4O7. Liquid Scintillation counting system was used for quantitation of the enzymatic product. In the assay of each GSL-GLT it is recommended to compare the selected method to be used with the exact conditions used by the authors published previously. As a test case for these assays the following kinetic parameters for Lactosylceramide Synthase, GalT-2 (UDP-Gal: Glc-Cer ß1-4-galactosyltransferase), (Km of glucosylceramide = 1.65 × 10-4 M; Km for UDP-Gal = 0.5 × 10-4 M; V max is determined in the presence of optimum detergent concentrations (2-15 mg/ml of Cutscum-Triton X-100, 2:1); Mn++ and Mg++, 10-20 mM) has been reported. The importance of use of GalT-2 assay method (as a model system) in the purified Golgi-rich membranes from 13-day-old embryonic chicken brains (13-ECB) is described.

Overview of the role of Shiga toxins in porcine edema disease pathogenesis.

Shiga toxin-producing Escherichia coli (STEC) have been implicated as the cause of enterotoxemias, such as hemolytic uremic syndrome in humans and edema disease (ED) of pigs. Stx1 and Stx2 are the most common types found in association with illness, but only Stx2e is associated with disease in the animal host. Porcine edema disease is a serious affection which can lead to dead causing great losses of weaned piglets. Stx2e is the most frequent Stx variant found in porcine feces and is considered the key virulence factor involved in the pathogenesis of porcine edema disease. Stx2e binds with higher affinity to Gb4 receptor than to Gb3 which could be due to amino acid changes in B subunit. Moreover, this subtype also binds to Forssman glycosphingolipids conferring upon Stx2e a unique promiscuous recognition feature. Manifestations of edema disease are caused by systemic effects of Stx2e with no significant morphologic changes in enterocytes. Endothelial cell necrosis in the brain is an early event in the pathogenesis of ED caused by Stx2e-producing STEC strains. Further studies are needed to generate techniques and tools which allow to understand the circulation and ecology of STEC strains in pigs even in resistant animals for diagnostic and epidemiological purposes.