Two glycosphingolipid sialyltransferases are localized in different sub-Golgi compartments in rat liver

A highly purified Golgi preparation from rat liver was fractionated on a sucrose density gradient and the activity of two sialyltransferases, CMP-NeuAc: Galβ1→4Glc-Cer (lactosylceramide) α-2→3sialyltransferase; Sat-1), and CMP-NeuAc: Galβ1→3GalNAcβ1→4(NeuAcα2→3)Galβ1→4Glc-Cer (GM1 ganglioside) α2 →3sialyltransferase; SAT-4), involved in the biosynthesis of gangliosides were assayed in the collected fractions. These two activities were recovered in different regions of the gradient; SAT-1 was found in a more dense region than SAT-4. This distribution coincided with that of two N-Asn linked oligosaccharide processing enzymes (UDP-GlcNAc:lysosomal enzyme precursor GlcNAc-1-phosphotransferase and UDP-Gal:ovalbumin galactosyltransferase), assumed as putative markers of eis- and trans-Golgi cisternae, respectively. These findings are consistent with the assembly of ganglioside oligosaccharide chains occurring in different sub-Golgi compartments

Suppression of beta1,3galactosyltransferase beta3Gal-T5 in cancer cells reduces sialyl-Lewis a and enhances poly-N-acetyllactosamines and sialyl-Lewis x on O-glycans

We investigated the role of beta 3 Gal-T5, a member of the beta 1,3galactosyltransferase (beta 1,3Gal-T) family, in cancer-associated glycosylation, focusing on the expression of sialyl-Lewis a (sLea, the epitope of CA19.9 antigen), poly N-acetyllactosamines, and sialyl-Lewis x (sLex) antigen. A clone permanently expressing an antisense fragment of beta 3Gal-T5 was obtained from the human pancreas adenocarcinoma cell line BxPC3 and characterized. Both beta 1,3Gal-T activity and sLea expression are dramatically impaired in the clone. Analysis of the oligosaccharides synthesized in cells metabolically labelled with tritiated galactose shows that a relevant amount of radioactivity is associated to large O-glycans. Endo-beta-galactosidase mostly releases NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal and NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal from such O-glycans of BxPC3 membranes, but GlcNAc beta 1-3Gal and type 2 chain oligosaccharides, including NeuAc alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-3Gal, from those of the antisense clone. Furthermore, BxPC3 cells secrete sLea in the culture media but not sLex, while antisense clone secretes mostly sLex, and accumulation of both antigens is prevented by benzyl-alpha-GalNAc. These data indicate that beta 3Gal-T5 suppression turns synthesis of type 1 chain O-glycans to poly N-acetyllactosamine elongation and termination by sLex. In other cell lines and clones, beta 3Gal-T5 transcript, beta 1,3Gal-T activity, and sLea antigen are also correlated, but quantitatively the relative expression ratios are very different from cell type to cell type. We suggest that beta 3Gal-T5 plays a relevant role in gastrointestinal and pancreatic tissues counteracting the glycosylation pattern associated to malignancy, and is necessary for the synthesis and secretion of CA19.9 antigen, whose expression still depends on multiple interacting factors

Topography of glycosyltransferases involved in the initial glycosylations of gangliosides.

We attempted to establish within which organelle UDP-Glc:ceramide beta 1----1'glucosyltransferase (GlcT) is located and moreover to obtain information about its orientation on intracellular membranes as well as that of UDP-Gal:glucosylceramide beta 1----4galactosyltransferase (GalT-2) and CMP-NeuAc:lactosylceramide alpha 2----3sialyltransferase (SAT-1). An extremely purified Golgi apparatus fraction was the only liver fraction where a ceramide-dependent formation of glucosylceramide could be demonstrated. This Golgi fraction, mainly constituted by stacks of intact cisternae which retained the same topographical orientation as in vivo, was then incubated with liposomal dispersions of glycosphingolipid-glycosyltransferase acceptors in reaction mixtures containing all the requirements for enzyme activity but no detergent. Under such conditions, SAT-1 and other late acting glycosyltransferases were over 90% latent, while both GlcT and GalT-2 were just as active as in the detergent-containing assay; they were still inhibited by EDTA. Sepharose-immobilized ceramide and Sepharose-immobilized glucosylceramide were found to be suitable acceptors for GlcT and GalT-2, respectively, still using intact Golgi cisternae as the enzyme source. Moreover, a part of GlcT and GalT-2 activity was released from intact Golgi cisternae upon cathepsin D treatment. These results provide strong evidence that GlcT and GalT-2 face the cytoplasmic side of the Golgi apparatus, whereas SAT-1 and the other late acting enzymes face the luminal side

Wideband digital phase comparator for high current shunts

A wideband phase comparator for precise measurements of phase difference of high current shunts has been developed at INRIM. The two-input digital phase detector is realized with a precision wideband digitizer connected through a pair of symmetric active guarded transformers to the outputs of the shunts under comparison. Data are first acquired asynchronously, and then transferred from on-board memory to host memory. Because of the large amount of data collected the filtering process and the analysis algorithms are performed outside the acquisition routine. Most of the systematic errors can be compensated by a proper inversion procedure. The system is suitable for comparing shunts in a wide range of currents, from several hundred of milliampere up to 100 A, and frequencies ranging between 500 Hz and 100 kHz. Expanded uncertainty (k=2) less than 0.05 mrad, for frequency up to 100 kHz, is obtained in the measurement of the phase difference of a group of 10 A shunts, provided by some European NMIs, using a digitizer with sampling frequency up to 1 MHz. An enhanced version of the phase comparator employs a new digital phase detector with higher sampling frequency and vertical resolution. This permits to decrease the contribution to the uncertainty budget of the phase detector of a factor two from 20 kHz to 100 kHz. Theories and experiments show that the phase difference between two high precision wideband digitizers, coupled as phase detector, depends on multiple factors derived from both analog and digital imprint of each sampling system.Comment: 20 pages, 9 figure

Capturing and Treating Unobserved Heterogeneity by Response Based Segmentation in PLS Path Modeling. A Comparison of Alternative Methods by Computational Experiments

Segmentation in PLS path modeling framework results is a critical issue in social sciences. The assumption that data is collected from a single homogeneous population is often unrealistic. Sequential clustering techniques on the manifest variables level are ineffective to account for heterogeneity in path model estimates. Three PLS path model related statistical approaches have been developed as solutions for this problem. The purpose of this paper is to present a study on sets of simulated data with different characteristics that allows a primary assessment of these methodologies.Partial Least Squares; Path Modeling; Unobserved Heterogeneity

A PART OF GLUCOSYLCERAMIDE FORMED FROM EXOGENOUS LACTOSYLCERAMIDE IS NOT DEGRADED TO CERAMIDE BUT RE-CYCLED AND GLYCOSYLATED IN THE GOLGI APPARATUS

Abstract The subcellular fate of glucosylceramide (GlcCer) formed from exogenous lactosylceramide (LacCer) in rat liver is investigated. LacCer radiolabeled on different positions of the molecule was intravenously administered to rats as a liposomal dispersion. A Golgi apparatus fraction 140-fold enriched in specific markers and constituted by intact cisternal stacks, as well as the lysosomal and plasma membrane fractions concurrently prepared from the same homogenate, were then studied in order to determine the time course of radioactive glycosphingolipids. LacCer quickly decreased with time in the plasma membrane, whereas in the lysosomes it increased up to 4 h and decreased thereafter. In both fractions results were regardless of the labeling position. In the Golgi apparatus, LacCer increased up to 12 h and then decreased. In this fraction, the radioactivity values of [Glc-3H]LacCer were over twice those of [Gal-3H]LacCer. GlcCer was found only after [Glc-3H]LacCer administration. In the lysosomes, its time course provided a peak similar in shape but delayed in timing with respect to that of LacCer. Conversely, in the Golgi apparatus GlcCer was earlier formed, but earlier consumed, than LacCer. Gangliosides increased in the Golgi apparatus until 4 h and then decreased after 12 h, whereas in the plasma membrane they were progressively accumulated. In both fractions the amount of [Glc-3H]gangliosides was over twice that of [Gal-3H]gangliosides was over twice that of [Gal-3H]gangliosides. Since we demonstrated that the sugars released in the course of LacCer degradation (LacCer----galactose + GlcCer----glucose + ceramide) are not incorporated into glycoconjugates, we conclude that a part of GlcCer formed during the lysosomal degradation of LacCer actually reaches the Golgi apparatus where it undergoes successive glycosylation

Sub-Golgi distribution in rat liver of CMP-NeuAc GM3- and CMP-NeuAc:GT1b alpha 2----8sialyltransferases and comparison with the distribution of the other glycosyltransferase activities involved in ganglioside biosynthesis.

Using a sucrose density gradient fractionation of a highly purified Golgi apparatus from rat liver, we determined the sub-Golgi distribution of CMP-NeuAc:GM3 ganglioside alpha 2----8sialyltransferase (GM3-SAT) and CMP-NeuAc:GT1b ganglioside alpha 2----8sialyltransferase (GT1b-SAT), in comparison with that of the other glycosyltransferase activities involved in ganglioside biosynthesis. While GM3-SAT was recovered in several density fractions, GT1b-SAT was mainly found on less dense sub-Golgi membranes; this indicates that these two activities are physically separate. Moreover, with regard to the monosialo pathway, CMP-NeuAc:lactosylceramide alpha 2----3sialyltransferase, UDP-GalNAc:GM3 ganglioside beta 1----4N-acetylgalactosaminyltransferase, UDP-Gal:GM2 ganglioside beta 1----3galactosyltransferase, and CMP-NeuAc:GM1 ganglioside alpha 2----3sialyltransferase were resolved from more dense to less dense fractions, respectively. In the disialo pathway, UDP-GalNAc:GD3 ganglioside beta 1----4N-acetylgalactosaminyltransferase, UDP-Gal:GD2 ganglioside beta 1----3galactosyltransferase and CMP-NeuAc:GD1b ganglioside alpha 2----3sialyltransferase co-distributed with the corresponding activities of the monosialo pathway. These last results indicate that many Golgi glycosyltransferases involved in ganglioside biosynthesis are localized in the order in which they act

Sialosignaling: Sialyltransferases as engines of self-fueling loops in cancer progression

Background Glycosylation is increasingly recognized as one of the most relevant postranslational modifications. Sialic acids are negatively charged sugars which frequently terminate the carbohydrate chains of glycoproteins and glycolipids. The addition of sialic acids is mediated by sialyltransferases, a family of glycosyltransferases with a crucial role in cancer progression. Scope of the review To describe the phenotypic and clinical implications of altered expression of sialyltransferases and of their cognate sialylated structures in cancer. To propose a unifying model of the role of sialyltransferases and sialylated structures on cancer progression. Major conclusions We first discuss the biosynthesis and the role played by the major cancer-associated sialylated structures, including Thomsen-Friedenreich-associated antigens, sialyl Lewis antigens, \u3b12,6-sialylated lactosamine, polysialic acid and gangliosides. Then, we show that altered sialyltransferase expression in cancer, consequence of genetic and epigenetic alterations, generates a flow of information toward the membrane through the biosynthesis of aberrantly sialylated molecules (inside-out signaling). In turn, the presence of aberrantly sialylated structures on cell membrane receptors generates a flow of information toward the nucleus, which can exacerbate the neoplastic phenotype (outside-in signaling). We provide examples of self-fueling loops generated by these flows of information. General significance Sialyltransferases have a wide impact on the biology of cancer and can be the target of innovative therapies. Our unified view provides a conceptual framework to understand the impact of altered glycosylation in cancer

The expanding roles of the Sda/Cad carbohydrate antigen and its cognate glycosyltransferase B4GALNT2

Background The histo-blood group antigens are carbohydrate structures present in tissues and body fluids, which contribute to the definition of the individual immunophenotype. One of these, the Sda antigen, is expressed on the surface of erythrocytes and in secretions of the vast majority of the Caucasians and other ethnic groups. Scope of review We describe the multiple and unsuspected aspects of the biology of the Sda antigen and its biosynthetic enzyme \u3b21,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) in various physiological and pathological settings. Major conclusions The immunodominant sugar of the Sda antigen is a \u3b21,4-linked N-acetylgalactosamine (GalNAc). Its cognate glycosyltransferase B4GALNT2 displays a restricted pattern of tissue expression, is regulated by unknown mechanisms - including promoter methylation, and encodes at least two different proteins, one of which with an unconventionally long cytoplasmic portion. In different settings, the Sda antigen plays multiple and unsuspected roles. 1) In colon cancer, its dramatic down-regulation plays a potential role in the overexpression of sialyl Lewis antigens, increasing metastasis formation. 2) It is involved in the lytic function of murine cytotoxic T lymphocytes. 3) It prevents the development of muscular dystrophy in various dystrophic murine models, when overexpressed in muscular fibers. 4) It regulates the circulating half-life of the von Willebrand factor (vWf), determining the onset of a bleeding disorder in a murine model. General significance The expression of the Sda antigen has a wide impact on the physiology and the pathology of different biological systems

Transcriptional control of the B3GALT5 gene by a retroviral promoter and methylation of distant regulatory elements

We focused on transcription factors and epigenetic marks that regulate the B3GALT5 gene through its retroviral long terminal repeat (LTR) promoter. We compared the expression levels of the B3GALT5 LTR transcript, quantitated by competitive RT-PCR, with those of the candidate transcription factors HNF1\u3b1/\u3b2 and Cdx1/2, determined by Western blot analysis, in colon cancer biopsies, various cell lines, and cell models serving as controls. We found that HNF1\u3b1/\u3b2 were easily detected, irrespective of the amount of LTR transcript expressed by the source, whereas Cdx1/2 were undetectable, and no sample lacking HNF1\u3b1/\u3b2 expressed the LTR transcript. On transfection in proper host cells, both HNF1\u3b1 and HNF1\u3b2 provided detectable LTR transcript, whereas shRNA-mediated silencing of HNF1\u3b2 impaired transcription. Treating cells with 5\u2032-aza-2\u2032-deoxycytidine (5AZA) strongly reduced expression, without affecting HNF1\u3b1/\u3b2, despite the lack of CpG islands in the LTR and proximal sequences. By electrophoresis mobility shift and luciferase reporter assays, the LTR promoter binding and activity did not correlate with the amounts of LTR transcript expressed in the cells and depended on the levels of the transcription factors. We conclude that HNF1\u3b1/\u3b2 are necessary but insufficient to activate and regulate B3GALT5 LTR transcription, which depends on unknown regulatory elements that are active when methylated and located outside of and far from the LTR promoter