Comparison of sialyl- and α-1,3-galactosyltransferase activity in NIH3T3 cells transformed with ras oncogene: increased β-galactoside α-2,6-sialyltransferase

Vandamme, V.; Cazlaris, Haris; Marer, Nadia Le; Laudet, Vincent; Lagrou, C.; Verbert, André; Delannoy, Philippe

doi:10.1016/0300-9084(92)90188-k

Cited by 34 publications

(17 citation statements)

References 39 publications

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“…We and others have shown that forced expression of oncogenic Ras alters ST6Gal-I expression in epithelial cells (7) and fibroblasts (22)(23)(24)(25). However, the current study describes regulation of ST6Gal-I by an endogenous multistep signaling cascade.…”

Section: Discussionmentioning

confidence: 47%

A Protein Kinase C/Ras/ERK Signaling Pathway Activates Myeloid Fibronectin Receptors by Altering β1 Integrin Sialylation

Seales

Shaikh

Woodard‐Grice

et al. 2005

Journal of Biological Chemistry

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Here we report that myeloid cells differentiating along the monocyte/macrophage lineage down-regulate the ST6Gal-I sialyltransferase via a protein kinase C/Ras/ERK signaling cascade. In consequence, the ␤1 integrin subunit becomes hyposialylated, which stimulates the ligand binding activity of ␣5␤1 fibronectin receptors. Pharmacologic inhibitors of protein kinase C, Ras, and MEK, but not phosphoinositide 3-kinase, block ST6Gal-I down-regulation, integrin hyposialylation, and fibronectin binding. In contrast, constitutively active MEK stimulates these same events, indicating that ERK is both a necessary and sufficient activator of hyposialylationdependent integrin activation. Consistent with the enhanced activity of hyposialylated cell surface integrins, purified ␣5␤1 receptors bind fibronectin more strongly upon enzymatic desialylation, an effect completely reversed by resialylation of these integrins with recombinant ST6Gal-I. Finally, we have mapped the N-glycosylation sites on the ␤1 integrin to better understand the potential effects of differential sialylation on integrin structure/function. Notably, there are three N-glycosylated sites within the ␤1 I-like domain, a region that plays a crucial role in ligand binding. Our collective results suggest that variant sialylation, induced by a specific signaling cascade, mediates the sustained increase in cell adhesiveness associated with monocytic differentiation.The U937 and THP-1 cell lines represent well accepted model systems for studying myeloid differentiation along the monocyte/macrophage lineage. Following treatment with phorbol myristate acetate (PMA), 4 these cells exhibit phenotypic changes that are characteristic of cell differentiation, including increased respiratory burst activity, enhanced phagocytotic capability, and markedly elevated cell adhesiveness to extracellular matrix ligands such as fibronectin. In vivo, the increased adhesiveness of monocytes/macrophages contributes to the extravasation of cells from the vasculature as well as tethering of cells within inflamed tissues.Differentiating myeloid cells bind to fibronectin through the integrin family of cell adhesion receptors, including the ␣5␤1 integrin species. The molecular mechanisms underlying PMA-dependent cell adhesion have not been well defined, although it has been reported that PMA increases the synthesis of both ␣5 and ␤1 integrin subunits (1-4). However, myeloid cells (U937 and THP-1) express an abundant amount of ␣5␤1 in the absence of PMA treatment, and yet these cells bind very poorly to fibronectin. This suggests that myeloid ␣5␤1 receptors are normally in an inactive state and that increased expression alone cannot account for the dramatically increased fibronectin binding induced by PMA.In our prior study (5) we observed that PMA stimulated a rapid but transient increase in fibronectin binding that was likely due to the activation of integrins already present on the cell surface. However, following this initial transient event there was a second phase of elevated fibronect...

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Section: Discussionmentioning

confidence: 47%

A Protein Kinase C/Ras/ERK Signaling Pathway Activates Myeloid Fibronectin Receptors by Altering β1 Integrin Sialylation

Seales

Shaikh

Woodard‐Grice

et al. 2005

Journal of Biological Chemistry

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“…This specificity could make this lectin an invaluable tool for glycobiological studies, especially for cancer research and diagnosis. For example, it has been well documented in NIH3T3 (or FR3T3) cells transformed with ras oncogene that there is an increased ␤-galactoside ␣-2,6-sialyltransferase activity (31,32) and a concomitant decreased CMP-Neu5Ac: Gal␤1,3GalNAc ␣-2,3-sialyltransferase activity (33). Furthermore, some tumors, e.g.…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of a Neu5Acα2–6Galβ1–4Glc/GlcNAc-specific Lectin from the Fruiting Body of the Polypore Mushroom Polyporus squamosus

Winter

Goldstein

2000

Journal of Biological Chemistry

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A lectin has been purified from the carpophores of the mushroom Polyporus squamosus by a combination of affinity chromatography on ␤-D-galactosyl-Synsorb and ion-exchange chromatography on DEAE-Sephacel. Gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and N-terminal amino acid sequencing indicated that the native lectin, designated P. squamosus agglutinin, is composed of two identical 28-kDa subunits associated by noncovalent bonds. P. squamosus agglutinin agglutinated human A, B, and O and rabbit red blood cells but precipitated only with human ␣ 2 -macroglobulin, of many glycoproteins and polysaccharides tested. The detailed carbohydrate binding properties of the purified lectin were elucidated using three different approaches, i.e. precipitation inhibition assay (in solution binding assay), fluorescence quenching studies, and glycolipid binding by lectin staining on high-performance thin layer chromatography (solid-phase binding assay). Based on the results obtained by these assays, we conclude that although the P. squamosus lectin binds ␤-D-galactosides, it has an extended carbohydratecombining site that exhibits highest specificity and affinity toward nonreducing terminal Neu5Ac␣2,6Gal␤1,4Glc/GlcNAc (6-sialylated type II chain) of N-glycans (2000-fold stronger than toward galactose). The strict specificity of the lectin for ␣2,6-linked sialic acid renders this lectin a valuable tool for glycobiological studies in biomedical and cancer research.

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“…Microsomes are prepared by differential or sucrose gradient centrifugation of crude subcellular membrane fractions obtained after homogenization of fresh or frozen tissues or cells. Sialyltransferase activity [25] may be used as marker enzyme of these membranes. Most experience in subcellular membrane preparation containing highest SOAT activity was obtained with rat liver [7,26], guinea pig liver [6] and bovine submandibular gland ( [9]; Srinivasan et al, unpublished).…”

Section: Soat Assay Using Endogenous Substratesmentioning

confidence: 99%

Assays of sialate-O-acetyltransferases and sialate-O-acetylesterases

Srinivasan

Schauer

2008

Glycoconj J

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The O-acetylation of sialic acids is one of the most frequent modifications of these monosaccharides and modulates many cell biological and pathological events. Sialic acid-specific O-acetyltransferases and O-acetylesterases are responsible for the metabolism of esterified sialic acids. Assays were developed for the analysis of the activities and specificities of these enzymes. The methods had to be varied in dependence on the substrate assayed, the kind of biological source, and the state of enzyme purity. With the new techniques the primary site of O-acetyl incorporation at C-7, catalyzed by the animal sialate-O-acetyltransferases studied, was ascertained. Correspondingly, this enzyme, for example from bovine submandibular gland, can be denominated as AcCoA:sialate-7-O-acetyltransferase (EC 2.3.1.45). Methods for assaying the activity of esterases de-O-acetylating sialic acids and their metabolic cooperation with the O-acetyltransferases are presented.

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Comparison of sialyl- and α-1,3-galactosyltransferase activity in NIH3T3 cells transformed with ras oncogene: increased β-galactoside α-2,6-sialyltransferase

Cited by 34 publications

References 39 publications

A Protein Kinase C/Ras/ERK Signaling Pathway Activates Myeloid Fibronectin Receptors by Altering β1 Integrin Sialylation

A Protein Kinase C/Ras/ERK Signaling Pathway Activates Myeloid Fibronectin Receptors by Altering β1 Integrin Sialylation

Purification and Characterization of a Neu5Acα2–6Galβ1–4Glc/GlcNAc-specific Lectin from the Fruiting Body of the Polypore Mushroom Polyporus squamosus

Assays of sialate-O-acetyltransferases and sialate-O-acetylesterases

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