Biomedical subjects

Assou El-Battari

Publications and source records attributed to Assou El-Battari.

9 recordsLinked to original sources

PubMed2006

Autofluorescent proteins for monitoring the intracellular distribution of glycosyltransferases.

To analyze the Golgi compartmentalization of glycosyltransferases (GTs), we generated versions of several enzymes fused to either the enhanced green fluorescent protein (EGFP) or the red fluorescent protein from Discosoma sp. reef coral (DsRed2) and examined their intracellular distribution by confocal fluorescence microscopy in living cells. In a previous work, we have shown that the N-terminal peptides of GTs, encompassing the cytosolic and the transmembrane domains (CTDs), can serve as Golgi-targeting signals to localize the enzymes to their corresponding compartments within the Golgi apparatus (Zerfaoui et al., 2002). Using sialyl-Lewis x synthesis and selectin binding as functional assays, we show here that by swapping CTDs between GTs, it is possible to mislocalize an enzyme from a Golgi compartment to another, thereby altering the overall cellular glycosylation. On the other hand, we demonstrate that the use of an autofluorescent tag such as EGFP offers numerous advantages including the possibility of (1) facilitating sorting by fluorescence-activated cell sorter (FACS) of stably transfected polyclonal cell population, (2) constantly monitoring the expression of the enzymes in live cells, (3) establishing a direct relationship between the fluorescence intensity and the enzyme activities in vivo and in vitro, (4) establishing a visual relationship between function and intracellular distribution of a given GT, as well as co-localization with cognate protein acceptors by confocal microscopy, and (5) detecting proteins on blots with highly sensitive commercially available antibodies.

Glycosyltransferases↗

PubMed2006

Visualizing intracellular distribution and activity of core2 beta(1,6)N-acetylglucosaminyltransferase-I in living cells.

The core2 beta(1,6)-N-acetylglucosaminyltransferase-I (C2GnT-I) is expressed by leukocytes and is involved in the synthesis of core2 O-glycans that carry sialyl-Lewis x (sLex) oligosaccharides. The core2-based sLex oligosaccharides (C2-O-sLex) have been demonstrated to be physiological selectin ligands that confer high affinity binding. The E-, P-, and L-selectins are adhesion proteins that direct leukocytes in the blood to lymphoid organs and sites of inflammation. They are also thought to be involved in the hematogenous dissemination of carcinoma cells expressing sialyl-Lewis glycans. Therefore, accumulation of data on structure-function relationships of this particular enzyme may represent an important part of investigations into pathologies involving selectins, such us inflammatory disorders and cancer progression. In this regard, studies of the intracellular distribution of C2GnT-I and its interaction with cognate substrates in vivo, as well as the knowledge of posttranslational modification (i.e., glycosylation, oligomerization, and proteolytic processing), may greatly aid in designing potential enzyme inhibitors. C2GnT-I fused to the green fluorescent protein is expressed to allow examination of the protein in living cells and to ease studies on structure-function relationships in vivo and in vitro.

Animals↗

PubMed2005

N-glycans of core2 beta(1,6)-N-acetylglucosaminyltransferase-I (C2GnT-I) but not those of alpha(1,3)-fucosyltransferase-VII (FucT-VII) are required for the synthesis of functional P-selectin glycoprotein ligand-1 (PSGL-1): effects on P-, L- and E-selectin binding.

C2GnT-I [core2 beta(1,6)-N-acetyglucosaminyltransferase-I] and FucT-VII [alpha(1,3)-fucosyltransferase-VII] are the key enzymes for the biosynthesis of sialyl-Lewis x determinants on selectin ligands and therefore they represent good drug targets for the treatment of inflammatory disorders and other pathologies involving selectins. In the present study, we examined the importance of N-glycosylation for the ability of C2GnT-I and FucT-VII to generate functional selectin ligands, particularly the PSGL-1 (P-selectin glycoprotein ligand-1). We found that (i) both enzymes have their two N-glycosylation sites occupied, (ii) for C2GnT-I, the N-glycan chain linked to Asn-95 significantly contributes to the synthesis of functional PSGL-1 and is required to localize the enzyme to the cis/medial-Golgi compartment, (iii) all N-glycosylation-deficient proteins of FucT-VII displayr a dramatic impairment of their in vitro enzymatic activities, but retain their ability to fucosylate the core2-modified PSGL-I and to generate P- and L-selectin binding, and (iv) the glycomutants of FucT-VII fail to synthesize sialyl-Lewis x or to generate E-selectin binding unless core2-modified PSGL-1 is present. All combined, our results show a differential functional impact of N-glycosylation on C2GnT-1 and FucT-VII and disclose that a strongly reduced FucT-VII activity retains the ability to fucosylate PSGL-1 on the core2-based binding site(s) for the three selectins.

Animals↗

PubMed2004

Role of injured endothelial cells in the recruitment of human pulp cells.

In restorative dentistry, deep cavity preparation may lead to partial destruction of the odontoblastic layer. However, newly formed odontoblast-like cells can replace the necrotic odontoblasts and secrete a reparative dentine matrix. While growth factors such as transforming growth factor beta1 (TGFbeta1) and bone morphogenetic proteins (BMP-2 and BMP-4) seem to be involved in the proliferation and differentiation of pulp cells, little is known about the migration of the newly proliferating stem cells to the injury site. Our hypothesis was that endothelial cell injury may be involved in directing these cells towards the injury site. For this study, human pulp fibroblasts and L929 cells were fluorescence-labeled by transduction with the Enhanced Green Fluorescent Protein (EGFP). Similarly, human umbilical vein endothelial cells (HUVEC) were labeled with the Discosoma Red Fluorescent Protein-2 (DsRed2). Cell migration was then studied in an insert cell culture system. The HUVEC cells were cultured in the lower compartment while the human pulp fibroblasts or L929 were in the upper compartment. After artificial injury to the HUVEC cells, only human pulp fibroblasts migrated to the lower compartment. At early time periods (4 days), migrating cells were randomly localized on the HUVEC layer. However, after 14 and 20 days, they were perfectly aligned along the injury site. In the absence of injury, no migration was observed. These results suggest that, the endothelial injury is involved in the recruitment of odontoblast-like cells at the injury site.

Cell Death↗

PubMed2004

Transgene expression of alpha(1,2)-fucosyltransferase-I (FUT1) in tumor cells selectively inhibits sialyl-Lewis x expression and binding to E-selectin without affecting synthesis of sialyl-Lewis a or binding to P-selectin.

During inflammation, E- and P-selectins appear on activated endothelial cells to interact with leukocytes through sialyl-Lewis x and sialyl-Lewis a antigens (sLe(x/a)). These selectins can also interact with tumor cells in a sialyl-Lewis-dependent manner and for this reason, they are thought to play a key role in metastasis. Diverting the biosynthesis of sialyl-Lewis antigens toward nonadhesive structures is an attractive gene therapy for preventing the hematogenous metastatic spread of cancers. We have previously shown that transfection of alpha(1,2)-fucosyltransferase-I (FUT1) in Chinese hamster ovary (CHO) cells had a slight effect on the overall sialylation while the synthesis of sLE(x) was dramatically prevented. We herein delivered the gene of FUT1 by a human immunodeficiency virus-derived lentiviral vector to three human cancer cell lines including pancreatic (BxPC3), hepatic (HepG2), and colonic (HT-29) cancer cells. We found that on FUT1 transduction, all cells exhibited a dramatic decrease in sLe(x) synthesis with a concomitant increase in Le(y) and Le(b) expression, without any detectable effect on the level of cell surface sLe(a) antigens. In parallel, FUT1-transduced HT-29 and HepG2 cells, but not BxPC3 cells, failed to interact with E-selectin as assessed by E-selectin-binding assay or dynamic adhesion to activated endothelial cells. We show also that transduced FUT1 efficiently fucosylates the P-selectin ligand PSGL-1 without altering P-selectin binding. These results have important implications for understanding cell-specific reactions underlying the synthesis of selectin ligands in cancer cells and may provide a basis for the development of anti-metastatic gene therapy.

Animals↗

PubMed2003

Different glycosyltransferases are differentially processed for secretion, dimerization, and autoglycosylation.

Modification of Golgi glycosyltransferases, such as formation of disulfide-bonded dimers and proteolytical release from cells as a soluble form, are important processes to regulate the activity of glycosyltransferases. To better understand these processes, six glycosyltransferases were selected on the basis of the donor sugars, including two N-acetylglucosaminyltransferases, core 1 beta1,3-N-acetylglucosaminyltransferase (C1-beta3GnT) and core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT-I); two fucosyltransferases, alpha1,2-fucosyltransferase-I (FucT-I) and alpha1,3-fucosyltransferase-VII (FucT-VII); and two sialyltransferases, alpha2,3-sialyltransferase-I (ST3Gal-I) and alpha2,6-sialyltransferase-I (ST6Gal-I). These enzymes were fused with enhanced green fluorescence protein and stably expressed in Chinese hamster ovary cells. Spectrofluorimetric detection and immunoblotting analyses showed that all of these glycosyltransferases except FucT-VII were secreted in the medium. By examining dimers formed in cells and culture media, we found that all of the enzymes, except ST3Gal-I, form a combination of monomers and dimers in cells, whereas the molecules released in the media are either exclusively monomers (C2GnT-I and ST6Gal-I), dimers (FucT-I) or a mixture of both (C1-beta3GnT). These results indicate that dimerization does not always lead to Golgi retention. Analysis of the N-glycosylation status of the enzymes revealed that the secreted proteins are generally more heavily N-glycosylated and sialylated than their membrane-associated counterparts, suggesting that the proteolytic cleavage occurs before the glycosylation is completed. Using FucT-I and ST6Gal-I as a model, we also show that these glycosyltransferases are able to perform autoglycosylation in the dimeric forms. These results indicate that different glycosyltranferases differ significantly in dimerization, proteolytic digestion and secretion, and autoglycosylation. These results strongly suggest that disulfide-bonded dimerization and secretion differentially plays a role in the processing and function of different glycosyltransferases in the Golgi apparatus.

Animals↗

PubMed2003

The impact of N- and O-glycosylation on the functions of Glut-1 transporter in human thyroid anaplastic cells.

It has been previously shown that glucose transporter Glut-1 expression was detectable by immunostaining in tissue sections from anaplastic carcinoma, but not in normal thyroid tissue. Using human thyroid anaplastic carcinoma cells, we studied the mechanism by which Glut-1 molecules are translocated from the endoplasmic reticulum to the cell surface. The contribution of N- and O-linked glycans for the translocation and activity of Glut-1 transporter is emphasized. The inhibition of N-glycosylation with tunicamycin (TM) led to a 50% decrease in glucose transport while glycosylated and unglycosylated forms of Glut-1 were found at the cell surface. However, the inhibition of N-linked oligosaccharide processing with deoxymannojirimycin (dMJ) and swainsonine (SW) influenced neither the intracellular trafficking nor the activity of the transporter. On the other hand, Glut-1 bound to the O-linked glycan-specific lectin jacalin and the O-glycosylation inhibitor benzyl-N-acetylgalactosamine dramatically inhibited glucose transport. These results show that O- and N-linked oligosaccharides arbored by Glut-1 are essential for glucose transport in anaplastic carcinoma cells. The quantitative and qualitative alterations of Glut-1 glycosylation and the increase in glucose transport are associated with the anaplastic phenotype of human thyroid cells.

Anti-Bacterial Agents↗

PubMed2003

Monitoring E-selectin-mediated adhesion using green and red fluorescent proteins.

Accumulating evidence suggests that E-selectin, which is physiologically involved in leukocyte recruitment during inflammation, plays an important role in the early stages of tumor cell interactions with vessel walls and contributes to the hematogenous spreading of cancer cells. Therapy designed to block this key step may provide an effective anti-inflammatory and anti-metastatic treatment. It is therefore critical to establish a safe, rapid and sensitive E-selectin adhesion assay. In this regard, we propose a simple and highly sensitive adhesion system based on CHO cells permanently co-expressing E-selectin and the enhanced green fluorescent protein EGFP or the red fluorescent protein DsRed2. This is an inverted adhesion assay in which tumor cells are maintained intact while fluorescent cells expressing E-selectin and EGFP (or DsRed2) are added to them. Adherent cells are then quantified by three different fluorescence-based techniques including spectrofluorimetry, ELISA-type cytofluorimetry and fluorescence microscopy coupled to digital image quantification. In this assay, a battery of cell lines can be analysed at once since only one cell line (fluorescent E-selectin-expressing cells) needs to be harvested. We used this approach to analyze a number of E-selectin-specific binding parameters of intestinal cancer cells in comparison with adhesion to activated endothelial cells or to plastic dishes coated with recombinant E-selectin. Besides the possibility of analyzing a battery of cell lines at once, this assay might be suitable for screening anti-metastatic compounds and could provide valuable information on the metastatic potential of human cancers.

Animals↗

PubMed2002

The cytosolic and transmembrane domains of the beta 1,6 N-acetylglucosaminyltransferase (C2GnT) function as a cis to medial/Golgi-targeting determinant.

The beta 1,6 N-acetylglucosaminyltransferase (C2GnT) has been recently mapped to the cis/medial-Golgi compartment. To analyze the Golgi-targeting determinants of C2GnT, we constructed various deletion mutants of the enzyme fused to the enhanced green fluorescent protein (EGFP) and localized these proteins by fluorescence microscopy in living cells. We found that the N-terminal peptide encompassing amino acids 1 to 32 represents the minimal Golgi-targeting signal sufficient to localize EGFP to the same compartment as the full-length C2GnT. This peptide makes up the cytoplasmic and the transmembrane domains of the enzyme and was referred to as CTd (cytoplasmic and transmembrane domains). We compared the Golgi-targeting efficiency of the C2GnT-derived CTd with its homologous domains from other glycosyltransferases, including the H-type alpha(1,2)-fucosyltransferase (FucTI), the polypeptide N-acetylgalactosaminyltransferase-I (GalNAcT-I), the alpha(1,3)-fucosyltransferase VII (FucTVII), and the alpha(2,6)-sialyltransferase (ST6Gal-I) and found that the Golgi-targeting determinants of these glycosyltransferases were also composed of their cytosolic and transmembrane domains. To investigate whether the CTd of C2GnT could serve as a cis to medial Golgi-specific signal, we tested its ability to mislocalize two late-Golgi acting glycosyltransferases FucTI and FucTVII. We show that fusing the C2GnT-derived CTd with the catalytic domain of FucTVII resulted in a complete mislocalization of the enzyme to the C2GnT compartment, with a parallel alteration of sialyl-Lewis x synthesis and P-selectin binding. The intracellular distribution and activity of FucTI, however, were not affected. Thus, CTds of either early or late-Golgi acting glycosyltransferases represent the Golgi-targeting domains of these enzymes. In addition, we show that C2GnT-derived CTd can function as a cis/medial Golgi-targeting determinant.

Animals↗