39 research outputs found
Infectivity and glycoprotein processing of herpes simplex virus type 1 grown in a ricin-resistant cell line deficient in N-acetylglucosaminyl transferase I
No full textWe report on the replication of herpes simplex virus type 1 (HSV-1) and viral glycoprotein processing in Ric(R)14 cells, a mutant ricin-resistant cell line defective in N-acetylglucosaminyl transferase I activity. In these cells HSV-1(MP) and (F) replicated to yields very similar to those in parental BHK cells. The kinetics of HSV-1 adsorption in mutant and in parent cells was also essentially identical. Progeny virions from ricin-resistant and wild-type cells displayed comparable specific infectivities. However, in the mutant cells the efficiency of plating of progeny virus from both Ric(R)14 and BHK cells was reduced. HSV-1(MP) failed to induce syncytia in Ric(R)14 cells either in a plaque assay or after a high-multiplicity infection. Moreover, the fully glycosylated forms of glycoproteins (gB, gC, and gD) were totally absent, and only the partially glycosylated precursors (pgC, pgD, and a triplet in the gB-gA region) accumulated in HSV-1 infected ricin-resistant cells and in herpesvirions made in these cells. Consistent with these results analysis of pronase glycopeptides from cells labeled with [14C]glucosamine showed a strong decrease of sialylated complex-type oligosaccharides and a dramatic accumulation of the neutral mannose-rich chains. The latter chains predominate in partially glycosylated precursors, whereas the complex acidic chains predominate in the fully processed forms of HSV glycoproteins. These results taken together indicate that (i) host-cell N-acetylglucosaminyl transferase I participates in the processing of HSV glycoproteins; and (ii) infectivity of herpesvirions does not necessarily require the mature form of gB. The absence of HSV-1(MP)-induced fusion in Ric(R)14 cells is discussed
Resistance to methotrexate is associated with selective changes of α2,6- and α2,3-sialyltransferase activities toward N-acetyllactosaminic sequences in human colon cancer cell line HT-29
No full textIn previous works we established that the α2,6-sialyltransferase acting on N-acetyllactosaminic sequences [α2,6(N)ST, E.C. 2.4.99.1] behaves, in colonic cells, as an onco-developmentally regulated enzyme. Subpopulations of the human colon cancer cell line HT-29 adapted to grow in 10-5 M methotrexate (MTX), permanently retain the ability to differentiate as mucus-secreting cells when kept confluent for extended periods of time [Lesuffleur et al. (1991) J. Cell Biol. 115, 1409-1418]. In this study we have compared the activities of five sialyltransferases acting on N- or O-linked chains of glycoproteins in parental HT-29 and in the 10-5 M MTX-resistant variant. Both cell lines were studies during the exponential phase of growth as well as after a long period of postconfluent culture (28-30 days). Regardless the culture conditions, resistance to 10-5 M MTX is associated with a virtual disappearance of α2,6(N)ST activity. This change results in a dramatic reduction of the reactivity of cell membranes with the fluorescent lectin from Sambucus nigra, specific for α2,6-sialylated structures. The activity of the α2,3-dialyltransferase which acts on N-acetyllactosaminic sequences increases about two times in postconfluent cultures of 10-5 M MTX-resistant cells, suggesting a close relationship with the differentiation degree. No significative changes were observed in the activity of other sialyltransferases. © 1993 Academic Press, Inc
Processing of herpes simplex virus-1 glycans in cells defective in glycosyl transferases of the golgi system: Relationship to cell fusion and virion egress
No full textWe studied herpes simplex virus-1 (HSV-1) glycan structure and the expression of HSV-1 functions regulated by viral glycoproteins in Ric21 cells (P. Vischer and R. C. Hughes, Eur. J. Bioch. 117, 275-284, 1981). This is a line of ricin-resistant mutant BHK cells defective in the enzymes of the Golgi system which add terminal sugars to N-linked glycans. Twp kinds of alterations were observed in the glycosylation of HSV glycoproteins in Ric21 cells. First, there was a defective processing of complex glycans leading to a reduction of biantennary and triantennary species and an increase of incompletely processed monosialylated oligosaccharides. Second, there was an overall reduction in the accumulation of HSV-1 glycoproteins. We found that (i) the release of herpesvirions from Ric21 cells was markedly lower than that from BHK cells, possibly reflecting reduced terminal sugar addition which, in turn, might affect the intracellular transport of glycoproteins. (ii) HSV-1 (MP)-infected Ric21 cells fused with a low efficiency. Furthermore, polycaryocytosis was reduced or abolished in BHK and in Ric21 cells exposed to neuraminidase, indicating that the presence of sialic acid residues in the cell surface glycans is essential for cells to interact in a fashion that brings cell fusion. (iii) Although capsid assembly was comparable, the rate of accumulation of infectious virus decreased in Ric21 cells. Infectivity of released virions from Ric21 and BHK cells was similar, in agreement with previous studies showing that complex-type glycans do not appear to be required for herpesvirion infectivity. The decrease in infectious HSV-1 yield seems to correlate with overall reduced ability to synthesize glycoproteins. © 1983
Effect of Ethanol on Human Colon Carcinoma CaCo‐2 and HT‐29 Cell Lines during the Maturation Process
No full textThe aim of the study was to ascertain whether the exposure to ethanol of human colon carcinoma CaCo‐2 and HT‐29 cell lines affects the differentiation process. As an index of enterocytic differentiation, the expression of sucrase, alkaline phosphatase, α2,6‐sialyltransferase toward the N‐acetyllactosaminic sequence, and β1,4‐N‐acetylgalactosaminyltransferase (β1,4GalNAc‐transferase) was examined. The latter enzyme is responsible for the biosynthesis of Sd* carbohydrate histo‐blood antigen, which mainly occurs in human colonic cells; its expression in CaCo‐2 cells depends strictly on the enterocytic differentiation. The addition of ethanol in the culture medium resulted in a significant increment of sucrase and α2,6‐sialyltransferase activities in both cell lines, as well as theβ1,4GalNAc‐transferase activity in CaCo‐2 cells and alkaline phosphatase activity in HT‐29 cells. The increment was dose‐dependent in the range between 50 and 200 mm ethanol and evident after 2 days of exposure in both cell systems. These results support the notion that, as occurs for cell lines of different origin, the ethanol in vitro positively affects the differentiation of intestinal cells, namely along the enterocytic lineage. The putative mechanism by which ethanol interferes with the maturation process of colonic cells is discussed. Copyright © 1994, Wiley Blackwell. All rights reserve
Effect of Acute and Chronic Ethanol Administration on Rat Liver α 2,6‐Sialyltransferase Activity Responsible for Sialylation of Serum Transferrin
No full textThe effect of a single administration and a 6‐week treatment with ethanol on rat liver sialyltransferase activity towards asialoglycoproteins and N‐acetyllactosamine (Galβ91,4GlcNAc) was studied. Since only the cα2,6‐sialyltransferase is involved in the in vivo sialylation of transferrin, GalβS1,4GlcNAc was chosen as an acceptor and α2,6‐sialyl‐N‐acetyllactosamine was separated from the corresponding α2,3‐sialyl isomer present in the sialyltransferase reaction mixture by high‐performance liquid chromatography. After a single ethanol administration there was a low (about 20%) but significant (p < 0.005) reduction of sialyltransferase activity towards asialotransferrin as well as a reduced α2,6‐sialyltransferase activity towards N‐acetyllactosamine. An opposite result was found in the chronically ethanol‐treated rats: in these animals either the total or α2,6‐sialyl‐transferase activity was slightly higher than in control animals. Blood ethanol concentration was significantly high (3.3 ±1.2 mg/ml) only in the acute‐treated animals, suggesting that the accumulation in the body of ethanol and/or its metabolites induces a reduction of liver α2,6‐sialyltransferase activity responsible for the transferrin sialylation. Current results are consistent with the finding (Stibler H, Hultcrantz R: Alcohol Clin Exp Res 11:468–473, 1987) that an enhanced level of hyposialylated transferrin isoforms is a marker of present but not previous alcohol abuse. Copyright © 1989, Wiley Blackwell. All rights reserve
Rapid isolation of Tamm-Horsfall glycoprotein (uromodulin) from human urine
No full textAn isolation method for Tamm-Horsfall protein is described which is based on the observation that a diatomaceous earth filter is able to retain most of the glycoprotein present in urine and that the glycoprotein is easily desorbed from the filter by deionized water. This behaviour depends on the tendency of Tamm-Horsfall glycoprotein at normal urinary concentrations to form a gel in a solution containing mono- and divalent ions. By means of two-step filtration, the glycoprotein was purified to homogeneity. The yield was of about 20 mg/l of urine, and the time required for the isolation was approximately 5-6 h. This procedure should be particularly useful for preparing large amounts of Tamm-Horsfall glycoprotein oligosaccharides in order to investigate their potential use as immunosuppressive agents both in vitro and in vivo. © 1989
Temporal aspects of O-glycosylation of glycoprotein C from herpes simplex virus type-1
No full textHerpes simplex virus type-1 glycoprotein C (gC1) contains several O-linked oligosaccharides clustered near N-linked chains, and Pronase digestion produces glycopeptides carrying both oligosaccharide types. We have taken advantage of this fact to investigate the temporal relationship between the initiation of O-linked chains and the processing of N-linked oligosaccharides. gC1 was isolated from herpes-simplex-virus-infected BHK (baby-hamster kidney) cells after short labelling periods with [3H]glucosamine, and the labelled Pronase-cleaved glycopeptides fractionated on concanavalin A-Sepharose. N-[3H]Acetylgalactosamine, mostly convertible into free N-[3H]acetylgalactosaminitol on mild alkaline-borohydride treatment, was found in glycopeptides with an affinity to concanavalin A-Sepharose corresponding to that of glycopeptides carrying Man8GlcNAc2 or larger N-linked chains. Since there is evidence that the processing of N-linked chains up to Man8GlcNAc2 involves enzymes located in the rough endoplasmic reticulum, current results strongly suggest that gC1 acquires O-linked N-acetylgalactosamine before the glycoprotein routing to the Golgi apparatus. The addition of the second sugar to the nascent O-linked chain appeared to occur after a relatively long lag time
Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC
No full textImmature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-mim pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-β-N-acetylglycosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-β-N-acetylglycosaminidase H-cleaved oligosaccharides, polymannosyl chains of different size, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies
Biosynthesis and oligosaccharide structure of human CD8 glycoprotein expressed in a rat epithelial cell line.
No full textExpression of UDP-GalNAc:NeuAc alpha 2,3Gal beta-R beta 1,4(GalNAc to Gal) N-acetylgalactosaminyltransferase involved in the synthesis of Sda antigen in human large intestine and colorectal carcinomas
No full textN-Acetylgalactosamine beta 1,4-linked to a galactose residue substituted in O-3 with one N-acetylneuraminic acid residue is the immunodominant sugar of the human blood group Sda antigen which is also largely present in the kidney medulla and colon mucosa. A beta 1,4-N-acetylgalactosaminyltransferase very similar to that previously described in urine of Sd(a+) individuals (F. Serafini-Cessi, N. Malagolini, and F. Dall'Olio. Arch. Biochem. Biophys., 266: 573-582, 1988) has been identified in cells released from human large intestine. The higher values of beta 1,4-N-acetylgalactosaminyltransferase activity were detected in proximal and medial segments of the large intestine, suggesting a proximal-distal gradient of the enzyme expression. When the beta 1,4-N-acetylgalactosaminyltranferase activity of colorectal carcinoma specimens from 18 patients was compared with that of the normal mucosa surrounding the tumor, a constant and in several cases drastic reduction of the activity was detected in tumor cells. Three human colorectal adenocarcinoma cell lines (Colo-205, SW-48, and SW-948) have been found to lack the beta 1,4-N-acetylgalactosaminyltransferase activity. Altogether, these results support the notion that the malignant transformation drastically affects the expression of this glycosyltransferase in large bowel cells
