Cell Surface Galactosyltransferase and Lectin Agglutination of Thymus and Spleen Lymphocytes

LaMont, J. Thomas; Perrotto, Joseph L.; Weiser, Milton M.; Isselbacher, Kurt J.

doi:10.1073/pnas.71.9.3726

Cited by 34 publications

(7 citation statements)

References 20 publications

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“…Adding Con A to spleen lymphocytes results in a fivefold stimulation of incorporation, with a maximum effect at 48 h. The increase in UDP-[l*C]galactose utilization coincides with the peak of [3H]thymidine incorporation. The results from these lymphocyte experiments confirm those from experiments on rat thymocytes in which Con A-stimulated blast transformation also resulted in increased galactose incorporation from exogenous UDP-galactose (8). We initiated studies to determine whether the incorporation into lymphocytes and fibroblasts was due to substrate degradation or ectogalactosyltransferases.…”

Section: Resultssupporting

confidence: 65%

Ectogalactosyltransferase studies in fibroblasts and concanavalin A-stimulated lymphocytes.

Patt¹,

Endres²,

Lucas³

et al. 1976

The Journal of Cell Biology

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Suspensions of intact animal cells are capable of transferring the sugar moiety of exogenous nucleotide sugars to endogenous glycoprotein and glycolipid acceptors (10,12,13). It has frequently been assumed that incorporation by suspended cells incubated with nucleotidr sugars indicates the presence of an ectoglycosyltransferase. Ectoenzymes are defined as membrane-bound enzymes whose active sites are accessible from the outside of the cell (3).Several recent publications have challenged the suggestion that glycosyltransferases are ectoenzymes. Evans found a nucleotide pyrophosphatase on the plasma membrane of hepatocytes which could degrade exogenously added nucleotide-sugar substrates (5). Deppert et al. (4) have reported evidence that all the incorporation by baby hamster kidney (BHK) monolayers is due to hydrolysis and intraceilular utilization of the labeled galactose released by degradation of exogenous UDPgalactose.In this communication, we show that while suspensions of mouse spleen cells incorporate label from exogenous UDP-[l~C]galactose, there are no ectogalactosyltransferases. The spleen cell preparations degrade the nucleotide sugar, releasing galactose which is utilized for complex carbohydrate synthesis within the cell. In contrast, BALB/c 3T3 and simian virus 40-transformed 3T3 cells in suspension possess an ectogalactosyltransferase capable of transferring the carbohydrate moiety of exogenous UDP-galactose to endogenous acceptor molecules. MATERIALS AND METHODS Cell CulturesBALB/c 3T3 cells (clone A31) and simian virus 40-transformed 3T3 cells (SVT2) were the gift of Dr. G. Todaro, National Institutes of Health, Bcthesda, Md. The procedures for fibroblast growth and suspension using 0.01 M EDTA in saline for studies of r tosyltransferases have been described (10).Cells were teased out of spleens from BALB/c or C3H mice into Hanks' balanced salts solution with 0.6% dextran (HBSS + dex). Cells were dispersed by expressing through a 20-G needle. They were then washed twice with HBSS + dex and resuspcnded in RPMI 1640 medium supplemented with 5% fresh human serum, 2 mM glutamine, 40 U/ml penicillin, and 50 ~g/ml streptomycin. Suspensions were diluted to 5 • 106 mononuclear cells per milliliter and 2-ml aliquots were placed in 17 • 100-ram plastic culture tubes (Falcon Plastics, Oxnard, Calif.). Concanavalin A (Con A) 1 from Calbiochem (San Diego, Calif.) was added in 0.2-ml aliquots of a 100 ~g/ml solution (final concentration l0 /~g/ml). Incubation was carried out for 48 h at 37~ in a humidified atmosphere of 5% CO2 in air. The 48-h point coincides with the maximum rate of [SH]thymidinc incorporation. After Con A stimulation, cells were centrifuged, resuspended, and washed once with TBS.Measurement of galactose incorporation into acidprecipitable material was performed essentially as described (10). The incubations with lymphocyte or fibroblast cell suspensions contained 80 pmol UDP-[I'C]galactose, 80 pmol [SH]galactose, 0.5 ~mol MnCls, and 1-3 • 106 cells in a final volume of 0.06 ml of TBS. We hav...

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Section: Resultssupporting

confidence: 65%

Ectogalactosyltransferase studies in fibroblasts and concanavalin A-stimulated lymphocytes.

Patt¹,

Endres²,

Lucas³

et al. 1976

The Journal of Cell Biology

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“…The activity of a Gal transferase on the surface of neonatal rat thymocytes and splenocytes (264) increased after mitogen stimulation of thymocytes. The presence of surface Gal transferase activity on lymphocytes was confirmed by Verbert and coworkers (265), and the same group (266,267) demonstrated the presence of surface NeuNAc transferase on rat spleen lymphocytes.…”

Section: Other Carbohydrate Binding Proteinsmentioning

confidence: 99%

Carbohydrate structure in tumor immunity

Reading

Hutchins

1985

Cancer Metast Rev

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Researchers have endeavored to define surface alterations associated with neoplasia for at least 25 years. In comparisons of normal tissues with animal and human tumors, cultured cells before and after transformation with oncogenic agents, tumorigenic and nontumorigenic transformed cells, metastatic and nonmetastatic tumor cells, high- and low-metastatic variants, and tumor cells before and after induction of differentiation to a less malignant phenotype, a consistent finding has been some form of alteration in surface carbohydrate structures. These changes in glycolipids, glycoproteins and glycosaminoglycans are reviewed, and their structures are illustrated. Both nucleotide sugar biosynthesis and glycosyltransferase changes have been associated with these alterations. In some cases, alterations in transformed cells were related to growth, rather than transformation. In others, the altered glycoconjugates are truly tumor-associated. There is evidence that cell surface glycoconjugates may function in growth control. Altered carbohydrate structures could also serve as receptors for growth promoting factors and be directly responsible for altered growth control. Recent studies with monoclonal antibodies indicate that the vast majority of antibodies recognizing tumor-associated antigens are detecting altered carbohydrate structures. Mechanisms by which the immune system can recognize these carbohydrate structures are considered, and immune recognition of tumor-associated carbohydrate structural alterations is explored. A number of these hypotheses relating to alterations in glycosylation, growth control, and tumor immunity deserve further investigation.

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“…We have also shown that surface galactosyltransferase of rat lymphocytes is markedly increased during in vitro blast transformation induced by concanavalin A (10). Elevation of surface glycosyltransferase during S phase has also been demonstrated by Bosmann (20) (2), but they did not study the effect of serum or viral transformation on release.…”

Section: Discussionmentioning

confidence: 81%

“…We also demonstrated a significant increase in cell-surface galactosyltransferase on lectin-transformed thymic lymphocytes compared to resting lymphocytes (10). In the current study, the relationship between cell-surface galactosyltransferase and cell division was explored in serum-stimulated NIL and BHK/C13 fibroblasts and their polyomatransformed counterparts.…”

mentioning

confidence: 99%

Cell-surface glycosyltransferases in cultured fibroblasts: increased activity and release during serum stimulation of growth.

LaMont¹,

Gammon²,

Isselbacher³

1977

Proc. Natl. Acad. Sci. U.S.A.

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Cell-surface galactosyltransferase was studied in suspensions of intact baby hamster kidney fibroblasts with both endogenous and exogenous glycoprotein acceptors. The cell-surface location of galactosyltransferase was demonstrated in experiments with the enzyme modifier a-lactalbumin, which does not enter the cell. The addition of a-lactalbumin to the assay medium for galactosyltransferase resulted in adcumulation of lactose in the medium but not in the cells. There was no detectable hydrolysis of UDP-galactose to free galactose by these cells, nor did a 100-fold molar excess of free galactose inhibit cell-surface galactosyltransferase. There was a marked increase in specific activity of cell-surface exogenous galactosyltransferase in serum-stimulated as compared to resting fibroblasts. Dividing but not resting fibroblasts released galactosyltransferase, but not sialyl-or fucosyltransferase, in soluble form into the tissue culture medium. The release of galactosyltransferase was greater from virally transformed than from nontransformed fibroblasts. Glycosyltransferases are a family of membrane-associated enzymes found primarily in the smooth endoplasmic reticulum and Golgi apparatus that catalyze transfer of monosaccharide residues from nucleotide sugars to incomplete acceptor glycoproteins. A number of recent studies have demonstrated that these enzymes are also present on the plasma membrane of various mammalian cells, where they may mediate cell-to-cell adhesion or recognition (1, 2). Cell-surface glucosyltransferase has been demonstrated on human platelets and evidence has been presented from several laboratories (3, 4) that this enzyme may be directly involved in platelet-collagen adhesion, an important first step in blood coagulation. Roth and White (5) (7) have also suggested that the apparent surface transferase activity of whole cell suspensions may reflect intracellular glycosylation or secretion of soluble glycosyltransferases into the incubation medium. However, the bulk of experimental evidence supports the localization of glycosyltransferases to the plasma membrane as well as internal membranes (8).In previous work from this laboratory (9) a correlation was observed between surface galactosyltransferase and concanavalin A agglutination in various normal and malignant mammalian cells. We also demonstrated a significant increase in cell-surface galactosyltransferase on lectin-transformed thymic lymphocytes compared to resting lymphocytes (10). In the current study, the relationship between cell-surface galactosyltransferase and cell division was explored in serum-stimulated NIL and BHK/C13 fibroblasts and their polyomatransformed counterparts. Rapidly dividing fibroblasts have increased levels of cell-surface galactosyltransferases, and release a soluble form of galactosyltransferase into the tissue culture medium. MATERIALS AND METHODSCell Cultures. BHK/C13 and NIL strains of baby hamster kidney fibroblasts (originally obtained from M. Stoker, Imperial Cancer Research Tund Laboratories, Lond...

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Cell Surface Galactosyltransferase and Lectin Agglutination of Thymus and Spleen Lymphocytes

Cited by 34 publications

References 20 publications

Ectogalactosyltransferase studies in fibroblasts and concanavalin A-stimulated lymphocytes.

Ectogalactosyltransferase studies in fibroblasts and concanavalin A-stimulated lymphocytes.

Carbohydrate structure in tumor immunity

Cell-surface glycosyltransferases in cultured fibroblasts: increased activity and release during serum stimulation of growth.

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