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

Abstract

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...

Purification and characterization of recombinant human β1–4 galactosyltransferase expressed in Saccharomyces cerevisiae

“…A difference that is important in comparison to higher eukaryotic cells related to the finding that recombinant gal-T was not secreted; even by applying a sensitive assay, activity in the yeast-culture supernatant could not be detected, which is in contrast to the supernatants of animal-cell cultures (LaMont et al, 1977). Therefore, our future efforts are aimed at the expression of a soluble form of active gal-T.…”

Homozygous deletions within human chromosome band 9p21 in melanoma.

“…The enzyme encoded by GGTB2 plays a role in glycosylation, and mutations in this protein could adversely affect cell adhesion and/or cell growth (10,11). This gene has been mapped to 9p13-p21 (6).…”

Molecular cloning and chromosomal localization of human 4-beta-galactosyltransferase.

“…Galactosyltransferase is normally found in the membrane of the Golgi apparatus but has also been reported to be a cell-surface constituent in certain cells (3,4,8 (2,37), but the biological significance of these observations has not been shown. It has been suggested that the cell-surface form of galactosyltransferase is involved in intercellular recognition or cell-cell adhesion (1,4), whereas the Golgi-bound form of the enzyme functions in posttranslational modification of proteins and lipids that eventually reach the cell surface (8).…”

“…Aberrations of these cell membrane constituents can lead to altered cell phenotypes due to the loss of cell anchorage and contact-inhibited cell growth (1,2). One group of tightly regulated cellular proteins that are involved in this complex biosynthetic pathway are the glycosyltransferases.…”