Retinoic acid-induced differentiation of the mouse teratocarcinoma cell line F9 is accompanied by an increase in the activity of UDP-galactose: beta-D-galactosyl-alpha 1,3-galactosyltransferase.

R D Cummings,S A Mattox

doi:10.1016/s0021-9258(19)57422-1

Abstract

We report here that both the mouse teratocarcinoma F9 cells and F9 cells induced to differentiate by treatment with retinoic acid contain cell surface glycoconjugates with terminal alpha-linked galactose residues, as shown by agglutination of cells with antisera to blood type B, but not to type A. In addition, both cell types contain high numbers of binding sites for Griffonia simplicifolia-I, a lectin which binds to terminal alpha-linked galactose residues, although differentiated F9 cells contain approximately 50% more binding sites/cell for this lectin. We have also confirmed that differentiation is accompanied by a decrease in the expression of the fucose-containing stage-specific embryonic antigen (SSEA)-1, as evidenced by the fact that F9 cells, but not differentiated F9 cells, are agglutinated by monoclonal antibody to this antigen. Since these results indicate that surface glycoconjugates contain terminal alpha-linked galactose residues, we assayed cell extracts for the enzyme UDP-Gal:beta-D-Gal-alpha 1,3-galactosyltransferase. We have found that F9 cell extracts contain this activity, and differentiation results in a significant increase in the specific activity of the enzyme, from approximately 2 nmol/mg h in F9 extracts to 7 nmol/mg h in RA/F9 extracts. It has been suggested that the loss of the SSEA-1 antigen upon differentiation of F9 cells is due to decreased activity of the enzyme GDP-Fuc:beta-D-GlcNAc-alpha 1, 3-fucosyltransferase. We therefore determined the activities of this fucosyltransferase and several other glycosyltransferases, which included UDP-GlcNAc:beta-D-Gal-beta 1,3-N-acetylglucosaminyltransferase, UDP-Gal:beta-D-GlcNAc-beta 1,4-galactosyltransferase, and GDP-Fuc:beta-D-GlcNAc-alpha 1,6-fucosyltransferase. We have found that extracts from both cell types contain these enzyme activities; differentiation, however, does not result in substantial changes in any of these activities.

Highlights

Retinoic Acid-induced Differentiation of the Mouse Teratocarcinoma Cell Line F9 Is Accompanied by an Increase in the Activitoyf UDP-galactose:~-D-Galactosyl-ar1,3-galactosyltransferase*
The radiola- Binding of Radioiodinated G. simplicifolia-Ito F9 and RA/F9 beled product of the reaction binds to the immobilized lectin and is Cells-We initially examined the surface glycoconjugates of eluted with 10 mM a-methylglucoside
The activity of the enzyme in extractsof both F9 andRA/F9 cells was high and in threange reported in othecrell types by other investigators [50]

Summary

RESULTS

Ucts by endogenous @-hexosaminidasesA. fter incubation, the mixture was boiled for 5 min and diluted to 1ml with water. The reaction mixtures included 45,000 by anti-blood type B sera (Fig. 1,A-D) These results suggest cpm of GDP-["C]fucose and were otherwise similar to thatdescribed that the cell surface glycoconjugates contain the terminal for the a-galactosyltransferase, except that fucose (15 mM) was in- sequence Galal,3Gal-R, which has previously been found in cluded to inhibit breakdown of products by endogenous a-fucosidases. To provide further evidence for the presence of the terminal a-linked galactose residues on surface glycoconjugates of F9 and R A P 9 cells, we determined the binding parameters of the cells with the plant lectin G. simplicifolia-I. This binding was inhibited greater than 95% by the inclusion of 50 mM raffinose in the assay (data notshown). Sites/cell with an apparent K, of 3.7 X lo7"' (Fig. 3B)

A 600 ug lectin

UDP-3H-ClcNAc

Findings

DISCUSSION