Interactions of cell-surface galactosyltransferase with immunoglobulins

Abstract

Detection of the activity of β-1,4-galactosyltransferase (β-1,4-GT) in suspensions of viable mouse hepatocytes, the human hepatoma cell line Hep G2, the human colonic adenocarcinoma cell line HT-29, the monocyte-like cell line U937, and human splenic B and T lymphocytes suggested the presence of β-1,4-GT, in an enzymatically active form, on plasma membranes. The presence of β-1,4-GT on cell surfaces was also indicated from the effect of trypsinization of live cells, which significantly reduced cell surface β-1,4-GT activity, but did not affect the activity associated with cytoplasmic membranes. Furthermore, the presence of β-1,4-GT on the cell surface was demonstrated by indirect immunofluorescence staining of cells with anti-β-1,4-GT antibody. The detection of radioactivity in immunoglobulins (Ig) and their component chains after incubation with suspensions of intact cells in the presence of Mn 2+ and UDP-[ 3H]-galactose, indicated that Ig molecules were galactosylated. In the absence of UDP-[ 3H]-galactose, β-1,4-GT on cell surfaces, or immobilized on Sepharose-4B, formed stable complexes with galactose acceptors, including Ig. The efficiency of binding decreased in the order: J chain > α chain > μ chain > polymeric IgA2 > monomeric/polymeric IgA1 > IgM > IgG. Thus, β-1,4-GT could act as a cell-surface receptor for Ig through a cation-dependent, lectin-like association of the β-1,4-GT with the carbohydrate moieties of the Ig. This was confirmed by indirect surface immunofluorescence and radiolabeled ligand binding assays. The binding was inhibitable by EDTA, α-lactalbumin (in the presence of glucose), GlcNAc, or uridine 3',5'dialdehyde. At 37°C, the apparent affinity constants and association rate constants of interaction between cell surface β-1,4-GT on glutaraldehyde-fixed HT-29 and U937 cells and α2 chain or monomeric IgA1 were in the range from 7.1 × 10 7 to 4.6 × 10 8 M −1 and from 1 × 10 5 to 3 × 10 6 M −1 s −1, respectively. The dissociation rate constants and half time of dissociation calculated from these data were in the range from 2.1 × 10 −2 to 5.0 × 10 −4 s −1 and from 33 to 1380 s, respectively. The number of α2 or IgA1 molecules bound per HT-29 and U937 cell were in the range from 1.9 × 10 5 to 1.3 × 10 6. The binding of IgA by the cell surface β-1,4-GT was not associated with internalization or the catabolic degradation of the ligand.