Detection of the activity of
beta-1,4-galactosyltransferase (beta-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 beta-1,4-GT, in an enzymatically active form, on plasma membranes. The presence of beta-1,4-GT on cell surfaces was also indicated from the effect of trypsinization of live cells, which significantly reduced cell surface beta-1,4-GT activity, but did not affect the activity associated with cytoplasmic membranes. Furthermore, the presence of beta-1,4-GT on the cell surface was demonstrated by indirect immunofluorescence staining of cells with anti-beta-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 Mn2+ and
UDP-[3H]-
galactose, indicated that Ig molecules were galactosylated. In the absence of
UDP-[3H]-
galactose, beta-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 > alpha chain > mu chain > polymeric
IgA2 > monomeric/polymeric
IgA1 >
IgM >
IgG. Thus, beta-1,4-GT could act as a
cell-surface receptor for Ig through a
cation-dependent,
lectin-like association of the beta-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,
alpha-lactalbumin (in the presence of
glucose), GlcNAc, or
uridine 3',5'dialdehyde. At 37 degrees C, the apparent affinity constants and association rate constants of interaction between cell surface beta-1,4-GT on
glutaraldehyde-fixed HT-29 and U937 cells and alpha 2 chain or monomeric
IgA1 were in the range from 7.1 x 10(7) to 4.6 x 10(8) M-1 and from 1 x 10(5) to 3 x 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 x 10(-2) to 5.0 x 10(-4) s-1 and from 33 to 1380 s, respectively. The number of alpha 2 or
IgA1 molecules bound per HT-29 and U937 cell were in the range from 1.9 x 10(5) to 1.3 x 10(6). The binding of
IgA by the cell surface beta-1,4-GT was not associated with internalization or the catabolic degradation of the
ligand.