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Ecto-sialyltransferase of human B lymphocytes reconstitutes differentiation
markers in the presence of exogenous CMP-N-acetyl neuraminic acid
HJ Gross, A Merling, G Moldenhauer and R Schwartz-Albiez
Institut fur klinische Chemie, Universitatsklinikum, Ulm, Germany.
The existence of an ecto-sialyltransferase (ecto-ST) on B lymphocytes with
increasing activity at late maturation stages is shown using a novel flow
cytometric enzyme assay. This ecto-ST is effective in reconstituting
different surface glycoconjugates on desialylated B cells in the presence
of exogenous CMP-NeuAc. We found that this ecto- ST is distinct in its
activity from soluble ST released into the culture supernatant. Surface
sialylation was independent of the amount of ST secreted into the culture
supernatant and followed different kinetics than sialylation of exogenous
substrate by soluble ST. Four human B-cell lines representing different
maturation stages were analyzed for secreted and ecto-ST activity. The
myeloma cell line U266 and the lymphoblastoid cell line JOK-1 showed higher
activity of both ST forms than the acute lymphoblastic leukemia B-cell line
Nalm-6. ST activity in culture supernatants of U266, JOK-1, and Nalm-6
cells consisted predominantly of the alpha 2,6 ST type with specificity for
N- linked oligosaccharides. As an exception, the myeloma cell line IM-9,
deficient of alpha 2,6 ST activity, secreted only small amounts of ST and
showed low activity of ecto-ST. Sialylation of surface-expressed
glycoconjugates by ecto-ST was measured by incubating B-cell lines in the
presence of fluorescent CMP-sialic acid. Surface structures labeled with
fluorescent sialic acid under this condition were visualized by confocal
laser scanning microscopy and fluorescent label was quantitatively assessed
by flow cytometric analysis on live cells. Incubation of cells in acidified
culture medium, to release possibly receptor-bound ST, did not alter the
intensity of cell surface sialylation. Inhibition of internalization and
membrane traffic by various approaches (reduced incubation temperature and
chloroquine or brefeldin A treatment) did not block surface sialylation.
Together, these observations point to cell surface sialylation in B
lymphocytes mediated by a cell surface-expressed ecto-ST distinct from the
secreted ST form. On desialylated JOK-1 cells, ecto-ST in the presence of
exogenous CMP-NeuAc was able to resialylate the B-cell surface sialoglycans
CDw75 and HB-6 and major surface glycoproteins of B cells, such as HLA
class I and II antigens, transferrin receptor, and surface IgM. In
contrast, cell surface glycans of coincubated desialylated erythrocytes
were not sialylated by the B-cell ecto-ST. Ecto-alpha 2,6 ST of B cells may
be involved in the sialylation of distinct differentiation glycan antigens.
Volume 87,
Issue 12,
pp. 5113-5126,
06/15/1996
Copyright © 1996 by The American Society of Hematology
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