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(Received for publication, June 7, 1995; and in revised form, September 6, 1995) We used a novel approach to study the role of the Asn-linked
oligosaccharides for human thyrotropin (hTSH) activity. Mutagenesis of
Asn(N) within individual glycosylation recognition sequences to Gln (Q)
was combined with expression of wild type and mutant hTSH in cell lines
with different glycosylation patterns. The in vitro activity
of hTSH lacking the Asn
Volume 270,
Number 49,
Issue of December 8, 1995 pp. 29378-29385
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CHARACTERIZATION OF A NOVEL ROLE FOR THE OLIGOSACCHARIDES IN THE IN VITRO AND IN VIVO BIOACTIVITY
oligosaccharide
(
Q52/TSH
) expressed in CHO-K1 cells (sialylated
oligosaccharides) was increased 6-fold compared with wild type, whereas
the activities of
Q78/TSH
and
/TSH
Q23 were
increased 2-3-fold. Deletion of the Asn oligosaccharide also increased the thyrotropic activity of human
chorionic gonadotropin, in contrast to previous findings at its native
receptor. The in vitro activity of wild type hTSH expressed in
CHO-LEC2 cells (sialic acid-deficient oligosaccharides), CHO-LEC1 cells
(Man
GlcNAc
intermediates), and 293 cells
(sulfated oligosaccharides) was 5-8-fold higher than of wild type
from CHO-K1 cells. In contrast to CHO-K1 cells, there was no difference
in the activity between wild type and selectively deglycosylated
mutants expressed in these cell lines. Thus, in hTSH, the
oligosaccharide at Asn and, specifically, its
terminal sialic acid residues attenuate in vitro activity, in
contrast to the previously reported stimulatory role of this chain for
human chorionic gonadotropin and human follitropin activity. The
increased thyrotropic activity of
Q52/CG
suggests that
receptor-related mechanisms may be responsible for these differences
among the glycoprotein hormones. Despite their increased in vitro activity,
Q52/TSH
, and
Q78/TSH
from CHO-K1
cells had a faster serum disappearance rate and decreased effect on
T
production in mice. These findings highlight the
importance of individual oligosaccharides in maintaining circulatory
half-life and hence in vivo activity of hTSH.
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