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J Biol Chem, Vol. 273, Issue 50, 33423-33428, December 11, 1998
From the Departments of The amino-terminal ectodomain of thyrotropin
(TSH) receptor (TSHR) is heavily glycosylated with asparagine-linked
(N-linked) oligosaccharides. The present studies were
designed to evaluate how acquisition and processing of N-linked
oligosaccharides play a role in the functional maturation of human
TSHR. A glycosylation inhibitor tunicamycin, which inhibits the first
step of N-linked glycosylation (acquisition of
N-linked oligosaccharides), and a series of mutant Chinese
hamster ovary (CHO)-Lec cells defective in the different steps of
glycosylation processing were used. Inhibition of acquisition of
N-linked oligosaccharides by tunicamycin treatment in CHO
cells stably expressing TSHR produced nonglycosylated TSHR, which was
totally nonfunctional. In contrast, all of the TSHRs synthesized in
mutant CHO-Lec1, 2, and 8 cells (mannose-rich, sialic acid-deficient,
and galactose-deficient oligosaccharides, respectively) bound TSH and
produced cAMP in response to TSH with an affinity and an
EC50 similar to those in TSHR expressed in parental CHO
cells (CHO-TSHR; sialylated oligosaccharides). However, Lec1-TSHR and
Lec2-TSHR were not efficiently expressed on the cell surface, whereas
the expression levels of Lec8-TSHR and CHO-TSHR were essentially
identical. All of the TSHRs expressed in CHO-Lec cells cleaved into two
subunits. Finally, anti-TSHR autoantibodies from Graves' patients
interacted with all of the TSHRs harboring different oligosaccharides
to a similar extent. These data demonstrate that acquisition and
processing of N-linked oligosaccharides of TSHR appear to
be essential for correct folding in the endoplasmic reticulum and for
cell surface targeting in the Golgi apparatus. We also show that
complex type carbohydrates are not crucially involved in the
interaction of TSHR with TSH and anti-TSHR autoantibodies.
Role of Asparagine-linked Oligosaccharides in Protein Folding,
Membrane Targeting, and Thyrotropin and Autoantibody Binding of the
Human Thyrotropin Receptor
,
,
Pharmacology 1, ¶ Nature
Medicine, and Internal Medicine 1, Nagasaki University
School of Medicine, Nagasaki, 852-8523, Japan
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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