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J Biol Chem, Vol. 273, Issue 33, 21232-21238, August 14, 1998
From Institut National de la Santé et de la Recherche
Médicale U 36, Collège de France, 3 rue d'Ulm,
75005 Paris, France
Human angiotensinogen, the specific substrate of
renin, is a heterogeneous glycoprotein constitutively secreted by the
liver. Different glycosylation levels may be responsible for its
heterogeneity. It contains four putative asparagine-linked
glycosylation sites (Asn-X-Ser/Thr). Systematic
site-directed mutagenesis (Asn replaced with Gln) of these four sites
was undertaken, and 11 (single, double, triple, and quadruple (N-4))
mutants were produced in COS-7 and/or CHO-K1 cells and characterized.
All of the sites were N-glycosylated with preferential
glycosylation of the Asn14 and the Asn271. The
suppression of the Asn14 glycosylation site led to 5 times
lower Km and a 10 times lower
kcat. Angiotensinogen heterogeneity was much
lower for the N-4 mutant protein, which produced a single form at 48 kDa. Pulse-chase experiments showed slight intracellular retention (15%) of the deglycosylated protein after 24 h. Interestingly, the N-4 mutant had a higher catalytic efficiency
(kcat/Km = 5.0 versus 1.6 µM
Role of N-Glycosylation in Human Angiotensinogen
1 · s1) than the
wild-type protein. The thermal stability of the N-4 protein was
unaffected by deglycosylation, suggesting that it was correctly folded.
This deglycosylated recombinant human angiotensinogen could be of value
for x-ray crystallography studies.
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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