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J Biol Chem, Vol. 273, Issue 33, 21232-21238, August 14, 1998

Role of N-Glycosylation in Human Angiotensinogen

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 K_m and a 10 times lower k_cat. 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 (k_cat/K_m = 5.0 versus 1.6 µM¹ · 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.

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