Format

Send to

Choose Destination
J Biol Chem. 1998 Aug 14;273(33):21232-8.

Role of N-glycosylation in human angiotensinogen.

Author information

1
Institut National de la Santé et de la Recherche Médicale U 36, Collège de France, 3 rue d'Ulm, 75005 Paris, France.

Abstract

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 microM-1 . s-1) 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.

PMID:
9694881
DOI:
10.1074/jbc.273.33.21232
[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for HighWire
Loading ...
Support Center