Role of N-glycosylation in human angiotensinogen.
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.
Institut National de la Santé et de la Recherche Médicale U 36, Collège de France, 3 rue d'Ulm, 75005 Paris, France.
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Gimenez-Roqueplo
J
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Célérier
G
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Lucarelli
P
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Corvol
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X
Jeunemaitre
D000808:Angiotensinogen / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D016466:CHO Cells; D019556:COS Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D006031:Glycosylation; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins / Q000378:metabolism; D012083:Renin / Q000378:metabolism
D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D000808:Angiotensinogen; D012083:Renin