ROLE OF N-GLYCOSYLATION IN HUMAN ANGIOTENSINOGEN

Human angiotensinogen, the specific substrate of renin, is a heterogen eous glycoprotein constitutively secreted by the liver. Different glyc osylation 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 Gin) of these four sites was undertaken, and 11 (single, double, triple, and quadrup le (N-4)) mutants were produced in COS-7 and/or CHO-K1 cells and chara cterized. All of the sites were N-glycosylated with preferential glyco sylation of the Asn(14) and the Asn(271). The suppression of the Asn(1 4) 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 experimen ts showed slight intracellular retention (15%) of the deglycosylated p rotein after 24 h, Interestingly, the N-4 mutant had a higher catalyti c efficiency (k(cat)/K-m = 5.0 versus 1.6 mu M-1 . s(-1)) than the wil d-type protein. The thermal stability of the N-4 protein was unaffecte d by deglycosylation, suggesting that it was correctly folded. This de glycosylated recombinant human angiotensinogen could be of value for x -ray crystallography studies.