Role of cysteine residues in human angiotensinogen - Cys(232) is required for angiotensinogen pro major basic protein complex formation

The M235T polymorphism of human angiotensinogen is associated with essentia l and pregnancy-induced hypertension. A covalent complex is formed between angiotensinogen and the proform of the eosinophil major basic protein (proM BP) during pregnancy. The sequence of human angiotensinogen contains four c ysteines. Their function was analyzed. Presence of free cysteines was demon strated by their alkylation with iodo[C-14]acetic:acid. A disulfide bond be tween Cys(18) and Cys(138) using a fully N-deglycosylated mutant of human a ngiotensinogen was identified by tryptic digestion and mass spectrometry. W e produced angiotensinogen proMBP complex by co-transfection of COS-7 cells and by co-culturing transfected CHO-K1 cells. Experiments with 8 mutated r ecombinant angiotensinogen, in which one or more of the four cysteines were replaced by alanine, demonstrated that Cys(232) in involved in complex for mation and could interact with the M235T variant. The angiotensinogen.proMB p complex was isolated by molecular sieving, Hydrolysis of the complex by h uman renin was 7 times slower than hydrolysis of monomeric form, whatever t he M235T genotype, The complex:monomeric angiotensinogen ratio was greater for Met(235) (72%) than for Thr(235) (58%) angiotensinogen. These data sugg est a new pathophysiological explanation for the genetic association betwee n M235T angiotensinogen polymorphism and pregnancy-induced hypertension.