Characterization of Precursor and Secreted Forms of Rat Angiotensinogen*

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Abstract
Angiotensinogen precursors synthesized by rabbit reticulocyte lysate primed with rat liver RNA were compared with angiotensinogen secreted by rat hepatoma cells and rat hepatocytes using immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibition of glycosylation with tunicamycin permittted identification of the nonglycosylated form of secreted angiotensinogen. While angiotensinogen secreted by hepatoma cells and hepatocytes showed electrophoretic heterogeneity (MW, 52-62 .times. 103), tunicamycin-treated cells secreted only a single angiotensinogen species [MW, 48.3 .+-. 0.7 .times. 103 (mean .+-. SD)], which could be cleaved by renin. Two putative angiotensinogen precursors were synthesized in the reticulocyte lysate: a major protein of 52.5 .+-. 1.0 .times. 103 MW II and a minor protein of 55.7 .+-. 1.3 .times. 103 MW. Evidence that these proteins represent separate angiotensinogen precursors includes the following. Both proteins were recognized by 5 different polyclonal antibodies and 2 monoclonal antibodies. Both proteins increased in parallel in reticulocyte lysates primed with liver RNA from rats nephrectomized and given hormones that increase liver angiotensinogen production. Both proteins were cleaved by renin to produce a single protein of 47.6 .+-. 0.8 .times. 103 MW. The des-angiotensin I-angiotensinogen generated by renin treatment of the lysate had an electrophoretic mobility identical to that of des-angiotensin I-angiotensinogen produced by renin treatment of nonglycosylated angiotensinogen secreted by tunicamycin-treated hepatoma cells and hepatocytes. Apparently rat liver synthesizes 2 separate angiotensinogen precursors which may differ only in the size of their prepro sequence. The heterogeneity of secreted angiotensinogen can be fully accounted for by differences in N-glycosylation of asparagine residues of the molecule. Glycosylation of angiotensinogen is not essential for its synthesis, processing and secretion or its hydrolysis by renin.