Structural and biological roles of glycosylations in pulmonary angiotensin I-converting enzyme

We enzymatically deglycosylated pig lung angiotensin I-converting enzyme (ACE) to study the involvement of its glycanic chains in its physicochemical and catalytic properties. The effects of endoglycosidases F2 and H, and of N-glycanase were assessed by ACE mobility in SDS-PAGE. N-Glycanase only was completely effective with or without previous denaturation, leading to a shift in ACE M(r) from 172 to 135 kDa; endoglycosidase F2 produced the same shift but only without previous denaturation. Deglycosylated ACE had the same kcat as native ACE for the substrate hippuryl-histidyl-leucine, and an identical Stokes radius as measured by size-exclusion high performance liquid chromatography. Neuraminidase had no effect on ACE Stokes radius but slightly decreased its kcat which could be related to variations in ionization of the active site. The isoelectric point of ACE, as, determined by isoelectric focusing, increased from 4.5-4.8 to 5.0-5.3 after either endoglycosidase F2 or neuraminidase digestion, but still with microheterogeneities which thus did not seem to be related to ACE glycans. Deglycosylated ACE did not bind onto agaroselectins in contrast to native ACE which bound strongly to concanavalin A showing interactions involving oligomannosidic or biantennary and sialylated N-acetyl-lactosaminic isoglycans. Finally, tunicamycin, an inhibitor of N-glycosylation, did not modify ACE secretion by endothelial cells. Thus, ACE glycans have no drastic effects on structural and biological properties of the protein, but they may have a functional role on intracellular targeting of both secreted and membrane-bound ACE isoforms, also for the protection of the soluble plasma form against hepatic lectins and the maintenance of its hydrosolubility.