STRUCTURAL AND BIOLOGICAL ROLES OF GLYCOSYLATIONS IN PULMONARY ANGIOTENSIN I-CONVERTING ENZYME

We enzymatically deglycosylated pig lung angiotensin I-converting enzy me (ACE) to study the involvement of its glycanic chains in its physic ochemical and catalytic properties, The effects of endoglycosidases F- 2 and H, and of N-glycanase were assessed by ACE mobility in SDS-PAGE, N-Glycanase only was completely effective with or without previous de naturation, leading to a shift in ACE M-r from 172 to 135 kDa; endogly cosidase F-2 produced the same shift but only without previous denatur ation, Deglycosylated ACE had the same kcat as native ACE for the subs trate hippuryl-histidyl-leucine, and an identical Stokes radius as mea sured by size-exclusion high performance liquid chromatography, Neuram inidase had no effect on ACE Stokes radius but slightly decreased its keat which could be related to variations in ionization of the active site, The isoelectric point of ACE, as, determined by isoelectric focu sing, increased from 4.5-4.8 to 5.0-5.3 after either endoglycosidase F , or neuraminidase digestion, but still with microheterogeneities whic h thus did not seem to be related to ACE glycans, Deglycosylated ACE d id not bind onto agaroselectins in contrast to native ACE which bound strongly to concanavalin A showing interactions involving oligomannosi dic or biantennary and sialylated N-acetyl-lactosaminic isoglycans. Fi nally, tunicamycin, an inhibitor of N-glycosylation, did not modify AC E secretion by endothelial cells, Thus, ACE glycans have no drastic ef fects on structural and biological properties of the protein, but they may have a functional role on intracellular targeting of both secrete d and membrane-bound ACE isoforms, also for the protection of the solu ble plasma form against hepatic lectins and the maintenance of its hyd rosolubitity.