For facilitating crystallization and structural studies of the testicular isozyme of angiotensin-converting enzyme (ACE), we attempted the production of enzymatically active ACE proteins which are unglycosylated or underglycosylated. Expression in Escherichia coli of the rabbit ACE cDNA resulted in the synthesis of an unglycosylated but inactive protein. Similarly, unglycosylated ACE synthesized in HeLa cells, by using a cDNA in which all five potential N-glycosylation sites had been mutated, was inactive and rapidly degraded. Several ACE variants carrying mutations in one or more of the potential N-glycosylation sites were used to examine the role of glycosylation at specific sites on ACE synthesis, transport to the cell surface, cleavage processing, and enzyme activity. These experiments demonstrated that allowing glycosylation only at the first or the second site, as counted from the NH terminus, was sufficient for normal synthesis and processing of active ACE. In contrast, ACEg3, which had only the third glycosylation site available, was unglycosylated, enzymatically inactive and rapidly degraded. N-Glycosylated ACE could also be produced in yeast. Surprisingly, the mutant ACEg3 was synthesized, N-glycosylated, and properly transported in yeast. Wild type and mutant ACE proteins were cleavage-secreted from yeast and enzymatically active.

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