Deglycosylation, processing and crystallization of human testis angiotensin-converting enzyme

Abstract

Angiotensin I-converting enzyme (ACE) is a highly glycosylated type I integral membrane protein. A series of underglycosylated testicular ACE (tACE) glycoforms, lacking between one and five N-linked glycosylation sites, were used to assess the role of glycosylation in tACE processing, crystallization and enzyme activity. Whereas underglycosylated glycoforms showed differences in expression and processing, their kinetic parameters were similar to that of native tACE. N-glycosylation of Asn-72 or Asn-109 was necessary and sufficient for the production of enzymically active tACE but glycosylation of Asn-90 alone resulted in rapid intracellular degradation. All mutants showed similar levels of phorbol ester stimulation and were solubilized at the same juxtamembrane cleavage site as the native enzyme. Two mutants, tACEΔ36-g1234 and -g13, were successfully crystallized, diffracting to 2.8 and 3.0Å resolution respectively. Furthermore, a truncated, soluble tACE (tACEΔ36NJ), expressed in the presence of the glucosidase-I inhibitor N-butyldeoxynojirimycin, retained the activity of the native enzyme and yielded crystals belonging to the orthorhombic P212121 space group (cell dimensions, a = 56.47Å, b = 84.90Å, c = 133.99Å, α = 90°, β = 90° and γ = 90°). These crystals diffracted to 2.0Å resolution. Thus underglycosylated human tACE mutants, lacking O-linked oligosaccharides and most N-linked oligosaccharides or with only simple N-linked oligosaccharides attached throughout the molecule, are suitable for X-ray diffraction studies.