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, tACEDelta36-g1234 and -g13, were successfully crystallized, diffracting to 2.8 and 3.0 A resolution respectively. Furthermore, a truncated, soluble tACE (tACEDelta36NJ), 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 P2(1)2(1)2(1) space group (cell dimensions, a=56.47 A, b=84.90 A, c=133.99 A, alpha=90 degrees, beta=90 degrees and gamma=90 degrees ). These crystals diffracted to 2.0 A 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.

A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme

“…The protein construct was also modified by truncation at Ser625 (corresponding to Ser1201 of somatic ACE), which eliminated part of the stalk region, the hydrophobic transmembrane domain, and the C-terminal intracellular domain. When this shortened protein was expressed in the presence of Nbutyldeoxynojirimycin, an a-glycosidase I inhibitor, and treated with endoglycosidase-H, it yielded crystals by vapor-diffusion hanging drop that were suitable for X-ray diffraction studies at a resolution of 2.0 A (Gordon et al, 2003;Natesh et al, 2003).…”

“…A major advance in this area was the successful determination of the crystal structure of human testis ACE. These studies were made possible by work from Riordan, Sturrock, and others who systematically investigated the role of glycosylation in the activity and stability of testis ACE (Yu et al, 1997;Gordon et al, 2003). With the exception of the N-terminal 36 amino acids, mature human testis ACE is identical to the C terminus of somatic ACE, and it was thought to be easier to crystallize than the larger somatic isozyme.…”

Crystal structures of sampatrilat and sampatrilat‐Asp in complex with human ACE – a molecular basis for domain selectivity

“…Minimally glycosylated N‐ and C‐domain human ACE proteins (N389 and G13 respectively) were generated by expression in cultured mammalian CHO cells and purified to homogeneity as described previously 20, 31. Synthesis and inhibition data for sampatrilat and samAsp analogue have been reported previously 18.…”

The Structure of Testis Angiotensin-Converting Enzyme in Complex with the C Domain-Specific Inhibitor RXPA380,

“…A variant of human tACE (tACE∆36NJ) was expressed in CHO cells and purified to homogeneity using lisinopril-Sepharose affinity chromatography, as described previously (23). We refer to this variant throughout the manuscript as tACE.…”