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.
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April 2003
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Research Article|
April 15 2003
Deglycosylation, processing and crystallization of human testis angiotensin-converting enzyme
Kerry GORDON;
Kerry GORDON
∗Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Observatory 7925, South Africa
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Pierre REDELINGHUYS;
Pierre REDELINGHUYS
∗Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Observatory 7925, South Africa
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Sylva L.U. SCHWAGER;
Sylva L.U. SCHWAGER
∗Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Observatory 7925, South Africa
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Mario R.W. EHLERS;
Mario R.W. EHLERS
1
∗Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Observatory 7925, South Africa
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Anastassios C. PAPAGEORGIOU;
Anastassios C. PAPAGEORGIOU
2
†Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, U.K.
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Ramanathan NATESH;
Ramanathan NATESH
†Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, U.K.
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K. Ravi ACHARYA;
K. Ravi ACHARYA
†Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, U.K.
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Edward D. STURROCK
Edward D. STURROCK
3
∗Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Observatory 7925, South Africa
‡MRC/UCT Liver Research Centre, Faculty of Health Sciences, University of Cape Town, Observatory 7925, South Africa
3To whom correspondence should be addressed, at the Division of Medical Biochemistry (e-mail sturrock@curie.uct.ac.za).
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Publisher: Portland Press Ltd
Received:
November 26 2002
Revision Received:
January 22 2003
Accepted:
January 24 2003
Accepted Manuscript online:
January 24 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 371 (2): 437–442.
Article history
Received:
November 26 2002
Revision Received:
January 22 2003
Accepted:
January 24 2003
Accepted Manuscript online:
January 24 2003
Citation
Kerry GORDON, Pierre REDELINGHUYS, Sylva L.U. SCHWAGER, Mario R.W. EHLERS, Anastassios C. PAPAGEORGIOU, Ramanathan NATESH, K. Ravi ACHARYA, Edward D. STURROCK; Deglycosylation, processing and crystallization of human testis angiotensin-converting enzyme. Biochem J 15 April 2003; 371 (2): 437–442. doi: https://doi.org/10.1042/bj20021842
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