Deglycosylation, processing and crystallization of human testis angiotensin-converting enzyme.
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.
Gordon
Kerry
K
Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Observatory 7925, South Africa.
Redelinghuys
Pierre
P
Schwager
Sylva L U
SL
Ehlers
Mario R W
MR
Papageorgiou
Anastassios C
AC
Natesh
Ramanathan
R
Acharya
K Ravi
KR
Sturrock
Edward D
ED
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003460:Crystallization; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008297:Male; D016297:Mutagenesis, Site-Directed; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013737:Testis / Q000201:enzymology*
D011994:Recombinant Proteins; D007703:Peptidyl-Dipeptidase A