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Zenda L. Woodman, Sylva L.U. Schwager, Pierre Redelinghuys, Anthony J. Chubb, Elizabeth L. van der Merwe, Mario R.W. Ehlers, Edward D. Sturrock
Homologous substitution of ACE C-domain regions with N-domain sequences: effect on processing, shedding, and catalytic properties
Keywords: cooperativity, domain selectivity, proteolytic cleavage, sheddase
Angiotensin-converting enzyme (ACE) exists as two isoforms: somatic ACE (sACE), comprised of two homologous N and C domains, and testis ACE (tACE), comprised of the C domain only. The N and C domains are both active, but show differences in substrate and inhibitor specificity. While both isoforms are shed from the cell surface via a sheddase-mediated cleavage, tACE is shed much more efficiently than sACE. To delineate the regions of tACE that are important in catalytic activity, intracellular processing, and regulated ectodomain shedding, regions of the tACE sequence were replaced with the corresponding N-domain sequence. The resultant chimeras C1–163Ndom-ACE, C417–579Ndom-ACE, and C583–623Ndom-ACE were processed to the cell surface of transfected Chinese hamster ovary (CHO) cells, and were cleaved at the identical site as that of tACE. They also showed acquisition of N-domain-like catalytic properties. Homology modelling of the chimeric proteins revealed structural changes in regions required for tACE-specific catalytic activity. In contrast, C164–416Ndom-ACE and C191–214Ndom-ACE demonstrated defective intracellular processing and were neither enzymatically active nor shed. Therefore, critical elements within region D164–V416 and more specifically I191–T214 are required for the processing, cell-surface targeting, and enzyme activity of tACE, and cannot be substituted for by the homologous N-domain sequence.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 387, 08/2006
Pages: 1043 - 1051