The N Domain of Human Angiotensin-I-converting Enzyme

Autor: Hazel R. Corradi, Edward D. Sturrock, Colin Anthony, Pierre Redelinghuys, Vincent Dive, Sylva L. U. Schwager, K. Ravi Acharya, Dimitris Georgiadis
Jazyk: angličtina
Rok vydání: 2010
Předmět:
Models
Molecular
Glycosylation
Protease Inhibitor
Thermal Stability
Crystallography
X-Ray
Biochemistry
Mass Spectrometry
Substrate Specificity
chemistry.chemical_compound
Protein structure
N-linked glycosylation
Cricetinae
Enzyme Stability
Mutagenesis Mechanisms
biology
Molecular Structure
Temperature
Recombinant Proteins
Protein Structure and Folding
Angiotensin-converting Enzyme
Phosphinic Peptide
Oligopeptides
medicine.drug
Protein Binding
Glycan
Protein Structure
Protein domain
Blotting
Western
CHO Cells
Peptidyl-Dipeptidase A
Metalloprotease
Cricetulus
Protein Domains
Hydrolase
medicine
Animals
Humans
Binding site
Molecular Biology
Binding Sites
Cell Biology
Phosphinic Acids
Protease inhibitor (biology)
Protein Structure
Tertiary
carbohydrates (lipids)
chemistry
Mutation
biology.protein
Biocatalysis
Zdroj: The Journal of Biological Chemistry
ISSN: 1083-351X
0021-9258
Popis: Angiotensin-I-converting enzyme (ACE) plays a critical role in the regulation of blood pressure through its central role in the renin-angiotensin and kallikrein-kinin systems. ACE contains two domains, the N and C domains, both of which are heavily glycosylated. Structural studies of ACE have been fraught with severe difficulties because of surface glycosylation of the protein. In order to investigate the role of glycosylation in the N domain and to create suitable forms for crystallization, we have investigated the importance of the 10 potential N-linked glycan sites using enzymatic deglycosylation, limited proteolysis, and mass spectrometry. A number of glycosylation mutants were generated via site-directed mutagenesis, expressed in CHO cells, and analyzed for enzymatic activity and thermal stability. At least eight of 10 of the potential glycan sites are glycosylated; three C-terminal sites were sufficient for expression of active N domain, whereas two N-terminal sites are important for its thermal stability. The minimally glycosylated Ndom389 construct was highly suitable for crystallization studies. The structure in the presence of an N domain-selective phosphinic inhibitor RXP407 was determined to 2.0 A resolution. The Ndom389 structure revealed a hinge region that may contribute to the breathing motion proposed for substrate binding.
Databáze: OpenAIRE
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