Role of Glycine 221 in Catalytic Activity of Hyaluronan-Binding Protein 2
| Autor: | Stavenuiter, Fabian, Ebberink, Eduard H T M, Mertens, Koen, Meijer, Sander, Pharmaceutics, Afd Pharmaceutics, Afd Biomol.Mass Spect. and Proteomics, Biomolecular Mass Spectrometry and Proteomics |
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| Přispěvatelé: | Pharmaceutics, Afd Pharmaceutics, Afd Biomol.Mass Spect. and Proteomics, Biomolecular Mass Spectrometry and Proteomics |
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
medicine.medical_treatment serine protease Mutant Allosteric regulation Glycine Mutation Missense 030204 cardiovascular system & hematology Biology Marburg I polymorphism Biochemistry Catalysis 03 medical and health sciences 0302 clinical medicine Protein Domains Fibrinolysis medicine enzyme mechanism coagulation factor factor seven activating protease (FSAP) Molecular Biology Serine protease Protease Serine Endopeptidases Cell Biology Urokinase-Type Plasminogen Activator Molecular biology Footprinting allosteric regulation N-terminus 030104 developmental biology Amino Acid Substitution hyaluronan-binding protein 2 (HABP2) Protein Structure and Folding biology.protein Calcium fibrinolysis sodium binding Plasminogen activator |
| Zdroj: | Journal of Biological Chemistry, 6381. American Society for Biochemistry and Molecular Biology Inc. STARTPAGE=6381;ISSN=0021-9258;TITLE=Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| Popis: | HABP2 (hyaluronan-binding protein 2) is a Ca2+-dependent serine protease with putative roles in blood coagulation and fibrinolysis. A G221E substitution, known as the Marburg I polymorphism, reportedly affects HABP2 function and has been associated with increased risk for cardiovascular disease. However, the importance of Gly-221 for HABP2 activity is unclear. Here, we used G221E, G221A, and G221S mutants to assess the role of Gly-221 in HABP2 catalysis. The G221E variant failed to activate the single-chain urokinase-type plasminogen activator, and the G221A and G221S variants displayed moderately reduced single-chain urokinase-type plasminogen activator activation. Activity toward the peptide substrate S-2288 was markedly decreased in all HABP2 variants, with G221E being the most defective and G221A being the least defective. In the absence of Ca2+, S-2288 cleavage by wild-type HABP2 was Na+-dependent, with Km decreasing from 3.0 to 0.6 mm upon titration from 0 to 0.3 m Na+. In the presence of 5 mm Ca2+, Km was further reduced to 0.05 mm, but without an appreciable contribution of Na+. At physiological concentrations of Na+ and Ca2+, the three HABP2 variants, and particularly G221E, displayed a major Km increase for S-2288. Chemical footprinting revealed that Ile-16 is significantly less protected from chemical modification in G221E than in wild-type HABP2, suggesting impaired insertion of the N terminus into the G221E protease domain, with a concomitant impact on catalytic activity. Homology modeling suggested that the Glu-221 side chain could sterically hinder insertion of the N terminus into the HABP2 protease domain, helping to explain the detrimental effects of Glu-221 substitution on HABP2 activity. |
| Databáze: | OpenAIRE |
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