title: Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.
abstract: Procarboxypeptidase U [proCPU, thrombin-activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability. The inherent instability of CPU is likely to be of major importance for the in vivo down-regulation of its activity, but the underlying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU. In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underlying the instability of CPU's activity. We have shown that single as well as a few 2-4 mutations in human CPU can prolong the half-life of CPU's activity at 37 degrees C from 0.2 h of wild-type CPU to 0.5-5.5 h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.
authors.affiliation: AstraZeneca R & D Mölndal, Mölndal, Sweden. wolfgang.knecht@astrazeneca.com
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Knecht
authors.forename: Johan
authors.initials: J
authors.lastname: Willemse
authors.forename: Hanna
authors.initials: H
authors.lastname: Stenhamre
authors.forename: Mats
authors.initials: M
authors.lastname: Andersson
authors.forename: Pia
authors.initials: P
authors.lastname: Berntsson
authors.forename: Christina
authors.initials: C
authors.lastname: Furebring
authors.forename: Anna
authors.initials: A
authors.lastname: Harrysson
authors.forename: Ann-Christin Malmborg
authors.initials: AC
authors.lastname: Hager
authors.forename: Britt-Marie
authors.initials: BM
authors.lastname: Wissing
authors.forename: Dirk
authors.initials: D
authors.lastname: Hendriks
authors.forename: Philippe
authors.initials: P
authors.lastname: Cronet
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation; D025901:Carboxypeptidase B2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015536:Down-Regulation; D004789:Enzyme Activation; D004795:Enzyme Stability; D005337:Fibrin / Q000235:genetics / Q000378:metabolism; D005342:Fibrinolysis; D006358:Hot Temperature; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D011489:Protein Denaturation; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D011498:Protein Precursors; D005337:Fibrin; D025901:Carboxypeptidase B2; D008239:Lysine