Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.
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.
Knecht
Wolfgang
W
AstraZeneca R & D Mölndal, Mölndal, Sweden. wolfgang.knecht@astrazeneca.com
Willemse
Johan
J
Stenhamre
Hanna
H
Andersson
Mats
M
Berntsson
Pia
P
Furebring
Christina
C
Harrysson
Anna
A
Hager
Ann-Christin Malmborg
AC
Wissing
Britt-Marie
BM
Hendriks
Dirk
D
Cronet
Philippe
P
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
D011498:Protein Precursors; D005337:Fibrin; D025901:Carboxypeptidase B2; D008239:Lysine