Post-translational Modifications of Human Thrombin-Activatable Fibrinolysis Inhibitor (TAFI):  Evidence for a Large Shift in the Isoelectric Point and Reduced Solubility upon Activation

Valnickova, Zuzana; Christensen, Trine; Skottrup, Peter Durand; Thøgersen, Ida B.; Højrup, Peter; Enghild, Jan J.

doi:10.1021/bi051956v

Cited by 31 publications

(45 citation statements)

References 43 publications

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“…31 Crystallization of plasma and fully glycosylated recombinant TAFI resulted in nicely shaped but poorly diffracting crystals. Therefore, we produced recombinant TAFI in a HEK293ES cell line, which has a mutation in Nacetylglucosaminyltransferase-I, resulting in the production of homogeneous N-linked glycans of the (Man) 5 (GlcNAc) 2 type.…”

Section: Crystallization and Overall Structurementioning

confidence: 99%

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

Marx

Brondijk²,

Plug

et al. 2008

Blood

115

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Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombincleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors. (Blood. 2008;112: 2803-2809) Introduction TAFI 1,2 is a pro-metallocarboxypeptidase that links the coagulation and fibrinolytic systems. TAFI is activated by thrombin, the thrombin-thrombomodulin complex or plasmin. 3 Activated TAFI (TAFIa) inhibits plasmin-mediated blood clot lysis by removing C-terminal lysine residues from partially degraded fibrin that are required for positive feedback in tissue plasminogen-activator dependent plasmin generation. In addition, TAFIa has been implicated in modulation of the inflammatory response by inactivating bradykinin and the anaphylatoxins C3a and C5a. 4,5 Although it is a powerful antifibrinolytic agent, there are no known physiologic inhibitors of TAFIa. Instead, the half-life of TAFIa activity is regulated by its intrinsic instability. The inactivation rate, 5 to 10 minutes at 37°C, is highly temperature-dependent, suggesting that inactivation involves a large conformational change. 6 This is also suggested by the susceptibility of the inactive enzyme, TAFIai to proteolytic cleavage by thrombin at Arg302, a site that is cryptic in TAFI and TAFIa. 6,7 The stability of TAFIa is an important determinant for its antifibrinolytic potential because TAFIa inhibits fibrinolysis through a threshold-dependent mechanism. [8][9][10] Full-length TAFI consists of 401 amino acids divided into 2 domains: the first 92 amino acids form the activation peptide; the next 309 amino acids form the catalytic domain. The activation peptide restricts substrate access to the catalytic cleft in the zymogen. TAFI is activated through cleavage at Arg92, which releases the activation peptide.TAFI is highly homologous to the pancreatic procarboxypeptidases with 42% sequence identity to human procarboxypeptidase B (pro...

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Section: Crystallization and Overall Structurementioning

confidence: 99%

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

Marx

Brondijk²,

Plug

et al. 2008

Blood

115

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“…Institute, Copenhagen, Denmark using plasminogendepleted plasma and plasminogen-Sepharose affinity chromatography as described previously [4,18]. Guinea pig liver (tissue) transglutaminase (EC 2.3.2.13), human fibrinogen and human thrombin (EC 3.4.21.5) were purchased from Sigma.…”

Section: Proteinsmentioning

confidence: 99%

“…Isoelectric focusing of bovine TAFI was performed essentially as described previously [18]. Briefly, 10 μg of saltfree protein was focused under native conditions in a Ready IEF gel using the MiniProtean III Cell (Biorad) according to the manufacturer's instructions.…”

Section: Isoelectric Focusingmentioning

confidence: 99%

“…A variety of trypsin-like proteinases have been shown to remove this peptide, generating the mature protein, TAFIa [4,[14][15][16][17]. The isoelectric point (pI) of this proteolytically cleaved protein is around pH 8.5, which is significantly more basic than that of TAFI (pI 5.5) [18]. TAFIa remains in circulation by forming complexes with α 2 -macroglobulin and pregnancy zone protein [19] but is highly unstable, a feature initially attributed to proteolytic cleavage.…”

Section: Introductionmentioning

confidence: 99%

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Biochemical Characterization of Bovine Plasma Thrombin-Activatable Fibrinolysis Inhibitor (TAFI)

Valnickova¹,

Thaysen‐Andersen²,

Højrup³

et al. 2011

Recent Advances in Biochemistry

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Background: TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa) in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombinactivatable fibrinolysis inhibitor (TAFI), and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI.

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“…The 92-residue pro-domain of TAFI contains four N-linked glycan structures (18). This is the only MCP pro-domain that is glycosylated, thus suggesting biological significance.…”

mentioning

confidence: 96%

Flexibility of the Thrombin-activatable Fibrinolysis Inhibitor Pro-domain Enables Productive Binding of Protein Substrates

Valnickova

Sanglas

Arolas

et al. 2010

Journal of Biological Chemistry

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We have previously reported that thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits intrinsic proteolytic activity toward large peptides. The structural basis for this observation was clarified by the crystal structures of human and bovine TAFI. These structures evinced a significant rotation of the prodomain away from the catalytic moiety when compared with other pro-carboxypeptidases, thus enabling access of large peptide substrates to the active site cleft. Here, we further investigated the flexible nature of the pro-domain and demonstrated that TAFI forms productive complexes with protein carboxypeptidase inhibitors from potato, leech, and tick (PCI, LCI, and TCI, respectively). We determined the crystal structure of the bovine TAFI-TCI complex, revealing that the pro-domain was completely displaced from the position observed in the TAFI structure. It protruded into the bulk solvent and was disordered, whereas TCI occupied the position previously held by the pro-domain. The authentic nature of the presently studied TAFI-inhibitor complexes was supported by the trimming of the C-terminal residues from the three inhibitors upon complex formation. This finding suggests that the inhibitors interact with the active site of TAFI in a substrate-like manner. Taken together, these data show for the first time that TAFI is able to form a bona fide complex with protein carboxypeptidase inhibitors. This underlines the unusually flexible nature of the prodomain and implies a possible mechanism for regulation of TAFI intrinsic proteolytic activity in vivo.Pancreatic carboxypeptidase A1 is the archetype of the M14 metallocarboxypeptidase (MCP) 3 family (1), also referred to as the funnelin tribe of metallopeptidases (2). The members of this family are subdivided into A/B or N/E MCPs on the basis of primary structure and structural homologies (3). They are also classified according to their substrate preference for C-terminal hydrophobic (A-type) or basic (B-type) amino acid residues. Thrombin-activatable fibrinolysis inhibitor (TAFI) (EC 3.4.17.20; UniProt Q96IY4) is a paralogue of pancreatic procarboxypeptidase B1 (pro-CPB1), pro-carboxypeptidases A1 (pro-CPA1), and A2 (pro-CPA2) and is also known as plasma pro-carboxypeptidase B2, R, or U (4 -9). Similarly to other B-type MCPs, TAFI possesses an N-terminal pro-domain that functions as a gatekeeper preventing substrate access to a preformed active site (for a review, see Refs. 2, 3). In human and porcine pancreatic pro-CPB1, access to the active site is completely shielded, resulting in no detectable intrinsic enzymatic activity of the zymogen (10). This is in contrast to human pro-CPA1 and pro-CPA2, which allow cleavage of small peptide substrates (11). Equally, pro-carboxypeptidase B from Pacific dogfish and African lungfish exhibit intrinsic activity toward small substrate molecules (12, 13). Likewise, TAFI interferes with carboxypetidase N assays (14), and is capable of cleaving larger peptide substrates due to its low but continuous carboxypeptidase activity...

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Post-translational Modifications of Human Thrombin-Activatable Fibrinolysis Inhibitor (TAFI): Evidence for a Large Shift in the Isoelectric Point and Reduced Solubility upon Activation

Cited by 31 publications

References 43 publications

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

Biochemical Characterization of Bovine Plasma Thrombin-Activatable Fibrinolysis Inhibitor (TAFI)

Flexibility of the Thrombin-activatable Fibrinolysis Inhibitor Pro-domain Enables Productive Binding of Protein Substrates

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