FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2146996104> ?p ?o ?g. }

• W2146996104 endingPage "32366" @default.
• W2146996104 startingPage "32359" @default.
• W2146996104 abstract "Thrombin-activable fibrinolysis inhibitor (TAFI) is a zymogen that inhibits the amplification of plasmin production when converted to its active form (TAFIa). TAFI is structurally very similar to pancreatic procarboxypeptidase B. TAFI also shares high homology in zinc binding and catalytic sites with the second basic carboxypeptidase present in plasma, carboxypeptidase N. We investigated the effects of altering residues involved in substrate specificity to understand how they contribute to the enzymatic differences between TAFI and carboxypeptidase N. We expressed wild type TAFI and binding site mutants in 293 cells. Recombinant proteins were purified and characterized for their activation and enzymatic activity as well as functional activity. Although the thrombin/thrombomodulin complex activated all the mutants, carboxypeptidase B activity of the activated mutants against hippuryl-arginine was reduced. Potato carboxypeptidase inhibitor inhibited the residual activity of the mutants. The functional activity of the mutants in a plasma clot lysis assay correlated with their chromogenic activity. The effect of the mutations on other substrates depended on the particular mutation, with some of the mutants possessing more activity against hippuryl-His-leucine than wild type TAFIa. Thus mutations in residues around the substrate binding site of TAFI resulted in altered C-terminal substrate specificity. Thrombin-activable fibrinolysis inhibitor (TAFI) is a zymogen that inhibits the amplification of plasmin production when converted to its active form (TAFIa). TAFI is structurally very similar to pancreatic procarboxypeptidase B. TAFI also shares high homology in zinc binding and catalytic sites with the second basic carboxypeptidase present in plasma, carboxypeptidase N. We investigated the effects of altering residues involved in substrate specificity to understand how they contribute to the enzymatic differences between TAFI and carboxypeptidase N. We expressed wild type TAFI and binding site mutants in 293 cells. Recombinant proteins were purified and characterized for their activation and enzymatic activity as well as functional activity. Although the thrombin/thrombomodulin complex activated all the mutants, carboxypeptidase B activity of the activated mutants against hippuryl-arginine was reduced. Potato carboxypeptidase inhibitor inhibited the residual activity of the mutants. The functional activity of the mutants in a plasma clot lysis assay correlated with their chromogenic activity. The effect of the mutations on other substrates depended on the particular mutation, with some of the mutants possessing more activity against hippuryl-His-leucine than wild type TAFIa. Thus mutations in residues around the substrate binding site of TAFI resulted in altered C-terminal substrate specificity. Carboxypeptidases (CPs) 1The abbreviations used are: CP, carboxypeptidase; CPI, potato carboxypeptidase inhibitor; TAFI, thrombin-activatable fibrinolysis inhibitor; TAFIa, activated TAFI; PPACK, d-Phe-Pro-Arg chloromethyl ketone.1The abbreviations used are: CP, carboxypeptidase; CPI, potato carboxypeptidase inhibitor; TAFI, thrombin-activatable fibrinolysis inhibitor; TAFIa, activated TAFI; PPACK, d-Phe-Pro-Arg chloromethyl ketone. are enzymes that catalyze the hydrolysis of the C-terminal peptide bond in peptides and proteins. Although it might seem that removal of one or a few amino acids from the C terminus of a peptide or protein would be of limited importance, it can have profound effects on their biological activity (1Skidgel R.A. Erdos E.G. Handbook of Proteolytic Enzymes (Barrett, A. J., Rawlings, N. D., and Woessner, J. F., eds). Academic Press, San Diego1998Google Scholar). CPs perform many diverse functions in the body. These include digestion and assimilation of dietary proteins, processing of peptide hormone precursors, regulation of peptide hormone activity, and regulation of protein binding. Thrombin-activable fibrinolysis inhibitor (TAFI, EC 3.4.17.20) is a plasma protein that has basic carboxypeptidase activity upon activation. It is also known as plasma carboxypeptidase B, carboxypeptidase U, and carboxypeptidase R. TAFI is synthesized by the liver and circulates in plasma as a zymogen within a concentration range of 50–150 nm. It has been shown to play a role in the regulation of fibrinolysis both in vitro and in vivo (2Bajzar L. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2511-2518Crossref PubMed Scopus (182) Google Scholar, 3Nesheim M. Walker J. Wang W. Boffa M. Horrevoets A. Bajzar L. Ann. N. Y. Acad. Sci. 2001; 936: 247-260Crossref PubMed Scopus (20) Google Scholar, 4Bouma B.N. Marx P.F. Mosnier L.O. Meijers J.C.M. Thromb. Res. 2001; 101: 329-354Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 5Schatteman K. Goossens F. Leurs J. Verkerk R. Scharpe S. Michiels J.J. Hendriks D. Clin. Appl. Thromb. Hemost. 2001; 7: 93-101Crossref PubMed Scopus (13) Google Scholar, 6Plow E.F. Allampallam K. Redlitz A. Trend. Cardiovas. Med. 1997; 7: 71-75Crossref PubMed Scopus (30) Google Scholar). TAFI is a glycoprotein with a M r ∼58,000. Following its activation by the thrombin/thrombomodulin complex, activated TAFI (TAFIa) cleaves C-terminal basic residues of fibrin that are newly exposed by plasmin cleavage. Since these C-terminal basic residues are high affinity binding sites for both plasminogen and plasminogen activators, they serve as an amplification system for plasmin production. When TAFIa removes these C-terminal basic residues, plasmin production is reduced, leading to slower lysis of the clot. TAFIa may also be involved in plasmin-mediated cell migration (6Plow E.F. Allampallam K. Redlitz A. Trend. Cardiovas. Med. 1997; 7: 71-75Crossref PubMed Scopus (30) Google Scholar). In vitro TAFIa can cleave a number of peptides with biological activity in the circulation such as bradykinin and anaphylatoxins (7Tan A.K. Eaton D.L. Biochemistry. 1995; 34: 5611-5816Crossref Scopus (138) Google Scholar, 8Shinohara T. Sakurada C. Suzuki T. Takeuchi O. Campbell W. Ikeda S. Okada N. Okada H. Int. Arch. Allergy. Immunol. 1994; 103: 400-404Crossref PubMed Scopus (63) Google Scholar, 9Campbell W. Okada N. Okada H. Immunol. Rev. 2001; 180: 162-167Crossref PubMed Scopus (90) Google Scholar). In vivo, these peptides are degraded rapidly by the second CP present in plasma, carboxypeptidase N (CPN, EC 3.4.17.3). CPN is a M r ∼280,000 tetrameric enzyme consisting of two small catalytic subunits and two large glycosylated subunits. It is synthesized by the liver and circulates in plasma at 100 nm in a constitutively active form (10Erdos E.G. Erdos E.G. Handbook of Experimental Pharmacology. Vol. 25. Springer-Verlag, Heidelberg, Germany1979: 427-487Google Scholar). Both TAFI and CPN belong to a class of metallocarboxypeptidases that catalyze the hydrolysis of the C-terminal peptide bond in peptides and proteins. Based on sequence analysis, metallocarboxypeptidases can be divided into two groups: 1) carboxypeptidases A1, A2, and B, TAFI, and mast cell carboxypeptidase A and 2) carboxypeptidases N, H/E, M, D, and Z (11Skidgel R.A. Hooper N.M. Zinc Metalloproteases in Health and Diseases. Taylor and Francis Ltd., London1996: 241-283Google Scholar). The sequence similarity is high within each group (40–58%) but much lower between the two groups (14–20%). CPs from the first group are synthesized as inactive zymogens and require removal of a propeptide before they exert carboxypeptidase activity, optimally at neutral pH. Active CPAs have a preference for aromatic or aliphatic residues, whereas active CPBs favor basic residues. A naturally occurring small protein, carboxypeptidase inhibitor from potato (CPI), and synthetic compounds such as guanidinoethylmercaptosuccinic acid inhibit the activity of these CPs. The three-dimensional structure and the mechanism of action for pancreatic carboxypeptidases A and B have been widely studied and are very similar (12Rees D.C. Lewis M. Lipscomb W.N. J. Mol. Biol. 1983; 168: 367-387Crossref PubMed Scopus (394) Google Scholar, 13Schmid M. Herriott J. J. Mol. Biol. 1976; 103: 175-190Crossref PubMed Scopus (95) Google Scholar, 14Aviles F.X. Vendrell J. Guasch A. Coll M. Huber R. Eur. J. Biochem. 1993; 211: 381-389Crossref PubMed Scopus (72) Google Scholar). On the other hand, carboxypeptidases from the second group are constitutively active toward basic residues at various pH optima and possess a long C-terminal extension whose functions are unknown (11Skidgel R.A. Hooper N.M. Zinc Metalloproteases in Health and Diseases. Taylor and Francis Ltd., London1996: 241-283Google Scholar). Although they are inhibited by guanidinoethylmercaptosuccinic acid, they are not susceptible to inhibition by CPI. They perform important functions in regulation of biologically active peptides such as processing of propeptide hormones (15Steiner D.F. Smeekens S.P. Ohagi S. Chan S.J. J. Biol. Chem. 1992; 267: 23435-23438Abstract Full Text PDF PubMed Google Scholar, 16Steiner D.F. Docherty K. Carroll R. J. Cell. Biochem. 1984; 24: 121-130Crossref PubMed Scopus (88) Google Scholar), inactivation of biologically active peptides (10Erdos E.G. Erdos E.G. Handbook of Experimental Pharmacology. Vol. 25. Springer-Verlag, Heidelberg, Germany1979: 427-487Google Scholar), and alteration of substrate specificity for receptor binding (17Regoli D. Barabe J. Pharmacol. Rev. 1980; 32: 1-4617Crossref PubMed Scopus (16) Google Scholar, 18Bhoola K.D. Figueroa C.D. Worthy K. Pharmacol. Rev. 1992; 44: 1-80PubMed Google Scholar, 19Bokisch V.A. Muller-Eberhard H.J. J. Clin. Invest. 1970; 49: 2427-2436Crossref PubMed Scopus (379) Google Scholar). Recently, the crystal structure of CPD has been elucidated and revealed an overall topological similarity to that of CPA and CPB but with unique structural features that may explain differences in the activity of the two groups of CPs (20Gomis-Ruth F.X. Companys V. Qian Y. Fricker L.D. Vendrell J. Aviles F.X. Coll M. EMBO J. 1999; 18: 5817-5826Crossref PubMed Scopus (71) Google Scholar). The current study was undertaken to investigate the structural basis for the differences in substrate specificity and susceptibility to CPI for CPs from the two groups. In this study, we employed site-directed mutagenesis to switch residues that may be involved in substrate binding between TAFI, a member of the first group of CPs, and CPN as a representative of the second group of CPs and then investigated the properties of the mutants. QuikChange site-directed mutagenesis kit was from Stratagene, La Jolla, CA. The mammalian expression vector, pCEP 4, Lipofectin, and Opti-MEM were from Invitrogen. Polyclonal sheep anti-TAFI antibody, human TAFI-deficient plasma, human carboxypeptidase N, and TAFI enzyme-linked immunosorbent assay kit were from Enzyme Research Laboratories, South Bend, IN. SP-Sepharose fast flow was from Amersham Biosciences. Centriprep 10 was from Amicon, Inc., Beverly, MA. SDS-PAGE gels were from BioWhittaker Molecular Applications, Rockland, ME. Human α-thrombin and TAFI Developer were from American Diagnostica Inc., Greenwich, CT. d-Phe-Pro-Arg chloromethylketone (PPACK) and hygromycin B were from Calbiochem-Novabiochem. Pancreatic carboxypeptidase A, cyanuric chloride, dioxane, hippuryl-arginine, hippuryl-lysine, hippuryl-His-leucine, and hippuryl-phenylalanine were from Sigma. Tissue plasminogen activator was from Genentech, South San Francisco, CA. Pefabloc SC was from Roche Applied Science. Construction of TAFI Mutants—All TAFI mutants were constructed based on the previously described wild type sequence (21Eaton D.L. Malloy B.E. Tsai S.P. Henzel W. Drayna D. J. Biol. Chem. 1991; 266: 21833-21838Abstract Full Text PDF PubMed Google Scholar), except with alanine at position 147 of the zymogen (22Zhao L. Morser J. Bajzar L. Nesheim M. Nagashima M. Thromb. Haemostasis. 1998; 80: 949-955Crossref PubMed Scopus (70) Google Scholar). Mutations were introduced into pCEP 4/TAFI, a plasmid coding for wild type TAFI cDNA, using a QuikChange site-directed mutagenesis kit according to the manufacturer's instructions. A pair of primers was used to construct each of four point mutants, one with Asp to Gln mutation at position 256 (TAFI D256Q) (numbering starts with the first alanine of activated TAFI), one with Ser to Asp mutation at position 207 (TAFI S207D), one with Leu to Trp mutation at position 248 (TAFI L248W), and one with Asp to Ala mutation at position 257 (TAFI D257A). The primers used for TAFID256Q were 5′-ATAGATCCAATCCTGCCCACCTCCAGG-3′ and 5′-CCTGGAGGTGGGCAGGATTGGATCTAT-3′; the primers used for TAFI-S207D were 5′-GGGACGATTGGATCTATCTATTGGGCATCAAATATTC-3′ and 5′-GAATATTTGATGCC CAATAGATAGATCCAATCGTCCC-3′; the primers used for TAFI-L248W were 5′-GGCTCAGAAACCTGGTACCTAGCTCCTG-3′ and 5′-CAGGAGCTAGGTACCAGGTTT CTGAGCC-3′; and the primers used for TAFI-D257A were 5′-TGGAGGTGGGGATGCTTGGATCTATGA-3′ and 5′-TCATAGATCCAAGCATCCCCACCTCCA-3′. A double mutant, TAFI D256Q/S207D, was constructed with the same pair of primers used for mutating Ser-207 to Asp using TAFI D256Q DNA as the template. All of the mutations were confirmed by DNA sequence analysis. Expression and Purification of Recombinant Wild Type and Mutant TAFI—Expression plasmids coding for wild type and mutant TAFI were transfected into human embryonic kidney 293 cells using Lipofectin. Clones that expressed TAFI stably were selected by culturing cells in the presence of 150 μg/ml hygromycin B. For production of recombinant wild type and mutant TAFI, stably expressing clones were cultured in Opti-MEM. Conditioned medium was harvested after either 48 h or 72 h, filtered through a 0.22-μm filter and stored at –20 °C in the presence of 0.4 mm Pefabloc SC. Expression of TAFI in the conditioned medium was confirmed by separation of proteins on SDS-PAGE followed by Western blotting with polyclonal sheep anti-TAFI antibody. To isolate wild type and mutant TAFI, typically 1 liter of conditioned medium was diluted 20-fold with H2O and adjusted to pH 6.8 with 0.1 m HCl. The sample was applied to a 25-ml SP-Sepharose column equilibrated with 20 mm phosphate buffer, pH 6.8, and washed extensively with the same buffer. Bound proteins were eluted from the column with a salt gradient of 0–0.5 m NaCl in 20 mm phosphate buffer, pH 6.8. Fractions were analyzed by Western blotting as above. Fractions containing recombinant TAFI were pooled and then concentrated using Centriprep 10. Purity was determined by silver staining of the gel following SDS-PAGE. The quantity of mutant and wild type TAFI was determined both by using the TAFI ELISA kit as well as by Western blotting with polyclonal sheep anti-TAFI antibody. For identification and characterization of the second TAFI species present in the purified preparations of TAFI-L248W and TAFI-S207D, Western blotting of an SDS-PAGE was performed using rabbit polyclonal anti-human TAFI peptide antibody raised against the peptide SEAVRAIEKTSKNT (residues 224–237). Reduction and alkylation of the samples was carried out by adding 10 mm dithiothreitol to samples of the TAFI mutants in SDS loading buffer and boiling for 10 min followed by addition of 50 mm iodoacetamide before analyzing by SDS-PAGE. For N-terminal sequencing, the samples were further purified on a plasminogen affinity column as described previously (22Zhao L. Morser J. Bajzar L. Nesheim M. Nagashima M. Thromb. Haemostasis. 1998; 80: 949-955Crossref PubMed Scopus (70) Google Scholar) to remove trace contaminants. The N-terminal sequence was determined by 241 protein sequencer (Agilent, Palo Alto, CA) that uses standard N-terminal chemistry. Activation of TAFI and Carboxypeptidase B Activity Assay—Recombinant wild type or mutant TAFI (17 nm) was activated with 15 nm thrombin, 50 nm thrombomodulin, and 5 mm CaCl2 in 20 mm HEPES, 0.15 m NaCl, pH 7.4, at room temperature for 5–20 min. Activation was stopped by addition of 1 μm PPACK and was confirmed by analyzing denatured samples on SDS-PAGE followed by Western blotting using polyclonal sheep anti-TAFI antibody. A non-activated control was prepared by preincubating PPACK with thrombin for 10 min before recombinant wild type or mutant TAFI and thrombomodulin were added. Carboxypeptidase B activity of TAFIa was measured in a chromogenic assay using hippuryl-arginine as the substrate. Carboxypeptidase activity of recombinant wild type and mutant TAFI was also measured using several other substrates: hippuryl-lysine, hippuryl-His-leucine, and hippuryl-phenylalanine. Carboxypeptidase N (3.6 nm) and pancreatic carboxypeptidase A (28.6 nm) were included in the assays as controls. Enzymes were incubated with these substrates for 30 min at room temperature, and the rate of hydrolysis was determined by conversion of the product, hippuric acid, to a chromogen with cyanuric chloride dissolved in dioxane. Briefly, in a 96-well microtiter plate, 12 μl of TAFIa, carboxypeptidase N, or carboxypeptidase A was mixed with 24 μl of 50 mm HEPES buffer, pH 7.8, 12 μl of H2O, and 12 μl of 25 mm substrate dissolved in 20 mm NaOH. The reaction was stopped by addition of 80 μlof0.2 m phosphate buffer, pH 8.3, and 60 μl of 3% (w/v) cyanuric chloride in dioxane. After mixing thoroughly by pipetting, the clear supernatant was transferred to a new well, and absorbance was measured at 382 nm. The background absorbance obtained in the absence of the enzyme was subtracted from all the measurements. The effect of CPI on the carboxypeptidase activity of TAFIa was determined by 5-min preincubation of the enzymes with CPI (1 μm final), which had been purified as described previously (23Nagashima M. Werner M. Wang M. Zhao L. Light D.R. Pagila R. Morser J. Verhallen P. Thromb. Res. 2000; 98: 333-342Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) prior to addition of the substrate. Plasma Clot Lysis Assay—In a 96-well microtiter plate, 30 μl of human TAFI-deficient plasma containing 12 μl of recombinant TAFI was mixed with 3 μlof0.8 μm thrombomodulin and 60 μl of assay buffer (40 mm HEPES, 150 mm NaCl, 0.02% Tween 80, pH 7.0). The mixture was immediately added to another well containing 4 μl of 75 NIH units/ml thrombin, 2 μl of 1 m CaCl2, and 4 μl of 29.4 nm tissue plasminogen activator in separate aliquots. The total volume of the mixture was made up to 120 μl with water. The final concentration of wild type or mutant TAFI in the solution was 3.4 nm. After mixing by pipetting, clot formation and lysis were monitored at 405 nm every 1 min at 37 °C using a SpectraMAX 250 microplate spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA). In some reactions, 5 μl of 24 μm CPI was added to demonstrate the effect of inhibition of TAFIa activity on clot lysis. Lysis time was defined as the time at which turbidity is one-half the difference between the plateau reached after clotting and the base line value achieved at complete lysis. Construction of TAFI Mutants—Comparison of the TAFI sequence with those of other CPs suggested several residues that could be important for the substrate specificity of TAFIa. Fig. 1 shows the amino acid sequence alignment between human TAFI and human CPN using SeqLab software based on secondary structure predictions. Numbering starts with the first alanine of the activated enzyme. Insertions and deletions were introduced to maximize the alignment. Although the overall identity between TAFI and CPN is only 14% (1Skidgel R.A. Erdos E.G. Handbook of Proteolytic Enzymes (Barrett, A. J., Rawlings, N. D., and Woessner, J. F., eds). Academic Press, San Diego1998Google Scholar), critical residues involved in zinc binding and catalysis, as well as many of the substrate binding residues, are conserved between the two proteins. From the alignment, we identified residues around the substrate binding site that may contribute to their differences in substrate specificity. We constructed five mutants of human TAFI to investigate these predictions. To test the importance of the negatively charged residues that define the P1′ pocket, Asp-256 in TAFI was mutated to Gln as in CPN. Similarly, Asp-257 was mutated to Ala. Ser-207 in TAFI was mutated to Asp as in CPN since this negatively charged residue in CPN is close to the P1′ pocket. Another mutation converted Leu-248 to Trp to examine the effect of introducing a bulky residue adjacent to Tyr-249 that is known to have an influence on substrate binding. A double mutant of Asp to Gln at residue 256 and Ser to Asp at residue 207 was also constructed to test whether this double mutation would result in cancellation of the effects of the charge reversal caused by either of the single mutations. Following site-directed mutagenesis, the DNA sequence of the plasmids was confirmed. The residues that were mutated are shown in Table I with the corresponding residues in the wild type, as well as in CPN and CPA.Table IA summary of the mutations made in human TAFI and corresponding residues in CPN and CPAResidueTAFI WTaWT, wild type.MutationCPNCPA207SerAspAspGly248LeuTrpTrpIle256AspGlnGlnIle257AspAlaAspAsp256AspGlnGlnIlea WT, wild type. Open table in a new tab Expression and Purification of TAFI and Mutants—To compare the properties of TAFI mutants with wild type TAFI, the recombinant proteins were expressed in 293 cells. Stable clones were selected, and recombinant proteins were purified by SPSepharose chromatography. Homogeneity of recombinant wild type TAFI was demonstrated using silver staining and Western blotting with polyclonal sheep anti-TAFI antibody after separation on SDS-PAGE (Fig. 2, A and B). The purification profile of recombinant TAFI mutants D256Q, D257A, S207D, L248W, and D256Q/S207D was indistinguishable from that of wild type TAFI, implying that these mutations had not lead to gross alterations in their overall properties. As an example, the purification of D256Q is shown in Fig. 2, C and D, in which it can be seen that the mutant TAFI was eluted at the same salt concentration as the wild type TAFI. Wild type TAFI and the D256Q, D257Q, and D256Q/S207D mutants comigrated at the expected molecular weight of ∼58,000, which is similar to that of TAFI purified from plasma. TAFI L248W and S207D both showed a second band with a slightly lower apparent molecular weight than 58,000, both of which differ slightly from each other. From SDS-PAGE, the molecular weight of the second band from L248W and S207D was 49,000 and 50,000, respectively. Western blotting analysis showed that both bands in TAFI S207D and L248W reacted with polyclonal sheep anti-TAFI antibody, indicating that both bands were TAFI (see Fig. 4, lanes 9 and 11). Only a single band was observed in preparations of wild type TAFI and other TAFI mutants (see Fig. 4, lanes 1, 3, 5, and 7). Since the M r 49,000 and M r 50,000 bands in L248W and S207D preparations, respectively, was observed in Western blots of the crude medium that contained protease inhibitors, these smaller TAFI species were not degradation products produced during purification. Furthermore, the finding that the corresponding band was absent in preparations of wild type TAFI and other mutants including the double mutant that had been purified in the same way supports our hypothesis that the formation of these smaller TAFI species were a specific consequence of these particular mutations. Further analysis showed that the smaller TAFI species in S207D and L248W were not due to altered folding since reduction and alkylation of the samples prior to SDS-PAGE did not alter the pattern of the Western blots (data not shown).Fig. 4Activation of wild type (WT) and mutant TAFI by thrombin/thrombomodulin complex. Ten μl of non-activated and activated wild type and mutant TAFI were run on 10% SDS-PAGE under denatured conditions followed by Western blot using a polyclonal sheep anti-TAFI antibody. Activation was performed as described under “Experimental Procedures.” Samples are non-activated and activated wild type TAFI (lanes 1 and 2), TAFI D256Q (lanes 3 and 4), TAFI D257A (lanes 5 and 6), TAFI D256Q/S207D (lanes 7 and 8), TAFI S207D (lanes 9 and 10), and TAFI L248W (lanes 11 and 12).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The possibility that the smaller TAFI species was the product of a proteolytic cleavage prior to harvesting of the conditioned medium due to altered susceptibility to proteolysis was tested as follows. We first tested the integrity of the N terminus of the mutant L248W by N-terminal sequencing. To do this, we further purified a TAFI L248W preparation by chromatography on plasminogen-Sepharose. Both M r 58,000 and M r 49,000 bands were detected, indicating that both TAFI species bound to plasminogen. The fractions containing the two TAFI species were pooled and concentrated prior to N-terminal amino acid sequence analysis. Only one sequence, FQSGQVLAA, was detected for this mixture of the two forms of TAFI, which is the N-terminal sequence of the zymogen. Thus the N terminus for both the M r 58,000 and the M r 49,000 TAFI species is the same, suggesting that the C terminus of the lower molecular weight form has been proteolytically cleaved. To investigate the possibility that the C-terminal sequence had been truncated, we analyzed the TAFI preparations with an antibody specific for a C-terminal sequence. TAFI L248W and S207D and wild type TAFI were run on a 4–20% Tris-glycine gel, and Western blotting was performed with an antibody raised against a peptide with the sequence SEAVRAIEKTSKNT that corresponds to residues 224–237 in the C terminus of TAFI. This antibody recognized wild type TAFI with M r 58,000 (Fig. 3A, lane 1), but it did not recognize the M r 50,000 species in the preparation of the S207D mutant (Fig. 3A, lane 2). Instead it bound to a band of M r 6,000. In the preparation of the L248W mutant, both the M r 58,000 and the M r 49,000 TAFI species were detected by the anti-peptide antibody (Fig. 3A, lane 3). Fig. 3B shows the same samples analyzed with polyclonal sheep anti-TAFI antibody in which the M r 58,000 species can be seen in all three samples and, in addition, the M r 49,000 species in the L248W mutant TAFI and the M r 50,000 species in the S207D TAFI. The intensity of the higher band with M r 58,000 in TAFI S207D (Fig. 3B, lane 2) was much lower than the lower band, indicating that the majority of S207D was in the lower form. This could explain the failure of the polyclonal antibody against the peptide to detect the M r 58,000 band (Fig. 3A, lane 2). These data are consistent with the hypothesis that the M r 50,000 and M r 49,000 TAFI species present in S207D and L248W preparations differ in their C terminus and that the former lacked C-terminal residues containing the sequence SEAVRAIEKTSKNT, whereas the latter still contained this sequence. It is of note that the TAFI species with the longer sequence is migrating faster in SDS-PAGE. Activation of TAFI and Mutants—To investigate whether mutations around the substrate binding site influenced activation, wild type and mutant TAFI were incubated with the thrombin/thrombomodulin complex at room temperature. After various times, aliquots were removed and analyzed by SDS-PAGE and Western blot using a polyclonal sheep anti-TAFI antibody. This antibody can only recognize the TAFI zymogen and TAFIa but not the activation peptide following its cleavage (data not shown). The time course of proteolytic activation of wild type and the TAFI mutants was similar when followed from 5 to 20 min, reaching the maximum after 10 min. The profiles after 10 min of activation are shown in Fig. 4. The TAFI zymogen with a molecular weight of 58,000 was cleaved to yield TAFIa with a molecular weight of 35,000 in all cases. Activation of the mutants was also calcium-dependent as shown for wild type TAFI, and activation was more efficient when 150 mm NaCl was used in the activation buffer rather than 300 mm NaCl (data not shown). Although all the mutants could be activated by the thrombin/thrombomodulin complex, TAFI D256Q and the double mutant were more readily activated than wild type TAFI. Carboxypeptidase Activity of Recombinant Wild Type and Mutant TAFIa—The effect of these mutations on TAFIa activity was measured using hippuryl-arginine as the substrate following their activation with the thrombin/thrombomodulin complex as described above. It is evident that the activity of all the mutants against hippuryl-arginine was greatly reduced (Fig. 5A). This reduction in activity is still significant when the data are normalized for differences in the extent of activation between wild type TAFI and TAFI mutants. Substrates with different C-terminal residues, hippuryl-lysine, hippuryl-His-leucine, and hippuryl-phenylalanine, were used to investigate whether the carboxypeptidase activities of the mutant TAFIa had altered substrate specificity (Fig. 5A). CPN and CPA were tested in the same assays as controls (Fig. 5B). Wild type TAFIa had high activity against hippuryl-arginine, lower activity against hippuryl-lysine, and even lower against hippuryl-His-leucine and hippuryl-phenylalanine, consistent with TAFIa being a basic carboxypeptidase with a preference for C-terminal arginine residues. All mutant TAFIas had greatly reduced activity toward both hippuryl-arginine and hippuryl-lysine as compared with that of wild type TAFIa. However, the magnitude of the reduction in activity toward hippuryl-arginine was greater, and consequently, they showed a slight preference for hippuryl-lysine, as is the case for CPN. CPA had no basic carboxypeptidase activity. Unlike CPA, the mutant TAFIas did not cleave hippury" @default.
• W2146996104 created "2016-06-24" @default.
• W2146996104 creator A5018985536 @default.
• W2146996104 creator A5019707796 @default.
• W2146996104 creator A5057686534 @default.
• W2146996104 creator A5068169767 @default.
• W2146996104 creator A5069848340 @default.
• W2146996104 date "2003-08-01" @default.
• W2146996104 modified "2023-09-28" @default.
• W2146996104 title "Mutations in the Substrate Binding Site of Thrombin-activatable Fibrinolysis Inhibitor (TAFI) Alter Its Substrate Specificity" @default.
• W2146996104 cites W1215858103 @default.
• W2146996104 cites W1514226843 @default.
• W2146996104 cites W168286673 @default.
• W2146996104 cites W1963133109 @default.
• W2146996104 cites W1964013037 @default.
• W2146996104 cites W1966721068 @default.
• W2146996104 cites W1970006317 @default.
• W2146996104 cites W1973605634 @default.
• W2146996104 cites W1976290526 @default.
• W2146996104 cites W1987577695 @default.
• W2146996104 cites W1987981908 @default.
• W2146996104 cites W1999372769 @default.
• W2146996104 cites W2005134321 @default.
• W2146996104 cites W2038723412 @default.
• W2146996104 cites W2048531341 @default.
• W2146996104 cites W2052534376 @default.
• W2146996104 cites W2053777314 @default.
• W2146996104 cites W2054169150 @default.
• W2146996104 cites W2097764843 @default.
• W2146996104 cites W2118270944 @default.
• W2146996104 cites W2122572489 @default.
• W2146996104 cites W2127536162 @default.
• W2146996104 cites W2154382678 @default.
• W2146996104 cites W4211156233 @default.
• W2146996104 doi "https://doi.org/10.1074/jbc.m300803200" @default.
• W2146996104 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12799375" @default.
• W2146996104 hasPublicationYear "2003" @default.
• W2146996104 type Work @default.
• W2146996104 sameAs 2146996104 @default.
• W2146996104 citedByCount "10" @default.
• W2146996104 countsByYear W21469961042013 @default.
• W2146996104 countsByYear W21469961042016 @default.
• W2146996104 crossrefType "journal-article" @default.
• W2146996104 hasAuthorship W2146996104A5018985536 @default.
• W2146996104 hasAuthorship W2146996104A5019707796 @default.
• W2146996104 hasAuthorship W2146996104A5057686534 @default.
• W2146996104 hasAuthorship W2146996104A5068169767 @default.
• W2146996104 hasAuthorship W2146996104A5069848340 @default.
• W2146996104 hasBestOaLocation W21469961041 @default.
• W2146996104 hasConcept C107824862 @default.
• W2146996104 hasConcept C126322002 @default.
• W2146996104 hasConcept C181199279 @default.
• W2146996104 hasConcept C185592680 @default.
• W2146996104 hasConcept C18903297 @default.
• W2146996104 hasConcept C2776825266 @default.
• W2146996104 hasConcept C2777289219 @default.
• W2146996104 hasConcept C2777292125 @default.
• W2146996104 hasConcept C2994592520 @default.
• W2146996104 hasConcept C55493867 @default.
• W2146996104 hasConcept C71924100 @default.
• W2146996104 hasConcept C86803240 @default.
• W2146996104 hasConcept C89560881 @default.
• W2146996104 hasConceptScore W2146996104C107824862 @default.
• W2146996104 hasConceptScore W2146996104C126322002 @default.
• W2146996104 hasConceptScore W2146996104C181199279 @default.
• W2146996104 hasConceptScore W2146996104C185592680 @default.
• W2146996104 hasConceptScore W2146996104C18903297 @default.
• W2146996104 hasConceptScore W2146996104C2776825266 @default.
• W2146996104 hasConceptScore W2146996104C2777289219 @default.
• W2146996104 hasConceptScore W2146996104C2777292125 @default.
• W2146996104 hasConceptScore W2146996104C2994592520 @default.
• W2146996104 hasConceptScore W2146996104C55493867 @default.
• W2146996104 hasConceptScore W2146996104C71924100 @default.
• W2146996104 hasConceptScore W2146996104C86803240 @default.
• W2146996104 hasConceptScore W2146996104C89560881 @default.
• W2146996104 hasIssue "34" @default.
• W2146996104 hasLocation W21469961041 @default.
• W2146996104 hasOpenAccess W2146996104 @default.
• W2146996104 hasPrimaryLocation W21469961041 @default.
• W2146996104 hasRelatedWork W1525234184 @default.
• W2146996104 hasRelatedWork W2017160385 @default.
• W2146996104 hasRelatedWork W2033637851 @default.
• W2146996104 hasRelatedWork W2037550082 @default.
• W2146996104 hasRelatedWork W2069134127 @default.
• W2146996104 hasRelatedWork W2087030184 @default.
• W2146996104 hasRelatedWork W2146996104 @default.
• W2146996104 hasRelatedWork W2165378040 @default.
• W2146996104 hasRelatedWork W2768904551 @default.
• W2146996104 hasRelatedWork W1832912762 @default.
• W2146996104 hasVolume "278" @default.
• W2146996104 isParatext "false" @default.
• W2146996104 isRetracted "false" @default.
• W2146996104 magId "2146996104" @default.
• W2146996104 workType "article" @default.