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Jens J Led - One of the best experts on this subject based on the ideXlab platform.
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solution structure and backbone dynamics of the human alpha3 chain type vi collagen c terminal Kunitz Domain
Biochemistry, 1997Co-Authors: Morten Dahl Sorensen, Soren E Bjorn, Kjeld Norris, Ole Hvilsted Olsen, Lars Christian Petersen, Thomas L James, Jens J LedAbstract:The solution structure and backbone dynamics of the 58-residue C-terminal Kunitz Domain fragment [α3(VI)] of human α3-chain type VI collagen has been studied by two-dimensional 1H−1H and 1H−15N nuclear magnetic resonance spectroscopy at 303 K. The solution structure is represented by an ensemble of 20 structures calculated with X-PLOR using 612 distance and 47 dihedral angle restraints. The distance restraints were obtained by a complete relaxation matrix analysis using MARDIGRAS. The root mean squared (rms) deviation is 0.91 A for the backbone atoms of the residues Thr2(8)−Gly12(18), Arg15(21)−Tyr35(41), and Gly40(46)−Pro57(63). The central β-sheet [residues Ile18(24)−Tyr35(41)] and the C-terminal α-helix [residues Gln48(54)−Cys55(61)] are better defined with a backbone rms deviation of 0.46 A. The solution structure of α3(VI) is virtually identical to the crystal structure of α3(VI) and to the solution structure of bovine pancreatic trypsin inhibitor (BPTI). The 15N spin−lattice and spin−spin relaxation...
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elucidation of the origin of multiple conformations of the human alpha 3 chain type vi collagen c terminal Kunitz Domain the reorientation of the trp21 ring
Journal of Biomolecular NMR, 1996Co-Authors: Morten Dahl Sorensen, Soren E Bjorn, Kjeld Norris, Ole Hvilsted Olsen, Soren M Kristensen, Jens J LedAbstract:The human α3-chain type VI collagen C-terminal Kunitz Domain fragment (α3(VI)) has been studied by two-dimensional 1H−1H and 1H−13C NMR spectroscopy at 303 K. It is shown that the secondary structure of the protein is strikingly similar to that of BPTI, and that a number of unusual Hα chemical shifts, which are highly conserved in Kunitz-Domain proteins, are also observed for α3(VI). Further-more a series of exchange cross peaks observed in 1H−1H spectra shows that a large number of protons in the central β-sheet exist in two different chemical environments, corresponding to two unequally populated conformations that are slowly exchanging on the NMR time scale. Several protons, including Ser47(53) Hα, Arg32(38) Hγ2, and Gln48(54) Hβ2, all located in the vicinity of the Trp21(27) ring in the crystal structure of α3(VI) [Arnoux, B. et al. (1995) J. Mol. Biol., 246, 609–617], have very different chemical shifts in the two conformations, the most affected being Gln48(54) Hβ2 (Δδ=1.53 ppm), which is placed directly above the Trp21(27) ring in the crystal structure of α3(VI). It is concluded that the origin of the multiple conformations of the central β-sheet is a reorientation of the Trp21(27) ring. From the intensities of corresponding signals in the two conformations, the population of the minor conformation was found to be 6.4±0.2% of that of the major conformation, while a rate constant kM=1.01±0.05 s-1 for the major to minor interconversion was obtained from a series of NOESY spectra with different mixing times. In addition, it is shown that Cys14(20)-Cys38(44) disulfide bond isomerization, previously observed in BPTI [Otting, G. et al. (1993) Biochemistry, 32, 3570–3582], is also likely to occur in α3(VI).
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Elucidation of the origin of multiple conformations of the human α3-chain type VI collagen C-terminal Kunitz Domain: The reorientation of the Trp^21 ring
Journal of Biomolecular NMR, 1996Co-Authors: Morten Dahl Sorensen, Kjeld Norris, Soren M Kristensen, Søren Bjørn, Ole Olsen, Jens J LedAbstract:The human α3-chain type VI collagen C-terminal Kunitz Domain fragment (α3(VI)) has been studied by two-dimensional ^1H−^1H and ^1H−^13C NMR spectroscopy at 303 K. It is shown that the secondary structure of the protein is strikingly similar to that of BPTI, and that a number of unusual H^α chemical shifts, which are highly conserved in Kunitz-Domain proteins, are also observed for α3(VI). Further-more a series of exchange cross peaks observed in ^1H−^1H spectra shows that a large number of protons in the central β-sheet exist in two different chemical environments, corresponding to two unequally populated conformations that are slowly exchanging on the NMR time scale. Several protons, including Ser^47(53) H^α, Arg^32(38) H^γ2, and Gln^48(54) H^β2, all located in the vicinity of the Trp^21(27) ring in the crystal structure of α3(VI) [Arnoux, B. et al. (1995) J. Mol. Biol. , 246 , 609–617], have very different chemical shifts in the two conformations, the most affected being Gln^48(54) H^β2 (Δδ=1.53 ppm), which is placed directly above the Trp^21(27) ring in the crystal structure of α3(VI). It is concluded that the origin of the multiple conformations of the central β-sheet is a reorientation of the Trp^21(27) ring. From the intensities of corresponding signals in the two conformations, the population of the minor conformation was found to be 6.4±0.2% of that of the major conformation, while a rate constant k_M=1.01±0.05 s^-1 for the major to minor interconversion was obtained from a series of NOESY spectra with different mixing times. In addition, it is shown that Cys^14(20)-Cys^38(44) disulfide bond isomerization, previously observed in BPTI [Otting, G. et al. (1993) Biochemistry , 32 , 3570–3582], is also likely to occur in α3(VI).
James J. Valdes - One of the best experts on this subject based on the ideXlab platform.
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Understanding the evolutionary structural variability and target specificity of tick salivary Kunitz peptides using next generation transcriptome data
BMC Evolutionary Biology, 2014Co-Authors: Alexandra Schwarz, Alejandro Cabezas Cruz, Jan Kopecky, James J. ValdesAbstract:Background Ticks are blood-sucking arthropods and a primary function of tick salivary proteins is to counteract the host’s immune response. Tick salivary Kunitz-Domain proteins perform multiple functions within the feeding lesion and have been classified as venoms; thereby, constituting them as one of the important elements in the arms race with the host. The two main mechanisms advocated to explain the functional heterogeneity of tick salivary Kunitz-Domain proteins are gene sharing and gene duplication. Both do not, however, elucidate the evolution of the Kunitz family in ticks from a structural dynamic point of view. The Red Queen hypothesis offers a fruitful theoretical framework to give a dynamic explanation for host-parasite interactions. Using the recent salivary gland Ixodes ricinus transcriptome we analyze, for the first time, single Kunitz-Domain encoding transcripts by means of computational, structural bioinformatics and phylogenetic approaches to improve our understanding of the structural evolution of this important multigenic protein family. Results Organizing the I. ricinus single Kunitz-Domain peptides based on their cysteine motif allowed us to specify a putative target and to relate this target specificity to Illumina transcript reads during tick feeding. We observe that several of these Kunitz peptide groups vary in their translated amino acid sequence, secondary structure, antigenicity, and intrinsic disorder, and that the majority of these groups are subject to a purifying (negative) selection. We finalize by describing the evolution and emergence of these Kunitz peptides. The overall interpretation of our analyses discloses a rapidly emerging Kunitz group with a distinct disulfide bond pattern from the I. ricinus salivary gland transcriptome. Conclusions We propose a model to explain the structural and functional evolution of tick salivary Kunitz peptides that we call target-oriented evolution . Our study reveals that combining analytical approaches (transcriptomes, computational, bioinformatics and phylogenetics) improves our understanding of the biological functions of important salivary gland mediators during tick feeding.
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Understanding the evolutionary structural variability and target specificity of tick salivary Kunitz peptides using next generation transcriptome data
BMC Evolutionary Biology, 2014Co-Authors: Alexandra Schwarz, Alejandro Cabezas Cruz, Jan Kopecky, James J. ValdesAbstract:Ticks are blood-sucking arthropods and a primary function of tick salivary proteins is to counteract the host’s immune response. Tick salivary Kunitz-Domain proteins perform multiple functions within the feeding lesion and have been classified as venoms; thereby, constituting them as one of the important elements in the arms race with the host. The two main mechanisms advocated to explain the functional heterogeneity of tick salivary Kunitz-Domain proteins are gene sharing and gene duplication. Both do not, however, elucidate the evolution of the Kunitz family in ticks from a structural dynamic point of view. The Red Queen hypothesis offers a fruitful theoretical framework to give a dynamic explanation for host-parasite interactions. Using the recent salivary gland Ixodes ricinus transcriptome we analyze, for the first time, single Kunitz-Domain encoding transcripts by means of computational, structural bioinformatics and phylogenetic approaches to improve our understanding of the structural evolution of this important multigenic protein family. Organizing the I. ricinus single Kunitz-Domain peptides based on their cysteine motif allowed us to specify a putative target and to relate this target specificity to Illumina transcript reads during tick feeding. We observe that several of these Kunitz peptide groups vary in their translated amino acid sequence, secondary structure, antigenicity, and intrinsic disorder, and that the majority of these groups are subject to a purifying (negative) selection. We finalize by describing the evolution and emergence of these Kunitz peptides. The overall interpretation of our analyses discloses a rapidly emerging Kunitz group with a distinct disulfide bond pattern from the I. ricinus salivary gland transcriptome. We propose a model to explain the structural and functional evolution of tick salivary Kunitz peptides that we call target-oriented evolution. Our study reveals that combining analytical approaches (transcriptomes, computational, bioinformatics and phylogenetics) improves our understanding of the biological functions of important salivary gland mediators during tick feeding.
Morten Dahl Sorensen - One of the best experts on this subject based on the ideXlab platform.
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Spin-state-selective coherence transfer via intermediate states of two-spin coherence in IS spin systems: Application to E.COSY-type measurement of J coupling constants
Journal of Biomolecular NMR, 1997Co-Authors: Morten Dahl Sorensen, Axel Meissner, Ole Winneche SørensenAbstract:It is demonstrated that a new pulse sequence element, Spin-State-SelectiveCoherence Transfer (S^3CT), via an intermediate state ofheteronuclear IS zero- or double-quantum coherence can transfer the twosingle-quantum coherences on one of the spins exclusively to any one of thetwo single-quantum coherences on the other spin. This fact is used for editinginto two subspectra that are most suitable for extraction of homo- orheteronuclear J coupling constants when S^3CT is combined withhomonuclear coherence transfer during a mixing period. Experimentalconfirmation is obtained using a ^15N-labeled 58-residue protein,the C-terminal Kunitz Domain from human type VI collagen. The J coupling con-stants determined include ^3J_HN-Hα and^3J_N-Hβ related to the φ andχ^1 angles, respectively.
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solution structure and backbone dynamics of the human alpha3 chain type vi collagen c terminal Kunitz Domain
Biochemistry, 1997Co-Authors: Morten Dahl Sorensen, Soren E Bjorn, Kjeld Norris, Ole Hvilsted Olsen, Lars Christian Petersen, Thomas L James, Jens J LedAbstract:The solution structure and backbone dynamics of the 58-residue C-terminal Kunitz Domain fragment [α3(VI)] of human α3-chain type VI collagen has been studied by two-dimensional 1H−1H and 1H−15N nuclear magnetic resonance spectroscopy at 303 K. The solution structure is represented by an ensemble of 20 structures calculated with X-PLOR using 612 distance and 47 dihedral angle restraints. The distance restraints were obtained by a complete relaxation matrix analysis using MARDIGRAS. The root mean squared (rms) deviation is 0.91 A for the backbone atoms of the residues Thr2(8)−Gly12(18), Arg15(21)−Tyr35(41), and Gly40(46)−Pro57(63). The central β-sheet [residues Ile18(24)−Tyr35(41)] and the C-terminal α-helix [residues Gln48(54)−Cys55(61)] are better defined with a backbone rms deviation of 0.46 A. The solution structure of α3(VI) is virtually identical to the crystal structure of α3(VI) and to the solution structure of bovine pancreatic trypsin inhibitor (BPTI). The 15N spin−lattice and spin−spin relaxation...
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elucidation of the origin of multiple conformations of the human alpha 3 chain type vi collagen c terminal Kunitz Domain the reorientation of the trp21 ring
Journal of Biomolecular NMR, 1996Co-Authors: Morten Dahl Sorensen, Soren E Bjorn, Kjeld Norris, Ole Hvilsted Olsen, Soren M Kristensen, Jens J LedAbstract:The human α3-chain type VI collagen C-terminal Kunitz Domain fragment (α3(VI)) has been studied by two-dimensional 1H−1H and 1H−13C NMR spectroscopy at 303 K. It is shown that the secondary structure of the protein is strikingly similar to that of BPTI, and that a number of unusual Hα chemical shifts, which are highly conserved in Kunitz-Domain proteins, are also observed for α3(VI). Further-more a series of exchange cross peaks observed in 1H−1H spectra shows that a large number of protons in the central β-sheet exist in two different chemical environments, corresponding to two unequally populated conformations that are slowly exchanging on the NMR time scale. Several protons, including Ser47(53) Hα, Arg32(38) Hγ2, and Gln48(54) Hβ2, all located in the vicinity of the Trp21(27) ring in the crystal structure of α3(VI) [Arnoux, B. et al. (1995) J. Mol. Biol., 246, 609–617], have very different chemical shifts in the two conformations, the most affected being Gln48(54) Hβ2 (Δδ=1.53 ppm), which is placed directly above the Trp21(27) ring in the crystal structure of α3(VI). It is concluded that the origin of the multiple conformations of the central β-sheet is a reorientation of the Trp21(27) ring. From the intensities of corresponding signals in the two conformations, the population of the minor conformation was found to be 6.4±0.2% of that of the major conformation, while a rate constant kM=1.01±0.05 s-1 for the major to minor interconversion was obtained from a series of NOESY spectra with different mixing times. In addition, it is shown that Cys14(20)-Cys38(44) disulfide bond isomerization, previously observed in BPTI [Otting, G. et al. (1993) Biochemistry, 32, 3570–3582], is also likely to occur in α3(VI).
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Elucidation of the origin of multiple conformations of the human α3-chain type VI collagen C-terminal Kunitz Domain: The reorientation of the Trp^21 ring
Journal of Biomolecular NMR, 1996Co-Authors: Morten Dahl Sorensen, Kjeld Norris, Soren M Kristensen, Søren Bjørn, Ole Olsen, Jens J LedAbstract:The human α3-chain type VI collagen C-terminal Kunitz Domain fragment (α3(VI)) has been studied by two-dimensional ^1H−^1H and ^1H−^13C NMR spectroscopy at 303 K. It is shown that the secondary structure of the protein is strikingly similar to that of BPTI, and that a number of unusual H^α chemical shifts, which are highly conserved in Kunitz-Domain proteins, are also observed for α3(VI). Further-more a series of exchange cross peaks observed in ^1H−^1H spectra shows that a large number of protons in the central β-sheet exist in two different chemical environments, corresponding to two unequally populated conformations that are slowly exchanging on the NMR time scale. Several protons, including Ser^47(53) H^α, Arg^32(38) H^γ2, and Gln^48(54) H^β2, all located in the vicinity of the Trp^21(27) ring in the crystal structure of α3(VI) [Arnoux, B. et al. (1995) J. Mol. Biol. , 246 , 609–617], have very different chemical shifts in the two conformations, the most affected being Gln^48(54) H^β2 (Δδ=1.53 ppm), which is placed directly above the Trp^21(27) ring in the crystal structure of α3(VI). It is concluded that the origin of the multiple conformations of the central β-sheet is a reorientation of the Trp^21(27) ring. From the intensities of corresponding signals in the two conformations, the population of the minor conformation was found to be 6.4±0.2% of that of the major conformation, while a rate constant k_M=1.01±0.05 s^-1 for the major to minor interconversion was obtained from a series of NOESY spectra with different mixing times. In addition, it is shown that Cys^14(20)-Cys^38(44) disulfide bond isomerization, previously observed in BPTI [Otting, G. et al. (1993) Biochemistry , 32 , 3570–3582], is also likely to occur in α3(VI).
Anindita Chakrabarty - One of the best experts on this subject based on the ideXlab platform.
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ets 2 and c ebp beta are important mediators of ovine trophoblast Kunitz Domain protein 1 gene expression in trophoblast
BMC Molecular Biology, 2007Co-Authors: Anindita Chakrabarty, Michael R RobertsAbstract:Background The trophoblast Kunitz Domain proteins (TKDPs) constitute a highly expressed, placenta-specific, multigene family restricted to ruminant ungulates and characterized by a C-terminal "Kunitz" Domain, preceded by one or more unique N-terminal Domains. TKDP-1 shares an almost identical expression pattern with interferon-tau, the "maternal recognition of pregnancy protein" in ruminants. Our goal here has been to determine whether the ovine (ov) Tkdp-1 and IFNT genes possess a similar transcriptional code.
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Ets-2 and C/EBP-beta are important mediators of ovine trophoblast Kunitz Domain protein-1 gene expression in trophoblast
BMC Molecular Biology, 2007Co-Authors: Anindita Chakrabarty, R Michael RobertsAbstract:Background The trophoblast Kunitz Domain proteins (TKDPs) constitute a highly expressed, placenta-specific, multigene family restricted to ruminant ungulates and characterized by a C-terminal "Kunitz" Domain, preceded by one or more unique N-terminal Domains. TKDP-1 shares an almost identical expression pattern with interferon-tau, the "maternal recognition of pregnancy protein" in ruminants. Our goal here has been to determine whether the ovine (ov) Tkdp-1 and IFNT genes possess a similar transcriptional code. Results The ovTkdp-1 promoter has been cloned and characterized. As with the IFNT promoter, the Tkdp-1 promoter is responsive to Ets-2, and promoter-driven reporter activity can be increased over 700-fold in response to over-expression of Ets-2 and a constitutively active form of protein Kinase A (PKA). Unexpectedly, the promoter element of Tkdp-1 responsible for this up-regulation, unlike that of the IFNT , does not bind Ets-2. However, mutation of a CCAAT/enhancer binding element within this control region not only reduced basal transcriptional activity, but prevented Ets-2 as well as cyclic adenosine 5'-monophosphate (cAMP)/PKA and Ras/mitogen-activated protein kinase (MAPK) responsiveness. In vitro binding experiments and in vivo protein-protein interaction assays implicated CCAAT/enhancer binding protein-beta (C/EBP-β) as involved in up-regulating the Tkdp-1 promoter activity. A combination of Ets-2 and C/EBP-β can up-regulate expression of the minimal Tkdp-1 promoter as much as 930-fold in presence of a cAMP analog. An AP-1-like element adjacent to the CCAAT enhancer, which binds Jun family members, is required for basal and cAMP/ C/EBP-β-dependent activation of the gene, but not for Ets-2-dependent activity. Conclusion This paper demonstrates how Ets-2, a key transcription factor for trophoblast differentiation and function, can control expression of two genes ( Tkdp-1 and IFNT ) having similar spatial and temporal expression patterns via very different mechanisms.
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rapid evolution of the trophoblast Kunitz Domain proteins tkdps a multigene family in ruminant ungulates
Journal of Molecular Evolution, 2006Co-Authors: Anindita Chakrabarty, Michael R Roberts, James A Maclean, Austin L Hughes, Jonathan A GreenAbstract:The trophoblast Kunitz Domain proteins (TKDPs) are products of the outer cells (trophoblasts) of the placenta of cattle, sheep, and related species. Most are expressed abundantly for only a few days during the time at which the ruminant conceptus is first establishing intimate contacts with the uterine lining. The TKDPs are secretory proteins that possess a carboxyl-terminal peptidase inhibitory Domain related to the Kunitz family of serine peptidase inhibitors. On the amino-terminal end are one or more highly unusual regions that are unique to the TKDP genes and have no apparent similarity to any other known sequences. The TKDPs are a rather divergent family that exhibits a good deal of variation among the members. To better understand the reason for such variation, the rates of synonymous (dS) and nonsynonymous (dN), as well as radical (p NR ) and conservative (p NC ), substitutions were assessed. Phylogenetic trees revealed that the Kunitz Domains represented three related groups, whereas the amino-terminal Domains formed four groupings. Pairwise comparisons between Kunitz and amino-terminal Domain groups demonstrated that dN was consistently greater than dS. In addition, nonsynonymous substitutions in the Kunitz Domains tended to be radical (changing charge or polarity), while those in the amino-terminal Domains exhibited neither a preponderance of conservative nor radical substitution rates. In summary, the rapid evolution of the TKDPs, coupled with their restricted temporal expression during development, likely reflects the establishment of protein-protein interactions that have evolved to serve the unusual synepitheliochorial placenta of ruminant ungulates.
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family of Kunitz proteins from trophoblast expression of the trophoblast Kunitz Domain proteins tkdp in cattle and sheep
Molecular Reproduction and Development, 2003Co-Authors: James A Maclean, Anindita Chakrabarty, Michael R Roberts, Sancai Xie, James A Bixby, Jonathan A GreenAbstract:Here we report the molecular cloning of several members of a family of novel proteins expressed by the ruminant trophoblast, known as the trophoblast Kunitz Domain proteins (TKDPs). Each contains a carboxyl-terminal module of ∼64 amino acids belonging to the Kunitz family of serine proteinase inhibitors. These Kunitz modules are preceded by one or more structurally related Domains, each about 80 amino acids long. The function of these Domains is unclear. The TKDPs differ considerably in sequence identity, with much of the diversity due to variability in the amino-terminal Domains. However, nine of the ten Kunitz Domains described here are themselves unique, ranging in amino acid sequence identity from 90% to 53% to each other and averaging only about 50% identity with bovine pancreatic trypsin inhibitor (BPTI). The “warhead” P1 residues, which govern specificity, are themselves variable and include some unusual amino acids, such as Asn, Thr, and Ile, as well as the more common Lys. The Kunitz Domains of three of the TKDPs lack the conserved cysteines at positions 14 and 38 (BPTI numbering) that normally contribute to the orientation of the inhibitory loop. Northern blotting and reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that the TKDP genes do not exhibit identical expression patterns during trophoblast development, although mostly are expressed maximally during early pregnancy. It is possible that the TKDPs provide a broad range of specificities against maternal proteinases that might be damaging to the trophoblast during pregnancy. Mol. Reprod. Dev. 65: 30–40, 2003. © 2003 Wiley-Liss, Inc.
Paul S Bajaj - One of the best experts on this subject based on the ideXlab platform.
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Decoy Plasminogen Receptor Containing a Selective Kunitz-Inhibitory Domain
2015Co-Authors: Yogesh Kumar, Godwin I Ogueli, Kanagasabai Vadivel, Amy E Schmidt, Nalaka Rannulu, Joseph A Loo, Sathya M Ponnuraj, Madhu S. Bajaj, Paul S BajajAbstract:Kunitz Domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2′ residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz Domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 μM) but not to KD1L17R-VT. Furthermore, KD1L17R-KT inhibited tPA-induced plasma clot fibrinolysis more efficiently than KD1L17R-VT. Additionally, compared to ε-aminocaproic acid KD1L17R-KT was more effective in reducing blood loss in a mouse liver-laceration injury model, where the fibrinolytic system is activated. In further experiments, the micro(μ)-plasmin–KD1L17R-KT complex inhibited urokinase-induced plasminogen activation on phorbol-12-myristate-13-acetate-stimulated U937 monocyte-like cells, whereas the μ-plasmin–KD1L17R-VT complex failed to inhibit this process. In conclusion, KD1L17R-KT inhibits the active site of plasmin as well as acts as a decoy receptor for the kringle Domain(s) of plasminogen/plasmin; hence, it limits both plasmin generation and activity. With its dual function, KD1L17R-KT could serve as a preferred agent for controlling plasminogen activation in pathological processes
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decoy plasminogen receptor containing a selective Kunitz inhibitory Domain
Biochemistry, 2014Co-Authors: Yogesh Kumar, Madhu S. Bajaj, Godwin I Ogueli, Kanagasabai Vadivel, Amy E Schmidt, Nalaka Rannulu, Joseph A Loo, Sathya M Ponnuraj, Paul S BajajAbstract:Kunitz Domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2′ residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz Domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 μ...
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antifibrinolytic efficacy of truncated l17r mutant of Kunitz Domain 1 kd1 of tissue factor pathway inhibitor 2 tfpi 2 comparison with lysine analogues
Blood, 2011Co-Authors: Madhu S. Bajaj, Godwin I Ogueli, Yogesh Kumar, Kanagasabai Vadivel, Amy E Schmidt, Nalaka Rannulu, Joseph A Loo, Paul S BajajAbstract:Abstract 855 Previously, we demonstrated that changing residue Leu17 (BPTI/Aprotinin numbering) to Arg in Kunitz Domain 1 (73-residue KD1-L17R) of TFPI-2 abolishes its anticoagulant functions and enhances its plasmin inhibition (Bajaj et al., J Biol Chem 286, 4329–4340, 2011). In that study we used the entire KD1 Domain, which in addition to the core structural homologous region of BPTI (58 residues) included 9 residues on the N-terminal and 6 residues on the C-terminal side of the protein. Conformation of these 15 residues may be different in the isolated KD1-Domain as compared to the complete TFPI-2 molecule. Thus, these residues could be potentially immunogenic. To address these concerns, we investigated weather N- and C-terminal regions of 73-residue KD1-L17R could be cleaved upon prolonged incubation with thrombin (IIa). Incubation of 73-residue KD1-L17R with IIa for 72 hrs yielded smaller version(s) of KD1-L17R as analyzed by SDS-PAGE. N-terminal sequence and MALDI-TOF/ESI mass spectrometry analyses revealed three closely related species present in the truncated KD1-L17R preparations (Fig. 1). Species 1 has Gly-Asn-Asn as the amino terminus and Val-Pro-Lys as the C-terminus. Species 2 and 3 are similar to species 1 except species 2 is produced after losing Gly and Asn from the N-terminus, whereas species 3 is produced after losing Val-Pro-Lys from the C-terminus. Thus, all three species have the intact core Kunitz Domain with minor variations at the N- and C-terminus regions. Further, these species are cleaved at the viable albeit very slow IIa-cleavage sites; herein, these species are collectively referred to as truncated KD1-L17R. A plausible mechanism for proteolysis at these cleavage sites is shown in Fig. 2. Similar to the 73-residue KD1-L17R, the truncated preparations did not inhibit (Ki > 3 μM) plasma kallikrein, factor (F) XIa, FVIIa/soluble tissue factor, FXa, activated protein C, tissue plasminogen activator (tPA), IIa and IIa/soluble thrombomodulin. Importantly, the truncated KD1-L17R preparations inhibited plasmin with Ki ∼1.2 nM. Further, the truncated KD1-L17R inhibited tPA-induced plasma clot fibrinolysis with an apparent IC 50 of ∼0.37 μM, a value similar to that obtained with the 73-residue KD1-L17R and BPTI. Two lysine analogues, Epsilon amino caproic acid (EACA) and tranexamic acid (TE) inhibited tPA-induced plasma clot fibrinolysis with an apparent IC 50 of ∼80 μM and ∼20 μM, respectively. Further, efficacy of truncated KD1-L17R was tested in a mouse liver laceration model of bleeding. As compared to saline, the amount of blood loss was reduced by ∼65% by truncated KD1-L17R (N=6, p 0.001), ∼70% by BPTI (N=10, p 0.003), ∼52% by TE (N=10, p 0.019) and ∼25% by EACA (N=16, p 0.03). We also observed seizures in four (25%) of the animals treated with a single dose of EACA. In conclusion, truncated KD1-L17R is an effective antifibrinolytic agent similar to the 73-residue KD1-L17R and BPTI/Aprotinin. Although lysine analogues are relatively effective in reducing blood loss, EACA caused seizures in our studies. These observations are consistent with recent reports that one of the major side effects of lysine analogues is seizures (Martin et al., J Cardiothorac Vasc Anesth 25, 20–25, 2011; Koster and Schirmer, Curr Opin Anaesthesiol 24, 92–97, 2011). We conclude that truncated KD1-L17R may serve as an excellent alternative to BPTI and lysine analogues in preventing blood loss during major surgeries including coronary artery bypass graft (CABG) surgery. We are currently expressing the 60-residue KD1-L17R (NH 2 Asn-Ala-Glu······Ile-Glu-Lys) protein for further efficacy studies. We are also generating additional mutant(s) on the 60-residue KD1-L17R molecule for achieving increased plasmin potency without provoking anticoagulant functions. Supported By HL89661 and HL36365. Disclosures: No relevant conflicts of interest to declare.
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engineering Kunitz Domain 1 kd1 of human tissue factor pathway inhibitor 2 to selectively inhibit fibrinolysis properties of kd1 l17r variant
Journal of Biological Chemistry, 2011Co-Authors: Madhu S. Bajaj, Godwin I Ogueli, Yogesh Kumar, Kanagasabai Vadivel, Gregory W Lawson, Sreejesh Shanker, Amy E Schmidt, Paul S BajajAbstract:Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein, and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz Domain (KD1) also inhibits plasmin; therefore, it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2′ position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2′ residue Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with ∼6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was ∼8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R.
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engineering Kunitz Domain of human tissue factor pathway inhibitor 2 to selectively inhibit fibrinolysis an antifibrinolytic agent with potential to replace aprotinin
Blood, 2008Co-Authors: Madhu S. Bajaj, Godwin I Ogueli, Sreejesh Shanker, Amy E Schmidt, Paul S BajajAbstract:Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz Domain (KD1) also inhibits plasmin and therefore it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this report, structure based investigations and analysis of the serine proteases profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2′ (nomenclature of Schechter and Berger, BBRC, 27:157–162, 1967) position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2′ residue Leu17 (bovine pancreatic trypsin inhibitor/aprotinin numbering) in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with the wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in E. Coli, folded and purified to homogeneity. Amino-terminal sequences and mass spectra revealed proper folding of the KD1-WT and KD1-L17R. As compared to KD1-WT, the KD1-L17R neither prolonged the activated partial thromboplastin time of normal plasma nor it inhibited factor XIa, plasma kallikrein or factor VIIa/tissue factor. Further, KD1-L17R inhibited plasmin with ~4-fold increased affinity. In a mouse liver laceration model of bleeding from small vessels, KD1-L17R reduced total blood loss by 84% compared with KD1-WT, which reduced total blood loss by 10%. Moreover, in this bleeding model, KD1-L17R was more effective than aprotinin (70% reduction), which has been used as an antifibrinolytic agent to decrease blood loss during major surgery. In this model, KD1-L17R was also more effective than the lysine analogue tranexamic acid (52% reduction). In additional studies, in a tail transection model of bleeding from a large vessel, KD1-L17R reduced total blood loss by 70% and was more effective than KD1-WT (46% reduction), aprotinin (43% reduction) and tranexamic acid (67% reduction). Notably, as compared to aprotinin, renal toxicity manifesting as multifocal tubular necrosis by histopathology was not observed with KD1-L17R or KD1-WT. In conclusion, KD1-L17R is a specific inhibitor of plasmin without anticoagulant properties and is more effective in reducing blood loss compared with known antifibrinolytic agents in clinical use.
