scholarly journals Identification of a phage display-derived peptide interacting with the N-terminal region of Factor VII activating protease (FSAP) enables characterization of zymogen activation.

2022 ◽  
Author(s):  
Sebastian Seidl ◽  
Nis V Nielsen ◽  
Michael Etscheid ◽  
Bengt-Erik Haug ◽  
Maria Stensland ◽  
...  
Keyword(s):  
Phage Display ◽  
Tissue Injury ◽  
Peptide Identification ◽  
Activation Mechanism ◽  
Factor Vii ◽  
Zymogen Activation ◽  
Binding Studies ◽  
Interaction Site ◽  
N Terminus ◽  
Contact Pathway

Increased Factor VII activating protease (FSAP) activity has a protective effect in diverse disease conditions as inferred from studies in FSAP-/- mice and humans deficient in FSAP activity due to a single nucleotide polymorphism. The activation of FSAP zymogen in plasma is mediated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear if this activation mechanism is specific and amenable to manipulation. Using a phage display approach we have identified a peptide, NNKC9/41, that activates pro-FSAP in plasma. Other commonly found zymogens in the plasma were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. Blocking the contact pathway of coagulation did not influence pro-FSAP activation by the peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30-35) in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41 and this was reversed by MA-FSAP-38C7 demonstrating the utility of this peptide. Identification of this peptide, and the corresponding interaction site, provides proof of principle that it is possible to activate a single protease zymogen in blood in a specific manner. Peptide NNKC/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP in more detail, elucidate its biological role.

scholarly journals Specificity and VH sequence of two monoclonal antibodies against the N-terminus of dystrophin

1995 ◽  
Vol 309 (1) ◽  
pp. 355-359 ◽  
Author(s):  
G E Morris ◽  
C Nguyen ◽  
Nguyen thi Man
Keyword(s):  
Monoclonal Antibodies ◽  
Point Mutations ◽  
Hypervariable Region ◽  
Variable Region ◽  
Binding Studies ◽  
Cdna Sequences ◽  
N Terminus ◽  
Random Library ◽  
Blast Cells

We have used a random library of 15-mer peptides expressed on phage to show that two monoclonal antibodies (mAbs) require only the first three amino acids of dystrophin (Leu-Trp-Trp) for binding. Since the mAbs recognize dystrophin in frozen muscle sections, the results suggest that this hydrophobic N-terminus of dystrophin is accessible to antibody in situ. Quantitative binding studies suggested minor differences in specificity between the two mAbs, so the Ig heavy-chain variable region (VH) sequences of the two hybridomas were determined by RT-PCR and cDNA sequencing. After elimination of PCR errors, the two cDNA sequences were found to be identical except for five somatic mutations which resulted in three amino acid changes in the second hypervariable region (CDR2). The results suggest that the two hybridomas originated from the same lymphocyte clone in a germinal centre of the spleen, but underwent different point mutations and subtype switches during clonal expansion to form blast cells.

scholarly journals Use of recombinant activated factor VII

Medicina ◽  
2009 ◽  
Vol 45 (3) ◽  
pp. 248
Author(s):  
Dagmara Reingardienė ◽  
Robertas Lažauskas
Keyword(s):  
Prothrombin Time ◽  
Liver Diseases ◽  
Hemophilia A ◽  
Tissue Injury ◽  
Factor Vii ◽  
Recombinant Activated Factor Vii ◽  
Congenital Deficiency ◽  
Activated Factor Vii ◽  
Life Threatening ◽  
Hemostatic Disorders

Recombinant activated factor VII (rFVIIa) has been used in the treatment of various congenital and acquired hemostatic disorders for more than 10 years. Hemostasis is initiated by the FVIIa bound to tissue factor (TF), which constitutes only approximately 1% of total amount of the FVII protein existing in the blood. rFVII becomes activated only after the binding to the TF, released at the site of tissue injury. The efficiency of rFVIIa in the treatment of such life-threatening hemorrhagic states like hemophilia reaches up to 76–84%. rFVIIa is successfully used in the treatment of congenital deficiency of factor VII. It normalizes prothrombin time in the patients with the liver diseases and in cases of overdose of indirect anticoagulants. It is also useful for patients suffering from thrombocytopenia, thrombocyte function disorders, hemophilia A and B with development of inhibitors. rFVIIa allows overcoming uncontrollable hemorrhages, etc. It is supposed that rFVIIa is becoming a universal hemostatic drug.

MECHANISM OF ACTIVATION OF BLOOD COAGULATION BY LEUKOCYTE PROCOAGULANT ACTIVITY

1987 ◽  
Author(s):  
N Narahara ◽  
H Sadakata ◽  
T Uchiyama ◽  
K Andoh ◽  
H Tanaka ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Calf Serum ◽  
Leukemia Cell ◽  
Specific Radioactivity ◽  
Activation Mechanism ◽  
Leukemia Cell Line ◽  
Coagulation Cascade ◽  
Factor Vii ◽  
Factor X ◽  
Viii And Ix

To investigate the process of activation mechanism of blood coagulation by leukocytes, binding of radiolabelled Factor X and the activation of Factor X on the cell surface of leukocytes were studied by using cultured leukemia cell line, Molt-4 cells. Cells were cultured in RPMI 1640 medium with 10% inactivated fetal, calf serum at a concentration of 1x106cells/ml. After 6 hours' stimulation with 1 ug/ml of endotoxin(LPS: Escherichia coli 026:B6), cells were separated by centrifugation, washed three times with Tris containing NaCl buffer(pH 7.5, TBS), and then suspended in TBS containing 0.5% bovine serum albumin(TBS-BSA) to the concentration of 5x106 cells/ml. Factors X, VII and IX were purified from fresh-frozen human plasma by the method of Bajaj in a modified version. Factor VIII was purified from cryoprecipitate as starting material. Factor X labelled with 1-125 by the method of McFarlane showed a single band on autoradiography. Specific radioactivity was 0.3 mCi/mg. For the study on binding of Factor X, TBS-BSA solution containing 4 mM of CaClp, various amounts of radiolabelled Factor X with/without purified Factors VII, VIII and IX were added to the LPS-stimulated washed cell suspension and mixed well. N-butyl-phtalate was layered over the reaction mixture after incubation at room temperature for various minutes. Total amount of bound Factor X was calculated from the radioactivity of the cell pellet separated by centrifugation of the reaction mixture. The Xa activity generated in the supernatants was assayed using S2222.Results: Factor X bound specifically to the LPS-stimulated Molt-4 cells. Amount of bound Factor X and the dissociation constant was 1.0 ng/5x10bcells (5.2x103sites/cell) and 5x106M, respectively. More amounts of Factor X bound when Factors VIII and IX were present in the reaction mixture than their absence. Five times more Factor Xa was generated when Factors VII, VIII and IX were present in the reaction mixture as compared with presence of Factor VII, alone. These results suggest that blood coagulation cascade proceeds on the LPS-stimulated leukocyte surface.

scholarly journals TFIIA induces conformational changes in TFIID via interactions with the basic repeat.

1992 ◽  
Vol 12 (11) ◽  
pp. 5189-5196 ◽  
Author(s):  
D K Lee ◽  
J DeJong ◽  
S Hashimoto ◽  
M Horikoshi ◽  
R G Roeder
Keyword(s):  
Dna Binding ◽  
Conformational Changes ◽  
Structural Changes ◽  
Inhibitory Effect ◽  
Binding Studies ◽  
Binding Interaction ◽  
Contact Points ◽  
Tata Element ◽  
N Terminus ◽  
Dna Binding Studies

DNA-binding studies with Saccharomyces cerevisiae TFIID point mutants indicated that TFIIA interacts with the basic repeat region of TFIID and induces structural changes. The latter was shown by the ability of TFIIA to compensate for TFIID point mutants defective for DNA binding. Interaction with TFIIA also rendered TFIID binding temperature independent, thus mimicking the effect of removing the nonconserved N terminus of TFIID. In addition, N-terminal truncation of the TFIID point mutants defective for DNA binding mimicked the ability of TFIIA to restore DNA binding of those mutants. Taken together, these results suggest that TFIIA enhances TFIID binding to DNA by eliminating an otherwise inhibitory effect of the nonconserved N terminus of TFIID. Furthermore, analyses of TFIID contact points on DNA and binding studies with TATA-containing oligonucleotide probes showed that TFIIA decreases the effect of sequences flanking the adenovirus major late TATA element on TFIID binding to DNA, suggesting a possible role of TFIIA in allowing TFIID to recognize a wider variety of promoters.

FOG-1 Directly Binds to a Chromatin Remodeling and Deacetylase-Containing Complex To Mediate Transcriptional Repression by GATA-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 355-355
Author(s):  
Wei Hong ◽  
Minako Nakazawa ◽  
Ying-Yu Chen ◽  
Rajashree Kori ◽  
Carrie Rakowski ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Histone Deacetylation ◽  
Gene Repression ◽  
Single Point Mutation ◽  
Mutant Form ◽  
Erythroid Cells ◽  
Binding Studies ◽  
Nucleosome Remodeling ◽  
N Terminus

Abstract Terminal erythroid maturation requires coordinated activation of erythroid marker genes and repression of genes associated with the undifferentiated state. These gene expression patterns are mediated by the concerted action of the erythroid transcription factor GATA-1 and its cofactor FOG-1 that can activate or repress transcription depending on promoter context. We and others showed previously that one mechanism by which FOG-1 functions is to facilitate GATA-1 association with certain DNA target sites in vivo. Using gene complementation studies of GATA-1-ablated erythroid cells, we show that at several GATA-1-repressed target genes (c-kit, c-myc and GATA-2) FOG-1 is dispensable for GATA-1 occupancy in vivo but essential for gene repression and histone deacetylation. To examine how FOG-1 functions as co-repressor we performed affinity chromatography, conventional protein purification and in vitro binding studies to identify proteins that bind FOG-1. We discovered that FOG-1 directly associates with the nucleosome remodeling and histone deacetylase complex NURD. This interaction is mediated by a small conserved domain at the N-terminus of FOG-1 and the MTA-1 subunit of NURD. Association of FOG-1 with NURD occurs in vivo and depends on an intact N-terminus of FOG-1. A series of point mutations across the N-terminus of FOG-1 revealed a tight correlation between NURD binding and transcriptional repression. In particular, a single point mutation at the N-terminus of FOG-1 that abrogated NURD binding also blocked gene repression by FOG-1. Finally, the ability of GATA-1 to repress transcription was impaired in erythroid cells expressing a mutant form of FOG-1 that is defective for NURD binding. Together, these studies show that FOG-1 and very likely other FOG proteins are bona fide co-repressors that link GATA proteins to histone deacetylation and nucleosome remodeling via a novel protein interaction module.

scholarly journals A key functional role for the insulin-like growth factor 1 N-terminal pentapeptide

1989 ◽  
Vol 259 (3) ◽  
pp. 665-671 ◽  
Author(s):  
C J Bagley ◽  
B L May ◽  
L Szabo ◽  
P J McNamara ◽  
M Ross ◽  
...  
Keyword(s):  
Growth Factor ◽  
Binding Protein ◽  
Biological Properties ◽  
Maximal Response ◽  
Insulin Like Growth Factor ◽  
Amino Acid Residues ◽  
Binding Studies ◽  
N Terminus ◽  
L6 Myoblast

In order to elucidate the role of the N-terminus of insulin-like growth factor 1 (IGF-1) with respect to its biological properties, we chemically synthesized analogues of IGF-1 truncated by one to five amino acid residues from the N-terminus. In a bioassay that measured the stimulation of protein synthesis in rat L6 myoblasts, the concentrations required to produce a half-maximal response were: IGF-1, 13 ng/ml; des-(1)-IGF-1, 10 ng/ml; des-(1-2)-IGF-1, 13 ng/ml; des-(1-3)-IGF-1, 1.5 ng/ml; des-(1-4)-IGF-1, 5.1 ng/ml; des-(1-5)-IGF-1, 1200 ng/ml. When tested for their abilities to compete with 125I-IGF-1 binding to L6 myoblasts at 3 degrees C, the concentrations required for 50% competition were: IGF-1, des-(1)-IGF-1 and des-(1-2)-IGF-1, 20 ng/ml; des-(1-3)-IGF-1, 14 ng/ml; des-(1-4)-IGF-1, 40 ng/ml; des-(1-5)-IGF-1, greater than 1000 ng/ml. Receptor-binding experiments at 25 degrees C, however, gave results suggesting that the myoblasts were secreting a binding protein selective for the three longest peptides. This interpretation was confirmed by binding studies with medium conditioned by the L6 myoblasts as well as binding protein purified from MDBK-cell-conditioned medium. In both cases IGF-1, des-(1)-IGF-1 and des-(1-2)-IGF-1 competed for tracer IGF-1 binding at least 60-fold better than did the three shorter peptides. The results obtained account for the increased potency of des-(1-3)-IGF-1 and des-(1-4)-IGF-1, since their activities are not attenuated by the binding protein, and the relatively lower potency of des-(1-4)-IGF-1 is a consequence of this peptide binding less well to the L6-myoblast receptor.

scholarly journals Quinine-dependent, platelet-reactive monoclonals mimic antibodies found in patients with quinine-induced immune thrombocytopenia

Blood ◽  
2009 ◽  
Vol 113 (5) ◽  
pp. 1105-1111 ◽  
Author(s):  
Daniel W. Bougie ◽  
Jessica Birenbaum ◽  
Mark Rasmussen ◽  
Mortimer Poncz ◽  
Richard H. Aster
Keyword(s):  
Molecular Basis ◽  
Immune Thrombocytopenia ◽  
Antibody Binding ◽  
Membrane Glycoproteins ◽  
Binding Studies ◽  
Platelet Destruction ◽  
Drug Induced ◽  
N Terminus ◽  
Sequencing Studies ◽  
Drug Dependent

Abstract Drug-induced immune thrombocytopenia (DITP) is caused by drug-dependent antibodies (DDAbs) that are nonreactive in themselves but bind tightly to specific platelet membrane glycoproteins (GP) when soluble drug is present at pharmacologic concentrations. This reaction takes place without covalent linkage of drug to the target, indicating that drug does not function as a classical hapten to promote antibody binding. Studies to define other mechanism(s) responsible for this interaction have been frustrated by the polyclonal nature of human DDAbs and limited quantities of antibody usually available. We produced 2 monoclonal antibodies (mAbs), 314.1 and 314.3, from a mouse immunized with purified human GPIIb/IIIa and quinine that recognize the N terminus of the GPIIb β propeller domain only when soluble quinine is present. Both monoclonals closely mimic the behavior of antibodies from patients with quinine-induced immune thrombo-cytopenia in their reactions at various concentrations of quinine and quinine congeners. Sequencing studies showed that the 2 mAbs are closely related structurally and that mAb 314.3 probably evolved from mAb 314.1 in the course of the immune response. These monoclonal reagents are the first of their kind and should facilitate studies to define the molecular basis for drug-dependent antibody binding and platelet destruction in DITP.

Mechanisms of polyamine catabolism-induced acute pancreatitis

2007 ◽  
Vol 35 (2) ◽  
pp. 326-330 ◽  
Author(s):  
M.T. Hyvönen ◽  
M. Merentie ◽  
A. Uimari ◽  
T.A. Keinänen ◽  
J. Jänne ◽  
...  
Keyword(s):  
Acute Pancreatitis ◽  
Cathepsin B ◽  
Tissue Injury ◽  
Necrotizing Pancreatitis ◽  
Acinar Cells ◽  
Pancreatic Tissue ◽  
Early Event ◽  
Zymogen Activation ◽  
Polyamine Catabolism ◽  
Trypsinogen Activation

Acute pancreatitis is an autodigestive disease, in which the pancreatic tissue is damaged by the digestive enzymes produced by the acinar cells. Among the tissues in the mammalian body, pancreas has the highest concentration of the natural polyamine, spermidine. We have found that pancreas is very sensitive to acute decreases in the concentrations of the higher polyamines, spermidine and spermine. Activation of polyamine catabolism in transgenic rats overexpressing SSAT (spermidine/spermine-N1-acetyltransferase) in the pancreas leads to rapid depletion of these polyamines and to acute necrotizing pancreatitis. Replacement of the natural polyamines with methylated polyamine analogues before the induction of acute pancreatitis prevents the development of the disease. As premature trypsinogen activation is a common, early event leading to tissue injury in acute pancreatitis in human and in experimental animal models, we studied its role in polyamine catabolism-induced pancreatitis. Cathepsin B, a lysosomal hydrolase mediating trypsinogen activation, was activated just 2 h after induction of SSAT. Pre-treatment of the rats with bismethylspermine prevented pancreatic cathepsin B activation. Analysis of tissue ultrastructure by transmission electron microscopy revealed early dilatation of rough endoplasmic reticulum, probable disturbance of zymogen packaging, appearance of autophagosomes and later disruption of intracellular membranes and organelles. Based on these results, we suggest that rapid eradication of polyamines from cellular structures leads to premature zymogen activation and autodigestion of acinar cells.

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