US20120165510A1 - novel application of fibrinogen-420 and its active domain - Google Patents

novel application of fibrinogen-420 and its active domain Download PDF

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Publication number
US20120165510A1
US20120165510A1 US13/254,987 US201013254987A US2012165510A1 US 20120165510 A1 US20120165510 A1 US 20120165510A1 US 201013254987 A US201013254987 A US 201013254987A US 2012165510 A1 US2012165510 A1 US 2012165510A1
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fibrinogen
protein
sequence
alpha
domain
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Luo Yongzhang
Huadong Tang
Yan Fu
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Tsinghua University
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Tsinghua University
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Assigned to TSINGHUA UNIVERSITY reassignment TSINGHUA UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FU, YAN, TANG, HUADONG, YONGZHANG, LUO
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/363—Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to a novel application of Fibrinogen-420 and its active domain.
  • Fibrinogen also known as coagulation factor I, is an important protein in the process of blood clotting. Fibrinogen has a molecular weight of 340,000 Daltons, and is composed of two subunits connected by disulfide bonds to form dimers. Each subunit respectively consists of three intertwined polypeptide chains, called A, B, C chain. In the process of clotting, fibrinogen digested by thrombin to generate fibrin and thus form insoluble fibrin polymers. And then the composition of fibrin polymer fibers and blood platelets will form solid tampon. Fibrinogen is also a stress protein, whose content in the blood is about 1.5-4 mg/ml. The content of fibrinogen is related to the immune status, which could also reflect the risk of cardiovascular disease.
  • Fibrinogen-420 is a subtype of fibrinogen, in which the C-terminal of A chain has an extension of globular domain than the ordinary fibrinogen. This extension of globular domain is called the alpha EC domain, which has high homology with the globular domain at the end of B, C chains.
  • the molecular weight of fibrinogen-420 is about 420,000 Daltons, which is different from the general tissue fibrinogen of 340,000 Dalton.
  • Protein misfolding disease is due to the conformational change of specific proteins in the tissue, during which proteins gather to produce amyloidosis and finally result in a class of diseases with pathological changes in tissues and organs, such as Alzheimer's disease and bovine spongiform encephalopathy.
  • pathological changes in tissues and organs such as Alzheimer's disease and bovine spongiform encephalopathy.
  • the existing methods such as monoclonal antibody technology, small molecules, synthetic peptides, et al. have many disadvantages including immune rejection reactions, lacking of broad-spectrum, significant side effects, and a short half-life in vivo.
  • the purpose of this invention is to provide novel applications of Fibrinogen-420 and its active domain.
  • Fibrinogen-420 has molecular chaperone activity and broad-spectrum, non-specific protective effects.
  • Fibrinogen-420 is an endogenous protein, so that it will not be quickly degraded in the body and does not arouse immune rejection. It can promote denatured proteins to refold properly and stabilize protein conformation and function. So it can be widely used in protein refolding, denatured protein testing in quality control, and prevention of protein denaturation et al.
  • the protein described could be a recombinant protein or natural protein.
  • Fibrinogen-420 contains alpha EC domain, and a separated alpha EC domain protein has the same or similar functions with intact fibrinogen-420.
  • the amino acid sequence of alpha EC domain is shown as SEQ ID NO. 1, and the fibrinogen-420 could specifically refer to human fibrinogen-420.
  • Fibrinogen-420 or alpha EC domain can be prepared into the protein reagent for use. Described protein reagents include at least fibrinogen-420 or alpha EC domain protein, and the protein reagents do not rule out other solvents and additives. Good results are expected when the ratio of other protein vs. fibrinogen-420 or alpha EC domain protein is in the range of 25:1 to 1:100, among which the ratio of 1:1 is included. The best ratio depends on the requirement of specific application.
  • the present invention also shows that fibrinogen-420 or alpha EC domain protein can inhibit the aggregation of degeneration protein, and protect the protein activity as well. Thus it can be used as drugs to treat protein conformation related diseases.
  • the drugs to treat protein misfolding diseases contain fibrinogen-420 or alpha EC domain protein as the active ingredients.
  • Therapeutic proteins as described can be used for the treatment of a variety of protein degenerative diseases, such as protein denaturation caused by fever, tobacco, alcohol, oxygen free radicals and other harmful substances.
  • the drugs for the treatment of protein misfolding diseases contain fibrinogen-420 or alpha EC domain protein as the active ingredients.
  • fibrinogen-420 or alpha EC domain protein can capture the protein unfolding process. By helping the protein to refold correctly or keeping it in a folded state, fibrinogen-420 or alpha EC domain protein can prevent protein aggregation and stabilize the activity and function of a protein. Thus, fibrinogen-420 or alpha EC domain protein can enhance the ability of a protein against denaturation, so that it can be used as a protein stabilizing agent in vitro.
  • the protein stabilizing agent described contains fibrinogen-420 or alpha EC domain protein as the active ingredient.
  • the protein stabilizing agent described can inhibit the precipitation of proteins which are easy to gather and form aggregation.
  • the protein stabilizing agent can also protect the enzyme activity, such as stabilize citrate synthase, luciferase, insulin and the activity of other enzymes.
  • fibrinogen-420 or alpha EC domain protein can stabilize the protein reagents especially the antibodies cross-linked with reporter enzymes (such as horseradish peroxidase, alkaline phosphatase or luciferase), and increase the shelf life and the quality of the products.
  • reporter enzymes such as horseradish peroxidase, alkaline phosphatase or luciferase
  • Fibrinogen-420 or alpha EC domain protein can also be used to identify unfolding and denatured proteins, so it can be used in the quality control of protein reagents.
  • Fibrinogen-420 or alpha EC domain protein can inhibit the thermal-induced denaturation and aggregation of citrate synthase.
  • FIG. 2 The alpha EC domain protein inhibits the chemical denaturation and aggregation of citrate synthase.
  • FIG. 3 Fibrinogen-420 inhibits the thermal-induced denaturation and inactivation of citrate synthase.
  • FIG. 4 The alpha EC domain protein inhibits the thermal-induced denaturation and inactivation of citrate synthase.
  • FIG. 5 The alpha EC domain protein specifically recognizes the denatured citrate synthase protein.
  • fibrinogen-420 The preparation of fibrinogen-420 started from the purification of a fibrinogen mixture from blood or cord blood, after which fibrinogen-420 can be further purified. Details are as follows:
  • Mono Q HR 10/10 anion exchange column (Pharmacia) is used as the chromatographic column.
  • Use pH step elution to elute the sample starting with 0.005 M Tris-phosphate buffer rapid transition to 0.2 M Tris-phosphate buffer, pH 6.0, and then maintain the 0.2 M Tris-phosphate buffer, pH 6.0 to elute 12 column volumes, and finally eluted using a linear gradient for 12 column volumes to 0.5 M Tris-phosphate buffer, pH 4.2.
  • Fibrinogen-420 is obtained in the last step of linear elution.
  • the protein can be stored after dialysis against 125 mM sodium chloride, 25 mM HEPES buffer (pH 7.4).
  • Alpha EC domain protein is obtained as follows:
  • Alpha EC domain protein refolding and purification Use the human liver cDNA library as a template for PCR amplification. Primers as follows:
  • Restriction sites of NdeI and XhoI are introduced in the primers.
  • the annealing temperature of PCR amplification is 55° C.
  • the sample loading buffer as follows: 8M urea, 20 mM Tris-HCl and pH 8.0, 30 mM BME; add 1M NaCl to loading buffer is elution buffer. Use a linear gradient elution, and collect elution peak step by step. Detect the protein purity by electrophoresis. Select the components of which purity greater than 80% to do the refolding experiments.
  • Citrate synthase is a key enzyme in the tricarboxylic acid cycle, but its thermal stability is poor. The temperature of 43° C. will make it denature, aggregate and precipitate. The process of citrate synthase aggregation can be examined by the changing of light scattering. The method is as follows:
  • the light scattering signal detecting result shown in the FIG. 1 indicates that during the process of heating for 200 s the citrate synthase in the control group 1 and 2 begin to aggregate and the intensity of light scattering will be increased. However, citrate synthase aggregation in the experiment group 1 and 2 will be reduced obviously. The effect of inhibition in the experiment group 2 is better than that of group 1. Moreover, 0.15 ⁇ M alpha EC domain protein can almost totally inhibit equal molarity of citrate synthase during the thermal denaturation and aggregation process.
  • ( ⁇ ) represents control group 1
  • (•) represents control group 2
  • ( ⁇ ) represents experiment 1
  • ( ⁇ ) represents adding 0.6 ⁇ M alpha EC.
  • the experiment group 1 is added 0.075 ⁇ M fibrinogen-420
  • the experiment group 2 is added 0.15 ⁇ M fibrinogen-420
  • the experiment group 3 is added 0.15 ⁇ M
  • the experiment group 4 is added 0.15 ⁇ M alpha EC domain protein
  • the control group is added an equal volume of HEPES buffer solution.
  • the method of detecting the activity of citrate synthase is as follows:
  • the determination result of the activity of citrate synthase shown in the FIGS. 6 and 7 indicates that with the time going on, the activity of citrate synthase in the control group decreases rapidly but all experiment groups can slow down the activity loss speed effectively.
  • ( ⁇ ) represents control group
  • ( ⁇ ) represents experiment group 1
  • ( ⁇ ) represents experiment group 2.
  • ( ⁇ ) represents control group
  • ( ⁇ ) represents experiment group 3
  • ( ⁇ ) represents experiment group 4.
  • Citrate synthase and alpha EC domain protein are incubated together at 43° C. After being heated for 5 min or 10 min, antibodies of citrate synthase and alpha EC domain protein are added into the supernatant to perform co-immunoprecipitation.
  • citrate synthase and alpha EC domain protein are incubated together at room temperature and antibodies of citrate synthase and alpha EC domain protein are added into the supernatant to perform co-immunoprecipitation.
  • Results shown in the FIG. 5 indicate that after adding the antibody of citrate synthase, the denatured citrate synthase can be precipitated and alpha EC domain protein can also be precipitated at the same time. After adding the antibody of alpha EC domain protein, both of alpha EC domain protein and citrate synthase can be precipitated.
  • the results above indicate that after being heated, citrate synthase and alpha EC domain protein can form complex so that the antibody of one protein can precipitate the other protein at the same time. In the control group, co-immunoprecipitation does not happen. The result illustrates that alpha EC domain protein can recognize and bind to the thermally denatured citrate synthase specifically.

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  • Proteomics, Peptides & Aminoacids (AREA)
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US13/254,987 2009-03-06 2010-03-05 novel application of fibrinogen-420 and its active domain Abandoned US20120165510A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CN200910079569.0 2009-03-06
CN2009100795690A CN101822823B (zh) 2009-03-06 2009-03-06 纤维蛋白原-420及其活性结构域的新用途
PCT/CN2010/000277 WO2010099703A1 (zh) 2009-03-06 2010-03-05 纤维蛋白原-420及其活性结构域的新用途

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EP (1) EP2404613B1 (pt)
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000009562A1 (en) * 1998-08-12 2000-02-24 The New York Blood Center, Inc. Novel cleaved fragments of fibrinogen
CN1712413A (zh) * 2004-06-14 2005-12-28 清华大学 组织纤维蛋白原的新用途

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000009562A1 (en) * 1998-08-12 2000-02-24 The New York Blood Center, Inc. Novel cleaved fragments of fibrinogen
CN1712413A (zh) * 2004-06-14 2005-12-28 清华大学 组织纤维蛋白原的新用途

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Lei, T. H. CN 1712413A (12/2005) Human assisted machine translation to English. *
Tang et al. Biochem. Biophys. Res. Comm. (2009/01/16 hard copy publication, and 2008/12/4 on line publication) 378 662-667. *
Tang et al., Biochemistry (2009/03/15 on line publication) 48, 3967-3976. *

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US9145452B2 (en) 2015-09-29
EP2404613B1 (en) 2020-07-01
JP5951257B2 (ja) 2016-07-13
WO2010099703A1 (zh) 2010-09-10
EP2404613A4 (en) 2013-07-31
CN101822823B (zh) 2013-02-13
CN101822823A (zh) 2010-09-08
JP2012519660A (ja) 2012-08-30
US20140087441A1 (en) 2014-03-27
EP2404613A1 (en) 2012-01-11

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