CN108570093B - Polypeptide capable of chelating copper ions and application thereof - Google Patents

Polypeptide capable of chelating copper ions and application thereof Download PDF

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CN108570093B
CN108570093B CN201810432470.3A CN201810432470A CN108570093B CN 108570093 B CN108570093 B CN 108570093B CN 201810432470 A CN201810432470 A CN 201810432470A CN 108570093 B CN108570093 B CN 108570093B
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polypeptide
copper ions
histidine
copper
chelating
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黄方
杜明娇
王晓娟
葛保胜
迟海霞
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Qingdao Langoo Oceantec Co ltd
China University of Petroleum East China
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Abstract

The invention discloses a copper ion chelating polypeptide which comprises m amino acids, wherein n histidine (H), m-n glutamic acid (E) or glycine (G) are contained in the amino acids, m is more than or equal to 3 and less than or equal to 6, and n is more than or equal to 1 and less than or equal to (m-1). The polypeptide is formed by mutating histidine in the polypeptide to glutamic acid or glycine on the basis of 3-6 histidine, and the number of the sites for mutating histidine to glutamic acid or glycine can be one or more. The polypeptide is used for treating diseases caused by excessive copper ions, particularly alleviating and/or treating Alzheimer's disease. The polypeptide has small toxic and side effect, good biocompatibility and strong binding force with copper ions, provides more choices for chelating the copper ions and provides a potential treatment strategy for diseases caused by the copper ions.

Description

Polypeptide capable of chelating copper ions and application thereof
Technical Field
The invention belongs to the technical fields of gene engineering, protein engineering, biochemistry and molecular biology, and particularly relates to a copper ion chelating polypeptide and application thereof.
Background
Metallic copper plays an important role in human life activities, and most of copper in the human body forms a complex with copper protein to play its physiological role. The copper protein mainly comprises ceruloplasmin (ceruloplasma) and albumin (albumin). The ceruloplasmin has the activity of ferrous oxidase, has the functions of removing toxicity caused by excessive copper ions and scavenging free radicals in vivo, and can carry 90% of copper ions in blood to participate in redox reaction to reduce bivalent copper into monovalent copper and oxidize bivalent iron into trivalent iron. If excessive free copper exists in human body, the copper can cause various oxidation-reduction reactions in the in vivo environment, generate toxic hydroxyl radical ions, generate toxicity to cells, and even cause diseases such as liver cirrhosis, motor and sensory nerve disorder and the like. In recent years, a great deal of research has found that excessive copper ions interfere with the normal folding of proteins, causing the wrong aggregation of proteins, and accompanied by the generation of free radical ions with greater cytotoxicity. These pathological processes may trigger various types of amyloidosis, such as, for example, Alzheimer's disease, type II diabetes, Parkinson's disease, Cutzeri-Jacob disease, and other systemic amyloid diseases. Therefore, the development of copper ion chelating agents for safely and effectively reducing excessive copper ions in human bodies is a potential therapeutic means for the diseases.
Currently, the design, synthesis and screening of drugs for metal chelators are receiving much attention. There are many kinds of small molecule inhibitors for chelating copper ions, such as 5-Chloro-7-iodo-8-hydroxyquinoline (5-Chloro-7-iodo-8-hydroxyquinoline, abbreviated as CQ), Ethylenediaminetetraacetic acid (EDTA), and 8-hydroxyquinoline derivatives. The 8-hydroxyquinoline derivative PBT-1 is the first chelating agent for Alzheimer's Disease resistance to enter clinical research and is one of few drugs with significant delay in the progression of Alzheimer's Disease (Journal of Alzheimer's Disease,2009,17(2): 423). However, PBT-1 has problems such as high toxicity and difficulty in synthesis, and thus PranaBiotech company has finally abandoned development of PBT-1. CQ showed a clear effect against Alzheimer's disease in transgenic mice Tg 2576. However, Clinical trials of CQ have been forced to be discontinued because drugs bring about iodine contamination when they are administered (European Journal of Clinical Investigation,2015,32(1): 51). The search for suitable effective copper ion chelating agents is of great practical significance. The polypeptide chelating agent is a good choice due to the advantages of small toxic and side effect, easy synthesis, good biocompatibility, strong binding force with copper ions and the like. Caballero et al explored His-Xaa-His type polypeptides (histidine-alanine-histidine, histidine-lysine-histidine and histidine-tryptophan-histidine) consisting of 3 amino acids to design potential drugs capable of chelating copper ions (Chemistry,2016,22(21): 7268). Chinese patent CN2015105795279 discloses a polypeptide capable of inhibiting the aggregation of Alzheimer's disease Abeta protein and application thereof. Our earlier studies also found that the binding ability of histidine (H) in the histidine polypeptide sequence to copper ion is greatly improved after mutation to aspartic acid (D) (a protein purification tag with stronger binding ability to metal ion and its application, chinese patent application No. CN 201610601945.8). Further research shows that the histidine in the histidine polypeptide is replaced by glutamic acid (E) or glycine (G), and the polypeptide synthesized according to the new sequence has good copper ion chelating function, and the polypeptide provides a potential treatment strategy for treating or relieving various diseases caused by excessive copper ions.
Disclosure of Invention
The invention aims to provide a polypeptide capable of chelating copper ions, which solves the problem of complexing of copper ions in vivo, and avoids the phenomenon that excessive copper ions interfere normal folding of protein to cause wrong aggregation of protein, thereby causing amyloidosis.
The invention relates to a polypeptide capable of chelating copper ions, which comprises m amino acids, wherein n histidine (H), m-n glutamic acid (E) or glycine (G) are contained in the amino acids, wherein m is more than or equal to 3 and less than or equal to 6, and n is more than or equal to 1 and less than or equal to (m-1). The polypeptide sequence is shown in SEQ ID NO 1-224.
Furthermore, the polypeptide is formed by mutating histidine in the polypeptide to glutamic acid or glycine on the basis of 3-6 histidine, wherein the number of sites for mutating histidine to glutamic acid or glycine can be one or more, and the mutation sites are uncertain.
Further, the polypeptide is capable of chelating copper ions well.
Further, the use of said polypeptide for the treatment of disorders caused by an excess of copper ions, in particular in the manufacture of a medicament for the alleviation and/or treatment of alzheimer's disease.
1-224 are specifically shown in the following table:
Figure BDA0001653783230000021
Figure BDA0001653783230000031
Figure BDA0001653783230000041
Figure BDA0001653783230000051
the beneficial results of the invention are:
(1) the polypeptide capable of chelating copper ions disclosed by the invention has the characteristics of small toxic and side effects, good biocompatibility, strong binding force with copper ions and the like.
(2) By mutating histidine in the histidine polypeptide into glutamic acid or glycine, the binding capacity of the polypeptide and copper ions is improved, more choices are provided for chelating the copper ions, and a potential treatment strategy is provided for diseases caused by the copper ions.
Drawings
FIG. 1 is a schematic structural diagram of a part of the polypeptide related to the present invention.
FIG. 2 shows the polypeptide EHHH (A) and the comparison polypeptides HHHHHH (B) and Cu according to example 1 of the present invention2+Combined isotherm titration calorimetry curve characterization plots.
FIG. 3 shows the polypeptide HHEHHHH (A) of example 2 of the present invention and comparative polypeptides HHHHHHHHHHH (B) and Cu2+Combined isotherm titration calorimetry curve characterization plots.
FIG. 4 shows the polypeptides GHHH and Cu according to example 3 of the present invention2+Combined isotherm titration calorimetry curve characterization plots.
FIG. 5 shows the polypeptides HHGHHH and Cu according to example 4 of the present invention2+Combined isotherm titration calorimetry curve characterization plots.
FIG. 6 shows the polypeptide fragments A beta 1-16 and Cu capable of chelating copper ions in beta-amyloid related to Alzheimer's disease according to comparative example 1 of the present invention2+Combined isotherm titration calorimetry curve characterization plots.
Detailed Description
The invention is further described below by means of specific embodiments.
The polypeptides referred to in the following examples were synthesized directly by the national institute of intense biotechnology, Suzhou.
Example 1:
the EHHH polypeptide sequence used in this example was obtained by mutating the first site of the polypeptide hhhhhh (histidine-histidine) containing four histidines to glutamic acid.
The binding constant of the polypeptide and copper ion was determined by directly titrating both the EHHH and hhhhhh polypeptides with copper ion using Isothermal Titration Calorimetry (ITC). The ITC is an experimental method which can continuously and accurately monitor and record a calorimetric curve of a change process through a high-sensitivity and high-automation micro calorimeter and simultaneously provide thermodynamic and kinetic information (including binding constants) in situ, on line and without damage.
In the experimental process, the concentration of the polypeptide used for titration is 0.2mM, and Cu is2+The concentration was 2 mM. The titration experiment used 50mM HEPES buffer (pH 7.5) with 100mM NaCl. To prevent the above Cu2+Precipitation occurred under this buffer system, we used 10mM glycine for protection, and the experimentally determined polypeptide/copper ion binding constant was the apparent binding constant. The ITC titration results are shown in table 1: the binding capacity of EHHH to copper ions is an order of magnitude stronger than that of HHHHHH of histidine polypeptide. It was shown that it is feasible to mutate histidine to glutamate in order to increase its binding capacity to copper ions.
Example 2:
the polypeptide sequence HHEHHH used in this example was obtained by mutating the third site of polypeptide HHHHHHHH (histidine-histidine) containing six histidines to glutamic acid.
The binding constants of both HHEHHH and HHHHHHHHHHHHHHHH polypeptides to copper ions were determined using the method used in example 1 and the results are shown in Table 1: the binding capacity of HHEHHH to copper ions is two orders of magnitude higher than that of the HHHHHHHH binding capacity of histidine polypeptides. It was shown that it is feasible to mutate histidine to glutamate in order to increase its binding capacity to copper ions.
Example 3:
the polypeptide sequence GHHH used in this example is obtained by mutating the first site of a polypeptide containing four histidines (histidine-histidine).
The binding constant of GHHH polypeptide to copper ions was determined using the method used in example 1 and the results are shown in table 1: the binding capacity of GHHH to copper ions is an order of magnitude higher than that of HHHHHH of histidine polypeptide. Indicating that it is feasible to mutate histidine to glycine to increase its binding ability to copper ions.
Example 4:
the polypeptide sequence HHGHHH used in this example was obtained by mutating the first site of polypeptide HHHHHHHHHH (histidine-histidine) containing six histidines to glycine,
the binding constants of HHGHHH polypeptides to copper ions were determined using the method used in example 1 and the results are shown in table 1: the binding capacity of HHGHHH to copper ions is two orders of magnitude higher than that of HHHHHHHH of histidine polypeptide. Indicating that it is feasible to mutate histidine to glycine to increase its binding ability to copper ions.
Comparative example 1:
the determination of the binding capacity of polypeptide fragment A beta 1-16 capable of chelating copper ions in beta-amyloid related to Alzheimer's disease to the copper ions. Abnormal aggregation of β 0-Amyloid (Amyloid- β 1, abbreviated as a β 2, and mainly present in vivo in the form of a β 40 and a β 42) in brain tissue is considered to be a major cause of the development of alzheimer's disease. A.beta.1-16 is the main site for binding of A.beta.to copper ions, and its binding constant is similar to A.beta.40 (or A.beta.42) (Journal of Physical Chemistry B,2008,112(27): 8160). Thus, the binding capacity of A.beta.1-16 to copper ions was determined and compared with the polypeptides according to the invention. The measurement method and experimental conditions were the same as in example 1. The concentration of the adopted A beta 1-16 is 1mM, Cu2+The concentration was 20 mM. The ITC titration results are shown in table 1.
As can be seen from Table 1, the binding constant of Abeta 1-16 to copper ion is 3.08 (+ -0.81). times.103M-1. The binding constants of the polypeptides HHEHHH, EHHHHH, HHGHHH and GHHH related to the invention and copper ions are respectively 7.19 (+ -0.91) × 106M-1、2.80(±0.72)×107M-1、6.38(±0.30)×106M-1And 1.98 (. + -. 0.37). times.107M-1And is 3-4 orders of magnitude stronger than A beta 1-16. The new polypeptide designed and synthesized according to the invention has stronger copper ion binding capacity, can reduce the copper ion concentration in a system, reduces the possibility of Abeta 1-16 aggregation, and provides a potential treatment strategy for treating or relieving various diseases caused by excessive copper ions.
TABLE 1 ITC titration results
Figure BDA0001653783230000071

Claims (1)

1. Use of a copper ion-chelating polypeptide in the preparation of a medicament for the alleviation and/or treatment of alzheimer's disease, wherein the copper ion-chelating polypeptide has the sequence: EHHH, glutamic-histidine.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003014292A2 (en) * 2001-08-06 2003-02-20 Technion Research And Development Foundation Ltd. Combinatorial library of bifunctional molecules
CN105175494A (en) * 2015-09-12 2015-12-23 复旦大学 Polypeptide capable of inhibiting AD (Alzheimer's disease) protein Abeta (beta-amyloid) accumulation activity and application thereof
CN106188234A (en) * 2016-07-27 2016-12-07 中国石油大学(华东) A kind of protein purification label and application thereof with metal ion with higher adhesion
CN106255701A (en) * 2014-03-06 2016-12-21 帝国改革有限公司 Can peptide hormone analog that in the past Proglucagon derives

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003014292A2 (en) * 2001-08-06 2003-02-20 Technion Research And Development Foundation Ltd. Combinatorial library of bifunctional molecules
CN106255701A (en) * 2014-03-06 2016-12-21 帝国改革有限公司 Can peptide hormone analog that in the past Proglucagon derives
CN105175494A (en) * 2015-09-12 2015-12-23 复旦大学 Polypeptide capable of inhibiting AD (Alzheimer's disease) protein Abeta (beta-amyloid) accumulation activity and application thereof
CN106188234A (en) * 2016-07-27 2016-12-07 中国石油大学(华东) A kind of protein purification label and application thereof with metal ion with higher adhesion

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