Conotoxin peptide κ-CPTx-btl03, preparation method therefor, and uses thereof

Abstract

Provided are a conotoxin peptide -CPTx-bt103 and a derivative polypeptide. The amino acid sequence of the conotoxin peptide is indicated by SEQ ID NO: 1. Also provided are a conotoxin peptide preparation method and uses of the conotoxin peptide in the treatment of diseases related to a calcium ion channel.

Claims

1. A conotoxin peptide -CPTx-btl03, the conotoxin peptide -CPTx-btl03 being derived from a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 by substitution of one or more amino acids in the amino acid sequence of SEQ ID NO: 1 of the polypeptide and having the function of the polypeptide, wherein the substitution of one or more amino acids is selected from the group consisting of: (i) substitution of the arginine at position 1 with lysine; (ii) substitution of the asparagine at position 3 with glutamine; (iii) substitution of the threonine at position 7 with serine; (iv) substitution of the leucine at position 9 or 29 with isoleucine or valine; (v) substitution of the glutamine at position 13 or 19 with asparagine; (vi) substitution of the valine at position 15 or 22 with leucine or isoleucine; (vii) substitution of the isoleucine at position 20 with leucine or valine; and (viii) substitution of the lysine at position 27 with arginine, and wherein the amino acid sequence of the polypeptide contains three pairs of disulfide bonds.

2. A polynucleotide encoding the conotoxin peptide -CPTx-btl03 according to claim 1.

3. A nucleic acid construct comprising the polynucleotide of claim 2, and one or more control sequences operably linked thereto and being able to direct the production of the polypeptide in an expression host.

4. An expression vector comprising the nucleic acid construct of claim 3.

5. A transformed cell into which the nucleic acid construct of claim 3 is transformed.

6. A transformed cell into which the expression vector of claim 4 is transformed.

7. A method for inhibiting a high-voltage activated calcium ion channel comprising administration, to a subject, of an effective amount of the conotoxin peptide -CPTx-btl03 according to claim 1.

8. A method for producing the conotoxin peptide -CPTx-btl03 according to claim 1, which comprises: (1) synthesizing the linear peptide of the conotoxin peptide -CPTx-btl03 according to its amino acid sequence by solid-phase chemical synthesis; (2) performing oxidative refolding of the linear peptide obtained in step (1) with glutathione method.

9. The method according to claim 8, wherein the solid-phase chemical synthesis is fluorenylmethoxycarbonyl solid-phase chemical synthesis.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the mass spectrometric identification result of the sequence of the conotoxin peptide -CPTx-btl03 of the present invention;

(2) FIG. 2 shows the inhibitory effect of 10 M conotoxin peptide -CPTx-btl03 on high-voltage activated calcium channels;

(3) FIG. 3 shows the inhibitory effect of 10 M Nifedipine on high-voltage activated calcium channels.

DETAILED DESCRIPTION

(4) For the purpose of understanding the present invention, the present invention is exemplified as follows. It will be apparent to those skilled in the art that the examples are merely illustrative of the invention and should not be construed as a specific limitation of the present invention.

EXAMPLE 1 Extraction and Identification of the Conotoxin Peptide -CPTx-btl03 of the Present Invention

(5) 1. Extraction and Reductive Alkylation of Conus Venom

(6) Four Conus betulinus native to Hainan were dissected after smashing their shells, and then the venom ducts thereof were clipped and the Conus venoms were collected. Bradford method was performed to determine the protein concentration in the venom, which was 6.48 mg/ml. The total amount of protein was 0.5 mg. DTT at a final concentration of 1 mM was added and reacted at 56 C. for 1 h. After reduction and cooling to room temperature, IAM at a final concentration of 55 mM was added to react in dark room at room temperature for 45 min.

(7) 2. Enrichment of Toxin Polypeptides

(8) The conotoxin polypeptides obtained as above were then subjected to Strata-X C18 column to enrich the polypeptides in Conus venoms. The enrichment by Strata-X C18 was performed according to the following standard procedure: 1) adding 1 ml of methanol to activate the column; 2) adding 1 ml 0.1% FA to balance the column; 3) loading 1 ml venom sample, washing with buffer (5% ACN+0.1% FA), repeating the wash for 3 times; 4) eluting with 100% ACN, collecting the eluent. The molecular weights of the enriched polypeptides were detected by MALDI-TOF-MS.

(9) 3. Sequence Identification of the Conotoxin Polypeptides

(10) 240 g peptide mixture was fractionated by a SCX-HPLC (Shimadzu) system: buffer A: 10 mM KH.sub.2PO.sub.4 in 25% ACN, pH 3.5; buffer B: further containing 500 mM potassium chloride on the basis of buffer A; Flow rate: 1 ml/min; elution procedure: 0-40% linear binary gradient of buffer B for 10 minutes, 40-90% of buffer B for 2 minutes, 90% of buffer B for 3 minutes; absorbance detection was performed at 214 nm; as a result, a total of 10 fractions were collected through gradient elution. The collected fractions were demineralized by C18 solid phase extraction column (Strata-X, Phenomenex) and then reconstituted with 30 l of 0.1% formic acid for nanoLC-MS/MS analysis.

(11) 4. NanoLC-MS/MS Analysis

(12) LC/MS was from Shimadzu's nano HPLC chromatograph system and AB Sciex's Triple TOF 5600 mass spectrometer system. Each pre-separated polypeptide component was separated by a self-made Ultimate capillary analysis column which had a length of 12 cm, an inner diameter of 75 m and was filled with Welch Materials brand XB-C18 column material with a particle size of 3 m and a pore diameter of 120 m at a flow rate of 300 nl/min. The injection volume for detection was 25 l and the concentration of Buffer B was evenly increased from 5% to 45% for 40 min for a gradient elution. For mass spectrum acquisition, the electrospray voltage was 2.5 kV, the auxiliary air pressure was 30 PSI, the sheath gas pressure was 15 PSI, and the source temperature was 150 C. The first-order mass spectrum was acquired using a high-resolution mode greater than or equal to 30000. The valence state of parent ions in the range of 2 charges to 5 charges was selected for acquisition of the second-order mass spectrum. After one scanning of the first-order mass spectrum, 30 second-order mass spectrometric fragmentations can be conducted continuously, so that 30 scannings of the second-order spectrum daughter ions can be completed in 250 ms, and more than 120 sheets of the second-order spectrums can be generated per second. The total cycle time was 3.3 seconds.

(13) In the obtained mass spectrometry raw data, the sequence search and alignment result of the mass spectrum corresponding to the conotoxin peptide -CPTx-btl03 of the present invention was shown in FIG. 1.

(14) 5. Data Analysis

(15) The mass spectrometry raw data obtained by nanoLC-MS/MS detection was format converted into MGF, which then was subjected to data search and identification by Mascot search software. In the obtained polypeptide sequences, the -CPTx-btl03 polypeptide having a full length amino acid sequence of RTNCGETCLKDEQCVGACQICVPSQLKCL was selected by sequence feature analysis.

EXAMPLE 2 Chemical Synthesis of the Conotoxin Peptide -CPTx-btl03 of the Present Invention

(16) The conotoxin linear peptide as shown in SEQ ID NO: 1 was synthesized by fluorenylmethoxycarbonyl (Fmoc) solid phase chemical synthesis (customized by GL Biochem (Shanghai) Ltd.).

(17) The chemically synthesized polypeptide was refolded by using glutathione oxidative refolding, i.e.:

(18) The polypeptide was dissolved in a solution at pH 7.4 containing 0.1 M Tris-HCl, 0.1 M NaCl, 5 mM GSH and 0.5 mM GSSG at a mass to volume ratio of 1:10 and reacted at 25 C. for 24 to 48 h. The refolding effect was detected by MALDI-TOF-MS.

EXAMPLE 3 Inhibitory Activity of the Conotoxin Peptide -CPTx-btl03 of the Present Invention on Calcium Ion Channels

(19) The inhibitory activity of the conotoxin peptide -CPTx-btl03 of the present invention on calcium ion channels was detected by patch-clamp technique. Specifically, the conotoxin peptide -CPTx-btl03 as prepared and refolded in Example 2 was weighed and detected for its effect on rat dorsal root ganglion (DRG) cell ion channels by whole-cell patch-clamp method. Nifedipine was used as a positive control.

(20) Intracellular fluid and extracellular fluid in patch-clamp method were configured as follows:

(21) Extracellular fluid: 140 mM NaCl, 4 mM KCl, 1 mM MgCl.sub.2, 2 mM CaCl.sub.2, 5 mM D-Glucose monohydrate, 10 mM HEPES (pH=7.4); intracellular fluid: 20 mM KCl, 110 mM potassium aspartate (KAspartic), 1 mM MgCl.sub.2, 5 mM EGTA, 10 mM HEPES (pH=7.2).

(22) DRG cells (dorsal root ganglion cells immediately separated from SD rats and cultured) in the thermostatic incubator were taken out, and the culture medium in the culture dish was replaced with a well-balanced extracellular fluid at room temperature to prevent drastic changes in the temperature of the solution. The extracellular fluid was gently added with a pipette along the wall of the dish to prevent the cells shedding from the bottom of the dish. The cells in the extracellular fluid were placed under an inverted microscope for observation, and those cells with smooth cell membrane and homogeneous cytoplasm were selected, and subjected to the patch clamp test at a room temperature of 20-25 C.

(23) 100 l borosilicate glass blank was used as glass microelectrode material. A two-step drawing was performed by a drawing instrument to make the diameter of the electrode tip be about 1.5-3.0 m, and the initial resistance of the glass microelectrode upon immersion into a liquid be 2-4 M. The electrode was filled and installed followed by being moved below the liquid surface, and then a continuous positive pressure was immediately applied to ensure that the electrode tip was clean, that is, conducting liquid junction potential compensation. Under the inverted microscope, a microelectrode was moved over the selected cell and close to the cell, the positive pressure was removed and a slight negative pressure was applied for attraction. After forming a high impedance Giga-Ohm (G) seal between the electrode and the cell membrane, an electrode fast capacitance compensation was conducted immediately. The cell was then clamped at 60 mV, and a short and strong negative pressure was applied, thereby breaking the cell membrane clamped in the microelectrode rapidly and then performing cell low capacitance compensation. After forming the whole cell recording pattern, the cell was clamped at 90 mV for 4-6 min, and then the cell was subjected to two depolarization stimuli: the first one was at from 90 mV to 30 mV, for 250 ms, and the second one was at from 60 mV to 0 mV, for 250 ms, the interval between the two stimuli was 500 ms, and the LVA calcium channel and the HVA calcium channel were recorded at the same time. A polypeptide sample was added to the extracellular fluid to give an effective concentration of 10 M. The changes in the recorded calcium channel current were observed simultaneously (the experiment was repeated three times and the result was an average of three replicates). The series resistance (Rs) was always constant within the range of <10 M during the experiment, and the system series resistance compensation (Rseries compensation) was between 30 and 70%.

(24) The detection results of the conotoxin peptide -CPTx-btl03 and Nifedipine were shown in FIG. 2 and FIG. 3 respectively, and the inhibition rate of 10 M of -CPTx-btl01 on HVA calcium channel currents in DRG neuronal cells was 49.64% (as shown in Table 1). The results showed that -CPTx-btl03 had a comparable inhibition rate on HVA calcium channel currents as compared to Nifedipine at the same concentration, suggesting that it can be used as a candidate drug for antihypertensive drugs.

(25) TABLE-US-00002 TABLE 1 Patch clamp detection results of the inhibitory rate of the conotoxin peptide -CPTx-btl03 on calcium ion channel cell current before current after inhibition tested drugs number loading (pA) loading (pA) rate (%) -CPTx-btl03 140903014 447.25 225.23 49.64 Nifedipine 140904007 1471.35 734.31 50.09

(26) The applicant states that the present invention illustrates the product, the detailed preparation process and the use thereof by the above examples, however, the present invention is not limited to the above-described detailed preparation process and use, and it is not meant that the present invention has to be carried out with respect to the above-described detailed manufacturing process and use described above. It will be apparent to those skilled in the art that any improvements to the present invention, equivalents of the raw materials of the present invention, addition of auxiliary ingredients, selection of specific means, etc., all fall within the protection scope and disclosure scope of the present invention.