A fusion polypeptide for treating cataracts and its application

Abstract

The present invention relates to a fusion polypeptide for treating cataract and application thereof. The fusion protein of the present invention comprises a cell-penetrating peptide, a crystallin core domain and the C-terminus of GRP78 (BiP) from the N-terminus to the C-terminus, namely, a CPP-αA-SBD fusion protein as shown in SEQ ID NO:5 and a CPP-αB-SBD fusion protein as shown in SEQ ID NO:6. The fusion protein can protect lens epithelial cells in a high-sugar environment, which is manifested in improving cell survival rate, reducing cell apoptosis and reducing oxidative stress level. At the same time, it has a significant effect on slowing down lens opacity and relieving the increase of lens thickness in cataract model mice.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a fusion polypeptide for treating cataracts and application thereof. Background Art

[0002] Crystallin (especially α-crystallin) plays an important role in the transparency and stability of the eye lens. α-crystallin belongs to the small heat shock protein family and has a molecular chaperone function. It maintains the transparency and normal function of the lens mainly by preventing the misfolding and aggregation of other proteins. However, under the influence of multiple factors such as diabetes, advanced age, and ultraviolet rays, crystallin may lose its chaperone function, resulting in misfolding and protein aggregation. These aggregates gradually form opaque areas in the lens, eventually leading to the formation of cataracts. Therefore, the maintenance of crystallin function is crucial for the prevention and treatment of cataracts.

[0003] GRP78 (also known as BiP) is a glucose-regulated protein in the endoplasmic reticulum and belongs to the heat shock protein 70 family. As an important molecular chaperone, GRP78 plays a key role in the regulation of protein folding, repair of misfolded proteins, and endoplasmic reticulum stress response. Studies have shown that GRP78 plays a role in stabilizing the protein folding environment in the endoplasmic reticulum of lens cells, especially in response to oxidative stress and high sugar environments, GRP78 can alleviate endoplasmic reticulum stress and prevent the accumulation of misfolded proteins. However, in diabetic cataracts, persistent high sugar stress may lead to dysregulation of GRP78 function, causing the protein folding environment in lens cells to lose control, further aggravating the misfolding and aggregation of lens proteins, and thus promoting the development of cataracts. Therefore, GRP78 is considered to be a potential target for regulating the occurrence of cataracts.

[0004] Based on the key roles of crystallin and GRP78 in cataracts, designing a fusion protein with targeting and repair capabilities can provide a new strategy for the treatment of cataracts. By constructing a fusion protein with a transmembrane peptide, a crystallin core domain, and the C-terminus of GRP78 from the N-terminus to the C-terminus, effective intervention in cataract formation may be achieved. Transmembrane peptides can help fusion proteins accurately locate in specific areas within cells, such as the endoplasmic reticulum or cell membrane, and the crystallin core domain can stabilize the correct folding of other proteins and prevent aggregation through the function of molecular chaperones. The C-terminus of GRP78 can further enhance the ability of cells to cope with stress and help repair proteins that have been misfolded. This fusion protein design can not only prevent protein aggregation, the main pathological mechanism of cataract formation, but also improve endoplasmic reticulum function and slow down cell damage caused by high sugar and oxidative stress, thereby effectively preventing or treating cataracts. Summary of the invention

[0005] In order to solve the above problems, the present invention first provides a fusion polypeptide for treating cataracts and its application.

[0006] In one aspect, the present invention provides a fusion polypeptide for treating cataracts. The fusion polypeptide comprises, from N-terminus to C-terminus, a cell-penetrating peptide, a crystallin core domain and a C-terminal substrate binding domain of GRP78 (BiP).

[0007] In certain embodiments, the amino acid sequence of the cell-penetrating peptide is shown as SEQ ID NO:4, the amino acid sequence of the crystallin core domain is shown as SEQ ID NO:1 or 2, and the amino acid sequence of the C-terminal substrate binding domain of GRP78 (BiP) is shown as SEQ ID NO:3.

[0008] In certain embodiments, the amino acid sequence of the fusion polypeptide is as shown in SEQ ID NO:5 or 6.

[0009] Another aspect of the present invention provides the use of the above fusion polypeptide in the preparation of a drug for treating cataracts.

[0010] Compared with the prior art, the present invention has at least the following beneficial effects:

[0011] The present invention constructs for the first time a fusion polypeptide for treating cataracts, wherein the fusion polypeptide comprises, from N-terminus to C-terminus, a transmembrane peptide, a crystallin core domain and a C-terminal substrate binding domain of GRP78 (BiP), and the amino acid sequence of the fusion polypeptide is as shown in SEQ ID NO: 5 or 6.

[0012] The fusion polypeptide prepared by the present invention as shown in SEQ ID NO: 5 or 6 has a protective effect on lens epithelial cells under a high sugar environment, which is manifested in improving cell survival rate, reducing cell apoptosis and reducing oxidative stress levels; at the same time, it has a significant effect on slowing down lens opacity and alleviating the increase of lens thickness in cataract model mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 SWISS-MODEL predicted the tertiary structure model of CPP-αA-SBD fusion protein and CPP-αB-SBD fusion protein;

[0014] Figure 2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot detection of CPP-αA-SBD fusion protein and CPP-αB-SBD fusion protein;

[0015] Figure 3Effects of CPP-αA-SBD fusion protein and CPP-αB-SBD fusion protein on HLEC cell survival rate;

[0016] Figure 4 Detection of ROS levels in HLEC cells by CPP-αA-SBD fusion protein and CPP-αB-SBD fusion protein. DETAILED DESCRIPTION

[0017] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0018] Example 1 Preparation of CPP-αA-SBD and CPP-αB-SBD fusion protein expression plasmids

[0019] (1) Gene construction and fusion protein design:

[0020] According to the NCBI search, the alpha crystallin A chain (GenBank: AMM63586.1) was found as follows: GISEVRSDRDKFVIFLDVKHFSPEDLTVKVQDDFVEIHGKHNERQDDHGYISREFHRRYRLPSNVDQSALSCSLSADGMLTFCGPK (SEQ ID NO: 1).

[0021] According to NCBI, the αB-crystallin core domain (Alpha-crystallin B chain, PDB: 2N0K_B) was retrieved: GLSEMRLEKDRFSVNLDVKHFSPEELKVKVLGDVIEVHGKHEERQDEHGFISREFHRKYRIPADVDPLTITSSLSSDGVLTVDGPRKQV (SEQ ID NO: 2).

[0022] According to NCBI, the C-terminal substrate binding domain (SBD, Substrate Binding Domain) of GRP78 (BiP) (endoplasmic reticulum chaperone BiP precursor [Homo sapiens], NCBI Reference Sequence: NP_005338.1) was retrieved: CPLTLGIETVGGVMTKLIPRNTVVPTKKSQIFSTASDNQPTVTIKVYEGERPLTKDNHLLGTFDLTGIPPAPRGVPQIEVT (SEQ ID NO: 3).

[0023] Cell-Penetrating Peptide (CPP): HIV TAT cell-penetrating peptide, a transmembrane sequence commonly used to deliver proteins or nucleic acids: YGRKKRRQRRR (SEQ ID NO: 4).

[0024] The CPP-αA-SBD / CPP-αB-SBD fusion protein consists of: a membrane-penetrating peptide, the core domain of αA / B-crystallin, and the C-terminal substrate binding domain of GRP78 from N-terminus to C-terminus; a flexible connecting peptide (Gly4Ser)3 (GGGGSGGGGSGGGGS) is introduced between the two functional domains to ensure the independence of the two functional domains and prevent mutual interference between the domains.

[0025] CPP-αA-SBD fusion protein: YGRKKRRQRRRGISEVRSDRDKFVIFLDVKHFSPEDLTVKVQDDFVEIHGKHNERQDDHGYISREFHRRYRLPSNVDQSALSCSLSADGML TFCGPKGGGGSGGGGSGGGGSCPLTLGIETVGGVMTKLIPRNTVVPTKKSQIFSTASDNQPTVTIKVYEGERPLTKDNHLLGTFDLTGIPPAPRGVPQIEVT (SEQ IDNO:5).

[0026] CPP-αB-SBD fusion protein: YGRKKRRQRRRGLSEMRLEKDRFSVNLDVKHFSPEELKVKVLGDVIEVHGKHEERQDEHGFISREFHRKYRIPADVDPLTITSSLSSDGVLTV DGPRKQVGGGGGSGGGGSGGGGSCPLTLGIETVGGVMTKLIPRNTVVPTKKSQIFSTASDNQPTVTIKVYEGERPLTKDNHLLGTFDLTGIPPAPRGVPQIEVT(SEQID NO:6).

[0027] (2) Construction of fusion protein gene:

[0028] Primer design: Appropriate primers were designed based on the above sequences with restriction endonucleases NotI and XhoI for PCR amplification of the transmembrane peptide, αA-crystallin core domain, αB-crystallin core domain, and the C-terminal substrate binding domain of GRP78.

[0029] (3) Connection and construction of fusion gene: After PCR amplification, the amplified transmembrane peptide, αA, αB and GRP78-SBD fragments and the expression vector pET-28a(+) were double-digested with restriction endonucleases NotI and XhoI, and then connected to form fusion genes CPP-αA-SBD and CPP-αB-SBD using T4 DNA ligase. After connection, the plasmid was transformed into Escherichia coli for amplification.

[0030] (4) Plasmid extraction and identification: The transformed colonies were screened by PCR and plasmids were extracted. Sanger sequencing was used to verify the correctness and sequence integrity of the fusion gene. The complete expression plasmid was provided by Sangon Biotech (Shanghai) Co., Ltd.

[0031] (5) Induction, expression and purification of fusion protein: The correct pET-28a(+)-CPP-αA-SBD or pET-28a(+)-CPP-αB-SBD plasmid was transformed into competent E. coli BL21(DE3) cells, coated with LB plates containing kanamycin, and cultured at 37°C for 16 hours. A single colony was picked and inoculated into 10 mL of LB liquid medium containing kanamycin, and cultured at 37°C with shaking at 200 rpm overnight. The next day, the overnight culture was inoculated into fresh LB medium containing kanamycin at a ratio of 1:100 and cultured at 37°C until OD600 reached 0.6-0.8. When the cells grew to OD600 0.6-0.8, IPTG was added at a final concentration of 0.5-1 mM for induction. During induction, the temperature was controlled at 16-20°C, and low-temperature induction was performed for 16 hours to promote the expression of soluble protein. After induction, cells were collected (4000 rpm, 10 minutes, 4°C), washed once with PBS, the supernatant was discarded, and the cell pellet was collected for protein extraction. The collected cell pellet was resuspended in lysis buffer (containing 50mM Tris-HCl, 300mM NaCl, 10mM imidazole, pH8.0), 1mM PMSF was added to inhibit protease, and the cells were disrupted by ultrasonication in an ice bath (30 seconds ultrasonication, 30 seconds off, a total cycle of 10 minutes). The supernatant was passed through a Ni²⁺ affinity chromatography column (purified using the 6×His tag in the pET-28a(+) vector), and the column equilibration buffer was a lysis buffer containing 10mM imidazole. After loading, non-specific binding proteins were gradually washed away with buffers containing 20mM and 50mM imidazole. Finally, the target protein was eluted with an elution buffer containing 250mM imidazole. After ultrasonication, centrifugation was performed at 12000g, 4°C for 20 minutes, and the supernatant was collected.

[0032] (6) Identification and analysis of fusion proteins: SWISS-MODEL predicted the tertiary structure models of CPP-αA-SBD fusion protein and CPP-αB-SBD fusion protein, such as Figure 1As shown; the eluted protein was detected by SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue to confirm the molecular weight of the target fusion protein; the purity of the target protein was detected by Western blot using His tag antibody and anti-GRP78 antibody, as shown Figure 2 shown.

[0033] Figure 1 The results showed that the functional domains of CPP-αA-SBD and CPP-αB-SBD fusion proteins were far away from each other and did not interfere with each other, so they could be used in subsequent functional verification experiments.

[0034] Figure 2 The results showed that the molecular weight of CPP-αA-SBD fusion protein was 21.10 kDa; the molecular weight of CPP-αB-SBD fusion protein was 21.35 kDa, the electrophoresis bands were clear, the purity was high and the specificity was strong.

[0035] Example 2 Experimental study on the effects of CPP-αA-SBD and CPP-αB-SBD fusion proteins on human lens epithelial cells

[0036] Human lens epithelial HLEC cell line SRA01 / 04 (Sciencell, USA) was cultured in DMEM medium containing 10% heat-inactivated NCS, 100 U / mL penicillin and 100 μg / mL streptomycin, pH = 7.4, at 37°C in an incubator containing 5% CO2, according to the following experimental group settings:

[0037] Blank control group: no galactose and fusion protein were added.

[0038] Galactose group: only galactose was added for treatment.

[0039] CPP-αA-SBD group: galactose and CPP-αA-SBD fusion protein were added.

[0040] CPP-αB-SBD group: galactose and CPP-αB-SBD fusion protein were added.

[0041] Galactose treatment: fresh culture medium was replaced and galactose was added at a final concentration of 50 mM to simulate the high-sugar environment of diabetic cataracts. Cells were collected after 48 hours of culture.

[0042] Fusion protein treatment: CPP-αA-SBD and CPP-αB-SBD fusion proteins were added to different experimental groups, with a final protein concentration of 30 μg / mL, and culture was continued for 24 hours.

[0043] Cell viability test: 4 h before measuring cell viability, add MTT solution with a final concentration of 0.5 mg / mL to each well, continue to culture for 4 h, then carefully aspirate the liquid in the culture wells, add 1 mL of DMSO to each well, fully dissolve for 10 min, and measure the absorbance (A) value of each well at a wavelength of 570 nm. Figure 3 shown.

[0044] Cell survival rate (%) = (A 实验组 -A 空白组 ) / (A 对照组 -A 空白组 )×100%

[0045] Figure 3 The results showed that compared with the blank control group, the cell survival rate in the galactose group was significantly decreased (P<0.01), indicating that galactose successfully induced a cataract cell model; compared with the galactose group, the cell survival rate was significantly increased after adding CPP-αA-SBD fusion protein or CPP-αB-SBD fusion protein (P<0.01).

[0046] Apoptosis detection: After the cells were cultured to 80%, the old culture medium was discarded and fresh serum-free culture medium was added for 24 hours. The cells were treated for 12 hours according to the experimental groups. After that, the cells were washed twice with cold PBS, and the cell pellets were collected. Then, they were fixed with 700 mL / L ethanol at -20°C for 4 hours, washed with PBS, and suspended in 0.9 mL buffer (containing 1% TritonX-100 and 100 μL 0.5 mg / mL RNaseA), incubated at 37°C for 30 minutes, and the cell density was adjusted to 1×10 6 / mL, react in 100μL 50μg / mL PI staining buffer at room temperature in the dark for 30min, then add 300μL PBS, and immediately detect the cell apoptosis rate by flow cytometry. The samples were tested within 1h, as shown in Table 1.

[0047] Table 1 Comparison of cell apoptosis rates in each group (x̅±s, %)

[0048] Note: Compared with the blank control group, a p<0.05, b p<0.01; compared with the galactose group, c p<0.05, d p<0.01.

[0049] The results in Table 1 showed that compared with the blank control group, the cell apoptosis rates in the galactose treatment group, CPP-αA-SBD group and CPP-αB-SBD group were significantly increased, and the difference was statistically significant (p<0.05); compared with the galactose group, the cell apoptosis rates in the CPP-αA-SBD group and CPP-αB-SBD group were significantly decreased, and the difference was statistically significant (p<0.01).

[0050] Detection of intracellular ROS levels: Take an appropriate amount of DCFH-DA probe, dissolve it with DMSO to a 10 mM stock solution, and then dilute it with serum-free culture medium to a 10 μM working solution. Be careful to avoid light to prevent the probe from spontaneously fluorescing. After 48 hours of treatment, remove the culture medium and gently wash the cells twice with PBS to remove the residual culture medium. Add the pre-prepared 10 μM DCFH-DA working solution to each well and shake gently to ensure that the probe is in full contact with the cells. Incubate at 37°C and 5% CO2 for 30-60 minutes, keeping it away from light. After incubation, remove the probe solution and wash the cells 3 times with PBS buffer to remove excess probes that have not entered the cells. Using a flow cytometer, trypsinize the cells, collect the cells and centrifuge them. Resuspend the cells with PBS, adjust to an appropriate concentration, use an excitation wavelength of 488 nm, and detect the DCF fluorescence signal at a wavelength of 530 nm. The fluorescence signal obtained by the flow cytometer can be analyzed by the dedicated software FlowJo to analyze the proportion of DCF-positive cells in each group, such as Figure 4 shown.

[0051] Figure 4 The results showed that compared with the blank control group, the ROS level in the galactose group was significantly increased (P<0.01), indicating that galactose successfully induced a cataract cell model; compared with the galactose group, the ROS level in the CPP-αA-SBD fusion protein group was significantly decreased (P<0.01), and the ROS level in the CPP-αB-SBD fusion protein group was significantly decreased (P<0.05); that is, CPP-αA-SBD and CPP-αB-SBD fusion proteins can reduce the ROS increase effect of high sugar in human lens epithelial cells and have a stronger antioxidant effect.

[0052] Example 3 Galactose-induced cataract mouse experimental model of CPP-αA-SBD and CPP-αB-SBD fusion proteins

[0053] Establishment of mouse model: Rats were grouped and C57BL / 6 mice were randomly divided into 4 groups, with 10 mice in each group, and treated according to the following experimental groups.

[0054] Blank control group: normal feeding, without any treatment.

[0055] Galactose group: only galactose was added for treatment.

[0056] CPP-αA-SBD group: At the same time as galactose treatment, CPP-αA-SBD fusion protein (5 mg / kg, twice a day) was injected intraperitoneally.

[0057] CPP-αB-SBD group: At the same time as galactose treatment, CPP-αB-SBD fusion protein (5 mg / kg, twice a day) was injected intraperitoneally.

[0058] Galactose treatment: Except for the blank control group, other groups were subcutaneously injected with 60% galactose solution at 200 mg / kg, twice a day, for 8 consecutive weeks to induce cataract formation.

[0059] Fusion protein treatment: Starting from the first day of the experiment, mice in the other groups, except the control group and the galactose group, were intraperitoneally injected with the corresponding fusion protein every day for 8 weeks.

[0060] Assessment of lens opacity (cataract grading): The lens of mice was examined weekly using a slit lamp microscope and scored based on the transparency of the lens. The scoring criteria were as follows:

[0061] Grade 0: The lens is completely transparent, without any opacity.

[0062] Grade 1: Mild posterior subcapsular opacity.

[0063] Grade 2: Moderate posterior subcapsular opacity affecting the visual axis.

[0064] Grade 3: Obvious lens opacity, with the visual axis completely opaque.

[0065] Grade 4: The lens is completely white and completely cloudy.

[0066] Table 2 Comparison of lens opacity in mice in each group after 8 weeks of treatment

[0067]

[0068] The results in Table 2 show that in the galactose group, the degree of lens opacity gradually worsened over time, and obvious cataract formation (grade 3-4) was observed in the 4th week. In the CPP-αA-SBD group and the CPP-αB-SBD group, due to the protective effect of the fusion protein, the progression of lens opacity was significantly slowed down, and the degree of opacity was maintained at grade 1-2.

[0069] Lens thickness measurement: The thickness of the lens of each group of mice was measured using an ophthalmic ultrasound device to evaluate the changes in lens morphology during cataract formation. Stepper motor OCT is a commonly used measurement method that can accurately measure the thickness of various tissue structures in the eyes of living mice, including lens thickness. Increased lens thickness is usually accompanied by lens fibrosis and degeneration.

[0070] Table 3 Comparison of lens thickness in each group of mice after 8 weeks of treatment

[0071]

[0072] The results in Table 3 show that the lens thickness of mice in the galactose group increased most significantly, while the treatment with CPP-αA-SBD and CPP-αB-SBD fusion proteins could significantly slow down the increase in thickness, especially CPP-αB-SBD, which showed a better protective effect.

[0073] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fusion polypeptide for treating cataracts, characterized in that: The fusion polypeptide comprises a transmembrane peptide, a crystallin core domain and a C-terminal substrate binding domain of GRP78 (BiP) from the N-terminus to the C-terminus, and the amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 5 or 6.

2. Use of the fusion polypeptide according to claim 1 in the preparation of a drug for treating cataracts.

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