Scallop polypeptide with anti-protein aggregation function and application thereof

Abstract

The application belongs to the field of small molecule polypeptides, and particularly relates to a scallop polypeptide with anti-protein aggregation function and application thereof. The application takes scallop as raw material, and extracts and separates a pure natural scallop polypeptide through steps of enzymatic separation, ultrafiltration purification and LC-MS / MS identification. 74 H 110 N 22 O 23 S, the average relative molecular mass is about 1707.88 Da, the theoretical isoelectric point is pH=6.41, and it is a hydrophilic polypeptide. The scallop polypeptide has functions of antioxidation, anti-protein aggregation and nerve protection, and can be further used for development of products such as medicines, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of small molecule peptides, specifically relating to a scallop peptide with anti-protein aggregation function and its application. Background Technology

[0002] Protein aggregation refers to the process by which proteins assemble into abnormal polymers or filaments inside or outside cells. This phenomenon usually involves structural abnormalities in proteins that prevent them from folding correctly or maintaining a stable state, causing these proteins to tend to aggregate. The toxicity caused by protein aggregation can place organisms under stress. First, the spatial structure of the aggregated proteins changes, affecting their normal physiological functions and hindering life activities; second, the aggregation process produces protein aggregates. Common forms of protein aggregation include amyloid filaments (such as β-amyloid in Alzheimer's disease) and neurofibrillary tangles (such as tau protein in Alzheimer's disease).

[0003] Neurodegenerative diseases are a general term for diseases characterized by the chronic, progressive decline and even death of neuronal structure and function, leading to functional impairment. Extracellular fibrillary amyloid deposition due to protein aggregation or abnormal intracellular proteofibrillar inclusions are common features of these neurodegenerative diseases, including Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and prion diseases. For example, Aβ oligomers, a class of small molecule polymers associated with Alzheimer's disease (AD), are composed of β-amyloid protein. Aβ is a protein fragment produced by the cleavage of amyloid precursor protein (APP) in the brains of Alzheimer's patients. Aβ oligomers can consist of 2 to 12 Aβ monomers; they appear earlier than long-chain amyloid plaques and are considered one of the toxic factors in Alzheimer's disease. These oligomers can interfere with the normal function of nerve cells, triggering oxidative stress, inflammatory responses, and synaptic damage, ultimately leading to cognitive decline and neurodegeneration. This toxicity is widely believed to be caused by protein aggregation.

[0004] In general, the toxicity of protein aggregates is mainly manifested in the following aspects: (1) Cytotoxicity: Aggregates can directly damage cell membranes, leading to cell dysfunction or death. They may interfere with normal protein function or enzyme activity within cells, thereby affecting normal physiological processes of cells. (2) Inflammatory response: Protein aggregates can trigger immune system responses and activate inflammatory pathways, which may further damage tissues and aggravate disease symptoms. (3) Intracellular toxicity: Aggregates may accumulate within cells, interfering with the normal function of organelles such as the endoplasmic reticulum and mitochondria, leading to oxidative stress and apoptosis. These toxic effects are closely related to a variety of neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease) and some other diseases (such as Huntington's disease). Missense mutations, oxidative modifications, and amino acid pairing errors during translation that occur in the human body during aging can lead to changes in protein structure and a weakening of cell repair capabilities, resulting in protein aggregation. Aging is one of the causes of such diseases, and the intensification of the aging process will weaken the organism's self-repair capabilities, thus aggravating the symptoms of the disease. Researchers are working hard to find effective treatments to slow down or prevent protein aggregation and its toxicity.

[0005] Bioactive peptides are protein fragments composed of short-chain amino acids with a variety of biological functions. They can be extracted from food, microorganisms, or cells and exert antioxidant, anti-inflammatory, blood pressure-lowering, and immunomodulatory effects in vivo. Bioactive peptides play an important role in anti-protein aggregation. Protein aggregation is a key process in the development of many diseases (such as Alzheimer's disease and Parkinson's disease), and bioactive peptides can intervene in protein aggregation through the following mechanisms: (1) Inhibiting the aggregation of abnormal proteins: Some peptides can directly bind to abnormally folded proteins, inhibiting them from forming aggregates. (2) Promoting normal protein folding: Bioactive peptides can help maintain the correct folding of proteins, thereby reducing the occurrence of misfolding and aggregation. (3) Clearing aggregates: Some peptides can promote intracellular autophagy and protein degradation pathways, helping to clear abnormal aggregates accumulated in the body. (4) Protecting cells: Through antioxidant and anti-inflammatory effects, bioactive peptides can protect cells from damage caused by protein aggregation. These mechanisms make bioactive peptides an important direction for researching potential treatments for diseases caused by protein aggregation.

[0006] The application of biopeptides in anti-protein aggregation research faces several limitations, which affect the feasibility of their large-scale production. The main limitations include: (1) High production costs: The synthesis of biopeptides typically requires complex technologies and equipment, especially when high purity and specific modifications are required. These costs increase significantly during large-scale production. (2) Complex production processes: The production of biopeptides involves multiple steps, such as peptide synthesis, purification, folding, and modification. Each step may introduce instability or efficiency issues, making large-scale production difficult. (3) Stability issues: Many biopeptides are prone to degradation or loss of activity during storage and handling. Maintaining their stability requires specific conditions and processing methods, which increases the complexity and cost of production. (4) Purification difficulties: Purifying biopeptides from complex mixtures is a technical challenge, especially during large-scale production, where purification efficiency may decrease, leading to product quality and consistency issues. These factors combined make the production of biopeptides face many challenges in large-scale applications. Although biopeptides have potential in anti-protein aggregation, further research and optimization are needed to overcome these limitations. Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a scallop polypeptide with anti-protein aggregation function, which has antioxidant, anti-protein aggregation and neuroprotective functions.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned scallop polypeptide.

[0009] Another object of the present invention is to provide the application of the above-mentioned scallop polypeptide.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] A scallop polypeptide with anti-protein aggregation function, the amino acid sequence of which is: TMYWTDVSNGQIHR.

[0012] The molecular formula of the scallop polypeptide is C 74 H 110 N 22 O 23 S has an average relative molecular mass of approximately 1707.88 Da and a theoretical isoelectric point of pH=6.41, making it a hydrophilic polypeptide.

[0013] The method for preparing the scallop polypeptide with anti-protein aggregation function includes the following steps:

[0014] The above-mentioned scallop polypeptide with anti-protein aggregation function can be prepared directly through solid-phase synthesis, or by using scallops as raw materials and obtaining the above-mentioned scallop polypeptide with anti-protein aggregation function through enzymatic hydrolysis, separation and extraction, and purification.

[0015] The enzymatic hydrolysis is preferably performed by sequentially using trypsin and papain.

[0016] The preferred specific operation for the enzymatic hydrolysis is as follows:

[0017] The freeze-dried scallop powder was mixed with water, and the pH of the system was adjusted to 7.5-8.0. Trypsin was added for enzymatic hydrolysis. Then the pH of the system was adjusted to 6.5-7.0, and papain was added for enzymatic hydrolysis. After enzymatic hydrolysis, the enzyme was inactivated to obtain scallop protein hydrolysate.

[0018] The preferred dosage of trypsin is 10,000 to 20,000 U of trypsin per gram of freeze-dried scallop powder.

[0019] The preferred dosage of papain is 10,000 to 20,000 U of papain per gram of freeze-dried scallop powder.

[0020] The preferred specific operation for the separation and extraction is as follows:

[0021] After cooling the scallop protein hydrolysate obtained by enzymatic hydrolysis, 95% ethanol was added to bring the ethanol concentration of the solution to 60-80%, and the solution was allowed to stand. Then, the precipitate was removed by vacuum filtration and centrifugation. The supernatant was then evaporated under reduced pressure to remove ethanol, and freeze-dried to obtain crude scallop polypeptide powder.

[0022] The purification is preferably at least one of ultrafiltration purification and gel chromatography column purification.

[0023] The ultrafiltration purification is preferably performed using an Ultracel-3 membrane to collect components with a molecular weight of less than 3 kDa.

[0024] The preferred specific procedure for the gel chromatography column purification is as follows:

[0025] The ultrafiltration purified components were desalted and then further purified using a Sephadex G-25 dextran gel chromatography column.

[0026] The purification process preferably further includes using reversed-phase high-performance liquid chromatography or size exclusion chromatography to further separate the components into individual peptides.

[0027] The application of the scallop polypeptide with anti-protein aggregation function in the preparation of products with anti-protein aggregation or anti-oxidation function.

[0028] The application of the scallop polypeptide with anti-protein aggregation function in the preparation of products with neuroprotective activity.

[0029] The application of the scallop polypeptide with anti-protein aggregation function in the preparation of products for the prevention and treatment of neurodegenerative diseases.

[0030] The neurodegenerative diseases mentioned include Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), or prions.

[0031] An antioxidant product comprising at least one of the following as active ingredients: the scallop polypeptide with anti-protein aggregation function, the protease hydrolysate containing the scallop polypeptide with anti-protein aggregation function, and the hydrolysate containing the scallop polypeptide with anti-protein aggregation function.

[0032] A drug for preventing and treating neurodegenerative diseases, comprising at least one of the following as active ingredients: scallop polypeptide with anti-protein aggregation function, protease hydrolysate containing scallop polypeptide with anti-protein aggregation function, and hydrolysate containing scallop polypeptide with anti-protein aggregation function.

[0033] The principle of this invention:

[0034] This invention uses scallops as raw material and extracts, isolates, and identifies a pure natural scallop polypeptide through enzymatic hydrolysis, ultrafiltration purification, and LC-MS / MS identification. The amino acid sequence of this scallop polypeptide is TMYWTDVSNGQIHR, and its molecular formula is C 74 H 110 N 22 O 23 S, with an average relative molecular mass of approximately 1707.88 Da and a theoretical isoelectric point of pH 6.41, is a hydrophilic polypeptide. This invention further utilizes the model organism *C. elegans* to discover that this scallop polypeptide possesses anti-protein aggregation and protein homeostasis regulating functions, and also exhibits neuroprotective activity, thus playing a role in preventing neurodegenerative diseases, as detailed below:

[0035] (1) ASH neuron survival test: This test verifies that the anti-protein aggregation peptide can effectively inhibit the toxic aggregation of pathogenic proteins and protect ASH neurons from damage.

[0036] (2) PolyQ (polyglutamine) aggregation inhibition test: This test verifies that the anti-protein aggregation peptide has the effect of inhibiting polyQ aggregation.

[0037] (3) Detection of reactive oxygen species level: This test verifies that the anti-protein aggregation peptide can reduce ROS level and protect nerve cells.

[0038] In summary, the present invention demonstrates through the above experiments that scallop polypeptides possess anti-protein aggregation function, meaning they can prevent or slow down the aggregation of proteins into insoluble deposits within cells or the body. The advantages of these anti-protein aggregation scallop polypeptides include strong antioxidant defense capabilities, good biocompatibility, low potential side effects, the ability to inhibit abnormal protein folding, helping proteins fold correctly and preventing aggregation, and effectively regulating protein homeostasis, thereby achieving a neuroprotective effect.

[0039] The present invention has the following advantages and effects compared with the prior art:

[0040] (1) This invention uses natural scallops as the target, and obtains scallop polypeptide components with neuroprotective activity through enzymatic hydrolysis and separation methods. Then, a single polypeptide is obtained through sequence identification, sequence comparison and polypeptide synthesis. Its amino acid sequence is Thr-Met-Tyr-Trp-Thr-Asp-Val-Ser-Asn-Gly-Gln-Ile-His-Arg (TMYWTDVSNGQIHR), and its molecular formula is C 74 H 110 N 22 O 23 S, with an average relative molecular mass of approximately 1707.88 Da and a theoretical isoelectric point of pH 6.41, is a hydrophilic polypeptide. This polypeptide can be isolated and purified from scallops or synthesized artificially.

[0041] (2) In this invention, the polyQ aggregation model AM141 nematode and the Huntington's disease model HA759 nematode were selected to conduct polyglutamine aggregation inhibition test and neuronal survival test, respectively, to study the neuroprotective effect of scallop polypeptide. The results showed that the scallop polypeptide can significantly inhibit the aggregation of abnormal proteins and polyQ, reduce neuronal damage, protect neurons, and improve neuronal survival rate. Therefore, the scallop polypeptide has a clear anti-protein aggregation effect and excellent neuroprotective activity.

[0042] (3) The scallop polypeptide provided by the present invention can reduce the reactive oxygen species level of nematodes that are elevated due to oxidative stress, and will not stimulate an abnormal increase in the reactive oxygen species level of nematodes under conditions without oxidative stress, thus having antioxidant activity.

[0043] (4) The scallop polypeptide provided by the present invention is derived from the enzymatic hydrolysate of natural food and medicine scallops. It has a small molecular weight, is easy to absorb, and has a clear source. As a drug, it can improve the efficiency and accuracy of treatment, reduce the occurrence of immune reactions, and ensure its long-term efficacy and safety.

[0044] (5) The scallop polypeptide provided by the present invention has antioxidant, anti-protein aggregation and neuroprotective functions, and can be further used in the development of pharmaceuticals and other products, with broad application prospects. Attached Figure Description

[0045] Figure 1 This is a graph showing the results of the analysis of the effect of scallop polypeptide My#9431 on the survival rate of neurons in HA759 nematodes.

[0046] Figure 2 This is a statistical graph showing the number of polyQ aggregation points in nematodes AM141 after treatment with scallop polypeptide My#9431.

[0047] Figure 3 This is a graph showing the distribution of polyQ aggregation points in *C. elegans* AM141 after 72 h of treatment with scallop polypeptide My#9431.

[0048] Figure 4 This is a standard curve for BCA protein content determination.

[0049] Figure 5 This is a standard curve for detecting catalase activity.

[0050] Figure 6 This is a graph showing the results of the analysis of the effect of scallop polypeptide My#9431 on the N2 reactive oxygen species level of wild-type nematodes.

[0051] Figure 7 This is a graph showing the regulatory effect of scallop polypeptide My#9431 on the N2 reactive oxygen species level of wild-type nematodes under low-dose (2 mM) paraquat-induced modeling.

[0052] Figure 8 This is a graph showing the effect of a low-dose (2 mM) paraquat-induced scallop polypeptide My#9431 on the N2 SOD enzyme activity of wild-type nematodes.

[0053] Figure 9 The figure shows the effect of low-dose (2 mM) paraquat-induced scallop polypeptide My#9431 on the N2 CAT enzyme activity of wild-type nematodes. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0055] The concentrated NA22 bacterial solution in the example is food for nematodes. The preparation method is as follows: activated Escherichia coli NA22 is inoculated into LB liquid medium and cultured with shaking. Then, the bacterial cells are collected by centrifugation to obtain the concentrated NA22 bacterial solution.

[0056] The N2, AM141, and HA759 Caenorhabditis elegans models all originated from the Caenorhabditis Genetics Center at the University of Minnesota.

[0057] Example 1 Isolation and Identification of Polypeptides from Scallops

[0058] 1. Enzymatic Hydrolysis and Isolation

[0059] (1) Take fresh scallops, remove the shells, wash them cleanly, chop them up and homogenize. After the homogenate is freeze-dried, weigh 5 g of scallop freeze-dried powder, add deionized water according to the solid-liquid ratio of 1:4, stir evenly with a glass rod, preheat in a constant temperature water bath at 45 °C, then adjust the pH of the solution to 7.5 with NaOH, add 1 mL of trypsin (15000 U / mL), and react at 45 °C for 4 h; then adjust the pH of the solution to 6.5 with HCl, add 1 mL of papain (15000 U / mL), and react at 65 °C for 4 h; boil to inactivate the enzymes to obtain scallop protease hydrolysate.

[0060] (2) After the scallop protease hydrolysate prepared in step (1) is naturally cooled to room temperature, slowly add 170 mL of ethanol with a volume fraction of 95% while stirring with a glass rod, let it stand overnight at room temperature, then perform vacuum filtration and centrifugation (4 °C, 8000 rpm, 5 min) to remove the precipitate. The supernatant is evaporated to remove ethanol by vacuum rotary evaporation, and then freeze-dried to obtain scallop crude polypeptide dry powder.

[0061] 2. Ultrafiltration Purification and Gel Chromatography Column Purification

[0062] (1) Prepare the scallop crude polypeptide dry powder obtained in step 1 into a solution with a concentration of 20 mg / mL, and use a centrifugal filter of Amicon Ultra-2 equipped with Ultracel-3, Ultracel-5, and Ultracel-10 membranes of Millipore for ultrafiltration. Use the membrane ultrafiltration method to obtain the retention components of scallop protease hydrolysates with MW>10 kDa, 5 kDa <MW<10 kDa, 3 kDa<MW<5 kDa, and MW<3 kDa; collect the components with a molecular weight less than 3 kDa, then pass through a 0.45 μm filter membrane to remove impurities, and use a desalting column to remove the salts in the polypeptide sample.

[0063] (2) Further separate the desalted polypeptide components in step (1) using a packed Sephadex G-25 dextran gel chromatography column, elute with primary deionized water at a flow rate of 1 mL / min, and monitor at 280 nm with a 785 UV / VIS detector while collecting the fractions of each absorption peak. A total of 5 polypeptide components are obtained, and these 5 polypeptide components are respectively freeze-dried to obtain the corresponding freeze-dried powders of polypeptides from scallops.

[0064] 3. LC-MS / MS Identification

[0065] The polypeptide composition and amino acid sequence of the scallop polypeptide freeze-dried powder obtained in step 2 were identified by LC-MS / MS.

[0066] 4. Database comparison and filtering

[0067] Based on the identified peptide sequences with antioxidant activity provided in the BIOPEP-UWM database, the scallop peptide sequences obtained in step 3 were compared with this database for virtual activity screening. The specific steps were as follows: Log in to the BIOPEP-UWM website, select the Bioactive peptides database, click the Analysis option, select Profile of potential biological activity in the tab, and then enter the scallop peptide sequences identified by liquid chromatography-mass spectrometry (LC-MS) in step 3 for comparison. Scallop peptide sequences with potential antioxidant and neuroprotective activities were selected for the following experiments. After screening, a scallop peptide with the sequence TMYWTDVSNGQIHR was obtained and named My#9431. The molecular formula was calculated using a specialized peptide calculator to be C0. 74 H 110 N 22 O 23 S, calculated using the online tool Expasy ComputepI / Mw, shows that the average relative molecular mass of the polypeptide is approximately 1707.88 Da, and its theoretical isoelectric point is pH=6.41, indicating that it is a hydrophilic polypeptide. The polypeptide provided by this invention has an isoelectric point close to 7, making it more widely applicable.

[0068] Example 2: Synthesis of scallop polypeptides using the Fmoc solid-phase method

[0069] The scallop polypeptide My#9431 was synthesized by Shanghai Taopu Biotechnology Co., Ltd., using the following specific method:

[0070] 1. Weigh out chlorotriphenylmethyl chloride resin and mix it with DCM (dichloromethane) solution, shake for 30 min to allow the resin to fully swell.

[0071] 2. Remove DCM solvent by core filtration. Add 3 molar excess of Fmoc- (the first amino acid at the C-terminus)-OH, 10 molar excess of N,N-diisopropylethylamine, and a small amount of dimethyl fumarate to dissolve the resin. Shake for 1 h to allow the first amino acid to bind to the resin. Then, wash 6 times alternately with dimethyl fumarate and DCM. Wash with a 20% (v / v) solution of piperidine dimethyl fumarate for 5 min to remove the protective effect of Fmoc-. Repeat this process for 15 min.

[0072] 3. Drain the solution, take out a dozen or so resin grains, wash them three times with ethanol, then add one drop each of ninhydrin, potassium cyanide and phenol solution in sequence, and heat at high temperature until a positive reaction occurs, turning a deep blue color.

[0073] 4. Add dimethyl fumarate, methanol, and dimethyl fumarate to the reaction tube in sequence according to the ratio. Wash twice with dimethyl fumarate. Then add 3 times the molar excess of Fmoc-(the second amino acid at the C-terminus)-OH, 3 times the molar excess of O-benzotriazole-tetramethylurea hexafluorophosphate, and a small amount of dimethyl fumarate to dissolve. Immediately afterward, add 10 times the molar excess of N,N-diisopropylethylamine and react for 40 min to carry out the amino acid condensation reaction.

[0074] 5. Repeat the above-described deprotection, ninhydrin detection, and condensation reaction procedures sequentially until all target amino acids are sequentially linked to the resin from right to left and deprotected from Fmoc-. Use ninhydrin to detect the reaction until a negative result indicates complete reaction. Finally, wash the resin three times with methanol solution and allow the liquid to evaporate at room temperature.

[0075] 6. Transfer the resin to a centrifuge tube, add the cleavage buffer to fully separate the peptide from the resin, and incubate at a constant temperature and shake for 120 min to cleave the peptide from the resin. The cleavage buffer was prepared according to the following ratio: 94.5% trifluoroacetic acid, 2.5% water, 2.5% 3,4-ethylenedioxythiophene, and 1% disulfide peptide (all volume fractions). Filter the above liquid using a sintering filter. Dry the resulting lysate as much as possible with nitrogen, then wash repeatedly with anhydrous diethyl ether 6 times, centrifuge at 4000 r / min for 3 min, collect the precipitate, and evaporate to dryness at room temperature to obtain crude scallop peptides. Purify the peptides by high performance liquid chromatography to obtain peptides with a purity of over 95%.

[0076] Example 3: Scallop polypeptide My#9431 exhibits neuroprotective activity.

[0077] The nematode model HA759 is a disease model of Huntington's disease (HD), capable of simulating the pathogenesis of HD. Simultaneously, through gene technology, it stably expresses green fluorescent protein (GFP), which not only facilitates the localization of ASH neurons but also allows for the assessment of neuronal survival status by observing GFP expression, providing an important observational window for studying the mechanisms of neuronal death in HD. Under strong drive from the osm-10 promoter, which is associated with osmotic sensing, this model specifically expresses the 150-glutamate-residue huntingtin protein Htn-Q150 in head ASH and ASI neurons, as well as tail PHA and PHB neurons. Htn-Q150 expression is most significant in ASH neurons, while expression is weaker in the other three types of neurons. Furthermore, the point-mutated pqe-1 gene enhances the sensitivity of ASH neurons to Htn-Q150 toxicity, accelerating the induction of Htn-Q150 toxicity. This results in the mass death of HA759 ASH neurons due to protein toxicity accumulation within approximately 3 days under 15°C culture conditions. When ASH neurons die, the GFP fluorescent spots attached to the ASH neurons near the nematode's head immediately disappear, decreasing from two spots to one or zero. Both of these cases are recorded as ASH neuron death. Only when the GFP fluorescence on two ASH neurons is simultaneously normal is the nematode considered alive. This invention uses HA759 as an example to study the neuroprotective activity of the scallop polypeptide My#9431. The specific method is as follows:

[0078] 1. Culture HA759 nematodes to L4 stage using conventional methods; wash L4 stage HA759 nematodes three times with S. Medium solution to remove NA22 bacterial culture; then adjust the nematode density to 40-50 nematodes / 10 μL with S. Medium solution.

[0079] 2. Using a 96-well plate, add 10 μL of concentrated NA22 bacterial culture, 1.5 μL of 5 mg / mL 5-FUdR, 2 μL of 5 mg / mL AMP solution, 10 μL of insect culture, and peptide stock solution to each well. The final peptide concentrations were set at 0, 0.5, 1, 2, and 4 mM. An equal volume of S. Medium solution was added as a control. Each well was then brought to a final volume of 100 μL with S. Medium solution. Three replicates were performed for each concentration. The outermost ring was sealed with S. Medium solution to reduce water evaporation, and the plate was finally sealed with sealing film. The 96-well plate was incubated at 15°C and 120 rpm for 72 h using a constant temperature shaker.

[0080] 3. After 72 hours of culture, aspirate the nematodes and place them on an agarose mat. Fix and paralyze them, then photograph them using a fluorescence microscope, taking pictures from top to bottom and left to right. Use a microscope with 20x magnification and the blue fluorescence channel to obtain images showing green GFP fluorescent dots within the nematodes.

[0081] The results are as follows Figure 1 As shown, the survival rate of ASH neurons in the control group was around 35-40%, while the survival rate of neurons in all treatment groups was significantly improved. p The scallop peptide My#9431 (<0.0001) showed the highest survival rate at peptide concentrations of 2 or 4 mM, increasing the survival rate of ASH neurons to approximately 60%, which was about 20% higher than the control group. Meanwhile, a concentration of 2 mM did not inhibit the normal growth and development of nematodes. These results indicate that the scallop peptide My#9431 can effectively inhibit the toxic accumulation of pathogenic proteins in neurons, protecting ASH neurons from damage and thus increasing their survival rate.

[0082] When the peptide concentration was 4 mM, peptide crystals were observed to precipitate in the liquid environment, and the culture environment in the well plates was significantly more viscous than in other groups. This inhibited the normal growth and development of nematodes, with some nematodes being significantly smaller and exhibiting developmental delays compared to other groups. In this situation, it is possible that shock or developmental delays prevented HA759 nematodes from properly entering the high-incidence phase of ASH neuronal apoptosis, leading to a higher neuronal survival rate and interfering with the experimental results. Furthermore, the ASH neuronal survival rate in the 4 mM peptide group was not significantly higher than that in the 2 mM group. In conclusion, the 2 mM scallop peptide effectively alleviated polyQ toxicity and accumulation while exhibiting optimal biocompatibility, validating that 2 mM is the optimal dosage concentration for in vivo validation of scallop active peptides. This result also suggests that a balance must be struck between efficacy and safety during the administration of scallop peptides.

[0083] Example 4: Inhibitory effect of scallop polypeptide My#9431 polyQ (polyglutamine) on aggregation.

[0084] The degree of protein aggregation can be evaluated by the number of aggregation points; an increase in the number of aggregation points indicates a deeper degree of aggregation. This experiment used the *C. elegans* polyQ aggregation model AM141 as the research subject. Immediately after the L1 stage, soluble Q40::YFP distribution was observed in the body wall muscle cells of AM141 nematodes. As they grow and develop, polyQ is expressed in the body wall muscle cells of AM141, gradually forming aggregation points from a diffuse state. When they reach adulthood, the AM141 nematode model will show a phenotype of complete Q40::YFP aggregation. The effect of the scallop polypeptide My#9431 on protein aggregation can be reflected by the change in the number of polyQ aggregation points. To determine the detection time of polyQ aggregation points, AM141 nematodes were cultured from the L1 stage to 24 h, 48 h, and 72 h, and the number of polyQ fluorescent aggregation spots in their bodies was measured. All nematode fluorescence images at 72 h were processed, and the distribution of polyQ protein aggregation points in the nematodes at this time was analyzed to evaluate the polyQ aggregation inhibitory effect of the scallop polypeptide My#9431. The specific methods are as follows:

[0085] 1. The nematode AM141 was synchronized using conventional methods to obtain L1 stage larvae. The larvae were washed three times with S. Medium solution to remove NA22. The nematode density was adjusted to 30-40 larvae / 10 μL by adding S. Medium solution.

[0086] 2. Add 10 μL of concentrated NA22 bacterial culture to each well of a 96-well plate (final OD per well). 570 Add 2 μL of 5 mg / mL AMP solution (between 0.5 and 0.6 μg / mL), 30 μL of the insect fluid prepared in step 1, and the peptide sample stock solution for drug administration. The treatment group received 2 mM scallop peptide My#9431, while the control group (C) received an equal volume of S. Medium solution. Then, each well was brought to a volume of 100 μL with S. Medium solution. At least 9 replicates were set up for each group. 100 μL of S. Medium solution was added around each well for liquid sealing. The edge of the plate cap was sealed with sealing film, and the cap was marked. The 96-well plate was incubated at 20°C and 120 rpm in a constant-temperature shaker until the appropriate time point.

[0087] 3. At 24 h, 48 h, and 72 h of culture, respectively, three wells of culture medium from each group were transferred from a 96-well plate to 1.5 mL centrifuge tubes. The nematodes were washed with M9 buffer until the supernatant was clear and thoroughly mixed. The nematodes were then aspirated onto an agarose pad and anesthetized with sodium azide. Fluorescence images were taken using a high-content imaging system, and the number of fluorescent spots around the nematodes was counted.

[0088] The test results are shown in Figure 2At 24 h, 2 mM of the scallop polypeptide My#9431 did not inhibit polyglutamine aggregation. This may be because the 24-hour period is not the peak phase for polyQ protein aggregation in the AM141 nematode model, and therefore, polypeptide treatment could not inhibit polyQ protein aggregation at this time. At 48 h, polyQ protein aggregation was somewhat inhibited; and at 72 h, it was effectively inhibited. Compared with the control group, this scallop polypeptide reduced polyQ protein aggregation in the somatic cell wall, thereby alleviating the neurodegenerative disease in nematodes, with a reduction of approximately 20%.

[0089] Based on this, this embodiment further processed all nematode fluorescence images at 72 h after peptide administration and analyzed the distribution of polyQ protein aggregation points in the nematodes at this time. The results are as follows: Figure 3 As shown in the figure, in the control group, the proportion of *C. elegans* with 90-110 fluorescent dots exceeded 50%, while only one nematode (3%) had 50-70 fluorescent dots. Compared with the control group, after treatment with 2 mM scallop peptide My#9431, the proportion of *C. elegans* with 90-110 fluorescent dots decreased to 17.14%, while the proportion of *C. elegans* with 50-70 fluorescent dots increased to 37.14%, and the proportion of *C. elegans* with 70-90 fluorescent dots remained within the range of 45-55%. These results indicate that scallop peptide My#9431 inhibits polyglutamine aggregation by reducing the proportion of *C. elegans* with high fluorescent dots and increasing the proportion of *C. elegans* with low fluorescent dots, thus maintaining protein aggregation at a moderate level. This example demonstrates that this active scallop peptide reduces the probability of neurodegenerative diseases caused by abnormal polyQ aggregation through its anti-abnormal protein aggregation activity.

[0090] Example 5: Scallop polypeptide My#9431 exhibits antioxidant activity.

[0091] As the primary site of cellular oxidative metabolism, mitochondria, with their membranes rich in unsaturated fatty acids, become a prime target for reactive oxygen species (ROS). Because the mitochondrial membrane is rich in important functional proteins, such as those involved in the respiratory chain electron transport system and oxidative phosphorylation, lipid membrane peroxidation damage inevitably affects the mitochondrial membrane structure, causing a shift in mitochondrial membrane potential and impacting ATP synthesis. Furthermore, the inherent structure and biological characteristics of mitochondria make mitochondrial DNA more susceptible to ROS attack, leading to oxidative damage and nucleic acid mutations. These functional impairments in mitochondria result in further increases in ROS levels, forming large amounts of peroxides. This vicious cycle, coupled with stimulation from the external environment or exogenous oxidants, can trigger excessive oxidative stress, ultimately causing neuronal damage and death. Under normal physiological conditions, antioxidants can scavenge reactive oxygen species produced in the body. In addition, the body's own antioxidant systems, such as superoxide dismutase (SOD) and catalase (CAT), also play a role in scavenging ROS. However, with aging or mitochondrial dysfunction caused by oxidative stress, the rate of ROS production gradually accelerates while the rate of ROS clearance slows down. This imbalance disrupts the dynamic equilibrium of ROS, leading to its accumulation in the body and triggering a series of oxidative stress diseases. This invention uses wild-type *C. elegans* N2 as the target organism and studies the antioxidant activity of the scallop polypeptide My#9431 by measuring ROS levels and SOD and CAT activities. The specific methods are as follows:

[0092] 1. Synchronize nematodes N2 according to standard methods and culture them to the L4 stage; wash the L4 stage nematodes with S. Medium solution three times to remove NA22 bacterial culture.

[0093] 2. In each well of a 24-well plate, concentrated NA22 bacterial suspension, 5.0 mg / mL 5-FUdR, 5.0 mg / mL AMP solution, and peptide sample stock solution were pre-added. The experiment was divided into four groups: control group, model group, My#9431 group, and My#9431+ model group. The My#9431 group and the My#9431+ model group were given peptide sample stock solution to achieve a final concentration of 2 mM in the system. The control group and the model group were given an equal volume of S. Medium solution to ensure consistent liquid volume in all wells. Finally, 100 μL of nematode solution was added to each well to ensure at least 1000 nematodes per well, with a final system volume of 500 μL per well. The final OD of NA22 in each well was ensured. 570 The concentrations were between 0.5 and 0.6, with a final concentration of 75 μg / mL for 5-FUdR and 100 μg / mL for AMP. Each treatment had four replicates. The mixture was then incubated at 120 rpm and 20°C for 24 h.

[0094] 3. Modeling: After 24 h of culture, 5 μL of 200 mM paraquat solution (final concentration 2 mM) was added to each well of the modeling group and the My#9431+ modeling group. 5 μL of S. Medium solution was added to the control group and the My#9431 group as controls. The group was cultured for another 48 h under the same conditions. Then, the liquid from each well was aspirated into a centrifuge tube, centrifuged, the supernatant was removed, and all nematodes were collected.

[0095] 4. Add 300 μL of PBST solution to the collected nematodes, resuspend thoroughly, and transfer to a 2 mL glass homogenizer. Homogenize on ice. Collect the homogenate lysate and centrifuge at low temperature (12,000 rpm for 4 min at 4°C) to remove the precipitate and collect the supernatant.

[0096] 5. Use the supernatant from step 4 as the test sample to detect its ROS level, SOD, and CAT activities. The specific method is as follows:

[0097] (1) Strictly follow the procedures outlined in the BCA Protein Quantitative Detection Kit (Beyotime Biotechnology Co., Ltd.) to prepare the protein content determination standard curve. Figure 4 In the equation, X represents the protein concentration, and Y represents the absorbance measured at 570 nm. The protein concentration X is calculated by reverse-calculating the equation based on the measured absorbance. The measured protein content of the sample was 1 mg / mL.

[0098] (2) The ROS level was determined according to the reactive oxygen species detection kit (Beyotime), as follows: First, the original 1 mM stock solution was diluted with PBS to prepare a 100 μM DCFH / DA probe, which was prepared immediately. Then, the samples were tested using a black 96-well plate. 50 μL of the sample prepared in step 4 and 50 μL of the DCFH / DA probe solution were added to each well and vortexed to mix. Finally, the fluorescence value was measured using a fluorescence microplate reader with the parameters set to emission wavelength 538 nm and excitation wavelength 480 nm. The measurement was performed every 20 min, starting from 0 min, for a total detection time of 120 min. The relative value of the DCF fluorescence intensity of the sample was obtained. Then, based on the protein concentration in step (1), the DCF fluorescence intensity (ROS intensity) per unit mass of protein was obtained to eliminate the error caused by the difference in the number of samples.

[0099] (3) SOD activity was measured according to the total SOD activity assay kit (WST-8 method, Beyotime), as follows: Prepare the WST-8 / enzyme working solution with a volume ratio of enzyme solution, WST-8 solution, and SOD detection buffer of 1:8:151; prepare the reaction initiation working solution with a volume ratio of reaction initiation solution (40×) and SOD detection buffer of 1:39. Add the corresponding solutions and the sample obtained in step 4 to the 96-well plate according to the reaction system set in Table 1. Shake to mix, incubate at 37℃ for 30 min, and then measure the OD activity using a microplate reader. 450 Then, based on the results, calculate the SOD enzyme activity per unit mass of protein sample; the inhibition percentage should be between 30% and 70%.

[0100] SOD enzyme activity units = Inhibition percentage / (1 - Inhibition percentage) units

[0101] Inhibition percentage = (OD) 450 Blank control 1-OD 450 (sample) / (OD) 450 Blank control 1-OD 450 Blank control 2) × 100%

[0102] Table 1 SOD Activity Assay System

[0103]

[0104] (4) CAT activity was determined using the CAT detection kit (Beyotime), as detailed below:

[0105] ① Prepare 250 mM and 5 mM hydrogen peroxide solutions separately. Quantitatively dilute the 5 mM hydrogen peroxide solution to prepare hydrogen peroxide standards with concentration gradients of 5, 3.75, 2.5, 1.25, and 0.625 mM. Prepare the chromogenic working solution (freshly prepared) at a peroxidase to chromogenic substrate volume ratio of 1:1000. In a 1.5 mL centrifuge tube, add 20 μL of the sample prepared in step 4 (with hydrogen peroxide detection buffer as a control), 20 μL of hydrogen peroxide detection buffer, and 10 μL of 250 mM hydrogen peroxide sequentially. Mix well and react at room temperature for 5 min. Quickly add 450 μL of catalase reaction termination solution to stop the reaction. Take a new centrifuge tube and add 40 μL of catalase detection buffer and 10 μL of the above reaction system sequentially, mixing well to obtain the test solution. Add 10 μL of the test solution or 4 μL of hydrogen peroxide standard at gradient concentrations to each well of a 96-well plate, then add 200 μL of the colorimetric working solution to each well. After mixing, react in a chamber for 30 min and measure the OD. 520 .

[0106] ② Finally, based on the catalase activity detection standard curve ( Figure 5The concentration of H2O2 remaining after the reaction of the sample mixture was calculated by combining the dilution factor. Then, referring to the H2O2 concentration of the blank control group, the number of moles of H2O2 consumed by the sample was calculated. Here, one catalase activity unit (1 unit) is clearly defined as the amount of enzyme required to convert 1 μmol of H2O2 in 1 minute at 25 ℃ and pH 7.0. Through the above calculations, the enzyme activity units contained in the sample and the enzyme activity units contained in a unit sample (unit mass of protein) were finally obtained.

[0107] ROS detection results showed that My#9431 could reduce the elevated ROS levels caused by oxidative stress, and it did not stimulate *C. elegans* under conditions without oxidative stress, leading to an abnormal increase in ROS levels. The ROS levels in the My#9431 + model group (i.e., scallop peptide pretreatment + 2 mM paraquat modeling) were between those of the control group and the model group, consistent with the experimental expectations. Figure 6 ).

[0108] In addition, this embodiment also compared the ROS levels of each group at the 7th detection using a fluorescent microplate reader when the detection time was 120 min. The results are as follows: Figure 7 As shown in the figure, in the group without paraquat modeling, administration of scallop peptides could slightly reduce the level of reactive oxygen species (ROS) in nematodes, but there was no significant difference among the components, and the effect was not obvious. However, in the modeling group, administration of scallop peptides 24 hours before paraquat modeling could significantly reduce the level of ROS in nematodes, thereby accelerating the ROS clearance rate in nematodes and helping the body to fight oxidative stress and protect the body against oxidative adversity.

[0109] The relative activity results of antioxidant enzymes SOD and CAT are as follows: Figure 8 and Figure 9 As shown. When paraquat was not used for modeling, treatment of *C. elegans* with the scallop peptide My#9431 did not affect the activity level of its own antioxidant enzymes, indicating that the scallop peptide did not affect SOD enzyme activity under non-stress conditions. When subjected to paraquat oxidative stress, the SOD activity in the control group of nematodes decreased significantly, but treatment with My#9431 effectively increased the SOD activity in *C. elegans* that had decreased due to oxidative stress. When paraquat was not used for modeling, My#9431 administration slightly increased CAT enzyme activity; however, after paraquat administration, the CAT enzyme activity in both the modeling group and the peptide-treated group (My#9431 + paraquat) was increased, indicating that modeling increases CAT enzyme activity, but peptide treatment does not further increase CAT activity after modeling.

[0110] A decrease in SOD enzyme activity indicates that SOD activity is inhibited. SOD enzyme activity inhibition means an increase in ROS levels and an exacerbation of the inflammatory response. On the other hand, the higher the SOD enzyme activity, the stronger the body / tissue's ability to scavenge free radicals and the faster the rate at which peroxides are cleared from the body.

[0111] The above results indicate that My#9431 possesses in vitro free radical scavenging capabilities. Simultaneously, My#9431 also alleviated the disorder of superoxide dismutase activity in *C. elegans* induced by paraquat oxidative stress and stimulated catalase activity in *C. elegans* to maintain reactive oxygen species levels and help the body resist oxidative stress.

[0112] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A scallop polypeptide with anti-protein aggregation function, characterized in that... Its amino acid sequence is: TMYWTDVSNGQIHR.

2. The method for preparing the scallop polypeptide with anti-protein aggregation function according to claim 1, characterized in that... It includes the following steps: Scallop polypeptides with anti-protein aggregation function were prepared directly through solid-phase synthesis.

3. The application of the scallop polypeptide with anti-protein aggregation function as described in claim 1 in the preparation of drugs for the prevention and treatment of neurodegenerative diseases, characterized in that: The neurodegenerative disease mentioned is Huntington's disease.

4. A drug for preventing and treating neurodegenerative diseases, characterized in that... It contains the scallop polypeptide with anti-protein aggregation function as described in claim 1.

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