CN109456405B - Depalmitoylation PD-L1 protein and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a dipalmitoylation modified PD-L1 protein, wherein cysteine of amino acid 272 in the PD-L1 protein is replaced by other amino acid; the dipalmitoylation modified PD-L1 protein is prepared by changing cysteine of a unique palmitoylation modification site (Cys272) of PD-L1 into other amino acids, constructing a eukaryotic expression vector by using a genetic engineering method, encoding the PD-L1 protein with Cys272 site mutation, and performing overexpression and purification in eukaryotic cells. The depalmitoylation modified PD-L1 protein can be used as a standard substance for verifying the accuracy of a palmitoylation detection method and the effectiveness of a method for regulating and controlling the palmitoylation of the PD-L1 protein, and has important application value in mechanism and intervention research of PD-L1 tumor immunity-related protein.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a dipalmitoylation PD-L1 protein and a preparation method and application thereof.
Background
Malignant tumors are a disease with leading morbidity and mortality in China, and in recent years, development of clinical diagnosis, operation, radiotherapy and chemotherapy enables a part of malignant tumor patients to be discovered and effectively treated at an early stage. However, worldwide, the search for new therapeutic methods and drugs has long been a focus in the field of tumor research. Unlike traditional treatment methods, tumor immunotherapy can activate or induce the body to establish a specific immune response to tumor antigens, eliminate primary or metastatic tumor cells, and establish immunological memory, organize tumor recurrence, drug resistance and metastasis. Programmed cell death molecule 1(PD-L1) is one of the immune checkpoint proteins that plays a major role in limiting T cell activity, which provides the major immune resistance mechanism by which tumor cells can evade immune surveillance. The interaction of PD-1 expressed on activated T cells with PD-L1 expressed on tumor cells down-regulates the immune response and attenuates anti-tumor immunity. The expression of PD-L1 on tumors is associated with a decreased survival rate for melanoma, lung, esophageal, and other types of cancer, and this pathway may serve as a new promising target for tumor immunotherapy.
Currently, pharmaceutical companies have developed antibodies targeting PD-L1 that show clinical activity in different tumor types. The PD-L1 antibody can block the combination with PD-1 and activate the immune response of T cells by combining PD-L1 on the cell surface, thereby improving the anti-tumor immunity. However, there are still significant challenges and deficiencies with current antibody-based immune checkpoint blockade therapies, such as poor response to PD-L1 antibody treatment in some tumor patients, and the efficacy of the treatment may also decrease with the duration of administration. There is therefore a need to develop new methods targeting PD-L1 in order to obtain more effective treatment of tumor patients.
The prior art does not disclose the existence of palmitoylation modifications of the PD-L1 protein, amino acid sites at which palmitoylation can occur, the influence of palmitoylation modifications on the expression and intracellular localization of the PD-L1 protein, and the like. Through integrated bioinformatics analysis, protein mass spectrometry detection, Click-IT palmitoylation specific immunoprecipitation, immunoblotting and other methods, the inventor finds and confirms that cysteine (Cys) at the 272 site can be palmitoylated and is the only site of PD-L1 protein which can be palmitoylated; meanwhile, palmitoylation has a remarkable influence on the increase of the expression level of PD-L1 and the positioning of cell membranes and Golgi apparatus. Therefore, the regulation of palmitoylation can become a new way for targeting PD-L1 tumor immune-related protein, and a new method is provided for the immunotherapy of tumors.
Disclosure of Invention
In view of the above-mentioned deficiencies of the prior art, the present invention is directed to the newly discovered palmitoylation modification of PD-L1 and its significant impact on PD-L1 expression and localization in cancer cells. Provides a depalmitoylated PD-L1 protein, a preparation method and application thereof.
To achieve the above object, the first aspect of the present invention provides a depalmitoylated PD-L1 protein, which is represented as follows:
a depalmitoylated PD-L1 protein, wherein the cysteine at amino acid position 272 in the amino acid sequence of the PD-L1 protein is replaced with another amino acid;
further, the dipalmitoylated PD-L1 protein has an amino acid sequence in which amino acids may be optionally substituted, deleted or added with one or more amino acids;
further, the other amino acid is selected from alanine, phenylalanine, leucine, isoleucine, methionine, valine, serine, proline, threonine, tyrosine, histidine, glutamine, asparagine, lysine, aspartic acid, glutamic acid, arginine, glycine;
further, the other amino acid at the 272 site in the amino acid sequence of the PD-L1 protein is alanine;
the second aspect of the invention provides a preparation method of the depalmitoylated PD-L1 protein, which comprises the following steps:
and 4, expressing the expression recombinant plasmid obtained in the step 3 to obtain the depalmitoylated PD-L1 protein.
The third aspect of the invention also provides application of the above-mentioned depalmitoylation PD-L1 protein in preparation of a drug for verifying the palmitoylation correlation of the PD-L1 protein.
The fourth aspect of the invention also provides a method for the depalmitoylation of the PD-L1 protein;
the invention also provides a method for inhibiting the expression of PD-L1 in tumor cells, which comprises the steps of administering the expression recombinant plasmid with the 272 mutant PD-L1 nucleotide sequence fragment to a subject in need, and inhibiting the PD-L1 of the tumor cells of the subject.
According to the technical scheme, palmitoylation modification and accurate sites of PD-L1 protein are analyzed and detected, palmitoylation of PD-L1 protein is completely blocked by changing amino acid of the modified sites, and the obtained debalmitoylated PD-L1 protein has the characteristic of no palmitoylation;
the method for preparing the depalmitoylated PD-L1 protein can be used for constructing a stable expression cell strain, has good repeatability, is beneficial to preparing a depalmitoylated PD-L1 protein standard product in a large scale, and is beneficial to improving the reliability of verification because the obtained depalmitoylated PD-L1 protein has high purity;
in conclusion, the invention provides PD-L1 protein without palmitoylation and a preparation method thereof, and fills the technical blank that no other known method can reliably prepare PD-L1 protein without palmitoylation at all and no larger-scale preparation method can be used at present. In the research of regulating and controlling the basis and application of PD-L1 palmitoylation modification, the depalmitoylated PD-L1 protein is used as a reliable negative control sample for verifying the accuracy of a palmitoylation detection method, can be used for verifying the effectiveness of a method for regulating and controlling the palmitoylation of the PD-L1 protein, and has important application value in the research of mechanism and intervention of a PD-L1 tumor immunity-related protein. Therefore, the technical scheme of the invention has obvious technical advantages and progress.
Drawings
FIG. 1 is a schematic diagram of the validation of the palmitoylation modification of wild-type PD-L1 protein by the method of non-radiolabelling (Click-IT palmitic acid labeling) in example 1;
FIG. 2 is a schematic diagram showing the result of analyzing the amino acid sequence of PD-L1 by the CSS-palm algorithm in example 1;
FIG. 3 is a schematic diagram of the pancreatin cleavage and tandem mass spectrometry detection of the presence of palmitoylation sites in PD-L1 protein in example 1;
FIG. 4 is a diagram showing the results of the electrophoresis of the endonuclease cleavage gel and the DNA sequencing after the amino acid at position 272 has been replaced with alanine in example 2;
FIG. 5 is a schematic diagram showing the results of example 3 in detecting that no palmitoylation occurred in the depalmitoylated PD-L1 protein;
FIG. 6 is a diagram showing the results of the localization detection of PD-L1 mutant C272A on the cell membrane in example 4;
in FIG. 1, Beads, IP are blank control samples, Click-IT, IP is to-be-verified wild type PD-L1 protein palmitoylation sample, and Input lysate is PD-L1 protein in cell lysate (no palmitoylation sample occurred);
in FIG. 6, the oval spots are DAPI-fluorescent cells, and the irregular spots surrounding DAPI are PD-L1, where the arrows indicate.
Detailed Description
The raw materials and equipment used in the embodiment of the present invention are known products and are obtained by purchasing commercially available products.
The invention provides a dipalmitoylation PD-L1 protein, wherein the amino acid at the 272 site in the amino acid sequence of the PD-L1 protein is alanine;
the amino acid sequence of the depalmitated PD-L1 protein is SEQ ID NO 1;
the nucleotide sequence for coding the depalmitoylated PD-L1 protein is SEQ ID NO. 2;
the invention provides a preparation method of a depalmitoylated PD-L1 protein, which comprises the following steps:
synthesizing a nucleotide sequence for coding the PD-L1 protein 272 site mutant according to the PD-L1 protein 272 site mutant amino acid sequence and selecting eukaryotic preference codons;
and 4, transfecting the mammalian cells by the expression recombinant plasmid obtained in the step 3, and constructing a stable cell strain to express so as to obtain the depalmitoylated PD-L1 protein.
In a preferred embodiment of the present invention,
in the step 2, other amino acids do not include cysteine;
in a preferred embodiment of the present invention,
in the step 2, other amino acid is alanine;
in a preferred embodiment of the present invention,
in the step 2, the amino acid sequence of the 272 site mutant of the PD-L1 protein is SEQ ID NO. 1;
the nucleotide sequence of the 272 site mutant of the encoded PD-L1 protein is SEQ ID NO. 2;
in a preferred embodiment of the present invention, in the step 2,
the nucleotide sequence encoding the PD-L1 protein mutant can be obtained by site-directed mutagenesis using wild-type PD-L1 as a template;
in a preferred embodiment of the present invention, in the step 4,
the target protein can also be expressed by using a transient transfection method;
in a preferred embodiment of the present invention, the preparation method,
optionally, step 5, purification of the target protein expressed by the cell is included.
The technical scheme of the invention provides the following uses or methods:
use of a depalmitoylated PD-L1 protein to verify the accuracy of a palmitoylation detection method.
Use of a depalmitoylated PD-L1 protein for validating the effectiveness of a method for modulating palmitoylation of a PD-L1 protein.
A method for verifying the palmitoylation-related PD-L1 protein using the above-described depalmitoylated PD-L1 protein;
a method for depalmitoylation of PD-L1 protein;
a method for inhibiting the expression of PD-L1 in tumor cells, which comprises administering the above expression recombinant plasmid having a nucleotide sequence fragment of 272 mutant PD-L1 to a subject in need thereof, and inhibiting PD-L1 in the tumor cells of the subject.
Reliable negative control samples (i.e., PD-L1 protein preparations that do not undergo palmitoylation) are needed in fundamental and applied studies that regulate the modification of PD-L1 palmitoylation. The depalmitoylation PD-L1 protein provided by the invention can be used as a standard substance for verifying the accuracy of a palmitoylation detection method and the effectiveness of a method for regulating and controlling the palmitoylation of the PD-L1 protein, and has important application value in mechanism and intervention research of PD-L1 tumor immunity-related protein.
The technical scheme of the invention is explained in detail by combining the following embodiments: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.
Example 1 determination of palmitoylation site of PD-L1 protein
1. The presence of palmitoylation of the PD-L1 protein was verified by a method of non-radiolabelling (Click-IT palmitic acid labeling):
adding Click-IT Azide-palmitic acid as a palmitic acid analogue into a cell culture medium, and marking all proteins modified by palmitoylation; carrying out Click-IT reaction of Alkyne-Biotin, and further carrying out Biotin labeling on palmitoylated protein; labeled protein was immunoprecipitated using Avidin microspheres, PD-L1 that was palmitoylated was detected using a PD-L1-specific antibody, and gel electrophoresis and immunoblotting were performed using PD-L1 protein in cell lysates as a control. The results are shown in FIG. 1, which indicates that wild type PD-L1 is modified by palmitoylation;
2. the bioinformatics method predicts the existence of palmitoylation sites in the PD-L1 protein; analysis of the PD-L1 amino acid sequence using the CSS-palm algorithm suggested that there was a palmitoylation modification at the cysteine at position 272, and that the amino acids near this position conform to the known palmitoylation motif, as shown in FIG. 2;
3. pancreatin cleavage and tandem mass spectrometry analysis are carried out on the wild type PD-L1 protein expressed in the tumor cell RKO purified by immunoprecipitation to detect the palmitoylation site existing in the PD-L1 protein, and the result shows that the Cys272 site has palmitoylation modification, as shown in FIG. 3;
the analysis and detection are combined to indicate that the PD-L1 protein has the only palmitoylation site which is Cys 272.
Example 2 preparation of depalmitoylated PD-L1 protein
1. Construction of eukaryotic expression vector encoding depalmitoylated PD-L1 protein
Substituting alanine for the amino acid at the 272 site in the amino acid sequence of the palmitoylated PD-L1 protein in example 1, and synthesizing a nucleotide sequence fragment for coding the 272 site mutant of the PD-L1 protein according to the 272 site mutant amino acid sequence of the PD-L1 protein by selecting eukaryotic preference codons;
connecting the nucleotide sequence fragment of the coding mutant PD-L1 into an eukaryotic expression vector to construct an expression recombinant plasmid;
the successful insertion of the nucleotide sequence fragment for coding the mutant PD-L1 is verified through restriction endonuclease cutting and DNA gel electrophoresis; DNA sequencing detection shows that the protein encoding the mutant PD-L1 has missense mutation at the 272 locus, which indicates that the amino acid at the 272 locus is replaced by alanine from the original cysteine, as shown in FIG. 4;
2. mammalian cells are transfected by the expression recombinant plasmid obtained above, and a stable cell strain is constructed and expressed to obtain the depalmitoylated PD-L1 protein.
Test example 3 test for detecting No palmitoylation of Depalmitoylated PD-L1 protein
The PD-L1 expression vector with 272 site mutation, the control empty vector and the wild type PD-L1 expression vector constructed in the example 2 are respectively transfected with SW480 human colorectal cancer cells which do not express endogenous PD-L1, and G418 is adopted to screen stable expression cell strains; adding Click-IT Azide-palmitic acid as a palmitic acid analogue into a cell culture medium, and marking all proteins modified by palmitoylation; carrying out Click-IT reaction of Alkyne-Biotin, and further carrying out Biotin labeling on palmitoylated protein; labeled proteins are immunoprecipitated using Avidin microspheres, gel electrophoresis and immunoblotting experiments are performed, PD-L1 protein in cell lysates is used as a control, and PD-L1 specific antibody is used to detect PD-L1 that is palmitoylated.
The results are shown in FIG. 5, which shows that wild-type PD-L1 is palmitoylated, whereas PD-L1 with a 272 mutation is not palmitoylated at all.
Test example 4 positioning test for detecting Depalmitoylated PD-L1
1. Human colorectal cancer cells RKO were transfected with PD-L1 of wild-type PD-L1 and mutant C272A, respectively, and after 48 hours, the cells were fixed with formaldehyde, the intracellular localization of PD-L1 was labeled with an antibody specific to PD-L1, and the DAPI immunofluorescence staining results showed that there was a significant membrane localization of wild-type PD-L1, while the localization of PD-L1 mutant C272A in the cell membrane disappeared, as shown in FIG. 6;
2. human colorectal cancer cells RKO were transfected with PD-L1 of wild type PD-L1 and mutant C272A, respectively. After 48 hours, the cells were fixed with formaldehyde, labeled with a 58k specific antibody for golgi and labeled with a PD-L1 specific antibody for intracellular localization of PD-L1, as shown by DAPI immunofluorescence staining results, similar to the cell membrane localization results, there was significant golgi localization of wild-type PD-L1, whereas localization of PD-L1 mutant C272A in golgi was significantly reduced or disappeared;
taken together, 1 and 2 show that the localization of PD-L1 mutant C272A is significantly reduced relative to the significant membrane localization and golgi localization present with wild-type PD-L1.
In conclusion, the cysteine of the only palmitoylation modification site (Cys272) of the PD-L1 is changed into other amino acid, the dipalmitoylated PD-L1 protein can be expressed and purified by transfecting eukaryotic cells or constructing a stable expression cell strain, and the standard of the dipalmitoylated PD-L1 protein can be prepared on a large scale. Has wide application value in the target and function research of PD-L1 protein and also has important application value in the mechanism and intervention research of PD-L1 tumor immunity related protein
Specific embodiments of the present invention have been described above in detail. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions obtained by logic analysis, reasoning or limited experiments based on the prior art according to the concepts of the present invention in the technical field should be within the scope of protection defined by the claims.
SEQUENCE LISTING
<110> Shanghai university of traffic medical college affiliated renji hospital
<120> depalmitoylation PD-L1 protein, and preparation method and application thereof
<130> 20210807
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Claims (6)
1. A depalmitoylated PD-L1 protein, characterized in that the cysteine at amino acid position 272 in the amino acid sequence of the PD-L1 protein is replaced with an alanine, resulting in the depalmitoylated PD-L1 protein; the amino acid sequence of the depalmitoylated PD-L1 protein is SEQ ID NO:1, and the nucleotide sequence coding the depalmitoylated PD-L1 protein is SEQ ID NO: 2.
2. A method of preparing the depalmitoylated PD-L1 protein of claim 1, comprising the steps of:
step 1, analyzing and detecting a palmitoylation site existing in PD-L1 protein, wherein the amino acid sequence of the PD-L1 protein is shown as a sequence table SEQ ID NO. 3;
step 2, constructing a mutant amino acid sequence according to the site obtained in the step 1, and further obtaining a nucleotide sequence of the PD-L1 protein mutant;
step 3, connecting the nucleotide sequence fragment of the coding mutant PD-L1 into an eukaryotic expression vector to construct an expression recombinant plasmid;
and 4, expressing the expression recombinant plasmid obtained in the step 3 to obtain the depalmitoylated PD-L1 protein.
3. The method of claim 2, further comprising the steps of:
step 1, analyzing an amino acid sequence of PD-L1 by using a CSS-palm algorithm, carrying out pancreatin cutting on PD-L1 protein, and detecting a palmitoylation site existing in the PD-L1 protein by tandem mass spectrometry, wherein the palmitoylation site existing in the PD-L1 protein is Cys 272;
step 2, replacing the palmitoylated 272 site amino acid obtained in the step 1 with other amino acids to obtain a 272 site mutant amino acid sequence of PD-L1 protein;
synthesizing a nucleotide sequence for coding the PD-L1 protein 272 site mutant according to the PD-L1 protein 272 site mutant amino acid sequence and selecting eukaryotic preference codons;
step 3, connecting the nucleotide sequence fragment of the coding mutant PD-L1 into an eukaryotic expression vector to construct an expression recombinant plasmid;
and 4, transfecting the mammalian cells by the expression recombinant plasmid obtained in the step 3, and constructing a stable cell strain to express so as to obtain the depalmitoylated PD-L1 protein.
4. The method as claimed in claim 3, wherein in the step 2, the 272 site mutant amino acid sequence of the PD-L1 protein is SEQ ID NO 1; the nucleotide sequence of the 272 site mutant of the encoded PD-L1 protein is SEQ ID NO. 2; the nucleotide sequence encoding the PD-L1 protein mutant is obtained by site-directed mutagenesis using wild-type PD-L1 as a template.
5. Use of the depalmitoylated PD-L1 protein of claim 1 for the preparation of validated PD-L1 protein
The use of the reagent or kit for palmitoylation of (a).
6. Use of the recombinant plasmid expressing a nucleotide sequence fragment of depalmitoylated PD-L1 as defined in claim 1 for the preparation of a reagent or kit for inhibiting PD-L1 in a cell.
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| WO2016176761A1 (en) * | 2015-05-01 | 2016-11-10 | Immunovaccine Technologies Inc., | Methods for potentiating an immune response using depot-forming and non-depot-forming vaccines |
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