CN116769015A - Linear ligand binding epitope of bovine IgG Fc receptor boFcgammaRIII - Google Patents
Linear ligand binding epitope of bovine IgG Fc receptor boFcgammaRIII Download PDFInfo
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Abstract
The application relates to a linear ligand binding epitope of bovine IgG Fc receptor boFcgammaRIII, the epitope sequence is AQRVVN, and the epitope sequence is positioned at the 98-103 positions of A-B loop of extracellular domain EC2 of boFcgammaRIII. The application designs and synthesizes a bofcγriii polypeptide in an EC2 domain of the bofcγriii, couples the polypeptide to carrier protein, and obtains a linear ligand binding epitope polypeptide specifically binding to bovine IgG1 by synthesizing N-terminal and C-terminal series truncated polypeptides; blocking ELISA and rosette inhibition test results show that the binding epitope polypeptide of the linear ligand of the bofcγRIII can effectively inhibit the binding of bovine IgG1 and the recombinant protein of the bofcγRIII and the expression of the bofcγRIII on the cell surface, and the polypeptide provides a new thought for the development of IgG Fc receptor target drugs.
Description
Technical Field
The application belongs to the technical field of bioengineering, relates to a ligand binding epitope of a cell surface receptor, and particularly relates to a linear ligand binding epitope of a bovine IgG Fc receptor boFcgammaRIII.
Background
IgG Fc receptors (fcγr) are cell surface receptors that bind to the immunoglobulin G constant region (Fc), are widely expressed on the surface of immune cells such as monocytes, macrophages, neutrophils, dendritic Cells (DCs), B cells, natural killer cells, and mast cells, and they play a key role in humoral and cellular immune responses through interactions with IgG Fc regions, becoming potential targets for developing novel immunotherapeutics against autoimmune diseases, infectious diseases, and tumors. Three different classes of receptors have been identified in humans, fcyri (CD 64), fcyriia/B/C (CD 32) and fcyriiia/B (CD 16), the fourth class of receptors being fcyriv found in mice, wherein fcyriii is a medium-low affinity IgG receptor whose extracellular region is similar to fcyrii and also contains two Ig-like domains. Human fcyriii is encoded by two homologous genes, hufcyriiia and hufcyriiib, whose extracellular regions are highly homologous, but the transmembrane and intracellular regions are distinct. hufcγriiia is a transmembrane receptor that binds human IgG1 and IgG3 monomers or complexes, but does not bind IgG2 or IgG4. FcRgamma chains or TCR zeta chains are necessary for the expression of FcγRIIIA on the cell surface, which can only be expressed by cotransfection of FcRgamma chains or TCR zeta chains. In contrast, fcγriiib does not have a transmembrane and intracellular region, nor is it coupled to the fcrγ chain or TCR ζ chain, but rather is anchored to the cell membrane by Glycosyl Phosphatidylinositol (GPI), with low affinity for binding to human IgG1 and IgG 3. The crystal structure of hufcγriii extracellular domain and IgG1Fc complex shows that the EC2 domain of low affinity fcγriii binds to the horseshoe opening of the Fc region of IgG homodimers, suggesting that it may be useful to design polypeptide molecules that inhibit IgG binding to fcγriii. In fact, polypeptides from human IgG have been shown to bind fcγr. Therefore, we have shown that Fc binding site polypeptides derived from low affinity fcγrs would be ideal candidates for modulating Fc receptor-initiated inflammatory responses.
We have cloned and identified FcgammaRIII molecules in cattle and sheep. Collins et al (1997) identified by RT-PCR from gamma/delta T cell clones the boFcGammaRIII gene, cDNA 1 071nt long, containing a single ORF (750 nt) encoding a 250aa transmembrane protein with 16aa signal peptide, 191aa extracellular region with 4 Cys distributed at positions 47, 89, 128 and 172, constituting two Ig-like domains, and 22aa transmembrane region with 21aa cytoplasmic tail and therefore homologous to human FcGammaRIIIA and not GPI-anchored receptor. However, the expression and distribution of bofcγriii in cells, and the interaction of the receptor with IgG, etc. have not been reported.
Disclosure of Invention
The application screens and identifies the linear ligand binding epitope of the bofcγRIII and key amino acid thereof, analyzes the specific binding of the linear ligand binding epitope polypeptide and bovine IgG1, discusses the regulation and control effect of the epitope polypeptide on the interaction of the IgG-bofcγRIII, and provides a new thought for the development of IgG Fc receptor targeted drugs.
The technical scheme of the application is as follows:
a linear ligand binding epitope of bovine IgG Fc receptor boFcgammaRIII, the sequence of the epitope is AQRVVN.
The linear ligand binding epitope is located at positions 98-103 of the A-B loop of the extracellular domain EC2 of boFcγRIII.
The linear ligand binding epitope polypeptide specifically binds bovine IgG1 but not bovine IgG2.
Ala of the linear ligand binding epitope polypeptide 98 、Gln 99 、Val 101 、Val 102 And Asn 103 To bind to a critical amino acid residue of bovine IgG1, mutation of either amino acid will result in the linear ligand binding epitope polypeptide losing its ability to bind to bovine IgG 1.
The linear ligand binding epitope polypeptide is effective in inhibiting binding of bovine IgG1 to a bofcγRIII recombinant protein and cell surface expressed bofcγRIII.
The application of the linear ligand binding epitope in preparation of FcgammaR targeted drugs.
In the application, the cDNA of the coding region of the bofcγRIII and the cDNA of the FcRγchain are respectively subcloned into an expression vector pcDNA3, receptor molecules are expressed on the surface of COS-7 cells, the ligand specificity of the bofcγRIII is measured by a rosette test, and IgG1-RBC can form obvious rosettes on the transfected cells of the bofcγRIII, so that the bofcγRIII specifically affins with bovine IgG1 but does not bind to bovine IgG2.
Subcloning the cDNA of extracellular region of bofcγriii into eukaryotic expression vector pcDNA3, secretory expressing extracellular region of bofcγriii in NS0 cells, inducing ascites in mice and purifying recombinant protein of bofcγrii.
Designing and synthesizing a bofcγriii polypeptide in an EC2 domain of the bofcγriii, screening by Dot-blot to obtain the shortest effective polypeptide 98-103 AQRVVN which specifically binds to bovine IgG1 but does not bind to bovine IgG2, wherein the shortest effective polypeptide is a linear ligand binding epitope of the bofcγriii and is positioned on the A-B ring of the receptor EC2 domain; analysis of the polypeptide mutations showed that bofcγriii binds to Ala of the epitope 98 、Gln 99 、Val 101 、Val 102 And Asn 103 Is a critical amino acid residue that binds bovine IgG1, and mutating either amino acid results in the linear ligand binding epitope polypeptide losing the ability to bind bovine IgG 1.
The application has the positive beneficial effects that:
the application utilizes synthetic polypeptide to screen and identify the linear ligand binding epitope of the bofcγRIII by expressing the bofcγRIII receptor molecule on the surface of COS-7 cells, and has important significance for deeply understanding the interaction of IgG-bofcγRIII.
(1) IgG Fc receptor function studies. The application constructs eukaryotic expression plasmid of full-length bofcγriii and FcRγchain cDNA, co-transfects COS-7 cells, and screens the eukaryotic expression plasmid by G418 to construct cell strain which stably expresses the bofcγriii molecules on the cell surface and construct a bofcγriii function research platform.
(2) Linear ligand binding epitope identification. The application designs and synthesizes a bofcγRIII extracellular domain polypeptide by referring to the crystal structure of a human fcγRIII-IgG complex, couples the polypeptide with carrier protein, screens and identifies polypeptide specifically combined with IgG by Dot-blot, and establishes a bofcγRIII linear ligand combined epitope identification method.
(3) Fcγr targeted drug design. The BofcγRIII linear ligand binding epitope and the polypeptide thereof screened and identified by the application have good regulation and control effects on the binding of BofcγRIII-IgG1, and provide a new idea for the design of FcgammaR targeted drugs.
Drawings
Fig. 1: alignment of fcγriii EC2 domain amino acid sequences.
Fig. 2: binding of the bofcγriii polypeptide to bovine IgG 1.
Fig. 3: binding of N-terminally truncated borii 1 polypeptides to bovine IgG 1.
Fig. 4: binding of the C-terminally truncated RIII1-N1 polypeptide to bovine IgG 1.
Fig. 5: binding of Ala-replacement 1N1-C8 polypeptide to bovine IgG 1.
Detailed Description
EXAMPLE 1 purification and labelling of bovine IgG
Separating and purifying bovine IgG from bovine anti-chicken Red Blood Cell (RBC) hyperimmune serum by sodium sulfate salting-out method, separating bovine IgG1 and IgG2 from bovine IgG by DEAE cellulose ion exchange chromatography, and further purifying bovine IgG1 or IgG2 protein by protein A affinity chromatography. Bovine IgG1 and IgG2 were labeled with horseradish peroxidase (HRP) using the modified sodium periodate method, and the hemagglutination titers of bovine IgG1 and IgG2 were determined on 0.5% (wt) chicken red blood cells RBC for the preparation of bovine IgG sensitized Red Blood Cells (RBCs).
Example 2 expression of bofcγriii on cell surface
According to the bofcγriii cDNA sequence (NM_ 174539), a synthetic primer is designed to amplify the full-length bofcγriii ORF and FcRγ chain cDNA ((containing a signal peptide sequence), cloned into eukaryotic expression vector pcDNA3, E.coli JM109 competent cells are transformed, eukaryotic expression plasmids pcboFcRIII and pcFcRγ are constructed through PCR and enzyme digestion identification, COS-7 cells are co-transfected with eukaryotic expression plasmids pcboFcRIII and pcFcRγ linearized by a loop or BglII, a rosette loop test is performed with bovine IgG1 or IgG2 sensitized erythrocytes (RBCs), expression of receptor molecules on the surface of the transfected cells is detected, and ligand specificity of the bofcγriii for bovine IgG1 and IgG2 binding is determined.
The results showed that bovine IgG1-RBC formed a distinct rosette around the transfected cells of bofcγRIII, whereas IgG2-RBC and the untransfected COS-7 control cells did not see rosette formation, indicating that bofcγRIII specifically affinities bovine IgG1, but did not bind to bovine IgG2, which is a specific receptor for bovine IgG 1.
EXAMPLE 3 secretory expression purification of the extracellular region of BofcγRIII in NS0 cells
Designing a primer to amplify cDNA (without a signal peptide sequence) of an extracellular region of the boFcGammaRIII, cloning into a eukaryotic expression vector pcDNA3, converting E.coli JM109 competent cells, and constructing eukaryotic expression plasmid pcsbofcRIII through PCR and enzyme digestion identification; transfecting NS0 cells with a linearized eukaryotic expression plasmid pcsbofcRII, and selectively culturing with G418; ascites is induced in the abdominal cavity of a mouse by using G418 resistant transfected cells, and the extracellular region recombinant protein of the bofcγRIII is secreted and expressed.
ELISA detection shows that the mouse ascites contains secreted protein specifically combined with HRP-IgG1, and the mouse ascites is purified by nickel chelate affinity chromatography to obtain the boFc gamma RIII recombinant protein.
Example 4 design synthesis of a bofcγriii polypeptide
The amino acid sequences of bofcγriiia (X99695) and hufcγriiia (np_000560), hufcγriiib (np_000561) and mofcγriii (np_ 034318) were aligned (fig. 1), and 6 polypeptides boRIII1, boRIII2, boRIII3, boRIII4, boRIII5, boRIII6, covering the a-B, B-C, C-C', D-E, E-F and F-G loops of their EC2 domains, respectively, were synthesized from the amino acid sequence fragments of the second domain (EC 2) of bofcγriiia by designing Cys from the crystal structure of hufcγriiib, with the N-termini of the remaining 4 polypeptides all incorporating Cys, except for the N-termini of the polypeptides boRIII2 and boRIII6, for carrier protein coupling (table 1).
Table 1 properties of the bofcγriii polypeptides
EXAMPLE 5 specific binding of the BofcγRIII polypeptide to bovine IgG1
Coupling of the bofcγriii polypeptides boRIII1, boRIII2, boRIII3, boRIII4, boRIII5, and boRIII6 to IgG-free bovine serum albumin (B) using the heterobifunctional reagent sulfosmccSA) carrier protein to increase the reactivity of the polypeptide. 1mg of Sulfo-SMCC (molecular weight: 436.37, spacer length:) In 50. Mu.l of dimethyl sulfoxide, then 4mg of BSA free of IgG were added and dissolved in 0.5ml of coupling buffer (0.1 mol/l PB pH 7.2,0.15mol/l NaCl,1mol/l EDTA), and after mixing, reacted at room temperature for 1 hour or 30 minutes at 37℃and then dialyzed overnight against coupling buffer at 4 ℃. Mu.l of peptide (100. Mu.g) was dissolved in 0.01mol/l PB buffer (pH 7.2) containing 5mM EDTA and 12.5% (v/v) Dimethylformamide (DMF), incubated with an equal volume of SMCC-activated BSA carrier protein (100. Mu.g) for 4 hours at room temperature, and then overnight at 4 ℃. The concentration of the conjugated carrier protein polypeptide was adjusted to 1mg/ml with 0.01M PB buffer (pH 7.2) and binding of the conjugated carrier protein polypeptide to HRP-IgG1 and IgG2 was detected by Dot-blot.
The results show that HRP-IgG1 specifically binds to the boRIII1 polypeptide without a color reaction with the other 5bofcγRIII polypeptides; whereas HRP-IgG2 did not bind to any of the bofcγriii polypeptides (fig. 2). In the Ig-binding blocking assay, both bovine serum and purified bovine IgG1 effectively blocked binding of HRP-IgG1 to the borII 1 polypeptide, indicating that the borII 1 polypeptide specifically affinities bovine IgG1, but not bovine IgG2.
Example 6 precise localization of BofcγRIII Linear ligand binding epitope
In order to accurately position the binding epitope of the linear ligand of the bofcγriii, the amino acid residues of the polypeptide of the boRIII1 are deleted from the N-terminal and the C-terminal one by one respectively, a series of polypeptides truncated at the N-terminal or the C-terminal are synthesized, the binding capacity of the polypeptides with bovine IgG1 is detected by Dot-blot, and the binding epitope of the linear ligand of the bofcγriii is accurately positioned. First, the bosii 1 polypeptide (except Cys) was shortened one by one from the N-terminus, and 8N-terminal truncated series of polypeptides RIII1-N1, RIII1-N2, RIII1-N3, RIII1-N4, RIII1-N5, RIII1-N6, RIII1-N7, RIII1-N8 (table 2) were synthesized.
IgG 1-binding assay results, val deleted 97 No influence on binding of the borII 1 polypeptide to HRP-IgG1 was seen, but Ala was deleted 98 The polypeptide of (2) completely loses binding activity, indicating Ala 98 N-terminal of linear ligand binding epitope for bofcγriiiTerminal residues (FIG. 3).
N-terminal truncated polypeptides of table 2borii 1 polypeptides
The RIII1-N1 polypeptides were shortened one by one from the C-terminus, and 8C-terminal truncated series of polypeptides 1N1-C1, 1N1-C2, 1N1-C3, 1N1-C4, 1N1-C5, 1N1-C6, 1N1-C7, 1N1-C8 were synthesized (Table 3). In the IgG 1-binding assay, the RIII1-N1 polypeptide is truncated from the C-terminus to Asn 103 Still maintaining good binding activity, suggesting that the C-terminal frost residue of the bofcγriii linear ligand binding epitope is Asn 103 (FIG. 4). Thus, AQRVVN is the shortest effective polypeptide for receptor binding to bovine IgG1, the linear ligand binding epitope of bofcγriii, located at positions 98-103 of the a-B loop of the receptor EC2 domain.
C-terminal truncated polypeptides of table 3RIII1-N1 polypeptides
Example 7 identification of key amino acid residues of a linear ligand binding epitope of BofcγRIII
Based on the 1N1-C8 polypeptide with bovine IgG1 binding activity, the amino acid residues of the polypeptide are replaced by Ala in sequence (except Cys), 6 Ala-mutant series polypeptides 1N1C8-A, 1N1C8-Q, 1N1C8-R, 1N1C8-V1, 1N1C8-V2, 1N1C8-N (Table 4) are synthesized, and key amino acid residues of the linear ligand binding epitope of boFcγRIII are identified.
Dot-blot results show that only Arg 100 Mutations did not affect binding of the 1N1-C8 polypeptide to HRP-IgG1, and mutations in other amino acid residues resulted in loss of bovine IgG1 binding activity of the polypeptide (FIG. 5), suggesting Ala 98 、Gln 99 、Val 101 、Val 102 And Asn 103 Is a critical amino acid residue of the linear ligand binding epitope of bofcγriii.
Ala-replacement polypeptides of Table 4 1N1-C8 polypeptides
Claims (6)
1. A linear ligand binding epitope of bovine IgG Fc receptor bofcγriii, characterized by: the sequence of the epitope is AQRVVN.
2. The linear ligand binding epitope of claim 1, wherein: the epitope is located at positions 98-103 of the A-B loop of the 2 nd extracellular domain EC2 of boFcγRIII.
3. The linear ligand binding epitope of claim 1, wherein: the linear ligand binding epitope polypeptide specifically binds bovine IgG1 but not bovine IgG2.
4. The linear ligand binding epitope of claim 1, wherein: ala of the linear ligand binding epitope polypeptide 98 、Gln 99 、Val 101 、Val 102 And Asn 103 To bind to a critical amino acid residue of bovine IgG1, mutation of either amino acid will result in the linear ligand binding epitope polypeptide losing its ability to bind to bovine IgG 1.
5. The linear ligand binding epitope of claim 1, wherein: the linear ligand binding epitope polypeptide is effective in inhibiting binding of bovine IgG1 to a bofcγRIII recombinant protein and cell surface expressed bofcγRIII.
6. Use of the linear ligand binding epitope of claim 1 in the preparation of an fcγr targeted drug.
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| CN112675293A (en) * | 2020-12-31 | 2021-04-20 | 吴伯骥 | Application of HrpNECb protein in pharmacy for recognizing and activating multiple types of receptors and/or membrane proteins and signal paths thereof |
| WO2022142977A1 (en) * | 2020-12-31 | 2022-07-07 | 吴伯骥 | Use of hrpz-type multi-mimotope epitope ligand protein in foods, cosmetics, health care products or pharmaceuticals |
| WO2022142976A1 (en) * | 2020-12-31 | 2022-07-07 | 昆明锐斯得科技有限公司 | Use of hrpn-type multi-mimotope epitope ligand protein in foods, cosmetics, health care products or pharmaceuticals |
| WO2022142978A1 (en) * | 2020-12-31 | 2022-07-07 | 吴伯骥 | Use of hrpw-type multi-mimotope ligandins in food products, cosmetics, health products or pharmaceuticals |
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2023
- 2023-06-07 CN CN202310670273.6A patent/CN116769015A/en active Pending
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|---|---|---|---|---|
| CN112675293A (en) * | 2020-12-31 | 2021-04-20 | 吴伯骥 | Application of HrpNECb protein in pharmacy for recognizing and activating multiple types of receptors and/or membrane proteins and signal paths thereof |
| WO2022142977A1 (en) * | 2020-12-31 | 2022-07-07 | 吴伯骥 | Use of hrpz-type multi-mimotope epitope ligand protein in foods, cosmetics, health care products or pharmaceuticals |
| WO2022142976A1 (en) * | 2020-12-31 | 2022-07-07 | 昆明锐斯得科技有限公司 | Use of hrpn-type multi-mimotope epitope ligand protein in foods, cosmetics, health care products or pharmaceuticals |
| WO2022142978A1 (en) * | 2020-12-31 | 2022-07-07 | 吴伯骥 | Use of hrpw-type multi-mimotope ligandins in food products, cosmetics, health products or pharmaceuticals |
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| WANG R等: "Identification of the Linear Fc-Binding Site on the Bovine IgG1 Fc Receptor (boFcγRIII) Using Synthetic Peptides.", 《VET SCI.》, vol. 11, no. 1, 8 January 2024 (2024-01-08), pages 24 * |
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