JP2021103947A - Thyroid-stimulating hormone receptor with mutation introduced - Google Patents

Abstract

To provide TSHR having an improved S/N ratio in order to solve following problems: the inventors have found out, through examination of methods for measuring autoantibody activity to TSHR, that wild-type TSHR has constitutive activity, has a low signal/noise ratio (S/N ratio), and results in decrease in measurement sensitivity; particularly, in one embodiment of measurement methods using a cAMP biosensor, the TSHR activation level is evaluated by a ratio of activation levels of a cAMP biosensor before and after contact with a sample, so that the decrease of sensitivity caused by the low S/N ratio of TSHR has a significant influence.SOLUTION: The inventors have conducted intense study and found out that introducing proper mutation into TSHR makes it possible to suppress constitutive activity of TSHR and improve its S/N ratio and, the invention has been finished by proceeding examination.SELECTED DRAWING: Figure 1

Description

The present invention relates to a mutated Thyroid Stimulating Hormone (TSH) receptor.

The TSH receptor (TSHR) is a TSH receptor present on the thyroid cell membrane and is a type of G protein-coupled receptor. When TSH secreted from the pituitary gland binds to TSH, the stimulation activates intracellular adenylate cyclase to produce cyclic adenosine monophosphate (cAMP), which receives the cAMP signal to secrete TSH and Synthesis takes place.

Graves' disease (also known as Graves' disease), which is a typical example of thyroid disease, is a disease caused by an autoantibody (Thyroid Stimulation Antibody; TSAb) that has stimulating activity against TSHR. Is. In patients with Graves' disease, TSAb overstimulates TSHR, resulting in enhanced thyroid function. In addition to Graves' disease, TSH-producing tumors, pregnancy thyrotoxicosis, hypothyroidism, Hashimoto's disease and the like are known as thyroid diseases caused by TSHR-stimulating or inhibitory autoantibodies.

In order to provide appropriate treatment to patients with thyroid disease, it is important to distinguish the patient's disease. For example, in Graves'disease, it is known that by measuring the TSAb activity in a patient's blood sample, it is possible to diagnose Graves' disease, grasp the pathogenesis, and analyze the etiology. As described above, the method for measuring the autoantibody activity against TSHR is industrially useful and is being developed.

Methods for measuring autoantibody activity against TSHR are roughly classified into two types based on the measurement principle. One is a method of measuring autoantibody activity from the rate of inhibition of TSH binding to TSH. As a specific method, patient serum and isotope-labeled TSH are added to the solubilized porcine thyroid cell membrane fraction and reacted, and the inhibition rate of binding of labeled TSH to TSH by the TSHR autoantibody in the test serum is determined by the TSHR autoantibody. A method of measuring as a value (Non-Patent Document 1) is known.

The other is to measure the amount of cAMP produced. Although the technical idea of measuring the amount of cAMP is common, several methods are known due to the difference in the method for quantifying cAMP. Typical ones are shown below. The amount of cAMP produced by incubating Chinese hamster ovary (CHO) cells in the presence of a test sample and the TSAb contained in the test sample stimulating the TSH receptor present on the CHO cell membrane is the enzyme of the reporter gene. A method for measuring via activity (Patent Document 1). The porcine thyroid cells are incubated in the presence of serum from the patient, and the amount of cAMP produced by the TSAb contained in the serum stimulating the TSHR present on the porcine thyroid cell membrane is used to lyse the cells. Method of measuring (Patent Document 2). Pig thyroid cells are incubated in the presence of serum from the patient, and the amount of cAMP produced by the TSAb contained in the serum stimulating the TSHR present on the porcine thyroid cell membrane is mediated by calcium ions. A method for measuring TSAb using a bioassay system that detects by luminescence (Patent Document 3).

In the methods described in Patent Documents 1 to 3, there are problems such as a long time for measurement and a need for pretreatment. Therefore, as one of the methods capable of rapidly measuring the autoantibody activity against TSHR in the sample, a method of measuring the activation level of the cAMP biosensor using mammalian cells expressing both the cAMP biosensor and TSHR is studied. (Patent Document 4). In this method, the autoantibody activity of the subject sample against TSHR can be calculated by performing the following steps (a) to (c).
(A) A step of incubating animal cells expressing both the cAMP biosensor and TSHR in the presence of a blood sample collected from a subject;
(B) After step (a), the step of measuring the activation level of the cAMP biosensor;
(C) A step of comparing the activation level measured in step (b) with the activation level in the control to calculate the TSHR activation level;

A method for producing human TSHR (Patent Document 5), dog TSHR (Patent Document 6), and pig TSHR (Patent Document 7) has been established for use by the methods described in Patent Documents 1 to 4. However, these studies were intended to easily and mass-produce TSHRs having the same performance as wild-type TSHRs, and did not provide TSHRs having higher performance than wild-type TSHRs.

Regarding TSHR, there are many previous studies on pathological conditions caused by mutations (Non-Patent Documents 2 to 7). However, all of these are studies for elucidating the mechanism of disease and the like, and no studies or studies have been conducted for applying them to a method for measuring autoantibody activity against TSHR.

Re-table 2004-500580 Japanese Unexamined Patent Publication No. 2016-75707 JP-A-2017-192396 Japanese Patent Application No. 2019-20595 Special Table No. 05-504683 Special Table No. 04-506752 JP-A-2007-314548

Methods in Enzymology, 74, 405-420 (1981), Endocr. Rev., 9, 106-120, (1988) Xiuyan Feng et al. Endocrinology 149 (4): 1705-1717 Ann-Karin Haas et al. Cell. Mol. Life Sci. (2011) 68: 159-167 Gunnar Kleinau et al. JBC (2007) 282-1: 518-525 Eneko Urizar et al. JBC (2005) 280-17: 17135-17141 Vanessa Chantreau et al. PLOS ONE. (2015) 10.1371 0142250 Gunner Kleinau et al. FASEBJ (2008) 22 (8): 2798-2808

While investigating a method for measuring autoantibody activity against TSHR, the present inventors have a constant activity in wild-type TSHR and a low signal / noise ratio (S / N ratio), resulting in measurement. We found that it caused a decrease in sensitivity. In particular, in one aspect of the measurement method using the cAMP biosensor, the S / N ratio of TSHR is evaluated in order to evaluate the TSHR activation level by the ratio of the activation level of the cAMP biosensor in the presence and absence of the TSHR stimulant. The effect of the decrease in sensitivity due to the low value was remarkable.

An object of the present invention is to solve the above problems. That is, it is an object to provide TSHR with an improved S / N ratio.

As a result of diligent studies to solve the above problems, the present inventors have found that it is possible to suppress the constitutive activity of TSHR and improve the S / N ratio by introducing an appropriate mutation into TSHR. I found it. Further, the present invention was completed by proceeding with the study.

That is, the present invention is as follows.
[1] A TSHR mutant characterized by having a mutation in at least one of the amino acids described below.
D460, L469, S479, N483, D487, R531, L552, S567, I568, M572, Y582, R625, L665
[2] A TSHR mutant characterized by having a mutation in at least one of the amino acids described below.
R531, L552, S567, Y582, R625, L665
[3] The TSHR mutant according to [1] or [2], wherein the mutation is a point mutation.
[4] The TSHR mutant according to any one of [1] to [3], which comprises at least one of the point mutations described below;
D460 (R, N, C, E, Q, G, H, K, S, T),
L469 (A, I, M, F, P, W, Y, V),
S479 (A, I, L, M, F, P, W, Y, V),
N483 (A, I, L, M, F, P, W, Y, V),
D487 (A, I, L, M, F, P, W, Y, V),
R531 (D, N, C, E, Q, G, H, K, S, T),
L552 (A, I, M, F, P, W, Y, V),
S567 (A, I, L, M, F, P, W, Y, V),
I568 (A, L, M, F, P, W, Y, V),
M572 (A, I, L, F, P, W, Y, V),
Y582 (A, I, L, M, F, P, W, V),
R625 (A, I, L, M, F, P, W, Y, V),
L665 (A, I, M, F, P, W, Y, V).
[5] The TSHR mutant according to any one of [1] to [4], which comprises at least one of the point mutations described below;
D460 (N, E, Q),
L469 (G, A, V, I, P),
S479 (A, V, L, I, P),
N483 (A, V, L, I, P),
D487 (A, V, L, I, P),
R531 (D, N, E, Q),
L552 (A, V, I, P),
S567 (A, V, L, I, P),
I568 (A, V, L, P),
M572 (A, V, L, I, P),
Y582 (F, W),
R625 (A, V, L, I, P),
L665 (A, V, I, P).
[6] The TSHR mutant according to [1] to [5], which has at least one of the point mutations described below;
D460N, L469P, S479A, N483A, D487A, R531Q, L552V, S567A, I568L, M572A, Y582F, R625A, L665V.
[7] The TSHR mutant according to any one of [1] to [6], which comprises at least one of the point mutations described below;
R531Q, L552V, S567A, Y582F, R625A, L665V.
[8] The TSHR mutant according to any one of [1] to [7], which comprises at least one of the point mutations described below;
S567A, L665V.
[9] A polynucleotide encoding the TSHR mutant according to any one of [1] to [8].
[10] An animal cell expressing the TSHR mutant according to any one of [1] to [8].
[11] A thyroid disease diagnostic kit containing the animal cells according to [10].
[12] The thyroid disease diagnostic kit according to [11], wherein the animal cell expresses a cAMP biosensor.
[13] The thyroid disease diagnostic kit according to [11] or [12], wherein the thyroid disease is Graves' disease.

The TSHR of the present invention has an improved S / N ratio, at least in a certain concentration range. Due to this property, it can be suitably used for measuring autoantibody activity against TSHR.

FIG. 1 is a schematic diagram showing a mutation point introduced into TSHR in Example 1 of the present specification. In the figure, the asterisk indicates the position of the mutation point. In the character string consisting of one letter of the alphabet, three letters of the alphabet, and one letter of the alphabet, the central three-digit number represents the number of the amino acid residue in human TSHR, and the leftmost alphabet represents the amino acid residue in wild-type TSHR. , The rightmost alphabet indicates the amino acid residue after the mutation. FIG. 2 shows constitutive cAMP levels, ie, RLUs in the absence of TSHR stimulants, in the wild-type TSHR and TSHR variants of Example 2 herein. The vertical axis shows the value of cAMP level (RLU). The horizontal axis shows each wild-type TSHR and TSHR mutant transiently expressed in HEK293 cells. FIG. 3 shows the signal-to-noise ratio (Fold change) of the wild-type TSHR and TSHR mutants when NIBSC08 / 204 was added at 31.25, 125, 500, and 2000 mIU / L. The vertical axis shows the S / N ratio (Fold change) after each concentration of NIBSC08 / 204 is added. The horizontal axis shows the concentration (mIU / L) of the added NIBSC08 / 204. FIG. 4 shows the TSHR activation level (Fold change) when a Graves' disease positive patient serum or a negative control (healthy person serum) was added to the transient expression strain of wild-type TSHR or TSHR mutant. The vertical axis shows the TSHR activation level, that is, the cAMP level after addition of each sample divided by the cAMP level of the negative control sample (1 negative serum addition, condition in the absence of TSHR stimulant). The horizontal axis indicates the number of the added sample. FIG. 5 shows the TSHR activation level (Fold change) in wild-type TSHR and TSHR mutant stable expression strains. In the figure, those indicated by ◇ represent wild-type strains, those indicated by ■ represent S567A, and those indicated by ▲ represent L665V. In the figure, * indicates that the p-value according to the t-test is p <0.03, and ** indicates that the p-value according to the t-test is p <0.01.

As used herein, the term "wild-type TSHR" means a TSHR in which no mutation has been introduced. In the present specification, the TSHR amino acid sequence in which the mutation has not been introduced is the TSHR amino acid sequence registered in the database of NCBI (http://www.ncbi.nlm.nih.gov/guide/). do. In the present specification, the origin of TSHR is not limited to humans, but unless the origin of TSHR is specifically described, it means that TSHR is derived from humans (SEQ ID NO: 1). Further, in the present specification, the wild-type TSHR may be referred to as "WT".

As used herein, the term "amino acid" is a general term for organic compounds having an amino group and a carboxyl group. Although not particularly limited, for example, alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, N), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid. (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine ( Includes Ph, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Val, V). In this specification, each amino acid may be abbreviated by one letter of the above alphabet.

As used herein, the term "TSHR variant" means a TSHR having a mutation that deletes, substitutes, inserts or adds an amino acid sequence from the wild-type TSHR, and its origin is not limited to humans. When one letter of the alphabet followed by a three-digit number is used to represent the variant, the first letter of the alphabet indicates the amino acid into which the mutation is introduced, and the following three-digit number is the amino acid sequence of human TSHR. The position of the amino acid residue in SEQ ID NO: 1) is shown. For example, "L665" means that the mutant has a mutation introduced at the position of leucine at position 665 of human TSHR.

When the origin of TSHR used in the present invention is other than human, the amino acid sequence of human TSHR (SEQ ID NO: 1) and the sequence of the target wild-type TSHR are set to NCBI (http://www.ncbi.nlm.nih.gov). /) Is aligned with BLASTalW (https://www.genome.jp/tools-bin/clustalw) so as to maximize the homology in BLAST, and the amino acid residue in the amino acid sequence of human TSHR (SEQ ID NO: 1). It means that the amino acid residue corresponding to is mutated.

In the present specification, when a mutant is represented by a character string in which one letter of the alphabet and a three-digit number are followed by one letter of the alphabet, the leftmost amino acid is the amino acid before the mutation, and the central three-digit number is the wild human TSHR. The position of the amino acid residue in the above is indicated by the alphabet at the right end indicating the amino acid after the mutation, and means that the amino acid at the left end is a mutant in which the amino acid at the left end is point-mutated to the amino acid at the right end. For example, "L665V" means that the leucine at position 665 is a mutant introduced with a point mutation in which it is replaced with valine.

In the present specification, when a plurality of alphabets are listed in parentheses following one letter and three digits of the alphabet, it means that the amino acid at the left end is a mutant that is point-mutated to one of the amino acids in parentheses. means. For example, in the case of L665 (A, V, I, P), it means that the leucine at position 665 is a mutant introduced with a point mutation in which leucine at position 665 is replaced with any of alanine, valine, isoleucine, and proline.

In the present specification, when the above-mentioned mutation site or mutation point is continuously represented by a slash (/), it means that the mutant is a mutant having both of the mutations described before and after the slash. .. For example, in the case of "R531Q / L665V", a point mutation in which arginine at position 531 is replaced with glutamine and a point mutation in which leucine at position 665 is replaced with valine are introduced. It means that it is a heavy mutant.

As used herein, the term "cAMP level" means the amount of cAMP produced in mammalian cells due to TSHR, and is defined as RLU as measured by the method described below.

[Animal cells used] Human fetal kidney cell-derived cell line (HEK293 cells) (obtained from ECACC [European Collection of Authenticated Cell Cultures], Cat no. 85120602).

[Vector used] GloSensor cAMP, that is, the amino acid residues 359 to 544 of firefly luciferase, the cAMP binding region of the regulatory subunit of protein kinase A (PKA), and the 4th to 355th of firefly luciferase. A plasmid vector (pGloSensor-22F, manufactured by Promega) expressing a protein containing an amino acid residue and pcDNA3.1 (+) (pcDNA3.1_TSHR) expressing TSHR. In the method described below, pcDNA3.1 (+) expressing a TSHR mutant may be appropriately used instead of pcDNA3.1_TSHR.

[Cell preparation method]
[1] 1.5 × 10 ⁴ HEK293 cells are seeded in D-MEM medium containing 10% FBS and cultured for 24 hours.
[2] pGloSenso-22F and pcDNA3.1_TSHR are transfected into HEK293 cells using FuGENE HD Transfection Reagent (manufactured by Promega) according to the protocol attached to the GloSensor cAMP Assay (manufactured by Promega).
[3] After culturing the transfected cells for 24 hours, those in which an increase in fluorescence intensity is observed by addition of a TSHR stimulant (for example, NIBSC08 / 204, which is an international standard for TSAb, or a TSAb-positive sample) are selected.

[Activity measurement conditions]
[1] After culturing the cells for 24 hours, the cells are replaced with a polyethylene glycol-containing incubation solution containing 2% (v / v) of GloSensor cAMP Reagent stock solution (manufactured by Promega), and equilibration treatment is performed at room temperature for 1 hour. ..
[2] After adding the TSHR stimulant and incubating for 20 minutes, RLU is measured using a luminometer. The measured RLU is defined as the cAMP level.

The cAMP level measurement method in the present specification can be appropriately modified as long as the equivalence with the above measurement method is guaranteed.

As used herein, the term "constant cAMP level" means the cAMP level indicated in the absence of a TSHR stimulant. In the present specification, the constitutive cAMP level can be measured under the activity measurement condition [2] of the cAMP level measurement method without adding a TSHR stimulant.

As used herein, the term "TSHR activation level" means the responsiveness of TSHR to a TSHR stimulant, and the ratio of the cAMP level to the constant cAMP level in mammalian cells in the presence of the TSHR stimulant (Fold change). Defined by.

As used herein, the term "TSHR stimulant" means a ligand that stimulates (activates) TSHR, and examples thereof include TSH itself and TSHR-stimulating autoantibodies.

As used herein, the term "TSHR-stimulating autoantibody" refers to an autoantibody capable of directly or indirectly stimulating (activating) TSHR (that is, an autoantibody produced in the body of a subject to be measured and in the body of the subject). It means an antibody that targets a substance such as a protein present in, for example, an agonist capable of binding to TSHR (for example, an anti-TSH antibody [TSAb] having an agonistic action).

As used herein, the term "TSHR-inhibiting autoantibody" refers to an autoantibody capable of directly or indirectly inhibiting (inactivating) TSHR, for example, directly or indirectly inhibiting the binding of TSH to TSHR. It means an antagonist (eg, an anti-TSH antibody [TSBAb] having an antagonistic effect).

In the present specification, "signal / noise ratio", "S / N ratio", and "S / N ratio" are TSAbs which are TSHR stimulants in a strain that transiently expresses TSHR according to the above-mentioned cAMP level measurement method. It means the level of TSHR activation when the international standard NIBSC08 / 204 is added. The TSHR activation level when NIBSC08 / 204 at a certain concentration X (mIU / L) is added is defined as the S / N ratio at X (mIU / L).

As used herein, "high (low) S / N ratio" means that at a certain NIBSC08 / 204 concentration, one S / N ratio is 1.2 times or more higher (low) than the other. That is, when the TSHR mutant has a higher S / N ratio than the wild-type human THSR at a certain concentration X (mIU / L), the S / N ratio of the TSHR mutant at X (mIU / L) is X ( It means that it is 1.2 times or more the S / N ratio of wild-type human TSHR in mIU / L). A high S / N ratio of TSHR means that when the TSHR is used in a measurement system for a TSHR stimulant, it is excellent in sensitivity and discrimination ability.

As used herein, the term "homology" generally means a calculation of the ratio of the same number of amino acids in an amino acid sequence between two or a plurality of amino acids. As used herein, "homology" is defined as a value measured by BLAST of NCBI (http://www.ncbi.nlm.nih.gov/). Blastp can be used by default for Algorithm when comparing amino acid sequences in BLAST. The measurement result is quantified as Positives or Ideas.

As used herein, the term "polynucleotide" includes nucleotides or bases, or their equivalents, which are composed of a plurality of bound forms thereof. Nucleotides and bases include DNA bases or RNA bases. The above equivalents include, for example, DNA or RNA bases that have undergone chemical modifications such as methylation, or nucleotide analogs. Nucleotide analogs include non-natural nucleotides.
The "DNA strand" represents a form in which two or more DNA bases or their equivalents are linked.
“RNA strand” refers to a form in which two or more RNA bases or their equivalents are linked.

In the present specification, the “vector” is, for example, an Escherichia coli-derived plasmid (for example, pBR322, pUC12, pET-Blue-2), a bacteriophage-derived plasmid (for example, pUB110, pTP5), a yeast-derived plasmid (for example, pSH19, pSH15), or an animal. Cell expression plasmids (eg, pA1-11, pcDNAI / Neo), bacteriophages such as λ phage, vectors derived from viruses such as adenovirus, retrovirus, and baculovirus can be used. These vectors may contain components required for protein expression, such as promoters, replication origins, or antibiotic resistance genes. The vector may be an expression vector.

As used herein, the term "cAMP biosensor" is a self-derived index (eg, enzyme activity) that is visible (imaging) and / or quantifiable, depending on the amount and / or concentration of cAMP produced in mammalian cells. It means a protein whose level, color development level, and emission [fluorescence] level) change. The cAMP biosensor usually has a cAMP binding region, and when cAMP binds to the cAMP binding region, the three-dimensional structure of the cAMP biosensor is changed, and the change from the inactivated state to the activated state is not performed. It has an allosteric effect such as a change from the visualized state to the visualized state.

One aspect of the present invention is a human TSHR mutant into which at least one of the following mutations has been introduced.
D460, L469, S479, N483, D487, R531, L552, S567, I568, M572, Y582, R625, L665
One aspect of the present invention is a non-human TSHR mutant into which at least one of the mutations corresponding to the mutation has been introduced.
Since the TSHR mutant of the present invention has the above-mentioned mutation, it has an extremely low concentration range (1 to 50 mIU / L), a low concentration range (50 to 300 mIU / L), a medium concentration range (300 to 1000 mIU / L), and a high concentration range. The S / N ratio is improved in at least one concentration range (1000 to 5000 mIU / L). Since the TSHR mutant can sensitively measure the TSHR activation level in the concentration range where the S / N ratio is improved, it can be preferably used for determining thyroid disease caused by TSHR-stimulating or inhibitory autoantibodies. can.

Among the mutations, since the S / N ratio is improved in a wider concentration range, the mutation is introduced into at least one of L469, S479, N483, D487, R531, L552, S567, Y582, R625, and L665. It is more preferable that the mutation is introduced into at least one of R531, L552, S567, Y582, R625, and L665. Further, as demonstrated in Examples described later, it is more preferable that a mutation is introduced into at least one of S567 and L665 because it can be suitably used for the diagnosis of Graves' disease.

The origin of TSHR that can be used in the present invention is not limited as long as it responds to a measurement target such as human TSAb. Since it exhibits a reactivity similar to that of human TSHR when expressed in animal cells, rodents such as mice, rats, hamsters and guinea pigs, lagomorphs such as rabbits, pigs, cows, goats, horses, sheep and the like. Cells derived from ungulates, cats such as dogs and cats, and primates such as humans, monkeys, red-tailed monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees can be exemplified. Among them, humans (SEQ ID NO: 1), mice (SEQ ID NO: 2), rats (SEQ ID NO: 3), and pigs (SEQ ID NO: 4) are the origins of cells that have been used for measuring TSAb activity in the past. Is preferable, and human (SEQ ID NO: 1) is more preferable because the TSAb activity for TSHR in the living body can be reflected as it is.

The TSHR of the present invention is characterized by having the above-mentioned mutation, but it does not necessarily have to have only one of the above-mentioned mutations, and may have a plurality of mutations among the above-mentioned mutations.

The mutation introduced into TSHR of the present invention is a mutation that deletes, substitutes, inserts or adds an amino acid sequence, and among them, a mutation that replaces an amino acid is preferable. This is because mutations that replace amino acids can change the constituent amino acids while preserving the basic three-dimensional structure of GPCRs as compared to amino acid deletions and insertions, and are suitable for functional modification.

Due to the nature of the amino acid side chain, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl group-containing side chains (S, T), amino acids with sulfur atom-containing side chains (G, A, V, L, I, P) C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), and amino acids with aromatic-containing side chains (D, N, E, Q) Classifications such as H, F, Y, W) are known (all in parentheses represent one-letter markings of amino acids). Substitutions between amino acids in each of these groups are collectively referred to as conservative substitutions, and it is generally known that they have little effect on protein functions and the like. Further, among the above-mentioned properties of amino acids, it is known that the hydrophobicity / hydrophilicity of amino acids has a great influence on the properties of proteins.

In light of the substitution mutations whose effects have been demonstrated in the examples described later and the classification of the amino acid group according to the nature of the side chain, in D460, the same hydrophobic amino acids R, N, C, E, It is preferably substituted with Q, G, H, K, S, T, and more preferably with N, E, Q having a carboxylic acid.

In L469, it is preferable that the amino acids are substituted with A, I, M, F, P, W, Y, V, which are also hydrophobic amino acids, and among them, G, A, V, which are amino acids having an aliphatic side chain, It is more preferable to replace with I and P.

In S479, N483, D487, S567, and R625, it is preferable that they are substituted with the hydrophobic amino acids A, I, L, M, F, P, W, Y, and V, and among them, they have an aliphatic side chain. It is preferably substituted with A, V, L, I, P.

In R531, it is preferable that the amino acid is substituted with D, N, C, E, Q, G, H, K, S, T, which are also hydrophilic amino acids, and among them, an amino acid having a carboxylic acid and an amide-containing side chain. It is more preferable to be replaced with a certain D, N, E, Q.

In L552 and L665, it is preferable that they are also substituted with the hydrophobic amino acids A, I, M, F, P, W, Y and V, and among them, the amino acids A, V, I and which also have an aliphatic side chain. It is preferably replaced with P.

In I568, it is preferable that the amino acids are substituted with the same hydrophobic amino acids A, L, M, F, P, W, Y and V, and among them, the amino acids A, V and L having an aliphatic side chain, It is more preferable to be replaced with P.

In M572, it is preferable to replace with A, I, L, F, P, W, Y, V which are also hydrophobic amino acids, and among them, A, V, L, I, P having an aliphatic side chain. It is more preferable to be replaced.

In Y582, it is preferably substituted with A, I, L, M, F, P, W, V, which are also hydrophobic amino acids, and above all, it is substituted with F, W having an aromatic-containing side chain. Is more preferable.

Among the mutations at the above mutation points, since it has been demonstrated in Examples that the effect of suppressing constitutive activity is exhibited, the mutation may be at least one of the amino acid substitutions shown below or the corresponding amino acid substitutions. preferable.
D460N, L469P, S479A, N483A, D487A, R531Q, L552V, S567A, I568L, M572A, Y582F, R625A, L665V
Above all, since the S / N ratio is improved in a wide concentration range, the mutation is introduced into at least one of L469P, S479A, N483A, D487A, R531Q, L552V, S567A, Y582F, R625A, and L665V. Preferably, it is more preferable that the mutation is introduced into at least one of R531Q, L552V, S567A, Y582F, R625A and L665V. Among them, mutations have been introduced into at least one of S567A and L665V because they show a strong constitutive cAMP level inhibitory effect and can be suitably used for the diagnosis of Graves' disease as demonstrated in Examples described later. Is more preferable.

In addition to the mutations described so far, the TSHR mutant of the present invention further deletes, substitutes, inserts or inserts an amino acid sequence within a range that does not cause a significant decrease in TSHR activity or a significant decrease in S / N ratio. It may have a mutation to be added. In general, a polypeptide that has been deleted, added, inserted, or replaced by another amino acid with one or several amino acid residues maintains its biological activity, especially at sites that are not directly involved in activity. Uru (Mark et al., Proc Natl Acad Sci US A.1984 Sep; 81 (18): 5662-5666., Zoller et al., Nucleic Acids Res. 1982 Oct 25; 10 (20): 6487-6500., Wang et al., Science. 1984 Jun 29; 224 (4656): 1431-1433.) Is known.

When the TSHR mutants of the present invention have further mutations, the smaller the number, the better. When the TSHR mutant of the present invention is compared with wild-type human TSHR (SEQ ID NO: 1), the homology is preferably 90% or more, and more preferably 95% or more.
This is because it is considered that the smaller the number of mutations in the amino acid sequence, the closer the characteristics are to those of wild-type human TSHR, and the more the TSAb activity for TSHR in the living body can be reflected as it is.

When the TSHR according to the embodiment of the present invention contains a specific amino acid sequence, any amino acid in the amino acid sequence may be chemically modified. Even in such a case, it can be said that the TSHR according to the embodiment of the present invention contains a specific amino acid sequence. In general, the chemical modifications that amino acids contained in proteins undergo in vivo include, for example, N-terminal modification (for example, acetylation, myristylation, etc.), C-terminal modification (for example, amidation, addition of glycosylphosphatidylinositol, etc.). Alternatively, side chain modification (for example, phosphorylation, glycosylation, etc.) is known.

One aspect of the present invention is a polynucleotide encoding the TSHR variant. One aspect of the invention is an animal cell that expresses the TSHR variant.

The polynucleotide according to one aspect of the present invention can be synthesized using a DNA / RNA synthesizer. In addition, it can be purchased from a contracted company for DNA base or RNA base synthesis (for example, Invitrogen, Takara Bio, etc.). The polynucleotide sequence of the present invention can be appropriately designed from the amino acid sequence of the TSHR mutant of the present invention and the genetic code table.

The vector containing the polynucleotide can be prepared by a generally known molecular biological method, and the preparation methods include, for example, TA cloning method, blunt-ended cloning method, ligation method, In-fusion cloning method, and Gateway. The cloning method, the assembly method and the like are exemplified.

The animal cell according to one aspect of the present invention may be any animal cell in which the TSHR mutant is transiently or stably expressed by the introduction of the vector. The cell line used is not particularly limited, but from the viewpoint of ease of purchase and experiment, mammalian thyroid cell line [FRTL-5, etc.], human fetal kidney cell-derived cell line [HEK293 cells, HEK293T cells, etc.], Chinese hamster ovary-derived cell lines [CHO cells] and human osteosarcoma cell lines [U2OS cells] are preferable. Among them, human fetal kidney cell-derived cell lines [HEK293 cells, HEK293T cells, etc.], Chinese from the viewpoint of gene transfer efficiency and stable proliferation. Hamster ovary-derived cell lines [CHO cells] are more preferred.

Since the animal cell of the present invention suppresses the constitutive activity and improves the S / N ratio by expressing the TSHR mutant, it can be suitably used for a method for measuring the autoantibody activity against TSHR. The method for measuring the autoantibody activity to be applied is not particularly limited, and the methods described in Patent Documents 2 to 4 can be exemplified, but among them, the measurement is completed in a short time without the need for pretreatment. The method described in Patent Document 4 is preferable.

The animal cell of the present invention can be produced by a genetic engineering technique generally used in this field. For example, promoters (eg, cytomegalovirus [CMV] IE [immediate early] gene promoter, SV40 [Simian virus 40] early promoter, retrovirus promoter, metallothioneine promoter, heat shock promoter, SRα promoter, NFAT promoter, etc. HIF promoters) and vectors containing genes encoding TSH receptors operably linked downstream of such promoters (eg, pcDNA3.1 (+), pcDM8, pAGE107, pAS3-3, pCDM8) It can be obtained by introducing (transfecting) into mammalian cells using a method such as a poration method, a calcium phosphate method, a lipofection method, a DEAE (Diethylaminoethyl) dextran method, or a virus infection method. In order to be used for measuring autoantibody activity, a gene other than the gene encoding the TSHR mutant may be appropriately introduced. For example, if the method described in Patent Document 4 using a cAMP biosensor is used, the cAMP biosensor The gene encoding the above may be introduced.

Examples of the cAMP biosensor that can be used in the present invention include a reporter protein (for example, a cAMP binding region derived from the control subunit of protein kinase A [PKA], a cAMP binding region derived from Epac1) including a cAMP binding region (for example, a cAMP binding region derived from Epac1). , HRP [horseradish peroxidase]; alkaline phosphatase; β-D-galactosidase; green luminescent luciferase [SLG], orange luciferase [SLO], red luciferase [SLR] and other luciferases; green fluorescent protein [GFP], red fluorescent protein Fluorescent proteins such as [DsRed] and green fluorescent protein [CFP]), specifically, GloSensor cAMP (manufactured by Promega), which is a luciferase containing a cAMP binding region derived from the regulatory subunit of PKA, and , Pink Flamindo (Pink Fluorescent cAMP indicator), which is a red fluorescent protein containing a cAMP binding region derived from Epac1 (Reference "Harada K., et al., Sci Rep. 2017 Aug 4; 7 (1): 7351. Doi: 10.1038 / s41598-017-07820-6. ”), And since its effect has been demonstrated in this example, GloSensor cAMP (manufactured by Promega) is preferable.

One aspect of the present invention is a thyroid disease diagnostic kit caused by a TSH receptor-stimulating or inhibitory autoantibody, which comprises an animal cell expressing the TSHR variant.

In the present specification, as thyroid disease caused by TSH receptor-stimulating or inhibitory autoantibodies, TSH receptors are stimulated or inhibited by TSH receptor-stimulating autoantibodies or TSH receptor-inhibiting autoantibodies in blood. Any thyroid disease caused by an increase or decrease in the concentration of free thyroid hormone in Can be preferably exemplified.

In the thyroid disease measurement kit according to one aspect of the present invention, commonly used reagents can be appropriately used. As an example, a kit configuration such as an animal cell solution, a luminescent substrate solution, a reaction plate, a standard solution, and a diluent can be considered.

Regarding the details of the cAMP level measurement method, the animal cell preparation method, and the self-antibody measurement kit according to the present invention, in addition to the contents described in the present specification, the documents described in the present specification, particularly Patent Document 4 , And the contents of the documents described in Patent Document 4 can be referred to as appropriate.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.

Example 1. Preparation of TSHR mutant A TSHR mutant in which one amino acid was substituted from wild-type TSHR was prepared according to the method shown below.

1-1. Design of TSHR mutants The following mutations described in Non-Patent Documents 2 to 7 were examined as mutations presumed to affect the activity of TSHR.
R531Q: Non-Patent Document 2
L552V, Y582F, L665V: Non-Patent Document 3
S567A, I568L, M572A: Non-Patent Document 4
D460N, R625A: Non-Patent Document 5
L469P: Non-Patent Document 6
S479A, N483A, D487A: Non-Patent Document 7
The location of each mutation in the protein structure of human TSHR is schematically shown in FIG.

1-2. Preparation of mutant TSHR expression vector 1-1. In order to design artificial primers of 20 to 30 mer on the 3'side and 5'side of the nucleotide corresponding to the mutation point, and to introduce the mutation with the 3'side primer of wild-type hTSHR so as to correspond to the mutation described in 1. Using the 5'side primer to which 1 to 3 bases were added and the vector pcDNA3.1_hTSHR expressing wild-type hTSHR, a point PCR method by Toyobo Co., Ltd. KOD-plus-Mutagenesis Kit (Code No. SMK-101) was applied to the TSHR sequence. The point mutation was introduced. A plasmid was purified from a single colony of Escherichia coli transformed with a point mutation introduction plasmid, and the introduction of a point mutation was confirmed by sequence analysis.

1-3. Acquisition of TSHR mutant transient expression cell line 1.5 × 10 ⁴ human fetal renal cell-derived cell lines (HEK293 cells) (obtained from ECACC [European Collection of Authenticated Cell Cultures], Cat no. 85120602), The cells were seeded on a 6-well multiplate with a D-MEM medium containing 10% FBS and cultured for 24 hours.
1-2. The TSHR variant vector 0.05 μg obtained in the above, the amino acid residues 359 to 544 of firefly luciferase, the cAMP binding region of the regulatory subunit of protein kinase A (PKA), and 4 of firefly luciferase. 2 μg of a plasmid vector (pGloSenso-22F, manufactured by Promega) expressing a protein containing ~ 355th amino acid residue is attached to the GloSensor cAMP Assay (manufactured by Promega) using FuGENE HD Transfection Reagent (Promega). HEK293 cells were transfected according to the protocol of. By the above operation, HEK293 cells that transiently express the TSHR mutant were obtained.

A wild-type human TSHR transient expression strain was also obtained from the wild-type human TSHR expression vector described in 1-2 for use as a control against the TSHR mutant transient expression strain.

Example 2. Measurement of activity of TSHR mutant on NIBSC08 / 204 To HEK293 cells that transiently express the TSHR mutant or wild-type TSHR obtained in Example 1, NIBSC08 / 204, which is an international standard for TSAb, was added to each of them. The S / N ratio of TSHR was measured.

The measurement method is as follows.
[Measurement condition]
After culturing HEK293 cells transiently expressing the TSHR mutant and HEK293 cells transiently expressing the wild-type TSHR for 24 hours, the cells were recovered and 2% (v / v) of GloSensor cAMP Reagent stock solution (manufactured by Promega). ) Was replaced with a polyethylene glycol-containing incubation solution, and the international standard NIBSC08 / 204 of TSAb was added to 0, 31.25, 125, 500, 2000 mIU / L, and the RLU value after 20 minutes of incubation was 96 wells. Measured with a multi-plate luminometer (manufactured by Promega). As an index of the constitutive activity of the TSHR mutant, the 0 mIU / L concentration of 08/204, that is, the RLU value under the condition of adding only the buffer solution was compared.

The RLU of each TSHR mutant without the addition of the international standard NIBSC08 / 204 is shown in FIG. It can be read that the constitutive activity is suppressed in any of the mutants.

The signal-to-noise ratio (Fold change) at each concentration of each TSHR mutant is shown in FIG. 3 and Table 1. In addition, each row in Table 1 shows the added NIBSC08 / 204 concentration, and each column shows the introduced mutation. "WT" indicates wild type. The upper row described as "S / N ratio" is after adding NIBSC08 / 204 to 0, 31.25, 125, 500, 2000 mIU / L in wild-type TSHR and each TSHR variant and incubating for 20 minutes. The lower row showing the S / N ratio and described as "to WT S / N ratio" represents the S / N ratio of each TSHR variant divided by the S / N ratio of the wild type TSHR.

From the above results, the S / N ratio at 2000 mIU / L was higher than that of wild-type TSHR in the mutants having mutations in D460, L469, S479, N483, D487, R531, L552, S567, I568, M572, Y582, R625, and L665. It can be read that is improving.

Among the above mutation points, the mutants having mutations in L469, S479, N483, D487, R531, L552, S567, Y582, R625, and L665 had an S / N ratio of 500 mIU / L and 2000 mIU / L compared to wild-type TSHR. It can be read that the S / N ratio in is improved.

Among the above mutation points, the mutants having mutations in R531, L552, S567, Y582, R625, and L665 are more preferable mutation points because the S / N ratio is improved at all four concentrations. Can be read.

Example 3. Measurement of TSHR mutant activity against Graves'disease-positive serum To HEK293 cells that transiently express the obtained TSHR mutant or wild-type TSHR, Graves' disease-patient-positive serum was added to measure the TSHR activation level of TSHR. did.

In Example 2, 25 μL each of 5 types of TSAbEIA-positive (> 120%) serum and 2 types of TSAbEIA-negative serum diluted 4-fold with a polyethylene glycol-containing incubation solution was added instead of the international standard NIBSC08 / 204. The TSHR activation level (Fold change) of TSHR was measured by the same method as described.

The TSHR activation levels of each TSHR mutant for negative and positive sera are shown in FIG. 4 and Table 2. In Table 2, each row represents the number of the added serum sample, and each column shows the introduced mutation. "WT" indicates that it is a wild-type TSHR. The upper row described as "TSHR activation level" indicates the TSHR activation level in wild-type TSHR and each TSHR mutant, and the lower row described as "vs. WT TSHR activation level" indicates the TSHR activation level of each TSHR mutant. Represents the level divided by the TSHR activation level of wild-type TSHR.

Among the mutants having improved TSHR activation levels in Example 2, mutants having mutations in R531, L552, S567, Y582, R625, and L665 had high TSHR activation levels in at least one concentration range. It can be read that it is.

Among the above mutations, it can be read that the TSHR activation level is improved in all the concentration ranges in the L552V and L665V mutations.

The results of this experimental example relate to the measurement of activity against positive serum of Graves' disease patients. From this result, it can be read that the TSHR mutant having an improved TSHR activation level can be suitably used for the diagnosis of Graves' disease, but on the other hand, the TSHR mutant having an improved TSHR activation level is industrially useful. Does not mean that it is not. This is because there is a possibility that other thyroid diseases such as TSH-producing tumors, pregnancy thyrotoxicosis, hypothyroidism, Hashimoto's disease, etc. can be measured with high sensitivity.

Example 4. Measurement of activity in stable expression strains of TSHR mutants We investigated whether the level of TSHR activation would be higher in cell lines that stably express TSHR mutants as well as transient expression.

Two types of vectors (pGloSensor-22F and pcDNA3.1_hTSHR, or TSHR mutants (S567A, L665V) vectors corresponding to pcDNA3.1_hTSHR) were transfected into HEK293 cells at 2 μg and 0.05 μg, respectively, and Hygromycin B GOLD (invivogen). (Manufactured) 400 μg / mL was added to isolate drug-resistant colonies, and a strain in which an increase in luciferase activity level was observed by adding NIBSC08 / 204, which is an international standard for TSAb, was selected as a stable expression strain. Seven or more stable expression strains were acquired.

In the wild-type TSHR expression strain, the S567A mutant TSHR stable expression strain, and the L665V mutant TSHR stable expression strain, RLU was measured by the same method as in Example 2, and the TSHR activation level was calculated. From the obtained results, the average value and variance of the TSHR activation level of each stable expression strain group were calculated. The result is shown in FIG.

The S567A mutant TSHR stable expression strain and the L665V mutant TSHR stable expression strain have improved TSHR activation levels as well as the transient expression strain, and show significantly higher TSHR activation levels than the wild-type TSHR stable expression strain. rice field.
From this, it was understood that the TSHR mutant strain of the present invention similarly improves the TSHR activation level in both the transient expression strain and the stable expression strain.

Example 5. Measurement of activity of TSHR mutants with multiple mutations against NIBSC08 / 204 In order to investigate how the S / N ratio of TSHR mutants changes when there are multiple mutations, double mutants are used by the method shown below. Was prepared, and the S / N ratio was measured.

Each TSHR double mutant was prepared according to the method described in Example 1 except that the mutation to be introduced was the double mutation shown below.
R531Q / L665V, L552V / L665V, S567A / L665V, Y582F / L665V

The S / N ratio of the TSHR double mutant obtained above, the L665V mutant as a control, and the wild-type TSHR was measured according to the method described in Example 2. The measurement results are shown in Table 3 below. In addition, each row in Table 3 shows the added NIBSC08 / 204 concentration, and each column shows the introduced mutation. "WT" indicates wild type.

The TSHR double mutant had an S / N ratio comparable to or higher than that of the single mutant (L665V). From this, it was understood that the TSHR mutant of the present invention may have a plurality of mutations.

The present invention contributes to the diagnosis and treatment of thyroid diseases caused by TSH receptor-stimulating or inhibitory autoantibodies.

Claims (13)

A TSHR mutant characterized by having a mutation in at least one of the amino acids described below.
D460, L469, S479, N483, D487, R531, L552, S567, I568, M572, Y582, R625, L665 A TSHR mutant characterized by having a mutation in at least one of the amino acids described below.
R531, L552, S567, Y582, R625, L665 The TSHR mutant according to claim 1 or 2, wherein the mutation is a point mutation. The TSHR mutant according to any one of claims 1 to 3, which is characterized by having at least one of the point mutations described below;
D460 (R, N, C, E, Q, G, H, K, S, T),
L469 (A, I, M, F, P, W, Y, V),
S479 (A, I, L, M, F, P, W, Y, V),
N483 (A, I, L, M, F, P, W, Y, V),
D487 (A, I, L, M, F, P, W, Y, V),
R531 (D, N, C, E, Q, G, H, K, S, T),
L552 (A, I, M, F, P, W, Y, V),
S567 (A, I, L, M, F, P, W, Y, V),
I568 (A, L, M, F, P, W, Y, V),
M572 (A, I, L, F, P, W, Y, V),
Y582 (A, I, L, M, F, P, W, V),
R625 (A, I, L, M, F, P, W, Y, V),
L665 (A, I, M, F, P, W, Y, V). The TSHR mutant according to any one of claims 1 to 4, which has at least one of the point mutations described below;
D460 (N, E, Q),
L469 (G, A, V, I, P),
S479 (A, V, L, I, P),
N483 (A, V, L, I, P),
D487 (A, V, L, I, P),
R531 (D, N, E, Q),
L552 (A, V, I, P),
S567 (A, V, L, I, P),
I568 (A, V, L, P),
M572 (A, V, L, I, P),
Y582 (F, W),
R625 (A, V, L, I, P),
L665 (A, V, I, P). The TSHR mutant according to claims 1 to 5, which comprises at least one of the point mutations described below;
D460N, L469P, S479A, N483A, D487A, R531Q, L552V, S567A, I568L, M572A, Y582F, R625A, L665V. The TSHR mutant according to any one of claims 1 to 6, which comprises at least one of the point mutations described below;
R531Q, L552V, S567A, Y582F, R625A, L665V. The TSHR mutant according to any one of claims 1 to 7, which has at least one of the point mutations described below;
S567A, L665V. A polynucleotide encoding the TSHR mutant according to any one of claims 1 to 8. An animal cell expressing the TSHR mutant according to any one of claims 1 to 8. A thyroid disease diagnostic kit containing the animal cell according to claim 10. The thyroid disease diagnostic kit according to claim 11, wherein the animal cells express a cAMP biosensor. The thyroid disease diagnostic kit according to claim 11 or 12, wherein the thyroid disease is Graves' disease.

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