WO2020090668A1 - Chimeric protein for visualizing sugar uptake of cells - Google Patents

Chimeric protein for visualizing sugar uptake of cells Download PDF

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WO2020090668A1
WO2020090668A1 PCT/JP2019/041946 JP2019041946W WO2020090668A1 WO 2020090668 A1 WO2020090668 A1 WO 2020090668A1 JP 2019041946 W JP2019041946 W JP 2019041946W WO 2020090668 A1 WO2020090668 A1 WO 2020090668A1
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protein
chimeric protein
glut4
cells
reporter
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義雄 加藤
祐一 古旗
崇之 秋本
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国立研究開発法人産業技術総合研究所
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to a chimeric protein for visualizing cellular glucose uptake, and an insulin responsiveness evaluation method using the same.
  • Insulin is a hormone secreted by the pancreas and controls blood sugar levels by acting on target organs such as skeletal muscle and adipose tissue to promote glucose uptake.
  • Insulin resistance which is a condition in which the target organ's insulin sensitivity is blunted and the target organ's glucose uptake capacity is reduced, is a rapidly increasing lifestyle-related disease worldwide such as obesity, diabetes, hyperlipidemia, and metabolic syndrome. It is considered to be one of the onset and exacerbation factors of.
  • insulin sensitizers such as pioglitazone or insulin secretagogues such as glinide are used as first-line drugs for insulin resistance, but none of them has been able to improve the prognosis of all patients. Not in.
  • Glucose uptake in skeletal muscle and adipose tissue is carried out via the glucose transporter protein GLUT4.
  • GLUT4 which is stored in intracellular vesicles, translocates to the cell membrane in response to insulin and functions as a transport carrier that takes up sugar into the cell. Insulin resistance is believed to result from the inhibition of translocation of GLUT4 from vesicles to the cell membrane as a result of impaired intracellular insulin signaling.
  • physical exercise is effective in improving insulin resistance.
  • Non-Patent Document 1 a report example in which a Myc tag was genetically inserted into the extracellular domain of GLUT4, and a change in the localization of GLUT4 was observed.
  • Non-Patent Document 1 a report example in which a Myc tag was genetically inserted into the extracellular domain of GLUT4, and a change in the localization of GLUT4 was observed.
  • Non-Patent Document 1 it is necessary to fix the cells and immunostain them in order to detect the Myc tag, and it is not possible to observe the real-time localization change of GLUT4 in the living cells.
  • Non-Patent Document 2 there is a report example in which the intracellular localization of GLUT4 in which the red fluorescent protein mCherry is fused to the C-terminus has been observed (Non-Patent Document 2).
  • a high-resolution imaging equipment capable of reducing background light such as a total internal reflection fluorescence microscope or a confocal fluorescence microscope is used.
  • any of the above methods can be applied only to cultured cells. In order to elucidate the mechanism of mechanical stress-dependent glucose uptake, it is essential to establish a method that can visualize the dynamics of GLUT4 in cells in vivo in real time.
  • the present invention visualizes, in real time, the dynamics of glucose transporter protein in cells, in particular, the dynamics of GLUT4 in contractible living skeletal muscle under conditions where blood flow is maintained in vivo, and insulin dependence And / or independent glucose uptake activity.
  • a chimeric protein having a fluorescent or luminescent reporter inserted in the extracellular domain of a glucose transporter protein can evaluate the sugar uptake activity of living cells.
  • the present invention provides a chimeric protein obtained by fusing a glucose transporter protein and a fluorescent or luminescent reporter, wherein the reporter is an extracellular loop of the glucose transporter protein. And the intensity and / or wavelength of fluorescence or luminescence changes when the chimeric protein is transferred to the cell membrane.
  • the glucose transporter protein is preferably GLUT4.
  • the extracellular loop is preferably extracellular loop 1 located between transmembrane domain 1 and transmembrane domain 2 of the glucose transporter protein.
  • the reporter is preferably a pH-responsive fluorescent protein.
  • the pH-responsive fluorescent protein is preferably pHluorin.
  • the reporter is preferably a pH-responsive luminescent enzyme protein.
  • the pH-responsive luminescent enzyme protein is luciferase.
  • the reporter is preferably a split fluorescent protein fragment or split luminescent enzyme protein fragment.
  • the present invention provides a nucleic acid containing a nucleotide sequence encoding the chimeric protein or an expression vector containing the nucleic acid.
  • the present invention also provides, according to one embodiment, a cell or transgenic non-human animal expressing the chimeric protein.
  • the present invention comprises: (1) exposing cells expressing the chimeric protein to insulin, and (2) detecting fluorescence of the chimeric protein or luminescence of the chimeric protein.
  • the present invention provides a method for evaluating insulin responsiveness, including:
  • the chimeric protein according to the present invention can visualize the dynamics of glucose transporter protein in living cells including skeletal muscle in vivo in real time. Therefore, detailed analysis of the mechanism of glucose uptake in cells and evaluation of insulin responsiveness of cells become possible.
  • FIG. 1 is a schematic diagram showing an outline of a method for evaluating insulin responsiveness using a GLUT4-SEP chimeric protein.
  • FIG. 2 is a diagram showing a three-dimensional structural model of the GLUT4-SEP chimeric protein.
  • FIG. 3 is a diagram showing the primary structures of four types of GLUT4-SEP chimeric proteins prepared in the examples.
  • FIG. 4 is a diagram showing a fluorescence microscope observation image confirming insulin responsiveness of C2C12 cells expressing the GLUT4-SEP chimeric protein.
  • FIG. 5 is a graph showing the results of FIG. 4 by fluorescence intensity.
  • FIG. 6 is a schematic diagram showing the outline of a method for evaluating insulin responsiveness using a GLUT4-split GFP fragment chimeric protein.
  • the present invention provides a chimeric protein in which a glucose transporter protein and a fluorescent or luminescent reporter are fused, wherein the reporter is an extracellular loop of the glucose transporter protein. And the intensity and / or wavelength of fluorescence or luminescence changes when the chimeric protein is transferred to the cell membrane.
  • the “glucose transporter protein” means a GLUT family protein that takes up glucose into cells by facilitated diffusion.
  • the glucose transporter protein in the present embodiment does not include the SGLT family protein that is a sodium-glucose cotransporter.
  • 14 isoforms of GLUT family proteins (hereinafter, also simply referred to as “GLUT”) have been identified, and they are classified into classes I to III based on the sequence similarity. (Class I: GLUT1 to 4 and GLUT14; Class II: GLUT5, 7, 9, 11; Class III: GLUT6, 8, 10, 12, 13). Sequence information for the human GLUT isoform is shown in Table 1.
  • the glucose transporter protein in the present embodiment may be a GLUT of any class / isoform, but is preferably GLUT1, GLUT4 or GLUT12, and particularly preferably GLUT4.
  • the glucose transporter protein in the present embodiment is not limited to the above-mentioned 14 types of GLUT isoforms and the physiological functions equivalent to them (translocation activity to cell membrane and / or sugar uptake activity) are maintained. Examples of these include mutants and homologs thereof. That is, the glucose transporter protein according to the present embodiment is not limited to the GLUT family protein as long as the physiological function (translocation activity to cell membrane and / or sugar uptake activity) equivalent to that of the GLUT family protein is maintained. Proteins consisting of amino acid sequences with 80% or more, preferably 90% or more, more preferably about 95% or more identity can be included. The amino acid sequence identity can be calculated using sequence analysis software or a program commonly used in the art (FASTA, BLAST, etc.).
  • the “fluorescent or luminescent reporter” means a protein or a split fragment thereof that can detect the translocation of the chimeric protein of the present embodiment to the cell membrane based on the change in fluorescence or luminescence. To do.
  • the intensity and / or wavelength of fluorescence or luminescence changes when the chimeric protein of this embodiment migrates to the cell membrane.
  • the reporter in this embodiment is preferably a pH-responsive fluorescent protein.
  • the “pH-responsive fluorescent protein” refers to a fluorescent protein whose fluorescence intensity and / or wavelength changes in response to a change in pH (preferably a change in pH in the range of pH 5.5 to 7.5). means.
  • a pH-sensitive fluorescent protein for example, in addition to GFP and its mutant pHluorin, DsRed and its mutants mNectarine, pHTomato, mOrange, pHoran variant, pHuji, etc. are known (Shen, Y. .Et al., J. Cell Biol., (2014), Vol. 207, No. 3, pp.
  • pH-responsive fluorescent protein that can be used in the present embodiment may include variants that maintain equivalent properties (for example, circular permutation mutant).
  • the pH-responsive fluorescent protein that can be used in the present embodiment is preferably pHluorin, pHoran variant or pHuji, particularly preferably pHluorin, and most preferably super-epictic pHluorin (SEP) (Sankaranayanan, S. et al., Biophys. J., (2000), Vol. 79, No. 4, pp. 2199-2208).
  • the reporter in the present embodiment is preferably a pH-responsive luminescent enzyme protein.
  • PH-responsive luminescent enzyme means an enzyme protein whose bioluminescent activity or chemiluminescent activity is changed in response to a change in pH (preferably a change in the range of pH 5.5 to 7.5).
  • the pH-responsive luminescent enzyme protein that can be used in the present embodiment is not particularly limited, and examples thereof include luciferase, alkaline phosphatase, ⁇ -galactosidase, ⁇ -glucuronidase and the like.
  • Examples of the luminescent substrate for each enzyme protein include luciferin, adamantyl methoxyphosphoryl phenyl dioxetane, adamantyl methoxyphosphoryl phenyl dioxetane galactopyranoside, adamantyl methoxyphosphoryl phenyl dioxetane glucuronide, and the like.
  • the pH-responsive luminescent enzyme protein that can be used in the present embodiment is preferably luciferase, and particularly preferably firefly luciferase.
  • the reporter in this embodiment is preferably a split fluorescent protein fragment or split luminescent enzyme protein fragment.
  • the “split fluorescent protein fragment” or “split luminescent enzyme protein fragment” (hereinafter, both are simply referred to as “split reporter fragment”) is a partial fragment of a fluorescent protein or a luminescent enzyme protein, Although it loses its fluorescence or luminescence activity by itself, it associates with a partial fragment of a fluorescent protein or a luminescent enzyme protein (hereinafter, referred to as “complementary split fragment”) that complements the partial fragment, so that a fluorescent protein or a luminescent enzyme is obtained. Means that the protein is reconstituted to restore fluorescence or luminescence activity.
  • the split reporter fragment in the present embodiment may be one of two split fragments obtained by splitting a fluorescent protein or a luminescent enzyme protein at an arbitrary position, and may include the N-terminal side or the C-terminal side of the original fluorescent protein or luminescent enzyme protein. Any of the split fragments of The split reporter fragment in this embodiment restores fluorescence or luminescence activity when associated with the extracellularly applied complementary split fragment. Therefore, the split reporter fragment and the complementary split fragment thereof in the present embodiment may be fused with an interactive peptide capable of mediating and supporting the association of split reporter fragment / complementary split fragment.
  • Such interacting peptides include a coiled-coil forming peptide capable of forming an antiparallel leucine zipper, a combination of calmodulin (CaM) and M13 peptide, a combination of FKBP and FRB, and the like.
  • the split fluorescent protein fragment in the present embodiment maintains fluorescent proteins such as GFP and its variants (CFP, YFP, etc.), and GFP-like proteins (mCherry, mStrawberry, tdTomato, etc.) and their equivalent properties. It may be a split fragment of a variant (eg circular permutation etc.). Fluorescent proteins have a ⁇ -barrel structure in common, and can preferably be split in loops between ⁇ -strands.
  • GFP has a ⁇ barrel structure composed of 11 ⁇ strands, and includes, for example, a split reporter fragment corresponding to ⁇ strands 1 to 5 and a complementary split fragment corresponding to ⁇ strands 6 to 11.
  • split fluorescent protein fragment in this embodiment is preferably a split GFP fragment, particularly preferably a split GFP fragment corresponding to ⁇ strands 1 to 3, 1 to 6, 1 to 7 or 1 to 8 and corresponding complements.
  • the target split fragments are ⁇ -strands 4-11, 7-11, 8-11 or 9-11, respectively.
  • split fluorescent proteins have already been reported, and they can also be used in the present embodiment.
  • Known split fluorescent proteins that can be used in the present embodiment include, for example, split-mKG (J. Immunol., 2008; 181: 629-640), sfCherry2 (1-10 / 11) and mNG2 (1-10 / 11). ) (Nat. Commun., 2017; 8: 370, doi: 10.10038 / s41467-017-00494-8, doi: 10.4049 / jimmunol.181.1.1629) and the like.
  • the split luminescent enzyme protein fragment in the present embodiment may be, for example, a split fragment such as luciferase or ⁇ -galactosidase.
  • Known split luciferases that can be used in the present embodiment include, for example, split NanoLuc luciferase (HiBiT / LgBiT, SmBit / LgBiT) (ACS Chem. Biol., 2016; 11: 400-8, doi: 10.1021 / acschembio. 5b00753), split Firefly luciferase (Fluc_N1-437 / C438-544) (Anal.
  • the chimeric protein of this embodiment is a fusion of a glucose transporter protein and a fluorescent or luminescent reporter.
  • the reporter is inserted into the extracellular loop of the glucose transporter protein.
  • GLUT which is a glucose transporter protein in the present embodiment, is 12-transmembrane type, has N-terminal and C-terminal located intracellularly, and has extracellular loops 1 to 6.
  • the reporter is one of extracellular loops 1 to 6 of the glucose transporter protein, as long as the physiological function of the glucose transporter protein (translocation activity to cell membrane and / or sugar uptake activity) is maintained. It may be inserted in any of them.
  • the reporter is an extracellular loop 1 located between transmembrane domain 1 and transmembrane domain 2 of glucose transporter protein, or between transmembrane domain 9 and transmembrane domain 10. It is particularly preferred that it can be inserted into the extracellular loop 5 located in the extracellular loop 1 located between the transmembrane domain 1 and the transmembrane domain 2 of the glucose transporter protein.
  • the reporter may be fused to the glucose transporter protein directly or via a linker.
  • the linker may further include an epitope tag such as Myc, HA, FLAG.
  • an epitope tag such as Myc, HA, and FLAG, or a fluorescent protein marker having a fluorescent wavelength different from that of the reporter protein may be added to the N-terminal and / or C-terminal of the chimeric protein of the present embodiment.
  • the chimeric protein of the present embodiment can be expressed in a target cell or non-human animal by introducing a nucleic acid containing a nucleotide sequence encoding an amino acid sequence designed according to the above or an expression vector containing the nucleic acid. ..
  • the present invention is a nucleic acid containing a nucleotide sequence encoding the above chimeric protein.
  • the nucleic acid of the present embodiment can be prepared by any conventionally known genetic engineering method based on the sequence information of glucose transporter protein and reporter protein obtained from a predetermined database or the like.
  • the present invention is an expression vector containing the above nucleic acid.
  • the expression vector of the present embodiment can be prepared by subcloning the nucleic acid of the second embodiment into the expression vector by any conventionally known genetic engineering method.
  • the type of expression vector that can be used in the present embodiment is not particularly limited, and examples thereof include viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, Sendai virus, and plasmids such as pcDNA3.1 and pCMV. It may be a vector.
  • the present invention is a cell expressing the above chimeric protein.
  • the cell in the present embodiment may be any vertebrate cell, but is preferably a mammalian cell such as mouse, rat, rabbit, dog, pig, bovine, non-human primate, human, and the like. Human cells are preferred.
  • the tissue from which the cells are derived is not particularly limited, and may be, for example, skeletal muscle, myocardium, brain, adipocyte, vascular endothelial cell, etc., preferably skeletal muscle or adipose tissue.
  • the cells in the present embodiment may be in vitro isolated from a living body or in vivo in an animal, that is, in vivo.
  • the cells in the present embodiment may be skeletal muscle cells or adipocytes prepared by differentiating pluripotent cells such as iPS cells. Moreover, the cells in the present embodiment may be immortalized. Alternatively, the cells in the present embodiment may be an already established skeletal muscle cell line, adipocyte cell line, or the like.
  • the skeletal muscle cell line for example, C2C12, L6, Sol8 or the like can be used.
  • the adipocyte cell line for example, 3T3-L1, ST13 or the like can be used.
  • the cell of this embodiment can be prepared by introducing the nucleic acid of the second embodiment or the expression vector of the third embodiment into the cell.
  • the nucleic acid or the expression vector can be introduced into cells by a method well known in the art depending on the type of cells, for example, lipofection, microinjection, electroporation and the like.
  • the present invention is a transgenic non-human animal expressing the above chimeric protein.
  • the “animal” of the present embodiment may be cells of any vertebrate except human, but is preferably a mammal such as mouse, rat, rabbit, dog, pig, cow, or non-human primate. Well, particularly preferably mouse or rat.
  • the transgenic non-human animal of this embodiment can be prepared by introducing the nucleic acid of the second embodiment or the expression vector of the third embodiment into the non-human animal by a method well known in the art. For example, by introducing the nucleic acid of the second embodiment or the expression vector of the third embodiment containing a suitable promoter sequence into a fertilized egg by microinjection or the like, a transgenic non-human animal expressing the above chimeric protein is obtained. It can be made.
  • the present invention provides an insulin comprising: (1) exposing insulin to a cell expressing the chimeric protein, and (2) detecting fluorescence or luminescence of the chimeric protein. This is an evaluation method of responsiveness.
  • the “cell” in this embodiment is the same as that defined in the fourth embodiment.
  • cells expressing the chimeric protein are exposed to insulin.
  • the preferred insulin that can be used in this embodiment may be insulin derived from any vertebrate, but is preferably derived from a mammal such as mouse, rat, rabbit, dog, pig, cow, non-human primate, or human. Insulin, particularly preferably human-derived insulin.
  • the insulin in this embodiment can be biosynthesized by a known method. Alternatively, since many insulin preparations are commercially available, they may be used. Preferred commercially available insulin preparations include, for example, glargine, degludec, detemir and the like.
  • the cells When the cells expressing the chimeric protein are in vitro, the cells can be exposed to insulin by adding insulin to the medium.
  • concentration of insulin can be appropriately selected within the range of 10 to 1000 nM, for example.
  • the medium may be selected and used appropriately depending on the cell type.
  • the cells When the cells expressing the chimeric protein are in vivo, the cells can be exposed to insulin by administering the insulin to an animal containing the cells in vivo.
  • the administration route of insulin is not particularly limited, and examples thereof include intravenous administration, intramuscular administration, subcutaneous administration, and intraperitoneal administration.
  • insulin may be administered once or repeatedly, and the dose can be appropriately selected within the range of, for example, 0.1 to 10 units / kg body weight.
  • the chimeric protein used in the present embodiment contains a split reporter fragment as a reporter
  • its complementary split fragment is applied to cells expressing the chimeric protein at the same time as or before exposure to insulin.
  • Complementary split fragments can be prepared by chemical or biosynthesis.
  • the concentration of the complementary split fragment is not particularly limited, but can be appropriately selected, for example, in the range of 1 pM to 10 mM.
  • Fluorescence or luminescence in cells in vitro can be detected by, for example, fluorescence microscopy.
  • fluorescence or luminescence in cells in vivo can be detected by an already established deep-body imaging technique such as a two-photon excitation microscope or IVIS Imaging System (Perkin Elmer).
  • GLUT4-SEP chimeric protein
  • SEP a chimeric protein
  • FIG. 1 The outline of the method for evaluating insulin responsiveness of the present embodiment using a chimeric protein (GLUT4-SEP) in which SEP is inserted into extracellular loop 1 of GLUT4 is shown in FIG.
  • GLUT4-SEP translocates from intracellular GLUT4 vesicles to the cell membrane in response to insulin stimulation.
  • the SEP located in the GLUT4 vesicle is exposed to the outside of the cell.
  • the pH inside the intracellular vesicles is less than 6, whereas the pH outside the cells is around 7.4, the fluorescence intensity of GLUT4-SEP transferred to the cell membrane increases. Therefore, the insulin responsiveness of cells can be evaluated by measuring the degree of change in fluorescence intensity of GLUT4-SEP.
  • FIG. 6 shows an outline of the insulin responsiveness evaluation method of the present embodiment using a chimeric protein (GLUT4-split GFP) in which a split GFP fragment is inserted into the extracellular loop 1 of GLUT4.
  • GLUT4-split GFP translocates from intracellular GLUT4 vesicles to the cell membrane in response to insulin stimulation.
  • the split GFP located in the GLUT4 vesicle is exposed to the outside of the cell and associates with the complementary split GFP fragment outside the cell to reconstitute GFP. Therefore, the insulin responsiveness of cells can be evaluated by measuring the fluorescence of GLUT4-split GFP.
  • SEP super-ecliptic pHluorin
  • Fig. 2 shows a three-dimensional structural model of a chimeric protein in which SEP and Myc tags are inserted in extracellular loop 1 located between transmembrane domain 1 and transmembrane domain 2 of GLUT4. It was predicted that when the SEP and Myc tags were placed in extracellular loop 1, no distortion would occur in the three-dimensional structure of GLUT4.
  • GLUT4-SEP Chimeric Protein Expression Vector a nucleic acid sequence encoding the GLUT4-SEP chimeric protein was prepared by inserting the nucleic acid sequence encoding the SEP gene sequence and the Myc tag into the nucleic acid sequence encoding the extracellular loop 1 of the mouse GLUT4 gene.
  • the SEP gene coding sequence was amplified by PCR and subcloned into the AgeI site of pLenti-myc-GLUT4-mCherry (Addgene, # 64049).
  • a primer containing / not containing a nucleic acid sequence encoding a linker consisting of 8 amino acids (GGSGGSGG) was used, and four types of GLUT4-containing or not containing a linker at the N-terminal and / or C-terminal of SEP were used.
  • An SEP chimeric protein expression vector plasmid DNA was prepared.
  • Table 2 shows the nucleotide sequences of the primers used for PCR. The underline indicates the nucleic acid sequence encoding the linker.
  • the amino acid sequences of the four GLUT4-SEP chimeric proteins are shown below. In the sequence, bold letters indicate SEP, single underline indicates Myc tag, double underline indicates extracellular loop 1 of GLUT4, and italics indicate linker.
  • the primary structure of the prepared GLUT4-SEP chimeric protein is shown in FIG.
  • the expression vector plasmid DNA was introduced into HEK293 cells to transiently express the GLUT4-SEP chimeric protein.
  • HEK293 cells were seeded in a 24-well plate, and after 24 hours, 50 ⁇ L of Opti-MEM1 (Thermo Fisher Scientific), 1.5 ⁇ L of TransIT-293 Reagent (Takara Bio), and 0.5 ⁇ g of expression vector plasmid DNA was added. Then, the cells were cultured in DMEM containing 10% FBS for 24 hours, and the expression of the chimeric protein was confirmed by the fluorescence of mCherry and SEP.
  • C2C12 cells were cultured for 5 days under low serum conditions (2% FBS / DMEM) to induce differentiation into myotube cells.
  • a final concentration of 100 nM insulin was added to the obtained myotube cells, and changes in the fluorescence intensity of SEP and mCherry before and after the addition of insulin were observed by a fluorescence microscope.
  • FIGS. 4 and 5 The results of myotube cells expressing SS-SEP-GLUT4-mCherry are shown in FIGS. 4 and 5.
  • the intensity of SEP-derived green fluorescence was significantly increased, while no significant change was observed in the intensity of mCherry-derived red fluorescence.
  • This result indicates that the GLUT4-SEP chimeric protein is translocated from the intracellular GLUT4 vesicles to the cell membrane, exposing the SEP located in the GLUT4 vesicles ( ⁇ pH6) to the outside of the cell membrane ( ⁇ pH7.4). It was thought to indicate that it was done.

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Abstract

Provided is a chimeric protein formed through fusion of a glucose transporter protein and a fluorescent or luminescent reporter protein having pH responsivity, wherein the reporter protein is inserted in an extracellular loop of the glucose transporter protein.

Description

細胞の糖取り込みを可視化するためのキメラタンパク質Chimeric protein for visualization of cellular glucose uptake
　本発明は、細胞の糖取り込みを可視化するためのキメラタンパク質、およびそれを用いたインスリン応答性の評価方法に関する。 The present invention relates to a chimeric protein for visualizing cellular glucose uptake, and an insulin responsiveness evaluation method using the same.
　インスリンは膵臓から分泌されるホルモンであり、骨格筋や脂肪組織などの標的臓器に作用して糖の取り込みを促進させることにより血糖値を制御する。標的臓器のインスリン感受性が鈍化し、標的臓器の糖取り込み能が低下した状態であるインスリン抵抗性は、肥満、糖尿病、高脂血症、メタボリックシンドロームなどの、世界的に急増している生活習慣病の発症・増悪因子のひとつと考えられている。現在、インスリン抵抗性に対する第一選択薬としては、ピオグリタゾンなどのインスリン抵抗性改善薬またはグリニド薬などのインスリン分泌促進薬が使用されているが、いずれもすべての患者の予後を改善するには至っていない。 Insulin is a hormone secreted by the pancreas and controls blood sugar levels by acting on target organs such as skeletal muscle and adipose tissue to promote glucose uptake. Insulin resistance, which is a condition in which the target organ's insulin sensitivity is blunted and the target organ's glucose uptake capacity is reduced, is a rapidly increasing lifestyle-related disease worldwide such as obesity, diabetes, hyperlipidemia, and metabolic syndrome. It is considered to be one of the onset and exacerbation factors of. Currently, insulin sensitizers such as pioglitazone or insulin secretagogues such as glinide are used as first-line drugs for insulin resistance, but none of them has been able to improve the prognosis of all patients. Not in.
　骨格筋や脂肪組織における糖取り込みは、グルコース輸送体タンパク質であるＧＬＵＴ４を介して行われる。ＧＬＵＴ４は、細胞内の小胞に格納されているが、インスリンに応答して細胞膜へと移行し、糖を細胞内に取り込む輸送担体として機能する。インスリン抵抗性は、細胞内におけるインスリンシグナル伝達が障害された結果として、ＧＬＵＴ４の小胞から細胞膜への移行が阻害されることにより生じると考えられている。一方、身体運動がインスリン抵抗性の改善に有効であることが古くから知られている。本発明者らを含むいくつかのグループによる研究結果は、筋収縮が、インスリンシグナル伝達経路とは独立した異なる経路を介してＧＬＵＴ４の細胞膜への移行を誘導し、糖取り込みを促進する可能性を示唆している。さらに、運動終了後２４時間程度まで、インスリン抵抗性の改善が維持されることが報告されている。しかし、こうしたメカニカルストレス依存性の糖取り込みのメカニズムは、現時点ではほとんど解明されていない。 Glucose uptake in skeletal muscle and adipose tissue is carried out via the glucose transporter protein GLUT4. GLUT4, which is stored in intracellular vesicles, translocates to the cell membrane in response to insulin and functions as a transport carrier that takes up sugar into the cell. Insulin resistance is believed to result from the inhibition of translocation of GLUT4 from vesicles to the cell membrane as a result of impaired intracellular insulin signaling. On the other hand, it has long been known that physical exercise is effective in improving insulin resistance. The results of studies by several groups, including the present inventors, suggest that muscle contraction may induce the translocation of GLUT4 to the plasma membrane via a pathway independent of the insulin signaling pathway and promote glucose uptake. Suggests. Further, it has been reported that improvement of insulin resistance is maintained up to about 24 hours after the end of exercise. However, the mechanism of such mechanical stress-dependent sugar uptake is currently unknown.
　ＧＬＵＴ４を標識することにより、ＧＬＵＴ４の細胞内動態を可視化し、細胞の糖取り込みをモニターしようとする試みがなされている。例えば、ＧＬＵＴ４の細胞外ドメインにＭｙｃタグを遺伝子工学的に挿入し、ＧＬＵＴ４の局在変化を観察した報告例がある（非特許文献１）。しかし、この手法では、Ｍｙｃタグを検出するために細胞を固定して免疫染色する必要があり、生きた状態の細胞におけるＧＬＵＴ４のリアルタイムの局在変化を観察することができない。また、最近では、Ｃ末端に赤色蛍光タンパク質ｍＣｈｅｒｒｙを融合させたＧＬＵＴ４の細胞内局在を観察した報告例がある（非特許文献２）。しかし、全反射照明蛍光顕微鏡や共焦点蛍光顕微鏡のような背景光を低減できる高解像度イメージング設備を使用しない限り、ｍＣｈｅｒｒｙ融合ＧＬＵＴ４の細胞内または細胞膜への局在を識別することは困難である。さらに、上記いずれの手法も、培養細胞にしか適用することができない。メカニカルストレス依存性の糖取り込みのメカニズムを解明するためには、生体内の細胞におけるＧＬＵＴ４の動態をリアルタイムに可視化できる手法の確立が不可欠である。 By labeling GLUT4, attempts have been made to visualize intracellular kinetics of GLUT4 and monitor glucose uptake in cells. For example, there is a report example in which a Myc tag was genetically inserted into the extracellular domain of GLUT4, and a change in the localization of GLUT4 was observed (Non-Patent Document 1). However, in this method, it is necessary to fix the cells and immunostain them in order to detect the Myc tag, and it is not possible to observe the real-time localization change of GLUT4 in the living cells. In addition, recently, there is a report example in which the intracellular localization of GLUT4 in which the red fluorescent protein mCherry is fused to the C-terminus has been observed (Non-Patent Document 2). However, it is difficult to identify the localization of mCherry-fused GLUT4 intracellularly or in the cell membrane unless a high-resolution imaging equipment capable of reducing background light such as a total internal reflection fluorescence microscope or a confocal fluorescence microscope is used. Furthermore, any of the above methods can be applied only to cultured cells. In order to elucidate the mechanism of mechanical stress-dependent glucose uptake, it is essential to establish a method that can visualize the dynamics of GLUT4 in cells in vivo in real time.
　本発明は、生体内において、血流が維持された条件下での、細胞におけるグルコース輸送体タンパク質の動態、特に、収縮可能な生きた骨格筋におけるＧＬＵＴ４の動態をリアルタイムに可視化し、インスリン依存性および／または非依存性の糖取り込み活性を評価することを目的としてなされたものである。 The present invention visualizes, in real time, the dynamics of glucose transporter protein in cells, in particular, the dynamics of GLUT4 in contractible living skeletal muscle under conditions where blood flow is maintained in vivo, and insulin dependence And / or independent glucose uptake activity.
　本発明者らは、鋭意研究の結果、グルコース輸送体タンパク質の細胞外ドメインに蛍光性または発光性のレポーターを挿入したキメラタンパク質により、生きた細胞の糖取り込み活性を評価できることを見出した。 As a result of intensive research, the present inventors have found that a chimeric protein having a fluorescent or luminescent reporter inserted in the extracellular domain of a glucose transporter protein can evaluate the sugar uptake activity of living cells.
　すなわち、本発明は、一実施形態によれば、グルコース輸送体タンパク質と、蛍光性または発光性のレポーターとが融合されてなるキメラタンパク質であって、前記レポーターが前記グルコース輸送体タンパク質の細胞外ループに挿入されており、前記キメラタンパク質が細胞膜に移行すると蛍光または発光の強度および／もしくは波長が変化する、キメラタンパク質を提供するものである。 That is, according to one embodiment, the present invention provides a chimeric protein obtained by fusing a glucose transporter protein and a fluorescent or luminescent reporter, wherein the reporter is an extracellular loop of the glucose transporter protein. And the intensity and / or wavelength of fluorescence or luminescence changes when the chimeric protein is transferred to the cell membrane.
　前記グルコース輸送体タンパク質は、ＧＬＵＴ４であることが好ましい。 The glucose transporter protein is preferably GLUT4.
　前記細胞外ループは、前記グルコース輸送体タンパク質の膜貫通ドメイン１と膜貫通ドメイン２の間に位置する細胞外ループ１であることが好ましい。 The extracellular loop is preferably extracellular loop 1 located between transmembrane domain 1 and transmembrane domain 2 of the glucose transporter protein.
　前記レポーターは、ｐＨ応答性の蛍光タンパク質であることが好ましい。 The reporter is preferably a pH-responsive fluorescent protein.
　前記ｐＨ応答性の蛍光タンパク質は、ｐＨｌｕｏｒｉｎであることが好ましい。 The pH-responsive fluorescent protein is preferably pHluorin.
　あるいは、前記レポーターは、ｐＨ応答性の発光酵素タンパク質であることが好ましい。 Alternatively, the reporter is preferably a pH-responsive luminescent enzyme protein.
　前記ｐＨ応答性の発光酵素タンパク質は、ルシフェラーゼであることが好ましい。 Preferably, the pH-responsive luminescent enzyme protein is luciferase.
　あるいは、前記レポーターは、スプリット蛍光タンパク質断片またはスプリット発光酵素タンパク質断片であることが好ましい。 Alternatively, the reporter is preferably a split fluorescent protein fragment or split luminescent enzyme protein fragment.
　また、本発明は、一実施形態によれば、上記キメラタンパク質をコードするヌクレオチド配列を含む核酸または前記核酸を含む発現ベクターを提供するものである。 Further, according to one embodiment, the present invention provides a nucleic acid containing a nucleotide sequence encoding the chimeric protein or an expression vector containing the nucleic acid.
　また、本発明は、一実施形態によれば、上記キメラタンパク質を発現する細胞またはトランスジェニック非ヒト動物を提供するものである。 The present invention also provides, according to one embodiment, a cell or transgenic non-human animal expressing the chimeric protein.
　また、本発明は、一実施形態によれば、（１）上記キメラタンパク質を発現する細胞にインスリンを曝露するステップと、（２）前記キメラタンパク質の蛍光または前記キメラタンパク質による発光を検出するステップとを含む、インスリン応答性の評価方法を提供するものである。 Moreover, according to one embodiment, the present invention comprises: (1) exposing cells expressing the chimeric protein to insulin, and (2) detecting fluorescence of the chimeric protein or luminescence of the chimeric protein. The present invention provides a method for evaluating insulin responsiveness, including:
　本発明に係るキメラタンパク質は、生体内の骨格筋を含む生きた細胞におけるグルコース輸送体タンパク質の動態をリアルタイムに可視化することができる。そのため、細胞における糖取り込みのメカニズムの詳細な解析や、細胞のインスリン応答性の評価が可能となる。 The chimeric protein according to the present invention can visualize the dynamics of glucose transporter protein in living cells including skeletal muscle in vivo in real time. Therefore, detailed analysis of the mechanism of glucose uptake in cells and evaluation of insulin responsiveness of cells become possible.
図１は、ＧＬＵＴ４－ＳＥＰキメラタンパク質によるインスリン応答性の評価方法の概略を示す模式図である。FIG. 1 is a schematic diagram showing an outline of a method for evaluating insulin responsiveness using a GLUT4-SEP chimeric protein. 図２は、ＧＬＵＴ４－ＳＥＰキメラタンパク質の立体構造モデルを示す図である。FIG. 2 is a diagram showing a three-dimensional structural model of the GLUT4-SEP chimeric protein. 図３は、実施例において作製した４種類のＧＬＵＴ４－ＳＥＰキメラタンパク質の一次構造を示す図である。FIG. 3 is a diagram showing the primary structures of four types of GLUT4-SEP chimeric proteins prepared in the examples. 図４は、ＧＬＵＴ４－ＳＥＰキメラタンパク質を発現させたＣ２Ｃ１２細胞のインスリン応答性を確認した蛍光顕微鏡観察像を示す図である。FIG. 4 is a diagram showing a fluorescence microscope observation image confirming insulin responsiveness of C2C12 cells expressing the GLUT4-SEP chimeric protein. 図５は、図４の結果を蛍光強度により表示したグラフである。FIG. 5 is a graph showing the results of FIG. 4 by fluorescence intensity. 図６は、ＧＬＵＴ４－スプリットＧＦＰ断片キメラタンパク質によるインスリン応答性の評価方法の概略を示す模式図である。FIG. 6 is a schematic diagram showing the outline of a method for evaluating insulin responsiveness using a GLUT4-split GFP fragment chimeric protein.
　以下、本発明を詳細に説明するが、本発明は本明細書中に説明した実施形態に限定されるものではない。 Hereinafter, the present invention will be described in detail, but the present invention is not limited to the embodiments described in the present specification.
　本発明は、第一の実施形態によれば、グルコース輸送体タンパク質と、蛍光性または発光性のレポーターとが融合されてなるキメラタンパク質であって、前記レポーターが前記グルコース輸送体タンパク質の細胞外ループに挿入されており、前記キメラタンパク質が細胞膜に移行すると蛍光または発光の強度および／もしくは波長が変化する、キメラタンパク質である。 According to the first embodiment, the present invention provides a chimeric protein in which a glucose transporter protein and a fluorescent or luminescent reporter are fused, wherein the reporter is an extracellular loop of the glucose transporter protein. And the intensity and / or wavelength of fluorescence or luminescence changes when the chimeric protein is transferred to the cell membrane.
　本実施形態において、「グルコース輸送体タンパク質」とは、促進拡散によってグルコースを細胞内に取り込むＧＬＵＴファミリーのタンパク質を意味する。なお、本実施形態におけるグルコース輸送体タンパク質には、ナトリウム－グルコース共輸送体であるＳＧＬＴファミリーのタンパク質は含まれない。現在のところ、ＧＬＵＴファミリータンパク質（以下、単に「ＧＬＵＴ」とも記載する）の１４種類のアイソフォームが特定されており、それらは、その配列の類似性に基づいて、クラスＩ～ＩＩＩに分類されている（クラスＩ：ＧＬＵＴ１～４およびＧＬＵＴ１４；クラスＩＩ：ＧＬＵＴ５、７、９、１１；クラスＩＩＩ：ＧＬＵＴ６、８、１０、１２、１３）。ヒトＧＬＵＴアイソフォームの配列情報を表１に示す In the present embodiment, the “glucose transporter protein” means a GLUT family protein that takes up glucose into cells by facilitated diffusion. The glucose transporter protein in the present embodiment does not include the SGLT family protein that is a sodium-glucose cotransporter. At present, 14 isoforms of GLUT family proteins (hereinafter, also simply referred to as “GLUT”) have been identified, and they are classified into classes I to III based on the sequence similarity. (Class I: GLUT1 to 4 and GLUT14; Class II: GLUT5, 7, 9, 11; Class III: GLUT6, 8, 10, 12, 13). Sequence information for the human GLUT isoform is shown in Table 1.
　表１．ヒトのＧＬＵＴファミリータンパク質
Figure JPOXMLDOC01-appb-T000001
 Table 1. Human GLUT family proteins
Figure JPOXMLDOC01-appb-T000001
　本実施形態におけるグルコース輸送体タンパク質は、いずれのクラス／アイソフォームのＧＬＵＴであってもよいが、好ましくは、ＧＬＵＴ１、ＧＬＵＴ４またはＧＬＵＴ１２であり、特に好ましくは、ＧＬＵＴ４である。 The glucose transporter protein in the present embodiment may be a GLUT of any class / isoform, but is preferably GLUT1, GLUT4 or GLUT12, and particularly preferably GLUT4.
　また、本実施形態におけるグルコース輸送体タンパク質には、上記１４種類のＧＬＵＴアイソフォームの他、これらと同等の生理機能（細胞膜への移行活性および／または糖取り込み活性）が維持されていることを限度として、これらの変異体やホモログなどが含まれる。すなわち、本実施形態におけるグルコース輸送体タンパク質には、上記ＧＬＵＴファミリータンパク質と同等の生理機能（細胞膜への移行活性および／または糖取り込み活性）が維持されていることを限度として、上記ＧＬＵＴファミリータンパク質と８０％以上、好ましくは９０％以上、より好ましくは約９５％以上の同一性を有するアミノ酸配列からなるタンパク質が包含され得る。アミノ酸配列の同一性は、配列解析ソフトウェアを用いて、または、当分野で慣用のプログラム（ＦＡＳＴＡ、ＢＬＡＳＴなど）を用いて算出することができる。 In addition, the glucose transporter protein in the present embodiment is not limited to the above-mentioned 14 types of GLUT isoforms and the physiological functions equivalent to them (translocation activity to cell membrane and / or sugar uptake activity) are maintained. Examples of these include mutants and homologs thereof. That is, the glucose transporter protein according to the present embodiment is not limited to the GLUT family protein as long as the physiological function (translocation activity to cell membrane and / or sugar uptake activity) equivalent to that of the GLUT family protein is maintained. Proteins consisting of amino acid sequences with 80% or more, preferably 90% or more, more preferably about 95% or more identity can be included. The amino acid sequence identity can be calculated using sequence analysis software or a program commonly used in the art (FASTA, BLAST, etc.).
　本実施形態において、「蛍光性または発光性のレポーター」とは、本実施形態のキメラタンパク質の細胞膜への移行を、蛍光または発光の変化に基づいて検出することができるタンパク質またはそのスプリット断片を意味する。本実施形態における蛍光性または発光性のレポーターは、本実施形態のキメラタンパク質が細胞膜に移行すると、蛍光または発光の強度および／もしくは波長が変化する。 In the present embodiment, the “fluorescent or luminescent reporter” means a protein or a split fragment thereof that can detect the translocation of the chimeric protein of the present embodiment to the cell membrane based on the change in fluorescence or luminescence. To do. In the fluorescent or luminescent reporter of this embodiment, the intensity and / or wavelength of fluorescence or luminescence changes when the chimeric protein of this embodiment migrates to the cell membrane.
　本実施形態におけるレポーターは、好ましくは、ｐＨ応答性の蛍光タンパク質である。「ｐＨ応答性の蛍光タンパク質」とは、ｐＨの変化（好ましくは、ｐＨ５．５～７．５の範囲におけるｐＨの変化）に応答して、その蛍光強度および／または波長が変化する蛍光タンパク質を意味する。そのようなｐＨ感受性蛍光タンパク質としては、例えば、ＧＦＰやその変異体であるｐＨｌｕｏｒｉｎの他、ＤｓＲｅｄやその変異体であるｍＮｅｃｔａｒｉｎｅ、ｐＨＴｏｍａｔｏ、ｍＯｒａｎｇｅ、ｐＨｏｒａｎバリアント、ｐＨｕｊｉなどが知られており（Ｓｈｅｎ，Ｙ．ｅｔ　ａｌ．，Ｊ．Ｃｅｌｌ　Ｂｉｏｌ．，（２０１４），Ｖｏｌ．２０７，Ｎｏ．３，ｐｐ．４１９－４３２）、それらのいずれのｐＨ感受性蛍光タンパク質を本実施形態におけるレポーターとして用いてよい。また、本実施形態において用いることができるｐＨ応答性の蛍光タンパク質には、同等の特性を維持するバリアント（例えば、円順列変異体など）が包含され得る。本実施形態において用いることができるｐＨ応答性の蛍光タンパク質は、好ましくはｐＨｌｕｏｒｉｎ、ｐＨｏｒａｎバリアントまたはｐＨｕｊｉであり、特に好ましくはｐＨｌｕｏｒｉｎであり、最も好ましくは、ｓｕｐｅｒ－ｅｃｌｉｐｔｉｃ　ｐＨｌｕｏｒｉｎ（ＳＥＰ）（Ｓａｎｋａｒａｎａｒａｙａｎａｎ，Ｓ．ｅｔ　ａｌ．，Ｂｉｏｐｈｙｓ．Ｊ．，（２０００），Ｖｏｌ．７９，Ｎｏ．４，ｐｐ．２１９９－２２０８）である。 The reporter in this embodiment is preferably a pH-responsive fluorescent protein. The “pH-responsive fluorescent protein” refers to a fluorescent protein whose fluorescence intensity and / or wavelength changes in response to a change in pH (preferably a change in pH in the range of pH 5.5 to 7.5). means. As such a pH-sensitive fluorescent protein, for example, in addition to GFP and its mutant pHluorin, DsRed and its mutants mNectarine, pHTomato, mOrange, pHoran variant, pHuji, etc. are known (Shen, Y. .Et al., J. Cell Biol., (2014), Vol. 207, No. 3, pp. 419-432), or any of these pH-sensitive fluorescent proteins may be used as the reporter in the present embodiment. In addition, the pH-responsive fluorescent protein that can be used in the present embodiment may include variants that maintain equivalent properties (for example, circular permutation mutant). The pH-responsive fluorescent protein that can be used in the present embodiment is preferably pHluorin, pHoran variant or pHuji, particularly preferably pHluorin, and most preferably super-epictic pHluorin (SEP) (Sankaranayanan, S. et al., Biophys. J., (2000), Vol. 79, No. 4, pp. 2199-2208).
　あるいは、本実施形態におけるレポーターは、好ましくは、ｐＨ応答性の発光酵素タンパク質である。「ｐＨ応答性の発光酵素」とは、ｐＨの変化（好ましくは、ｐＨ５．５～７．５の範囲における変化）に応答して、生物発光活性または化学発光活性が変化する酵素タンパク質を意味する。本実施形態において用いることができるｐＨ応答性の発光酵素タンパク質としては、特に限定されないが、例えば、ルシフェラーゼ、アルカリホスファターゼ、βガラクトシダーゼ、βグルクロニダーゼなどが挙げられる。それぞれの酵素タンパク質に対する発光基質としては、例えば、ルシフェリン、アダマンチルメトキシホスホリルフェニルジオキセタン、アダマンチルメトキシホスホリルフェニルジオキセタンガラクトピラノシド、アダマンチルメトキシホスホリルフェニルジオキセタングルクロニドなどが挙げられる。本実施形態において用いることができるｐＨ応答性の発光酵素タンパク質は、好ましくはルシフェラーゼであり、特に好ましくはホタルルシフェラーゼである。 Alternatively, the reporter in the present embodiment is preferably a pH-responsive luminescent enzyme protein. “PH-responsive luminescent enzyme” means an enzyme protein whose bioluminescent activity or chemiluminescent activity is changed in response to a change in pH (preferably a change in the range of pH 5.5 to 7.5). .. The pH-responsive luminescent enzyme protein that can be used in the present embodiment is not particularly limited, and examples thereof include luciferase, alkaline phosphatase, β-galactosidase, β-glucuronidase and the like. Examples of the luminescent substrate for each enzyme protein include luciferin, adamantyl methoxyphosphoryl phenyl dioxetane, adamantyl methoxyphosphoryl phenyl dioxetane galactopyranoside, adamantyl methoxyphosphoryl phenyl dioxetane glucuronide, and the like. The pH-responsive luminescent enzyme protein that can be used in the present embodiment is preferably luciferase, and particularly preferably firefly luciferase.
　あるいは、本実施形態におけるレポーターは、好ましくは、スプリット蛍光タンパク質断片またはスプリット発光酵素タンパク質断片である。本実施形態において、「スプリット蛍光タンパク質断片」または「スプリット発光酵素タンパク質断片」（以下、両者を単に「スプリットレポーター断片」とも記載する）とは、蛍光タンパク質または発光酵素タンパク質の部分断片であって、それ自体は蛍光または発光活性を失っているが、前記部分断片を補完する蛍光タンパク質または発光酵素タンパク質の部分断片（以下、「相補的スプリット断片」という）と会合することにより、蛍光タンパク質または発光酵素タンパク質が再構成されて、蛍光または発光活性を回復するものを意味する。 Alternatively, the reporter in this embodiment is preferably a split fluorescent protein fragment or split luminescent enzyme protein fragment. In the present embodiment, the “split fluorescent protein fragment” or “split luminescent enzyme protein fragment” (hereinafter, both are simply referred to as “split reporter fragment”) is a partial fragment of a fluorescent protein or a luminescent enzyme protein, Although it loses its fluorescence or luminescence activity by itself, it associates with a partial fragment of a fluorescent protein or a luminescent enzyme protein (hereinafter, referred to as “complementary split fragment”) that complements the partial fragment, so that a fluorescent protein or a luminescent enzyme is obtained. Means that the protein is reconstituted to restore fluorescence or luminescence activity.
　本実施形態におけるスプリットレポーター断片は、蛍光タンパク質または発光酵素タンパク質が任意の位置で分割された２つのスプリット断片の一方であってよく、元の蛍光タンパク質または発光酵素タンパク質のＮ末端側もしくはＣ末端側のスプリット断片のいずれであってもよい。本実施形態におけるスプリットレポーター断片は、細胞外から適用された相補的スプリット断片と会合したとき、蛍光または発光活性を回復する。そのため、本実施形態におけるスプリットレポーター断片およびその相補的スプリット断片は、スプリットレポーター断片／相補的スプリット断片の会合を仲介および支援し得る相互作用ペプチドが融合されていてよい。そのような相互作用ペプチドとしては、例えば、逆平行ロイシンジッパーを形成し得るコイルドコイル形成ペプチドや、カルモジュリン（ＣａＭ）とＭ１３ペプチドの組み合わせや、ＦＫＢＰとＦＲＢの組み合わせなどが挙げられる。 The split reporter fragment in the present embodiment may be one of two split fragments obtained by splitting a fluorescent protein or a luminescent enzyme protein at an arbitrary position, and may include the N-terminal side or the C-terminal side of the original fluorescent protein or luminescent enzyme protein. Any of the split fragments of The split reporter fragment in this embodiment restores fluorescence or luminescence activity when associated with the extracellularly applied complementary split fragment. Therefore, the split reporter fragment and the complementary split fragment thereof in the present embodiment may be fused with an interactive peptide capable of mediating and supporting the association of split reporter fragment / complementary split fragment. Examples of such interacting peptides include a coiled-coil forming peptide capable of forming an antiparallel leucine zipper, a combination of calmodulin (CaM) and M13 peptide, a combination of FKBP and FRB, and the like.
　本実施形態におけるスプリット蛍光タンパク質断片は、例えば、ＧＦＰおよびその改変体（ＣＦＰ、ＹＦＰなど）、ならびにＧＦＰ様タンパク質（ｍＣｈｅｒｒｙ、ｍＳｔｒａｗｂｅｒｒｙ、ｔｄＴｏｍａｔｏなど）などの蛍光タンパク質や、それらと同等の特性を維持するバリアント（例えば、円順列変異体など）のスプリット断片であってよい。蛍光タンパク質は共通してβバレル構造を有しており、好ましくは、βストランド間のループにおいて分割され得る。例えば、ＧＦＰは、１１本のβストランドにより構成されるβバレル構造を有しており、例えば、βストランド１～５に対応するスプリットレポーター断片とβストランド６～１１に対応する相補的スプリット断片とに分割されてよく、または、例えば、βストランド１～１０に対応するスプリットレポーター断片とβストランド１１に対応する相補的スプリット断片とに分割されてもよい。本実施形態におけるスプリット蛍光タンパク質断片は、好ましくはスプリットＧＦＰ断片であり、特に好ましくはβストランド１～３、１～６、１～７または１～８に対応するスプリットＧＦＰ断片であり、対応する相補的スプリット断片は、それぞれ、βストランド４～１１、７～１１、８～１１または９～１１である。 The split fluorescent protein fragment in the present embodiment maintains fluorescent proteins such as GFP and its variants (CFP, YFP, etc.), and GFP-like proteins (mCherry, mStrawberry, tdTomato, etc.) and their equivalent properties. It may be a split fragment of a variant (eg circular permutation etc.). Fluorescent proteins have a β-barrel structure in common, and can preferably be split in loops between β-strands. For example, GFP has a β barrel structure composed of 11 β strands, and includes, for example, a split reporter fragment corresponding to β strands 1 to 5 and a complementary split fragment corresponding to β strands 6 to 11. Or may be split into a split reporter fragment corresponding to β-strands 1-10 and a complementary split fragment corresponding to β-strand 11, for example. The split fluorescent protein fragment in this embodiment is preferably a split GFP fragment, particularly preferably a split GFP fragment corresponding to β strands 1 to 3, 1 to 6, 1 to 7 or 1 to 8 and corresponding complements. The target split fragments are β-strands 4-11, 7-11, 8-11 or 9-11, respectively.
　また、種々のスプリット蛍光タンパク質がすでに報告されており、本実施形態においては、それらを用いることもできる。本実施形態において使用できる既知のスプリット蛍光タンパク質としては、例えば、ｓｐｌｉｔ－ｍＫＧ（Ｊ．Ｉｍｍｕｎｏｌ．，２００８；１８１：６２９－６４０）、ｓｆＣｈｅｒｒｙ２（１－１０／１１）およびｍＮＧ２（１－１０／１１）（Ｎａｔ．Ｃｏｍｍｕｎ．，２０１７；８：３７０，ｄｏｉ：１０．１０３８／ｓ４１４６７－０１７－００４９４－８，ｄｏｉ：１０．４０４９／ｊｉｍｍｕｎｏｌ．１８１．１．６２９）などが挙げられる。 Also, various split fluorescent proteins have already been reported, and they can also be used in the present embodiment. Known split fluorescent proteins that can be used in the present embodiment include, for example, split-mKG (J. Immunol., 2008; 181: 629-640), sfCherry2 (1-10 / 11) and mNG2 (1-10 / 11). ) (Nat. Commun., 2017; 8: 370, doi: 10.10038 / s41467-017-00494-8, doi: 10.4049 / jimmunol.181.1.1629) and the like.
　本実施形態におけるスプリット発光酵素タンパク質断片は、例えば、ルシフェラーゼやβガラクトシダーゼなどのスプリット断片であってよい。本実施形態において使用できる既知のスプリットルシフェラーゼとしては、例えば、スプリットＮａｎｏＬｕｃルシフェラーゼ（ＨｉＢｉＴ／ＬｇＢｉＴ，ＳｍＢｉｔ／ＬｇＢｉＴ）（ＡＣＳ　Ｃｈｅｍ．Ｂｉｏｌ．，２０１６；１１：４００－８，ｄｏｉ：１０．１０２１／ａｃｓｃｈｅｍｂｉｏ．５ｂ００７５３）、スプリットＦｉｒｅｆｌｙルシフェラーゼ（Ｆｌｕｃ＿Ｎ１－４３７／Ｃ４３８－５４４）（Ａｎａｌ．Ｃｈｅｍ．，２００１；７３：２５１６－２１，ｄｏｉ：１０．１０２１／ａｃ００１３２９６）、スプリットＲｅｎｉｌｌａルシフェラーゼ（ｓＲＬ９１）（Ａｎａｌ．Ｃｈｅｍ．，２００３；７５：４１７６－８１，ｄｏｉ：１０．１０２１／ａｃ０３００８００）、スプリットＥｍｅｒａｌｄルシフェラーゼ（ＥＬｕｃ＿Ｎ１－４１５／Ｃ３９４－５４２）（Ａｎａｌ．Ｃｈｅｍ．，２０１０；８２：２５５２－６０，ｄｏｉ：１０．１０２１／ａｃ１００１０４ｑ）、スプリットＧａｕｓｓｉａルシフェラーゼ（ＧＬｕｃ＿Ｎ１－９３／Ｃ９４－１６９）（Ｎａｔ．Ｍｅｔｈｏｄｓ，２００６；３：９７７－９，ｄｏｉ：１０．１０３８／ｎｍｅｔｈ９７９）が挙げられる。βガラクトシダーゼのスプリット断片／相補的スプリット断片としては、αフラグメント／ωフラグメントを用いることができる（α相補性）。 The split luminescent enzyme protein fragment in the present embodiment may be, for example, a split fragment such as luciferase or β-galactosidase. Known split luciferases that can be used in the present embodiment include, for example, split NanoLuc luciferase (HiBiT / LgBiT, SmBit / LgBiT) (ACS Chem. Biol., 2016; 11: 400-8, doi: 10.1021 / acschembio. 5b00753), split Firefly luciferase (Fluc_N1-437 / C438-544) (Anal. Chem., 2001; 73: 2516-21, doi: 10.1021 / ac0013296), split Renilla luciferase (sRL91) (Anal. Chem.,. 2003; 75: 4176-81, doi: 10.1021 / ac0300800), split Emerald luciferase ( Luc_N1-415 / C394-542) (Anal. Chem., 2010; 82: 2552-60, doi: 10.1021 / ac100104q), split Gaussia luciferase (GLuc_N1-93 / C94-169) (Nat. Methods, 2006; 3: 977-9, doi: 10.1038 / nmeth979). As the β-galactosidase split fragment / complementary split fragment, α fragment / ω fragment can be used (α complementarity).
　本実施形態のキメラタンパク質は、グルコース輸送体タンパク質と、蛍光性または発光性のレポーターとが融合されてなる。ここで、レポーターは、グルコース輸送体タンパク質の細胞外ループに挿入される。本実施形態におけるグルコース輸送体タンパク質であるＧＬＵＴは、１２回膜貫通型であり、Ｎ末端およびＣ末端が細胞内に位置しており、細胞外ループ１～６を有する。本実施形態のキメラタンパク質において、レポーターは、グルコース輸送体タンパク質の生理機能（細胞膜への移行活性および／または糖取り込み活性）が維持される限りにおいて、グルコース輸送体タンパク質の細胞外ループ１～６のいずれに挿入されてもよい。好ましくは、本実施形態のキメラタンパク質において、レポーターは、グルコース輸送体タンパク質の膜貫通ドメイン１と膜貫通ドメイン２の間に位置する細胞外ループ１、または膜貫通ドメイン９と膜貫通ドメイン１０の間に位置する細胞外ループ５に挿入されることができ、グルコース輸送体タンパク質の膜貫通ドメイン１と膜貫通ドメイン２の間に位置する細胞外ループ１に挿入されることが特に好ましい。 The chimeric protein of this embodiment is a fusion of a glucose transporter protein and a fluorescent or luminescent reporter. Here, the reporter is inserted into the extracellular loop of the glucose transporter protein. GLUT, which is a glucose transporter protein in the present embodiment, is 12-transmembrane type, has N-terminal and C-terminal located intracellularly, and has extracellular loops 1 to 6. In the chimeric protein of the present embodiment, the reporter is one of extracellular loops 1 to 6 of the glucose transporter protein, as long as the physiological function of the glucose transporter protein (translocation activity to cell membrane and / or sugar uptake activity) is maintained. It may be inserted in any of them. Preferably, in the chimeric protein of this embodiment, the reporter is an extracellular loop 1 located between transmembrane domain 1 and transmembrane domain 2 of glucose transporter protein, or between transmembrane domain 9 and transmembrane domain 10. It is particularly preferred that it can be inserted into the extracellular loop 5 located in the extracellular loop 1 located between the transmembrane domain 1 and the transmembrane domain 2 of the glucose transporter protein.
　本実施形態のキメラタンパク質において、レポーターは、直接またはリンカーを介してグルコース輸送体タンパク質と融合されてよい。また、リンカーを用いる場合には、リンカー中に、Ｍｙｃ、ＨＡ、ＦＬＡＧなどのエピトープタグをさらに含んでもよい。さらに、本実施形態のキメラタンパク質のＮ末端および／またはＣ末端に、Ｍｙｃ、ＨＡ、ＦＬＡＧなどのエピトープタグや、レポータータンパク質とは異なる蛍光波長の蛍光タンパク質マーカーが付加されていてもよい。 In the chimeric protein of this embodiment, the reporter may be fused to the glucose transporter protein directly or via a linker. When a linker is used, the linker may further include an epitope tag such as Myc, HA, FLAG. Furthermore, an epitope tag such as Myc, HA, and FLAG, or a fluorescent protein marker having a fluorescent wavelength different from that of the reporter protein may be added to the N-terminal and / or C-terminal of the chimeric protein of the present embodiment.
　本実施形態のキメラタンパク質は、上記にしたがって設計されたアミノ酸配列をコードするヌクレオチド配列を含む核酸または前記核酸を含む発現ベクターを導入することにより、目的の細胞または非ヒト動物に発現させることができる。 The chimeric protein of the present embodiment can be expressed in a target cell or non-human animal by introducing a nucleic acid containing a nucleotide sequence encoding an amino acid sequence designed according to the above or an expression vector containing the nucleic acid. ..
　すなわち、本発明は、第二の実施形態によれば、上記キメラタンパク質をコードするヌクレオチド配列を含む核酸である。本実施形態の核酸は、所定のデータベースなどから入手したグルコース輸送体タンパク質およびレポータータンパク質の配列情報を元に、従来公知の任意の遺伝子工学的方法により調製することができる。 That is, according to the second embodiment, the present invention is a nucleic acid containing a nucleotide sequence encoding the above chimeric protein. The nucleic acid of the present embodiment can be prepared by any conventionally known genetic engineering method based on the sequence information of glucose transporter protein and reporter protein obtained from a predetermined database or the like.
　本発明は、第三の実施形態によれば、上記核酸を含む発現ベクターである。本実施形態の発現ベクターは、従来公知の任意の遺伝子工学的方法により、第二の実施形態の核酸を発現ベクターにサブクローニングして調製することができる。本実施形態において用いることができる発現ベクターの種類は、特に限定されないが、例えば、レトロウイルス、レンチウイルス、アデノウイルス、アデノ随伴ウイルス、センダイウイルスなどのウイルスベクターや、ｐｃＤＮＡ３．１、ｐＣＭＶなどのプラスミドベクターであってよい。 According to the third embodiment, the present invention is an expression vector containing the above nucleic acid. The expression vector of the present embodiment can be prepared by subcloning the nucleic acid of the second embodiment into the expression vector by any conventionally known genetic engineering method. The type of expression vector that can be used in the present embodiment is not particularly limited, and examples thereof include viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, Sendai virus, and plasmids such as pcDNA3.1 and pCMV. It may be a vector.
　本発明は、第四の実施形態によれば、上記キメラタンパク質を発現する細胞である。 According to the fourth embodiment, the present invention is a cell expressing the above chimeric protein.
　本実施形態における細胞は、任意の脊椎動物の細胞であってよいが、好ましくは、マウス、ラット、ウサギ、イヌ、ブタ、ウシ、非ヒト霊長類、ヒトなどの哺乳動物の細胞であり、特に好ましくはヒトの細胞である。細胞が由来する組織も特に限定されず、例えば、骨格筋、心筋、脳、脂肪細胞、血管内皮細胞などであってよく、好ましくは、骨格筋または脂肪組織である。本実施形態における細胞は、生体から単離されたインビトロのものであってもよいし、動物の生体内の、すなわちインビボのものであってもよい。 The cell in the present embodiment may be any vertebrate cell, but is preferably a mammalian cell such as mouse, rat, rabbit, dog, pig, bovine, non-human primate, human, and the like. Human cells are preferred. The tissue from which the cells are derived is not particularly limited, and may be, for example, skeletal muscle, myocardium, brain, adipocyte, vascular endothelial cell, etc., preferably skeletal muscle or adipose tissue. The cells in the present embodiment may be in vitro isolated from a living body or in vivo in an animal, that is, in vivo.
　本実施形態における細胞は、ｉＰＳ細胞などの多能性細胞を分化させて調製した骨格筋細胞や脂肪細胞などであってもよい。また、本実施形態における細胞は、不死化されていてもよい。あるいは、本実施形態における細胞は、すでに確立された骨格筋細胞株や脂肪細胞株などであってもよい。骨格筋細胞株としては、例えば、Ｃ２Ｃ１２、Ｌ６、Ｓｏｌ８などを用いることができる。また、脂肪細胞株としては、例えば、３Ｔ３－Ｌ１、ＳＴ１３などを用いることができる。 The cells in the present embodiment may be skeletal muscle cells or adipocytes prepared by differentiating pluripotent cells such as iPS cells. Moreover, the cells in the present embodiment may be immortalized. Alternatively, the cells in the present embodiment may be an already established skeletal muscle cell line, adipocyte cell line, or the like. As the skeletal muscle cell line, for example, C2C12, L6, Sol8 or the like can be used. As the adipocyte cell line, for example, 3T3-L1, ST13 or the like can be used.
　本実施形態の細胞は、第二の実施形態の核酸または第三の実施形態の発現ベクターを細胞に導入することにより調製することができる。核酸または発現ベクターの細胞への導入は、細胞の種類に応じて、当分野において周知の方法により行うことができ、例えば、リポフェクション、マイクロインジェクション、エレクトロポレーションなどにより行うことができる。 The cell of this embodiment can be prepared by introducing the nucleic acid of the second embodiment or the expression vector of the third embodiment into the cell. The nucleic acid or the expression vector can be introduced into cells by a method well known in the art depending on the type of cells, for example, lipofection, microinjection, electroporation and the like.
　本発明は、第五の実施形態によれば、上記キメラタンパク質を発現するトランスジェニック非ヒト動物である。 According to a fifth embodiment, the present invention is a transgenic non-human animal expressing the above chimeric protein.
　本実施形態の「動物」は、ヒトを除く任意の脊椎動物の細胞であってよいが、好ましくは、マウス、ラット、ウサギ、イヌ、ブタ、ウシ、非ヒト霊長類などの哺乳動物であってよく、特に好ましくは、マウスまたはラットである。 The “animal” of the present embodiment may be cells of any vertebrate except human, but is preferably a mammal such as mouse, rat, rabbit, dog, pig, cow, or non-human primate. Well, particularly preferably mouse or rat.
　本実施形態のトランスジェニック非ヒト動物は、第二の実施形態の核酸または第三の実施形態の発現ベクターを、当分野において周知の方法により非ヒト動物に導入して調製することができる。例えば、適切なプロモーター配列を含む第二の実施形態の核酸または第三の実施形態の発現ベクターを、マイクロインジェクションなどにより受精卵に導入することにより、上記キメラタンパク質を発現するトランスジェニック非ヒト動物を作製することができる。 The transgenic non-human animal of this embodiment can be prepared by introducing the nucleic acid of the second embodiment or the expression vector of the third embodiment into the non-human animal by a method well known in the art. For example, by introducing the nucleic acid of the second embodiment or the expression vector of the third embodiment containing a suitable promoter sequence into a fertilized egg by microinjection or the like, a transgenic non-human animal expressing the above chimeric protein is obtained. It can be made.
　本発明は、第六の実施形態によれば、（１）上記キメラタンパク質を発現する細胞にインスリンを曝露するステップと、（２）前記キメラタンパク質の蛍光または発光を検出するステップとを含む、インスリン応答性の評価方法である。 According to a sixth embodiment, the present invention provides an insulin comprising: (1) exposing insulin to a cell expressing the chimeric protein, and (2) detecting fluorescence or luminescence of the chimeric protein. This is an evaluation method of responsiveness.
　本実施形態における「細胞」は、第四の実施形態において定義したものと同様である。 The “cell” in this embodiment is the same as that defined in the fourth embodiment.
　本実施形態の方法では、上記キメラタンパク質を発現する細胞にインスリンを曝露する。本実施形態において使用できる好ましいインスリンは、任意の脊椎動物由来のインスリンであってよいが、好ましくは、マウス、ラット、ウサギ、イヌ、ブタ、ウシ、非ヒト霊長類、ヒトなどの哺乳動物由来のインスリンであり、特に好ましくはヒト由来のインスリンである。本実施形態におけるインスリンは、公知の手法により生合成することができる。あるいは、インスリン製剤が多数市販されているため、それらを用いてもよい。好ましい市販のインスリン製剤としては、例えば、グラルギン、デグルデク、デテミルなどが挙げられる。 In the method of the present embodiment, cells expressing the chimeric protein are exposed to insulin. The preferred insulin that can be used in this embodiment may be insulin derived from any vertebrate, but is preferably derived from a mammal such as mouse, rat, rabbit, dog, pig, cow, non-human primate, or human. Insulin, particularly preferably human-derived insulin. The insulin in this embodiment can be biosynthesized by a known method. Alternatively, since many insulin preparations are commercially available, they may be used. Preferred commercially available insulin preparations include, for example, glargine, degludec, detemir and the like.
　キメラタンパク質を発現する細胞がインビトロのものである場合には、培地にインスリンを添加することにより、前記細胞にインスリンを曝露することができる。インスリンの濃度は、例えば、１０～１０００ｎＭの範囲で適宜選択することができる。なお、培地は、細胞の種類に応じて適切なものを選択して使用すればよい。 When the cells expressing the chimeric protein are in vitro, the cells can be exposed to insulin by adding insulin to the medium. The concentration of insulin can be appropriately selected within the range of 10 to 1000 nM, for example. The medium may be selected and used appropriately depending on the cell type.
　キメラタンパク質を発現する細胞がインビボのものである場合には、前記細胞を生体内に含む動物にインスリンを投与することにより、前記細胞にインスリンを曝露することができる。インスリンの投与経路は、特に限定されないが、例えば、静脈内投与、筋肉内投与、皮下投与、腹腔内投与などが挙げられる。また、インスリンは、単回または反復投与されてよく、投与量は、例えば、０．１～１０単位／体重ｋｇの範囲で適宜選択することができる。 When the cells expressing the chimeric protein are in vivo, the cells can be exposed to insulin by administering the insulin to an animal containing the cells in vivo. The administration route of insulin is not particularly limited, and examples thereof include intravenous administration, intramuscular administration, subcutaneous administration, and intraperitoneal administration. In addition, insulin may be administered once or repeatedly, and the dose can be appropriately selected within the range of, for example, 0.1 to 10 units / kg body weight.
　本実施形態において用いられるキメラタンパク質が、レポーターとしてスプリットレポーター断片を含むものである場合には、その相補的スプリット断片を、インスリンの曝露と同時またはそれより前に、キメラタンパク質を発現する細胞に適用すればよい。相補的スプリット断片は、化学合成または生合成により調製することができる。相補的スプリット断片の濃度は、特に限定されないが、例えば、１ｐＭ～１０ｍＭの範囲で適宜選択することができる。 When the chimeric protein used in the present embodiment contains a split reporter fragment as a reporter, its complementary split fragment is applied to cells expressing the chimeric protein at the same time as or before exposure to insulin. Good. Complementary split fragments can be prepared by chemical or biosynthesis. The concentration of the complementary split fragment is not particularly limited, but can be appropriately selected, for example, in the range of 1 pM to 10 mM.
　次いで、キメラタンパク質の蛍光またはキメラタンパク質による発光を検出する。インビトロの細胞における蛍光または発光は、例えば蛍光顕微鏡観察などにより検出することができる。また、インビボの細胞における蛍光または発光は、すでに確立された生体深部イメージング手法、例えば、二光子励起顕微鏡やＩＶＩＳ　Ｉｍａｇｉｎｇ　Ｓｙｓｔｅｍ（パーキンエルマー）などにより検出することができる。 Next, the fluorescence of the chimeric protein or the luminescence of the chimeric protein is detected. Fluorescence or luminescence in cells in vitro can be detected by, for example, fluorescence microscopy. In addition, fluorescence or luminescence in cells in vivo can be detected by an already established deep-body imaging technique such as a two-photon excitation microscope or IVIS Imaging System (Perkin Elmer).
　ＧＬＵＴ４の細胞外ループ１にＳＥＰを挿入したキメラタンパク質（ＧＬＵＴ４－ＳＥＰ）を用いた、本実施形態のインスリン応答性の評価方法の概略を図１に示す。細胞にインスリンを曝露すると、ＧＬＵＴ４－ＳＥＰは、インスリン刺激に応答して、細胞内のＧＬＵＴ４小胞から細胞膜へと移行する。それに伴い、ＧＬＵＴ４小胞内に位置していたＳＥＰは、細胞外へと露出される。ここで、細胞内小胞の内部はｐＨ６未満であるのに対し、細胞外のｐＨは７．４前後であることから、細胞膜へと移行したＧＬＵＴ４－ＳＥＰの蛍光強度が増大する。したがって、ＧＬＵＴ４－ＳＥＰの蛍光強度変化の程度を測定することにより、細胞のインスリン応答性を評価することができる。 The outline of the method for evaluating insulin responsiveness of the present embodiment using a chimeric protein (GLUT4-SEP) in which SEP is inserted into extracellular loop 1 of GLUT4 is shown in FIG. Upon exposure of cells to insulin, GLUT4-SEP translocates from intracellular GLUT4 vesicles to the cell membrane in response to insulin stimulation. Along with that, the SEP located in the GLUT4 vesicle is exposed to the outside of the cell. Here, while the pH inside the intracellular vesicles is less than 6, whereas the pH outside the cells is around 7.4, the fluorescence intensity of GLUT4-SEP transferred to the cell membrane increases. Therefore, the insulin responsiveness of cells can be evaluated by measuring the degree of change in fluorescence intensity of GLUT4-SEP.
　また、ＧＬＵＴ４の細胞外ループ１にスプリットＧＦＰ断片を挿入したキメラタンパク質（ＧＬＵＴ４－スプリットＧＦＰ）を用いた、本実施形態のインスリン応答性の評価方法の概略を図６に示す。細胞にインスリンを曝露すると、ＧＬＵＴ４－スプリットＧＦＰは、インスリン刺激に応答して、細胞内のＧＬＵＴ４小胞から細胞膜へと移行する。それに伴い、ＧＬＵＴ４小胞内に位置していたスプリットＧＦＰは細胞外へと露出され、細胞外において相補的スプリットＧＦＰ断片と会合し、ＧＦＰを再構成する。したがって、ＧＬＵＴ４－スプリットＧＦＰの蛍光を測定することにより、細胞のインスリン応答性を評価することができる。 Further, FIG. 6 shows an outline of the insulin responsiveness evaluation method of the present embodiment using a chimeric protein (GLUT4-split GFP) in which a split GFP fragment is inserted into the extracellular loop 1 of GLUT4. Upon exposure of cells to insulin, GLUT4-split GFP translocates from intracellular GLUT4 vesicles to the cell membrane in response to insulin stimulation. Accordingly, the split GFP located in the GLUT4 vesicle is exposed to the outside of the cell and associates with the complementary split GFP fragment outside the cell to reconstitute GFP. Therefore, the insulin responsiveness of cells can be evaluated by measuring the fluorescence of GLUT4-split GFP.
　以下に実施例を挙げ、本発明についてさらに説明する。なお、これらは本発明を何ら限定するものではない。 The present invention will be further described with reference to examples. These do not limit the present invention in any way.
＜１．ＧＬＵＴ４－ＳＥＰキメラタンパク質の立体構造モデル＞
　グルコース輸送体としてマウスＧＬＵＴ４、レポーターとしてｓｕｐｅｒ－ｅｃｌｉｐｔｉｃ　ｐＨｌｕｏｒｉｎ（ＳＥＰ）を用いて、グルコース輸送体－レポーターキメラタンパク質を作製するにあたり、キメラタンパク質の立体構造モデルを構築し、ＳＥＰを挿入するのに最適なＧＬＵＴ４の部位を予測した。最初に、ＧＬＵＴ４との配列相同性を有し、かつ、立体構造が既知であるヒトＧＬＵＴ３のデータ（ＰＤＢ　ＩＤ：４ＺＷ９）を、ＰｙＭｏｌソフトウェア（ｈｔｔｐｓ：／／ｐｙｍｏｌ．ｏｒｇ／）で表示することにより、細胞膜外ドメインの候補をリスト化した。ヒトＧＬＵＴ３とマウスＧＬＵＴ４のアミノ酸配列を比較し、立体構造の割り当てを行ったところ、ＧＬＵＴ４の膜貫通ドメイン１（ＶＬＡＶＦＳＡＶＬＧＳＬＱＦＧＹＮＩＧＶＩＮＡＰ：配列番号１）と膜貫通ドメイン２（ＴＬＴＴＬＷＡＬＳＶＡＩＦＳＶＧＧＭＩＳＳＦＬＩＧＩＩ：配列番号２）の間に位置する細胞外ループ１（ＧＲＱＧＰＧＧＰＤＳＩＰＱＧ：配列番号３）において最も柔軟な構造が見られた。なお、構造の柔軟性は、αヘリックスの形成を妨げることが知られるグリシン残基およびプロリン残基に基づいて推定された。特に、グリシン残基が連続する部位において構造の柔軟性が最も高くなることから、この位置にＳＥＰを挿入することが最適であることが推察された。 <1. Three-dimensional structural model of GLUT4-SEP chimeric protein>
Optimal for constructing a three-dimensional structure model of a chimeric protein and inserting SEP when preparing a glucose transporter-reporter chimeric protein using mouse GLUT4 as a glucose transporter and super-ecliptic pHluorin (SEP) as a reporter The site of GLUT4 was predicted. First, by displaying the data (PDB ID: 4ZW9) of human GLUT3 having sequence homology with GLUT4 and having a known three-dimensional structure by PyMol software (https://pymol.org/). , A list of extracellular domain candidates was listed. When the amino acid sequences of human GLUT3 and mouse GLUT4 were compared and the three-dimensional structure was assigned, it was located between transmembrane domain 1 (VLAVFSAVLGSLQFGYNIGVINAP: SEQ ID NO: 1) and transmembrane domain 2 (TLTTLWALSVAIFSVGMISSFLIGII: SEQ ID NO: 2) of GLUT4. The most flexible structure was found in the extracellular loop 1 (GRQGPGPGPDSIPQG: SEQ ID NO: 3) that operates. The structural flexibility was estimated based on glycine and proline residues that are known to prevent the formation of α-helix. In particular, since the structural flexibility is highest at the site where glycine residues are continuous, it was speculated that inserting SEP at this position is optimal.
　ＧＬＵＴ４の膜貫通ドメイン１と膜貫通ドメイン２の間に位置する細胞外ループ１にＳＥＰおよびＭｙｃタグを挿入したキメラタンパク質の立体構造モデルを図２に示す。ＳＥＰおよびＭｙｃタグを細胞外ループ１に配置した場合には、ＧＬＵＴ４の立体構造に歪みが生じないものと予測された。 Fig. 2 shows a three-dimensional structural model of a chimeric protein in which SEP and Myc tags are inserted in extracellular loop 1 located between transmembrane domain 1 and transmembrane domain 2 of GLUT4. It was predicted that when the SEP and Myc tags were placed in extracellular loop 1, no distortion would occur in the three-dimensional structure of GLUT4.
＜２．ＧＬＵＴ４－ＳＥＰキメラタンパク質発現ベクターの構築＞
　以下の手順により、マウスＧＬＵＴ４遺伝子の細胞外ループ１をコードする核酸配列中に、ＳＥＰ遺伝子配列およびＭｙｃタグをコードする核酸配列を挿入し、ＧＬＵＴ４－ＳＥＰキメラタンパク質をコードする核酸を調製した。ｐＣＩ－ＳＥＰ－ＧｌｕＲ１（Ａｄｄｇｅｎｅ，＃２４０００）を鋳型として、ＳＥＰ遺伝子コード配列をＰＣＲにより増幅して、ｐＬｅｎｔｉ－ｍｙｃ－ＧＬＵＴ４－ｍＣｈｅｒｒｙ（Ａｄｄｇｅｎｅ，＃６４０４９）のＡｇｅＩサイトにサブクローニングした。ＰＣＲには、８アミノ酸からなるリンカー（ＧＧＳＧＧＳＧＧ）をコードする核酸配列を含む／含まないプライマーを用いて、ＳＥＰのＮ末端および／またはＣ末端にリンカーを含む、または含まない、４種類のＧＬＵＴ４－ＳＥＰキメラタンパク質の発現ベクタープラスミドＤＮＡを調製した。ＰＣＲに用いたプライマーのヌクレオチド配列を表２に示す。なお、下線はリンカーをコードする核酸配列を示す。 <2. Construction of GLUT4-SEP Chimeric Protein Expression Vector>
By the following procedure, a nucleic acid sequence encoding the GLUT4-SEP chimeric protein was prepared by inserting the nucleic acid sequence encoding the SEP gene sequence and the Myc tag into the nucleic acid sequence encoding the extracellular loop 1 of the mouse GLUT4 gene. Using the pCI-SEP-GluR1 (Addgene, # 24000) as a template, the SEP gene coding sequence was amplified by PCR and subcloned into the AgeI site of pLenti-myc-GLUT4-mCherry (Addgene, # 64049). For PCR, a primer containing / not containing a nucleic acid sequence encoding a linker consisting of 8 amino acids (GGSGGSGG) was used, and four types of GLUT4-containing or not containing a linker at the N-terminal and / or C-terminal of SEP were used. An SEP chimeric protein expression vector plasmid DNA was prepared. Table 2 shows the nucleotide sequences of the primers used for PCR. The underline indicates the nucleic acid sequence encoding the linker.
　表２．ＰＣＲのためのプライマー
Figure JPOXMLDOC01-appb-T000002
 Table 2. PCR primers
Figure JPOXMLDOC01-appb-T000002
　４種類のＧＬＵＴ４－ＳＥＰキメラタンパク質（ｍＣｈｅｒｒｙの配列を除く）のアミノ酸配列を以下に示す。なお、配列中、太字はＳＥＰ、一重下線はＭｙｃタグ、二重下線はＧＬＵＴ４の細胞外ループ１、斜体はリンカーを示す。また、作製したＧＬＵＴ４－ＳＥＰキメラタンパク質の一次構造を図３に示す。 The amino acid sequences of the four GLUT4-SEP chimeric proteins (excluding the mCherry sequence) are shown below. In the sequence, bold letters indicate SEP, single underline indicates Myc tag, double underline indicates extracellular loop 1 of GLUT4, and italics indicate linker. The primary structure of the prepared GLUT4-SEP chimeric protein is shown in FIG.
　表３．ＳＳ－ＳＥＰ－ＧＬＵＴ４（リンカーを含まない）（配列番号８）
Figure JPOXMLDOC01-appb-T000003
 Table 3. SS-SEP-GLUT4 (without linker) (SEQ ID NO: 8)
Figure JPOXMLDOC01-appb-T000003
　表４．ＳＬ－ＳＥＰ－ＧＬＵＴ４（ＳＥＰのＣ末端にリンカーを含む）（配列番号９）
Figure JPOXMLDOC01-appb-T000004
 Table 4. SL-SEP-GLUT4 (including a linker at the C-terminus of SEP) (SEQ ID NO: 9)
Figure JPOXMLDOC01-appb-T000004
　表５．ＬＳ－ＳＥＰ－ＧＬＵＴ４（ＳＥＰのＮ末端にリンカーを含む）（配列番号１０）
Figure JPOXMLDOC01-appb-T000005
 Table 5. LS-SEP-GLUT4 (including a linker at the N-terminus of SEP) (SEQ ID NO: 10)
Figure JPOXMLDOC01-appb-T000005
　表６．ＬＬ－ＳＥＰ－ＧＬＵＴ４（ＳＥＰのＮ末端およびＣ末端にリンカーを含む）（配列番号１１）
Figure JPOXMLDOC01-appb-T000006
 Table 6. LL-SEP-GLUT4 (including a linker at the N-terminal and C-terminal of SEP) (SEQ ID NO: 11)
Figure JPOXMLDOC01-appb-T000006
　ＧＬＵＴ４－ＳＥＰキメラタンパク質のｐＨ応答性を確認するために、発現ベクタープラスミドＤＮＡをＨＥＫ２９３細胞に導入し、ＧＬＵＴ４－ＳＥＰキメラタンパク質を一過的に発現させた。ＨＥＫ２９３細胞を２４ウェルプレートに播種し、２４時間後、５０μＬのＯｐｔｉ－ＭＥＭ１（サーモフィッシャー・サイエンティフィック）、１．５μＬのＴｒａｎｓＩＴ－２９３　Ｒｅａｇｅｎｔ（タカラバイオ）、および０．５μｇの発現ベクタープラスミドＤＮＡの混合液を添加した。その後、１０％ＦＢＳ含有ＤＭＥＭ中で２４時間培養し、ｍＣｈｅｒｒｙおよびＳＥＰの蛍光により、キメラタンパク質の発現を確認した。その後、ｐＨの異なる培地（ＤＭＥＭ－ｐＨ７．５およびＤＭＥＭ－ｐＨ５．５）を細胞に添加したところ、ＤＭＥＭ－ｐＨ７．５を添加した細胞ではＳＥＰの蛍光強度が維持されたのに対し、ＤＭＥＭ－ｐＨ５．５を添加した細胞ではＳＥＰの蛍光強度の低下が見られた。この結果から、ＧＬＵＴ４－ＳＥＰキメラタンパク質がｐＨ応答性であることが確認された（データは省略）。 In order to confirm the pH responsiveness of the GLUT4-SEP chimeric protein, the expression vector plasmid DNA was introduced into HEK293 cells to transiently express the GLUT4-SEP chimeric protein. HEK293 cells were seeded in a 24-well plate, and after 24 hours, 50 μL of Opti-MEM1 (Thermo Fisher Scientific), 1.5 μL of TransIT-293 Reagent (Takara Bio), and 0.5 μg of expression vector plasmid DNA Was added. Then, the cells were cultured in DMEM containing 10% FBS for 24 hours, and the expression of the chimeric protein was confirmed by the fluorescence of mCherry and SEP. Then, when mediums having different pHs (DMEM-pH7.5 and DMEM-pH5.5) were added to the cells, the fluorescence intensity of SEP was maintained in the cells to which DMEM-pH7.5 was added, whereas DMEM- A decrease in SEP fluorescence intensity was observed in cells to which pH 5.5 was added. From this result, it was confirmed that the GLUT4-SEP chimeric protein was pH-responsive (data not shown).
＜３．ＧＬＵＴ４－ＳＥＰキメラタンパク質による筋肉細胞のインスリン応答性の評価＞
　ＧＬＵＴ４－ＳＥＰキメラタンパク質の発現ベクタープラスミドＤＮＡ（ｐＬｅｎｔｉ－ｍｙｃ－ＳＳ－ＳＥＰ－ＧＬＵＴ４－ｍＣｈｅｒｒｙ）およびパッケージングプラスミド（ｐＣＭＶ－ＶＳＶ－Ｇ）をＰｌａｔ－Ａ細胞にリポフェクションし、レンチウイルスを調製した。Ｃ２Ｃ１２細胞を６ウェルプレートに１×１０６の濃度で播種し、２０％ＦＢＳ／ＤＭＥＭ中でコンフルエントになるまで培養した。その後、Ｃ２Ｃ１２細胞に上で調製したレンチウイルスを感染させ、２日間培養した。その後、培地を交換し、Ｃ２Ｃ１２細胞を低血清条件（２％ＦＢＳ／ＤＭＥＭ）下で５日培養し、筋管細胞へと分化誘導した。得られた筋管細胞に、終濃度１００ｎＭのインスリン（サーモフィッシャー・サイエンティフィック）を添加し、インスリン添加前後でのＳＥＰおよびｍＣｈｅｒｒｙの蛍光強度の変化を蛍光顕微鏡により観察した。 <3. Evaluation of insulin responsiveness of muscle cells by GLUT4-SEP chimeric protein>
An expression vector plasmid DNA (pLenti-myc-SS-SEP-GLUT4-mCherry) of the GLUT4-SEP chimeric protein and a packaging plasmid (pCMV-VSV-G) were lipofected into Plat-A cells to prepare a lentivirus. C2C12 cells were seeded in a 6-well plate at a concentration of 1 × 10 6 and cultured in 20% FBS / DMEM until confluent. Then, C2C12 cells were infected with the lentivirus prepared above and cultured for 2 days. Then, the medium was exchanged, and C2C12 cells were cultured for 5 days under low serum conditions (2% FBS / DMEM) to induce differentiation into myotube cells. A final concentration of 100 nM insulin (Thermo Fisher Scientific) was added to the obtained myotube cells, and changes in the fluorescence intensity of SEP and mCherry before and after the addition of insulin were observed by a fluorescence microscope.
　ＳＳ－ＳＥＰ－ＧＬＵＴ４－ｍＣｈｅｒｒｙを発現させた筋管細胞における結果を図４および図５に示す。インスリンの添加により、ＳＥＰ由来の緑色蛍光の強度は顕著に増大したのに対し、ｍＣｈｅｒｒｙ由来の赤色蛍光の強度には有意な変化は見られなかった。この結果は、ＧＬＵＴ４－ＳＥＰキメラタンパク質が細胞内のＧＬＵＴ４小胞から細胞膜へと移行することにより、ＧＬＵＴ４小胞内（＜ｐＨ６）に位置していたＳＥＰが細胞膜外（～ｐＨ７．４）に露出されたことを示すものと考えられた。なお、ｍＣｈｅｒｒｙはＧＬＵＴ４の移行に関係なく細胞質に位置するため、蛍光強度が変化しないものと考えられた。以上の結果から、ＧＬＵＴ４－ＳＥＰキメラタンパク質を用いて、細胞のインスリン応答性を可視化できることが示された。また、ＳＳ－ＳＥＰ－ＧＬＵＴ４－ｍＣｈｅｒｒｙ以外の、リンカーを含む３種類のＧＬＵＴ４－ＳＥＰキメラタンパク質を発現させた細胞でも、同様の結果が得られた（データは省略）。 The results of myotube cells expressing SS-SEP-GLUT4-mCherry are shown in FIGS. 4 and 5. By the addition of insulin, the intensity of SEP-derived green fluorescence was significantly increased, while no significant change was observed in the intensity of mCherry-derived red fluorescence. This result indicates that the GLUT4-SEP chimeric protein is translocated from the intracellular GLUT4 vesicles to the cell membrane, exposing the SEP located in the GLUT4 vesicles (<pH6) to the outside of the cell membrane (~ pH7.4). It was thought to indicate that it was done. Since mCherry is located in the cytoplasm regardless of the transfer of GLUT4, it was considered that the fluorescence intensity did not change. From the above results, it was shown that the insulin responsiveness of cells can be visualized by using the GLUT4-SEP chimeric protein. Similar results were also obtained in cells expressing three types of GLUT4-SEP chimeric proteins containing a linker other than SS-SEP-GLUT4-mCherry (data not shown).

Claims (13)

  1. 　グルコース輸送体タンパク質と、蛍光性または発光性のレポーターとが融合されてなるキメラタンパク質であって、前記レポーターが前記グルコース輸送体タンパク質の細胞外ループに挿入されており、前記キメラタンパク質が細胞膜に移行すると蛍光または発光の強度および／もしくは波長が変化する、キメラタンパク質。 A chimeric protein obtained by fusing a glucose transporter protein and a fluorescent or luminescent reporter, wherein the reporter is inserted into the extracellular loop of the glucose transporter protein, and the chimeric protein is transferred to the cell membrane. A chimeric protein in which the intensity and / or wavelength of fluorescence or luminescence changes.
  2. 　前記グルコース輸送体タンパク質がＧＬＵＴ４である、請求項１に記載のキメラタンパク質。 The chimeric protein according to claim 1, wherein the glucose transporter protein is GLUT4.
  3. 　前記細胞外ループが、前記グルコース輸送体タンパク質の膜貫通ドメイン１と膜貫通ドメイン２の間に位置する細胞外ループ１である、請求項１または２に記載のキメラタンパク質。 The chimeric protein according to claim 1 or 2, wherein the extracellular loop is extracellular loop 1 located between transmembrane domain 1 and transmembrane domain 2 of the glucose transporter protein.
  4. 　前記レポーターがｐＨ応答性の蛍光タンパク質である、請求項１～３のいずれか１項に記載のキメラタンパク質。 The chimeric protein according to any one of claims 1 to 3, wherein the reporter is a pH-responsive fluorescent protein.
  5. 　前記ｐＨ応答性の蛍光タンパク質がｐＨｌｕｏｒｉｎである、請求項４に記載のキメラタンパク質。 The chimeric protein according to claim 4, wherein the pH-responsive fluorescent protein is pHluorin.
  6. 　前記レポーターがｐＨ応答性の発光酵素タンパク質である、請求項１～３のいずれか１項に記載のキメラタンパク質。 The chimeric protein according to any one of claims 1 to 3, wherein the reporter is a pH-responsive luminescent enzyme protein.
  7. 　前記ｐＨ応答性の発光酵素タンパク質がルシフェラーゼである、請求項６に記載のキメラタンパク質。 The chimeric protein according to claim 6, wherein the pH-responsive luminescent enzyme protein is luciferase.
  8. 　前記レポーターがスプリット蛍光タンパク質断片またはスプリット発光酵素タンパク質断片である、請求項１～３のいずれか１項に記載のキメラタンパク質。 The chimeric protein according to any one of claims 1 to 3, wherein the reporter is a split fluorescent protein fragment or a split luminescent enzyme protein fragment.
  9. 　請求項１～８のいずれか１項に記載のキメラタンパク質をコードするヌクレオチド配列を含む核酸。 A nucleic acid comprising a nucleotide sequence encoding the chimeric protein according to any one of claims 1 to 8.
  10. 　請求項９に記載の核酸を含む発現ベクター。 An expression vector containing the nucleic acid according to claim 9.
  11. 　請求項１～８のいずれか１項に記載のキメラタンパク質を発現する細胞。 A cell expressing the chimeric protein according to any one of claims 1 to 8.
  12. 　請求項１～８のいずれか１項に記載のキメラタンパク質を発現するトランスジェニック非ヒト動物。 A transgenic non-human animal expressing the chimeric protein according to any one of claims 1 to 8.
  13. 　（１）請求項１～８のいずれか１項に記載のキメラタンパク質を発現する細胞にインスリンを曝露するステップと、
    　（２）前記キメラタンパク質の蛍光または前記キメラタンパク質による発光を検出するステップと
    を含む、インスリン応答性の評価方法。
          (1) exposing insulin to cells expressing the chimeric protein according to any one of claims 1 to 8;
    (2) Detecting fluorescence of the chimeric protein or luminescence of the chimeric protein, the method for evaluating insulin responsiveness.
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Citations (3)

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