JP2014135947A - Fluorescent biosensor for detecting ectodomain shedding - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a useful method for evaluating ectodomain shedding activity of a cell.SOLUTION: There is provided a method for evaluating extracellular domain shedding activity (ectodomain shedding activity) with respect to a factor which is synthesized as a membrane-bound precursor having an extracellular domain and an intracellular domain and cleaved into a secretory type on a cell surface by protease. A cell is used which has an expressed membrane-bound factor in which a first fluorescent protein is fused with an extracellular domain and a second fluorescent protein is fused with an intracellular domain, wherein the first fluorescent protein and the second fluorescent protein have wavelength characteristics which can be discriminated from each other, and the activity is evaluated based on fluorescence derived from the first fluorescent protein and fluorescence derived from the second fluorescent protein. Thus, observation of shedding activity in individual cells over time is made possible.

Description

  Heparin binding epidermal growth factor-like growth factor HB-EGF (heparin binding-epidermal growth factor-like growth factor) is EGF, amphiregulin, transforming growth factor (TGF) -α, betacelulin and neuregulin Is one of the members of the EGF family. HB-EGF expression has been detected in most parts of the body, particularly in the lung, heart, brain and skeletal muscle. HB-EGF is involved in various physiological and pathological events including wound healing, eyelid formation, blastocyst transplantation, pulmonary hypertension, restenosis after balloon injury, atherosclerosis and tumor progression . Knockout of the HB-EGF gene impairs the development of several neural crest cell-derived tissues, including craniofacial structures, heart and enteric nerve development. Brain-specific knockout mice show that HB-EGF also contributes to mental disorders.

  HB-EGF, like other members of the EGF family, is first synthesized as a transmembrane precursor called proHB-EGF. proHB-EGF is cleaved on the cell surface by proteases such as disintegrin and metalloproteases (ADAMs) to release soluble HB-EGF. The process is known as ectodomain shedding. The ectodomain shedding of proHB-EGF includes 12-O-tetradecanoylphorborl-13-estate, calcium ionophore, GPCR ligands such as lysophosphatidic acid and growth factors Stimulated by various physiological or pharmacological stimuli. The ectodomain shedding of proHB-EGF is an essential event for the growth factor activity of HB-EGF. Mice expressing an uncleavable mutant of proHB-EGF show severe heart failure as shown by HB-EGF null mice. It has also been reported that proHB-EGF non-cleavable form induces myocardial apoptosis under hypoxic conditions. On the other hand, mice expressing mutants truncating the transmembrane domain of proHB-EGF (always released from cells) show severe hyperplasia in the heart and skin. Furthermore, proHB-EGF shedding is also known to affect tumor progression.

  In order to measure the ectodomain shedding activity of proHB-EGF in cells, so far, (1) separated proHB-EGF as a protein from the cell extract and detected by Western blot, or (2) ProHB-EGF fused with alkaline phosphatase (AP) in the extracellular region is expressed on the cell, and AP activity of AP fusion ligand released into the medium by shedding has been detected (Non-patent Document 1) , 2). They are very useful methods to know the shedding activity of a cell population under various conditions.

Dethlefsen, SM, Raab, G., Moses, MA, Adam, RM, Klagsbrun, M., and Freeman, MR (1998) Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C. J Cell Biochem. 69, 143-153 Tokumaru, S., Higashiyama, S., Endo, T., Nakagawa, T., Miyagawa, JI, Yamamori, K., Hanakawa, Y., Ohmoto, H., Yoshino, K., Shirakata, Y., Matsuzawa , Y., Hashimoto, K., and Taniguchi, N. (2000) Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing.J Cell Biol. 151, 209-220

  However, all of the conventional methods for measuring ectodomain shedding activity are (1) a system that evaluates as a cell group, and thus it is difficult to evaluate individual cells and cell localities. In addition, since it is necessary to collect the cells themselves or the culture supernatant for measurement, it is not possible to observe the same cells over time while maintaining the same state, and for the same reason, There was a problem that evaluation could not be performed.

  ProHB-EGF ectodomain shedding has been shown to affect tumor progression and is important for studying tumor cell dynamics and in the research and development of oncology drugs. From previous reports, evaluation of shedding activity is considered to be important for understanding normal or pathological tissue status.

  The present inventors have intensively studied a probe that can visualize shedding. The inventors of the present invention stably expressed in cells the proHB-EGF fused with a red fluorescent protein outside the cell and a green fluorescent protein in the intracellular region. And when these cells were treated with a substance known as an ectodomain shedding stimulator, the intensity ratio of the two types of fluorescence changed over time, allowing chronological observation of shedding activity in individual cells. As a result, the present invention was completed.

The present invention provides the following.
[1] An extracellular domain cleavage activity (ectodomain shedding activity) for a factor that is synthesized as a membrane-bound precursor having an extracellular region and an intracellular region and is cleaved by a protease on the cell surface to become a secreted type A method of evaluating;
A cell expressing a membrane-bound factor, in which the first fluorescent protein is fused to the extracellular region and the second fluorescent protein is fused to the intracellular region, is used. The fluorescent proteins of the above have different wavelength characteristics from each other;
A method of evaluating the activity based on fluorescence derived from a first fluorescent protein and fluorescence derived from a second fluorescent protein.
[2] The method according to [1], wherein the factor is heparin-binding epidermal growth factor-like growth factor (HB-EGF).
[3] Evaluation based on the fluorescence derived from the first fluorescent protein and the fluorescence derived from the second fluorescent protein is based on the intensity of the fluorescence derived from the first fluorescent protein and the fluorescence derived from the second fluorescent protein. The method according to [1] or [2], wherein a ratio with the intensity is calculated and evaluated by a change in the ratio.
[4] The method according to any one of [1] to [3], wherein the evaluation is performed by image analysis.
[5] The method according to any one of [1] to [4], which is performed in a living body.
[6] A method of visualizing ectodomain shedding in living cells for a factor that is synthesized as a membrane-bound precursor having an extracellular region and an intracellular region and is cleaved by a protease on the cell surface to become a secreted type. And
In the cell, a membrane-bound factor in which the first fluorescent protein is fused to the extracellular region and the second fluorescent protein is fused to the intracellular region is expressed. At this time, the first fluorescent protein and the second fluorescent protein are expressed. Fluorescent proteins have wavelength characteristics that are distinguishable from each other;
When the extracellular part containing the first fluorescent protein is cleaved by ectodomain shedding, the fluorescence derived from the first fluorescent protein changes and / or the intensity of the fluorescence derived from the first fluorescent protein A method wherein the ratio of intensity to fluorescence from a second fluorescent protein is varied to visualize ectodomain shedding.
[7] The method described in [6], for visualizing ectodomain shedding for a precursor of HB-EGF (proHB-EGF).
[8] In a factor that is synthesized as a membrane-bound precursor having an extracellular region and an intracellular region and is cleaved by a protease on the cell surface to become a secreted type, the first fluorescent protein is fused to the extracellular region. A second fluorescent protein is fused to the intracellular region;
When expressed in cells, the first fluorescent protein is placed in the intracellular region, the second fluorescent protein is placed outside the cell, and the extracellular part containing the first fluorescent protein is cleaved by ectodomain shedding. Fluorescence biosensor capable of detecting ectodomain shedding by changing fluorescence derived from at least one fluorescent protein.
[9] The fluorescent biosensor according to [8], wherein the first fluorescent protein is a red fluorescent protein, the second fluorescent protein is a green fluorescent protein, and they are fused to proHB-EGF. .
[10] The fluorescent biosensor according to [9], comprising the following (a), (b) or (c):
(A) a polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing;
(B) It consists of a sequence having at least 80% sequence identity with the polypeptide described in (a), and when expressed in cells, the first fluorescent protein is in the intracellular region and the second fluorescent protein is extracellular. (C) a polypeptide that is arranged in the structure and is capable of detecting a change in fluorescence derived from at least one fluorescent protein by cleaving the extracellular portion containing the first fluorescent protein by ectodomain shedding; The amino acid sequence of the polypeptide described in (a) consists of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added, and when expressed in a cell, the first fluorescent protein is an intracellular region. The second fluorescent protein is placed outside the cell, and the extracellular part containing the first fluorescent protein is cleaved by ectodomain shedding. By a change in the fluorescence from at least one fluorescent protein is detectable polypeptide.
[11] A polynucleotide encoding the fluorescent biosensor described in [8] to [10].
[12] A vector comprising the polynucleotide described in [11].
[13] Method for observing ectodomain shedding of at least one factor in a cell, comprising the following steps: A fluorescent biosensor obtained by transformation with the vector described in [12] is provided on the membrane Cells are exposed to environmental changes (eg, hypoxic environment, oxidative environment, changes in tissue environment such as ultraviolet irradiation) to detect the presence or absence of changes in fluorescence in the cell or its vicinity. Detecting the resulting fluorescence change means that the candidate substance has affected the efficiency of ectodomain shedding of the factor.
[14] A method for screening a substance that can affect the efficiency of ectodomain shedding of at least one factor, comprising the following steps: Fluorescence biosensor by transforming cells with the vector described in [12] And a candidate substance is donated to the prepared cell to detect the presence or absence of a change in fluorescence in the cell or the vicinity thereof. At this time, the fluorescence due to the provision of the candidate substance is detected. A detected change means that the candidate substance has affected the efficiency of ectodomain shedding of the factor.
[15] The method described in [14], wherein the method is for screening a substance that can affect the efficiency of ectodomain shedding of proHB-EGF.
[16] The method described in [15], wherein the method is for screening a substance capable of affecting tumor progression.

[17] An apparatus for carrying out the method according to any one of [1] to [6].
[18] A cell image analysis apparatus comprising: an image input unit that inputs a plurality of cell images in which fluorescence emitting cells are interval-imaged; and a calculation unit that calculates a fluorescence change from the input cell images.
[19] A threshold storage unit that stores in advance a threshold value related to a fluorescence change, and compares the calculated fluorescence change with the threshold value to identify that there is ectodomain shedding or that its efficiency has changed. The cell image analysis device according to [18], further comprising a coding identification unit.
[20] An image input step of inputting, to a computer, a plurality of cell images obtained by interval imaging of cells emitting fluorescence, and a calculation step of calculating a fluorescence change from the plurality of input cell images. Program to let you.
[21] The computer includes a threshold storage unit that previously stores a threshold relating to fluorescence change, and the calculated fluorescence change is compared with the threshold to determine whether there is ectodomain shedding or the efficiency thereof has changed. The program according to [20], for further executing the identifying step.

Biochemical characteristics and subcellular localization of Fluhemb (Fluorescent proHB-EGF-based metalloprotease biosensor). (A) Structure of Fluhemb. (BD) Confocal laser microscope analysis of Fluhemb expressing HT1080 cells. The cells were fixed with 4% paraformaldehyde in PBS for 15 minutes, and the fluorescence signal of Fluhemb was detected with a confocal laser microscope A1R. (B) mAG1, (C) mCherry, (D) Superposition. Lower panel: XZ cross section, right panel: YZ cross section. The scale bar indicates 20 μm. (E) After serum starvation, Fluhemb-expressing HT1080 cells were stimulated with 50 nM 12-o-tetradecanoylphorbol-13-acetate (TPA) for 60 minutes. For metalloprotease inhibition, cells were treated with 20 μM GM-6001 or KB-R7785 for 30 minutes prior to TPA stimulation. SDS-PAGE and Western blot analysis of whole cell lysates was performed. Fluhemb was detected with anti-mCherry or anti-monomer Azami Green (mAG) antibody. Beta actin was also detected as a loading control. TPA stimulation on Fluhemb-expressing HT1080 cells. (A and B) After serum starvation, Fluhemb expressing HT1080 cells were stimulated with 50 nM 12-o-tetradecanoylphorbol-13-acetate (TPA) for 60 minutes. For metalloprotease inhibition, cells were treated with 20 μM GM-6001 or KB-R7785 for 30 minutes prior to TPA stimulation. Time-lapse images were obtained for 1 hour every minute by Biostation IM. The ratio of the fluorescence intensity of mAG1 to mCherry was calculated by NIS-elements (ratio range: 0 to 2). (B) Ratio image of high magnification field of view in (A). Arrows indicate the leading ledges of the cell. Scale bars indicate 100 μm (A) and 20 μm (B). Other types of shedding stimuli on HT1080 or HaCaT cells. Fluhemb-expressing HT1080 (A) or HaCaT (B) cells were treated with 20 μM calcium ionophore A23187. (C) Fluhemb-expressing HaCaT cells were stimulated with 10 nM EGF after serum starvation. After the stimulation, a time-lapse assay was performed with Biostation IM for 1 hour every minute, fluorescence images were obtained with Biostation IM, and the ratio of the fluorescence intensity of mAG1 to mCherry was calculated with NIS-elements (ratio range: 0 ~ 2). Scale bars indicate 10 μm (A and B) and 20 μm (C). Physiological stimulation of shedding on Fluhemb expressing cells. (A) Fluhemb-expressing HaCaT cells were scratched with a yellow chip, and a time-lapse assay was performed with Biostation IM. The time indicates the elapsed time after the cell is scratched. (BD) Fluhemb-expressing HT1080 cells are seeded onto one chamber of μ-slide 3D chemotaxis and infiltration into BD Matrigel is induced by medium containing serum on the opposite side of the chamber, Time lapse assay was performed with Biostation IM. (D) Actin was detected with Alexa Fluor 647 conjugated phalloidin. The arrow indicates a pseudoleg rich in actin. The ratio of the fluorescence intensity of mAG1 to mCherry was calculated by NIS-elements (ratio range: 0 to 2). Scale bars indicate 50 μm (A and B) and 20 μm (C and D). ProHB-EGF shedding imaging of Fluhemb-expressing HT1080 cells in balb / c nu / nu mice. Ten 6 6 Fluhemb-expressing HT1080 cells were implanted subcutaneously into 6-week-old female balb / c nu / nu mice. Ten days after transplantation, tumor imaging was performed with a confocal microscope A1R (Nikon). Tumor blood vessels were detected by Angiosense 680 (Visen). (A) Fluorescence ratio image of Fluhemb in HT1080 cells. (B) High magnification field image of (A). Broken lines indicate blood vessels. Scale bars indicate 100 μm (A) and 50 μm (B). The map of the vector which inserted the fluorescence biosensor Fluhemb (Fluorescent proHB-EGF-based metalloprotease biosensor) used in the Example. Fluhemb was inserted into the EcoRI and NotI sites of the lentiviral vector pCSII-IRES2-Bsd. The nucleotide sequence of the fluorescent biosensor Fluhemb used in the examples.

In the present invention, when the range is represented by “n _{1 to} n ₂ ”, values at both ends are included unless otherwise specified.
In the present invention, “cell” refers to a living cell unless otherwise specified. The cell may be a cultured cell, a part of a tissue, and / or a part of a living body.

  The present invention provides a method for evaluating extracellular domain cleavage activity (ectodomain shedding activity) for at least one factor in a cell.

  The method of the present invention is suitable for evaluating the ectodomain shedding of the EGF family, particularly HB-EFG, but in addition, as a membrane-bound precursor having an extracellular region and an intracellular region. Various growth factors that are synthesized and cleaved by a protease on the cell surface to become a secreted form can also be used. Typical examples of membrane-bound factors are TNF-alpha (tumor necrosis factor), c-kit ligand (Sl factor, stem cell factor), CSF-1, IL-1, and Ephrin-B, except for the EGF family. is there. In cells, various factors that are ectodomain-shedded in vivo can be evaluated. In this specification, the case where the membrane-bound factor is HB-EFG may be described as an example. However, the explanation is also applied when other membrane-bound factors are used, unless otherwise specified. apply.

  The method of the present invention uses a cell expressing a membrane-bound factor in which a first fluorescent protein is fused to an extracellular region and a second fluorescent protein is fused to an intracellular region.

  The first and second fluorescent proteins can be selected from existing ones used for cell evaluation, for example, the following ones. Examples of fluorescent proteins: Sirius, EBFP, ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, CFP, TurboGFP, AcGFP, TagGFP, Azami Green 1 (mAG1), ZsGreen, EmGFP, EGFP, GFP2, HyPer, TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana, KusabiraOrange, mOrange, TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry, TurboFP602, mRFP1, RedRed, Red, Red KeimaRed, mRasberry, mPlum, PS-CFP, Dendra2, Kaede, EosFP, and KikumeGR.

  In the present invention, ectodomain shedding is evaluated based on the fluorescence derived from the first fluorescent protein and the fluorescence derived from the second fluorescent protein. The evaluation based on fluorescence includes the presence / absence or degree of change in fluorescent color tone as a whole, the presence / absence or degree of change in fluorescence intensity of at least one substance, the intensity of fluorescence derived from the first fluorescent protein, and the second It means that a ratio with the intensity of fluorescence derived from a fluorescent protein is calculated and the presence or absence or degree of change in the ratio is detected and used as an index.

  In the present invention, when ectodomain shedding occurs, the first fluorescent protein can be released from the membrane outside the cell. In the cell, the second fluorescent protein can be decomposed and metabolized after staying on the membrane for a while. Therefore, the fluorescence resulting from each fluorescent protein can change.

  The change in fluorescence can be captured by obtaining a time-lapse image using an existing apparatus and analyzing the obtained image using appropriate software as desired.

  In the present invention, the first fluorescent protein and the second fluorescent protein are preferably a pair having wavelength characteristics that are distinguishable from each other. In general, in a method using a plurality of types of fluorescent substances, fluorescence resonance energy transfer (FRET) may be used. On the other hand, in the present invention, since ectodomain shedding is visualized by measuring each wavelength independently, the evaluation of the two types of fluorescent proteins is more accurate when the wavelengths of the fluorescent signals are far apart. Can be expected.

  An example of a preferred pair of fluorescent proteins used in the present invention consists of mAG1 and mCherry. In this specification, there may be a case where mAG1 and mCherry are used as a fluorescent protein pair as an example, but the explanation is also applied when other fluorescent pairs are used unless otherwise specified. apply.

  In the present invention, the first fluorescent protein is fused to the intracellular region, and the second fluorescent protein is fused to the extracellular region. Fluorescent biosensors (sometimes referred to as “fluorescent probes”) obtained by applying such fusion to the membrane-bound precursor of HB-EGF (proHB-EGF), especially Fluhemb (Fluorescent proHB- EGF-based metalloprotease biosensor).

  Fusion can be achieved by inserting or substituting a protein encoding a fluorescent protein at an appropriate position in the polynucleotide encoding the factor and expressing the resulting modified polynucleotide. In a particularly preferred embodiment, the EGF region is replaced with a fluorescent protein in the extracellular region. When the EGF region is left, characteristics such as cell proliferation can be enhanced by the introduced biosensor, that is, the characteristics of the introduced cells can be changed, but such inconvenience can be prevented by the replacement. In a particularly preferred embodiment, further, in the intracellular region, the fluorescent protein is inserted rather than substituted. By inserting, intracellular regions involved in functions such as membrane localization can be maintained.

  Those skilled in the art can appropriately select the positions for substitution or insertion of the two fluorescent protein genes. In the extracellular region, it may be considered that the insertion of a molecule near the shedding site may change the shedding activity. In addition, in the intracellular region, for the proHB-EGF, the fluorescent protein is placed between the 193rd and 194th amino acids in proHB-EGF, which contributes to membrane localization and protein stability. It turns out to be excellent. The structure of Fluhemb prepared from such a viewpoint is shown in FIG. 1 (A), and its nucleotide sequence is shown in FIG. 7 and SEQ ID NO: 1 in the sequence listing.

According to the present invention, a fluorescent biosensor Fluhemb or a variant thereof is provided. Fluhemb or a variant thereof consists of any of the following (a) to (c):
(A) a polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing;
(B) at least 70%, preferably 80% or more, more preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, more preferably 97.% or more with the polypeptide described in (a). It consists of a sequence having 5% or more, more preferably 99% or more sequence identity, and when expressed in cells, the first fluorescent protein is placed in the intracellular region and the second fluorescent protein is placed outside the cell, A polypeptide capable of detecting a change in fluorescence derived from at least one of the fluorescent proteins by cleaving the extracellular portion containing the first fluorescent protein by the bonding; or (c) described in (a) The amino acid sequence of a polypeptide consists of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added, and when expressed in a cell, the first The photoprotein is derived from at least one fluorescent protein by arranging the photoprotein in the intracellular region, the second fluorescent protein outside the cell, and cleaving the extracellular part containing the first fluorescent protein by ectodomain shedding. A polypeptide that can detect a change in fluorescence.

  The present invention also provides a polynucleotide encoding the fluorescent biosensor Fluhemb or a variant thereof.

  Regarding the amino acid sequence in the present invention, the number of amino acids to be substituted when referred to as “amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added” is the number of any amino acid except those specifically described. The peptide is not particularly limited as long as the polypeptide comprising the amino acid sequence has a desired function, but more if it is a substitution with an amino acid of about 1 to 9, 1 to 4, or similar properties. There may be a number of substitutions. Means for preparing a polynucleotide according to such an amino acid sequence are well known to those skilled in the art.

  In the present invention, when referring to `` identity '' with respect to amino acid sequences, except when specifically described, when the two sequences are aligned in an optimal manner, the percentage of the number of matched amino acids shared between the two sequences is calculated. means. That is, identity = (number of matched positions / total number of positions) × 100, which can be calculated using a commercially available algorithm. Such an algorithm is also described in Altschul et al. , J .; Mol. Biol. 215 (1990) 403-410, incorporated into the NBLAST and XBLAST programs. More specifically, search / analysis regarding the identity of amino acid sequences can be performed by algorithms or programs (for example, BLASTN, BLASTP, BLASTX, ClustalW) well known to those skilled in the art. Those skilled in the art can appropriately set parameters when using a program, and the default parameters of each program may be used. Specific techniques for these analysis methods are also well known to those skilled in the art.

  A person skilled in the art can prepare the polypeptide of the present invention and the polynucleotide encoding the same, and the fluorescent biosensor Fluhemb or a variant thereof using conventional techniques.

  The present invention also provides a vector comprising the polynucleotide described above. A preferred example of the vector is a retroviral vector, more specifically a lentiviral vector. An example of a vector constructed according to the present invention is shown in FIG. The vector may contain various regions other than the region encoding the fluorescent biosensor of the present invention. For example, it may contain a selection marker that can function in the host cell for amplification, a region for incorporating a desired portion into the genome of the target cell, and a promoter that controls the expression of a specific gene.

  Specifically, the vector can be prepared as follows. Using a commercially available cloning kit, the EGF-like region of the proHB-EGF cDNA was replaced with mCherry cDNA, and the monomeric mAG1 cDNA was inserted between the 579th and 580th nucleic acids of the proHB-EGF cDNA. Fluhemb cDNA is obtained. The cDNA-containing fragment is inserted into a lentiviral vector using a restriction enzyme region or homologous recombination. The resulting lentivirus containing Fluhemb cDNA is amplified using a host cell for vector amplification.

  The obtained virus vector can infect target cells to obtain transformed cells in which a fluorescent biosensor is expressed on the membrane. The target cells can be derived from various organisms, for example, mammals. Examples of mammals include rodents (such as mice and rats) and primates (such as monkeys and humans). The tissue from which the cells are derived is not limited, for example, skin, liver, kidney, adrenal gland, testis (including sperm cells), lung, muscle, heart, intestine, ovary and spleen, brain, embryo or embryo tissue, adipose tissue It can be.

  Cells expressing the fluorescent biosensor can be cultured under the same conditions as the original cells. For the culture, for example, a medium containing a non-essential amino acid, L-glutamine and / or fetal bovine serum can be used. A specific method for performing an assay using cells can be performed with reference to the conditions and procedures described in the Examples section of this specification.

  The fluorescent biosensor obtained by the present invention can be shed similarly to the factor from which it is derived. In addition, by using the ratio of two types of fluorescent signals, information on the activity of shedding in individual cells can be obtained.

  Furthermore, according to the present invention, the influence of various substances that can stimulate shedding can be observed. Although proHB-EGF is known to be cleaved by various stimuli such as calcium ionophore and EGF, according to the study by the present inventors, after stimulation of calcium ionophore A23187, Fluhemb-expressed HT1080 and HaCaT In both, the fluorescence ratio of mAG1 / mCherry increased in a time course dependent manner. In addition, Fluhemb also showed that the response to A23187 is much faster in HaCaT than in HT1080. Furthermore, EGF stimulation also increased the mAG1 / mCherry ratio in Fluhemb-expressing HT1080. In particular, the shedding tended to start in the intercellular adhesion region. Such information obtained by the present invention indicates that the present invention may be useful for the evaluation of various substances as shedding stimulants and for understanding the shedding response in cells.

  According to the present invention, the local cellular activity of shedding in vivo can be observed. In order to evaluate whether Fluhemb also works in vivo, the present inventors subcutaneously transplanted Fluhemb-expressed HT1080 into a female mouse of balb / cnu / nu, and observed it 10 days later with a confocal laser microscope. And most tumor cells did not show an increased mAG1 / mCherry ratio of Fluhemb, but a local increase in the ratio was observed in some HT1080 cells on the blood vessels. From this information, it is clear that tumor cells on blood vessels may be more active in proHB-EGF shedding than tumor cells that are not in contact with blood vessels. The present invention is also suitable for in vivo assessment of shedding.

The present invention also provides an apparatus and a computer program for carrying out the method of the present invention.
The apparatus includes: an image input unit that inputs a plurality of cell images obtained by interval imaging of cells that emit fluorescence; a calculation unit that calculates a fluorescence change from the plurality of input cell images;
Can be provided. Furthermore, a threshold storage unit that stores in advance a threshold relating to fluorescence change
It is preferable to provide an ectodomain shedding specifying unit that compares the calculated fluorescence change with a threshold value and specifies that ectodomain shedding is present or its efficiency is changing. The program executes an image input step for inputting a plurality of cell images obtained by interval imaging of cells emitting fluorescence, and a calculation step for calculating a fluorescence change from the plurality of input cell images. It can be. Furthermore, the computer is provided with a threshold value storage unit that preliminarily stores a threshold value related to the fluorescence change, and compares the calculated fluorescence change with the threshold value to identify that there is ectodomain shedding or that the efficiency has changed. It is preferable that the step to perform can be performed.

  The present invention has various advantages. Although the present invention provides a fluorescent biosensor for visualizing shedding, it is possible to temporally visualize the shedding in cells by using the fluorescent biosensor. In particular, since proHB-EGF shedding is known to be important for tumor progression, the method of the present invention is useful in evaluating the action of drugs to develop proHB-EGF shedding inhibitors as anticancer drugs. Can be expected. In addition, it can be used to evaluate whether changes in tissue environment such as hypoxic environment, oxidizing environment, and ultraviolet irradiation affect the shedding activity in cells.

  In addition, the EGF family is known to have several components whose shedding is controlled by several proteases, such as ADAMs, but the functional correlation between ADAMs remains unclear. By preparing and using a fluorescent biosensor according to the present invention, further research development of the EGF family of shedding systems can be expected.

1. Experimental procedure
1.1. Materials
12-o-tetradecanoylphorbol-13-acetate (TPA) was purchased from Sigma Aldrich. Epidermal growth factor (EGF), A23187 and GM6001 were purchased from Calbiochem. KB-R7785 was provided by Carnabio co.

1.2. Construction of Fluhemb expression lentiviral vector and infection of cells
In order to construct the cDNA of Fluhemb (Fluorescent proHB-EGF-based metalloprotease biosensor), the EGF-like region of the cDNA of proHB-EGF was replaced with mCherry cDNA (Invitrogen) using the In-Fusion HD cloning kit (Clontech). Monomeric Azami Green 1 (mAG1) cDNA (Medical and Biological Laboratories Co., LTD (MBL)) was inserted between the 579th and 580th nucleic acids of the proHB-EGF cDNA. Fragments containing the cDNAs of these two fluorescent proteins were inserted into the EcoRI and NotI sites of the lentiviral vector pCSII-IRES2-Bsd (provided by Dr. Miyoshi). Lentivirus containing Fluhemb cDNA was produced in 293T cells (RIKEN BioResource Center) and cells were infected at a multiplicity of infection (moi) of 20.

1.3. Cell culture and shedding stimulant assays The human fibroblast sarcoma cell line HT1080 cells and the human keratinocyte cell line HaCaT are provided by the American Tissue and Cell Collection (ATCC). It was. HT1080 cells were maintained in Eagle's minimum essential medium (Wako) containing non-essential amino acids (Life Technologies), L-glutamine (Life Technologies) and 10% fetal calf serum. HaCaT cells were cultured in Dalbeco's minimum essential medium (Wako) containing L-glutamine (Life Technologies) and 10% fetal calf serum. Prior to stimulation with TPA or EGF, HT1080 cells or HaCaT cells were cultured in serum-free medium for 30 minutes. The time-lapse assay was performed with Biostation IM (Nikon), and the fluorescence intensity ratio of mAG1 to mCherry was analyzed with NIS-elements software (Nikon).

1.4. Identification of Fluhemb localization in HT1080 cells
HT1080 cells were fixed with 4% paraformaldehyde (Nacalai Tesque) in PBS for 15 minutes. Fluorescent 3D images of the cells were obtained with a confocal laser microscope A1R (Nikon). The image data was analyzed by NIS-elements software.

1.5. Western blotting assay samples were subjected to SDS-PAGE, transferred to a PVDF membrane (Bio-rad) and examined with appropriate antibodies. The antibodies are anti-mCherry (clone: 1C51, abcam), anti-Azami Green (MBL), and anti-beta actin (clone: AC-15, Sigma-Aldrich) as a loading control.

1.6. Matrigel invasion assay
Fluhemb-expressing HT1080 cells were seeded onto one chamber of 3D chemotactic μ-slide (ibidi) with serum-free medium. Medium containing serum was placed in a separate chamber. The space between the two chambers was filled with BD Matrigel (Becton, Dickinson and Company). On the next day, time-lapse images of HT1080 cells infiltrated in matrigel were performed every 10 minutes for 24 hours by Biostation IM, and the fluorescence intensity ratio of mAG1 to mCherry was analyzed by NIS-elements software.
48 hours after seeding of Fluhemb-expressing HT1080 cells, the cells were fixed with 4% PFA and stained with Alexa Fluor 647-conjugated phalloidin (Invitrogen). Fluhemb and actin were detected by confocal laser microscope A1R, and the fluorescence intensity ratio of mAG1 to mCherry was analyzed by NIS-elements software.

1.7. Scratch assay
Fluhemb-expressing HaCaT cells were seeded on μ-Dish 35 mm (ibidi). The next day, the cells were scratched with a yellow tip, a time-lapse assay was performed every 10 minutes for 24 hours with Biostation IM, and the fluorescence intensity ratio of mAG1 to mCherry was analyzed with NIS-elements software.

1.8. Transplantation assay of Fluhemb-expressing HT1080 cells into nude mice
Ten 6 6 Fluhemb-expressing HT1080 cells were implanted subcutaneously into 6-week-old female balb / c nu / nu mice, and the mice were raised for 10 days. On the day of image analysis, fluorescence images of tumor cells in mice anesthetized with isoflurane (Mylan) were obtained percutaneously with A1R, and the fluorescence intensity ratio of mAG1 to mCherry was analyzed with NIS-elements software.

2. Results
2.1. Fluorescent proHB-EGF-based metalloprotease biosensor (Fluhemb) inserts a monomeric Azami Green cDNA, shed similarly to proHB-EGF, into the C-terminal region of the proHB-EGF gene In order to avoid any effect of the expressed probe on cell proliferation, the epidermal growth factor region was replaced with mCherry cDNA. The shedding biosensor was named Fluhemb (Figure 1A). Fluhemb was stably introduced into the human fibroblast sarcoma cell line HT1080 by a lentiviral infection system. Confocal microscopy analysis revealed that the intracellular localization of full-length Fluhemb is predominantly in the cell membrane like wild-type proHB-EGF (FIGS. 1B-D). Fluhemb was detected around 70 kDa in steady state Fluhemb-expressing HT1080. However, when Fluhemb-expressed HT1080 was stimulated with 12-o-tetradecanoylphorbol-13-acetate (TPA), a strong shedding inducer, its 70 kDa band decreased and contained the remaining fragment, mAG1 A 30 kDa band was clearly detected (FIG. 1E). Furthermore, treatment with GM-6001 (Non-patent document 7) and KB-R7785 (Non-patent document 8), which are proHB-EGF sheddase inhibitors, counteracted the action of TPA stimulation (FIG. 1E). The data indicate that Fluhemb was cleaved in a manner similar to proHB-EGF by activated metalloproteases such as ADAMs.

2.2. The ratio of the two fluorescent signals in Fluhemb showed activation of shedding by TPA stimulation in individual cells , followed by a time-lapse assay under treatment of TPA with Fluhemb-expressing HT1080. The mCherry fluorescence signal gradually decreased with time after TPA stimulation, and the fluorescence ratio of mAG1 to mCherry (mAG1 / mCherry) increased (FIGS. 2A and B). In particular, the ratio tended to be higher at the leading edge (FIG. 2B). Treatment with GM6001 or KB-R7785 inhibited the increase in the ratio of mAG1 / mCherry by TPA stimulation (FIG. 2A). These data suggest that the change in fluorescence ratio indicates shedding activation in individual cells.

2.3. Various stimulators of proHB-EGF shedding also induced an increase in the Fluhemb mAG1 / mCherry ratio in various cell lines
proHB-EGF is cleaved by various stimuli such as calcium ionophore and EGF (Non-patent Document 1) (Non-patent Document 4). Treatment of calcium ionophore or EGF was attempted to induce higher ratios of mAG1 / mCherry in Fluhemb expressing HT1080 or human human keratinocyte cell line HaCaT. After stimulation of the calcium ionophore A23187, the fluorescence ratio of mAG1 / mCherry increased in a time course manner in both Fluhemb-expressing HT1080 and HaCaT (FIGS. 3A and B). Fluhemb also showed that the response to A23187 is much faster in HaCaT than in HT1080. Furthermore, EGF stimulation also increased the mAG1 / mCherry ratio in Fluhemb-expressing HT1080 (FIG. 3C). In particular, the shedding tended to start in the intercellular adhesion region. These data indicate that Fluhemb may be useful for the evaluation of various factors as proHB-EGF shedding stimulators and for understanding intracellular proHB-EGF shedding responses.

2.4. Fluhemb uses Fluhemb-expressing HT1080 or HaCaT to show proHB-EGF shedding activity in physical conditions that showed local cellular activity of proHB-EGF shedding in physical conditions in vivo. Cell culture experiments were performed. Initially, a scratch assay was performed using Fluhemb-expressed HaCaT. The mAG1 / mCherry ratio was highest in HaCaT in the area that was scratched immediately after the cell was scratched (FIG. 4A). Subsequently, Fluhemb's mAG1 / mCherry ratio gradually decreased in HaCaT at the edge of the scratched cell sheet. On the other hand, the proportion of cells inside the edge increased (Figure 4A). Next, infiltration assay of Fluhemb-expressed HT1080 into matrigel was performed. The ratio of mAG1 / mCherry increased predominantly in the actin-rich pseudopod formation region of HT1080 in matrigel (FIGS. 4B, C and D). The results suggest that proHB-EGF shedding activity is high in infiltrating processes.
Infiltrating tumor cells may be detected in vivo by Fluhemb.
In order to evaluate whether Fluhemb works also in vivo, Fluhemb-expressing HT1080 was implanted subcutaneously into female mice of balb / cnu / nu and observed 10 days later with a confocal laser microscope. Most tumor cells did not show an increased mAG1 / mCherry ratio for Fluhemb (FIG. 5A). However, a local increase in the ratio was shown in some HT1080 cells on blood vessels (FIGS. 5A and 5B). These data reveal that tumor cells on blood vessels may be more active in proHB-EGF shedding than tumor cells that are not in contact with blood vessels. When taken in conjunction with FIGS. 4B, C and D, it may indicate that cells with higher activity of proHB-EGF shedding are more invasive.
The map of the vector used in this example (CSII-EF-MCS-IRES2-Bsd was introduced with a polynucleotide encoding Fluhemb) is shown in FIG. 6, and the nucleotide sequence of Fluhemb is shown in FIG. 7 and the sequence listing. The number 1 is shown.

3. Discussion Recent advances in fluorescent biosensors have allowed studies of enzyme activity and distribution in living cells (35-37). Metalloprotease biosensors have also been developed to visualize activation by fluorescence resonance energy transfer (FRET) (38,39). Transmembrane metalloproteases such as ADAMs perform EGF family ectodomain shedding. However, it seems difficult to create biosensors based on ectodomain shedding FRET. Since ectodomain shedding of the EGF family, including proHB-EGF, is performed in a limited region close to the cell membrane, any modification in this jaxtamembrane region affects the efficiency of ectodomain shedding (40, 41). Simply generate a fluorescent probe based on proHB-EGF consisting of mCherry instead of the EGF domain and mAG1 inserted in the cytoplasmic domain (Figure 1A), and the fluorescence intensity of mAG1 to mCherry after various stimuli on ectodomain shedding Expected change in ratio. TPA is one of the strongest inducers for proHB-EGF shedding (Non-Patent Document 3). Initially, it was demonstrated that Fluhemb is capable of responding to TPA stimulation in HT1080 cells (FIGS. 2A and B). The fluorescence intensity ratio began to change approximately 10 minutes after TPA stimulation. Fluhemb's response to TPA seems to be reasonable in terms of visualization of proHB-EGF ectodomain shedding compared to a report by Goishi et al. In order to minimize the proliferation effect of proHB-EGF based probes on cells, the EGF domain was replaced with mCherry. The data also suggests that this replacement seems to have little effect on the control of ectodomain shedding.

  Next, we confirmed that Fluhemb works in other cell lines using different stimulators for proHB-EGF shedding. Calcium ionophore A23187 has been reported as a stimulant of proHB-EGF ectodomain shedding (Non-Patent Document 1). Fluhemb responded to A23187 stimulation in both HT1080 and HaCaT cells (FIGS. 3A and B). In addition, the results showed a difference in response speed between these cell lines. Previously, growth factors such as EGF or basic fibroblast growth factor (bFGF) have been reported to induce ectodomain shedding of proHB-EGF through growth factor receptor-mediated signaling in keratinocytes (non- Patent Document 4). In this experiment, Fluhemb expressed in HaCaT cells also responded to EGF stimulation. These data are not only able to work under the treatment of chemical or humoral stimulants where Fluhemb has been reported as a stimulant for proHB-EGF shedding, but also for various stimulators of proHB-EGF shedding Or / and that differences in responses in various types of cells could also be conveyed.

  The response of Fluhemb to physiological stimuli was also confirmed. HB-EGF has been reported to have a strong activity related to cell motility (Non-patent Document 9). In the development of eyelids and healing of skin wounds, HB-EGF contributes to cell motility rather than cell proliferation (Non-Patent Documents 10 and 11). In MDCK cells, the expression of uncleavable proHB-EGF mutants promotes cell-matrix and cell-cell interactions, cell migration, hepatocyte growth factor (HGF) -induced cell scattering and tubular Structure formation was reduced (Non-patent Document 12). In contrast, soluble HB-EGF expression induced and promoted increased cell migration, cell-matrix production and decreased cell-cell interaction. Inhibition of proHB-EGF shedding also inhibited wound-induced EGF receptor activation and caused suppression of keratinocyte migration (Non-Patent Document 2). The data suggest that ectodomain shedding of proHB-EGF plays an important role in cell migration. An in vitro wound healing assay using Fluhemb-expressing HaCaT cells was also performed, and the results in FIG. 4A were reported previously (Non-Patent Document 2), and the formation of wounds was ectodomain shedding of proHB-EGF. It showed strong induction. The local ectodomain shedding activity of proHB-EGF was also demonstrated in a Matrigel invasion assay using Fluhemb-expressing HT1080 cells. The activity of proHB-EGF shedding appears to be strong in the infiltrating process of the infiltrating cells in FIG. 4B. The results suggest that proHB-EGF shedding contributes to the formation of infiltrating protrusions in squamous cell carcinoma of the head and neck (Non-patent Document 13), which may make sense. Finally, proHB-EGF shedding was visualized in tumor tissue in vivo (FIGS. 5A and B). It was extremely difficult to identify infiltrating tumor cells in living animals. However, this method may be useful for in vivo identification and tracking of invading tumor cells.

  In this study, we developed a fluorescent biosensor Fluhemb to visualize proHB-EGF shedding. By using Fluhemb, proHB-EGF shedding in cells can be visualized temporally. ProHB-EGF shedding is known to be important for tumor progression (Non-patent Document 6). Therefore, this method may have advantages in evaluating the action of drugs to develop proHB-EGF shedding inhibitors as anticancer drugs. In the EGF family, there are several components whose shedding is controlled by several proteases such as ADAMs (Non-patent Document 4). However, the functional correlation between ADAMs in each EGF family shedding remains unclear. In addition, fluorescent biosensors such as Fluhemb based on other family components will provide a deep understanding of the EGF family of shedding systems.

4. References cited in the Examples section
(Non-Patent Document 3) Goishi, K., Higashiyama, S., Klagsbrun, M., Nakano, N., Umata, T., Ishikawa, M., Mekada, E., and Taniguchi, N. (1995) Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity.Mol Biol Cell.6, 967-980
(Non-Patent Document 4) Higashiyama, S., Nanba, D., Nakayama, H., Inoue, H., and Fukuda, S. (2011) Ectodomain shedding and remnant peptide signaling of EGFRs and their ligands.J Biochem. 150 , 15-22
(Non-Patent Document 5) Nanba, D., Inoue, H., Shigemi, Y., Shirakata, Y., Hashimoto, K., and Higashiyama, S. (2008) An intermediary role of proHB-EGF shedding in growth factor -induced c-Myc gene expression.Journal of cellular physiology.214, 465-473
(Non-Patent Document 6) Miyazono, K. (2012) Ectodomain shedding of HB-EGF: a potential target for cancer therapy.J Biochem. 151, 1-3
(Non-Patent Document 7) Xu, KP, Ding, Y., Ling, J., Dong, Z., and Yu, FS (2004) Wound-induced HB-EGF ectodomain shedding and EGFR activation in corneal epithelial cells.Investigative ophthalmology & visual science. 45, 813-820
(Non-Patent Document 8) Asakura, M., Kitakaze, M., Takashima, S., Liao, Y., Ishikura, F., Yoshinaka, T., Ohmoto, H., Node, K., Yoshino, K. , Ishiguro, H., Asanuma, H., Sanada, S., Matsumura, Y., Takeda, H., Beppu, S., Tada, M., Hori, M., and Higashiyama, S. (2002) Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: metalloproteinase inhibitors as a new therapy.Nature medicine.8, 35-40
(Non-Patent Document 9) Higashiyama, S., Abraham, JA, and Klagsbrun, M. (1993) Heparin-binding EGF-like growth factor stimulation of smooth muscle cell migration: dependence on interactions with cell surface heparan sulfate.J Cell Biol 122, 933-940
(Non-Patent Document 10) Shirakata, Y., Kimura, R., Nanba, D., Iwamoto, R., Tokumaru, S., Morimoto, C., Yokota, K., Nakamura, M., Sayama, K. , Mekada, E., Higashiyama, S., and Hashimoto, K. (2005) Heparin-binding EGF-like growth factor accelerates keratinocyte migration and skin wound healing. Journal of cell science. 118, 2363-2370
(Non-Patent Document 11) Mine, N., Iwamoto, R., and Mekada, E. (2005) HB-EGF promotes epithelial cell migration in eyelid development.Development. 132, 4317-4326
(Non-Patent Document 12) Singh, AB, Tsukada, T., Zent, R., and Harris, RC (2004) Membrane-associated HB-EGF modulates HGF-induced cellular responses in MDCK cells.Journal of cell science.117, 1365-1379
(Non-Patent Document 13) Hayes, KE, Walk, EL, Ammer, AG, Kelley, LC, Martin, KH, and Weed, SA (2012) Ableson kinases negatively regulate invadopodia function and invasion in head and neck squamous cell carcinoma by inhibiting an HB-EGF autocrine loop. Oncogene.

SEQ ID NO: 1: Nucleotide sequence of Fluhemb

Claims (16)

A method for evaluating extracellular domain cleavage activity (ectodomain shedding activity) for a factor synthesized as a membrane-bound precursor having an extracellular region and an intracellular region and cleaved by a protease on the cell surface to become a secreted type Because;
A cell expressing a membrane-bound factor, in which the first fluorescent protein is fused to the extracellular region and the second fluorescent protein is fused to the intracellular region, is used. The fluorescent proteins of the above have different wavelength characteristics from each other;
A method of evaluating the activity based on fluorescence derived from a first fluorescent protein and fluorescence derived from a second fluorescent protein.   The method according to claim 1, wherein the factor is heparin-binding epidermal growth factor-like growth factor (HB-EGF).   The evaluation based on the fluorescence derived from the first fluorescent protein and the fluorescence derived from the second fluorescent protein is based on the intensity of the fluorescence derived from the first fluorescent protein and the intensity of the fluorescence derived from the second fluorescent protein. The method according to claim 1, wherein the ratio is calculated and evaluated by a change in the ratio.   The method according to claim 1, wherein the evaluation is performed by image analysis.   The method according to claim 1, wherein the method is performed in a living body. A method of visualizing ectodomain shedding in a living cell for a factor that is synthesized as a membrane-bound precursor having an extracellular region and an intracellular region and is cleaved by a protease on a cell surface to become a secreted type;
In the cell, a membrane-bound factor in which the first fluorescent protein is fused to the extracellular region and the second fluorescent protein is fused to the intracellular region is expressed. At this time, the first fluorescent protein and the second fluorescent protein are expressed. Fluorescent proteins have wavelength characteristics that are distinguishable from each other;
When the extracellular part containing the first fluorescent protein is cleaved by ectodomain shedding, the fluorescence derived from the first fluorescent protein changes and / or the intensity of the fluorescence derived from the first fluorescent protein A method wherein the ratio of intensity to fluorescence from a second fluorescent protein is varied to visualize ectodomain shedding.   The method according to claim 6, which is for visualizing ectodomain shedding for a precursor of HB-EGF (proHB-EGF). In a factor that is synthesized as a membrane-bound precursor having an extracellular region and an intracellular region, and is cleaved by a protease on the cell surface to become a secreted type, the first fluorescent protein is fused to the extracellular region, A second fluorescent protein is fused to the region;
When expressed in cells, the first fluorescent protein is placed in the intracellular region, the second fluorescent protein is placed outside the cell, and the extracellular part containing the first fluorescent protein is cleaved by ectodomain shedding. Fluorescence biosensor capable of detecting ectodomain shedding by changing fluorescence derived from at least one fluorescent protein.   The fluorescent biosensor according to claim 8, wherein the first fluorescent protein is a red fluorescent protein, the second fluorescent protein is a green fluorescent protein, and they are fused to proHB-EGF. The fluorescent biosensor according to claim 9, comprising the following (a), (b), or (c):
(A) a polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing;
(B) It consists of a sequence having at least 80% sequence identity with the polypeptide described in (a), and when expressed in cells, the first fluorescent protein is in the intracellular region and the second fluorescent protein is extracellular. (C) a polypeptide that is arranged in the structure and is capable of detecting a change in fluorescence derived from at least one fluorescent protein by cleaving the extracellular portion containing the first fluorescent protein by ectodomain shedding; The amino acid sequence of the polypeptide described in (a) consists of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added, and when expressed in a cell, the first fluorescent protein is an intracellular region. The second fluorescent protein is placed outside the cell, and the extracellular part containing the first fluorescent protein is cleaved by ectodomain shedding. By a change in the fluorescence from at least one fluorescent protein is detectable polypeptide.   A polynucleotide encoding the fluorescent biosensor according to claim 8.   A vector comprising the polynucleotide of claim 11. A method for observing ectodomain shedding of at least one factor in a cell comprising the following steps:
A cell having a fluorescent biosensor obtained by transformation with the vector according to claim 12 on a membrane is exposed to a change in the environment to detect the presence or absence of a change in fluorescence in the cell or the vicinity thereof.
At this time, a method in which a change in fluorescence due to provision of a candidate substance is detected means that the candidate substance has affected the efficiency of ectodomain shedding of the factor. A method of screening for substances that can affect the efficiency of ectodomain shedding of at least one factor comprising the following steps:
A cell is transformed with the vector of claim 12 to prepare a cell having a fluorescent biosensor on a membrane; and a candidate substance is donated to the prepared cell to detect a change in fluorescence in or near the cell. Detect presence or absence,
At this time, detecting a change in fluorescence due to the provision of the candidate substance means that the m candidate substance has influenced the efficiency of ectodomain shedding of the factor.   15. The method according to claim 14, wherein the method is for screening a substance capable of affecting the efficiency of proHB-EGF ectodomain shedding.   The method according to claim 15, wherein the method is for screening for a substance capable of affecting tumor progression.