JP5682795B2 - APP Localization Detection Method - Google Patents

Description

  The present invention relates to a method for detecting the localization of APP using a specific antibody.

Alzheimer's disease (also called Alzheimer's disease, which may be simply abbreviated as AD hereinafter) is estimated based on the Ministry of Health, Labor and Welfare Ministry of Health data and patient survey results. The number of Alzheimer-type dementia patients as of 2010 is about 116. It is estimated that the number will reach 1.40 million in 2015 and 2.2 million in 2025. In the United States, 5.3 million people are said to be affected, and it is predicted that the disease will be the fifth leading cause of death at the age of 65 and over, and that the number of patients will be the highest in the world within 30 years.
Here, other diseases with a large number of patients, such as hypertension, diabetes, and hyperlipidemia, have become possible to control without progressing the disease, even if they cannot be completely cured. As for the part, healing or its pathology is becoming controllable. However, the cause and mechanism of action of AD are unknown, and the progression of the disease state is hardly controlled. (Although the true onset is still 10 to 20 years) The family's mental and economic burden from diagnosis to AD until death (usually 5 to 12 years) is one of the biggest social problems among the diseases It is. Therefore, AD is a disease with extremely high social demands for early diagnosis, prevention, and treatment.
As AD progresses, it is known that brain regression occurs due to the disappearance of brain cells as a symptom, and amyloid βA4 precursor protein (hereinafter abbreviated as APP) is known as a central molecule related thereto. The mechanism of AD has been studied for more than 30 years, including the function, role, and metabolic regulation of APP, but there are still many parts that have not been elucidated, and at least the accumulation of Aβ42 or Aβ43 and the correlation between the onset of AD Is known to be extremely high, but not all (Non-Patent Document 1). Until now, the location of APP has not been completely grasped, and has been considered to exist in the cytoplasm (Non-Patent Documents 2 to 7).

Experimental Medicine October 2008 Vol.26 No.16 Abnormal Protein Accumulation and Treatment Strategy for Alzheimer's Disease Elucidation of Proteolysis Mechanism and Challenge to Establish Diagnosis by Biomarker Takeshi Iwatsubo / Planning TE Golde, S. Estus, M. Usiak, LH Younkin, SG Younkin SG, Expression of beta amyloid protein precursor mRNAs: Recognition of a novel alternatively spliced form and quantitation in Alzheimer's disease using PCR, Neuron. 4 (1990) 253-67 . G. Thinakaran, E.H.Koo, Amyloid precursor protein trafficking, processing, and function, J. Biol. Chem. 283 (2008) 29615-2969. WT Kimberly, JB Zheng, SY Guenette, DJ Selkoe, The intracellular domain of the beta-amyloid precursor protein is stabilized by Fe65 and translocates to the nucleus in a notch-like manner, J. Biol. Chem. 276 (2001) 40288- 40292. RC von Rotz, BM Kohli, J. Bosset, M. Meier, T. Suzuki, RM Nitsch, U. Konietzko, The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor, J. Cell Sci. 117 ( 2004) 4435-4448. H. Yamaguchi, T. Yamazaki, K. Ishiguro, M. Shoji, Y. Nakazato, S. Hirai, Ultrastructural localization of Alzheimer amyloid beta / A4 protein precursor in the cytoplasm of neurons and senile plaque-associated astrocytes, Acta Neuropathol. 85 (1992) 15-22. GL Caporaso, K. Takei, SE Gandy, M. Matteoli, O Mundigl, P. Greengard, P. De Camilli, Morphologic and biochemical analysis of the intracellular trafficking of the Alzheimer beta / A4 amyloid precursor protein, J. Neurosci. 14 ( 1994) 3122-3138.

  An object of the present invention is to provide a method for obtaining the localization information of APP that seems to be useful for elucidating the function, role, metabolic regulation and the like of APP.

As a result of intensive studies on the APP detection method, when a specific antibody that recognizes the N-terminal side of APP is used under specific conditions, APP is present in the cytoplasm, nucleus, or nucleolus of neuronal cancer cells, etc. I discovered that. Among many years of enormous research, the knowledge that the N-terminal side of APP is localized in the nucleus is not known at all and is a novel knowledge. Moreover, what this knowledge means is an involvement in important regulation that affects the rise and fall of cells.
Specifically, the present invention has the following configuration.
[1] A method for detecting APP including the following steps.
(A) reacting APP in a cell with an antibody that recognizes any part of the APP amino acid sequence at positions 1-210 (excluding 66-81);
(B) forming a complex of the antibody and APP;
(C) detecting the complex; and (d) identifying a detection site of the complex and obtaining APP localization information. [2] The method for detecting APP according to the above [1], wherein a site is selected by processing and the detection is performed based on the presence or absence of detection at the selected site.
[3] The above-mentioned [1], wherein the antibody recognizing any part of APP amino acid sequence 1-210 (excluding 66-81) is an antibody recognizing part 44-63. Or the method of detecting APP as described in [2].
[4] An antibody recognizing any part of APP amino acid sequence 1-210 (excluding 66-81) is GS4463 (Genscript No. A00692, anti-APP antibody (44-63)) or The method for detecting APP according to any one of [1] to [3], which is 10D1 (IBL, No. 11090, anti-human APP (N) antibody (10D1)) (FIG. 6).
[5] An antibody that recognizes any part of the APP amino acid sequence 1-210 (excluding 66-81) is GS4463 (Genscript No. A00692, anti-APP antibody (44-63)). The method for detecting APP according to any one of [1] to [4] above, which is a competing antibody or an antibody competing with 10D1 (IBL, No. 11090, anti-human APP (N) antibody (10D1)).
[6] The above [1] to [5], wherein the antibody recognizing any part of the APP amino acid sequence 1-210 (excluding 66-81) is an antibody recognizing HMP-20. A method for detecting APP according to any one of the above.
[7] The method for detecting APP according to any one of [1] to [6], wherein the detection site is any one or more of cytoplasm, nucleus, or nucleolus.
[8] The method for detecting APP according to any one of [1] to [7], wherein the detection site is a nucleolus and further comprises the following steps.
(E) a step of detecting a nucleolus with a fibralin antibody or elF6 [9] a method for determining the presence or absence of APP localization abnormality in a cell,
(A) contacting APP in a cell with an antibody that recognizes any part of the APP amino acid sequence at positions 1-210 (excluding the 66-81th positions);
(B) forming a complex of the antibody and APP;
(C) detecting the complex; and (d) obtaining APP localization information by specifying a detection site of the complex, and comparing the APP localization of the cell with a previously obtained normal state. A step of determining the presence or absence of sex abnormality
Including methods.
[10] An antibody that recognizes any part of the APP amino acid sequence 1-210 (excluding 66-81), and the localization information of the complex of the antibody and APP and the presence or absence of abnormality A test kit for determining the presence / absence of an abnormality in the localization of APP, including an instruction manual explaining the association.

  According to the present invention, new localization information of APP can be known. And this information leads to obtaining information on new metabolic regulation pathways and functions of APP, and is extremely useful for research to explore the original physiological action of APP and AD onset mechanism. For example, only the information that APP is localized in the nucleus limits the functions related to APP to replication, DNA modification, transcription, and the like. Similarly, the nucleolus is an rRNA synthesis factory and is involved in the control of cell dormancy, growth, and disappearance. Therefore, only the information that APP is localized in the nucleolus can be used as a function related to APP. Can be squeezed. Furthermore, by examining the correlation between the abnormal localization of APP and the disease, it becomes possible to diagnose a disease that correlates with the abnormal localization of APP.

It is a figure which shows the immunofluorescence microscope observation result of APP by each antibody in human neurocytoma SK-N-SH cell. (1) Antibody 10D1 (2) Antibody GS4463 (3) Antibody 22C11 Each left column of (1) to (3) is a fluorescently labeled secondary antibody that visualizes the binding site of each primary antibody, and the center column is DAPI The right column is a diagram in which only the nuclei are visualized by the above, and the right column is a superposition of them. It is a comparison result when stained with GS4463 (left figure) and when antigen peptide HMP20 is reacted with GS4463 antibody in advance before the primary reaction (right figure). It is a figure which shows the immunofluorescence microscope observation result of APP immunostained with the antibody GS4463 and the fibralin antibody or elF6 in the SK-N-SH cell. It is a figure which shows the immunofluorescence microscope observation result of APP by each antibody in a human normal renal epithelial cell (HREC). (1) is antibody 10D1 (2) is antibody GS4463 It is a figure which shows the immunofluorescence microscope observation result of APP by each antibody in a human normal brain stellate cell (NHA). (1) Antibody 10D1 (2) Antibody GS4463 It is a figure which shows the result of having isolate | separated the cytoplasm fraction (Cyto) and the nuclear fraction (NE)) by SDS-PAGE, and carrying out the western blotting by the anti- GAPDH antibody and the anti- fibralin antibody. It is a figure which shows the result of having separated the cytoplasm fraction (Cyto) and the nuclear fraction by SDS-PAGE, and carrying out the Western blotting by each antibody. (1) Antibody 10D1 (2) Antibody 22C11 (3) Antibody GS4463 (4) When antibody GS4463 and peptide HMP20100 μg / ml are preincubated (5) Concentration of antibody GS4463 is increased to twice that of (3) (4 μg / ml), when the detection conditions are strengthened by reacting overnight FIG. 3 is a schematic diagram showing recognition sites for three types of antibodies that recognize the N-terminal side of APP in the APP amino acid sequence.

  The present invention is a method for examining the localization of APP using a specific antibody. The localization of APP refers to detecting how and where APP is present in which part of the cell. Which part is the cell organelle, nucleus, endoplasmic reticulum, mitocondotria, Golgi apparatus, centrosome, nucleus, nuclear membrane, nucleolus, nuclear speckles, PML body, etc. It is meant to include internal structures. In addition, not only the above-mentioned site but also the existence state includes all existence states such as abundance, range of existence, deviation of existence, molecular weight distribution state, modification state, and coexistence with a specific protein. Intention to include. In the examples described later, the presence / absence of APP in the cytoplasm, nucleus, and nucleolus in a specific cell is exemplified as APP localization information and coexistence information.

(detection)
Detection is performed by reacting APP in cells with an antibody. The reaction between the APP in the cell and the antibody is performed by, for example, visualizing and detecting a complex formed by the APP and the antibody in the cell by bringing the cell and the antibody into contact with each other under specific conditions. Visualization is possible, for example, by sensitizing the primary antibody with a fluorescent label or a fluorescently labeled secondary antibody. The cells are fixed, and the complex of the antibody and APP is visualized by fluorescent staining or the like, so that the localization in the whole cell can be immediately detected by observation with a fluorescence microscope. In addition to the above, for example, by pre-treating cells, extraction and separation into cytoplasm, nuclei, and nucleolus components are made into samples of each organ. By forming a body and detecting the complex, the detection site of the complex can also be specified. Furthermore, the detection site may be specified by selectively detecting and sorting the organs without extracting and separating the organs. Here, in addition to quantitative detection of the complex, if it can be quantitatively detected (measured), it can be determined which amount is present in which organ, and the abundance ratio of APP in each organ is also clarified. Can be more detailed information. In addition, coexistence information can also be obtained by simultaneously using other specific organelle marker antibodies.

(Detection conditions)
Detection in cells can be performed by treatments such as washing, immobilization, contact with an antibody, and blocking. Washing is preferably performed with PBS, and immobilization is preferably performed with paraformaldehyde. For the blocking, goat serum is desirable, and it is desirable to use in combination with treatment with Triton X-100.
Specifically, as shown in the examples described below, washing with PBS, fixation: 4% paraformaldehyde, 0 ° C. for 20 minutes (or −20 ° C. to −30 ° C. methanol, 5 minutes) PBS washing, 0. Examples include treatment of 25 to 0.4% Triton X-100 / PBS at 0 ° C for 15 minutes, PBS washing, blocking with 5% goat serum PBS, and at 0 ° C for 1 hour or more. Such a process makes it possible to detect APP in nuclei and nucleolus, which could not be detected in the past, and to examine the localization of APP in cells.
In addition, when detecting the APP in the nucleolus, more accurate APP detection information in the nucleolus can be obtained by using the antibody of the present invention together with the fibralin antibody or elF6 used for the detection of the nucleolus. desirable.

(antibody)
The antibody used in the present invention needs to be an antibody recognizing any part of the APP amino acid sequence 1-210 (excluding 66-81) among APP antibodies. Of these, antibodies that recognize the 44th to 63rd positions are desirable, and specifically, GS4463 (Genscript No. A00692, anti-APP antibody (44-63)), 10D1 (IBL, No. # 11090, anti-human APP). (N) (10D1)) An antibody that competes with GS4463, an antibody that competes with 10D1, or an antibody that recognizes peptide HMP-20 is desirable (FIG. 6).

(Judgment method)
The method for determining the presence or absence of APP localization abnormality in the cell of the present invention is a method comprising the following steps (a) to (d).
(A) contacting the cell with an antibody that recognizes any part of the APP amino acid sequence from the 1-210th position (excluding the 66th to 81st positions);
(B) forming a complex of the antibody and APP;
(C) detecting the complex; and (d) obtaining APP localization information by specifying a detection site of the complex, and comparing the APP localization of the cell with a previously obtained normal state. A step of determining the presence or absence of sexual abnormality,

The step (d) is, for example, a step of quantitatively or qualitatively detecting the presence of APP in the cytoplasm, nucleus, or nucleolus and determining the presence or absence of APP localization abnormality based on the information. is there. The comparison with the normal state obtained in advance refers to, for example, comparison with the APP detection site in the normal state and the APP detection amount at each detection site. For example, for certain types of cells, APP is usually present in a predetermined amount in the nucleus, nucleolus, and cytoplasm, and is present in a large amount in the nucleus and nucleolus and almost absent in the cytoplasm or vice versa. It is judged that the case is abnormal. In addition, it is possible to determine the presence or absence of a cell abnormality and to determine a disease by investigating the type of cell and the disease of the patient from whom the cell originates, obtaining statistical data, and associating the statistical data. . The determination may be made by a person, or the image can be easily analyzed by a computer using pattern recognition software. Pattern recognition software has progressed until human faces can be identified. For example, it will be easy to identify how much additional fluorescence is present in the nucleus stained with DAPI. The shape of the normal pattern could also be stored.

APP is a precursor of amyloid β (Aβ) that is closely related to the pathology of Alzheimer's disease, and is one of the responsible gene products of this disease (Non-patent Document 1).
APP is a single transmembrane protein, and at least three types of splicing molecular species composed of 695 amino acids, 751 amino acids, and 770 amino acids have been reported (Non-patent Document 2). APP has a processing site by α-secretase, β-secretase, and γ-secretase inside (Non-patent Document 3). APP is cleaved in the membrane by γ-secretase to release dissolved APP (sAPP). Furthermore, sAPP is metabolized by an amyloid-generating pathway that is cleaved by α-secretase and a amyloid-generating pathway that is cleaved by β-secretase to produce amyloid Aβ. Intracellular APP is presumably present in the Golgi apparatus and endoplasmic reticulum (Non-patent Documents 6-12). In the cell, the presence of APP in the endoplasmic reticulum or the Golgi is consistent with the secretion of sAPP extracellularly.
There have been some reports suggesting that a short fragment of about 50 amino acids at the C-terminus of APP called AICD is present in the nucleus (Non-patent Document 13). When the C-terminal 59 amino acids of APP695 were expressed from a recombinant gene, it was localized in the nucleus and coexisted with Fe65, a transcriptional regulatory factor. In an expression experiment using a fusion protein of AICD and fluorescent protein, formation of a complex with Tip60, a transcriptional regulator of Fe65 and histone acetylase subunits, was suggested (Non-patent Document 14). These results suggest that a nuclear translocation signal or modification is included in the AICD sequence. However, in the Gal4-Tip60 transcriptional activation experiment, transcriptional activation was observed when expressed from the full-length APP plasmid, but not when AICD was expressed. It was suggested that this is different from the Notch and NICD system. These series of AICD localization experiments are not the behavior of APP originally in the cell, but the observation results of forced expression of AICD fragments by a large amount of external genes, denying the nuclear localization of full-length APP. It is not a thing. If a nuclear translocation signal is included in the AICD sequence, it is possible that full-length APP including AICD is translocated into the nucleus.
APP and its family APLP1 and APLP2 simultaneous knockout mice were lethal (Non-patent Document 15). Since APP knockout mice exhibit motor and memory impairment, it can be easily inferred that APP has important functions. For this reason, not only the vicinity of Aβ42 but also mutations in other important sites of the amino acid sequence of APP may be a disease. Although APP is an important protein related to the onset of Alzheimer, there are still many unclear points regarding its metabolic details and functions. In the present invention, as a result of intensive studies on the intracellular localization of APP using three types of antibodies near the amino terminus of APP, it is shown for the first time that a part of APP is localized in the nucleus and further in the nucleolus. It was. In addition, the significance of localization in the nucleolus and the amino acid sequence homology of the first half of APP suggested the possibility of having RNA binding activity as part of the function.
As the APP behavior revealed this time, it was found that a part of intracellular APP colocalizes with fibrillarin and eIF6 in the nucleolus. Fibrillarin is rRNA 2'-O-methyltransferase and is a component of nucleolar small ribonucleoprotein (snRNA) that participates in the first step of preribosomal rRNA processing (Non-patent Document 16). eIF6 binds to pre-60S ribosomal particles m) and plays a role in transporting ribosomes from the nucleolus to the cytoplasm (Non-patent Document 17).
In the present invention, the immunostaining signal of GS4463 co-localizes with the signals of anti-fibrillarin antibody and anti-eIF6 antibody, suggesting that APP may be involved in ribosome production and cell proliferation. Yes. The role of APP in cell proliferation has been previously suggested by several reports. In one report, fibroblasts in which APP expression was reduced by an antisense RNA expression plasmid had a slower growth rate (Non-patent Document 18). When APP695 was added to the medium from the outside, the growth rate was recovered (Non-patent Document 18). The region essential for this growth recovery was the amino acid at positions 403-407 on the N-terminal side (Non-patent Document 19). From these results, the function of APP in cell proliferation is similar to the autocrine or paracrine mode of growth factors. Soluble APPα has been shown to be extremely important for the elongation of nerve axons via Reelin and integrin α3β1 in vitro and in vivo (Non-patent Documents 20-22).
Nucleolin is well known as a protein that can see the nucleolus and cell surface. Nucleolin is an intranuclear non-histone protein that is ubiquitous, and has been reported to be involved in logarithmic cell proliferation, ribosomal RNA synthesis and processing, and cell death (Non-patent Document 23). Nucleolin is known to be involved in the transfer of ribosomal proteins from the nucleus to the cytoplasm (Non-patent Document 24). In addition, nucleolin has been reported to interact with APP mRNA (Non-patent Documents 25 and 26). Because of these important physiological roles, nucleolin, which is also lethal to nucleolin knockout cells, has many stretches of negatively charged amino acids of various sequences that are not conserved on the amino-terminal side. In this respect, it is similar to the HMG protein that electrostatically interacts with the acidic amino acid portion of histone to relax chromatin. APP also has a highly conserved negative charge amino acid stretch composed of glutamic acid and aspartic acid in the 190-260 amino acid region, and is similar to nucleolin in this respect in addition to intracellular behavior.
As a result of analyzing the APP amino acid sequence homology by the method named OLIGOBLAST in the present invention, the first half of APP 1-500th includes RNA such as tRNA synthetase, ribosomal protein, RNase, transcription factor of various organisms. A partial homology with a protein that can interact with APP suggested that APP interacts with rRNA (Table 1). It was also found that there was partial homology in the negative charge amino acid stretch with nucleolin (Table 1). CDEBP, a DNA binding protein identified in mice, showed sequence homology and domain organization homology with APP over the entire length, and was detected in the nucleus at the cell division interface (Non-patent Document 27).

Consider a case where knowledge of localization information in the nucleolus of APP is obtained.
A nucleolus is a structure that actively constructs ribosomes with rRNA, which is a protein production plant of cells, and is an intracellular organelle that is directly involved in the control of cell life activity, growth, and rest. Since AD is the final phenotype of the decrease and disappearance of the number of neurons in the cranial nerve, the possibility of involvement of nucleolus is also considered.
If a cell is in a state where APP is partly transported into the nucleolus and is involved in the regulation of ribosomal rRNA synthesis, for example, it may affect the movement control of APP and its function in the nucleolus. Mutations can lead to AD. Therefore, to investigate the correlation between the presence / absence and concentration of APP in the nucleolus and the state of cells (growth, differentiation, cessation, apoptosis) and the onset of dementia is the mechanism of AD onset. May lead to investigation. Therefore, the detection method of the present invention is a very important technique for clarifying the cause or action mechanism of AD.

(kit)
The test kit of the present invention is a kit containing at least the following (A) and (B).
(A) An antibody that recognizes any part of the APP amino acid sequence 1-210 (excluding 66-81) (B) Localization information of the complex of the antibody and APP and the presence or absence of abnormality Instructions explaining the association of here,
The instruction manual (B) refers to, for example, an instruction manual describing the presence of APP examined in the cytoplasm, nucleus, and nucleolus, that is, the relationship between the localization of APP and the presence or absence of abnormality. More specifically, for certain types of cells, it is normal that APP is present in a predetermined amount in the nucleus, nucleolus, and cytoplasm, and is abnormal when it is almost absent in the nucleus and nucleolus, or In the opposite case, an explanation is given that describes the abnormality. In addition, it is possible to determine the presence or absence of a cell abnormality and to determine a disease by investigating the type of cell and the disease of the patient from whom the cell originates, obtaining statistical data, and associating the statistical data. .

(Target cell)
The cell to which the detection method of the present invention can be applied is not particularly limited, and any cell can be a detection target.

I Material (1) Primary antibody (i) GS4463 (named GS4463 herein) (FIG. 6)
Genscript No. A00692, anti-APP antibody (44-63)
It is an affinity purified rabbit polyclonal anti-peptide antibody whose immunogen is amino acid residues 44-63 of human APP. Used at 2 μg / ml.
(Ii) 10D1 (FIG. 6)
IBL, No. 11090, anti-human APP (N) antibody (10D1)
The epitope is located at the N-terminal 1-210 amino acid of APP, as obtained by immunizing a GST fusion of the N-terminal 1-210 amino acid residue of human APP. Mouse monoclonal antibody. Used at 5 μg / ml.
(Iii) 22C11 (FIG. 6)
Millipore Corporation MAB348, an anti-APP antibody immunogen, is a 66-81 amino acid residue peptide of APP. Mouse monoclonal antibody. Used at 1 μg / ml. A schematic diagram showing the recognition site in the APP amino acid sequence of the antibodies (i) to (iii) above is shown in FIG.
(Iv) Fibrillarin antibody Abcam No. ab4566, fibrillarin antibody [38F3] mouse IgG monoclonal antibody. Used at 1000-fold dilution.
(V) Anti-eIF6 antibody Cell Signaling Technology Anti-eIF6 antibody (D16E9) 3833, a rabbit monoclonal antibody. Used at 1000-fold dilution.
(Vi) Anti-GAPDH antibody Cell Signaling Technology, No. 2118, anti-GAPDH antibody (14C10), rabbit monoclonal antibody. Used at 1000-fold dilution.
(2) Secondary antibody (2-1) For fluorescent staining Both were used at 1000-fold dilution.
(I) Alexa Fluor® 488 (No. A11001) goat anti-mouse IgG
(Ii) Alexa Fluor® 488 (No. A11008) goat anti-rabbit IgG (H + L)
(Iii) Alexa Fluor® 555 (No. A21422) goat anti-mouse IgG (H + L)
(2-2) For Western blot analysis All were used at 5000 times dilution.
(I) Rabbit polyclonal anti-mouse immunoglobulin alkaline phosphatase conjugate (DAKO No. D0314)
(Ii) Goat polyclonal anti-rabbit immunoglobulin alkaline phosphatase conjugate (DAKO No. D0487)
(3) Human APP synthetic peptide (HMP20)
A peptide (HMP20) consisting of the N-terminal 44-63rd amino acid sequence of human APP (Ac-HMNVQNGKWDSDPSGKTCI-CONH2 (purity: 92%))
According to the BLAST search, there is no protein other than APP that has 70% or more homology with this sequence in humans. Obtained from Operon Biotechnologies (Tokyo).
(4) Cell lines and cell culture conditions (i) Human neuroblast SK-N-SH
DS Pharma Biomedical Co. , Ltd., Ltd. Product No. EC86012802
Culture in MEM-α (ii) Normal human astrocytes CC-2565
Obtained from Lonza (AGM BulletKit and REGM ™ BulletKit) cultured in (iii) human kidney epithelial cells CC-2554
(5) Cell extract (i) Protein for Western blotting Cells cultured in a 90 mm dish were washed twice with PBS and detached with a cell detachment solution (Sigma Aldrich No. C5914). Cells precipitated by centrifugation at 1000 rpm for 5 minutes at 4 ° C. were resuspended in 400 μL ice-cold PBS containing protease inhibitors (Roche No. 697 498 001 11). 5 μL of Benzonase nuclease (Merck No. 70644-3) and 100 μL of 5 × SDS sample buffer were added with rapid mixing and used as protein for Western blotting.
(Ii) Cytoplasmic fraction The cells separated from the 90 mm dish by washing and centrifuging were collected by 300 μL of buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, 0. Resuspended in 1 mM APMSF, protease inhibitor (Roche No. 11 697 498 001)) and left on ice for 15 minutes. Next, 18.8 μL of 10% Nonidet P-40 was added with vortexing and centrifuged at 2500 rpm (500 × g) for 3 minutes. The supernatant was used as the cytoplasmic fraction.
(Iii) Nuclear fraction The precipitate of (ii) above was resuspended in 300 μL of buffer A using a pipette and centrifuged at 2500 rpm for 1 minute. The pellet was resuspended in 300 μL buffer B (20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM APMSF, protease inhibitor (Roche)) and incubated on ice for 15 minutes. And centrifuged at 15,000 rpm. The supernatant was used as the nuclear fraction.

II Immunostaining and immunofluorescence microscopy In all experiments, cells (3 × 10 ⁵ ) were cultured at 37 ° C., 5% CO _{2 in} 0.3 mL of the medium shown in the figure in 8354688 BD BioCoat PDL / Laminin 8-well culture slides. For 24 hours. Immunostaining was performed at 0 ° C. on ice. Cells were washed twice with PBS (5 minutes each), fixed with 4% paraformaldehyde diluted in PBS for 20 minutes, washed twice (5 minutes each), and 5% goat serum diluted in PBS for 1 hour. Blocked. The fixed cells were cultured overnight at 4 ° C. in 250 μL blocking buffer containing the first antibody. The concentration of the first antibody was 2 μg / mL (GS4463), 5 μg / mL (10D1), and 1 μg / mL (22C11). After incubation with the first antibody, cells were washed 3 times with PBS (5 minutes each) and blocked with 5% goat serum diluted in PBS for 30 minutes. The blocking buffer was replaced with the same buffer containing the second antibody, and Alexa488 or Alexa555 (Invitrogen) conjugate was added at a 1000-fold dilution, followed by incubation for 1 hour. After washing 3 times (5 minutes each), Prolong Gold containing DAPI (Invitrogen) was added to the cells. Immunostained slides were analyzed with a Carl Zeiss LSM510 META Confocal. In a competition experiment using GS4463, HMP20, human APP synthetic peptide (44-63) (Ac-HMNVQNGKWDSDPSGKTCI-CONH2), before performing the staining reaction, GS4463 was added to PBS containing 5% goat serum and 100 μg / mL of the peptide. And preincubation for 1 hour at room temperature.

III Western Blot Analysis The protein concentration of the SDS-PAGE sample was diluted 10-fold with pure water (DW) and then measured by the BIORAD protein assay. Add 10 μg SDS-PAGE cell extract protein sample or 6 μL molecular weight marker (BIORAD Precision Plus Dual Color) to 4-20% SDS-polyacrylamide gel (Cosmobio Co. Ltd., No. 414879) (20 mA). Blotted onto a nitrocellulose membrane (No. IB3010-02) by the iBlot system (Invitrogen No. IB1001). Membranes were blocked with 3% skim milk diluted in PBST (TBS with 0.1% Tween-20) for 1 hour at room temperature or overnight at 4 ° C. and in 0.3% skim milk / PBST at the concentrations indicated above. Incubated with primary antibody. Next, the membrane was washed 3 times with 0.3% skim milk / PBST (5 minutes each), and incubated with a secondary antibody (alkaline phosphatase conjugate) (dilution rate: 5000 times) for 1 hour. After washing three times, an immune reaction band was detected by reacting with an alkaline phosphatase substrate solution (1-step NBT / BCIP substrate, Thermo Scientific No. PI34042) for 30 minutes.

[Example 1] Detection of APP in human neuroblastoma cell line SK-N-SH (1)
1. Test Method For human neuroblastoma cell line SK-N-SH, cells were immunostained using antibodies 10D1, GS4463, 22C11 and observed with an immunofluorescence microscope.
2. Test results The results of staining are shown in FIG.
10D1 stained the cytoplasm and whole nucleus of the cells. GS4463 also stained cytoplasm and nucleus. The punctate staining signal from within the nucleus by GS4463 clearly showed nucleoli or small spot-like structures. On the other hand, the staining pattern by 22C11 was different from the above two antibodies, and most existed in the cytoplasm and tubulin-like network structure. Preincubation with GS4463 and 100 [mu] g / mL human APP synthetic peptide (44-63) abolished cytoplasmic and nuclear staining signals. These results indicate that the immunostaining signal from GS4463 appears from an antibody that recognizes the sequence of amino acids 44-63 of APP.

[Example 2] Detection of APP in human neuroblastoma cell line SK-N-SH (2)
1. Test Method According to Example 1 above, it was found by GS4463 that APP was present in the cytoplasm and nucleus, and it was further examined whether or not the APP is present in the nucleus in the nucleolus. SK-N-SH cells were stained with antibodies to nucleolar markers fibrillarin and eIF6 and GS4463.
2. Test results The results are shown in FIG.
The signal from GS4463 in the nucleus was completely consistent with the signals from fibrillarin and eIF6 antibody, confirming the co-localization of APP and nucleolar markers. On the other hand, the signal from GS4463 did not colocalize with the antibody of small spot marker SC35 antibody (ab11826) (data not shown). These results indicate that a fraction of APP is located in the nucleolus (at least in the cultured neuroblast cell line SK-N-SH).

[Example 3] Detection of APP in primary cultured cells collected from normal human tissue, brain and kidney. Primary cultured cells collected from normal human tissues, brain and kidney were immunostained using antibodies 10D1 and GS4463 and observed with an immunofluorescent microscope.
2. Test results The results are shown in FIGS.
10D1 stained the nucleus of human kidney epithelial cells (HREC) as with SK-N-SH cells, but stained normal cytoplasm in normal human astrocytes (NHA). GS4463 showed nucleolus-like staining in both NHA and HREC, similar to SK-N-SH cells. These signals from 10D1 and GS4463 were not observed by preculture with human APP synthetic peptide (44-63). Therefore, it is shown that a part of the APP fraction is localized in the nucleolus even in normal cells collected from the brain and kidney.

[Example 4] Detection of APP in cytoplasmic extract and nuclear extract Test Method To examine the distribution and molecular weight of APP processing products in the cytoplasmic fraction (cytoplasmic extract) and nuclear fraction (nuclear extract), the cytoplasmic fraction of SK-N-SH cells cultured in MEM-α and The nuclear fraction was analyzed by Western blot using antibody GS4463. Concentration of the extract from each fraction was confirmed by antibodies against GAPDH and fibrillarin (FIG. 5-1).
2. Test results The results are shown in Fig. 5-2.
A band of 110-120 Kd, which appears to be full-length APP, was observed in the cytoplasmic fraction by all antibodies (FIGS. 5-2 (1)-(3)). Higher molecular weight smear bands such as glycosidation reactions were observed using 10D1 or 22C11. A 30 Kd band specific for the nuclear fraction was observed with 10D1. No major immunological staining band in the nuclear fraction was observed by 22C11. In addition to the 120 Kd product, a 50 kd product was observed in the cytoplasmic fraction (major band) and the nuclear fraction by GS4463, while the 110 kd band observed in the cytoplasmic fraction was not detected in the nuclear fraction. When the amount of protein prepared by the protocol described above is the same, the molar ratio of 120 kd product to 50 Kd product appears to be higher in the nuclear fraction. The 120 kd and 50 Kd products disappear upon preincubation with human APP synthetic peptide (44-63), indicating that these signals are derived from an antibody that recognizes the 44-63 amino acid sequence of APP. In addition, a 70 Kd product was detected in both fractions, and a 60 Kd product was observed only in the nuclear fraction under strong staining conditions (FIGS. 5-2 (5)).

[Example 5] APP amino acid sequence and DNA or RNA binding protein amino acid sequence homology A functional protein is considered to be a combination of sequences of about 15 amino acids called a motif, which is the minimum functional unit of a polypeptide. Based on this, the amino acid sequence 1-770 of human APP was divided every 30 to 50 amino acids and searched by the homology search algorithm BLAST ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). . Usually, in BLAST, the longer the homology region, the better the score, and the hit target is listed from the score with high homology. For this reason, even if a long sequence is subjected to a BLAST search as it is, hit targets with short sequences even if the homology is high have a very low score and are not visible in the top 250 in the list. However, by applying a short sequence to BLAST, a short but highly homologous hit object can be seen. Such hits have high E values and low individual reliability, but when various proteins with similar functions are hit or the hit positions are concentrated, the function of the region is reduced. Analogy is possible. Such a method is referred to herein as OLIGOBLAST. As a result, as shown in Table 1, 1 to 500 of APP has a short but 60-100% homology with tRNA synthetase, ribosomal protein, RNase, transcription factor, DNA binding protein, etc. of various microorganisms. It turned out that there was something, especially concentrated on the 400th to 500th. In addition, homology with nucleolin, which binds to rRNA and participates in ribosome construction and travels between the cell surface and the nucleolus, was also found. This search method is useful because these hits are not found in a normal motif search algorithm.

According to the present invention, new localization information of APP can be known. This information leads to the acquisition of information on APP's new metabolic regulation pathways and functions, and is extremely useful for research that explores the original physiological actions of APP and the onset mechanism of AD. For example, only the information that APP is localized in the nucleus narrows the functions related to APP to replication, DNA modification, and transcription. Similarly, the nucleolus is an rRNA synthesis plant that is involved in the control of cell quiescence, growth, and disappearance. Can be squeezed.
Furthermore, by examining the correlation between the abnormal localization of APP and the disease, it becomes possible to diagnose a disease that correlates with the abnormal localization of APP.

[Non-patent literature]
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Claims (10)

A method for detecting APP comprising the following steps.
(A) reacting APP in a cell with an antibody that recognizes any part of the APP amino acid sequence at positions 1-210 (excluding 66-81);
(B) forming a complex of the antibody and APP;
(C) detecting the complex; and (d) specifying a detection site of the complex and obtaining localization information of APP. The method for detecting APP according to claim 1, wherein the detection site of the complex is identified by pre-treating cells to select a site, and detecting whether or not the selected site is detected. The antibody recognizing any one part of the amino acid sequence of APP (excluding the 66th to 81st positions) of APP is an antibody recognizing a part of the 44th to 63rd aspects. To detect the APP. An antibody that recognizes any part of the APP amino acid sequence 1-210 (excluding 66-81) is GS4463 (Genscript No. A00692, anti-APP antibody (44-63)) or 10D1 (IBL). No. 11090, anti-human APP (N) antibody (10D1)). An antibody that recognizes any part of the APP amino acid sequence 1-210 (excluding 66-81) is bound to GS4463 (Genscript No. A00692, anti-APP antibody (44-63)) and APP binding. The method for detecting APP according to any one of claims 1 to 4, which is an antibody that competes with a competing antibody or 10D1 (IBL, No. 11090, anti-human APP (N) antibody (10D1)) in APP binding. An antibody that recognizes any part of the APP amino acid sequence 1-210 (excluding 66-81) is a peptide consisting of the N-terminal 44-63 amino acid sequence of human APP ( HMP-20). The method for detecting APP according to any one of claims 1 to 5, wherein The method for detecting APP according to any one of claims 1 to 6, wherein the detection site is one or more of cytoplasm, nucleus, or nucleolus. The method for detecting APP according to claim 1, wherein the detection site is a nucleolus and further comprises the following steps.
(E) a step of detecting nucleolus with an anti - fibrallin antibody or an anti- elF6 antibody A method for determining the presence or absence of APP localization abnormality in a cell,
(A) contacting APP in a cell with an antibody that recognizes any part of the APP amino acid sequence at positions 1-210 (excluding the 66-81th positions);
(B) forming a complex of the antibody and APP;
(C) detecting the complex; and (d) obtaining APP localization information by specifying a detection site of the complex, and comparing the APP localization of the cell with a previously obtained normal state. A step of determining the presence or absence of sex abnormality
Including methods. An antibody that recognizes any part of the amino acid sequence of APP (excluding positions 66-81), and the association between the localization information of the complex of the antibody and APP and the presence or absence of abnormality Instruction manual,
A test kit for determining the presence / absence of abnormalities in the localization of APP.