JP5773415B2 - Method for detecting glycated protein and biosensor chip for detecting glycated protein - Google Patents

Description

  The present invention relates to a method for detecting a glycated protein and a biosensor chip for detecting the glycated protein. The present invention also relates to a method for purifying glycated protein and a column for purification.

  With the diversification of dietary habits in recent years, various lifestyle-related diseases have spread and become a social problem. In order to prevent lifestyle-related diseases, various studies such as metabolome-related research have been energetically advanced, but the clues of prevention are hardly visible. One reason for this is that a system for objectively evaluating parameters that define individual lifestyle habits caused by various lifestyle-related diseases has not been established.

  Among lifestyle-related diseases, diabetes has a large number of patients, and according to the 2007 demographic statistics by the Ministry of Health, Labor and Welfare, the number of deaths per year is about 14,000 in Japan. About 15,000 people start artificial dialysis due to kidney problems, and about 3,000 people are suffering from visual impairment due to diabetes each year.

  It is known that proteins in the body undergo glycation non-enzymatically when hyperglycemia persists for a long time, such as in diabetes. In a recent study, various glycated proteins formed by the “sustained hyperglycemic state” characteristic of diabetes have been found in cardiovascular diseases such as arteriosclerosis (Selvin E., et.al., “Glycated hemoglobin, diabetes , and cardiocascular risk in nondiabetic adults ”, N. Engl. J. Med., 2010, 362 (9): 800-811), Alzheimer's disease (Takeuchi M. et. al., Curr. Alzheimer Res., 2004, 1 : 39-46, Sato T. et. Al., Am. J. Alzheimers Dis. Other Dement., 2006, 21: 197-208), cancer growth and metastasis (Abe R., J. Ivest. Dermatol. , 2004, 122: 461-467), it is becoming clear that it can be an important cause of complications such as inflammatory reactions. In addition, it has been found that various proteins undergo glycation with aging even in a normal blood glucose state.

  In order to measure a glycated protein, it is necessary to measure the glycated protein separately from the non-glycated protein. At present, measurement by high performance liquid chromatography (HPLC-MS / MS) connected to a mass spectrometer, measurement by ELISA using a glycated protein-specific antibody, and the like are mainly used. However, the method using HPLC is difficult to use for a simple test because the apparatus is heavy and long and takes time. In addition, the method using a glycated protein-specific antibody makes it difficult to obtain a glycated protein-specific antibody, and therefore, testing using an antibody is only used in limited cases. Therefore, establishment of a method that can easily measure glycated protein has been strongly desired.

  By the way, it is known that galectin existing in a living body regulates cell adhesion and cell information transmission by crosslinking a complex carbohydrate containing β-galactoside (Non-patent Document 1). Moreover, the relationship between galectin and glycosaminoglycan which is a kind of sugar chain has been reported (Patent Document 1). However, no relationship between galectin and glycated protein has been reported so far.

JP 2007-332136 A

Leffler, H. “Introduction to Galectins” Trends in Glycoscience and Glycotechnology, Vol.9, No.45, pp9-19 (1997)

  An object of the present invention is to provide a method for easily detecting a glycated protein and to provide a biosensor chip for detecting a glycated protein. It is another object of the present invention to provide a method for purifying glycated protein and a column for purifying glycated protein.

  The present inventor has found that galectin-3 belonging to the galectin family specifically recognizes certain glycated protein groups. The present inventor has also found that a glycated protein can be accurately measured by a surface plasmon resonance method using a biosensor chip on which galectin is immobilized. The present invention is based on these findings.

That is, according to the present invention, the following inventions are provided.
(1) A method for detecting a glycated protein comprising measuring the amount of glycated protein bound to galectin or a carbohydrate recognition domain (Carbohydrate Recognition Domain; hereinafter simply referred to as “CRD”) "The detection method of the present invention").
(2) The method according to (1) above, wherein the galectin is galectin-3.
(3) Early reaction product of glycation reaction, late reaction product of saccharification reaction added with carboxymethyllysine or carboxyethyllysine, or late reaction product of glycation reaction derived from glucose, methylglyoxal or glyceraldehyde The method according to (1) or (2) above, which is a product.
(4) The amount of glycated protein bound to galectin or its CRD is measured by surface plasmon resonance method, quartz crystal microbalance measurement method, reflection interference spectroscopy, protein chip method, semiconductor contact type biosensor method or electrochemical method The method according to any one of (1) to (3) above.
(5) The method according to any one of (1) to (4) above, which is a method for measuring a blood glucose level, glycated LDL or glycated Aβ.
(6) A biosensor chip comprising a solid support on which galectin or its CRD is immobilized.
(7) The biosensor chip according to (6), wherein the galectin is galectin-3.
(8) The biosensor according to (6) or (7) above, which is used for detecting a glycated protein.
(9) Early reaction product of saccharification reaction with glycated protein, late reaction product of saccharification reaction with addition of carboxymethyllysine or carboxyethyllysine, or late reaction product of saccharification reaction derived from glucose, methylglyoxal or glyceraldehyde The biosensor chip according to (8), wherein
(10) The biosensor chip according to any one of (6) to (9), which is used for measuring a blood glucose level.
(11) An affinity column obtained by binding galectin or its CRD to a carrier.
(12) A method for purifying a glycated protein using the affinity column described in (11) above.

  In the present invention, the glycated protein can be detected by utilizing the binding ability of the glycated protein to galectin. Since the blood concentration of the glycated protein is related to the blood glucose level as described later, the present invention can be used for the measurement of the blood glucose level. The present invention can also be used to separate specific glycated proteins from a sample. Since glycated proteins are related to diseases such as lifestyle-related diseases, the present invention can be used as equipment and techniques for elucidating in vivo mechanisms of glycated proteins.

FIG. 1 is a schematic diagram showing a typical route through which AGE is generated from D-glucose. FIG. 2 is a view obtained by analyzing the binding and dissociation between galectin-3 immobilized on the sensor chip and each AGE by SPR. FIG. 2-A shows an SPR sensorgram for binding to glucose-AGE-BSA, FIG. 2-B shows an SPR sensorgram for binding to CEL-HSA, and FIG. 2-C shows glyceraldehyde-AGE-BSA. FIG. 2-D shows the SPR sensorgram of binding with methylglyoxal-AGE-BSA. In FIGS. 2A, B, C and D, a, b, c, d, e and f represent AGE concentrations of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL and 12.5 μg / mL, respectively. The sensorgram when it is 6.25 μg / mL is shown. FIG. 3 is a view obtained by analyzing the binding / dissociation between CRD of galectin-3 immobilized on the sensor chip and each AGE by SPR. FIG. 3-A shows an SPR sensorgram for binding to glucose-AGE-BSA, FIG. 3-B shows an SPR sensorgram for binding to CEL-HSA, and FIG. 3-C shows glyceraldehyde-AGE-BSA. The SPR sensorgram for binding to and glyoxal-AGE-BSA is shown in FIG. 3-D. In FIGS. 3A, B, C and D, a, b, c, d, e and f represent AGE concentrations of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL and 12.5 μg / mL, respectively. The sensorgram when it is 6.25 μg / mL is shown. FIG. 4 shows the association rate constant (K _on ) and dissociation rate constant (K _off ) between galectin-3 and each AGE. FIG. 4-A shows the association rate constant (K _on ) between galectin-3 and each AGE, and FIG. 4-B shows the dissociation rate constant (K _off ). FIG. 5 shows the association rate constant (K _on ) and dissociation rate constant (K _off ) between the CRD of galectin-3 and each AGE. FIG. 5-A shows the association rate constant (K _on ) between CRD of galectin-3 and each AGE, and FIG. 5-B shows the dissociation rate constant (K _off ). FIG. 6 shows the binding characteristics of galectin-3 to glyceraldehyde-AGE-BSA. 6A, a shows an SPR sensorgram of the binding between glycolaldehyde and galectin-3, and b shows an SPR sensorgram of the binding between glycolaldehyde-AGE-BSA and galectin-3. In FIG. 6-B, c shows an SPR sensorgram of binding between glyceraldehyde and galectin-3, and d shows an SPR sensorgram of binding between glyceraldehyde-AGE-BSA and galectin-3.

Detailed Description of the Invention

  The method for detecting glycated protein of the present invention comprises measuring the amount of glycated protein bound to galectin. Since the amount of glycated protein bound to galectin indicates the presence or content of the glycated protein in the sample, the glycated protein detection method of the present invention can determine the presence or absence of the glycated protein in the sample. In addition, the glycated protein in the sample can be quantified.

  As used herein, “glycated protein” refers to a series of proteins produced by non-enzymatic binding of protein and glucose by an aminocarbonyl reaction called saccharification reaction or Maillard reaction. Glycated proteins are roughly classified into early reaction products and late reaction products.

  The pre-reaction product is a product of the pre-reaction of the Maillard reaction. In Maillard reaction, monosaccharides such as glucose, galactose, and fructose react with proteins to form a Schiff base, and then a stable Amadori transfer product is formed. Pre-reaction products include Schiff bases and Amadori rearrangement products.

  Late reaction products were collectively called AGE (Advanced glycation end-product) and were found as products from the late reaction of Maillard reaction. Subsequent research has revealed that late reaction products (hereinafter sometimes simply referred to as “AGE”) are also produced in various reaction pathways that do not go through the early reaction. FIG. 1 shows a typical pathway for producing various AGEs from D-glucose. In FIG. 1, AGE-1 produced from D-glucose via a Schiff base, Amadori transfer product, AGE-2 produced from glyceraldehyde, AGE-3 produced from glycolaldehyde, produced from methylglyoxal AGE-4 and carboxyethyllysine (hereinafter simply referred to as “CEL”), AGE-5 and carboxymethyllysine (hereinafter sometimes simply referred to as “CML”) produced from glyoxal are shown as AGE Has been.

  Examples of the AGE detected by the present invention include CML, pentosidine, pyralin, croslin, CEL, pyropyridine, imidazolone, argyprimidine, GA-pyridine, MG-imidazolone, 3DG-imidazolone, GOLD (glyoxal-derived lysine dimer). AGE to which MOLD (methylglyoxal-derived lysine dimer), DOLD (3-deoxyglucosone-derived lysine dimer), vesperlysine, and MRX (Maillard reaction product X) are added.

  The AGE detected by the present invention is preferably a late reaction product of a saccharification reaction to which CML or CEL is added, or a late reaction product of a glycation reaction derived from methylglyoxal, glyceraldehyde or glucose. it can.

Examples of proteins that undergo glycation in vivo include extracellular matrix proteins such as collagen, myelin, fibronectin, fibrin, and amyloid β (Aβ); erythrocyte glucose transport protein, erythrocyte spectrin, erythrocyte membrane protein, and endothelial cell membrane protein. Membrane proteins; intracellular proteins such as hemoglobin A, lens crystallin, tubulin, calmodulin; cathepsin B, lysozyme, pancreatic riboase, copper / zinc superoxide dismutase, carbonic acid dehydratase, β-N-acetylhexosaminidase, alcohol Enzymes such as dehydrogenase, aldose reductase, aldehyde reductase, sorbitol dehydrogenase, Na ⁺ / K ⁺ -ATPase; albumin, immunoglobulin, apo AI Apo A-II, apo B, apo C-I, apo E, haptoglobin, ferritin, transferrin, α1-antitrypsin, plasminogen, plasminogen activator, fibrinogen, fibrin, antithrombin III, β2-microglobin, Plasma proteins such as ceruloplasmin, thrombomodulin, lipoprotein; and hormones such as thyroid hormone, insulin. Any glycated protein derived from such a protein is included in the glycated protein detected by the present invention.

  In the present invention, galectin is used for detection of glycated protein. In mammals, 15 types of galectins from galectin-1 to galectin-15 and their isoforms are known (Non-patent Document 1). In addition to mammals, galectins have been found in birds, amphibians, fish, nematodes, sponges, and the like (Non-patent Document 1). Therefore, “galectins” in this specification should be understood to include all galectins of mammals, birds, amphibians, fish, nematodes, and sponges. That is, in the present invention, mammals, birds, amphibians, fish, nematodes, sponge galectins can be used for detection of glycated proteins, preferably mammalian galectins can be used, and more preferably, Mammalian galectin-3 can be used, most preferably human galectin-3. Human galectin-3 corresponds to the amino acid sequence of SEQ ID NO: 2.

  In the present invention, CRD of galectin may be used for detection of glycated protein. CRD is a region conserved across species, and those skilled in the art can easily identify the region (Non-patent Document 1). That is, in the present invention, CRDs of mammals, birds, amphibians, fish, nematodes, and sponge galectins can be used for detection of glycated proteins. Preferably, CRDs of mammalian galectins can be used. More preferably, mammalian galectin-3 CRD can be used, and most preferably, human galectin-3 CRD can be used. The CRD of human galectin-3 corresponds to the amino acid region of Nos. 114 to 250 of SEQ ID NO: 2.

  As used herein, “galectin” includes a modified galectin as long as its glycated protein binding ability is not impaired. Examples of the modified galectins include galectins having a high binding ability to a specific glycated protein or all glycated proteins, and galectins having different glycated protein selectivity. As a method for preparing a modified galectin, for example, by using a method of molecular evolution in vitro, a galectin with enhanced binding characteristics to glycated protein or a galectin with altered binding characteristics is prepared, and the obtained galectin is glycated. Examples include a method of binding to a protein. Specifically, artificially introduces mutations into DNA encoding galectin using DNA synthase, etc., and obtains DNA encoding galectin that exhibits higher binding ability to one or more specific glycated proteins. can do. If the desired binding ability cannot be obtained sufficiently, galectin with enhanced binding properties can be obtained by further in vitro molecular evolution. Techniques for obtaining a protein having higher binding ability to a specific substance or DNA encoding the protein by molecular evolution in vitro are well known to those skilled in the art. Further, the evaluation of the bonding characteristics can be performed according to the procedure described in the examples described later.

  Similarly, in the present invention, a CRD of a modified galectin can be used as long as its glycated protein binding ability is not impaired.

  Examples of the modified galectin-3 include a protein consisting of the amino acid sequence of SEQ ID NO: 2 having one or more modifications. This protein can have a function equivalent to the amino acid sequence of SEQ ID NO: 2, and for example, it can be assumed that the glycated protein binding ability is not impaired.

  Examples of the modified CRD of galectin-3 include a protein consisting of amino acid region 114 to 250 of SEQ ID NO: 1 having one or more modifications. This protein can have a function equivalent to the amino acid sequence of SEQ ID NO: 2, and for example, it can be assumed that the glycated protein binding ability is not impaired.

  Whether the modified amino acid sequence has a glycated protein binding ability can use the method of molecular evolution in vitro as described above. Here, the “modification” of the amino acid sequence is selected from substitution, deletion, insertion and addition, and the “substitution” can be a “conservative substitution” which does not substantially affect the function of the protein. When there are a plurality of modifications, they may be the same or different. The number of modifications is preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to several, and most preferably 1, 2, 3 or 4.

  As used herein, “conservative substitution” refers to substitution of one or more amino acid residues with another chemically similar amino acid residue so as not to substantially alter the function of the protein. means. For example, when a certain hydrophobic residue is substituted by another hydrophobic residue, a certain polar residue is substituted by another polar residue having the same charge, and the like. Functionally similar amino acids that can make such substitutions are well known to those skilled in the art for each amino acid. Specifically, examples of nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of the basic amino acid having a positive charge include arginine, histidine, and lysine. Examples of acidic amino acids having a negative charge include aspartic acid and glutamic acid.

  The modified galectin-3 also includes a protein comprising an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity with the amino acid sequence of SEQ ID NO: 2. This protein can have a function equivalent to the amino acid sequence of SEQ ID NO: 2, and for example, it can be assumed that the glycated protein binding ability is not impaired.

  As a modified CRD of galectin-3, a protein comprising an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity with the amino acid region of SEQ ID NO: 2 from No. 114 to No. 250 Is mentioned. This protein can have a function equivalent to the amino acid sequence of SEQ ID NO: 2, and for example, it can be assumed that the glycated protein binding ability is not impaired.

  Here, any numerical value of the identity of amino acid sequences may be a numerical value calculated using a homology search program known to those skilled in the art. For example, the homology algorithm BLAST of the National Center for Biotechnology Information (NCBI) It can be calculated by using parameters of (Basic local alignment search tool) (initial setting).

  The modified galectin-3 further includes a protein comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a complementary strand of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 2. This protein can have a function equivalent to the amino acid sequence of SEQ ID NO: 2, and for example, it can be assumed that the glycated protein binding ability is not impaired. Examples of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 include a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1.

  The modified CRD of galectin-3 consists of an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a complementary strand of the polynucleotide encoding the amino acid region of amino acids 114 to 250 of SEQ ID NO: 2. Examples include proteins. This protein can have a function equivalent to the amino acid sequence of SEQ ID NO: 2, and for example, it can be assumed that the glycated protein binding ability is not impaired.

  In the present specification, “hybridizes under stringent conditions” means that the membrane is washed after hybridization in a low salt concentration solution at a high temperature. Here, “stringent conditions” refers to a washing solution in which a reaction is performed at a temperature of 40 ° C. to 70 ° C. or the like in a hybridization buffer that can be commonly used by those skilled in the art, and a salt concentration of 15 to 300 mmol / L or the like. Those who are skilled in the art should appropriately set conditions under which polynucleotides having 90% or more identity with the target sequence will hybridize and polynucleotides with less than 90% identity will not hybridize. Can do. The stringent conditions are, for example, washing conditions of 0.5 × SSC concentration (1 × SSC: 15 mM trisodium citrate, 150 mM sodium chloride), 60 ° C., and 15 minutes. Hybridization can be performed according to a known method.

  In the present invention, two or more types of galectins selected from galectins and galectin CRDs can be used in combination for detection of glycated proteins. The galectin used may be used in the presence of another protein that forms a complex with galectin in vivo (for example, OST48 and / or 80K-H), or may be used alone.

  The galectin and galectin CRD used in the present invention can be prepared by various known methods such as genetic engineering techniques and synthesis methods. Specifically, in the case of genetic engineering techniques, a galectin or CRD-encoding polynucleotide is introduced into an appropriate host cell, and the resulting transformant is cultured under conditions that allow expression, and the expressed protein is separated. And can be prepared by separating and purifying the desired polypeptide from the culture by methods commonly used for purification. In the case of the synthesis method, it can be synthesized according to a conventional method such as a liquid phase method or a solid phase method, and an automatic synthesizer can be usually used. The synthesis of the chemically modified product can be performed by a conventional method.

  The polynucleotide encoding galectin or its CRD may be naturally derived or fully synthesized, and in addition to this, it was synthesized using a part of naturally derived. May be. As a typical method for obtaining the polynucleotide encoding the galectin and its CRD of the present invention, for example, from a commercially available library or cDNA library, a method commonly used in the field of genetic engineering, for example, galectin or its CRD is used. And a method of screening using an appropriate DNA probe prepared based on the partial amino acid sequence information.

  In the detection of the glycated protein of the present invention, any method may be used as long as it can detect an interaction between proteins. For example, methods for detecting interactions between proteins such as biosensor methods, immunoprecipitation methods, column methods, determination methods based on protein colocalization, electrochemical methods, fluorescent labeling methods, etc. Any method can be used, which is well known to those skilled in the art. A biosensor method is preferred in that not only the presence or absence of protein but also the amount of binding thereof can be monitored quickly and easily and the interaction between proteins can be detected without labeling.

  Biosensors that can be used for detection of glycated protein in the present invention are not limited to the following, but include, for example, surface plasmon resonance (hereinafter sometimes simply referred to as “SPR”), quartz crystal microbalance measurement method ( Hereinafter, it may be simply referred to as “QCM”), reflection interference spectroscopy, protein chip method, or semiconductor contact biosensor method.

  In surface plasmon resonance, when galectin immobilized on a sensor chip and glycated protein are combined, a change occurs in the resonance angle that occurs when light is irradiated, and the glycated protein can be detected by the change in the resonance angle. It is. In the quartz crystal microbalance measurement method, when galectin immobilized on the sensor chip and glycated protein are combined, the resonance frequency of the quartz crystal is changed (decreased). Can be detected. In reflection interferometry, when galectin and glycated protein immobilized on a sensor chip are combined, a physical thickness change and a light refractive index change occur on the upper surface of the sensor chip, where light By monitoring the change in the interference spectrum reflected by irradiating, the bound glycated protein can be detected. In the protein chip method, the labeled glycated protein stays on the sensor chip via the galectin when the galectin immobilized on the sensor chip is bound to the labeled glycated protein, so the label is monitored. Thus, it is possible to detect the conjugated glycated protein. Furthermore, when a semiconductor contact-type biosensor binds galectin and glycated protein immobilized on the sensor chip, the glycated protein is adsorbed on the sensor chip surface connected to the gate of the field effect transistor, thereby causing the sensor chip surface to be absorbed. By monitoring the change in the current value of the source and drain, which changes in accordance with the surface potential, it is possible to detect the bound glycated protein.

  In addition to the above, biosensors based on various principles are currently being developed, but any biosensor using a principle capable of detecting an interaction between substances can be used for detecting a glycated protein in the present invention. In the present specification, the “biosensor” refers to a chemical sensor that detects a biomolecule using a molecular recognition mechanism of a living body.

When detection of glycated protein by galectin is performed using surface plasmon resonance, it occurs when galectin is immobilized on an SPR sensor chip by an ordinary method, washed, contacted with a sample, and irradiated with light. This is done by detecting the change in resonance angle. When a change in resonance angle of 0.1 ° is defined as 1,000 resonance units (RU), it has been confirmed that 1,000 units correspond to a mass change of about 1 ng / mm ² of protein. The amount of protein bound can be estimated from the change. According to SPR, the process of binding and dissociation can be monitored, so that not only the presence or absence of glycated protein to be bound, but also the binding rate constant (K _on ), the dissociation rate constant (K _off ) and the equilibrium constant (K _d ) can also be known.

  When detection of glycated protein by galectin is performed using a quartz crystal microbalance measurement method, galectin is immobilized on a QCM sensor chip by a usual method, washed, and then contacted with a sample. This is performed by detecting a change (decrease) in the resonance frequency of the crystal resonator. The QCM is becoming more portable and is advantageous in that it can be inspected at the bedside.

  Any method using other biosensors can accurately detect the amount of binding between galectin immobilized on the sensor chip and the glycated protein in the measurement target, and can be used in the detection method of the present invention. it can. SPR and QCM are preferred because they are generally widely used as protein detection methods and have established detection methods.

  In an electrochemical method capable of measuring protein-protein interaction, for example, it occurs when galectin-3 is bound to the tip of a microelectrode and then a glycated protein is bound to galectin-3 by applying a voltage and then contacting the sample. By monitoring the potential change, glycated protein can be detected.

  Since the glycated protein can be an indicator of various diseases as described later, the detection method of the present invention can be carried out on a sample isolated from mammals including humans. Examples of such a sample isolated from a living body include blood, tears, sweat, saliva, urine and the like.

  The relationship between the increase in blood glucose level and the progress of protein glycation has been clarified so far, and it is possible to estimate blood glucose level by measuring the amount of glycated protein in the blood (Bleyer JM., RI Med. J., 1979, 62 (4): 131-133). Therefore, the detection method of the present invention can be used for blood glucose level measurement and diabetes diagnosis.

  The glycation of protein in blood is gradually caused over a long period of time, and is hardly affected by a short-term change in blood glucose level immediately before. On the other hand, as described above, a glycated protein can be detected by using galectin. Therefore, according to the present invention, it is possible to provide a highly reliable method for measuring a blood glucose level that is not easily influenced by a change in blood glucose level immediately before. As shown in the examples described later, since galectin recognizes a plurality of glycated proteins, the detection method of the present invention is a method for measuring a blood glucose level that is more reliable than measurement of a specific glycated protein. This method is useful, for example, when an average blood glucose state in the past 2 to 4 weeks is examined when glycated hemoglobin (HbA1c) or the like cannot be measured due to anemia or the like.

  In addition to the above, it is known that non-physiological cross-linking between collagen molecules by AGE-LDL (glycated LDL) or AGE can cause atherosclerosis (Fu MX, et. Al., J. Biol. Chem). ., 1996, 271: 9982-9986). It is also known that glycated amyloid β (glycated Aβ) can cause Alzheimer's disease (Yan SD., Et. Al., Nature, 1996, 382: 685-691; Takeuchi M., J. Neuropathol. Exp. Neurol., 2000, 59: 1094-1105). Therefore, the detection method of the present invention can be used for diagnosis of atherosclerosis and Alzheimer's disease and determination of susceptibility to these diseases.

  Since a biosensor can be used in the method for detecting a glycated protein of the present invention, according to another aspect of the present invention, a biosensor chip on which galectin is immobilized is provided. On the solid support on the biosensor chip of the present invention, galectin CRD may be immobilized instead of galectin, or galectin or CRD thereof or a mixture of two or more thereof may be immobilized. As the galectin immobilized on the biosensor chip of the present invention and the glycated protein to be detected, those described in the detection method of the present invention can be used. The biosensor chip of the present invention can be implemented in the same manner as the detection method of the present invention.

  The biosensor chip is not particularly limited as long as it is a biosensor chip capable of detecting the interaction between substances. For example, surface plasmon resonance method, quartz crystal microbalance measurement method, reflection interference spectroscopy, protein A biosensor chip for a chip method or a semiconductor contact biosensor method can be used. Preferably, a surface plasmon resonance biosensor chip or a quartz vibrator microbalance measurement biosensor chip can be used.

  According to another aspect of the present invention, there are provided an affinity column to which galectin or galectin CRD or a mixture thereof is bound, and a method for purifying glycated protein using the affinity column. For example, by using a column to which galectin-3 is bound, it is possible to purify, among glycated proteins, a pre-reaction product derived from glucose, a protein added with AGE and CEL, an AGE derived from methylglyoxal, and an AGE derived from glyceraldehyde. In addition to glucose-derived pre-reaction products and proteins added by AGE and CEL, methylglyoxal-derived AGEs and glyceraldehyde-derived AGEs, CML-added proteins Can be purified. Moreover, the protein which CML added can be refine | purified by utilizing the difference in the binding ability of CRD between galectin-3 and galectin-3. Since glycated proteins are related to diseases such as lifestyle-related diseases, the affinity column of the present invention and the purification method of the present invention can be used as equipment and techniques for elucidating the in vivo mechanism of glycated proteins.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by the following Example.

Example 1: Detection of glycated protein with galectin (1) Preparation of CRD of galectin-3 and galectin-3 Escherichia coli BL-21 (DE3) strain transformed with galectin-3 expression plasmid was treated with 10% (weight / weight). Cultured at 37 ° C. with 1 L of 2 × YT medium supplemented with (volume) glucose and 100 μg / mL ampicillin. D. 600 was set to 0.7. Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 0.1 mM to induce the expression of the fusion protein, and further cultured at 37 ° C. for 2 hours. . The medium was removed by centrifugation, and the E. coli pellet was removed from 100 mL of a cell disruption solution (10 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 5 mM β-mercaptoethanol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF)). ) And sonicated for 4 minutes on ice. Next, Triton X-100 was added to a final concentration of 1% (weight / volume), and the mixture was stirred at 4 ° C. for 30 minutes. After centrifugation, the supernatant was further purified by lactose-agarose affinity chromatography (Seikagaku Corporation, Tokyo, Japan) (Matsushita et. Al. 2000; Nishi et. Al., 2003). The galectin-3 expression plasmid is a polynucleotide derived from human Jurkat cells encoding galectin-3 (SEQ ID NO: 1) or a polynucleotide encoding CRD of galectin (nucleotide sequence from 340 to 750 of SEQ ID NO: 1). PET11-a vector containing

(2) AGE used in the examples
In this example, N ^ε- (carboxymethyl) lysine-HSA (CML-HSA) (catalog number: CY-R2066), N ^ε- (carboxyethyl) purchased from Cyclex (Nagano, Japan) was used as AGE. ) Lysine-HSA (CEL-HSA) (Catalog Number: CY-R2067), Glyoxal-AGE-BSA (Catalog Number: CY-R2064), Glycolaldehyde-AGE-BSA (Catalog Number: CY-R2060), Glyceraldehyde -AGE-BSA (catalog number: CY-R2058), methylglyoxal-AGE-BSA (catalog number: CY-R2062), glucose-AGE-BSA (catalog number: CY-R2056) were used.

(3) Preparation of Galectin-3 Immobilized Sensor Chip and Galectin-3 CRD-immobilized Sensor Chip Galectin-3 (SEQ ID NO: 2) or Galectin-3 CRD (amino acid sequence of SEQ ID NO: 2) prepared in (1) above 114-250) was immobilized on a sensor chip CM5 (research grade) for Birecore 3000 (Biacore, Piscataway, NJ) by amino coupling reaction according to the manufacturer's instructions. The amount of protein to be immobilized was 5000 resonance units (RU). Immobilized by N-hydroxy-succinimide / 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide coupling reaction in 10 mM sodium acetate (pH 5.0) at a flow rate of 10 μL / min. did.

(4) Analysis conditions for interaction between CRD and AGE of galectin-3 and galectin-3 The binding characteristics of CRD and AGE of galectin-3 and galectin-3 are based on an interaction analysis device based on the surface plasmon resonance (SPR) phenomenon. Analysis was performed using a Birecore 3000. SPR analysis was performed at 30 ° C. and a flow rate of 10 μL / min. All samples were subjected to analysis at 20 μL, and contacted for 120 seconds on a galectin-3 immobilized CM5 sensor chip and / or a galectin-3 CRD immobilized CM5 sensor chip to obtain CRD and AGE of galectin-3 or galectin-3. The binding of was monitored. Thereafter, the sensor chip was treated with a 70 μL 20 mM lactic acid solution dissolved in a running buffer (10 mM Hepes (pH 7.4), 3 mM EDTA, 150 mM NaCl, and 0.005% surfactant P20) for 120 seconds. AGE dissociation was monitored. The amount of residual AGE after elution treatment was measured as% RU. Also, association rate constants (K _on ) and dissociation rate constants (K _off ) were determined using the BIAevaluation 4.1 software package (GE Healthcare, Piscataway, NJ). The equilibrium constant (K _d ) was obtained by dividing K _off by K _on .

(5) Evaluation of binding between galectin-3 and AGE A sensorgram of binding between galectin-3 and each AGE is shown in FIG. Galectin-3 is glucose-AGE-BSA (AGE-1, FIG. 2-A), CEL-HSA (FIG. 2-B), glyceraldehyde-AGE-BSA (AGE-2, FIG. 2-C), methyl It was found that all of glyoxal-AGE-BSA (AGE-4, FIG. 2-D) had binding ability. The respective association rate constants (K _on ) and dissociation rate constants (K _off ) are shown in FIG. Further, when the binding ability between galectin-3 and various AGEs was examined, galectin-3 was bound to glucose-AGE-BSA, CEL-HSA, methylglyoxal-AGE-BSA, and glyceraldehyde-AGE-BSA. However, it did not bind to glyoxal-AGE-BSA, CML-HSA, or glycolaldehyde-AGE-BSA (Table 1). Thus, it was suggested that galectin-3 has a binding specificity with a specific AGE.

Table 1 shows the equilibrium constant (K _d ) between galectin-3 or galectin-3 CRD obtained by SPR and each AGE. In order to evaluate the affinity between galectin-3 and AGE, asialofetuin known to bind to galectin-3 was used as a positive target. The smaller the value of the equilibrium constant (K _d ), the stronger the bond.

(6) Evaluation of binding between CRD of galectin-3 and AGE FIG. 2 shows a sensorgram of binding between CRD of galectin-3 and each AGE. The CRD of galectin-3 is glucose-AGE-BSA (AGE-1, FIG. 2-A), CEL-HSA (FIG. 2-B), glyceraldehyde-AGE-BSA (AGE-2, FIG. 2-C). , Methylglyoxal-AGE-BSA (AGE-4, FIG. 2-D) was found to have binding ability. The respective association rate constants (K _on ) and dissociation rate constants (K _off ) are shown in FIG.

  Further, when the binding ability of galectin-3 CRD and various AGEs was examined, CRD of galectin-3 was found to be glucose-AGE-BSA, CML-HSA, CEL-HSA, methylglyoxal-AGE-BSA, glycerin. Although it showed binding ability with aldehyde-AGE-BSA, it did not show binding ability with glyoxal-AGE-BSA and glycolaldehyde-AGE-BSA (Table 1). Thus, even if galectin-3 was only CRD, it retained the binding ability to each AGE and the binding specificity to each AGE. However, the binding ability to CML-HSA was different between full-length galectin-3 and CRD of galectin-3 (Table 1).

(7) Evaluation of Galectin-3 Binding Characteristics In order to identify the site of AGE recognized by galectin-3, the binding characteristics of galectin-3 and glyceraldehyde were analyzed by SPR. Galectin-3 binds to glyceraldehyde-AGE-BSA (AGE-2) (FIG. 6-B, d) but does not bind to glyceraldehyde itself (FIG. 6-B, c). I understood. Galectin-3 has no binding ability with BSA itself (data not shown). This suggests that both a protein moiety and an additional moiety are required for the binding of galectin-3 and AGE, that is, galectin-3 can widely recognize only glycated proteins.

Claims (8)

A method for detecting a late glycation reaction product of a glycated protein, comprising measuring the amount of the late reaction product of the glycation protein to galectin- 3 or its carbohydrate recognition region (CRD). The late reaction product of the glycation protein is derived from the late reaction product of glycation reaction added with carboxyethyl lysine or glucose, methylglyoxal or glyceraldehyde when measuring the amount of binding to galectin-3. Late reaction product of saccharification reaction, when measuring the amount of galectin-3 bound to the hydrocarbon recognition region (CRD), the late reaction product of saccharification reaction added with carboxymethyllysine or carboxyethyllysine, or glucose After saccharification reaction derived from methylglyoxal or glyceraldehyde The reaction product, method. The amount of glycated protein late reaction product bound to galectin- 3 or its CRD is determined by surface plasmon resonance method, quartz crystal microbalance measurement method, reflection interference spectroscopy method, protein chip method or semiconductor contact biosensor method or The method according to claim 1, which is measured by an electrochemical method. The method according to any one of claims 1 and 2 , which is a method for measuring a blood glucose level, glycated LDL or glycated amyloid β. The method according to any one of claims 1 to 3 , wherein the CRD has an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 from 114 to 250. Galectin-3, or a hydrocarbon recognition region (CRD) is made from the immobilized solid support is a biosensor chip for use in the detection of glycation late reaction product of a sugar protein, glycated proteins The late reaction product of the saccharification reaction is a saccharification reaction product obtained by adding carboxyethyl lysine, or saccharification reaction derived from glucose, methylglyoxal or glyceraldehyde when galectin-3 is immobilized on a solid support. Late reaction product, when the carbohydrate recognition region (CRD) of galectin-3 is immobilized on a solid support, the late reaction product of saccharification reaction added with carboxymethyllysine or carboxyethyllysine, or glucose After saccharification reaction derived from methylglyoxal or glyceraldehyde The reaction product, the biosensor chip. The method according to any one of claims 1 to 4, wherein a late reaction product of a glycated protein is detected using the biosensor chip according to claim 5 . Carrier to bind the galectin-3 or a hydrocarbon recognition region (CRD) comprising, a affinity column for the purification of saccharification late reaction product of a sugar protein, glycation late reaction product of a glycated protein Is a late saccharification reaction product added with carboxyethyllysine when galectin-3 is bound to an affinity column, or a late saccharification reaction product derived from glucose, methylglyoxal or glyceraldehyde, and galectin -3 hydrocarbon recognition region (CRD) bound to an affinity column, the late reaction product of saccharification reaction added with carboxymethyllysine or carboxyethyllysine, or saccharification derived from glucose, methylglyoxal or glyceraldehyde After reaction The reaction product, affinity column. Using an affinity column according to claim 7, a method for purifying a saccharification late reaction product of a sugar protein, glycation late reaction product of glycated protein, galectin-3 bound to the affinity column In this case, it is a late saccharification reaction product to which carboxyethyllysine has been added, or a late saccharification reaction product derived from glucose, methylglyoxal or glyceraldehyde, and has a hydrocarbon recognition region (CRD) of galectin-3. The method, when bound to an affinity column, is a late saccharification reaction product to which carboxymethyllysine or carboxyethyllysine has been added, or a late saccharification reaction product derived from glucose, methylglyoxal or glyceraldehyde .

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