WO2004036216A1 - Procede de mesure d'interactions entre des chaines du sucre et une proteine de liaison a la chaine du sucre et utilisation associee - Google Patents

Procede de mesure d'interactions entre des chaines du sucre et une proteine de liaison a la chaine du sucre et utilisation associee Download PDF

Info

Publication number
WO2004036216A1
WO2004036216A1 PCT/JP2003/013239 JP0313239W WO2004036216A1 WO 2004036216 A1 WO2004036216 A1 WO 2004036216A1 JP 0313239 W JP0313239 W JP 0313239W WO 2004036216 A1 WO2004036216 A1 WO 2004036216A1
Authority
WO
WIPO (PCT)
Prior art keywords
sugar chain
sugar
chain
binding protein
lectin
Prior art date
Application number
PCT/JP2003/013239
Other languages
English (en)
Japanese (ja)
Inventor
Kazuaki Kakehi
Kazuki Nakajima
Mitsuhiro Kinoshita
Original Assignee
Japan Science And Technology Agency
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Japan Science And Technology Agency filed Critical Japan Science And Technology Agency
Priority to JP2004544967A priority Critical patent/JPWO2004036216A1/ja
Publication of WO2004036216A1 publication Critical patent/WO2004036216A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis

Definitions

  • the present invention relates to a method for measuring the interaction between a sugar chain and a ⁇ chain-binding protein, and a method for using the same.
  • the present invention relates to a clinical diagnosis such as a genetic disease or cancer diagnosis based on a sugar chain abnormality, and a sugar chain.
  • Method for measuring the overall action of sugar-chain-glycoprotein binding proteins that can be applied to the development of new drugs targeting glycan-binding proteins, and screening of sugar chains and glycolytic binding proteins using the same
  • the present invention relates to a method, a measuring reagent used in the measuring method, and a measuring kit. Background art
  • sugar chain-related enzymes which are expressed by being controlled by genes, such as glycosyltransferases and sugar hydrolases, involved in the biosynthesis of sugar chains. Therefore, delicate regulation of sugar chain functions, that is, measurement of delicate changes in the interaction between glycosides and glycan-binding proteins, cannot be elucidated only by studies at the gene level.
  • the sugar chains bound to glycoproteins consist of a mixture of a plurality of complex sugar chains.
  • the sugar chains bound to glycoproteins consist of a mixture of a plurality of complex sugar chains.
  • a glycosylation mixture is used as a library, and a disease or a genetic abnormality involving a carbohydrate-binding protein present in a living body. It is expected to be able to measure
  • the present invention has been made in view of the above-mentioned problems, and its purpose is to reliably measure the interaction of a sugar chain-single-chain binding protein, and to use it for drug development such as development of a new drug.
  • a screening method suitably used in the test industry and the reagent industry such as industry and clinical tests, and a measurement reagent and a measurement kit suitably used in the pharmaceutical industry, the test industry and the reagent industry. It is in. Disclosure of the invention
  • the present inventors have proposed a sugar chain-tan for which a measurement method has not been established until now.
  • the method of measuring the interaction between proteins was studied diligently.
  • the interaction between glycans and glycans-binding proteins can be performed simultaneously, with high sensitivity and specificity.
  • a new measurement method that can be measured has been found.
  • the method for measuring the interaction between a sugar chain and a sugar chain-binding protein comprises the steps of separating a mixture of one or more sugar chains, It is characterized in that the interaction between a sugar chain and a sugar chain-binding protein is measured based on a comparison with a separation result of a reaction mixture obtained by reacting the protein with a chain-binding protein. -In the measurement method of the present invention, the reaction mixture obtained by reacting the sugar chain mixture with the sugar chain binding protein is separated.
  • the interaction between the sugar chain and the sugar chain-binding protein increases, and the sugar chain-to-sugar chain binding property increases.
  • the measurement method of the present invention utilizes such a difference in the separation results to measure the interaction between the ⁇ -glycan-binding protein. That is, the measurement method of the present invention can simultaneously measure the interaction between the sugar chain and the sugar chain-binding protein while separating the sugar chain and the bran-binding protein. As described above, the measurement method of the present invention measures the interaction by comparing the separation result of only the ⁇ chain with the separation result of the mixture obtained by reacting the sugar chain with the sugar chain-binding protein.
  • the interaction can be measured simultaneously even for a complex mixture of multiple sugar chains.
  • the interaction between each sugar chain in the sugar chain mixture and the sugar chain binding protein, or between the sugar chain binding protein and each sugar chain in the sugar chain mixture, the interaction between the sugar chain and the sugar chain-binding protein is highly sensitive. This allows quick and easy simultaneous measurement.
  • the measuring method of the present invention can be applied to, for example, the following screening method.
  • the method for screening a sugar chain of the present invention is a method for screening a sugar chain using the above-described measurement method of the present invention.
  • a glycosylation reaction step in which a protein is reacted with one or more kinds of sugar chain mixtures that are not known to bind to the sugar chain-binding protein ', a separation result of only the sugar chain mixture, and the sugar chain reaction
  • a sugar chain determination step of determining whether or not the sugar chain binding to the sugar chain binding protein is present in the sugar chain mixture based on a comparison with the separation result of the reaction mixture obtained in the step. It is a feature.
  • the method for screening a sugar chain-binding protein according to the present invention is a method for screening a sugar chain-binding protein using the above-described measurement method according to the present invention, wherein one or more types of sugars having a clear structure are included.
  • Chain mixture and specific sugar chain A sugar chain-binding protein reaction step in which one or more sugar chain-binding proteins are unknown to be recognized by the above, the separation result of only the sugar chain, and the sugar chain-binding protein reaction step
  • the separation result of only the ⁇ chain mixture is compared with the separation result of the reaction mixture after the completion of the sugar chain determination step or the sugar chain binding protein determination step. If there is a difference, the presence or absence of a sugar chain that binds to the sugar chain-binding protein present in the sugar chain mixture, and the specificity of the sugar chain present in a complex sugar chain-binding protein, for example, a biologically-derived protein mixture It is possible to determine the presence or absence of a sugar chain-binding protein that is specifically recognized. In this way, screening of sugar chains and screening of sugar chain-binding proteins can be performed.
  • the presence or absence of a sugar chain or a sugar chain binding protein is determined based on a difference in electrophoresis time by capillary electrophoresis. Good to do Good.
  • the accuracy and reliability of the screening method of the present invention can be further improved, and further, automatic measurement can be performed.
  • the sugar chains of the sugar chain mixture may be labeled.
  • sugar chains, sugar chain-binding proteins, and sugar chain-sugar chain-binding protein complexes can be more easily separated.
  • the method may include a step of removing the sialic acid residue.
  • the interaction between the ⁇ chain and the sugar chain-binding protein may be further strengthened.
  • sugar chains, sugar chain-binding proteins, and sugar chain-sugar chain-binding proteins can be more accurately separated. That is, the reliability of the determination in the sugar chain determination step and the sugar chain binding protein determination step is further improved.
  • the above-mentioned one or more kinds of sugar chain-binding proteins whose sugar chains specifically recognized or a mixture of one or more kinds of sugar chains whose structure is clear are added to a support. It may be fixed.
  • the one or more sugar chain-binding proteins whose sugar chains specifically recognized or the mixture of one or more sugar chains whose structure is apparent are, for example,
  • It may be a microchip, microarray, or macroarray fixed at high density to a substrate. This allows large-scale screening to be performed simultaneously. Further, if reagents and media used for separation, such as gel media for electrophoresis, are simultaneously fixed to the substrate, the time required for screening is further reduced.
  • reagents and media used for separation such as gel media for electrophoresis, are simultaneously fixed to the substrate, the time required for screening is further reduced.
  • novel sugar chains and novel sugar chain-binding proteins obtained by the above screening method are also included.
  • the sugar chain determination step and the sugar chain binding protein determination step are, in other words, a step of classifying a sugar chain or a sugar chain binding protein (a sugar chain classification step and a sugar chain binding protein classification step). It is. Therefore, the screening method of the present invention can be said to be a method for classifying, discriminating, selecting, confirming, or detecting a sugar chain or a sugar chain-binding protein.
  • the reagent for measuring the interaction between a sugar chain and a sugar chain-binding protein according to the present invention is a measuring reagent used in the above-described measuring method of the present invention, wherein one or more types of sugar chains specifically recognized are clearly identified. It is characterized by containing a sugar-binding protein or a mixture of one or more sugar chains whose structure is apparent.
  • the measuring reagent of the present invention similarly to the effect of the above-described measuring method, even in the case of a complex mixture of a plurality of sugar chains, the interaction of the sugar chain-single sugar chain binding protein can be simultaneously measured.
  • Possible measurement methods can be provided as measurement reagents.
  • the above-mentioned measuring method can be easily implemented, and the measuring method can be simplified. Therefore, measurement time can be reduced.
  • the measurement kit of the present invention is characterized by containing the reagent for measuring the interaction between the sugar chain and the sugar chain-binding protein of the present invention.
  • a measurement method that can simultaneously measure the interaction between a sugar chain and a sugar chain-binding protein can be provided as a measurement kit. Furthermore, the above-mentioned measuring method can be easily implemented, and the measuring method can be simplified. Therefore, measurement time can be reduced.
  • the “sugar chain-binding protein” can be arbitrarily and variously selected from, for example, 100 or more known sugar chain-binding proteins. It is preferable to use the six lectins used in Examples described later. That is, Tachinata bean lectin (ConA: Concanavalin A), wheat germ lectin: wheat germ agglutinin, tulip lectin (TGA: T.
  • gesneriana agglutinin RSL: Rizopus stronipher lectin
  • RSL Rizopus stronipher lectin
  • nicotine collectin SSA: Samb.ucus sieboldiana lectin
  • nuenshi lecten MAM: Maackia anraurensis lectin
  • lectin set Among these naturally occurring lectins, remarkable differences in electrophoresis results are observed due to specific binding to sugar chains. For this reason, the lectin set is particularly suitable for comprehensively analyzing most sugar chains contained in various organisms.
  • the six sugar chain binding proteins used in the examples were used. It is preferred to use at least one. Further, in order to further improve the determination accuracy in the sugar chain determination step, it is preferable to analyze a plurality of combinations of the separation results obtained by the above six sugar chain binding proteins. As a result, the accuracy of sugar chain determination is remarkably improved, A chain detection rate of 95% or more can be secured.
  • the “sugar chain mixture” can be arbitrarily and widely selected from more than 1000 kinds of known ⁇ chains. It is preferable to use a five-sugar-chain mixture library derived from a glycoprotein, which is used in Examples described later. Specifically, ⁇ 1 acidic glycoprotein (AGP: a 1-acid glycoprotein), fetuin, 'vomucoid, ovomucoid), immunoglobulin G (IgG), and thyroglobulin It is preferred to use a mixture of sugar chains derived from,.
  • the above-mentioned sugar chain library shows a remarkable difference in the electrophoresis results by specifically binding to the sugar chain-binding protein among many sugar chains. Therefore, the above-mentioned sugar chain library is particularly suitable for the analysis of naturally occurring sugar chain binding proteins.
  • the glycolytic mixture derived from glycoprotein can be easily obtained by, for example, appropriate enzyme treatment.
  • sugar chain mixture library By using the above-mentioned sugar chain mixture library and the sugar chain binding protein, not only the presence or absence of sugar chain and bran chain binding protein but also the structures of sugar chains and sugar chain binding proteins whose structures are unknown are known. Clarification and classification are also possible. Moreover, naturally occurring sugar chains or sugar chain binding proteins The quality can be easily analyzed with high throughput.
  • FIGS. 1 (a) to 1 (c) show the results of electrophoresis of glycans derived from a1 acidic glycoprotein with and without lectin addition in Example 1.
  • FIG. 1 shows the results of electrophoresis without the addition of lectin.
  • FIG. 1 (b) is a schematic diagram of a sugar chain derived from an acidic glycoprotein
  • FIG. 1 (c) is a diagram showing three types of lectins
  • FIG. 4 is a diagram showing the results of electrophoresis when WGA, ConA, and TGA) were added.
  • FIG. 2 is a diagram showing a structure of a sugar chain derived from ⁇ 1 acidic glycoprotein used in Example 1.
  • FIGS. 3 (a) to 3 (c) show the results of electrophoresis of fetuin-derived sugar chains with and without lectin addition in Example 2, and FIG. Fig. 3 (b) is a schematic diagram of a sugar chain derived from fetuin, and Fig. 3 (c) is a diagram showing the results of electrophoresis when no lectin was used.
  • FIG. 4 shows the results of electrophoresis when TGA) was added.
  • FIG. 4 is a diagram showing a structure of a sugar chain derived from fetuin used in Example 2.
  • FIGS. 5 (a) to 5 (c) show the results of electrophoresis of ovomucoid-derived heavy chain in Example 3 with and without the addition of lectin.
  • FIG. 5 (a) is a diagram showing the results of electrophoresis when no lectin was added
  • FIG. 5 (b) is a schematic diagram of ovomucoide-derived sugar chains
  • FIG. 5 (c) is a diagram showing three types of lectins
  • FIG. 4 is a view showing the results of electrophoresis when PHA—E 4 , WGA, and Con A) were added.
  • FIG. 6 is a diagram showing the structure of a sugar chain derived from ovomucoid used in Example 3.
  • FIG. 7 is a diagram showing the results of electrophoresis when lectin (WGA) was added to the sugar chain mixture in Example 4 while changing the concentration.
  • Figure 8 is a result of (t 7 - - 1 [P] - 1 and the relationship is a graph flop Lock bets.
  • FIGS. 9 (a) to 9 (b) are diagrams showing the results of Example 5, and FIG. 9 (a) is a schematic diagram of a sugar chain derived from Psi IgG, and FIG. 9 (b) FIG. 3 is a view showing the results of electrophoresis with and without the addition of a crude viscous extract to sugar chains derived from Psi IgG.
  • FIG. 10 is a diagram showing high-throughput functional classification of glycoprotein-derived sugar chains based on the results of each example.
  • FIG. 11 is a diagram showing the concentration of lectin used in each Example.
  • FIGS. 12 (a) to 12 (c) show the results of electrophoresis of fetuin-derived sugar chains with and without the addition of lectin in Example 6, and FIG. 12 (a) shows the results.
  • Fig. 12 (b) is a schematic diagram of a sugar chain derived from fetuin
  • Fig. 12 (c) is a diagram showing the results of electrophoresis without the addition of lectin. , MAM, SSA) are shown.
  • FIG. 13 is a diagram showing the structure of a phtophin-derived sugar chain used in Example 6. It is.
  • FIGS. 14 (a) to 14 (b) show the results of Example 7.
  • FIG. 14 (a) shows the presence or absence of the addition of lectin (RSL) to the sugar chain derived from Psi IgG.
  • FIG. 14 (b) is a schematic diagram of a sugar chain derived from Psi IgG.
  • FIG. 15 is a diagram showing the results of Example 8, and is a schematic diagram of the results of electrophoresis of a sugar chain derived from porcine thyroglobulin with and without the addition of lectin (RSL) and a sugar chain derived from porcine tigrogopenin. .
  • FIG. 16 is a diagram showing a structure of a glycan derived from butyroglobulin used in Example 8.
  • FIGS. 17 (a) to 17 (b) show the results of Example 9.
  • FIG. 17 (a) shows the results of the addition of lectin (AAL) to the sugar chain derived from ⁇ 1 acidic glycoprotein. It is a figure which shows the result of the electrophoresis with or without
  • FIG. 17 (b) is a schematic diagram of a 1-acid glycoprotein.
  • FIG. 18 is a diagram showing a structure of a sugar chain derived from ⁇ 1 acidic glycoprotein used in Example 9. BEST MODE FOR CARRYING OUT THE INVENTION
  • FIGS. 1 (a) to 18 One embodiment of the present invention will be described below with reference to FIGS. 1 (a) to 18. Note that the present invention is not limited to this.
  • the screening method of the present invention comprises: (1) a method for screening a sugar chain; and (2) a method for screening a sugar chain-binding protein on a large scale using a method for measuring the interaction between a sugar chain and a protein.
  • This is a method that enables simultaneous screening. Both screening methods are similar in that both are based on the interaction of glycans-monosaccharide-binding proteins. However, the only difference is whether the target of screening is a sugar chain or a sugar chain-binding protein.
  • the method for screening a sugar chain in the above (1) includes a sugar chain-binding protein whose specificity for a sugar chain is known, and a ⁇ chain specifically recognized by the sugar chain-binding protein.
  • the sugar chain reaction step in which a sample containing a possible sugar chain mixture is combined, and the separation results of the sugar chain mixture alone and the reaction mixture after the sugar chain reaction step are compared.
  • a sugar chain determination step of determining the presence or absence of a sugar chain that binds to the chain-binding protein.
  • the method for screening a sugar chain-binding protein of the above (2) is characterized in that a sugar chain library obtained from an arbitrary oligosaccharide mixture or a sugar protein having a known sugar chain composition is used as a sugar chain library.
  • a sugar chain-binding protein reaction step of combining a sample obtained from cells, tissues, and the like, which may contain sugar chain-binding proteins, with this library, By comparing the results of separation of only the glycan mixture with the separation of the reaction mixture after the glycan-binding protein reaction step, the presence or absence of glycan-binding proteins that bind to a glycan library is determined.
  • a binding protein determination step is characterized in that a sugar chain library obtained from an arbitrary oligosaccharide mixture or a sugar protein having a known sugar chain composition.
  • the “sugar chain-binding protein” specifically recognizes a specific sugar chain And a protein that binds.
  • Typical examples of specific sugar chains with known specificity include lectins and sugar chain antibodies.
  • the “arbitrary oligosaccharide mixture” is not particularly limited, but may be, for example, a mixture of double-stranded, triple-stranded, and quadruple oligosaccharides, and hyal sulfonic acid. ⁇ ⁇ Glycosamino-dalican-derived oligosaccharide mixtures obtained by digestion of glycosamino-glycans such as chondroitin sulfate can be mentioned.
  • the “mixture of sugar chains prepared from glycoproteins” is not particularly limited, but includes, for example, double-stranded, triple-stranded, quadruple-chain, and Lewis X-type sugar chains.
  • a sugar chain mixture prepared by treating a1 acidic glycoprotein consisting of the above mixture with an appropriate glycosidase treatment or the like.
  • the origin of the sugar chains, sugar chain-binding proteins and oligosaccharides is not particularly limited, and may be prepared from animals, plants, microorganisms and the like according to the purpose.
  • the conditions for performing the sugar chain reaction step and the sugar chain binding protein reaction step are such that the sugar chain and the sugar chain binding protein can sufficiently react with each other to form a sugar chain-single sugar chain binding protein complex. If there is, there is no particular limitation.
  • the sugar chain reaction step was carried out in an example in which the interaction of the sugar chain of the ⁇ 1 acidic glycoprotein described later was measured (see FIGS. 1 (a) to 1 (c)). As shown in), first, the sugar chains of ⁇ 1 acidic glycoprotein are analyzed in the absence of lectin. Then, wheat germ 7
  • Analysis of the glycan mixture obtained from the target glycan-binding protein was performed in a fused silica capillary or a surface-modified silica capillary filled with a buffer containing lectin (WGA), or in a microchip.
  • the analysis is performed using a buffer solution containing lectin (Tonata) derived lectin (C on A) and tulip derived lectin (TGA).
  • the analysis was performed using the lectin concentrations shown in Fig. 1 (c), but the analysis is not limited to these concentrations, and may be changed as appropriate depending on the combination of sugar chains or the type of lectin used. do it. Generally, it can be used in a wide range from nanomolar (nM) to millimolar (mM) concentrations.
  • the type of glycolytic protein ⁇ lectin known to have specificity for a sugar chain used in the present invention is not particularly limited. It is also desirable to use a plurality of types of lectins in any combination. That is, in the sugar chain determination step, it is preferable to determine a combination of a plurality of separation results when lectin is added. For example, the separation results obtained when wheat germ lectin (WG A), tuliplectin (TGA), and tatita bean lectin (Concanapalin A (C on A)) are added may be combined. Thus, by combining the separation results obtained when a plurality of lectins are added, the composition, structure, function, and the like of the sugar chain in the sugar chain mixture can be divided in more detail.
  • a sugar chain mixture serving as a sample for analyzing whether or not it reacts with such a lectin may be prepared by a conventionally known method.
  • a sample obtained by extracting a glycoprotein of interest after separation by affinity chromatography or separation by gel electrophoresis may be used.
  • those in which sugar chains are separated from the extracted glycoprotein by glycosidase treatment or the like may be used.
  • the screening method (1) for example, it is possible to comprehensively screen the change in the sugar chain composition ratio due to the change in the physiological state. Therefore, it is possible to easily analyze changes in the structure of the sugar chain of the ⁇ protein in a disease and changes in the composition of each sugar chain of the sugar chain mixture over time. In addition, it is possible to classify sugar chains by function, which is useful for analyzing a wide variety of physiological activities and functions such as glycoproteins and proteasomes, which are products of post-translational modification by sugar chains.
  • the sugar chain-binding protein reaction step may include sugar chain-binding protein such as blood, cell extract, or microorganism-derived extract.
  • sugar chain-binding protein such as blood, cell extract, or microorganism-derived extract.
  • the glucose solution mixture containing a buffer solution containing blood, a cell extract, or a microorganism-derived extract, and a buffer solution containing no such solution is used. Separation of Each Oligosaccharide Mixture 9
  • the electrophoresis buffer contains a lectin (RSL) derived from Rhizopus spp.
  • the following processing may be performed.
  • the presence or absence of a sugar chain or a sugar chain-binding protein is determined based on a difference in electrophoresis time by capillary electrophoresis. It is preferable.
  • Capillary electrophoresis has extremely high separation accuracy among various types of electrophoresis.
  • the reason for this is that capillary electrophoresis performs electrophoresis in very fine stone, glass, or synthetic capillary tubes.
  • the diameter of the capillary is 1 to several hundreds / cm.
  • heat is dissipated, so that a voltage approximately 10 times higher than that of other electrophoresis methods can be applied.
  • the sample can be separated in a short time.
  • band spreading due to diffusion is small, and extremely good separation is possible.
  • capillary electrophoresis like HPLC, allows for automated measurements. Therefore, capillary electrophoresis is particularly suitable for automating the present invention.
  • the accuracy and reliability of the screening method of the present invention can be further improved, and furthermore, automatic measurement is possible. Become.
  • the sugar chains can be easily obtained by comparing the chatters obtained as a result of the capillary electrophoresis. And the presence or absence of sugar chain binding protein becomes clear. Moreover, by comparing the charts, it is possible to clarify the unknown sugar chains and the sugar chains recognized by the sugar chain-binding protein.
  • a step of removing the sialic acid residue when the sugar chain or the sugar chain binding protein contains a sialic acid residue is included. Is also good. — In other words, a step of converting the sugar chain or sugar chain-binding protein into an ash mouth sugar chain or an asharoprotein may be included. Some sugar chains and sugar chain-binding proteins are stably present by containing sialic acid. By removing this sialic acid, the properties of sugar chains and sugar chain-binding proteins may be significantly changed. Therefore, the present invention also includes a step of removing a sialic acid residue of a sugar chain or a sugar chain-binding protein, thereby providing a sugar chain-sugar chain binding protein.
  • Protein interactions may be even more robust. Thereby, sugar chains, sugar chain-binding proteins, and sugar chain-single sugar chain-binding proteins can be separated with higher accuracy. That is, the reliability of the determination in the sugar chain determination step and the sugar chain binding protein determination step is further improved.
  • the method can be applied to the development of a drug targeting such an acyloglycoprotein receptor. .
  • the sugar chain of the sugar chain mixture may be labeled.
  • the method of labeling is not particularly limited, and may be set as appropriate depending on the separation method.
  • a fluorescent substance, an enzyme, a radioisotope, a luminescent substance, an ultraviolet absorbing substance, a spin labeling agent, etc. may be bound to a sugar chain and then separated.
  • labeling substances conventionally known reagents can be used.
  • the proteins are fluorescently labeled with APTS (9-aminovirene-1,4,6 trisulfonate).
  • an aminobenzene derivative such as 2-aminobenzoic acid and a 2′-aminopyridine amide nononaphthalene derivative may be used. it can.
  • a derivative of a labeled sugar chain can be used, for example, in a fluorescence detection device. It can be easily detected with high sensitivity using a laser beam excitation laser detector. That is, if a method is used in which a sugar chain is labeled to form a derivative and electrophoresis and laser-induced fluorescence (LIF) are combined, a sugar chain, a sugar chain-binding protein, a sugar chain-sugar chain-binding protein can be obtained. The protein complex can be separated and detected more easily.
  • LIF laser-induced fluorescence
  • the above-mentioned one or more glycemic-binding proteins or the mixture of one or more glycans having a clear structure are immobilized on a support. Is also good.
  • the one or more sugar chain-binding proteins or the sugar chain mixture of which the sugar chain specifically recognizing is clearly identified are, for example, immobilized on the substrate at a high density.
  • the screening method of the present invention has been described above.
  • a sugar chain with a sugar chain specifically recognized is known.
  • a protein or a sugar chain mixture having a known structure may be used.
  • it is more preferably prepared as a measurement reagent that improves the reactivity between the sugar chain mixture and the glycolytic protein.
  • the measurement reagent of the present invention can easily measure the interaction between sugar chains and sugar chain-binding proteins, it can be used in the field of analyzing the physiological activities and functions of sugar chains and sugar chain-binding proteins. It can be used as a reagent for basic research. It is also useful in the development of foods, pharmaceuticals, and diagnostics in the field of chain engineering.
  • the measurement reagent of the present invention further includes a reagent for easily reacting the ⁇ ⁇ chain and the ⁇ chain binding protein; a reagent for improving the accuracy and reliability of the separation; and a reagent for measurement. It may contain a reagent or the like for improving the convenience and preservability of the product.
  • AAL hilocha wantake lectin
  • mushroom lectin which has specificity for galactose
  • lentil lectin that shows specificity for the terminal part of glycans
  • saffron lectin that shows specificity for core pentasaccharide
  • SSA dinitro lectin
  • MAM innugen lectin
  • rhizopsuka lectin Lectins that have been used, known lectins whose sugar chains are clearly recognized, and the like can be used.
  • a mixture of sugar chains obtained by enzymatic degradation of a double-stranded sugar chain that can be obtained in large quantities can be used as a library '.
  • the origin of the sugar chain binding protein and the sugar chain library is not particularly limited.
  • the measurement reagent may be combined with another drug, a reagent for separation, a medium for separation, or the like to provide a kit for measuring the interaction between a glycoprotein and a sugar chain-binding protein. Very preferred.
  • the measuring method of the present invention can be easily carried out simply by obtaining a sample containing a sugar chain or a sugar-binding protein whose function is unknown. Can be applied. As a result, the time required for the measurement method of the present invention can be significantly reduced, and more samples can be measured in a short time.
  • a microphone mouth chip may be used.
  • the present invention provides a sugar chain binding that specifically recognizes a sugar chain. It is characterized by simultaneous separation of a mixture of sugar chains having a complex structure and structural analysis of each sugar chain by using an amphoteric protein.
  • the present invention is characterized in that a natural sugar chain-binding protein that specifically binds to the sugar chain mixture is analyzed using the sugar chain mixture. Further, the present invention is characterized in that a naturally-occurring sugar chain-binding protein or sugar chain is simply and easily subjected to high-throughput analysis.
  • the present invention can provide a method of classifying sugar chains by function and screening a sugar chain mixture by measuring the interaction of sugar chain-single sugar chain binding proteins. This makes it possible to measure changes in sugar chains on the cell surface and sugar chains of glycoproteins present in body fluids extremely simply and accurately. Therefore, it is expected that this method will lead to the discovery of clinical test methods using changes in sugar chains as indicators and changes in glucose levels in cell surface membrane components as indicators. In addition, if changes in sugar chains during a disease state are used as indicators, and changes in sugar chains are tracked over time under the conditions of the addition of a drug candidate compound, it is a very important method for developing new drugs. Become.
  • the present invention can be extended to the analysis of ⁇ proteins that specifically bind to sugar minerals present in blood, cells, and the like using the sugar chain measurement method.
  • ⁇ proteins that specifically bind to sugar minerals present in blood, cells, and the like using the sugar chain measurement method.
  • an unknown sugar chain that specifically binds to a sugar chain in a library by using a sugar chain mixture of glycoproteins with a known composition or an arbitrary mixture of oligosaccharides as a library Binding proteins can be easily found.
  • sugar chain-binding proteins that recognize these sugar chains. According to the simple screening method for sugar chain-binding proteins of the present invention, more detailed analysis of functions and functions of such sugar chain-binding proteins can be performed.
  • the present invention is a technique required next to genome-based protein analysis, and is a technique relating to sugar chain engineering, in which it is difficult to finely adjust the biosynthesis directly by controlling genes.
  • the combination of the library and lectin of the sugar chain used in the present invention is not limited to the examples of the present invention, but can be separated and phased in a solution using some basic combinations! : Reflects subtle differences in glycans by combining the unique method of simultaneous measurement of action with the simultaneous interaction measurement method using a microphone-mouth chip with multiple analysis circuits. The observed sugar-monosaccharide-binding protein interaction can be observed.
  • the measurement of the interaction between a sugar chain and a sugar chain-binding protein using the lectin of the present invention makes it possible to diagnose a genetic disease, cancer or inflammation based on a sugar chain abnormality, which has not been known until now.
  • the sugar chain library it is possible to accurately track changes in the amount and composition ratio of sugar chain binding proteins related to the cell surface, blood, intercellular tissues, and the like.
  • each analysis can be performed even if the analysis of a standard sample is included. Within minutes, rapid separation and classification of glycans is completed within at most 3 hours.
  • sugar chains are purified from a sugar chain mixture, and each of the purified sugar chains is linked to a sugar chain binding protein. The interaction with the protein was measured. Therefore, there was a problem that it was impossible to measure the interaction of the difficult-to-purify bran chains, or that a measurement period of every month was required.
  • the present invention can also be applied as follows.
  • the present invention is a technique devised as a result of long-term basic research to overcome such a problem, and is based on the use of the above-mentioned sugar chain-binding protein library and ⁇ chain library. It can be used for analysis and classification of almost all sugar chains present in living organisms, and for confirming the binding between sugar chain-binding proteins and ⁇ chains.
  • the affinity of the interaction between sugar chains and sugar-binding proteins ranges from mM to nM
  • the weak affinity between sugar chains and sugar chain-binding proteins is important for subtle functional regulation of living organisms.
  • the interaction between the sugar chain and the sugar chain-binding protein can be widely supported from a strong affinity to a weak affinity.
  • even a mixture of sugar chains having a weak affinity and a strong affinity has an advantage that the interaction between individual sugar chains and a sugar chain-binding protein can be analyzed simultaneously as a mixture.
  • microarrays In these fields, the application of microarrays is also expected, but using the above-mentioned sugar chain library and glycolytic binding protein library (lectin library-1) makes it impossible to track microarray technology. Because it can respond to subtle changes, more precise analysis is possible.
  • sugar chains and sugar chain-binding proteins to be arranged on the microarray can be used as effective means for selecting an appropriate set of sugar chain-binding proteins and sugar chains by using the present invention. Also.
  • fluorescence excited by laser is detected by capillary electrophoresis in the presence of different concentrations of sugar chain-binding proteins.
  • the technology provided in this way can simultaneously detect (1) to (3).
  • kinetic data eg, binding constant of each chain.
  • it is useful for elucidating biological phenomena expressing sugar chain changes on the cell surface. It is also particularly important in evaluating genetically modified biological agents. This is because products obtained from different cell lines or different culture conditions can sometimes contain a wide variety of sugar chains.
  • the detection method that combines electrophoresis and laser excitation fluorescence (LIF) is a powerful method for analyzing sugar chains in glycoprotein samples with ultra-high sensitivity.
  • Reagents for fluorescently labeling sugar chains in a sample include those used for derivatization and analysis of reducing sugars used for capillary electrophoresis with LIF detection, for example, 8-aminovinylene 1.1.
  • APTS Trisulfonate
  • the glycans to be analyzed should be categorized according to their function. ⁇ For example, the interaction between a protein capable of binding to a glycoconjugate and a sugar chain is considerably promoted by the binding constant to a specific protein. The reason is that sugar chains are mediators of the transmission of biological information.
  • the present inventors analyzed AGP sugar chains after separating AGP molecular species based on the sugar chain components by affinity column chromatography using lectin as a stationary phase. Furthermore, it was clarified that each fraction showed a characteristic amount of sugar chains generated. To analyze the interaction between glycans and lectins, Three
  • a method has been developed. Most of them are based on the interaction between one protein and one sugar chain. For example, plasmon ringing, fluorescence polarization, and time-resolved fluorometry.
  • the affinity chromatography is a FAC ZMS which combines an immobilized lectin column and an MS connected thereto (Kasai K., et al., J. Chromatogr. Biomed. Appl., 1986, 376, 33-47.) D This is applied to the interaction between glycosides and monosaccharide-binding proteins. According to this FACZ MS, the respective binding constants existing as a mixture can be calculated.
  • FA CZM S uses an affinity column with immobilized lectin.
  • a sample containing a mixture of sugar chains is continuously injected into the column.
  • Components with low affinity for immobilization or lectins elute earlier, and components with higher affinity elute later.
  • the dissociation constant (K d ) of the glycan composition in the mixture can be detected simultaneously.
  • affinity chromatography is a method of determining the interaction of a sample with "immobilized” receptor molecules. However, it may be necessary to determine the interaction in "solution” state.
  • Capillary affinity electrophoresis can measure the interaction between molecules of sugar chains and proteins in solution when the electrophoretic mobility differs between sugar chains and lectins.
  • CAE Capillary affinity electrophoresis
  • this method shows kinetic studies on the binding reaction using one type of oligosaccharide and lectin. Simultaneous determination of the binding constant of a mixture of simple oligosaccharides to lectins by capillary electrophoresis has been reported (Taga A, et al., J.
  • the present invention proposes a method for high-throughput functional classification of a complex mixture of sugar chains.
  • the mixture or sugar chain is fluorescently labeled in advance.
  • analysis is performed by capillary electrophoresis.
  • the present inventor has succeeded in classifying sugar chains based on the migration mode of each sugar chain.
  • the 'firefly-light labeling reagent is proposed labeled child and power s using Rereru having a charge (Shimura., Et al, Anal . Biochem., 1995, 227, 186-194.).
  • the charge of the labeled sugar chain is the driving force for the migration of sugar chains that bind to proteins or sugar chains that do not.
  • the reducing end of the sugar chain is modified by reductive amination using APTS as shown in the following chemical formula.
  • APS-labeled glycoprotein samples such as monosaccharides and oligosaccharides and chemically modified capillaries shows good separation ability.
  • the negative charge of the aminovirene residue by the sulfonic acid is suitable for studying binding reactions.
  • lectins classified as C on A mannose, G 1 c NA WGA classification of c, TGA classification of complex type sugar chains, Baisekuti ring G lc NA c residues classified into PHA-E 4 of fucose RSL and AAL were selected for the classification of glycoconjugates, and SSA and MAM were selected for the classification of carbohydrate chains.
  • Combinations of lectins at different concentrations provide efficient and sensitive ⁇ 1 acidic glycoproteins, fetuin, ovomucoid, stomach IgG, porcine thyroglobulin, and glycans of ⁇ 1 acidic glycoprotein from cancer patients Enabled classification.
  • the samples used were ⁇ 1 acidic glycoprotein and fetuin (both manufactured by Shimadzu Aldrich Tsuchi Japan).
  • Chicken egg white ovomucoid was purified from female egg white according to the method described in Waheed A, Biochem. J., 1972, 128, 49p.
  • Konkanapari down A (C on A), wheat germ lectin (WGA), PHA- E 4 was used those made of Seikagaku Corporation.
  • Kito oligosaccharide N-Acetyl darcosamine oligosaccharide; G1cNAc oligomers was also used from Seikagaku Corporation.
  • Tuliplectin was isolated and purified according to the method described in Oda Y., et al., Eur. J. Biochem., 1987, 165, 297-302-.
  • Peptide-1 N4- (acetyl) 3-D-dalcosaminol) Asparagine amidase was from Roche Molecular Biochemistry.
  • the high-purity APTS used was manufactured by Beckman-Coulter (Fulleton, CA). All other samples and reagents were of commercial grade or HPLC grade. Purified water was used for all aqueous solutions.
  • a glycoprotein (1 mg) as a sample was dissolved in a 2 O mM phosphate buffer (pH 7.0, 0 ⁇ L).
  • N-glycosidase F (5 mU, 5 ⁇ ) was added, and the solution was incubated overnight at 37 ° C. The solution was maintained in a boiling bath for 5 minutes and centrifuged at 1000 O'g for 10 minutes. Next, the supernatant containing oligosaccharide was evaporated to dryness using a centrifugal vacuum evaporator.
  • the obtained residue was dissolved in a 2 M aqueous acetic acid solution (50 L), and the mixture was maintained at 80 ° C for 3 hours to remove sialic acid from the oligosaccharide. The residue is then washed with 10OmM? The mixture was dissolved in a 15% aqueous acetic acid solution (5 // L) containing D3, and a freshly prepared THF solution (5 ⁇ L) of 1M sodium cyanoborohydride was added to the mixture. The mixture contains mineral oil (100 ⁇ L, nD1.67, d0.8).
  • Capillary affinity electrophoresis was performed on a P / ACE MDQ glycoprotein system (Beckraan Coulter) equipped with a system to detect fluorescence excited by an argon laser. Detection was performed using an argon laser with a fluorescence wavelength of 520 nm and an excitation wavelength of 488 nm.
  • a capillary with an inner wall coated with e-CAPN-CHO (effective length: 10 cm (length: 30 cm); 50 ⁇ m (Beckman Coulter)) Using.
  • the same size cabillary (GL Science Co., Ltd.) coated with dimethinopolysiloxane (DB-1) can also be used. Separation was performed at 25 ° C at all times during the operation, and injection was performed by the pressurized method (0.5 psi). The data obtained was analyzed on Windows 2000® using standard 32 Karat software.
  • the electrophoresis solution used was 100 mM Tris acetate buffer (pH 7.4). Prior to capillary affinity electrophoresis, sugar chains labeled with APTS were labeled by capillary electrophoresis at an applied voltage of 10 kV as described above. analyzed. Subsequently, the above electrophoresis running solution containing the lectin at the concentration shown in FIG. 11 was injected into the cavities. Prior to analysis, the cells were washed for 1 min with the buffer for kinetics. Next, the cells were washed with the same buffer containing lectin for 1 minute. The lab can process the same solution in a 96-well plate, thus automatically performing a series of binding reactions.
  • AGP fetuin
  • chicken egg white vomucoid were used as glycoproteins.
  • the sugar chains of AGP are two-, three-, and four-chain oligosaccharides. Some of the three- and four-stranded oligosaccharides are replaced by fucose (FIGS. 1 (b) and 2).
  • Fetuin contains double- and triple-stranded oligosaccharides.
  • oligosaccharide of the triplet a part of the G a1 -j31-4-G1cNAc branch is changed to Gal- ⁇ 1-3-G1cNAc (Fig. 3 (b) and Figure 4).
  • Egg ovomucoid contains a fairly complex mixture of oligosaccharides. Some of the oligosaccharides have been replaced with bisecting GlcNAc residues. Obomuco 'A typical oligosaccharide found in Eid is shown in Figure 6'. Oligosaccharides with a small molecular size (01) having an N-glycan structure as a core are also found in chicken egg white ovomucoid.
  • lectins that specifically recognize and bind to the following sugar chains were used. That is, (1) a lectin that specifically recognizes high mannose-type oligosaccharides, (2) a lectin that specifically recognizes N-acetylglucosamine (GlcNAc) or its oligomer. 3) Galactose ( Lectin recognizing G a1) or lactosamine G a1 ⁇ 1-4 / 3G1c ⁇ A c (4) Lectin recognizing sialic acid (5) Lectin recognizing fucose.
  • the lectin is stable and binds to a specific sugar chain with high specificity.
  • ConA, WGA, TGA, PHA-E4, SSA, MAM, RSL, and AAL which show sugar chain specificity were selected as lectins. These lectins are relatively stable during analysis and are suitable for pre-analytical storage.
  • a1 acidic glycoprotein contains double-chain, triple-chain, and 4-chain sugar chains as shown in FIG. 1 (b) and FIG. Furthermore, among the three- and four-chain sugar chains, there are sugar chains containing a fucose residue in the non-reducing terminal G a1 ⁇ 1-4 G 1 c NA c residue. .
  • the mobility of the mixture of asia mouth-glucose obtained from AGP was examined. The result is shown in Fig. 1 (c).
  • Double glycans were observed at the earliest transit time (5.2 minutes).
  • the three-chain ( ⁇ ⁇ ) and four-chain IMV) sugar chains were observed at 6.2 minutes and 7.- 2 minutes, respectively.
  • the peaks observed at 6.5 minutes and 7.5 minutes are the three-chain sugar chain ( ⁇ ) and the four-chain sugar chain (AV), both of which have a fucose residue added (Fig. 1 ( a))).
  • Tulip bulbs contain two types of lectins, one of which binds to yeast. Another type of lectin (TGA) specifically binds to mouse erythrocytes, and this binding is specifically inhibited by swine tyroglobulin. Addition of TGA was interesting—results in the transfer of oligosaccharides from AGP. 'Three-glycans (All and ⁇ )' showed high specificity for this lectin (TGA). 2 [On the 11th floor, the group of All and ⁇ moved to 7.0 minutes. In the 12 / ⁇ TGA, All and ⁇ eluted as a single broad peak and were observed the latest (about 9.3 minutes). On the other hand, the affinity between TGA and double- and quadruplex sugar chains was weak.
  • Example 2 Classification of sugar chains derived from Futuin-1 (when sialic acid residues were removed) As shown in FIG. 4, fetuin contains one type of double-chain sugar chain (AI) and two types of three-chain sugar chain (All and FII). One of the three glycans (FII) contains a G a1 ⁇ 1-3G1cNAc branch. In this example, as shown in FIG. 4, a mixture of ⁇ chains from which sialic acid residues had been removed was used.
  • the main three-chain ⁇ chain (All) was observed slightly later (about 8.9 minutes) in the 12 M WGA than in the absence of WGA. No delay in the transit time of the other three-chain sugar chain (FII) having the G a1 ⁇ 1-3G1cNAc branch was observed, but was observed at 7.5 minutes. With the / M WGA, All and FII could be completely separated.
  • TGA strongly recognizes the sugar chain of triplet.
  • TGA showed different affinities for All and FII.
  • FII sugar chain
  • FII containing the G a 131-3G 1 c NA c partial chain
  • a delay in the migration time was observed.
  • the transfer order of All and FII was reversed.
  • the difference in affinity between All and FII is remarkable, and the three glycans can be completely separated.
  • Ovomucoid derived from chicken egg white has five sugar chain binding sites and constitutes 20 to 25% of glycoproteins. More than 20 sugar chains have been reported in chicken egg white ovomucoid, and a bisexual G1cNAc residue is also present.
  • Figure 6 shows typical oligosaccharides found in Ovomucoid. The separation of this oligosaccharide is shown in Fig. 5 (a) and Fig. 5 (c).
  • sugar chains could not be identified due to the complexity of the sugar chains. However, it was confirmed that the sugar chain exhibited a different movement mode based on the structural characteristics of the sugar chain due to the presence of the lectin.
  • the stoichiometry of the coupling reaction must be determined.
  • the interaction between WGA and an APSTS-derivatized GlcNAc oligomer is shown. This model was chosen because the kinetics and mechanism of this lectin binding have been well studied.
  • the G1cNAc oligosaccharides showed interesting changes in transit time in the presence of various concentrations of WGA—.
  • Trisaccharides (3 in Fig. 7, and so on) showed weak affinity for WGII even at a high concentration of 3 ⁇ M WGA.
  • the tetrasaccharide (4) is 0.8 3 ⁇ 4 1 of 0
  • Trisaccharide, tetrasaccharide and pentasaccharide showed concentration and good linearity of the WG A, coupling constants, respectively, 0. 5 6 X 1 0 6 M- 1 - 5 6 X 1 0 6 M ⁇ 1 2 5 4 X 10 6 M— 1 .
  • the results obtained by the present invention are similar to those described in Daro T., et al., Chem. Rev., 2002, 102, 387-429. Asensio JL, Chemistry & Biology 2000, 7, 529-543, also reports a chitin-binding motif of WGA that binds polyvalently to the G1cNAc oligomer.
  • the glycan mixture was analyzed using lectin as the glycan-binding protein, and the glycan mixture was analyzed and classified.
  • a glycan of Lisopus genus was used as a sugar chain, and a crude glycan extracted from a liposome of the genus Lysopus was extracted with a buffer solution for electrophoresis.
  • the reaction mixture reacted with the extract was examined for fluctuations in electrophoresis time.
  • Add R S L Rostronipher lectin
  • FIG. 9 (a) shows a schematic diagram of a sugar chain derived from a mouse IgG.
  • the electrophoresis time of the sugar chain containing the crude mold extract is significantly slower than the swimming time without it, as shown in Fig. 9 (b). .
  • a sugar chain-binding protein which specifically exists in the crude mold extract and specifically binds to glycan derived from P. IgG was confirmed.
  • a sugar chain mixture having a known structure the presence or absence of a sugar chain-binding protein in a sample can be confirmed. This method leads to the discovery of novel sugar-chain-binding proteins and the development of new drugs using them.
  • the fetuin of this example is composed of one double-stranded sugar chain (SAI) and three triple-stranded sugar chains (SFI, SFII and SFII). ⁇ S Fill).
  • SAI single-stranded sugar chain
  • SFI, SFII and SFII triple-stranded sugar chains
  • SFI and SFII contain NeuAca2-6Gal
  • SFIII has 3 ⁇ 11. Includes 2-30 & 1.
  • SFIII contains Ga1 ⁇ 1-3G1cNAc, and the other contains Ga1 ⁇ 1-4G1cNAc.
  • TGA strongly recognizes three-chain sugar chains, and has different affinities between SFI and SFII and SFIII. Indicated. That is, the sugar chain (SFII) containing the G a1 1-3G 1cNAc branch chain exhibited a delay in the migration time. In the 12 ⁇ TGA, the order of movement between SFIII, SFI and SFII was reversed. At 12 ⁇ of TGA, the difference in affinity between All and FII was remarkable, and each three-chain sugar chain could be completely separated. (2) Addition of MAM
  • SSA recognizes ⁇ 2,6 sialyl lactosamine more strongly, and ⁇ recognizes 0: 2,3 sialyl lactosamine more strongly. Subtle differences could be distinguished.
  • a sugar chain with a known structure derived from Psi IgG was used as a sugar chain library, and lip scabilelectin (RSL) was added, as shown in FIG. 14 (b).
  • IgG derived from Psi contains a sugar chain having a fucose (indicated by ⁇ in FIG. 14 (b)) at the end of the fluorescent substance (1-1 V).
  • the addition of RSL markedly increased the migration time of these fucose-linked rice bran (fucosylated sugar chains) (I to EV) depending on the amount added. It was confirmed that it was late. That is, in this example, fucose-linked sugar chains could be detected efficiently.
  • glycans derived from butyroglobulin contain double-stranded (TI) and small amounts of triple-stranded (TI) sugar chains, and high-mannose-type sugar chains (HM).
  • TI double-stranded
  • TI triple-stranded
  • HM high-mannose-type sugar chains
  • the double- and triple-chain sugar chains are fucose-linked sugar chains in which fucose has been added to the terminal G1cNAc.
  • a sugar chain derived from butyroglobulin was used as a sugar chain library, and TGA and ConA were added.
  • TGA fucose-linked sugar chains
  • putatyroglobulin is a useful library monosaccharide containing a hymannose monosaccharide and a complex oligosaccharide.
  • the sugar chains of AGP are glycoproteins whose sugar chains change with physiological changes such as canceration and inflammation.
  • changes in fucosylated sugar chains (AIII, AV, AVI) associated with canceration have attracted attention in recent years.
  • the sugar chain analysis of AGP derived from a cancer patient and the effect of adding a fucose recognition lectin were examined.
  • FIG. 17 (a) normal AGP glycans were found at 5.0 minutes (AI), 5.7 minutes (AII), 5.9 minutes (AIII), 6.5 minutes (AIV), and 6.7 minutes (AV).
  • AVI a peak
  • AAL Aleuria aurantia lectin
  • Fig. 17 (a) a fucose-recognition lectin
  • AAL Aleuria aurantia lectin
  • Fig. 17 (a) a fucose-recognition lectin
  • a decrease (disappearance) in the peak intensity of the fucose-linked bran chain (fucosylated sugar chain) was confirmed. Due to the specificity of AAL, this fucose-linked sugar chain is It is a fucosylated sugar chain having an antigen.
  • high-throughput function classification of glycoprotein-derived sugar chains is possible. More specifically, in Kakumi ⁇ to jar good of the above, to classify the sugar chain, eight types of lectins, ie, C on A, WGA, P HA- E 4, T GA, RSL, AA L, SSA and MAM were selected. The characteristics of these lectins are shown below.
  • ConA recognizes a double-stranded sugar chain. High mannose and hybrid oligosaccharides are also recognized by ConA.
  • WGA affects the translocation time of double, triple and quadruple bran chains. That is, in the presence of WGA, the order of movement of the three- and four-chain sugar chains to which fucose residues have been added and the order of movement of the respective sugar chains that do not contain fucose residues change.
  • TGA is very useful for discriminating triple glycans. As shown in FIG. 3 (c), the triple sugar chain is specifically recognized by TGA.
  • P HA_ E 4 is Remind as in FIG. 5 (a) ⁇ FIG 5 (c), can be applied to the recognition of the sugar chain having Baisekute queuing G 1 c NA c residues.
  • the inventors succeeded in identifying an oligosaccharide having a bisexual GlcNAc residue from a complex mixture of oligosaccharides derived from chicken egg white ovomucode.
  • RSL and AAL show specificity for fucose
  • SSA and MAM show specificity for sialic acid
  • sugar chains classified into the above four types C on A, WGA, T GA, and PHA-E 4
  • lectins showed binding to sugar chains in two different ways. That is, in the binding between C on A in ovomucoid and the core pentasaccharide (OI in FIGS. 5 (a) to 5 (c)), disappearance of the peak was observed. Other In this case, the binding between the lectin and the sugar chain resulted in a slower translocation time. To date, the reasons for these different binding modes have not been elucidated. In addition, although kinetic studies are needed, the kinetics of binding should be considered.
  • the affinity constant (K a) at which the ⁇ chain binds to the lectin can be determined simultaneously. This is because, as shown in FIG. 7, it is not necessary to measure the concentration of a fluorescently labeled ligand (for example, a sugar chain), unlike the binding between chito-oligosaccharide and protein. Therefore, the method of the present invention is very useful for the kinetic measurement of a complex mixture of sugar chains from a biological sample, whose concentration is difficult to determine.
  • sugar chains were successfully classified using a combination of selected lectins at a predetermined concentration.
  • the total analysis time required for one sugar chain sample is within 4 hours.
  • the total amount of lectin and glycan samples used in the study of WGA and AGP was 20 // g (500 pm01) and 2 ⁇ g (50 pmol), respectively.
  • the main feature is to identify the characteristics of the sugar chain skeleton.
  • the present invention can be applied to a method utilizing the characteristics of a sugar chain containing sialic acid.
  • the technology of the present invention is based on electrophoresis of a known and / or unknown glycan library using a solubilized solution, gel or sol of a known or unknown protein as a medium. Based on the difference in the electrophoresis of the sugar chain-single sugar chain binding protein complex formed below, we classify the sugar chains and analyze the interaction between the ⁇ chain and the protein.
  • the sugar chain library and the electrophoresis medium can be provided as a kit, and the electrophoresis gel medium can be provided in the form of a microchip or the like.
  • the sugar chain after translation of the protein It is very useful in elucidating modifications. It is also useful for studying the pathology of glycoprotein diseases due to sugar chain deficiency.
  • By characterizing sugar chains on the cell surface using the present invention it can be useful for molecular-level pathological analysis.
  • the method for measuring the interaction between a sugar chain and a ⁇ chain-binding protein comprises the step of reacting the separation result of one or more sugar chain mixtures with the sugar chain mixture and the sugar chain-binding protein.
  • This is a method for measuring the interaction between a sugar chain and a sugar chain-binding protein based on comparison with the separation result of the reaction mixture. Therefore, for example, a mixture of a plurality of sugar chains can be used as a library to measure subtle changes in the use of sugar chains, sugar chain bonds, and the properties of proteins.
  • a plurality of known sugar chain-binding proteins for example, lectins
  • the binding reaction with a complex mixture of sugar chains can be efficiently and simultaneously measured.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé de mesure d'interactions entre des chaînes du sucre et une protéine de liaison à la chaîne du sucre. Ce procédé comprend la comparaison du résultat de la séparation d'un mélange d'au moins une chaîne du sucre et du résultat de la séparation d'un mélange de réaction du mélange susmentionné avec une protéine de liaison à la chaîne du sucre, afin de mesurer les interactions entre les chaînes du sucre et la protéine de liaison à la chaîne du sucre qui n'ont jamais été établies jusqu'ici. Selon ce procédé, des interactions entre des chaînes du sucre et une protéine peuvent être mesurées de manière exhaustive simultanément, même dans un mélange de chaînes du sucre compliqué.
PCT/JP2003/013239 2002-10-18 2003-10-16 Procede de mesure d'interactions entre des chaines du sucre et une proteine de liaison a la chaine du sucre et utilisation associee WO2004036216A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2004544967A JPWO2004036216A1 (ja) 2002-10-18 2003-10-16 糖鎖−糖鎖結合性タンパク質の相互作用の測定方法およびその利用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002-305086 2002-10-18
JP2002305086 2002-10-18

Publications (1)

Publication Number Publication Date
WO2004036216A1 true WO2004036216A1 (fr) 2004-04-29

Family

ID=32105154

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2003/013239 WO2004036216A1 (fr) 2002-10-18 2003-10-16 Procede de mesure d'interactions entre des chaines du sucre et une proteine de liaison a la chaine du sucre et utilisation associee

Country Status (2)

Country Link
JP (1) JPWO2004036216A1 (fr)
WO (1) WO2004036216A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009036577A (ja) * 2007-07-31 2009-02-19 Institute Of Physical & Chemical Research 糖鎖分析方法
JP2017203762A (ja) * 2016-05-09 2017-11-16 グリコボンド・アクチボラゲットＧｌｙｃｏｂｏｎｄ Ａｂ 新規の結合アッセイ
WO2018233618A1 (fr) * 2017-06-20 2018-12-27 江苏先思达生物科技有限公司 Méthode d'établissement d'un modèle de profil de glycome de glycoprotéine sérique de cirrhose du foie
JP2019148564A (ja) * 2018-02-28 2019-09-05 学校法人近畿大学 糖鎖解析方法、糖鎖解析システム、糖鎖解析用プログラム、及び糖鎖解析用キット

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62253396A (ja) * 1986-04-16 1987-11-05 アンステイテユ−・メリウ− アフイニテイ・クロマトグラフイ−材料及びこれを用いてのボルデテラ属細菌の蛋白質抗原の精製法
JPH10267931A (ja) * 1997-03-27 1998-10-09 Takara Shuzo Co Ltd 糖と標的物との相互作用の測定方法
WO2000068688A1 (fr) * 1999-05-06 2000-11-16 Glycodata Ltd. Structure polysaccharidique et determination de la sequence
JP2002500358A (ja) * 1997-12-24 2002-01-08 セテク コーポレイション 標的に結合する配位子を検出し、その相対親和力を決定するための毛管電気泳動法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62253396A (ja) * 1986-04-16 1987-11-05 アンステイテユ−・メリウ− アフイニテイ・クロマトグラフイ−材料及びこれを用いてのボルデテラ属細菌の蛋白質抗原の精製法
JPH10267931A (ja) * 1997-03-27 1998-10-09 Takara Shuzo Co Ltd 糖と標的物との相互作用の測定方法
JP2002500358A (ja) * 1997-12-24 2002-01-08 セテク コーポレイション 標的に結合する配位子を検出し、その相対親和力を決定するための毛管電気泳動法
WO2000068688A1 (fr) * 1999-05-06 2000-11-16 Glycodata Ltd. Structure polysaccharidique et determination de la sequence

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KINOSHITA ET AL.: "Capillary DNA sequencer o mochiiru to-tanpaku-shitsu tosa no cho biryo bunseki", DAI 21 KAI PROCEEDINGS OF THE SYMPOSIUM ON CAPILLARY ELECTROPHORESIS KOEN YOSHISHU, 12 December 2001 (2001-12-12), pages 95 - 96, XP002983864 *
NAKAJIMA ET AL.: "Capillary denki eidoho o shiyo suru tosa kongobutsu-tanpaku-shitsu kan sogo sayo no issei kaiseki", DAI 23 KAI THE JAPANESE SOCIETY OF CARBOHYDRATE RESEARCH NENKAI YOSHISHU, 25 July 2002 (2002-07-25), pages 97, XP002983863 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009036577A (ja) * 2007-07-31 2009-02-19 Institute Of Physical & Chemical Research 糖鎖分析方法
JP2017203762A (ja) * 2016-05-09 2017-11-16 グリコボンド・アクチボラゲットＧｌｙｃｏｂｏｎｄ Ａｂ 新規の結合アッセイ
WO2018233618A1 (fr) * 2017-06-20 2018-12-27 江苏先思达生物科技有限公司 Méthode d'établissement d'un modèle de profil de glycome de glycoprotéine sérique de cirrhose du foie
JP2019148564A (ja) * 2018-02-28 2019-09-05 学校法人近畿大学 糖鎖解析方法、糖鎖解析システム、糖鎖解析用プログラム、及び糖鎖解析用キット
US11371997B2 (en) 2018-02-28 2022-06-28 Shimadzu Corporation Glycan analysis method, glycan analysis system, program for glycan analysis, and kit for glycan analysis
JP2022186876A (ja) * 2018-02-28 2022-12-15 学校法人近畿大学 糖鎖解析方法、糖鎖解析システム、糖鎖解析用プログラム、及び糖鎖解析用キット
JP7469780B2 (ja) 2018-02-28 2024-04-17 学校法人近畿大学 糖鎖解析方法、糖鎖解析システム、糖鎖解析用プログラム、及び糖鎖解析用キット

Also Published As

Publication number Publication date
JPWO2004036216A1 (ja) 2006-02-16

Similar Documents

Publication Publication Date Title
US10317393B2 (en) Alkynyl sugar analogs for labeling and visualization of glycoconjugates in cells
Safina Application of surface plasmon resonance for the detection of carbohydrates, glycoconjugates, and measurement of the carbohydrate-specific interactions: A comparison with conventional analytical techniques. A critical review
Hendrickson et al. Analytical application of lectins
JP5552421B2 (ja) エキソグリコシダーゼを用いるｎ−グリカンの特性決定法
Horlacher et al. Carbohydrate arrays as tools for research and diagnostics
Laurent et al. Glycoarrays—tools for determining protein–carbohydrate interactions and glycoenzyme specificity
JP4988889B2 (ja) 多糖類の構造および配列決定
EP1910838B1 (fr) Utilisations de glycanes spécifiques du cancer
JP5116837B2 (ja) グリカンを評価するためのｍｓ法
CN101308141B (zh) 一种分析糖蛋白的方法
WO2008128222A1 (fr) Analyse de glycanes, glycopeptides ou glycoprotéines phosphorylés par imac
Smith et al. History and future of shotgun glycomics
CA2588260A1 (fr) Analyse de glycanes utilisant le glucose deutere
Wu et al. Lectins and ELLSA as powerful tools for glycoconjugate recognition analyses
Grün et al. One-step biotinylation procedure for carbohydrates to study carbohydrate–protein interactions
JP2002544485A5 (fr)
Yue et al. Microarrays in glycoproteomics research
WO2001040796A2 (fr) Matrices de molecules glycanes (glycomatrices) sur la surface de biopuces (glycopuces) et leurs utilisations
AU2006257904B2 (en) Rapid saccharide biomarker assay
Yamamoto et al. Measurement of the carbohydrate-binding specificity of lectins by a multiplexed bead-based flow cytometric assay
WO2004036216A1 (fr) Procede de mesure d'interactions entre des chaines du sucre et une proteine de liaison a la chaine du sucre et utilisation associee
Brooks Lectin Histochemistry: Historical perspectives, state of the art, and future directions
JP2004506874A (ja) 炭水化物ポリマーを分析するための方法および組成物
Petrović et al. Lectin and Liquid Chromatography-Based Methods for Immunoglobulin (G) Glycosylation Analysis
Huang et al. Using the glycoarray technology in biology and medicine

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): DE FR GB

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2004544967

Country of ref document: JP

122 Ep: pct application non-entry in european phase