Compositions and methods for detecting Fucα1-3GlcNAc structures

Abstract

To specifically detect a specific sugar chain structure using a lectin.SOLUTION: Disclosed is a method for detecting an Fucα1-3GlcNAc structure as a sugar chain structure in a sample according to the present invention, which includes the steps of: bringing a CLA lectin into contact with a sample; quantifying the CLA lectin bound to the Fucα1-3GlcNAc structure; and determining the presence of the Fucα1-3GlcNAc structure in the sample when the amount of the CLA lectin bound to the Fucα1-3GlcNAc structure is equal to or greater than a predetermined threshold value.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition and method for detecting a Fucα1-3GlcNAc structure, in which fucose is α1,3-linked to N-acetylglucosamine, among other sugar chain structures, and in particular to a composition and method for detecting a Fucα1-3GlcNAc structure using a lectin. [Background technology]

[0002] Sugar chains of glycoconjugates, such as glycoproteins and glycolipids, present on cell surfaces and in body fluids function as information devices and are deeply involved in important biological phenomena, such as development, immunity, cancer, and infection. Meanwhile, many types of lectins, which are glycan-binding proteins, have been isolated from marine algae and algae (freshwater cyanobacteria). They function as glycan-recognizing molecules and, like glycans, play important biological roles. It is known that different lectins recognize different glycans. For example, Patent Document 1 discloses Hypnin A, isolated from the red alga Hypnea japonica, as a lectin that strongly interacts with fucose (α-1,6-linked to N-acetylglucosamine at the reducing end of N-linked glycans (asparagine-linked glycans). Patent Document 1 also discloses a method for detecting α-1,6-fucose glycans using Hypnin A.

[0003] In addition, some lectins are known to recognize and bind to sugar chains on the surface of various viruses and cancer cells, and for this reason, it is thought that lectins may also be used to detect viruses, etc. (see Patent Documents 2 and 3, etc.). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 5109001 [Patent Document 2] Japanese Patent Publication No. 2020-117445 [Patent Document 3] Patent Publication No. 2021-013375 Summary of the Invention [Problem to be solved by the invention]

[0005] However, it cannot be said that sufficient substances are currently known that can specifically bind to and detect glycans present on the surfaces of various viruses, cancer cells, etc., and because the number of such substances is limited, there is no sufficient supply of such substances. Therefore, it is necessary to discover more substances that specifically recognize specific glycans and to clarify their properties.

[0006] The present invention has been made in view of the above problems, and its object is to enable specific detection of a particular sugar chain structure using a lectin. [Means for solving the problem]

[0007] To achieve the above-mentioned objective, the present inventors conducted extensive research and discovered that a CLA lectin isolated from the green alga Codium latum specifically recognizes the Fucα1-3GlcNAc structure, in which fucose is α1,3-linked to N-acetylglucosamine, among other sugar chain structures, and thus completed the present invention.

[0008] Specifically, the composition of the present invention is a composition for detecting Fucα1-3GlcNAc structures, characterized by containing a CLA lectin. As described above, CLA lectin can specifically recognize Fucα1-3GlcNAc structures, and therefore, the composition of the present invention containing a CLA lectin is useful for detecting Fucα1-3GlcNAc structures in glycans in a given sample. Therefore, it can be applied, for example, to detecting cancer cells or viruses that express glycans containing Fucα1-3GlcNAc structures on their cell surfaces.

[0009] In the composition of the present invention, the CLA lectin may be conjugated to a labeling molecule, which allows for the use of a binding molecule that recognizes and binds to the labeling molecule, for example, to quantify or detect the CLA lectin bound to the Fucα1-3GlcNAc structure, thereby simplifying the quantification and detection.

[0010] The method of the present invention for detecting Fucα1-3GlcNAc structures in a sample comprises the steps of contacting the sample with CLA lectin, quantifying the amount of CLA lectin bound to the Fucα1-3GlcNAc structures, and determining the presence of Fucα1-3GlcNAc structures in the sample if the amount of CLA lectin bound to the Fucα1-3GlcNAc structures is equal to or greater than a predetermined threshold. As described above, CLA lectin can specifically recognize Fucα1-3GlcNAc structures, and the method of the present invention allows the detection of Fucα1-3GlcNAc structures in the sample by contacting the sample with CLA lectin and assessing the amount or presence of CLA lectin binding to Fucα1-3GlcNAc structures in the sample.

[0011] In the method of the present invention for detecting a Fucα1-3GlcNAc structure in a sample, a labeling molecule may be bound to the CLA lectin, as described above.

[0012] In the method of the present invention, the quantification step can include contacting the CLA lectin bound to the Fucα1-3GlcNAc structure with a binding molecule that binds to the CLA lectin or the labeled molecule, and can also include quantifying the CLA lectin bound to the Fucα1-3GlcNAc structure by quantifying the binding molecule. [Effects of the Invention]

[0013] The composition and method for detecting the Fucα1-3GlcNAc structure are extremely useful because they enable specific detection of the Fucα1-3GlcNAc structure among sugar chain structures. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows Lewis X structures and sialyl Lewis X structures as sugar chain structures. [Figure 2] FIG. 1 shows the results of an analysis of sugar chain binding specificity of CLA by centrifugal ultrafiltration-HPLC method in an example. [Figure 3] FIG. 1 shows the results of an analysis of sugar chain binding specificity of CLA by centrifugal ultrafiltration-HPLC method in an example. [Figure 4] FIG. 1 shows N-linked glycans used in the glycan array in the Examples. [Figure 5] FIG. 1 shows N-linked glycans used in the glycan array in the Examples. [Figure 6] FIG. 1 shows N-linked glycans used in the glycan array in the Examples. [Figure 7] FIG. 1 shows O-linked glycans used in the glycan array in the Examples. [Figure 8] FIG. 1 shows O-linked glycans used in the glycan array in the Examples. [Figure 9] FIG. 1 shows glycosphingolipid sugar chains used in the glycan array in the Examples. [Figure 10] 1 is a graph showing the results of a glycan array targeting N-linked glycans in an example. [Figure 11] 1 is a graph showing the results of a glycan array targeting O-linked glycans in an example. [Figure 12] 1 is a graph showing the results of a glycan array targeting glycosphingolipid sugar chains in an example. DETAILED DESCRIPTION OF THE INVENTION

[0015] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following description of preferred embodiments is merely exemplary in nature and is not intended to limit the present invention, its application, or its uses.

[0016] One embodiment of the present invention is a composition for detecting Fucα1-3GlcNAc structures, comprising CLA lectin (hereinafter referred to as CLA). As will be explained in detail in the Examples below, the present inventors have now discovered that CLA can specifically bind to Fucα1-3GlcNAc structures. CLA is a type of glycoprotein derived from the green alga Codium latum and is a protein of approximately 16 kDa having the amino acid sequence of SEQ ID NO: 1. In particular, CLA preferably consists of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, more preferably 95%, 96%, 97%, 98%, or 99% or more identity. Most preferably, CLA consists of the amino acid sequence of SEQ ID NO: 1.

[0017] The CLA in this embodiment is derived from the green alga Codium latum as described above, but in addition to natural purified products, it also includes recombinant CLA as a product of chemical synthesis procedures and a product produced by recombinant technology from a prokaryotic or eukaryotic host (including, for example, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells).

[0018] In this embodiment, the term "sugar chain" refers to a linear or branched oligosaccharide or polysaccharide. The sugar chains are broadly classified into N-glycoside-linked sugar chains (hereinafter referred to as "N-glycan" or "N-glycan") that are linked to asparagine and O-glycoside-linked sugar chains (hereinafter referred to as "O-glycan" or "O-glycan") that are linked to serine, threonine, or the like, depending on the mode of linkage with the protein. N-glycan includes high-mannose sugar chains, complex sugar chains, and hybrid sugar chains.

[0019] Oligosaccharides are formed by the dehydration of 2 to 10 monosaccharides or substituted derivatives of monosaccharides. Carbohydrates with a larger number of monosaccharides bound together are called polysaccharides. Polysaccharides vary depending on the type of sugar they contain, but sugars containing a large amount of uronic acid or ester sulfates are called acidic polysaccharides, while those containing only neutral sugars are called neutral polysaccharides. Among polysaccharides, a group of polysaccharides called mucopolysaccharides are mostly bound to proteins and are called proteoglycans. Monosaccharides are the building blocks of sugar chains and are basic substances that cannot be broken down into simpler molecules by hydrolysis.

[0020] Monosaccharides are further divided into three main categories: acidic sugars with acidic side chains such as carboxyl groups, amino sugars in which hydroxyl groups are replaced with amino groups, and other neutral sugars. Monosaccharides present in living organisms include acidic sugars such as sialic acids (N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (Neu5Gc)) and uronic acids, amino sugars such as N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc), and neutral sugars such as glucose (Glc), mannose (Man), galactose (Gal), and fucose (Fuc).

[0021] All N-glycans share a common core structure called the "trimannosyl core" (Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc). High-mannose glycans contain only α-mannose residues in the branched structures, in addition to the trimannosyl core. These glycans share a common core structure: a heptasaccharide (Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc). Hybrid glycans are also called hybrid glycans because they share characteristics of both complex and high-mannose glycans. One or two α-mannosyl groups are attached to the Manα1-6 arm of the trimannosyl core, as in the high-mannose glycans, and the same side chains as in complex glycans are attached to the Manα1-3 arm of the core.

[0022] The presence or absence of a fucose bond at the C-6 position of the GlcNAc at the reducing end of the trimannosyl core, and the presence or absence of a β-GlcNAc bond at the C-4 position of the β-mannosyl residue (called a bisecting GlcNAc), contribute to the structural diversity of complex and hybrid glycans. Among the three types of N-glycans, the complex type contains the most diverse structures.

[0023] This diversity is mainly due to two factors: one to five side chains attached to a trimannosyl core at different linkage positions to form mono-, di-, tri-, tetra-, or penta-antennary glycans. Two isomers of the triantennary complex glycan have been found, containing either [GlcNAcβ1-4(GlcNAcβ1-2)Manα1-3] or [GlcNAcβ1-6(GlcNAcβ1-2)Manα1-6].

[0024] The Fucα1-3GlcNAc structure to which the CLA of the present embodiment specifically binds is a sugar chain structure in which Fuc is specifically linked to GlcNAc via an α1,3 bond. Representative structures of this type are Lewis X and sialyl Lewis X, shown in Figure 1. As shown in Figure 1, Lewis X is a structure in which Gal is linked via a β1-4 bond to GlcNAc, which is linked via an α1,3 bond to Fuc. Sialyl Lewis X is a structure in which NeuAc is linked via an α1-3 bond to Gal. The boxed area in Figure 1 represents the Fucα1-3GlcNAc structure. Lewis X and sialyl Lewis X are known to be expressed on the surface of various cancer cells. Specifically, Lewis X is expressed on the surface of cancer cells such as colon cancer and blood cancer, while sialyl Lewis X is expressed on the surface of cancer cells such as lung cancer, breast cancer, and pancreatic cancer. Therefore, a composition containing the CLA of the present embodiment can specifically bind to the above-mentioned cancer cells and potentially be used to detect these cancer cells.

[0025] Whether or not CLA binds to a glycan can be assessed by, for example, passing the test CLA through a column onto which the target glycan or glycoprotein bound to the glycan is immobilized, and then measuring the amount of CLA in the flow-through or the amount of CLA eluted from the column with a specific eluent. Alternatively, the binding of the target glycan-bound glycoprotein can be assessed by immobilizing the target glycan on a membrane or the like, and detecting it using CLA labeled with biotin, fluorescein isothiocyanate, peroxidase, or the like, using Western blotting (see Forensic Medicine Practice and Research, 37, 155, 1994) or dot blotting (see Analytical Biochemistry, 204(1), 198, 1992).

[0026] Alternatively, the affinity between the target glycan or glycoprotein bound to the glycan and the CLA to be tested can be measured using surface plasmon resonance (SPR). This method is preferable because it can measure not only the presence or absence of affinity but also its strength. The binding constant (affinity constant) (K A ), but 10(M -1 ) or more, more preferably 10 3 (M -1 ) or more, most preferably 10 4 (M -1 ) or higher, it can be determined that CLA is bound to the sugar chain.

[0027] The CLA in this embodiment may be bound to a labeling molecule. The labeling molecule is not particularly limited as long as it can label the CLA to facilitate detection of the CLA, and examples thereof include fluorescent proteins, luciferase, biotin, avidin, His tag peptide, GST tag peptide, and FLAG tag peptide. Binding such a target molecule to the CLA is advantageous because it allows for easy evaluation of the presence or absence of the CLA.

[0028] The composition according to this embodiment contains CLA and a solvent, and may also contain other additives, for example, for improving the stability of CLA in the solvent.

[0029] Another embodiment of the present invention is a method for detecting Fucα1-3GlcNAc structures in a sample, which comprises the steps of contacting the sample with CLA, quantifying the amount of CLA bound to the Fucα1-3GlcNAc structures, and determining the presence of Fucα1-3GlcNAc structures in the sample if the amount of CLA bound to the Fucα1-3GlcNAc structures is equal to or greater than a predetermined threshold.

[0030] In this embodiment, the sample is an object to be evaluated for the presence or absence of a glycan containing a Fucα1-3GlcNAc structure, and may be, for example, a solution containing an artificially prepared specific glycan, or a biological sample such as body fluid, cells, or tissue.

[0031] In this embodiment, the step of contacting the sample with CLA is carried out by, for example, adding a solution containing CLA to the above-mentioned solution or body fluid as the sample and mixing it. When the sample is a tissue, a portion of the tissue may be removed from the body and used as the sample. Alternatively, the tissue may be treated with CLA in vivo, and the Fucα1-3GlcNAc structure bound to the CLA in vivo may be detected using an endoscope or the like.

[0032] In this embodiment, the step of quantifying the CLA bound to the Fucα1-3GlcNAc structure can be carried out using, for example, the centrifugal ultrafiltration-HPLC method used in the Examples below, or by using a conventional method used in the art that uses an antibody or binding molecule that specifically binds to CLA or a labeling molecule that binds to it. For example, but not limited to, when a tag peptide is bound to CLA as a labeling molecule, the CLA can be detected using an antibody that targets the tag peptide, and the antibody can be labeled with a fluorescent molecule or the like, thereby quantifying the amount of CLA bound to the Fucα1-3GlcNAc structure based on its fluorescence intensity. Such methods also allow the detection and quantification of Fucα1-3GlcNAc structures.

[0033] In this embodiment, a threshold value (reference value) is set in advance in the step of determining the presence of Fucα1-3GlcNAc structures in a sample. This reference value is not limited to the following, but for example, a sample not containing Fucα1-3GlcNAc structures can be used as a reference, and the quantitative value of CLA obtained from this reference in a step similar to the above-mentioned step of quantifying CLA can be used as the reference value. In this case, if the quantitative value of CLA obtained from the sample to be determined is greater than the reference value, it can be determined that Fucα1-3GlcNAc structures are present in the sample.

[0034] As described above, the method of this embodiment allows for the simple detection of glycans containing Fucα1-3GlcNAc structures in a sample using CLA that can specifically bind to the Fucα1-3GlcNAc structure, and therefore may be applicable to the detection of cancer cells that express glycans containing Fucα1-3GlcNAc structures on their cell surface. [Example]

[0035] The following examples are provided to provide a detailed description of the compositions and methods for detecting Fucα1-3GlcNAc structures according to the present invention.

[0036] Example 1: Preparation of His-tagged recombinant CLA (Construction of E. coli strain for expressing His-tagged recombinant CLA) A synthetic DNA encoding CLA was designed based on the sequence information of the translated region encoded by the CLA cDNA (see SEQ ID NO: 2) and was produced by Integrated DNA Technologies. The CLA-encoding DNA fragment was amplified by PCR using primers with restriction enzyme recognition sites at the 5' end (forward primer: NheI, reverse primer: XhoI) and PrimeSTAR HS DNA Polymerase (Takara Bio). The amplified DNA fragment was digested with restriction enzymes NheI and XhoI and subcloned into the vector pET-28a(+) (Merck) that had also been digested with both restriction enzymes to obtain the CLA expression plasmid pET28a-rCLA. Furthermore, this pET28a-rCLA was used to transform the expression strain E. coli SHuffle T7 Express (New England Biolabs) to obtain the His-tag fused recombinant CLA expression strain pET28a-rCLA / SHuffle T7 Express.

[0037] (Expression of His-tagged recombinant CLA) The E. coli strain (pET28a-CLA / SHuffle T7 Express) for expressing His-tagged recombinant CLA (His-rCLA) obtained as described above was inoculated into 3 mL of kanamycin-containing LB liquid medium and cultured overnight at 37°C. The culture was then added to 250 mL of kanamycin-containing LB liquid medium and cultured with shaking at 37°C until the medium reached mid-logarithmic growth phase. When the OD600 reached 0.5, expression induction was initiated by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM, and the culture was then cultured with shaking at 20°C for 16 hours. The cells were collected by centrifugation (10,000 × g, 4°C, 20 min), and 1 / 20 volume of the culture medium was added to the sonication buffer (20 mM phosphate buffer (PB) (pH 7.4), 500 mM NaCl, 20 mM imidazole) and suspended. Then, the cells were sonicated while cooled on ice. The sonication conditions were "sonication for 1 minute, rest for 1 minute," and seven cycles were performed. After the sonication, the cells were centrifuged (10,000 × g, 4°C, 20 min), and the supernatant was used as the soluble fraction.

[0038] (Purification of His-tagged recombinant CLA) A nickel chelate column (Vt = 1 mL, His GraviTrap, GE Healthcare) was equilibrated with 10 mL of the same buffer, and the soluble fraction was loaded onto the column to adsorb the His-tagged recombinant lectin. To wash away impurities nonspecifically adsorbed to the column, the column was washed with a buffer containing 150 mM imidazole (20 mM PB (pH 7.4), 500 mM NaCl). Subsequently, 3 mL and 2 mL of elution buffer (20 mM PB (pH 7.4), 500 mM NaCl, 500 mM imidazole) were added and collected, yielding purified His-tagged recombinant CLA. The following tests were performed using this purified His-tagged recombinant CLA.

[0039] [Example 2: Glycosylation specificity of CLA] (Glycan binding specificity analysis by centrifugal ultrafiltration-HPLC method) The glycan-binding specificity of CLA was measured using centrifugal ultrafiltration-HPLC. This method does not require immobilization of lectins or glycans, and is a simple method for analyzing the interaction between lectins and glycans. In this method, lectins are mixed with fluorescently labeled glycans in a buffer, and the unbound glycans recovered by centrifugal ultrafiltration are separated and quantified by HPLC. The binding activity is expressed as the ratio of the amount of bound glycan to the amount of added glycan, and the amount of bound glycan is calculated by subtracting the amount of unbound glycan from the amount of added glycan. The glycan-binding specificity of a lectin is determined by comparing the binding activity of multiple glycans. The specific method and results are described below.

[0040] First, 90 μL of 5 μM His-tagged recombinant CLA and 10 μL of 300 nM pyridylamino (PA)-oligosaccharides (Takara Bio) were mixed in 50 mM Tris-HCl (pH 7.0) and incubated for 1 h at room temperature. The mixture was then centrifuged at 10,000 × g for 30 s at room temperature using a centrifugal ultrafilter (10 kDa molecular weight cutoff, PALL, NY, USA). 20 μL of the filtrate was loaded onto a TSKgel ODS-80™ column (4.6 × 150 mm, Tosoh) equilibrated with 15% methanol-containing 0.1 M ammonium acetate buffer and eluted with the same solution. The HPLC analysis was performed at a flow rate of 1 mL / min at 40°C using a Waters Alliance HPLC system (Waters, Alliance e2695 Separations Module). The eluate was monitored at an excitation wavelength of 320 nm and an emission wavelength of 400 nm, and the glycan peaks were analyzed and quantified using Empower3 (Waters). Separately, 90 μL of 50 mM Tris-HCl (pH 7.0) containing no His-tagged recombinant CLA was mixed with the PA-oligosaccharides and subjected to centrifugal ultrafiltration as described above. The filtrate was used as a blank. The amount of PA-oligosaccharide bound to CLA [O bound ] is the amount of PA-oligosaccharides added [O added ] to the amount of unbound PA-oligosaccharides [O unbound ] was obtained by subtracting [O added ] to [O boundThe ratio of the total PA-oligosaccharides to the total PA-oligosaccharides was defined as the binding activity (%). The 32 PA-oligosaccharides used and their binding activities are shown in Figures 2 and 3.

[0041] As shown in Figures 2 and 3, the glycans that showed the highest binding activity were No. 10 (89.2%), No. 7 (77.7%), and No. 22 (45.1%). These glycans share the Galβ1-4(Fucα1-3)GlcNAc structure, which is also found in Lewis X and sialyl Lewis X antigens. Furthermore, they did not bind to Fucα1-2Gal (No. 20, 28, 30, 31, and 32), Fucα1-4GlcNAc (No. 21, 28, and 30), or Fucα1-6GlcNAc (No. 14), a high-mannose glycan. They showed extremely weak binding activity (approximately 10%) to Fucα1-6GlcNAc (No. 4, 6, and 9), a complex glycan. These results suggest that CLA binds strongly to fucose-containing glycans, particularly Fucα1-3GlcNAc. Furthermore, the greater the number of antennae (number of non-reducing terminal branches) in the glycan, the stronger the binding to the glycan.

[0042] (Glycan-binding specificity analysis using glycan arrays) Furthermore, a glycan array was performed to evaluate the binding specificity of CLA to various glycans. Glycan arrays are a tool for investigating interactions between glycans and biomolecules, allowing for the analysis of interactions between multiple glycans in a single experiment. The principle is as follows: first, biomolecules are added to a glass slide spotted with multiple types of glycans, allowing the biomolecules to react with the glycans. Then, a fluorescently labeled secondary antibody is bound to the biomolecules, and the fluorescent signal is quantified to evaluate the binding affinity between the glycans and biomolecules. In this example, the binding activity of CLA to 100 types of N-linked glycans, 94 types of O-linked glycans, and 58 types of glycosphingolipid glycans was comprehensively analyzed using a glycan array. Specifically, we used the N-Glycan Array (8-subarray slide), a glycan-immobilized microarray slide from Z Biotech, which is equipped with 100 types of N-linked glycans as shown in Figures 4 to 6, the O-Glycan Array, a glycan-immobilized microarray slide from Z Biotech, which is equipped with 94 types of O-linked glycans as shown in Figures 7 and 8, and the Glycosphingolipid-Glycan Array, a glycan-immobilized microarray slide from Z Biotech, which is equipped with 58 types of glycosphingolipid glycans as shown in Figure 9. The methods and results are described below.

[0043] First, the slides were mounted in the provided cassette. 200 μl of 1% BSA-containing Hydrazide Blocking Buffer (Z Biotech) was added to each well. The wells were covered with the provided adhesive film to prevent buffer evaporation, and the plates were incubated for 60 minutes at 85 rpm in a shaking incubator. After incubation, the buffer was removed, and 200 μl of 1% BSA-containing Glycan Array Assay Buffer (Z Biotech) containing 5 μg / mL CLA was added to each well. The wells were covered with adhesive film and incubated for 60 minutes at 100 rpm. After incubation, the CLA solution was removed, and 200 μl of wash buffer (50 mM Tris-HCl, 137 mM NaCl, 0.05% Tween 20, pH 7.6) was added to each well. The wells were covered with adhesive film and incubated for 5 minutes at 85 rpm. After incubation, the wash buffer was removed, and 200 μl of 1% BSA-containing Glycan Array Assay Buffer containing 1 μg / ml antibody (Alexa Flour 555-labeled anti-His-tag mouse antibody) was added to each well. The wells were covered with adhesive film and incubated at 85 rpm for 60 minutes. The glass slides were shielded from light with aluminum foil from further steps. After incubation, the antibody solution was removed, and 200 μl of wash buffer was added to each well and immediately removed. This procedure was repeated once more, and the cassette was removed from the slide. The slide was then placed in a container filled with wash buffer (Coplin staining chamber) and incubated at 60 rpm for 10 minutes. After incubation, the slide was placed in a container filled with ultrapure water and incubated at 60 rpm for 2 minutes. After incubation, the slide was removed and thoroughly dried. After the reaction, the slides were photographed using GlycoLite 2200 to capture fluorescent images, and the fluorescence intensity was quantified using Image Studio Lite Ver 5.2 (LI-COR) to quantify the binding between the glycans and CLA. The results for 100 N-linked glycans are shown in Figure 10, those for 94 O-linked glycans in Figure 11, and those for 58 glycosphingolipid glycans in Figure 12.

[0044] As shown in Figure 10, CLA showed high binding activity to N-linked glycans N004, N005, N014, N015, N024, N025, N034, N035, N044, N045, N054, N055, N114, N115, N124, N125, N134, N135, N144, N155, N214, N215, N224, N225, N234, N244, N255, N6144, N6244, N3004, N015G and N025G. Furthermore, as shown in Figure 11, CLA exhibited high binding activity to O-linked glycans O19, O20, O32, O33, O44, O45, O46, O54, O60, O66, O67, O73, O76, O78, O85, O86, and O90. Furthermore, as shown in Figure 12, CLA exhibited high binding activity to glycosphingolipid glycans 36, 45, and 46.

[0045] As shown in Figures 4 to 9, all of the above sugar chains that showed high binding activity to CLA were Fucα1-3GlcNAc. That is, CLA showed strong binding to all sugar chains containing Fucα1-3GlcNAc. On the other hand, it showed almost no binding activity to sugar chains that did not contain Fucα1-3GlcNAc.

[0046] These results suggest that CLA can specifically bind to the Fucα1-3GlcNAc structure, and therefore, such CLA lectins can be used to detect glycans containing the Fucα1-3GlcNAc structure in samples, suggesting that they can be used to detect cancer cells that express glycans containing the Fucα1-3GlcNAc structure on their cell surface.

Claims

1. A composition for detecting a Fucα1-3GlcNAc structure, comprising CLA lectin.

2. The composition according to claim 1 , wherein the CLA lectin is bound to a labeling molecule.

3. contacting the sample with CLA lectin; Quantifying the amount of CLA lectin bound to the Fucα1-3GlcNAc structure; determining that a Fucα1-3GlcNAc structure is present in the sample when the amount of CLA lectin bound to the Fucα1-3GlcNAc structure is equal to or greater than a predetermined threshold.

4. The method according to claim 3 , wherein the CLA lectin is bound to a labeling molecule.

5. The method of claim 3, wherein the quantifying step comprises contacting the CLA lectin bound to the Fucα1-3GlcNAc structure with a binding molecule that binds to the CLA lectin.

6. The method according to claim 4, wherein the quantifying step includes a step of contacting a binding molecule that binds to the labeled molecule with CLA lectin bound to the Fucα1-3GlcNAc structure.

7. The method according to claim 5 or 6, wherein the step of quantifying comprises a step of quantifying the CLA lectin bound to the Fucα1-3GlcNAc structure by quantifying the binding molecule.

Images