JP7744004B2 - Compositions and methods for detecting Fucα1-3GlcNAc structures - Google Patents

Description

The present invention relates to a composition and method for detecting Fucα1-3GlcNAc structures, in which fucose is α1,3-linked to N-acetylglucosamine, among glycan structures, and in particular to a composition and method for detecting Fucα1-3GlcNAc structures using lectins.

Sugar chains of glycoconjugates such as glycoproteins and glycolipids present on cell surfaces and in body fluids function as a type of information device 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-recognition molecules and, like glycans, play important biological roles. It is known that different lectins recognize different glycans depending on the type. For example, Patent Document 1 discloses Hypnin A, isolated from the red alga Hypnea japonica, as a lectin that strongly interacts with fucose (α1,6-linked to N-acetylglucosamine at the reducing end of N-linked glycans (asparagine-linked glycans). Patent Document 1 also discloses a method for detecting α1,6-fucose glycans using Hypnin A.

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 could also be used to detect viruses, etc. (See Patent Documents 2 and 3, etc.).

Patent No. 5109001 Japanese Patent Application Laid-Open No. 2020-117445 Japanese Patent Application Laid-Open No. 2021-013375

However, at present, it cannot be said that sufficient substances are 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.

The present invention was made in consideration of the above problems, and its purpose is to enable the specific detection of specific glycan structures using lectins.

To achieve the above-mentioned objective, the inventors conducted extensive research and discovered that CLA lectin isolated from the green alga Codium latum specifically recognizes the Fucα1-3GlcNAc structure, a glycan structure in which fucose is α1,3-linked to N-acetylglucosamine, thereby completing the present invention.

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

In the composition of the present invention, a labeling molecule may be bound to the CLA lectin. In this case, for example, to quantify or detect CLA lectin bound to a Fucα1-3GlcNAc structure, it becomes possible to use a binding molecule that recognizes and binds to the labeling molecule, allowing for convenient quantification and detection.

A method for detecting Fucα1-3GlcNAc structures in a sample according to the present invention comprises the steps of contacting a sample with CLA lectin, quantifying the amount of CLA lectin bound to the Fucα1-3GlcNAc structures, and determining that a Fucα1-3GlcNAc structure is present 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. Therefore, according to the method according to the present invention, Fucα1-3GlcNAc structures in a sample can be detected by contacting a sample with CLA lectin and evaluating the amount or presence of CLA lectin binding to Fucα1-3GlcNAc structures in the sample.

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

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 labeling molecule. Furthermore, in the method of the present invention, the quantification step can include quantifying the CLA lectin bound to the Fucα1-3GlcNAc structure by quantifying the binding molecule.

The composition and method for detecting Fucα1-3GlcNAc structures are extremely useful because they enable specific detection of Fucα1-3GlcNAc structures among glycan structures.

FIG. 1 shows Lewis X structures and sialyl Lewis X structures as sugar chain structures. FIG. 1 shows the results of an analysis of sugar chain binding specificity of CLA by centrifugal ultrafiltration-HPLC in an example. FIG. 1 shows the results of an analysis of sugar chain binding specificity of CLA by centrifugal ultrafiltration-HPLC in an example. FIG. 1 shows N-linked glycans used in the glycan array in the Examples. FIG. 1 shows N-linked glycans used in the glycan array in the Examples. FIG. 1 shows N-linked glycans used in the glycan array in the Examples. FIG. 1 shows O-linked glycans used in the glycan array in the Examples. FIG. 1 shows O-linked glycans used in the glycan array in the Examples. FIG. 1 shows glycosphingolipid sugar chains used in the glycan array in the Examples. 1 is a graph showing the results of a glycan array targeting N-linked glycans in an example. 1 is a graph showing the results of a glycan array targeting O-linked glycans in an example. 1 is a graph showing the results of a glycan array targeting glycosphingolipid sugar chains in an example.

The following describes an embodiment of the present invention with reference to the drawings. The following description of the preferred embodiment is merely exemplary in nature and is not intended to limit the present invention, its application, or its uses.

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 recently 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 that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1, and more preferably consists of an amino acid sequence that is 95%, 96%, 97%, 98%, or 99% or more identical. Most preferably, CLA consists of the amino acid sequence of SEQ ID NO: 1.

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).

In this embodiment, "glycan" refers to a linear or branched oligosaccharide or polysaccharide. Furthermore, the above-mentioned glycans are broadly classified according to the mode of binding to proteins into N-glycoside-linked glycans (hereinafter referred to as "N-glycans" or "N-glycans") that are linked to asparagine, and O-glycoside-linked glycans (hereinafter referred to as "O-glycans" or "O-glycans") that are linked to serine, threonine, etc., and N-glycans include high-mannose glycans, complex glycans, and hybrid glycans.

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

Monosaccharides are further broadly divided into three 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. Among the monosaccharides present in the body, acidic sugars include sialic acids such as N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (Neu5Gc) and uronic acid; amino sugars include N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc); and neutral sugars include glucose (Glc), mannose (Man), galactose (Gal), and fucose (Fuc).

All N-glycans share a common core structure called the "trimannosyl core" consisting of Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc. High-mannose glycans contain only α-mannose residues in the branched structure in addition to the trimannosyl core. These glycans share a common core structure consisting of 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. As with high-mannose glycans, one or two α-mannosyl groups are attached to the Manα1-6 arm of the trimannosyl core, and the same side chains found in complex glycans are attached to the Manα1-3 arm of the core.

The presence or absence of fucose attached to the C-6 position of the GlcNAc at the reducing end of the trimannosyl core, and the presence or absence of β-GlcNAc attached to the C-4 position of the β-mannosyl residue (called 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.

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

The Fucα1-3GlcNAc structure to which the CLA of this embodiment specifically binds is a glycan structure in which Fuc, in particular, is α1,3-linked to GlcNAc. 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 β1-4-linked to GlcNAc that is α1,3-linked to Fuc, and sialyl Lewis X is a structure in which NeuAc is α1-3-linked to the Gal. The boxed area in Figure 1 is 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 this embodiment can specifically bind to the above-mentioned cancer cells and may be applicable to detecting these cancer cells.

Whether or not CLA binds to glycans can be assessed, for example, by passing the test CLA through a column onto which the target glycan or glycoprotein bound to the glycan has been immobilized, and determining whether or not CLA has bound to the column by 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 it on a membrane or the like, and detecting it with CLA labeled with biotin, fluorescein isothiocyanate, peroxidase, or the like, using the Western blot method (see Forensic Medicine Practice and Research, 37, 155, 1994) or the dot blot method (see Analytical Biochemistry, 204(1), 198, 1992).

Alternatively, the affinity between a chip on which a target sugar chain or a sugar chain-bound glycoprotein or the like is immobilized and the test subject CLA 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. If the binding constant (affinity constant) (K _A ) obtained in this manner is 10 (M ⁻¹ ) or more, more preferably 10 ³ (M ⁻¹ ) or more, and most preferably 10 ⁴ (M ⁻¹ ) or more, it can be determined that CLA and sugar chain are bound.

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, but examples include fluorescent proteins, luciferase, biotin, avidin, His tag peptides, GST tag peptides, and FLAG tag peptides. Binding such target molecules to the CLA advantageously allows for easy evaluation of the presence or absence of CLA.

The composition according to this embodiment contains CLA and a solvent, and may also contain additives, for example, to improve the stability of the CLA in the solvent.

Another embodiment of the present invention is a method for detecting Fucα1-3GlcNAc structures in a sample. The method includes the steps of contacting a sample with CLA, quantifying the amount of CLA bound to the Fucα1-3GlcNAc structures, and determining that a Fucα1-3GlcNAc structure is present in the sample if the amount of CLA bound to the Fucα1-3GlcNAc structures is equal to or greater than a predetermined threshold.

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 a body fluid, cell, or tissue.

In this embodiment, the step of contacting the sample with CLA is carried out by, for example, adding and mixing a solution containing CLA with the above-mentioned solution or body fluid as the sample. When using tissue as the sample, a portion of the tissue removed from the body may be used as the sample. Alternatively, 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.

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. Alternatively, a conventional method used in this technical field using an antibody or binding molecule that specifically binds to CLA or a labeling molecule that binds to it can be used. For example, but not limited to, if a tag peptide is bound to CLA as a labeling molecule, 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. This method can also be used to detect and quantify the Fucα1-3GlcNAc structure.

In this embodiment, a threshold value (reference value) is set in advance in the step of determining the presence of a Fucα1-3GlcNAc structure in a sample. This reference value is not limited to the following, but for example, a sample that does not contain a Fucα1-3GlcNAc structure can be used as a reference, and the quantitative value of CLA obtained from this reference in a step similar to the step of quantifying CLA described above 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 a Fucα1-3GlcNAc structure is present in the sample.

As described above, the method according to this embodiment makes it possible to easily detect glycans containing Fucα1-3GlcNAc structures in a sample by utilizing CLA that can specifically bind to Fucα1-3GlcNAc structures. Therefore, this method may be applicable to detecting cancer cells that express glycans containing Fucα1-3GlcNAc structures on their cell surface.

The following examples provide a detailed explanation of the compositions and methods for detecting Fucα1-3GlcNAc structures according to the present invention.

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

(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 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 ultrasonic disruption buffer (20 mM phosphate buffer (PB) (pH 7.4), 500 mM NaCl, 20 mM imidazole) was added to the culture medium and suspended, followed by ultrasonic disruption while cooling on ice. The ultrasonic disruption conditions were a set of "1 minute of ultrasonic disruption - 1 minute of rest" repeated 7 times. After the disruption treatment, the cells were centrifuged (10,000×g, 4°C, 20 min), and the supernatant was used as the soluble fraction.

(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 applied to 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 the purified His-tagged recombinant CLA as CLA.

[Example 2: Glycosylation specificity of CLA]
(Analysis of glycan binding specificity by centrifugal ultrafiltration-HPLC method)
The glycan-binding specificity of CLA was measured by 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 glycans to the amount of added glycans, and the amount of bound glycans is calculated by subtracting the amount of unbound glycans from the amount of added glycans. 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.

First, 90 μL of 5 μM His-tagged recombinant CLA and 10 μL of 300 nM pyridylaminated (PA)-oligosaccharides (Takara Bio) were mixed with 50 mM Tris-HCl (pH 7.0) and incubated at room temperature for 1 hour. The mixture was then centrifuged at 10,000 × g for 30 seconds at room temperature using a centrifugal ultrafilter (molecular weight cutoff: 10 kDa, 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. This HPLC (Waters Alliance HPLC system (Waters, Alliance e2695 Separations Module)) was performed at a flow rate of 1 mL/min at 40°C. The eluate was monitored at an excitation wavelength of 320 nm and an emission wavelength of 400 nm, and the sugar chain peaks were analyzed and quantified using Empower3 (Waters). Separately, 90 μL of 50 mM Tris-HCl (pH 7.0) not containing His-tag fused recombinant CLA was mixed with the above PA-oligosaccharide, and the mixture was subjected to the above centrifugal ultrafiltration, and the filtrate was used as a blank. The amount of PA-oligosaccharide bound to CLA [O _bound ] was obtained by subtracting the amount of unbound PA-oligosaccharide [O _unbound ] from the amount of PA-oligosaccharide added [O _added ]. The ratio of [O _bound ] to [O _added ] was defined as the binding activity (%). The 32 PA-oligosaccharides used and their binding activities are shown in Figures 2 and 3.

As shown in Figures 2 and 3, the glycans that showed high 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, and glycans with this structure include Lewis X antigen and sialyl Lewis X antigen. Furthermore, they showed no binding to Fucα1-2Gal found in Nos. 20, 28, 30, 31, and 32, Fucα1-4GlcNAc found in Nos. 21, 28, and 30, or Fucα1-6GlcNAc found in the high-mannose glycan No. 14. However, they showed extremely weak binding activity of approximately 10% to Fucα1-6GlcNAc found in the complex-type glycans Nos. 4, 6, and 9. This indicates that CLA binds strongly to fucose-containing glycans, particularly to 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.

(Glycan binding specificity analysis using glycan arrays)
Furthermore, a glycan array was performed to evaluate the binding specificity of CLA for 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 test. The principle is as follows: first, biomolecules are added to a glass slide on which multiple types of glycans have been spotted, and the biomolecules are allowed 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 the biomolecules. In this example, the binding activity of CLA with 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, the following glycan-immobilized microarray slides were used: N-Glycan Array (8-subarray slide), a glycan-immobilized microarray slide from Z Biotech, which is equipped with 100 types of N-linked glycans shown in Figures 4 to 6; O-Glycan Array, a glycan-immobilized microarray slide from Z Biotech, which is equipped with 94 types of O-linked glycans shown in Figures 7 and 8; and Glycosphingolipid-Glycan Array, a glycan-immobilized microarray slide from Z Biotech, which is equipped with 58 types of glycosphingolipid glycans shown in Figure 9. The methods and results are described below.

First, the slide was mounted in the provided cassette, and 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 slides 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 then covered with adhesive film, and the slides were 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 at 85 rpm for 5 minutes. 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 slide was shielded from light with aluminum foil from this point onward. 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, the cassette was removed from the slide, and the slide was placed in a container filled with wash buffer (Coplin staining tank) 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, a fluorescent image of the slide was captured using a GlycoLite 2200, and the fluorescence intensity was quantified using Image Studio Lite Ver. 5.2 (LI-COR), and the binding affinity between the glycan and CLA was quantified. The results for 100 types of N-linked glycans are shown in Figure 10, those for 94 types of O-linked glycans are shown in Figure 11, and those for 58 types of glycosphingolipid glycans are shown in Figure 12.

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 O19, O20, O32, O33, O44, O45, O46, O54, O60, O66, O67, O73, O76, O78, O85, O86, and O90 in O-linked glycans. Furthermore, as shown in Figure 12, CLA exhibited high binding activity to 36, 45, and 46 in glycosphingolipid glycans.

As shown in Figures 4 to 9, all of the above glycans that showed high binding activity with CLA were Fucα1-3GlcNAc. In other words, CLA showed strong binding to all glycans containing Fucα1-3GlcNAc. On the other hand, it showed almost no binding activity to glycans that did not contain Fucα1-3GlcNAc.

From the above, it is believed that CLA can specifically bind to the Fucα1-3GlcNAc structure, and therefore such a CLA lectin can be used to detect glycans containing the Fucα1-3GlcNAc structure in a sample. This suggests that it can be used to detect cancer cells that express glycans containing the Fucα1-3GlcNAc structure on their cell surface.

Claims (7)

A composition for detecting Fucα1-3GlcNAc structures, characterized by containing CLA lectin. The composition of claim 1, wherein the CLA lectin is bound to a labeling molecule. 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. The method of claim 3, wherein the CLA lectin is bound to a labeling molecule. 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. The method according to claim 4, wherein the quantifying step comprises a step of contacting a binding molecule that binds to the labeled molecule with the CLA lectin bound to the Fucα1-3GlcNAc structure. 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.