JP6360738B2 - Method for determining the sequence structure of glycosaminoglycan sugar chains - Google Patents

Description

  The present invention relates to a method for determining the sequence structure of glycosaminoglycan sugar chains such as chondroitin sulfate.

  Glycosaminoglycans, which are the main acidic polysaccharides widely distributed in the living body, are chondroitin sulfate / dermatan sulfate and heparan sulfate / heparin. Glycosaminoglycans exert diverse and important physiological functions by binding and interacting with growth factors and cell receptors. Glycosaminoglycans usually have a molecular weight of several thousand to several tens of thousands of Da, and in principle have a linear structure. Glycosaminoglycans basically have a structure in which disaccharides of uronic acid and amino sugar are repeated, and a polysaccharide that has a complex and diverse modified structure with many sulfate groups bonded to the sugar hydroxyl group. It is.

  Chondroitin sulfate, which is one of the major glycosaminoglycans, has a basic skeleton of a repeating disaccharide structure of D-glucuronic acid (GlcA) as uronic acid and N-acetyl-D-galactosamine (GalNAc) as amino sugar. Chondroitin sulfate has a structure in which a large number of sulfate groups are bound in a complex and diverse manner. As shown in FIG. 1, the chondroitin sulfate main disaccharide units include chondroitin disaccharide (O) having no sulfate group, chondroitin sulfate A disaccharide (A) in which the 4-position of GalNAc residue is sulfated, and GalNAc. There is chondroitin sulfate C disaccharide (C) in which the residue 6 is sulfated.

  Physiological functions of glycosaminoglycans are thought to depend on unique and complex sulfated sugar chain sequences present within the linear polysaccharide structure. However, as shown in Non-Patent Document 1, the structure analysis method is a method for analyzing a disaccharide composition after complete decomposition with a degrading enzyme or the like, and determining a short sugar chain sequence formed from several sugars. Only the way to do was known.

Anal. Biochem. (1989) vol. 177, pp 327-332

  However, the method of Non-Patent Document 1 has a problem that the sequence of the active sugar chain cannot be determined. In addition, even when other conventional methods are used, there is a problem in that it is not possible to determine a long sugar chain sequence that particularly exceeds decasaccharide.

  As a result, the physiological function analysis and structure-activity relationship research of polysaccharides were delayed compared to other in vivo macromolecules such as nucleic acids and proteins. If a method for determining the glycosaminoglycan sequence is developed, basic research such as identification of the active structure will be promoted, as well as clinical development such as the development of pharmaceuticals and diagnostics using the active sugar sequence. Application is also promoted.

  The present invention has been made in view of such circumstances, and an object of the present invention is to provide a method for determining the sequence structure of glycosaminoglycan sugar chains.

  In order to solve the above-mentioned problem, a method for determining the sequence structure of a glycosaminoglycan sugar chain according to an aspect of the present invention includes the step of binding a first labeled compound to the reducing end of a glycosaminoglycan sugar chain to label glycosaminoglycan sugar chain. A step of generating a saminoglycan sugar chain, a step of limitedly degrading a labeled glycosaminoglycan sugar chain using a hydrolase, and a limited glycosaminoglycan sugar chain that has been decomposed into two sugars Separating each of the glycosaminoglycan sugar chains having different lengths, and desorbing each of the separated labeled glycosaminoglycan sugar chains into disaccharides using a desorption enzyme; Generating a first labeled disaccharide labeled with the labeled compound of step (a), forming a second labeled disaccharide by binding the reducing end of the unlabeled disaccharide with the second labeled compound, 2 Determining the composition of the disaccharide and the first labeled disaccharide for each glycosaminoglycan sugar chain having a different sugar chain length for each disaccharide, and for each determined glycosaminoglycan sugar chain Determining the sequence of glycosaminoglycan sugar chains based on the composition.

  According to this embodiment, the sequence structure of the glycosaminoglycan sugar chain can be easily determined.

  It is preferable to use different compounds as the first labeling compound and the second labeling compound. According to this aspect, the efficiency of determination of the sugar chain sequence can be increased by facilitating detection.

  In the method for determining the sequence structure of a glycosaminoglycan sugar chain, the glycosaminoglycan sugar chain may be chondroitin sulfate. According to this embodiment, the sugar chain sequence can be determined more easily.

  In the method for determining the sequence structure of a glycosaminoglycan sugar chain, the glycosaminoglycan sugar chain may include a plurality of sugar chains having different sequences. According to this embodiment, the sequence of each sugar chain can be determined collectively.

  According to the present invention, the sequence structure of a glycosaminoglycan sugar chain can be easily determined.

It is a figure which shows the main disaccharide unit structure of chondroitin sulfate. It is a conceptual diagram which shows the sequence structure determination method of the chondroitin sulfate oligo dodecasaccharide concerning this Embodiment. FIG. 2A shows a CSA 12 that is a sample. FIG. 2B is a diagram showing a state in which labeled CSA12 is formed by binding the first labeled compound a (PA) to the reducing end of CSA12. FIG. 2 (C) shows separation of decasaccharide, octasaccharide, and hexasaccharide PA-labeled products that are cleaved by disaccharides from the non-reducing end by detecting the decomposed labeled CSA12 by fluorescence of the modifying group PA. It is a figure which shows a procedure. FIG. 2 (D) is a diagram showing a state in which labeled CSA12 before decomposition is completely decomposed using a desorption enzyme and second labeled compound b (AB) is bound thereto. FIG. 2 (E) is a diagram showing a state in which the limited degradation product decasaccharide (PA-decasaccharide) of labeled CSA12 is completely decomposed using a desorbing enzyme and the second labeled compound b (AB) is bound thereto. It is. FIG. 2 (F) is a diagram showing a state where the limited degradation product octasaccharide (PA-modified octasaccharide) of labeled CSA12 is completely decomposed using a desorbing enzyme and the second labeled compound b (AB) is bound thereto. It is. FIG. 2 (G) is a diagram showing a state in which the limited degradation product hexasaccharide (PA-hexasaccharide) of labeled CSA12 is completely decomposed using a desorbing enzyme and the second labeled compound b (AB) is bound thereto. It is. It is a figure which shows the fluorescence HPLC pattern of AB labeled body of a saturated and unsaturated disaccharide standard.

  We conducted sincere research to establish a method for determining the glycosaminoglycan sugar chain, which has been impossible until now. As a result, the present researchers should make full use of limited hydrolysis of hydrolase, complete decomposition by desorbing enzyme, multiple sugar chain reducing end labeling methods, and technology for separating and quantifying saturated and unsaturated sugars. Thus, a method for sequencing glycosaminoglycan sugar chains was established.

  The present inventors have repeated research to develop a method for determining a sugar chain by utilizing the difference in the structure of a cleaved sugar chain formed by hydrolase and lyase, which are degrading enzymes. . Specifically, a sugar hydrolase is an enzyme that cleaves a glycosidic bond, which is a dehydration bond that connects sugar and sugar, by hydrolysis. A normal saturated sugar chain appears at the site cleaved by the hydrolase. On the other hand, a desorption enzyme (lyase) is a degrading enzyme that does not involve hydrolysis. In the case of glycosaminoglycan sugar chains, the elimination enzyme (lyase) cleaves the glycosidic bond between amino sugar and uronic acid. At that time, the uronic acid residue at the cleavage site undergoes a dehydration reaction between positions 4-5, a double bond is formed, and a degradation product having an unsaturated uronic acid residue at the non-reducing end is generated.

  Here, since sugar chains generally have little visible and ultraviolet absorption, low detection sensitivity is a problem. Therefore, analysis can be performed in a very small amount by binding a labeled molecule that enhances detection sensitivity to the sugar chain reducing end. Furthermore, by labeling the sugar chain reducing end, it is possible to distinguish and separate a reducing end free sugar degradation product generated after enzymatic degradation and a reducing end labeled sugar chain degradation product including the original sugar chain reducing end. In addition, the original long-chain glycosaminoglycan sugar chain and the disaccharide after degradation are labeled with different molecules, and each labeled product can be identified by changing the detection method even for mixed samples of each labeled product. Can be analyzed.

  The present invention provides a method for determining the sequence structure of a glycosaminoglycan sugar chain by combining such a degrading enzyme with a molecular labeling and analysis method.

  Hereinafter, a method for determining the sequence structure of the glycosaminoglycan sugar chain according to the present embodiment will be described in detail.

The method for determining the sequence structure of a glycosaminoglycan sugar chain includes the following steps.
(Step 1) A step of producing a labeled glycosaminoglycan sugar chain by binding the first labeled compound a to the reducing end of the glycosaminoglycan sugar chain.
(Step 2) A step of limitedly decomposing the labeled glycosaminoglycan sugar chain using a hydrolase.
(Step 3) A step of separating the limited glycosaminoglycan sugar chain subjected to limited decomposition into glycosaminoglycan sugar chains each having a different sugar chain length for each disaccharide.
(Step 4) Using the desorption enzyme (lyase), each of the separated labeled glycosaminoglycan sugar chains is decomposed into disaccharides, and the first labeled with the unlabeled disaccharide and the first labeled compound a Producing a labeled disaccharide.
(Step 5) A step of binding the second labeled compound b to the reducing end of the unlabeled disaccharide to form a second labeled disaccharide.
(Step 6) determining the composition of the second labeled disaccharide and the first labeled disaccharide for each glycosaminoglycan sugar chain having a different sugar chain length for each disaccharide.
(Step 7) A step of determining the sequence of the glycosaminoglycan sugar chain based on the determined composition of each glycosaminoglycan sugar chain.

  Hereinafter, these steps will be described in order. As an example, using a chondroitin sulfate A dodecasaccharide (CSA12) prepared by a synthetic enzyme as a model molecule of a glycosaminoglycan sugar chain to be analyzed, the sequence structure of the glycosaminoglycan sugar chain according to the present embodiment A method for determining the value is specifically shown (FIG. 2). CSA12 is composed of six disaccharide units.

(Step 1)
First, the labeled CSA12 is formed by binding the first labeled compound a to the reducing end of the sample CSA12 (FIG. 2A) (FIG. 2B). As the first labeling compound a, a fluorescent label 2-aminopyridine (PA) can be suitably used. Other substances that can be used as the first labeling compound a will be described later together with the second labeling compound b.

(Step 2)
Next, the labeled CSA 12 is limitedly decomposed using a hydrolase. As the hydrolase, hyaluronidase (derived from testicles) that cleaves glycosaminoglycan sugar chains into endo-types can be preferably used. It is preferable to separate the reducing end labeled degradation product and the reducing end free degradation product from the reaction mixture using, for example, reducing sugar adsorption chromatography.

(Step 3)
Next, the limited degradation product of labeled CSA12 is separated into labeled glycosaminoglycan sugar chains each having a different sugar chain length for each disaccharide. That is, a degradation product of a sugar chain in which the reducing end is labeled with the first labeling compound a (reducing end a-labeled degradation product) is separated and purified according to the sugar chain length. Thereby, each of the reducing end a-labeled degradation products in which the sugar chains are excised from the non-reducing end of the sugar chain sample by two sugars can be obtained. In these fractions, each non-reducing end is saturated uronic acid. On the other hand, the reducing end is a sugar chain (a-labeled limited degradation sugar chain) modified with the first labeling compound a and having different sugar chain lengths for each disaccharide.

  Specifically, the degradation product of labeled CSA12 is separated by, for example, gel filtration chromatography and detected by the fluorescence of the modifying group PA (FIG. 2C). As a result, a decasaccharide, an octasaccharide, a hexasaccharide, and, if necessary, a tetrasaccharide PA-labeled product obtained by cleaving disaccharides from the non-reducing end can be separated.

(Step 4) Using a desorption enzyme (lyase), each of the separated glycosaminoglycan sugar chains with different chain lengths is decomposed into disaccharides and labeled with unlabeled disaccharide and the first labeled compound a A first labeled disaccharide is produced. The desorbing enzyme (lyase) is an enzyme that specifically completely desorbs and decomposes a sugar chain that is labeled with the first labeling compound a and limitedly decomposed by a hydrolase. As a result, the first label of the unsaturated disaccharide derived from the non-reducing end saturated disaccharide and the intermediate elimination reaction derived from the respective a-labeled limited degradation sugar chains, and the unsaturated disaccharide derived from the reducing end A labeled product (unsaturated disaccharide a labeled product) with compound a is produced.

  Specifically, the separated and purified PA-decomposed degradation product (PA-decasaccharide, PA-octasaccharide, and PA-hexasaccharide) and the PA label of the dodecasaccharide before degradation are desorbed and degraded (chondroitin lyase C). -Complete decomposition with ACII (hereinafter also simply referred to as ACII) (FIGS. 2D to 2G). Thereby, a saturated disaccharide and an intermediate unsaturated disaccharide at each non-reducing end, and an unsaturated disaccharide PA label derived from the reducing end are obtained.

(Step 5)
Next, the second labeled compound b is bound to the reducing end of the unlabeled disaccharide to form a second labeled disaccharide. That is, a reaction for binding the second labeled compound b to the reducing end of the disaccharide that is the degradation product is performed. As a result, all the disaccharide degradation products other than the reducing end disaccharide already labeled with the first labeling compound a are labeled with the second labeling compound b. As the second labeling compound b, 2-aminobenzamide (AB) can be preferably used.

The first labeling compound a and the second labeling compound b are preferably different in order to facilitate detection, but may be the same compound. When the same compound is used, it is not possible to distinguish between the intermediate sugar chain and the reducing terminal sugar chain, so in order to determine the complete sequence, it is necessary to analyze the tetrasaccharide degradation products in addition to the hexasaccharide or more. . Further, as the first labeling compound a and the second labeling compound b, fluorescent labels such as 2-aminobenzoic acid, 2-aminoacidone, and 1-pyrenebutanoic acid hydride can be used. In addition, the first labeling compound a and the second labeling compound b may be substances other than the fluorescent label as long as they are detectable labels after modification at the reducing end. For example, at least one of the first labeling compound a and the second labeling compound b may be a group containing a radioisotope ( ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹²⁵ I). In addition, a reducing end [ ³ H] labeling with [ ³ H] -NaBH ₄ is also suitable.

(Step 6)
Next, the composition of the second labeled disaccharide to which the second labeled compound b is bound and the first labeled disaccharide labeled with the first labeled compound a are converted into glycosami having different sugar chain lengths for each disaccharide. Determine for each of the noglycan sugar chains. That is, the saturated disaccharide labeled with the second labeling compound b (saturated disaccharide b-labeled product) and the unsaturated disaccharide labeled with the second labeling compound b (unsaturated disaccharide b-labeled product) can be separated. The labeled compound b is detected using an analytical method. This makes it possible to identify and quantify the saturated disaccharide b-labeled product and the intermediate unsaturated disaccharide b-labeled product derived from the non-reducing end of each limited degradation product having a different sugar chain length for each disaccharide. Further, by using a separation method capable of separating the unsaturated disaccharide a-labeled product from the disaccharide degradation product and a method for detecting the first labeling compound a, the unsaturation labeled with the first labeling compound a The structure of the disaccharide can be identified. It is the reducing terminal disaccharide structure of the original glycosaminoglycan sugar chain. By the above method, the entire disaccharide sequence of the glycosaminoglycan sugar chain sample can be determined.

  Specifically, by performing fluorescence high performance liquid chromatography (HPLC) in which the mixture of the decomposed labeled bodies is detected at the fluorescence wavelength of AB by elution using a salt concentration gradient, the position and number of sulfation are determined. AB labels of different saturated and unsaturated disaccharides are clearly separated and detected (FIG. 3). At this time, since the PA-ized disaccharide is not detected because it is measured at the fluorescence excitation wavelength of AB, the analysis of the AB labeled body is not hindered by PA. The reducing end unsaturated PA-disaccharide can be detected by performing chromatography with a concentration gradient of potassium phosphate on an HPLC column packed with aminated silica gel and measuring at the fluorescence excitation wavelength of PA.

(Step 7)
Finally, the sequence of the glycosaminoglycan sugar chain is determined based on the determined composition of each glycosaminoglycan sugar chain. Each non-reducing terminal disaccharide unit is detected and identified as a saturated disaccharide from each degradation product, and what is detected and identified as a PA-labeled disaccharide is a reducing terminal disaccharide. By combining these results, the entire sugar chain sequence structure of the original sulfated chondroitin sulfate dodecasaccharide can be determined.

  That is, a 2n sugar glycosaminoglycan composed of n disaccharide units is prepared by the method of the present embodiment to prepare limited-decomposed a-labeled sugar chains having different sugar chain lengths for each disaccharide. After desorption and complete decomposition, they are labeled with b, their non-reducing end b-labeled saturated disaccharide units are each identified as a single structure, and the reducing end a-labeled unsaturated disaccharide is also identified as a single structure. In this case, the structures of non-reducing terminal b-labeled saturated disaccharide units identified in the long order from the sugar chain before degradation are arranged, and finally the sequence ending with the reducing-terminal a-labeled unsaturated disaccharide unit structure is the original sequence. It is the entire sugar chain sequence of glycosaminoglycan of 2n sugar.

  The labor of this step depends on the degree of sulfation of glycosaminoglycan sugar chains and how many kinds of sugar chains are mixed in the sample. Considering the labor of this step, when a plurality of sequences having different sequences are included in the sample, as shown in Examples 1 and 2 described later, their lengths and the degree of sulfation are the same. It is preferable. It is preferable that the degree of sulfation at least from the reducing end to the predetermined length is the same.

  For example, when there are X specific sulfate group-modified disaccharide structures S in a sample having a 2n sugar chain length, among the sugar chain sequences identified by the method of the present embodiment, Na, Nb ,..., Suppose that the Nm-th (m <n) disaccharide structure is a mixture of unsulfated disaccharide O and sulfated structure S. In addition, when the ratio of each S structure is Sa, Sb,..., Sm and the disaccharide structure at other positions can be determined to be only O or S, the number of determined S is all It is assumed that this is a case where (X-1) is smaller than the number X of the S structure. At this time, the sugar chain in the sample is composed of m kinds of sugar chains in which any one of sugar chain positions Na, Nb,. The abundance ratio of each sugar chain is Sa, Sb,..., Sm (provided that Sa + Sb +... + Sm = 1). Further, when n sugar chain sequences having n candidate sites for sulfation are contained in the sample, the ratio of these sugar chain sequences contained in the sample is calculated by solving the n-ary linear equation. be able to. Further, when the number of candidate sites for sulfation is different from the number of sugar chain sequences, it can be calculated in the same manner.

  On the other hand, if the degree of sulfation differs between multiple sugar chain sequences contained in the sample, the same degree of sulfation can be achieved using ion exchange chromatography or gel filtration chromatography as shown in the Examples. It is preferable to carry out the sugar chain sequencing method of this step after separating the sugar chains.

(Target)
There is no limitation on the sugar chain length of the glycosaminoglycan sugar chain sample to be analyzed as long as it is possible to separate limited degradation products having different lengths for each disaccharide. In view of the ability to separate two sugar chains having different lengths in the current gel filtration chromatography, the sugar chain length is preferably about 6 to about 30 sugars, and about 12 sugars to about 20 sugars. More preferably. The sugar chain does not need to be determined as a full-length sequence. For example, only a partial sequence may be determined from the reducing end side. Alternatively, only a part of the sequence may be determined from the non-reducing terminal side.

Alternatively, for a sugar chain X that is so long that the length cannot be determined at once (cannot be separated by gel filtration chromatography), the reducing end side labeled with the first labeling compound a after the operation of FIG. separating the non-reducing terminal of the sugar chains which are not sugar X ₁ and labeled. Next, the non-reducing end of the sugar chains which are not labeled, by performing the operation of FIG. 2 (A) ~ (B) , obtaining a sugar chain X ₂ in the reducing end side. By repeating this operation according to the length of the original sugar chain, a sugar chain _Xn including the non-reducing end of the original sugar chain is obtained. The sequences of these sugar chains X _{1 to} X _n are determined by the operations shown in FIGS. 2 (C) to (G), respectively, and finally these sequences are connected to determine the original sugar chain X sequence. May be. In this case, multiple types of sugar chain sequences with different sequences may be included.

  When a sample includes a plurality of sugar chain sequences, the sugar chain length of the glycosaminoglycan sugar chain sample is not necessarily single. This is because the reducing end of the sample is labeled, so that the sequence structure based on the reducing end can be determined. That is, in the case of a sugar chain sample having a reduced end as a sequence, the sequence structure from the reducing end side of samples having different chain lengths can be determined. Further, even if the sugar chain length of the sample is not uniform, the non-reducing terminal disaccharide of the sample can be identified by the method of the present embodiment.

  Furthermore, as described above, the sample may not be a completely uniform molecule, that is, a mixture in which a plurality of sugar chains having different sequences and lengths are mixed. Even in this case, it is possible to determine the proportion of different disaccharide units present at a specific sugar chain sequence position, so multiple sugar chain sequences contained in the sample are identified and their quantitative ratio is also calculated. be able to.

  The method of the present embodiment is applicable to all glycosaminoglycan sugar chains. As glycosaminoglycans other than chondroitin sulfate, it can be applied to dermatan sulfate, heparan sulfate / heparin sugar chain, hyaluronic acid and the like.

  In the case of determining the sequence of dermatan sulfate, chondroitin lyase ABC or chondroitin lyase B is used in combination as the degrading enzyme in the chondroitin sulfate analysis method. When determining the sequence of heparan sulfate / heparin, in the analysis method of chondroitin sulfate, a hydrolase (heparanase) that specifically reacts with heparan sulfate / heparin and a degrading enzyme (heparitinase, heparitin lyase) And combine. When the sequence of hyaluronic acid is determined, an animal-derived hydrolase hyaluronidase and a desorption enzyme hyaluronic acid lyase (microorganism-derived hyaluronidase) are combined. In general, hyaluronic acid is not modified with a sulfate group, so the significance of sequencing is low, but it can be applied to the analysis of hyaluronic acid that has undergone side chain modification after artificial or biosynthesis.

  As described above, when the method for determining the sequence structure of glycosaminoglycan sugar chains according to the present embodiment is used, the sequence of glycosamine sugar chains that interact with physiologically active molecules such as growth factors and cytokines and exert physiological functions can be obtained. By examining this, it is possible to determine a sequence structure that specifically binds to a physiologically active molecule. In addition, structures of binding sequences with bacteria and viruses that infect via glycosaminoglycan sugar chains, and sugar chain sequences that interact with cell surfaces such as neurons and immune cells that have glycosaminoglycan-specific receptors The structure can be determined. In addition, by determining the sequence structure of the sugar chain prepared by artificially acting on glycosaminoglycan synthase such as sulfate transferase, the position and priority of enzyme reaction such as sulfate transferase should be clearly indicated. Can lead to the elucidation of the biosynthetic mechanism.

(Usefulness)
Glycosaminoglycan sugar chains such as chondroitin sulfate exhibit strong binding properties to many physiologically active molecules such as growth factors and cell receptors, and exhibit diverse and important physiological functions. As these physiological functions, for example, a cancer suppressing action, a central nerve regeneration controlling action, an immune cell activating action, a virus infection protecting action and the like are known. Glycosaminoglycan sugar chains are expected to be clinically applied as pharmaceuticals or diagnostics. However, since there was no conventional method for analyzing the sugar chain sequence structure of the active structure, the development of active sugar chains linked to clinical application did not progress. By determining the sequence structure of the glycosaminoglycan sugar chain exhibiting the above physiological function using the method according to the present embodiment, it is possible to link to clinical applications such as development of pharmaceuticals and diagnostic agents.

(Synthesis of chondroitin sulfate A dodecasaccharide)
Chondroitin dodecasaccharide (CH-12) synthesized using the method of Sugiura et al. (Glycoconj. J. (2008) vol. 25, pp521-530) Biol. Chem. (2012) vol. 287, pp 43390-43400, an enzyme-synthesized chondroitin sulfate A dodecasaccharide (CSA12) was synthesized using recombinant chondroitin 4-sulfotransferase-1 (C4ST-1). This sample has a structure in which a sulfate group is modified at the 4-position hydroxyl group of six GalNAc residues, and is a mixture of sugar chains having different numbers of modified sulfate groups and modified positions in the sugar chain sequence. This sample was applied to an ion exchange resin column (mono Q HR 5/10, manufactured by GE Healthcare) and changed from 50 mM sodium acetate buffer (pH 4.0) to the same buffer containing 0.5 M sodium chloride. Was eluted by linear gradient chromatography. Then, the sample CSA12 was separated into a plurality of fractions, and the main fractions were designated CSA12-a and CSA12-b in order from the elution of the low salt concentration. From the results of disaccharide composition analysis, the number of sulfate group modifications in each fraction was estimated to be 1 or 2 per 12 sugars.

[Example 1]
(Reductive end fluorescent labeling of chondroitin sulfate A dodecasaccharide (PA))
0.025 mg / μL of 2-aminopyridine containing an enzyme-synthesized chondroitin sulfate A dodecasaccharide fraction (CSA12-a, 9.4 nmol) in which one sulfate group is bonded in the molecule separated by the ion exchange chromatography (PA) and 0.024 mg / μL of sodium cyanoborohydride (NaBH ₃ CN) in 30% acetic acid-dimethylsulfoxide (DMSO) solution (5 μL) were added and dissolved, followed by heating at 60 ° C. for 2 hours. After cooling the reaction solution to room temperature, it was applied to a Superdex Peptide HR 10/300 column (manufactured by GE Healthcare) equilibrated with 0.2 M aqueous ammonium acetate solution, and subjected to gel filtration chromatography. The eluate is detected at an excitation wavelength (Ex) of 310 nm and an emission wavelength (Em) of 370 nm, and the fractions are collected to obtain a reducing end fluorescently labeled chondroitin sulfate dodecasaccharide fraction a (PA-CSA12-a). It was.

(Limited degradation of PA chondroitin sulfate A dodecasaccharide by hydrolase)
The PA chondroitin sulfate A dodecasaccharide fraction a (PA-CSA12-a) was dissolved in 20 μL of 50 mM sodium phosphate buffer (pH 6.8) containing 150 mM sodium chloride, and sheep testicle-derived hyaluronidase (manufactured by Sigma) was dissolved. In addition, it was incubated at 37 ° C. for 30 minutes. After heat treatment at 100 ° C. for 1 minute, the mixture was cooled to room temperature, the reaction solution was passed through a BlotGlyco column (manufactured by Sumitomo Bakelite Co., Ltd.), and 200 μL of water was passed twice through the column to collect PA sugar. At this time, the free sugar chain at the reducing end produced by enzymatic degradation is adsorbed to the column. The passing solution and the washing solution were collected, applied to a Superdex 30HR 16/600 column (manufactured by GE Healthcare), and eluted with a 0.2 M aqueous ammonium acetate solution. The eluate was detected at a fluorescence wavelength of Ex 310 nm and Em 370 nM, and fractionated PA oligosaccharides (decasaccharide, octasaccharide, hexasaccharide) were fractionated.

(Degradation of PA-oligosaccharides by desorbing enzyme and fluorescent labeling (AB conversion))
Each PA oligosaccharide (decasaccharide, octasaccharide, hexasaccharide) and pre-degradation PA dodecasaccharide fraction a (PA-CSA12-a) (each 50 to 200 pmol) are each a chondroitin lyase that is a desorption enzyme. (Chondroitinase) ACII (manufactured by Seikagaku Corporation) was dissolved in 50 μL of 50 mM sodium acetate buffer (pH 6.0) containing 10 mU and reacted at 37 ° C. for 1 hour. The reaction solution was concentrated under reduced pressure, and 2-aminobenzamide (AB) and a 30% acetic acid-DMSO solution (5 μL) of NaBH ₃ CN were added and dissolved, followed by reaction at 60 ° C. for 2 hours. 200 μL of water was added to the reaction solution, and extraction was performed 4 times with ethyl acetate (200 μL) to remove excess reaction reagent. The aqueous layer was concentrated under reduced pressure and lyophilized, and redissolved with 50 μL of water. The redissolved solution contains non-reducing terminal AB-saturated disaccharides, intermediate AB-saturated disaccharides, and reducing-terminal PA-unsaturated disaccharides derived from the respective limited degradation oligosaccharide fractions.

  Using the Senshu PAK DC-1151 column (4.6 × 150 mm, manufactured by Senshu Kagaku Co., Ltd.), the obtained redissolved solution was 1.2 mM tetra-n-butylammonium hydrogen sulfate (TBAS), 8.5%. Separation by HPLC system with acetonitrile containing 2 mM to 140 mM NaCl gradient. The HPLC eluate was detected at a fluorescence wavelength of Ex 330 nm and Em 420 nm to detect a disaccharide degradation product AB label. Separately, saturated and unsaturated disaccharide AB fluorescent labels, saturated chondroitin disaccharide-AB (sO-AB), unsaturated chondroitin disaccharide-AB (ΔO-AB), saturated chondroitin sulfate A disaccharide-AB (sA-AB) ), Unsaturated chondroitin sulfate A disaccharide-AB (ΔA-AB), saturated chondroitin sulfate C disaccharide-AB (sC-AB), and unsaturated chondroitin sulfate C disaccharide-AB (ΔC-AB) (each 20 pmol) Was analyzed using the same HPLC system as a standard product (FIG. 3), and the AB-labeled disaccharide component of this product was fractionally determined based on the elution time and fluorescence intensity of the standard product. The results are shown in Table 1. PA-CSA10-a, PA-CSA8-a, and PA-CSA6-a represent the PA-ized decasaccharide fraction a, the PA-octasaccharide fraction a, and the PA-hexasaccharide fraction a after degradation, respectively.

  As explained using FIG. 2, the reducing terminal disaccharide of each limited degradation oligosaccharide is PA-ized. For this reason, PA-modified reducing terminal disaccharides were not detected by the difference in fluorescence wavelength in the analysis of AB-ized disaccharides. As described with reference to FIG. 2, the saturated disaccharide detected in the analysis of the AB-ized disaccharide corresponds to the non-reducing terminal disaccharide of each limited degradation oligosaccharide. The detected unsaturated disaccharide is an intermediate disaccharide unit, which corresponds to 4 for the dodecasaccharide, 3 for the decasaccharide, 2 for the octasaccharide, and 1 for the hexasaccharide. Based on the number of saturated sugars and unsaturated sugars that can be derived from the principle of FIG. 2, the composition ratio of saturated / unsaturated disaccharides shown in Table 1 was divided into saturated sugars and unsaturated sugars and converted to the number of analytical sugar units. Values are shown in Table 2.

  For detection of PA-unsaturated disaccharide at the reducing end, a lyase enzyme degradation product was used with a PolyAmine column (4.6 × 250 mm, manufactured by YMC) or a UK amino column (4.6 × 250 mm, manufactured by Intact). In addition, the eluate was measured at a fluorescence wavelength of Ex 310 nm and Em 370 nm with an HPLC system based on a concentration gradient of 40 mM to 800 mM potassium dihydrogen phosphate aqueous solution containing 1 mM TBAS. As a result of the measurement, the reducing end disaccharide was all PA-unsaturated chondroitin disaccharide (ΔO-PA) that was not sulfated. This chondroitin sulfate fraction CSA12-a has one sulfate group modified in the dodecasaccharide. From the above data, assuming that the disaccharide unit of the dodecasaccharide is I, II, III, IV, V, VI from the non-reducing end, the respective ratios can be estimated as shown in Table 3.

Therefore, in the sugar chain sequence of CSA12-a, which is the chondroitin sulfate dodecasaccharide fraction, any of the third to fourth disaccharide units from the non-reducing end is sulfated as follows: It was found to be composed of one kind of monosulfated dodecasaccharide. Here, chondroitin disaccharide having no sulfate group is represented by O, and chondroitin sulfate A disaccharide in which the 4-position of the GalNAc residue is sulfated is represented by A.
I II III IV V VI
Glycan sequence 1: O—O—A—O—O—O
Glycan sequence 2: O—O—O—A—O—O

  From Table 3, the abundance ratio of the sugar chain sequences 1 and 2 is 0.47: 0.36, that is, the CSA12-a contains about 57 of the sugar chain sequence 1: O—O—A—O—O—O. %, Sugar chain sequence 2: it was calculated that about 43% of O—O—O—A—O—O was contained.

[Example 2]
(Reductive end fluorescent labeling of chondroitin sulfate A dodecasaccharide (PA))
For the fraction b (CSA12-b) of the enzyme-synthesized chondroitin sulfate A dodecasaccharide in which two sulfate groups exist in the molecule separated by the ion exchange chromatography of Example 1, the reducing end was obtained in the same manner as in Example 1. PA labeling was performed. Subsequently, gel filtration chromatography was performed using a Superdex Peptide HR 10/300 column to obtain a reducing end fluorescently labeled (PA-ized) chondroitin sulfate dodecasaccharide fraction b (PA-CSA12-b).

(Limited degradation of PA chondroitin sulfate A dodecasaccharide by hydrolase)
The obtained PA-ized chondroitin sulfate A dodecasaccharide fraction b (PA-CSA12-b) was subjected to limited digestion with sheep testicle-derived hyaluronidase in the same manner as in Example 1, and the PA-ized sugar was recovered using a BlotGlyco column. Furthermore, the partially decomposed oligosaccharide (decasaccharide, octasaccharide, hexasaccharide) of PA-ized chondroitin sulfate A dodecasaccharide fraction b was fractionated using a Superdex 30 HR 16/600 column.

(Degradation of PA-oligosaccharides by desorbing enzyme and fluorescent labeling (AB conversion))
Each PA oligosaccharide and PA dodecasaccharide (PA-CSA12-b) before decomposition were decomposed with chondroitin lyase ACII in the same manner as in Example 1, and then AB labeling reaction was performed. The obtained sample was subjected to separation and quantification of AB fluorescent labels of saturated and unsaturated disaccharides using a Senshu PAK DC-1151 column. The results are shown in Table 4.

  Saturated sugar corresponding to 1 unit of non-reducing terminal disaccharide of each oligosaccharide chain, disaccharide in 4 sugar chains, 3 sugars, 2 sugars, 2 sugars and 1 sugar Table 5 shows the converted sugar chain values of unsaturated sugar corresponding to the unit.

  The PA-unsaturated disaccharide at the reducing end was a PA-unsaturated chondroitin disaccharide (ΔO-PA) that was not 100% sulfated. From the above results, assuming that the disaccharide unit of the chondroitin sulfate fraction CSA12-b is I, II, III, IV, V, VI from the non-reducing end, the ratio of the disaccharide unit composition is estimated as shown in Table 6, respectively. it can.

Accordingly, the chondroitin sulfate dodecasaccharide fraction CSA12-b has three types of sequence structures in which any two sugars at positions II, III, and IV are sulfated as shown below. It was found to be composed of dodecasaccharide (α, β, γ).
I II III IV V VI
Sugar chain sequence α: O-A-A-O-O-O
Glycan sequence β: O-A-O-A-O-O
Glycan sequence γ: O—O—A—A—O—O

From Table 6, since the ratio that II is CSA is 0.45, the ratio that III is CSA is 0.60, and the ratio that IV is CSA is 0.57, the following formulas (1) to (3 ) Holds.
(Α + β) ÷ 2 = 0.45 (1)
(Α + γ) ÷ 2 = 0.60 (2)
(Β + γ) ÷ 2 = 0.57 (3)
From these equations, values of α, β, and γ are obtained. First, the following equations (4) and (5) are obtained by modifying equations (1) and (2).
β = 0.90−α (4)
γ = 1.20−α (5)
When equations (4) and (5) are introduced into equation (3), α is calculated as follows.
α = (0.90 + 1.20-1.14) ÷ 2 = 0.48
Further, β and γ are calculated from the equations (4) and (5) as follows.
β = 0.90-0.48 = 0.42
γ = 1.20−0.48 = 0.72

  Therefore, the abundance ratio (α: β: γ) of the three constituent sugars of CSA12-b is 0.48: 0.42: 0.72, that is, the sugar chain sequence α: O-A-A-O-O. -O is about 30%, sugar chain sequence [beta]: O-A-O-A-O-O is about 26%, sugar chain sequence [gamma]: O-O-A-A-O-O is about 44%. And calculated.

  The present invention has been described with reference to the above-described embodiment and each evaluation test. However, the present invention is not limited to the above-described embodiment and each evaluation test, and the configurations of the embodiments are appropriately combined. Those that have been replaced or replaced are also included in the present invention. In addition, it is possible to appropriately change the combinations and the order of steps in the embodiments based on the knowledge of those skilled in the art and to add various modifications such as various design changes to the embodiments. The described embodiments can also be included in the scope of the present invention.

Claims (4)

Binding a first labeled compound to the reducing end of the glycosaminoglycan sugar chain to produce a labeled glycosaminoglycan sugar chain;
Limited hydrolysis of the labeled glycosaminoglycan sugar chain using a hydrolase;
Separating the labeled glycosaminoglycan sugar chain that has been limitedly decomposed into each glycosaminoglycan sugar chain having a different sugar chain length for each disaccharide;
Using a desorption enzyme, each of the separated labeled glycosaminoglycan sugar chains is decomposed into disaccharides to produce a first labeled disaccharide labeled with an unlabeled disaccharide and the first labeled compound. And steps to
Attaching a reducing end of the unlabeled disaccharide to a second labeled compound to form a second labeled disaccharide;
Determining the composition of the second labeled disaccharide and the first labeled disaccharide for each glycosaminoglycan sugar chain having a different sugar chain length for each disaccharide;
Determining the sequence of the glycosaminoglycan sugar chain based on the determined composition of each glycosaminoglycan sugar chain, and determining the sequence structure of the glycosaminoglycan sugar chain.   The method for determining a sequence structure of glycosaminoglycan sugar chains according to claim 1, wherein different compounds are used as the first labeling compound and the second labeling compound.   The method for determining the sequence structure of a glycosaminoglycan sugar chain according to claim 1 or 2, wherein the glycosaminoglycan sugar chain is chondroitin sulfate.   The method for determining the sequence structure of a glycosaminoglycan sugar chain according to any one of claims 1 to 3, wherein the glycosaminoglycan sugar chain includes a plurality of sugar chains having different sequences.

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