JP2011137754A - Method for determining prostate cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting a sugar chain of a prostate specific antigen (PSA) of a subject affected with a prostate cancer (PC) and accurately determining the PC, based on the sugar chain structure of the PSA. <P>SOLUTION: The method for determining the prostate cancer includes the steps of analyzing the sugar chain structure of the PSA, in a sample deriving from the subject and determining the prostate cancer, when there are three or more sugar chains having LacdiNAc (N-acetyl galactosamine-N-acetyl glucosamine); and further, analyzing the sugar chain structure of the PSA by a method whereby lectin is made to act on the PSA, a method of making an antibody act on the PSA, a high-performance liquid chromatography or a mass spectrometry, or an analysis means that combines these methods. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a novel method for determining prostate cancer. More specifically, the present invention relates to a novel method for determining prostate cancer based on the difference in the sugar chain structure of prostate specific antigen (PSA).

  Prostate cancer (hereinafter referred to as “PC”) is one of the leading causes of death in men. Prostate specific antigen (hereinafter referred to as “PSA”) is recognized as a main tumor marker for PC (see, for example, Non-Patent Document 1). PSA has a molecular weight of about 30 kDa composed of a protein part consisting of 237 amino acid residues having a molecular weight of about 26 kDa and a sugar chain part bound to the 45th amino acid residue (asparagine) of the protein part. Or a derivative thereof.

  Although the usefulness of serum PSA concentration tests for the early diagnosis of PC has already been described in many literatures, serum PSA concentrations of 4 ng / mL to 10 ng / mL are called gray zones and are associated with PC. A concentration region that cannot be determined to be present (see, for example, Non-Patent Document 2). Several attempts have been made to solve this problem so far (eg, determination by PSA density, PSA slope (annual increase), free PSA / total PSA ratio, etc.) A diagnostic method has not yet been established.

  Recently, in a study by continuous lectin affinity chromatography combined with concanavalin A, plant agglutinin E4 (PHA-E4) and PHA-L4, the structure of the sugar chain that binds to asparagine of PSA is related to PC tissue and prostatic hypertrophy. The result that it differs with a structure | tissue (or normal tissue) was reported (refer nonpatent literature 3). In this non-patent document 3, N-linked sugar chains in PSA change during canceration in the human prostate, and N-linked sugar chains in PSA may be useful as a diagnostic tool for PC. It is described.

  On the other hand, as a method for discriminating between PC and benign prostatic hyperplasia (hereinafter referred to as “BPH”) by detecting the structure of PSA, there are several methods using a binding molecule that binds to the sugar chain of PSA. Immunological methods have been proposed. For example, a method for determining PC and BPH by contacting PSA with a lectin and measuring PSA separated based on the affinity between the sugar chain of PSA and the lectin has been reported (see Patent Document 1). ). In this report, it is described that the difference in affinity between the sugar chain of PSA and a lectin (such as a mackeria amurensis lectin) is based on the orientation of sialic acid added to the end of the sugar chain. However, the specific structure of the PSA sugar chain of a subject suffering from PC has not been determined.

  A method for determining PC based on whether or not at least three-branched sugar chains exist in PSA has been reported (see Patent Document 2). In this method, a binding molecule that binds to at least three-branched sugar chains but does not bind to one-branched and two-branched sugar chains is used. Binding molecules that can be used include lectins (such as PHA-L) and antibodies. However, this report does not show a structure in which LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine) is present (added) in the three-branched sugar chain.

  Furthermore, there has been a report focusing on the content of N-acetylgalactosamine (GalNAc) in PSA (see Non-Patent Document 4). This report examines the structure of PSA sugar chain in detail, and the content of GalNAc in the seminal plasma-derived PSA sugar chain is 25%, whereas the PSA sugar chain of prostate cancer cell line (LNCaP) has a GalNAc of 65 % Is included. These authors have also observed the same change in the pancreatic cancer-derived cell line (Capan-1) (see Non-Patent Document 5), from which the change in the content of GalNAc is due to a cancerous mutation. Analogy.

  In addition, there has been reported a method for comprehensively analyzing sugar chains of a target protein including PSA using an array composed of a specific antibody and a large number of lectins (see Non-Patent Document 6). In this method, it has been clarified that there are many LacdiNAc structures (N-acetylgalactosamine-N-acetylglucosamine structures) at the non-reducing ends on the sugar chain structure of PSA derived from prostate cancer cell lines. However, it does not target PSA sugar chains of subjects suffering from PC.

  Relatedly, a LacdiNAc structure is disclosed as an oligosaccharide sequence that is specifically expressed in cancer cells, and a patent application has been filed for a method for detecting cancer using its presence detected in a biological sample as an indicator. (See Patent Document 3). However, a specific method relating to the detection is not described, and further, it does not target PC determination.

  Alternatively, a method for evaluating the clinical state of a subject by determining a sugar profile of a sugar chain of a target glycoprotein containing PSA has been proposed (see Patent Document 4). This document describes that in an analysis using the MALDI-MS method, a PSA sugar chain of a subject suffering from PC is different from a normal PSA sugar chain. However, the specific structure of the PSA sugar chain of a subject suffering from PC is not described.

  As described above, many events have been reported in which the PSA sugar chain structure changes depending on the disease, but there are still few examples of comprehensive analysis of PSA sugar chains in PC patients. It has not been done so far to determine PC based on chain structure.

JP 2002-055108 A Special Table 2000-514919 JP 2005-500057 Gazette JP-T-2006-515927

Stamey et al., N. Engl. J. Med., 317, 909-916 (1987) Catalona et al., J. Am. Med. Assoc, 279, 1542-1547 (1998) Sumi et al., J. Chromatogr. B, 727, 9-14 (1994) Peracaula et al., Glycobiology, 13 (6), 457-470 (2003) Peracaula et al., Glycobiology, 13 (4), 227-244 (2003) Kuno et al., Mol. Cell. Proteomics, 8 (1) 99-108 (2009) Tabares et al., Glycobiology, 16 (2), 132-145 (2006) Bindukumar et al., J. Chromatogr. B, Analyt. Technol. Biomed. Life. Sci., 813 (1-2), 113-120 (2004) Zhang et al., Clin. Chem., 41 (11), 1567-1573 (1995) Okada et al., Biochim. Biophys. Acta, 1525 (1-2), 149-160 (2001) Jacques et al., J. Biol. Chem., 254 (7), 2400-2407 (1979)

  Therefore, an object of the present invention is to provide a method for detecting a PSA sugar chain of a subject suffering from PC and accurately determining PC based on the sugar chain structure of PSA.

  Based on the fact that the present inventors have discovered for the first time in the world that a PSA sugar chain of a subject suffering from PC has a three-chain sugar chain having a LacdiNAc structure, prostate cancer can be detected using its presence as an index. The present invention was completed by finding that it can be determined.

［式（１）中、Ｇａｌはガラクトース、Ｍａｎはマンノース、Ｆｕｃはフコース、ＧａｌＮＡｃはＮ−アセチルガラクトサミン、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを示す。括弧は３ヶ所の非還元末端の何れかに結合していることを示す。「ＧａｌＮＡｃ−ＧｌｃＮＡｃ−」又は「Ｇａｌ−ＧｌｃＮＡｃ−」を含む残基は、Ｍａｎα１−６Ｍａｎ側にあっても、Ｍａｎα１−３側にあってもよく、Ｍａｎへの結合様式は、β１−２、β１−４又はβ１−６の何れであってもよい。「Ｐｅｐｔｉｄｅ」とは、ＩＲＮＫＳ（Ｉｌｅ−Ａｒｇ−Ａｓｎ−Ｌｙｓ−Ｓｅｒ）であり、ＧｌｃＮＡｃはＮに結合している。］ That is, the present invention is the following method.
[1]
It includes the step of analyzing the sugar chain structure of PSA in a sample derived from a subject, and is determined to be prostate cancer when there are three or more sugar chains having LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine) A method for determining prostate cancer, comprising:
[2]
The sugar chain structure of the PSA is analyzed by a method in which a lectin is allowed to act on the PSA, a method in which an antibody is allowed to act on the PSA, a high performance liquid chromatography method or a mass spectrometry method, or an analysis means combining these. The method for determining prostate cancer according to [1], which is performed.
[3]
(1) a step of purifying PSA from a subject-derived sample;
(2) preparing a PSA derivative from the PSA purified in step (1);
(3) a step of labeling the PSA derivative obtained in step (2);
(4) including the step of analyzing the labeled PSA derivative obtained in step (3) by mass spectrometry, and determining that the cancer is prostate cancer when there are three or more sugar chains having LacdiNAc. The method for determining prostate cancer as described in [2], which is characterized by the following.
[4]
The method for determining prostate cancer according to [3], wherein the PSA derivative prepared in the step (2) is a PSA-derived sugar chain.
[5]
The method for determining prostate cancer according to [3], wherein the PSA derivative prepared in the step (2) is a PSA-derived glycopeptide.
[6]
The PSA purified in the above step (1) is contacted with concanavalin A lectin (ConA) to remove PSA having a sugar chain structure that binds to ConA from the PSA, and a sugar chain that does not bind to ConA. Including a step of obtaining a PSA having a structure, and in the step (2), a PSA derivative is prepared from a PSA having a sugar chain structure that does not bind to ConA obtained in the step [3]. To determine prostate cancer.
[7]
By contacting the PSA derivative prepared in the above step (2) with concanavalin A lectin (ConA), the PSA derivative having a sugar chain structure that binds to ConA is removed from the derivative and does not bind to ConA. Including a step of obtaining a PSA derivative having a sugar chain structure, and in the above step (3), the PSA derivative having a sugar chain structure that does not bind to ConA obtained in the step is labeled [3] A method for determining prostate cancer as described.
[8]
An isolated PSA comprising a glycopeptide represented by the following formula (1):

[In the formula (1), Gal represents galactose, Man represents mannose, Fuc represents fucose, GalNAc represents N-acetylgalactosamine, and GlcNAc represents N-acetylglucosamine. The parentheses indicate binding to any of the three non-reducing ends. Residues containing “GalNAc-GlcNAc-” or “Gal-GlcNAc-” may be on the Manα1-6Man side or on the Manα1-3 side, and the mode of binding to Man is β1-2, β1 -4 or β1-6. “Peptide” is IRNKS (Ile-Arg-Asn-Lys-Ser), and GlcNAc is bonded to N. ]

  According to the present invention, a method for solving the above problems and problems, detecting the sugar chain structure of prostate specific antigen (PSA), and accurately determining whether or not it is prostate cancer based on the difference in the structure Can provide. Furthermore, it is possible to provide a method for accurately determining whether the cancer is prostate cancer (PC) or benign prostatic hypertrophy (BPH).

  That is, in the method for measuring the PSA concentration in serum, which is widely used for screening of current PCs, a high biopsy was shown (for example, 4 ng / mL or more), and a needle biopsy, which is a painful secondary test, was received By applying the present invention to the subject, unnecessary examination can be avoided.

  Further, it is known that the PSA concentration in serum or the like tends to increase with age. Therefore, in the future aging society in particular, the method of the present invention for determining whether or not a PC is highly accurate from among subjects exhibiting a high concentration of PSA is not only a medical contribution but also a medical economy. In addition, it is expected to have a good effect.

  Furthermore, even for subjects whose PSA concentration is almost the same level as normal, PCs can be identified and determined with high accuracy by detecting three or more sugar chains having LacdiNAc contained in the PSA sugar chain. It is possible to avoid missing a person suffering from PC.

2 is a diagram showing a positive ion MS spectrum obtained in Example 1. FIG. In Example 1, it is a figure which shows the positive ion MS / MS spectrum obtained by using signal P1 obtained by m / z 2792 as a precursor ion. 6 is a diagram showing a positive ion MS spectrum obtained in Example 2. FIG.

  Hereinafter, although this invention is demonstrated, this invention is not limited to the specific aspect of the following implementation, It can implement arbitrarily deform | transforming.

  The method for determining PC of the present invention is a method for determining whether or not a subject is a PC, and includes a step of analyzing the sugar chain structure of PSA contained in the “sample derived from the subject”. In this step, it is confirmed whether or not there are three or more sugar chains having LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine). When such “LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine) has at least three sugar chains having at least one chain” is determined, the subject is determined to have prostate cancer.

In the present invention, “LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine)” is a chemical structure represented by the following formula (a) located at the end of a sugar chain.
GalNAc-GlcNAc- (a)
[In the formula (a), “Gal” represents galactose, “Glc” represents glucose, “Ac” represents an acetyl group, and “NAc” represents an acetyl group in N (nitrogen atom) of glucosamine or galactosamine. Indicates that they are joined. ]

  In the present invention, since “Gal-Glc” is Lac (lactose), the chemical structure represented by the formula (a) may be abbreviated as “LacdiNAc”.

  Further, the “sugar chain having LacdiNAc” includes a case where another chemical structure such as sialic acid or fucose is bonded to “LacdiNAc”. There may be cases where another chemical structure such as sialic acid or fucose is bound to the sugar chain of PSA in the sample derived from the subject, and these may be excluded in the step of analyzing the sugar chain structure.

“There are three or more sugar chains” means that PSA is composed of a protein part and a sugar chain part, and at least three of the sugar chain parts are represented by the above formula (a) or the following. It means that it has a chemical structure represented by the formula (b). In addition, the case where another chemical structure such as sialic acid or fucose is bonded to the following chemical structure is included.
Gal-GlcNAc- (b)
[In the formula (b), “Gal” represents galactose, “Glc” represents glucose, “Ac” represents an acetyl group, and “GlcNAc” represents an acetyl group bonded to N (nitrogen atom) of glucosamine. Indicates that ]

  The present invention includes a step of analyzing the sugar chain structure of PSA in a sample derived from a subject, and the terminal of the sugar chain of the PSA contains three or more “GalNAc-GlcNAc-” or “Gal-GlcNAc-”. A method for determining prostate cancer, characterized in that it is determined to be prostate cancer when it is composed of residues and at least one of them is a residue containing "GalNAc-GlcNAc-" is there. As described above, the sugar chain of PSA in a sample derived from a subject may be modified with sialic acid, fucose, etc., and may be excluded in the analysis step. It was clarified that this case is also included in the present invention.

  In the present invention, there are three or more sugar chains containing a structure represented by the formula (a) or the formula (b) at the terminal, and one or more of them are represented by the formula (a). When there is a PSA containing the chemical structure to be determined, it is determined as PC. For example, when there are three sugar chains, if only one of them contains the chemical structure represented by the above formula (a), it is determined as PC, and the double chain is represented by the above formula (a). ) Is also determined as PC, and when all three strands include the chemical structure represented by the above formula (a), it is also determined as PC. Further, for example, when a four-chain sugar chain is present, one, two, three, or four (that is, one or more) includes the chemical structure represented by the above formula (a). In the case, it is determined as PC. The same applies to the case of more sugar chains. Residues containing “GalNAc-GlcNAc-” or “Gal-GlcNAc-” may be on the Manα1-6Man side or the Manα1-3 side, and the binding mode to Man is β1-2. , Β1-4, or β1-6.

  The “sample derived from a subject” used in the present invention is a body fluid such as blood (including serum, plasma, etc.), urine, semen (sperm), cell / tissue, and the like.

In the method for determining prostate cancer of the present invention, the analysis of the sugar chain structure of the PSA is performed as follows:
* A method of causing a lectin to act on the PSA,
* A method of causing an antibody to act on the PSA,
* High-performance liquid chromatography or
* Mass spectrometry,
Or
* It is preferable to carry out by an analysis means combining these.

  That is, in the PC determination method according to the present invention, PSA in a sample derived from a subject is analyzed by a method using a specific binding molecule such as a lectin or an antibody, by high performance liquid chromatography, by mass spectrometry, or a combination thereof. In these steps, when a sugar chain structure having three or more chains having LacdiNAc is detected in the subject's PSA, it is preferable to determine that the subject is prostate cancer (PC).

  Among these, more preferable is a method of determining a PC when a sugar chain structure having three or more chains having LacdiNAc is detected by mass spectrometry.

Even in mass spectrometry, a particularly preferable method for determining PC according to the present invention is the step of analyzing by mass spectrometry,
(1) a step of purifying PSA from a subject-derived sample;
(2) preparing a PSA derivative from the PSA purified in step (1);
(3) a step of labeling the PSA derivative obtained in step (2);
(4) analyzing the labeled PSA derivative obtained in step (3) by mass spectrometry;
Including
It is determined that the cancer is prostate cancer when three or more sugar chains having LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine) are present, that is, when three or more sugar chains having LacdiNAc are detected. It is a method to do.

  In step (1), PSA is purified from a sample derived from a subject. Step (1) can be performed by any method known in the art.

  Alternatively, when serum derived from a subject is used as a sample in step (1), a method in which several types of affinity chromatography are combined can be used. For example, for serum derived from a subject, an affinity carrier based on adsorption of sulfur, such as Fractogel (registered trademark) EMD TA, Cibacron Blue 3GA, or protein A Sepharose CL-4B, HiTrap heparin column HPLC, etc. (See Non-Patent Document 7). The eluent is treated with ethanolamine to give free PSA. The obtained PSA is treated using the immunoprecipitation method described above to obtain a PSA derivative. According to the above-mentioned document, about 63 μg of PSA was required in the sample derived from the subject in order to analyze the obtained PSA derivative.

  Further, when a sample other than the subject-derived serum is used as the sample in the step (1), the PSA can be purified using an affinity carrier based on the one-step parent sulfur adsorption (see Non-Patent Document 8). For example, PSA is purified from human semen, prostate cancer patient serum, prostate cancer cell (LNCaP) culture supernatant by chromatography using 3S, T-gel slurry (Fructgel® EMD TA) and Western Blotting can be used to identify complexes with PSA alone or α1-antichymotrypsin (ACT).

  Alternatively, when semen derived from a subject is used as a sample, PSA may be purified by combining affinity chromatography based on parent sulfur adsorption and gel filtration (see Non-Patent Document 8). For example, Fractogel (registered trademark) TA 650 (S) can be combined with Ultragel AcA-54, which is a gel filtration carrier. In this combination method, free PSA could be recovered in a yield of 72%.

  Alternatively, when semen derived from a subject is used as the sample in step (1), PSA can be purified using the following method (see Non-Patent Documents 9 and 10). For example, by performing precipitation by adding ammonium sulfate, hydrophobic interaction chromatography (Phenyl-Sepharose-HP column), gel filtration (Sephacryl S-200 column), and anion exchange chromatography (Resource Q column) in sequence, PSA Can be purified. In this method, it was reported that the final PSA recovery was 30.1% when the sample contained 33.9 mg of PSA.

  Furthermore, when using a subject-derived androgen-dependent LNCaP as a sample in step (1), PSA can be purified by a method combining ultrafiltration and various chromatographies (see Non-Patent Document 5). For example, ultrafiltration (5 kDa cut-off polysulfone membrane (Millipore)), affinity chromatography (Cibacron Blue 3GA), gel filtration (Biogel P60), affinity chromatography (Cibacron) containing LNCaP cultured by appropriate means. Blue 3GA) and reverse phase chromatography (214TP-RP C4, using HPLC) can be used to purify the PSA.

  Next, in step (2), a PSA derivative is prepared from the purified PSA. The “PSA derivative” in the present invention means a sugar chain or glycopeptide derived from PSA, and specifically means a sugar chain or glycopeptide separated from PSA. For example, for PSA, endoprotease (thermolysin, endoproteinase Arg-C, endoproteinase Lys-C, trypsin, chymotrypsin, pepsin, proline-specific endopeptidase, protease V8, proteinase K, aminopeptidase M, carboxypeptidase B, etc. ) Or a peptide bond cleaving agent to form a glycopeptide, and the glycopeptide may be used in step (3).

  Alternatively, PSA may be treated enzymatically to form free sugar chains. Alternatively, the aforementioned glycopeptide derived from PSA may be enzymatically treated to form a free sugar chain. The sugar chain thus obtained may be used in step (3). In this enzymatic treatment, for example, peptide N-glycanase (PNGase F, PNGase A), endoglycosidase (EndoH, EndoF), and / or one or more proteases (trypsin, endoproteinase Lys-C, etc.) are used. be able to. Alternatively, PSA or a glycopeptide derived from PSA may be treated with chemical means (such as degradation with anhydrous hydrazine, reductive or non-reductive β-elimination) to form a free sugar chain. Good. The free sugar chain thus obtained may be used in the following step (3). Alternatively, a sugar chain or glycopeptide from which sialic acid has been removed by sialidase treatment or acid hydrolysis may be used.

  In addition, when electrophoresis is performed at the final stage of the step (1) and a PSA band is cut out, the above-mentioned reactant is allowed to act on the cut out band to perform in-gel digestion, and glycopeptide and / or sugar chain May be generated.

  In step (3), the PSA derivative is labeled. Examples of the labeling compound include a condensed polycyclic hydrocarbon moiety such as naphthalene, anthracene, and pyrene, a reactive functional group capable of binding to the molecule to be analyzed, and the condensed polycyclic hydrocarbon moiety as necessary. A compound having a spacer moiety that links the reactive functional group can be used.

  The fused polycyclic hydrocarbon moiety is preferably pyrene. The reactive functional group has reactivity with the carboxy group of sialic acid or the reducing end of sugar, and includes, for example, a diazomethyl group, an amino group, a hydrazide group, and the like. The spacer portion includes, for example, a linear or branched alkylene group. Labeling compounds that can be used in the present invention include 1-pyrenyldiazomethane (PDAM), 1-pyrenebutanoic acid hydrazide (PBH), 1-pyreneacetic acid hydrazide, 1-pyrenepropionic acid hydrazide, and aminopyrene (including structural isomers). 1-pyrenemethylamine, 1-pyrenepropylamine, 1-pyrenebutylamine and the like. The most preferably used labeling compound is PDAM.

  Step (3) can be performed by mixing the PSA derivative and the labeling compound and heating. Heating can be carried out at a temperature in the range of 40 to 80 ° C., for example. Optionally, labeling may be performed in the presence of a promoter such as water soluble carbodiimide or N-hydroxysuccinimide. More preferably, the labeling can be carried out by dropping a solution of the PSA derivative on the target plate used in the MALDI method and drying it, and dropping the solution of the labeling compound on the target plate and drying by heating. .

  The present invention relates to a method for analyzing a labeled PSA derivative by mass spectrometry. Hereinafter, “analysis by mass spectrometry” may be abbreviated as “MS analysis”. The ionization part that can be used in the MS analysis in the step (4) includes a matrix-assisted laser desorption ionization (MALDI) type or an electrospray ionization (ESI) type device.

  In the present invention, by coupling a condensed polycyclic hydrocarbon group such as pyrene, the ionization efficiency of the labeled PSA derivative in the MALDI method is improved as compared with the unlabeled PSA derivative. In addition, the labeled PSA derivative of the present invention is easier to apply the ESI method. Because the PSA derivative has high hydrophilicity and it is difficult to obtain an organic solution of the sample used in the ESI method, the labeled PSA derivative of the present invention can be obtained by introducing a condensed polycyclic hydrocarbon group. This is because it is soluble in an organic solvent.

The separation unit that can be used in the MS analysis in the step (4) includes any device known in the art such as a time-of-flight type (TOF type), a double focusing type, a quadrupole focusing type, or the like. In particular, it is convenient to use an apparatus having an ion trap in consideration of performing MS ⁿ (n ≧ 2) analysis. A particularly suitable device is the quadrupole ion trap-time of flight (QIT-TOF). Here, as a time-of-flight device, any of a linear device, a reflectron device, or a circulating device may be used.

  Next, in the obtained MS spectrum, a signal derived from a sugar chain structure having three or more chains having LacdiNAc is searched, and when the detection is confirmed, it is determined that the cancer is prostate cancer.

  In the MS spectrum obtained in this step, the sugar chain is selected by a signal from which some sugars such as a labeled compound, sialic acid, galactose, fucose, and GlcNAc are eliminated in the mass spectrometer, and the above determination procedure is performed. May be.

  ConA (Concanavalin A (Concanavalin A lectin)) has specificity for a structure containing two or more residues of alpha-D-mannose in which the hydroxyl groups at positions C-3, C-4 and C-6 are unsubstituted. Therefore, in the N-linked sugar chain, a mother nucleus structure (trimannosyl core) in which other mannose is bonded to the 6th and 3rd positions of mannose is recognized, and a high mannose sugar chain, a mixed sugar chain, Although it has binding ability to single-chain sugar chains, it has weak binding ability to multi-branched complex type sugar chains such as three- and four-strands, and its binding is significantly inhibited by the presence of GlcNAc bound to mannose by β1-4. (Non-Patent Document 11).

  Accordingly, a further preferred embodiment of the present invention has a sugar chain structure that binds to ConA from among the derivatives by contacting the PSA derivative prepared in the above step (2) with concanavalin A lectin (ConA). Including a step of removing the PSA derivative to obtain a PSA derivative having a sugar chain structure that does not bind to ConA. In the step (3), the PSA derivative having a sugar chain structure that does not bind to ConA obtained in the step is labeled. A method for determining prostate cancer as described above.

  That is, when the PSA derivative is subjected to ConA after the step (2) and the step (3) and subsequent steps are performed on the fraction that does not bind, the main sugar chain structure contained in the PSA derivative is excluded and the LacdiNAc structure 3 There is an advantage that sugar chains larger than this chain can be easily detected.

  From the same viewpoint, before preparing the PSA derivative in step (2), the PSA has a sugar chain structure that binds to ConA from the PSA by contacting the PSA with a concanavalin A lectin (ConA). It is also preferred to remove the PSA followed by derivatization.

  Therefore, a further preferred embodiment of the present invention is a PSA having a sugar chain structure that binds to ConA from the PSA by contacting the PSA purified in the above step (1) with concanavalin A lectin (ConA). In the above step (2), a PSA derivative is prepared from the PSA having a sugar chain structure that does not bind to ConA obtained in the step (2). This is a method for determining prostate cancer.

  That is, the contact of concanavalin A lectin (ConA) may be performed between step (1) and step (2), or may be performed between step (2) and step (3).

  Usually, when using a lectin, it is common to focus on the sugar chain that binds, so the above method of using a lectin focusing on the sugar chain that does not bind is very characteristic, and those skilled in the art can easily It is thought that it cannot be conceived.

  As described above, the present invention is a prostate cancer determination method for analyzing the sugar chain structure of PSA in a sample derived from a subject by mass spectrometry, and the analysis of the sugar chain structure of PSA is performed using the above PSA. It is also a method for determining prostate cancer by a method in which a lectin is acted on, a method in which an antibody is acted on the above-mentioned PSA, a high-performance liquid chromatography method or the mass spectrometry described above, or an analysis means combining these.

  PSA contained in a sample derived from a subject may be measured as it is, or may be measured after extraction or purification of PSA. PSA contained in the sample or extracted or purified PSA may form a complex with antichymotrypsin or may be free, but if the mixture remains as it is, the complex and free form are separated. In some cases, the complex is made free. Furthermore, sugar chains or glycopeptides may be prepared from those PSA as PSA derivatives.

  The present invention relates to a method of using high performance liquid chromatography (sometimes abbreviated as “HPLC”) as a means for analyzing PSA. For example, separation depending on the molecular weight of the detection target is performed by normal phase HPLC represented by Amide column or the like, or separation depending on the orientation of the constituent sugar chain of detection target is performed by reverse phase HPLC represented by ODS column or the like. Determination is made by detecting a signal resulting from a sugar chain structure of three or more chains having LacdiNAc in the chromatogram obtained by the analysis. For detection, an arbitrary absorption wavelength or a specific fluorescence wavelength for the selected labeling compound can be used.

  Alternatively, the present invention relates to a method of using an analysis technique based on a combination of specific binding molecules acting on PSA. Specific binding molecules include several lectins, antibodies and fragments thereof. For example, a specific binding molecule is reacted with a detection target immobilized on an ELISA plate, a polyvinylidene fluoride (PVDF) film, a glass or silicon array chip, and the target is detected.

  Detection uses a lectin or antibody conjugated with an enzyme such as alkaline phosphatase or horseradish peroxidase, and chemical coloration using bromochloroindolyl phosphate (BCIP) or nitroblue tetrazolium (NBT) via an enzymatic reaction, or a luminol reagent Chemiluminescence using can be used, or surface plasmon resonance can also be used. Relatedly, the detection target can be bound to a solid-phased specific binding molecule, and another type of lectin or antibody can be reacted therewith to perform a series of detections. In the present invention, “specific antibody” may be simply abbreviated as “antibody”, and “specific lectin” may be simply abbreviated as “lectin”.

  When a lectin is used as a specific binding molecule, for example, a LacdiNAc sugar chain can be detected by allowing a WFA lectin (Wisteria floribunda agglutinin) that recognizes non-reducing terminal GalNAc to act on PSA. When using an antibody as a specific binding molecule, a commercially available polyclonal antibody or monoclonal antibody can be used as an antibody against PSA.

  In connection with the method of using an antibody that recognizes a specific sugar chain structure as a specific binding molecule, the present invention relates to the next step of producing a polyclonal or monoclonal antibody that recognizes a LacdiNAc structure. First, a sugar chain structure containing a LacdiNAc structure, an analog or derivative thereof is prepared from a biological sample, or chemically or biochemically synthesized to produce a multivalent conjugate having the same structure. The animal is then immunized with a multivalent conjugate along with an immune response activator. At this time, the multivalent conjugate is preferably bound to an immune reaction activator, and the conjugate is used for immunization alone or together with a further immune reaction activator. More preferably, the multivalent conjugate is injected into the antibody-producing organism together with at least one adjuvant molecule or administered to the mucosa.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples as long as the gist thereof is not exceeded.

Example 1
Serum (337.9 ng PSA / mL) of one subject (N-315) diagnosed with PC by biopsy and given an identification code was used. The procedure was approved by a medical institution for ethical review and informed consent was given to the subjects.

(A) Step 1 Isolation of PSA First, immunoglobulin was removed from serum. 5 mL of protein A agarose carrier (PIERCE) was equilibrated with phosphate buffered saline (PBS). This was packed in a disposable plastic column (PIERCE), a mixture of the subject's serum and PBS was added, and the mixture was shaken at 4 ° C. for 30 minutes. The fraction containing PSA that does not bind to the carrier is collected, and the fraction obtained by washing the protein A agarose carrier with 3 times the volume of the carrier (3 CV) PBS is collected. Na ₂ SO ₄ was added to 1M.

Next, albumin, which is a major protein in serum, was removed from the PSA-containing fraction. 2.5 mL of Fractogel ™ EMD TA (S) (Merck) was loaded onto a disposable plastic column and equilibrated with 20 mM phosphate buffer (pH 7.4, containing Na ₂ SO ₄ (1M)). The fraction obtained above was added to the fructogel-filled column, and 7 CV of the same buffer was sent using a pump to remove albumin.

Next, the protein adsorbed on Fractogel (trademark) was eluted with 20 mM phosphate buffer (pH 7.4, not containing Na ₂ SO ₄ ) to obtain a fraction containing PSA. Subsequently, this fraction was added to 2 mL of protein A agarose carrier equilibrated with PBS, and shaken at 4 ° C. for 30 minutes to remove immunoglobulin that could not be removed. A mixed solution of the fraction containing PSA not bound to the protein A agarose carrier and the fraction washed with 3 CV PBS was used for the next immunoprecipitation.

  Subsequently, anti-PSA antibody beads used for immunoprecipitation were prepared. NHS-activated Sepharose 4 Fast Flow (GE Healthcare Bioscience) was washed 3 times with 1 mM HCl. Next, a commercially available anti-human PSA / rabbit polyclonal antibody (DAKO) was added to the washed beads, and the antibody and beads were cross-linked by shaking for 30 minutes at room temperature.

  Subsequently, a 0.5 M monoethanolamine aqueous solution containing 0.5 M NaCl was added, and the remaining active groups were inactivated by shaking at room temperature for 30 minutes. The obtained anti-PSA antibody beads were alternately exchanged with 0.1 M sodium acetate buffer (pH 4.0) containing 0.5 M NaCl and 1 M Tris buffer (pH 9.0) containing 0.5 M NaCl. Washed 3 times. The anti-PSA antibody beads were then washed 3 times with PBS. Finally, the beads were washed with 0.02% sodium azide-containing PBS and stored at 4 ° C. until use.

  Subsequently, immunoprecipitation was performed. To the mixed solution fraction containing PSA described above, 40 μL of anti-PSA antibody beads were added and shaken at 4 ° C. for 1 hour. Subsequently, the anti-PSA antibody beads were transferred to a Micro Bio-Spin chromatography column (Bio-Rad Laboratories). The anti-PSA antibody bead column was washed 4 times with PBS, 3 times with PBS containing 0.02% Tween-20, and 2 times with distilled water. Further, a 1M aqueous propionic acid solution was added to the anti-PSA antibody bead column, and the protein adsorbed on the beads was eluted and collected. Elution with an aqueous propionic acid solution was performed 5 times in total, and the eluates were mixed and dried using a centrifugal concentrator.

  A part of the obtained protein fraction was subjected to Western blotting and subjected to semi-quantitative analysis. As a result, it was found that 70% or more of PSA contained in the first serum could be recovered.

(B) Step 2 Preparation of glycopeptide 50 mM aqueous solution of ammonium bicarbonate (pH 8.0) containing 50 units of thermolysin (Calbio), 100 μL to the tube containing the vacuum-dried sample obtained in step (a). Was directly added and allowed to stand at 56 ° C. for 18 hours. After the reaction mixture was dried by a centrifugal concentrator, the obtained solid was dissolved in 0.8% aqueous trifluoroacetic acid solution and allowed to stand at 80 ° C. for 40 minutes to carry out a desialic acid reaction. The reaction sample was dried with a centrifugal concentrator.

  Subsequently, the glycopeptide was roughly purified and desalted from the obtained sample using Intersep GC 50 mg (GL Science), which is a carbon graphite filled cartridge. First, a 1M NaOH aqueous solution, distilled water, 30% acetic acid aqueous solution, and an aqueous solution containing 5% acetonitrile and 1% trifluoroacetic acid are sequentially fed to the cartridge to wash and equilibrate the carbon graphite. went.

  Next, the dried glycopeptide sample was dissolved in “an aqueous solution containing 5% acetonitrile and 1% trifluoroacetic acid” and sent to the cartridge. Distilled water and “an aqueous solution containing 5% acetonitrile and 1% trifluoroacetic acid” were sequentially fed to the glycopeptide adsorbed on the cartridge for washing. Thereafter, “an aqueous solution containing 80% acetonitrile and 1% trifluoroacetic acid” was fed to the cartridge, and the adsorbed glycopeptide was eluted to obtain a crude purified glycopeptide fraction. The resulting solution was dried using a centrifugal concentrator.

  The glycopeptide was purified using Cellulose, fibrous (Sigma Aldrich). Cellulose was washed 5 times with 50% aqueous ethanol before use, and then washed 5 times with butanol: distilled water: ethanol = 4: 1: 1. The dried crude crude glycopeptide fraction was dissolved in the same mixed solution, and 20 μL of the washed Cellulose solution was added to the solution, and the glycopeptide was adsorbed by shaking at room temperature for 1 hour. Subsequently, Cellulose was washed 6 times using the above-mentioned mixed solution.

  The glycopeptide was eluted by adding 50% ethanol aqueous solution to Cellulose adsorbed with the glycopeptide and shaking for 30 minutes at room temperature. The solution containing the eluted glycopeptide was collected and dried to dryness with a centrifugal concentrator. Next, the dried glycopeptide was dissolved in 4 μL of 5% acetonitrile aqueous solution.

(C) Step 3 Labeling of Glycopeptide (PSA Derivative) On the MALDI target plate, 1.0 μL of the glycopeptide solution obtained in Step 2 was dropped and allowed to air dry. Next, 0.25 μL of a DMSO solution of 1-pyrenyldiazomethane (PDAM, 500 pmol) was dropped on the target plate, heated to 80 ° C. for about 5 minutes, and dried. By this operation, a glycopeptide labeled with PDAM was obtained on the target plate.

(D) Step 4 MS analysis On the target plate carrying the labeled glycopeptide obtained in Step 3, 1.0 μL of a 60% acetonitrile solution (concentration 10 mg / mL) of 2,5-dihydroxybenzoic acid (DHBA) was added. Dropped and dried at room temperature.

  The obtained target plate was installed in a MALDI-QIT-TOFMS apparatus AXIMA-QIT (manufactured by Shimadzu Corporation / Kratos) and subjected to MS analysis. In order to make a quantitative comparison, a raster scan was performed on the area surrounded by the outer circumference larger than the spread of the sample on the target plate, and all significant signals were accumulated.

  A typical example of the obtained MS spectrum is shown in FIG. In FIG. 1, as indicated by P1, a three-chain sugar chain represented by the following formula (1) having LacdiNAc was found in PSA-derived peptide 43-47. This structure is a novel structure that has not been reported so far in PSA, and was first found in PC patients according to the present invention.

［式（１）中、Ｇａｌはガラクトース、Ｍａｎはマンノース、Ｆｕｃはフコース、ＧａｌＮＡｃはＮ−アセチルガラクトサミン、ＧｌｃＮＡｃはＮ−アセチルグルコサミンを示す。括弧は３ヶ所の非還元末端の何れかに結合していることを示す。「ＧａｌＮＡｃ−ＧｌｃＮＡｃ−」又は「Ｇａｌ−ＧｌｃＮＡｃ−」を含む残基は、Ｍａｎα１−６Ｍａｎ側にあっても、Ｍａｎα１−３側にあってもよく、Ｍａｎへの結合様式は、β１−２、β１−４又はβ１−６の何れであってもよい。「Ｐｅｐｔｉｄｅ」とは、ＩＲＮＫＳ（Ｉｌｅ−Ａｒｇ−Ａｓｎ−Ｌｙｓ−Ｓｅｒ）であり、ＧｌｃＮＡｃはＮに結合している。］ [P1] (m / z 2792)

[In the formula (1), Gal represents galactose, Man represents mannose, Fuc represents fucose, GalNAc represents N-acetylgalactosamine, and GlcNAc represents N-acetylglucosamine. The parentheses indicate binding to any of the three non-reducing ends. Residues containing “GalNAc-GlcNAc-” or “Gal-GlcNAc-” may be on the Manα1-6Man side or on the Manα1-3 side, and the mode of binding to Man is β1-2, β1 -4 or β1-6. “Peptide” is IRNKS (Ile-Arg-Asn-Lys-Ser), and GlcNAc is bonded to N. ]

  Therefore, the PSA of the subject diagnosed with PC is a natural product, but the PSA having a glycopeptide represented by the following formula (1) present on the target plate for mass spectrometry is in a novel state. . In addition, as shown in Example 3 below, since the PSA can be isolated, the isolated PSA characterized by having the glycopeptide represented by the above formula (1) is novel.

FIG. 2 shows the results of MS ² analysis using m / z 2792 ions as precursor ions. As shown in FIG. 2, it was confirmed from the fragment ion that the structure was represented by the above formula (1). Fragment ions m / z 701, 821, and 967 show the peptide IRNKS to which the 83 Da GlcNAc cross-ring fragment, GlcNAc, and GlcNAc-Fuc were bound, respectively, demonstrating that they are bound on the PSA protein. This method, which was analyzed as a glycopeptide without releasing the sugar chain, was not affected by contaminating proteins, and extremely accurate results were obtained.

  As shown in Example 1, in the glycopeptide of cancer patient-derived PSA, a three-chain sugar chain having LacdiNAc that was not found in PSA derived from healthy subjects could be detected. This finding not only shows the effectiveness of this case of determining the presence or absence of cancerous changes related to the prostate based on the difference in the sugar chain structure of PSA, but also for cancer biological elucidation and therapeutic application. This is a meaningful discovery.

Example 2
The same serum as the sample used in Example 1 was subjected to the thermolysin reaction and desialic acid reaction in Step 1 and Step 2, and then a solid-phased concanavalin A (ConA) carrier was used. By preparing the fraction, sugar chains of three or more chains were selectively detected.

  First, 10 μL of 10 mM ammonium acetate buffer adjusted to pH 7.4 was aspirated into Dia Chrom ConA Tip (Glygen corp) and discharged to equilibrate the chip. The equilibration operation was performed three times. Next, after carrying out the thermolysin reaction and desialic acid reaction in step 1 and step 2, the sample dried under reduced pressure was dissolved in 10 μL of ammonium acetate buffer, and suction and discharge were performed on the chip after equilibration. Repeated 20 times.

  Thereafter, the sample solution was allowed to stand for 30 minutes in a state of being sucked into the chip, whereby a specific type of glycopeptide was reacted with ConA. Subsequently, the chip was washed by sucking and discharging 10 μL of distilled water, and further, the operation of leaving still for 10 minutes while sucking 10 μL of distilled water and then discharging was repeated three times. The sample solution after being subjected to ConA and distilled water used for washing were collected as a non-binding fraction for ConA, and dried using a centrifugal concentrator.

  Subsequently, the unbound fraction solution that had been roughly purified and desalted by Intersep GC was dried using a centrifugal concentrator. The dried and unbound fraction was dissolved in 4 μL of 5% acetonitrile aqueous solution without being subjected to Cellulose, and the operation after Step 3 was carried out in the same manner as in Example 1 and MS analysis was performed.

  The obtained MS spectrum is shown in FIG. In FIG. 3, as indicated by P1, a three-chain sugar chain (structure represented by the above formula (1)) having LacdiNAc was found in PSA-derived peptide 43-47. On the other hand, m / z 2386 ion, which is a double-stranded structure, and m / z 2600 ion, which is a pyrene-labeled derivative thereof, also present in PSA derived from subjects other than cancer patients were hardly detected. By performing MS / MS analysis of other signals, a glycopeptide having a sugar chain bound to PSA-derived peptide 43-47 other than the structure represented by the above formula (1) was not detected. Furthermore, a signal different from the signal seen in the MS spectrum of Example 1 was not detected, and it was found that the step shown in Example 2 is preferable in that the cancer-specific sugar chain structure is selectively detected.

Example 3
After performing Step 1 on the same serum as the sample used in Example 1, a solid phase concanavalin A (ConA) carrier is used to prepare a fraction that does not bind to the carrier, so that the formula (1) PSA having a glycopeptide represented by the formula can be selectively isolated.

  First, as in Example 2, a Dia Chrom ConA Tip (Glygen corp) chip is equilibrated using a 10 mM ammonium acetate buffer. Next, after the step 1 has been carried out, the sample dried under reduced pressure is dissolved in 10 μL of ammonium acetate buffer, and suction and discharge are repeated 20 times with the tip having been equilibrated.

  Thereafter, the sample solution is allowed to react with Con A by allowing it to stand for 30 minutes while being sucked into the chip. Subsequently, the chip is washed by sucking and discharging 10 μL of distilled water, and is further allowed to stand for 10 minutes while sucking 10 μL of distilled water, and then discharged three times. The sample solution after being subjected to ConA and the distilled water used for washing are collected as a non-binding fraction for ConA and lyophilized. It can be confirmed as follows that this fraction is an isolated PSA having the glycopeptide represented by the formula (1).

  That is, a PSA glycopeptide is prepared by performing the operations in and after step 2 in the same manner as in Example 1. Finally, the dried sample is dissolved in 4 μL of a 5% acetonitrile aqueous solution, and the operation after Step 3 is carried out in the same manner as in Example 1 to perform MS analysis.

  By subjecting the sample obtained by the above operation to MS analysis, a mass spectrum in which only the structure represented by the above formula (1) is detected, similar to FIG. 3, is obtained, and the above ConA non-binding fraction is It can be confirmed that it is an isolated PSA having a glycopeptide represented by the formula (1).

  According to the method for determining prostate cancer of the present invention, it is possible to accurately determine whether or not it is prostate cancer (PC), and it is possible to avoid a painful needle biopsy that is a secondary test. In addition, even for a subject with a normal PSA concentration, it is possible to identify and determine PC with high accuracy by detecting sugar chains of three or more chains having LacdiNAc, so that PC is determined with high accuracy. The present invention is widely used.

Claims (8)

  It includes the step of analyzing the sugar chain structure of PSA in a sample derived from a subject, and is determined to be prostate cancer when there are three or more sugar chains having LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine) A method for determining prostate cancer, comprising:   The sugar chain structure of the PSA is analyzed by a method in which a lectin is allowed to act on the PSA, a method in which an antibody is allowed to act on the PSA, a high performance liquid chromatography method or a mass spectrometry method, or an analysis means combining these. The method for determining prostate cancer according to claim 1, wherein the method is performed. (1) a step of purifying PSA from a subject-derived sample;
(2) preparing a PSA derivative from the PSA purified in step (1);
(3) a step of labeling the PSA derivative obtained in step (2);
(4) including a step of analyzing the labeled PSA derivative obtained in step (3) by mass spectrometry, wherein there are three or more sugar chains having LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine) 3. The method for determining prostate cancer according to claim 2, wherein it is sometimes determined that the cancer is prostate cancer.   The method for determining prostate cancer according to claim 3, wherein the PSA derivative prepared in the step (2) is a PSA-derived sugar chain.   The method for determining prostate cancer according to claim 3, wherein the PSA derivative prepared in the step (2) is a glycopeptide derived from PSA.   The PSA purified in the above step (1) is contacted with concanavalin A lectin (ConA) to remove PSA having a sugar chain structure that binds to ConA from the PSA, and a sugar chain that does not bind to ConA. 4. The method according to claim 3, further comprising the step of obtaining a PSA having a structure, wherein in the step (2), a PSA derivative is prepared from a PSA having a sugar chain structure that does not bind to ConA obtained in the step. To determine prostate cancer.   By contacting the PSA derivative prepared in the above step (2) with concanavalin A lectin (ConA), the PSA derivative having a sugar chain structure that binds to ConA is removed from the derivative and does not bind to ConA. The method includes the step of obtaining a PSA derivative having a sugar chain structure, and in the step (3), the PSA derivative having a sugar chain structure that does not bind to ConA obtained in the step is labeled. A method for determining prostate cancer as described. An isolated PSA comprising a glycopeptide represented by the following formula (1):

[In the formula (1), Gal represents galactose, Man represents mannose, Fuc represents fucose, GalNAc represents N-acetylgalactosamine, and GlcNAc represents N-acetylglucosamine. The parentheses indicate binding to any of the three non-reducing ends. Residues containing “GalNAc-GlcNAc-” or “Gal-GlcNAc-” may be on the Manα1-6Man side or on the Manα1-3 side, and the mode of binding to Man is β1-2, β1 -4 or β1-6. “Peptide” is IRNKS (Ile-Arg-Asn-Lys-Ser), and GlcNAc is bonded to N. ]

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