JP2023174447A - Use of sugar chain structures of o-glycosylated proteins - Google Patents
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Abstract
Description
本発明は、O-型糖鎖修飾タンパク質の糖鎖構造の利用に関し、より詳細には、O-型糖鎖修飾タンパク質の糖鎖構造の腫瘍マーカーとしての利用に関する。 The present invention relates to the use of the sugar chain structure of an O-type glycosylated protein, and more particularly, to the use of the sugar chain structure of an O-type glycosylated protein as a tumor marker.
現在、大腸がんの１次スクリーニング検査として「便潜血検査免疫法」と「全腸内視鏡検査」、「腫瘍マーカー法」が実施されている。便潜血検査免疫法の感度（大腸がん患者を陽性と判定する確率）は、がんの発生部位や病変の進行部位によって差があり30-90％（平均すると65%程度）、全腸内視鏡検査の感度は95%以上とされている（非特許文献１）。腫瘍マーカー法では、血清中の糖鎖（シアリルルイスa）を抗原とするCA19-9が用いられている。その感度は30-50％であり、早期がんではさらに低下するため、主に治療再発マーカーとして用いられている。一方、膵がんの１次スクリーニング検査としては「腫瘍マーカー法」と「腹部超音波検査」がある。腫瘍マーカー法では、大腸がんと同じCA19-9が主に用いられている。その感度は70-80%であり、早期膵がんでは50%程度である。腹部超音波検査の膵がん診断の感度は48-89%であり、対象や術者間で大きな差異がある（非特許文献２）。 Currently, ``fecal occult blood test immunoassay,'' ``whole intestine endoscopy,'' and ``tumor marker assay'' are being conducted as primary screening tests for colorectal cancer. The sensitivity of the immunological test for fecal occult blood (probability of determining a positive colorectal cancer patient) varies depending on the site of cancer occurrence and the site of progression of the disease, and is 30-90% (about 65% on average). The sensitivity of endoscopy is said to be 95% or more (Non-Patent Document 1). The tumor marker method uses CA19-9, which uses sugar chains (sialyl Lewis a) in serum as an antigen. Its sensitivity is 30-50% and is even lower in early stage cancers, so it is mainly used as a therapeutic recurrence marker. On the other hand, primary screening tests for pancreatic cancer include the ``tumor marker method'' and the ``abdominal ultrasound examination.'' In tumor marker methods, CA19-9, which is the same as colorectal cancer, is mainly used. Its sensitivity is 70-80%, and about 50% for early pancreatic cancer. The sensitivity of abdominal ultrasonography for pancreatic cancer diagnosis is 48-89%, and there are large differences between subjects and operators (Non-Patent Document 2).
がんにおける糖タンパク質の糖鎖変化を利用した腫瘍マーカーとしては肝細胞がんにおけるAFP（alpha-Fetoprotein）コアフコシル化の亢進を利用したAFP-L3（AFPレクチン分画比%）がマーカーとして利用されている。また、大腸がんと膵がんにおけるHP（Haptoglobin）コアフコシル化の亢進を利用したレクチンELISAキットの開発も進められている（大阪大学、タカラバイオ、免疫生物研究所）。両者の感度は共に40-50%である。さらに、近年、既存の前立腺がんマーカーPSAの診断精度向上を目指し、PSA糖鎖の変化（LacdiNAc構造の有無）を新たな診断指標に加えたPSA G-indexも開発されている（がん研究会、LSIメディエンス）。これらは全てN-型糖鎖の糖鎖変化を利用しており、主に抗タンパク質抗体と糖鎖結合タンパク質レクチンを用いたサンドイッチELISA系で検出している。 AFP-L3 (AFP lectin fraction ratio %), which utilizes increased AFP (alpha-Fetoprotein) core fucosylation in hepatocellular carcinoma, is used as a tumor marker that utilizes changes in the sugar chains of glycoproteins in cancer. ing. Additionally, a lectin ELISA kit is being developed that utilizes increased HP (Haptoglobin) core fucosylation in colon cancer and pancreatic cancer (Osaka University, Takara Bio, and Immunobiological Research Institute). The sensitivity of both is 40-50%. Furthermore, in recent years, with the aim of improving the diagnostic accuracy of the existing prostate cancer marker PSA, the PSA G-index, which adds changes in PSA sugar chains (presence or absence of LacdiNAc structure) as a new diagnostic index, has been developed (Cancer Research Association, LSI Medience). All of these utilize sugar chain changes in N-glycans, and are mainly detected using a sandwich ELISA system using anti-protein antibodies and sugar chain-binding protein lectins.
大腸がん診断における「便潜血検査免疫法」については、食事制限等の前処置の必要が無く、患者負担の少ない方法であるが、がんの発生部位や病変の進行部位によって感度（30-90％）に著しい差がみられる点が問題である。「全腸内視鏡検査」は優れた感度（95%以上）を有するものの、前処置で使用される下剤や食事制限による脱水症、およびそれに伴う血栓症が高齢者（患者）にとって大きな問題となっている。 The ``fecal occult blood test immunotherapy method'' for colorectal cancer diagnosis does not require pretreatment such as dietary restrictions, and is a less burdensome method for patients. The problem is that there is a significant difference in the number of respondents (90%). Although "whole bowel endoscopy" has excellent sensitivity (over 95%), dehydration due to the laxatives used in pretreatment and dietary restrictions, and associated thrombosis are major problems for elderly patients (patients). It has become.
「腫瘍マーカー法」は、便潜血検査免疫法と同様に患者負担が比較的少ない方法であるが、偽陽性の高いことが課題となっている。偽陽性率は平均20-30%（早期膵がんでは52％)である。原因として、シアリルルイスaをはじめとする糖鎖腫瘍抗原ががんと無関係のタンパク質にも結合しており、検査用抗体ががんと無関係のタンパク質上の糖鎖抗原も認識していることが考えられる。 The "tumor marker method" is a method with a relatively low burden on patients, similar to the fecal occult blood test immunotherapy method, but the problem is that it has a high rate of false positives. The average false-positive rate is 20-30% (52% for early pancreatic cancer). The cause is thought to be that sugar chain tumor antigens such as sialyl Lewis A also bind to proteins unrelated to cancer, and that the testing antibodies also recognize sugar chain antigens on proteins unrelated to cancer. It will be done.
本発明は、新規な腫瘍マーカーを提供することを目的とする。 The present invention aims to provide a novel tumor marker.
本発明は、血清中に存在するタンパク質へのO-型糖鎖修飾の有無に着目したものである。上記背景技術における腫瘍マーカーはいずれも、コアフコシル化の亢進をはじめがんにおける“N-型糖鎖の構造変化”に着目しており、修飾されていることを前提としている。そのため、N-型糖鎖の部分的な構造変化を利用したマーカーとなっており、検出には複雑な糖鎖構造の微細な違いを識別する方法を構築する必要がある。一方、O-型糖鎖修飾では、細胞の状態により、修飾の有無そのものに違いが生じることがある。また、N型糖鎖に比べて構造の多様性が少ないため、糖鎖構造の違いを容易に識別できるといった利点もある。しかし、O-型糖タンパク質の網羅解析には技術的な課題が多く難しかった。 The present invention focuses on the presence or absence of O-type sugar chain modification on proteins present in serum. All of the tumor markers in the background art described above focus on "structural changes in N-glycan chains" in cancer, including enhancement of core fucosylation, and are based on the premise that they are modified. Therefore, it is a marker that utilizes partial structural changes in N-glycans, and detection requires building a method to distinguish minute differences in complex glycan structures. On the other hand, in O-glycan modification, the presence or absence of modification may vary depending on the state of the cell. Additionally, since there is less structural diversity than N-type sugar chains, it has the advantage that differences in sugar chain structure can be easily identified. However, comprehensive analysis of O-type glycoproteins was difficult due to many technical issues.
発明者らは、独自にO型糖鎖修飾解析プラットフォームの開発に成功し、供与血清から得られたタンパク質より、大腸がん患者血清群でのみ同定されたO-型糖タンパク質をリスト化した。その中から、患者群における同定頻度が高い６種（FBLN2、ANGPTL3、MAN2A1、CSF1、VCAN、C1QAのO型グリコフォーム）及び非ラベル化定量法により統計学的有意性を示した２種（MRC1、FGA）を腫瘍マーカーとした。患者血清中では、これらのタンパク質の特定領域に特定のO-型糖鎖修飾（T抗原、Sialyl T抗原、di- Sialyl T抗原）が結合したグリコフォームが著しく増加していることを見出し、本発明を完成させるに到った。 The inventors succeeded in independently developing an O-type glycosylation analysis platform, and created a list of O-type glycoproteins that were identified only in colorectal cancer patient serogroups from proteins obtained from donor serum. Among them, 6 types are frequently identified in the patient group (FBLN2, ANGPTL3, MAN2A1, CSF1, VCAN, O-type glycoforms of C1QA) and 2 types showed statistical significance by non-labeled quantitative method (MRC1 , FGA) was used as a tumor marker. We discovered that glycoforms with specific O-type glycosylation (T antigen, Sialyl T antigen, di-Sialyl T antigen) attached to specific regions of these proteins were significantly increased in patient serum. He has completed his invention.
本発明の要旨は以下のとおりである。
（１）被験者由来の生物学的試料におけるO-型糖鎖で修飾されたタンパク質のレベルを測定することを含む、がんの診断を補助するための方法であって、タンパク質が、FBLN2(Fibulin-2)、ANGPTL3 (Angiopoietin-related protein 3)、MAN2A1 (Alpha-mannosidase 2)、CSF1(Macrophage colony-stimulating factor 1)、VCAN (Versican core protein)、C1QA (Complement C1q subcomponent subunit A)、MRC1（Macrophage mannose receptor 1 ）及びFGA（Fibrinogen alpha chain）からなる群より選択される少なくとも１つである前記方法。
（２）O-型糖鎖で修飾されたタンパク質のレベルが高い場合に、がんに罹患している可能性が高いことを示し、O-型糖鎖で修飾されたタンパク質のレベルが低い場合に、がんに罹患している可能性が低いことを示す（１）記載の方法。
（３）O-型糖鎖が、T抗原：Hex(1)HexNAc(1)、Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)、di-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)、フコシル化T抗原：Hex(1)HexNAc(1)dHex(1)又はそれらの組み合わせである（１）又は（２）に記載の方法。
（４）O-型糖鎖で修飾されたタンパク質が、下記の(i)～(viii)からなる群より選択される少なくとも１つである（３）記載の方法。
(i) T抗原：Hex(1)HexNAc(1)が付加されたFBLN2、
(ii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたANGPTL3、
(iii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)が付加されたMAN2A1、
(iv) T抗原：Hex(1)HexNAc(1)が付加されたCSF1及びdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたCSF1、
(v)T抗原：Hex(1)HexNAc(1)が付加されたVCAN、
(vi) フコシル化T抗原：Hex(1)HexNAc(1)dHex(1)が付加されたC1QA
(vii) T抗原：Hex(1)HexNAc(1)が付加されたMRC1
(viii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)が付加されたFGA
（５）O-型糖鎖で修飾されたタンパク質が、下記の(ia)～(viiia)からなる群より選択される少なくとも１つである（４）記載の方法。
(ia) aa330-349、aa350-364及びaa365-378のうち少なくとも１つの領域内の部位にT抗原：Hex(1)HexNAc(1)が付加されたFBLN2、
(iia) aa206-221及びaa222-235のうち少なくとも１つの領域内の部位にSialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原 Hex(1)HexNAc(1)NeuAc(2)が付加されたANGPTL3、
(iiia) aa211-221領域内の部位にSialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原 Hex(1)HexNAc(1)NeuAc(2)が付加されたMAN2A1、
(iva) aa356-395領域内の部位にT抗原：Hex(1)HexNAc(1)が付加されたCSF1及び370-395領域内の部位にdi-Sialyl T抗原 Hex(1)HexNAc(1)NeuAc(2)が付加されたCSF1、
(va)aa1415-1430及びaa2468-2475のうちの少なくとも１つの領域内の部位にT抗原：Hex(1)HexNAc(1)が付加されたVCAN、並びに
(via) aa101-121領域内の部位にフコシル化T抗原：Hex(1)HexNAc(1)dHex(1)が付加されたC1QA
(viia) aa1215-1229領域内の部位にT抗原：Hex(1)HexNAc(1)が付加されたMRC1、並びに
(viiia) aa354-367及びaa511-527のうち少なくとも１つの領域内の部位にSialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原 Hex(1)HexNAc(1)NeuAc(2)が付加されたFGA
（６）生物学的試料が、細胞、組織、体液、腹腔液、腹腔洗浄液、糞便又は尿である（１）～（５）のいずれかに記載の方法。
（７）体液が血液である（６）記載の方法。
（８）血液が、全血、血清、血漿又は血漿交換外液のいずれかである（７）記載の方法。
（９）がんが大腸がんである（１）～（８）のいずれかに記載の方法。
（１０）下記の(i)～(viii)からなる群より選択される少なくとも１つのO-型糖鎖修飾タンパク質を特異的に検出することができる試薬を含む、検査キット。
(i) T抗原：Hex(1)HexNAc(1)が付加されたFBLN2、
(ii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたANGPTL3、
(iii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたMAN2A1、
(iv) T抗原：Hex(1)HexNAc(1)が付加されたCSF1及びdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたCSF1、
(v) T抗原：Hex(1)HexNAc(1)が付加されたVCAN、並びに
(v) フコシル化T抗原：Hex(1)HexNAc(1)dHex(1)が付加されたC1QA
(vii) T抗原：Hex(1)HexNAc(1)が付加されたMRC1
(viii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)が付加されたFGA
The gist of the present invention is as follows.
(1) A method for assisting cancer diagnosis, including measuring the level of a protein modified with an O-glycan in a biological sample derived from a subject, the method comprising: -2), ANGPTL3 (Angiopoietin-related protein 3), MAN2A1 (Alpha-mannosidase 2), CSF1 (Macrophage colony-stimulating factor 1), VCAN (Versican core protein), C1QA (Complement C1q subcomponent subunit A), MRC1 (Macrophage mannose receptor 1) and FGA (Fibrinogen alpha chain).
(2) If the level of proteins modified with O-type sugar chains is high, it indicates that there is a high possibility of suffering from cancer, and if the level of proteins modified with O-type sugar chains is low. The method according to (1), which shows that the possibility of suffering from cancer is low.
(3) O-type sugar chains are T antigen: Hex(1)HexNAc(1), Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1), di-Sialyl T antigen: Hex(1)HexNAc (1) The method according to (1) or (2), which is NeuAc (2), fucosylated T antigen: Hex (1) HexNAc (1) dHex (1), or a combination thereof.
(4) The method according to (3), wherein the O-glycan modified protein is at least one selected from the group consisting of (i) to (viii) below.
(i) T antigen: FBLN2 with Hex(1)HexNAc(1) added,
(ii) Sialyl T antigen: ANGPTL3 with Hex(1) HexNAc(1) NeuAc(1) and/or di-Sialyl T antigen Hex(1) HexNAc(1) NeuAc(2) added;
(iii) Sialyl T antigen: Hex(1) HexNAc(1) NeuAc(1) and/or di-Sialyl T antigen: MAN2A1 with Hex(1) HexNAc(1) NeuAc(2) added;
(iv) T antigen: CSF1 with Hex(1) HexNAc(1) added and CSF1 with di-Sialyl T antigen Hex(1) HexNAc(1) NeuAc(2) added,
(v) T antigen: VCAN with Hex(1) HexNAc(1) added;
(vi) Fucosylated T antigen: C1QA with Hex(1) HexNAc(1) dHex(1) added
(vii) T antigen: MRC1 with Hex(1)HexNAc(1) added
(viii) FGA with Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1) and/or di-Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(2)
(5) The method according to (4), wherein the protein modified with an O-type sugar chain is at least one selected from the group consisting of (ia) to (viiia) below.
(ia) FBLN2 with T antigen: Hex(1) HexNAc(1) added to at least one region of aa330-349, aa350-364 and aa365-378;
(iia) Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1) and/or di-Sialyl T antigen Hex(1)HexNAc( 1) ANGPTL3 with NeuAc(2) added,
(iiia) Sialyl T antigen: Hex(1) HexNAc(1) NeuAc(1) and/or di-Sialyl T antigen Hex(1) HexNAc(1) NeuAc(2) is added to the site within the aa211-221 region. MAN2A1,
(iva) T antigen in the aa356-395 region: CSF1 with Hex(1)HexNAc(1) and di-Sialyl T antigen in the region 370-395 Hex(1)HexNAc(1)NeuAc CSF1 with (2) added,
(va) VCAN with T antigen: Hex(1) HexNAc(1) added to at least one region of aa1415-1430 and aa2468-2475, and
(via) C1QA with fucosylated T antigen: Hex(1)HexNAc(1)dHex(1) added to the aa101-121 region
(viia) MRC1 with T antigen: Hex(1) HexNAc(1) added to the site within the aa1215-1229 region, and
(viiia) Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1) and/or di-Sialyl T antigen Hex(1)HexNAc( 1)FGA with NeuAc(2) added
(6) The method according to any one of (1) to (5), wherein the biological sample is a cell, tissue, body fluid, peritoneal fluid, peritoneal wash, feces, or urine.
(7) The method according to (6), wherein the body fluid is blood.
(8) The method according to (7), wherein the blood is whole blood, serum, plasma, or a plasma exchange solution.
(9) The method according to any one of (1) to (8), wherein the cancer is colon cancer.
(10) A test kit comprising a reagent capable of specifically detecting at least one O-type glycosylated protein selected from the group consisting of (i) to (viii) below.
(i) T antigen: FBLN2 with Hex(1)HexNAc(1) added,
(ii) Sialyl T antigen: ANGPTL3 with Hex(1) HexNAc(1) NeuAc(1) and/or di-Sialyl T antigen Hex(1) HexNAc(1) NeuAc(2) added;
(iii) Sialyl T antigen: MAN2A1 with Hex(1) HexNAc(1) NeuAc(1) and/or di-Sialyl T antigen Hex(1) HexNAc(1) NeuAc(2) added;
(iv) T antigen: CSF1 with Hex(1) HexNAc(1) added and CSF1 with di-Sialyl T antigen Hex(1) HexNAc(1) NeuAc(2) added,
(v) T antigen: VCAN with Hex(1) HexNAc(1) added, and
(v) Fucosylated T antigen: C1QA with Hex(1) HexNAc(1) dHex(1) added
(vii) T antigen: MRC1 with Hex(1)HexNAc(1) added
(viii) FGA with Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1) and/or di-Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(2)
本発明により、新たな腫瘍マーカーが同定された。これらを単独、または組み合わせで用いることにより、糖鎖部分のみを認識抗原とする従来の腫瘍マーカーと比べ、感度と特異度に優れた診断が補助される。 A new tumor marker has been identified according to the present invention. By using these alone or in combination, diagnosis with superior sensitivity and specificity can be aided compared to conventional tumor markers that recognize only sugar chain moieties as antigens.
以下、本発明を詳細に説明する。 The present invention will be explained in detail below.
本発明は、被験者由来の生物学的試料におけるO-型糖鎖で修飾されたタンパク質のレベルを測定することを含む、がんの診断を補助するための方法であって、タンパク質が、FBLN2(Fibulin-2)、ANGPTL3(Angiopoietin-related protein 3)、MAN2A1(Alpha-mannosidase 2)、CSF1(Macrophage colony-stimulating factor 1)、VCAN(Versican core protein)、C1QA(Complement C1q subcomponent subunit A) 、MRC1 （Macrophage mannose receptor 1 ）及びFGA（Fibrinogen alpha chain）からなる群より選択される少なくとも１つである前記方法を提供する。からなる群より選択される少なくとも１つである前記方法を提供する。 The present invention is a method for assisting in cancer diagnosis, which includes measuring the level of a protein modified with an O-glycan in a biological sample derived from a subject, and the method comprises: measuring the level of a protein modified with an O-glycan; Fibulin-2), ANGPTL3 (Angiopoietin-related protein 3), MAN2A1 (Alpha-mannosidase 2), CSF1 (Macrophage colony-stimulating factor 1), VCAN (Versican core protein), C1QA (Complement C1q subcomponent subunit A), MRC1 ( Macrophage mannose receptor 1) and FGA (Fibrinogen alpha chain). The method is at least one selected from the group consisting of:
FBLN2(Fibulin-2)は、フィブリンファミリーに属する細胞外マトリックスタンパク質である。さまざまな細胞外リガンドやカルシウムに結合する。がん遺伝子としても知られており、FBLN2遺伝子の発現変動は癌の発生と進行と関連しているとされる。[UniProtKB - P98095] FBLN2 (Fibulin-2) is an extracellular matrix protein belonging to the fibrin family. Binds to various extracellular ligands and calcium. It is also known as an oncogene, and fluctuations in the expression of the FBLN2 gene are said to be associated with the development and progression of cancer. [UniProtKB - P98095]
ANGPTL3(Angiopoietin-related protein 3)は、主に肝臓で発現する多機能分泌タンパク質である。脂質代謝や血管新生に関与することで発癌との関連性が指摘されている。[UniProtKB - Q9Y5C1] ANGPTL3 (Angiopoietin-related protein 3) is a multifunctional secreted protein mainly expressed in the liver. It has been pointed out that it is related to carcinogenesis due to its involvement in lipid metabolism and angiogenesis. [UniProtKB - Q9Y5C1]
MAN2A1(Alpha-mannosidase 2)は、ゴルジ体に局在し、N-型糖鎖合成経路の最終的な加水分解ステップを触媒する酵素である。[UniProtKB - Q16706] MAN2A1 (Alpha-mannosidase 2) is an enzyme localized in the Golgi apparatus that catalyzes the final hydrolysis step of the N-glycan synthesis pathway. [UniProtKB - Q16706]
CSF1(Macrophage colony-stimulating factor 1)は、マクロファージの産生、分化、および機能を制御するサイトカインである。前駆体は膜アンカー型であるが、シェディングにより細胞外へ分泌される（分泌型；活性型）。抗腫瘍マクロファージ上のCSF1レセプターと結合することで腫瘍関連マクロファージへの分化が促進される。がんの予後不良との関連性が指摘されている。[UniProtKB - P09603] CSF1 (Macrophage colony-stimulating factor 1) is a cytokine that controls macrophage production, differentiation, and function. Although the precursor is a membrane-anchored type, it is secreted outside the cell by shedding (secretory type; active type). Binding to the CSF1 receptor on anti-tumor macrophages promotes their differentiation into tumor-associated macrophages. It has been pointed out that it is associated with poor prognosis of cancer. [UniProtKB - P09603]
VCAN(Versican core protein)は、aggrecan/versicanプロテオグリカンファミリーに属するコンドロイチン硫酸プロテオグリカンであり、細胞外マトリックスの主成分として知られる。細胞接着、増殖、増殖、遊走、血管新生に関与し、組織の形態形成と維持に中心的な役割を果たす。[UniprotKB- P13611] VCAN (Versican core protein) is a chondroitin sulfate proteoglycan that belongs to the aggrecan/versican proteoglycan family and is known as a main component of the extracellular matrix. It is involved in cell adhesion, proliferation, proliferation, migration, and angiogenesis, and plays a central role in tissue morphogenesis and maintenance. [UniprotKB-P13611]
C1QA(Complement C1q subcomponent subunit A) は、補体タンパク質であり細胞外に分泌される。さまざまな種類のヒト腫瘍の微小環境で高度に発現しており、腫瘍の成長に密接に関与することが知られる。[UniProtKB - P02745] C1QA (Complement C1q subcomponent subunit A) is a complement protein and is secreted outside the cell. It is highly expressed in the microenvironment of various types of human tumors and is known to be closely involved in tumor growth. [UniProtKB - P02745]
MRC1 （Macrophage mannose receptor 1 ）は、マクロファージによる糖タンパク質のエンドサイトーシスを媒介する I 型膜受容体である。MRC1を発現する腫瘍関連マクロファージは、腫瘍の免疫抑制、血管新生、転移、および再発に関与することが知られている。[UniProtKB - P22897] MRC1 (Macrophage mannose receptor 1) is a type I membrane receptor that mediates endocytosis of glycoproteins by macrophages. Tumor-associated macrophages expressing MRC1 are known to be involved in tumor immunosuppression, angiogenesis, metastasis, and recurrence. [UniProtKB - P22897]
FGA（Fibrinogen alpha chain）は、フィブリノーゲンベータ (FGB) およびフィブリノーゲンガンマ (FGG) と共に不溶性フィブリンマトリックスを形成する、血栓の主要成分として知られている。[UniProtKB - P02671] FGA (Fibrinogen alpha chain) is known as a major component of blood clots, forming an insoluble fibrin matrix together with fibrinogen beta (FGB) and fibrinogen gamma (FGG). [UniProtKB - P02671]
本発明において、O-型糖鎖で修飾されたタンパク質のレベルが高い場合に、がんに罹患している可能性が高いことを示し、O-型糖鎖で修飾されたタンパク質のレベルが低い場合に、がんに罹患している可能性が低いことを示しうる。 In the present invention, when the level of a protein modified with an O-type sugar chain is high, it indicates that there is a high possibility of suffering from cancer, and when the level of a protein modified with an O-type sugar chain is low. In some cases, it may indicate that the possibility of having cancer is low.
O-型糖鎖は、T抗原：Hex(1)HexNAc(1)、Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)、di-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)、フコシル化T抗原：Hex(1)HexNAc(1)dHex(1)又はそれらの組み合わせであるとよい。 O-type sugar chains include T antigen: Hex(1)HexNAc(1), Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1), di-Sialyl T antigen: Hex(1)HexNAc(1) NeuAc(2), fucosylated T antigen: Hex(1) HexNAc(1) dHex(1), or a combination thereof.
O-型糖鎖で修飾されたタンパク質は、下記の(i)～(v)からなる群より選択される少なくとも１つでありうる。
(i) T抗原：Hex(1)HexNAc(1)が付加されたFBLN2、
(ii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)が付加されたANGPTL3、
(iii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)が付加されたMAN2A1、
(iv) T抗原：Hex(1)HexNAc(1)が付加されたCSF1及びdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたCSF1、
(v) T抗原：Hex(1)HexNAc(1)が付加されたVCAN、並びに
(vi) フコシル化T抗原：Hex(1)HexNAc(1)dHex(1)が付加されたC1QA
(vii) T抗原：Hex(1)HexNAc(1)が付加されたMRC1
(viii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)が付加されたFGA
上記(i)のT抗原：Hex(1)HexNAc(1)が付加されたFBLN2は、aa330-349、aa350-364及びaa365-378のうち少なくとも１つの領域内の部位にT抗原が付加されたFBLN2でありうる。FBLN2のaa330-349、aa350-364及びaa365-378の各領域内の部位には、それぞれ、１個（箇所）のT抗原が付加しうる。例えば、aa330-349領域内の部位に1個（箇所）のT抗原が付加されたFBLN2などががん患者の血清において同定されている(Table 1)。
The protein modified with an O-type sugar chain may be at least one selected from the group consisting of (i) to (v) below.
(i) T antigen: FBLN2 with Hex(1)HexNAc(1) added,
(ii) Sialyl T antigen: Hex(1) HexNAc(1) NeuAc(1) and/or di-Sialyl T antigen: ANGPTL3 with Hex(1) HexNAc(1) NeuAc(2) added;
(iii) Sialyl T antigen: Hex(1) HexNAc(1) NeuAc(1) and/or di-Sialyl T antigen: MAN2A1 with Hex(1) HexNAc(1) NeuAc(2) added;
(iv) T antigen: CSF1 with Hex(1) HexNAc(1) added and CSF1 with di-Sialyl T antigen Hex(1) HexNAc(1) NeuAc(2) added,
(v) T antigen: VCAN with Hex(1) HexNAc(1) added, and
(vi) Fucosylated T antigen: C1QA with Hex(1) HexNAc(1) dHex(1) added
(vii) T antigen: MRC1 with Hex(1)HexNAc(1) added
(viii) FGA with Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1) and/or di-Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(2)
The FBLN2 to which T antigen: Hex(1) HexNAc(1) in (i) above has T antigen added to at least one region of aa330-349, aa350-364, and aa365-378. Could be FBLN2. One T antigen can be added to each site in the aa330-349, aa350-364, and aa365-378 regions of FBLN2. For example, FBLN2, which has one T antigen added to the aa330-349 region, has been identified in the serum of cancer patients (Table 1).
上記(ii)のSialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)が付加されたANGPTL3は、aa206-235のうち少なくとも１つの部位にSialyl T抗原及び/又はdi-Sialyl Tが付加されたANGPTL3でありうる。ANGPTL3のaa206-221及びaa222-235の各領域の部位には、それぞれ、１個～３個（箇所）のSialyl T抗原又は1個（箇所）di-Sialyl T抗原が付加しうる。例えば、aa206-221領域内の部位に１個（箇所）のSialyl T抗原が付加されたANGPTL3、aa222-235領域内の部位に2個（箇所）のSialyl T抗原が付加されたANGPTL3などががん患者の血清において同定されている(Table 1)。 ANGPTL3 to which Sialyl T antigen of (ii) above: Hex(1) HexNAc(1) NeuAc(1) and/or di-Sialyl T antigen: Hex(1) HexNAc(1) NeuAc(2) is added is aa206 It may be ANGPTL3 with Sialyl T antigen and/or di-Sialyl T added to at least one site of -235. One to three Sialyl T antigens or one di-Sialyl T antigen can be added to each of the aa206-221 and aa222-235 regions of ANGPTL3. For example, ANGPTL3 with one Sialyl T antigen added to the site within the aa206-221 region, ANGPTL3 with two Sialyl T antigens added to the site within the aa222-235 region, etc. It has been identified in the serum of patients with cancer (Table 1).
上記(iii)のSialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)が付加されたMAN2A1は、aa211-221領域内の部位にSialyl T抗原及び/又はdi-Sialyl T抗原が付加されたMAN2A1でありうる。MAN2A1のaa211-221領域内の部位には、0個～2個（箇所）のSialyl T抗原、0個～1個（箇所）のdi-Sialyl T抗原が付加しうる。例えば、aa211-221領域内の部位に1個（箇所）のSialyl T抗原が付加されたMAN2A1や1個（箇所）のdi-Sialyl T抗原が付加されたMAN2A1などががん患者の血清において同定されている(Table 1)。 MAN2A1 to which Sialyl T antigen of (iii) above: Hex(1) HexNAc(1) NeuAc(1) and/or di-Sialyl T antigen: Hex(1) HexNAc(1) NeuAc(2) is added is aa211 It may be MAN2A1 with Sialyl T antigen and/or di-Sialyl T antigen added to the -221 region. 0 to 2 (positions) Sialyl T antigen and 0 to 1 (position) di-Sialyl T antigen can be added to sites within the aa211-221 region of MAN2A1. For example, MAN2A1 with one Sialyl T antigen attached to the aa211-221 region and MAN2A1 with one di-Sialyl T antigen attached in the aa211-221 region were identified in the serum of cancer patients. (Table 1).
上記(iv)のT抗原：Hex(1)HexNAc(1) 及び/又はdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたCSF1は、aa356-395領域内の部位にT抗原が付加されたCSF1、又はaa370-395領域内の部位にdi-Sialyl T抗原が付加されたCSF1でありうる。CSF1のaa356-395領域内の部位には、1個～3個（箇所）のT抗原、aa370-395領域内の部位には、0~1個（箇所）のdi-Sialyl T抗原が付加しうる。例えば、aa356-369領域内の部位に1~2個（箇所）のT抗原が付加されたCSF1、aa370-386領域内の部位に1個（箇所）のdi-Sialyl T抗原が付加されたCSF1、aa370-395領域内の部位にT抗原とdi-Sialyl T抗原が各1個（箇所）ずつ付加されたCSF1などががん患者の血清において同定されている(Table 1)。 The T antigen in (iv) above: Hex(1) HexNAc(1) and/or di-Sialyl T antigen Hex(1) HexNAc(1) NeuAc(2) is added to CSF1 at a site within the aa356-395 region. It may be CSF1 with T antigen added to, or CSF1 with di-Sialyl T antigen added to a site within the aa370-395 region. 1 to 3 T antigens are added to the aa356-395 region of CSF1, and 0 to 1 di-Sialyl T antigen is added to the aa370-395 region. sell. For example, CSF1 with 1 to 2 T antigens added to a site within the aa356-369 region, CSF1 with 1 di-Sialyl T antigen added to a site within the aa370-386 region , CSF1, which has one T antigen and one di-Sialyl T antigen added to the aa370-395 region, has been identified in the serum of cancer patients (Table 1).
上記(v)のT抗原：Hex(1)HexNAc(1)が付加されたVCANは、aa1415-1430及びaa2468-2475のうちの少なくとも１つの領域内の部位にT抗原：Hex(1)HexNAc(1)が付加されたVCANでありうる。VCANのaa1415-1430領域内の部位には、1個（箇所）のT抗原が付加しうる。VCANのaa2468-2475領域内の部位には、1個（箇所）のT抗原が付加しうる。例えば、aa1415-1430領域内の部位に１個（箇所）のT抗原が付加されたVCAN、aa2468-2475領域内の部位に１個（箇所）のT抗原が付加されたVCANなどががん患者の血清において同定されている(Table 1)。 The above (v) T antigen: Hex(1) HexNAc(1) is added to the VCAN at a site within at least one region of aa1415-1430 and aa2468-2475. It can be a VCAN with 1) added. One T antigen can be added to a site within the aa1415-1430 region of VCAN. One T antigen can be added to a site within the aa2468-2475 region of VCAN. For example, VCAN with one T antigen added to a site within the aa1415-1430 region, VCAN with one T antigen added to a site within the aa2468-2475 region, etc. are cancer patients. have been identified in the serum of patients (Table 1).
上記の(vi)のフコシル化T抗原：Hex(1)HexNAc(1)dHex(1)が付加されたC1QAは、aa101-121領域内の部位にフコシル化T抗原が付加されたC1QAでありうる。C1QAの、aa101-121領域内の部位には、２個（箇所）のフコシル化T抗原が付加しうる。aa101-121領域内の部位に２個（箇所）のフコシル化T抗原が付加されたC1QAががん患者の血清において同定されている(Table 1)。 The above (vi) fucosylated T antigen: C1QA to which Hex(1) HexNAc(1) dHex(1) is added may be C1QA to which fucosylated T antigen is added to the site within the aa101-121 region. . Two fucosylated T antigens can be added to sites within the aa101-121 region of C1QA. C1QA, which has two fucosylated T antigens added to the aa101-121 region, has been identified in the serum of cancer patients (Table 1).
上記の（vii）のT抗原：Hex(1)HexNAc(1)が付加されたMRC1は、aa1215-1229領域内の部位にT抗原が付加されたMRC1でありうる。MRC1の、aa1215-1229領域内の部位には、1個（箇所）のT抗原が付加しうる。aa1215-1229領域内の部位に１個（箇所）のT抗原が付加されたMRC1ががん患者の血清において同定されている。（Table ２） The MRC1 to which T antigen: Hex(1)HexNAc(1) in (vii) above is added may be MRC1 to which T antigen is added to a site within the aa1215-1229 region. One T antigen can be added to a site within the aa1215-1229 region of MRC1. MRC1 with one T antigen attached at a site within the aa1215-1229 region has been identified in the serum of cancer patients. (Table 2)
上記の（viii）のSialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)が付加されたFGAは、aa354-367及びaa511-527のうち少なくとも１つの領域内の部位にSialyl T抗原及び/又はdi-Sialyl T抗原が付加されたFGAであり得る。FGAの、aa354-367領域内の部位には、少なくとも1個（箇所）のSialyl T抗原が付加しうる。aa511-527領域内の部位には、少なくとも1個（箇所）のdi-Sialyl T抗原が付加しうる。例えば、aa354-367領域内の部位に１個（箇所）のSialyl T抗原が付加されたFGA、aa511-527領域内の部位に１個（箇所）のdi-Sialyl T抗原が付加されたFGAなどががん患者の血清において同定されている(Table 2)。 The above (viii) Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1) and/or di-Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(2) is added to FGA, It may be an FGA in which Sialyl T antigen and/or di-Sialyl T antigen is added to a site within at least one region of aa354-367 and aa511-527. At least one Sialyl T antigen can be added to a site within the aa354-367 region of FGA. At least one di-Sialyl T antigen can be added to a site within the aa511-527 region. For example, FGA with one Sialyl T antigen added to a site within the aa354-367 region, FGA with one di-Sialyl T antigen added to a site within the aa511-527 region, etc. have been identified in the serum of cancer patients (Table 2).
本発明の方法は、被験者由来の生物学的試料におけるO-型糖鎖で修飾されたタンパク質のレベルを測定することを含む。 The method of the present invention includes measuring the level of a protein modified with an O-glycan in a biological sample derived from a subject.
本発明の方法において、被験者はがんへの罹患（がんの再発も含む）が疑われる患者であるが、発病の危険性が考えられるすべてのヒトを対象としてもよい。被験者は、がんの治療中あるいは治療後の患者であってもよい。 In the method of the present invention, the subject is a patient who is suspected of having cancer (including cancer recurrence), but the subject may be any human who is considered to be at risk of developing the disease. The subject may be a patient undergoing or following cancer treatment.
本発明の方法において、生物学的試料は、細胞、組織、体液、腹腔液、腹腔洗浄液、糞便又は尿でありうる。体液は、血液であるとよく、血液は、全血、血清、血漿又は血漿交換外液のいずれかであるとよい。 In the method of the invention, the biological sample can be a cell, tissue, body fluid, peritoneal fluid, peritoneal wash, feces or urine. The body fluid may be blood, and the blood may be whole blood, serum, plasma, or plasmapheresis fluid.
O-型糖鎖で修飾されたタンパク質のレベルの測定は、O-型糖鎖で修飾されたタンパク質の有無を検出してもよいし、O-型糖鎖で修飾されたタンパク質の量（濃度）を測定してもよい。 The level of protein modified with O-glycans can be measured by detecting the presence or absence of proteins modified with O-glycans, or by detecting the amount (concentration) of proteins modified with O-glycans. ) may be measured.
O-型糖鎖で修飾されたタンパク質のレベルの測定には、糖タンパク質を検出できるいかなる方法を用いてもよく、例えば、抗タンパク質抗体と糖鎖検出用レクチンを用いたサンドイッチELISA系、質量分析法などを用いることができる。 To measure the level of proteins modified with O-glycans, any method that can detect glycoproteins may be used, such as a sandwich ELISA system using an anti-protein antibody and a lectin for detecting sugar chains, mass spectrometry. The law can be used.
抗タンパク質抗体と糖鎖検出用レクチンを用いたサンドイッチELISA系により、O-型糖鎖修飾タンパク質のレベルを測定するには、例えば、固定化されたレクチンに試料中のO-型糖鎖修飾タンパク質を反応させて、複合体を形成させる。次いで蛍光標識抗タンパク質抗体と反応させ、レクチン－O-型糖鎖修飾タンパク質－抗タンパク質抗体複合体を蛍光で検出する。 To measure the level of O-type glycosylated proteins using a sandwich ELISA system using an anti-protein antibody and lectin for sugar chain detection, for example, inject O-type glycosylated proteins in the sample onto immobilized lectin. react to form a complex. Next, it is reacted with a fluorescently labeled anti-protein antibody, and the lectin-O-type sugar chain modified protein-anti-protein antibody complex is detected by fluorescence.
抗FBLN2抗体、抗ANGPTL3抗体、抗MAN2A1抗体、抗CSF1抗体、抗C1QA抗体、抗VCAN抗体、抗MRC1抗体および抗FGA抗体は市販されており、その市販の抗体を用いてもよいし、公知の方法によりモノクローナル抗体あるいはポリクローナル抗体を作製して、それを用いてもよい。T抗原検出用レクチンとしては、オーサージオレンジレクチン (Maclura pomifera lectin; MPL/MPA)やピーナッツレクチン (Peanut lectin; PNA) 、Sialyl T抗原検出用レクチンとしては、ジャックフルーツレクチン（Artocarpus integrifolia lectin ; Jacalin）又はエルダベリーレクチン(Sambucus Nigra lectin; SNL/SNA/EBL)、di-Sialyl T抗原検出用レクチンとしては、Jacalinやイヌエンジュレクチン（Maackia amurensis; MAH）などを用いることができる。 Anti-FBLN2 antibodies, anti-ANGPTL3 antibodies, anti-MAN2A1 antibodies, anti-CSF1 antibodies, anti-C1QA antibodies, anti-VCAN antibodies, anti-MRC1 antibodies, and anti-FGA antibodies are commercially available, and these commercially available antibodies may be used, or known A monoclonal antibody or a polyclonal antibody may be produced by a method and used. Lectins for detecting T antigen include Osage orange lectin (Maclura pomifera lectin; MPL/MPA) and peanut lectin (PNA); lectins for detecting Sialyl T antigen include jackfruit lectin (Artocarpus integrifolia lectin; Jacalin). Alternatively, as the lectin for detecting elderberry lectin (Sambucus Nigra lectin; SNL/SNA/EBL), di-Sialyl T antigen, Jacalin, dog lectin (Maackia amurensis; MAH), etc. can be used.
質量分析法により、O-型糖鎖修飾タンパク質のレベルを測定するには、例えば、図１に示した通り、MPL、Jacalin、SNLレクチンを用いて連続的に精製されたO-型糖鎖修飾タンパク質のトリプシン消化物から、アセトン濃縮法とGlycOCATCH法によりO-型糖ペプチドを濃縮し、Combineされた濃縮物のLC/MS/MSを異なる開裂エネルギーで２回実施し、スペクトルデータを得て、O-型糖ペプチド由来のシグナルのPeak Area値を出すとよい。Peak Area値がカットオフ値よりも高ければ、がんに罹患している可能性が高く、Peak Area値がカットオフ値よりも低ければ、がんに罹患している可能性が低いと判定することができる。カットオフ値は、ROC曲線を用い、感度と特異度を考慮して、設定することができる。質量分析法においては、例えば、m/z204 (HexNAc)、274/292 (NeuAc)、366(HexNAc-Hex)を糖鎖の診断イオンとして利用できる。 To measure the levels of O-glycan modified proteins by mass spectrometry, for example, as shown in Figure 1, O-glycan modified proteins that were sequentially purified using MPL, Jacalin, and SNL lectin. O-type glycopeptides were concentrated from tryptic digests of proteins using the acetone concentration method and the GlycOCATCH method, and LC/MS/MS of the combined concentrate was performed twice at different cleavage energies to obtain spectral data. It is best to calculate the Peak Area value of the signal derived from O-type glycopeptide. If the Peak Area value is higher than the cutoff value, it is determined that the possibility of having cancer is high, and if the Peak Area value is lower than the cutoff value, it is determined that the possibility of having cancer is low. be able to. The cutoff value can be set using an ROC curve and taking sensitivity and specificity into consideration. In mass spectrometry, for example, m/z 204 (HexNAc), 274/292 (NeuAc), and 366 (HexNAc-Hex) can be used as diagnostic ions for sugar chains.
本発明において、診断を補助する対象となるがんは、大腸がんであるとよい。がんは進行性あるいは再発がんであってもよい。 In the present invention, the cancer to be assisted in diagnosis is preferably colon cancer. The cancer may be an advanced or recurrent cancer.
本発明の方法により、がんに罹患している可能性が高いことが示された場合、被験者にはがんの治療を施すとよい。がんの治療法としては、手術（外科治療）、薬物療法、放射線治療、内視鏡治療、造血幹細胞移植、免疫療法、がんゲノム医療、リハビリテーション、緩和ケアなどがあり、これらの治療法を単独で、あるいは組み合わせて治療するとよい。大腸がんの治療には、内視鏡治療、手術、薬物療法、放射線治療などを単独で、あるいは組み合わせて行うとよい。大腸がんに対する薬物療法には、１）手術後の再発を防ぐ目的で行う「補助化学療法」、２）手術による治癒が難しく、症状を緩和する目的で行う「切除不能進行・再発大腸がんに対する薬物療法」の2つがある。補助化学療法としては、カペシタビン（ゼローダ）、テガフール・ウラシル配合剤（UFT：ユーエフティ）、テガフール・ギメラシル・オテラシルカリウム配合剤（TS-1：ティーエスワン）や、点滴で行うフォルフォックス（FOLFOX）療法（5-FU（点滴）とレボホリナート（l-ロイコボリン、アイソボリン）にオキサリプラチンを組み合わせる）、飲み薬と点滴を組み合わせるカポックス（CAPOX：カペシタビンとオキサリプラチンの併用）療法が推奨されている。切除不能進行・再発大腸がんに対する薬物療法には、一次治療から五次治療まであり、まずは一次治療から開始し、効果が低下した場合は二次、三次と順に治療を続けていくとよい。一次治療、二次治療に使用する薬としては、フルオロウラシル（5-FU：点滴）、カペシタビン（ゼローダ）、テガフール・ギメラシル・オテラシルカリウム配合剤（TS-1）、フォルフォックス（FOLFOX）療法、カポックス（CAPOX）療法、ソックス（SOX）療法（オキサプラチンとS-1の組み合わせ）、フォルフィリ（FOLFIRI）療法（オキサプラチン、フルオロウラシル、レボホリナート、イリノテカンの組み合わせ）、アイリス（IRIS）療法（TS-1とイリノテカンの組み合わせ）、カピリ（CAPIRI）療法（カペシタビンとイリノテカンの組み合わせ）、ベバシズマブ（アバスチン）、ラムシルマブ（サイラムザ）、アフリベルセプト（ザルトラップ）、セツキシマブ（アービタックス）/パニツムマブ（ベクティビックス）などがあり、三次治療以降に使用する薬としては、トリフルリジン/チピラシル塩酸塩（ロンサーフ）、レゴラフェニブ（スチバーガ）、ペムブロリズマブ（キイトルーダ）などの免疫チェックポイント阻害剤などがある。 If the method of the present invention indicates that the subject is likely to be suffering from cancer, the subject may be treated for cancer. Treatment methods for cancer include surgery (surgical treatment), drug therapy, radiation therapy, endoscopic therapy, hematopoietic stem cell transplantation, immunotherapy, cancer genomic medicine, rehabilitation, and palliative care. May be treated alone or in combination. For the treatment of colorectal cancer, endoscopic therapy, surgery, drug therapy, radiation therapy, etc. may be performed alone or in combination. Drug therapy for colorectal cancer includes 1) "adjuvant chemotherapy" to prevent recurrence after surgery, and 2) "unresectable advanced/recurrent colorectal cancer" to treat symptoms that are difficult to cure and alleviate symptoms. There are two types of drug therapy. Adjuvant chemotherapy includes capecitabine (Xeloda), tegafur/uracil combination (UFT), tegafur/gimeracil/oteracil potassium combination (TS-1), and intravenous FOLFOX. (5-FU (intravenous) and levofolinate (l-leucovorin, isovorin) combined with oxaliplatin) and CAPOX (capecitabine and oxaliplatin combination) therapy, which combines oral medication and infusion, are recommended. Drug therapy for unresectable advanced/recurrent colorectal cancer ranges from first-line to fifth-line treatment, and it is best to start with first-line therapy and, if the efficacy decreases, continue with second- and third-line treatments. Medications used for primary and secondary treatment include fluorouracil (5-FU (intravenous)), capecitabine (Xeloda), tegafur/gimeracil/oteracil potassium combination (TS-1), FOLFOX therapy, and capox. (CAPOX) therapy, SOX therapy (combination of oxaplatin and S-1), FOLFIRI therapy (combination of oxaplatin, fluorouracil, levofolinate, and irinotecan), IRIS therapy (TS-1 and irinotecan), CAPIRI therapy (a combination of capecitabine and irinotecan), bevacizumab (Avastin), ramucirumab (Cyramza), aflibercept (Zaltrap), and cetuximab (Erbitux)/panitumumab (Vectibix). Third-line and subsequent treatments include immune checkpoint inhibitors such as trifluridine/tipiracil hydrochloride (Lonsurf), regorafenib (Stivarga), and pembrolizumab (Keytruda).
本発明の方法は、がんの治療効果、がんの再発や進行、がんの転移リスクの評価のために実施してもよく、がんの治療方針の決定などにも利用することができる。 The method of the present invention may be carried out to evaluate cancer treatment effects, cancer recurrence and progression, and cancer metastasis risk, and can also be used to determine cancer treatment policies. .
また、本発明は、下記の(i)～(v)からなる群より選択される少なくとも１つのO-型糖鎖修飾タンパク質を特異的に検出することができる試薬を含む、検査キットを提供する。
(i) T抗原：Hex(1)HexNAc(1)が付加されたFBLN2、
(ii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1) 及び/又はdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたが付加されたANGPTL3、
(iii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたMAN2A1、
(iv) T抗原：Hex(1)HexNAc(1)が付加されたCSF1及びdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたCSF1、
(v) T抗原：Hex(1)HexNAc(1)が付加されたVCAN、
(vi) フコシル化T抗原：Hex(1)HexNAc(1)dHex(1)が付加されたC1QA、
(vii) T抗原：Hex(1)HexNAc(1)が付加されたMRC1、並びに
(viii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)が付加されたFGA
(i)、(ii)、(iii)、(iv)、(v)、(vi)、(vii)及び(viii)の糖鎖修飾タンパク質については、上述した。
Furthermore, the present invention provides a test kit containing a reagent capable of specifically detecting at least one O-type glycosylated protein selected from the group consisting of (i) to (v) below. .
(i) T antigen: FBLN2 with Hex(1)HexNAc(1) added,
(ii) Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1) and/or ANGPTL3 with di-Sialyl T antigen Hex(1)HexNAc(1)NeuAc(2) added;
(iii) Sialyl T antigen: MAN2A1 with Hex(1) HexNAc(1) NeuAc(1) and/or di-Sialyl T antigen Hex(1) HexNAc(1) NeuAc(2) added;
(iv) T antigen: CSF1 with Hex(1) HexNAc(1) added and CSF1 with di-Sialyl T antigen Hex(1) HexNAc(1) NeuAc(2) added,
(v) T antigen: VCAN with Hex(1)HexNAc(1) added;
(vi) Fucosylated T antigen: C1QA with Hex(1) HexNAc(1) dHex(1) added,
(vii) T antigen: MRC1 with Hex(1)HexNAc(1) added, and
(viii) FGA with Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1) and/or di-Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(2)
The glycosylated proteins (i), (ii), (iii), (iv), (v), (vi), (vii) and (viii) have been described above.
本発明の検査キットに含まれるO-型糖鎖修飾タンパク質を特異的に検出することができる試薬としては、抗タンパク質抗体と糖鎖検出用レクチンの組み合わせ、LC/MS/MSに用いる試薬などを挙げることができる。さらに、本発明の検査キットには、コントロール（例えば、がん患者と健常者由来の生物学的試料において差異がないことが期待される公知のタンパク質など）、希釈液、洗浄液、検出液、反応停止液、96-wellマイクロプレート、還元剤、アルキル化剤、消化酵素、レクチン、取扱説明書などが含まれてもよい。 Reagents that can specifically detect O-type glycan-modified proteins included in the test kit of the present invention include combinations of anti-protein antibodies and lectins for sugar chain detection, reagents used for LC/MS/MS, etc. can be mentioned. Furthermore, the test kit of the present invention includes a control (for example, a known protein that is expected to show no difference between biological samples derived from cancer patients and healthy individuals), a diluent, a washing solution, a detection solution, and a reaction solution. A stop solution, a 96-well microplate, a reducing agent, an alkylating agent, a digestive enzyme, a lectin, an instruction manual, and the like may be included.
本発明の検査キットは、がんの診断を補助することができ、がんの治療効果、がんの再発や進行、がんの転移リスクの評価、がんの治療方針の決定などにも利用することができる。
The test kit of the present invention can assist in cancer diagnosis, and can also be used to evaluate cancer treatment effects, cancer recurrence and progression, cancer metastasis risk, and determine cancer treatment policies. can do.
以下、実施例により本発明を更に詳細に説明する。
〔実施例１〕
1. Introduction
がんの再発と進行は大腸がん（CRC）の主な死因である。CRC再発・進行を伴う患者の予後は非常に多様である。転移リスクが高い患者の特定や、患者の状態に合わせて効果的な治療法選択を支援するための新たなバイオマーカーの導入が促されている。
Hereinafter, the present invention will be explained in more detail with reference to Examples.
[Example 1]
1. Introduction
Cancer recurrence and progression are the main causes of death in colorectal cancer (CRC). The prognosis of patients with CRC recurrence/progression is highly variable. New biomarkers are being introduced to help identify patients at high risk of metastasis and select effective treatments tailored to the patient's condition.
糖鎖は最も重要な翻訳後修飾の1つであり、コンセンサス配列（N-X-S/T, X≠P）のAsnに結合するN-型糖鎖と、Ser/Thrに結合するO-型糖鎖に大別される。O-型糖鎖（ムチン型糖鎖）付加はSer/ThrにN-アセチルガラクトサミン（GalNAc）が付加することで始まる。最初のO-GalNAc生合成ステップは20個のGalNAcトランスフェラーゼ（T1～T20）が担い、様々なトランスフェラーゼの介入により糖鎖の複雑性が増す。これらムチン型の糖鎖修飾は、血清に含まれる分泌型タンパク質でしばしば認められ、タンパク質の機能を微調整している(1, 2)。癌では、これらムチン型糖鎖修飾の変化が頻繁に観察される。特に短いムチン型糖鎖であるTn抗原、Sialyl Tn抗原、T抗原などの新発現（neo-expression）や過剰発現（over expression）は、タンパク質のコンフォメーションやシグナル伝達の変化と関連し、大腸がんを含む多くのがんで腫瘍の進行や悪性化に重要な役割を果たすことが示されている(3-9)。 Glycans are one of the most important post-translational modifications, with N-type glycans binding to Asn in the consensus sequence (N-X-S/T, X≠P) and O-type glycans binding to Ser/Thr Broadly classified. O-type glycan (mucin-type glycan) addition begins with the addition of N-acetylgalactosamine (GalNAc) to Ser/Thr. The first O-GalNAc biosynthesis step is carried out by 20 GalNAc transferases (T1 to T20), and the complexity of the glycan increases with the intervention of various transferases. These mucin-type glycosylation modifications are often observed on secreted proteins found in serum and fine-tune protein function (1, 2). Changes in mucin-type glycosylation are frequently observed in cancer. In particular, neo-expression and overexpression of short mucin-type sugar chains such as Tn antigen, Sialyl Tn antigen, and T antigen are associated with changes in protein conformation and signal transduction, and the large intestine It has been shown to play an important role in tumor progression and malignant transformation in many cancers, including cancer (3-9).
癌における糖鎖の変化は、癌の診断マーカーとして利用されてきた。その代表例が肝細胞がんマーカーalpha fetoprotein-L3 (AFP-L3)であろう。肝細胞がん特異的にコアフコシル化されたAFPに着目し、コアフコース認識レクチンLCAと抗AFP抗体を組み合わせて検出することが可能である(10-12)。また、AFPと同様、コアフコシル化されたhaptoglobinsは膵がんの有望な診断マーカー候補である(13)。大腸がんに関していえば、高度に糖鎖修飾されたタンパク質である癌胎児性抗原（carcinoembryonic antigen: CEA）と、シアリルルイスaを認識するCA19-9（carbohydrate antigen 19-9）が主に術前・術後診断マーカーとして利用されている。しかし、両者は共に感度が低く、診断における信頼性が大幅に制限されている(14)のが現状である。 Changes in sugar chains in cancer have been used as diagnostic markers for cancer. A typical example is the hepatocellular carcinoma marker alpha fetoprotein-L3 (AFP-L3). Focusing on AFP, which is core fucosylated specifically for hepatocellular carcinoma, it is possible to detect it by combining core fucose recognition lectin LCA and anti-AFP antibody (10-12). Also, like AFP, core fucosylated haptoglobins are promising diagnostic marker candidates for pancreatic cancer (13). Regarding colorectal cancer, carcinoembryonic antigen (CEA), which is a highly glycosylated protein, and CA19-9 (carbohydrate antigen 19-9), which recognizes sialyl Lewis a, are mainly used preoperatively. It is used as a postoperative diagnostic marker. However, both have low sensitivity and their diagnostic reliability is currently severely limited (14).
グライコプロテオミクス分野の多くの目標は、近年の質量分析技術の開発と改善によって達成されてきた。親水性相互作用クロマトグラフィー（HILIC）(15-16)やレクチン(17-19)、アセトン(20, 21)などで濃縮されてきたインタクトN-型糖ペプチドのLC/MS/MSは、糖鎖の組成と共に、付加部位とそのペプチド配列、タンパク質を網羅的に明らかにすることができる。その一方で、癌におけるムチン型糖鎖の変化、すなわち、非還元末端側へのシアル酸の付加のような繊細な変化や糖鎖修飾のダイナミックな変化をタンパク質と同時に特定した例は驚くほど少ない。これは、O-型糖鎖の不均一性、O-型糖鎖コンセンサス配列の欠如、およびO-型糖ペプチドを効率よく濃縮する方法が無かったことに起因する。臨床血清検体からのO-グライコプロテオミクス実現により、がんで新発現（neo-expression）や過剰発現したO-型糖鎖修飾タンパク質を明らかにできれば、転移リスクや治療効果の評価に大きな利益を与えるに違いない。 Many goals in the field of glycoproteomics have been achieved through the development and improvement of mass spectrometry techniques in recent years. LC/MS/MS of intact N-glycopeptides, which have been enriched using hydrophilic interaction chromatography (HILIC) (15-16), lectins (17-19), acetone (20, 21), etc. In addition to the composition, the addition site, its peptide sequence, and protein can be comprehensively clarified. On the other hand, there are surprisingly few cases in which changes in mucin-type glycans in cancer, such as delicate changes such as the addition of sialic acid to the non-reducing end, or dynamic changes in glycan modification, have been simultaneously identified in proteins. . This is due to the heterogeneity of O-glycans, the lack of an O-glycan consensus sequence, and the lack of a method to efficiently concentrate O-glycans. If O-glycoproteomics from clinical serum samples could reveal neo-expression or overexpressed O-glycosylated proteins in cancer, it would bring great benefits to the evaluation of metastatic risk and therapeutic efficacy. Must.
これらの洞察に基づいて、我々は、独自のO-型糖鎖解析プラットフォームを用いて、ムチン型糖鎖修飾がダイナミックに変化した血清タンパク質をスクリーニングし、進行・再発大腸がんにおける治療効果や患者層別化を可能にする新たな検査指標開発に向けた分子的根拠を得る。 Based on these insights, we used our unique O-glycan analysis platform to screen serum proteins with dynamically changed mucin-type glycosylation, and to improve treatment efficacy and patient outcomes in advanced and recurrent colorectal cancer. Obtain the molecular basis for developing new test indicators that enable stratification.
2. Materials and methods
2.1. General Information
使用した試薬はすべて質量分析グレードまたはそれに準ずるグレードであり、全てプロメガ、和光、関東化学から購入した。 ストレプトアビジン（SA）修飾磁気ビーズは、Vector Laboratories（Burlingame、UK）から購入した。 ビオチン標識MPL、Jacalin、SNLレクチンはVector Laboratoriesから入手した。
2. Materials and methods
2.1. General Information
All reagents used were mass spectrometry grade or equivalent, and were purchased from Promega, Wako, and Kanto Kagaku. Streptavidin (SA) modified magnetic beads were purchased from Vector Laboratories (Burlingame, UK). Biotin-labeled MPL, Jacalin, and SNL lectins were obtained from Vector Laboratories.
2.2. Clinical Specimens
再発・進行大腸がん関連O-型糖鎖修飾タンパク質のスクリーニングは、平均年齢68歳の患者から前向きに収集された血清試料10検体を用いて実施された。すべての手順は、患者の同意を得た後、横浜市立大学倫理委員会の承認の下で実施された。すべての臨床病理学的情報は、患者の臨床記録から得られた。
2.2. Clinical Specimens
Screening for O-glycosylated proteins associated with recurrent/advanced colorectal cancer was performed using 10 serum samples prospectively collected from patients with an average age of 68 years. All procedures were performed under the approval of the Yokohama City University Ethics Committee after obtaining patient consent. All clinicopathological information was obtained from the patients' clinical records.
2.3. Lectin affinityによるO-型糖タンパク質の精製
全ての血清タンパク質はBCA法で定量された。80μg相当の血清タンパク質はまずMPLレクチンで精製された。500 μlのTBS/Triton-x 100（TBS-Tx）で繰り返し洗浄された100 μlのストレプトアビジン―磁性粒子（Streptavidin magnetic beads: SAビーズ）に対し、195 μlのTBS-Txで希釈された10 μgのビオチン化MPL (5 μl slurry; Vector Laboratories)を加えて４℃, 1400×rpmで30 min反応させた。800 μlのTBS-Txで3回洗浄後、TBS-Txで希釈された400 μl のpre-clear済みの血清タンパク質を加え、４℃、1400×rpm で16h反応させた。80 μg相当の血清タンパク質のpre-clearは、50 μlのSAビーズに対して４℃、1400×rpmで30 min反応させることで実施した。MPL-非結合画分（上清）は新たなチューブに回収した。MPLビーズに結合した血清タンパク質は400 μlのTBS-Txで繰り返し洗浄された後、100 μlのTBS/0.2%SDS中で95℃加熱することで回収された。
2.3. Purification of O-type glycoproteins by lectin affinity All serum proteins were quantified using the BCA method. Serum proteins equivalent to 80 μg were first purified with MPL lectin. 10 μg diluted in 195 μl TBS-Tx to 100 μl streptavidin magnetic beads (SA beads) washed repeatedly with 500 μl TBS/Triton-x 100 (TBS-Tx). Biotinylated MPL (5 μl slurry; Vector Laboratories) was added and reacted at 4° C. and 1400×rpm for 30 min. After washing three times with 800 μl of TBS-Tx, 400 μl of pre-cleared serum protein diluted with TBS-Tx was added and reacted at 4° C. and 1400×rpm for 16 hours. Pre-clearing of serum proteins equivalent to 80 μg was performed by reacting 50 μl of SA beads at 4° C. and 1400×rpm for 30 min. The MPL-unbound fraction (supernatant) was collected in a new tube. Serum proteins bound to MPL beads were washed repeatedly with 400 μl of TBS-Tx and then recovered by heating at 95°C in 100 μl of TBS/0.2% SDS.
一方、回収されたMPL-非結合画分は、2nd Jacalin精製に供された。197.4 μlのTBS-Txで希釈された13 μgのビオチン化Jacalin (2.6μl slurry; Vector Laboratories)を加えて４℃, 1400×rpmで30 min反応させた。さらに、MPL/Jacalin非結合画分は3rd SNL精製に供した。197.5 μlのTBS-Txで希釈された7μgのビオチン化SNL (2.5μl slurry; Vector Laboratories)を加えて４℃、1400×rpmで30min反応させた。ビーズの洗浄およびレクチン結合タンパク質の回収はMPLと同じ条件で実施した。全ての溶出画分は同じチューブに結合された。 On the other hand, the recovered MPL-unbound fraction was subjected to 2nd Jacalin purification. 13 μg of biotinylated Jacalin (2.6 μl slurry; Vector Laboratories) diluted with 197.4 μl of TBS-Tx was added and reacted at 4° C. and 1400×rpm for 30 min. Furthermore, the MPL/Jacalin-unbound fraction was subjected to 3rd SNL purification. 7 μg of biotinylated SNL (2.5 μl slurry; Vector Laboratories) diluted with 197.5 μl of TBS-Tx was added and reacted at 4° C. and 1400× rpm for 30 min. Bead washing and lectin-binding protein recovery were performed under the same conditions as MPL. All elution fractions were combined into the same tube.
2.4. Glycopeptide enrichment
アセトンによる糖ペプチド濃縮法 [20]とGlycOCATCHのコンビネーション（Genovis）を用いた。3種類のLectinで連続的に精製されたO-型糖タンパク質は、3倍容のアセトンで－30℃、16h反応させた後に12,000×gで10 min遠心することで沈殿された。沈殿されたO-型糖タンパク質は、10 mM dithiothreitol (DTT)で56℃, 30 min処理することで還元され、20 mM ヨードアセトアミドにより25°C 、40 min 暗所でアルキル化された。その後、1.5 μg のトリプシンで37°C (800×rpm)、16h消化された。5倍容の冷アセトンを加えて激しく攪拌し、－30℃で16h静置した。Ｏ型糖ペプチドは、12,000×g で 10 min遠心し、沈殿させた。 上清は別チューブに回収しspeed vac.の後、TBS-Txに再溶解されGlycOCATCH（Genovis）精製に供された。
2.4. Glycopeptide enrichment
A combination of the glycopeptide enrichment method using acetone [20] and GlycOCATCH (Genovis) was used. O-type glycoproteins that were sequentially purified with three types of lectins were precipitated by reacting with 3 volumes of acetone at -30°C for 16 h and then centrifuging at 12,000 x g for 10 min. The precipitated O-glycoproteins were reduced by treatment with 10 mM dithiothreitol (DTT) at 56°C for 30 min and alkylated with 20 mM iodoacetamide at 25°C for 40 min in the dark. It was then digested with 1.5 μg of trypsin at 37°C (800× rpm) for 16 h. Five times the volume of cold acetone was added, stirred vigorously, and allowed to stand at -30°C for 16 hours. O-type glycopeptides were precipitated by centrifugation at 12,000×g for 10 min. The supernatant was collected into a separate tube and after speed vac., redissolved in TBS-Tx and subjected to GlycOCATCH (Genovis) purification.
GlycOCATCH によるＯ型糖ペプチド濃縮は、添付のプロトコルに準拠して実施した。アフィニティーレジンは、300 μlのTBSを用いて20°C、200 ×gで1 min遠心することで３回洗浄した。アセトンによる糖ペプチド濃縮で得られた上清（非糖ペプチド画分）は、speed vac.の後、TBS-Txに再溶解され、10 unitのSialEXOとともにGlycOCATCHアフィニティーレジンと2h室温で反応させた。1000×gで1min遠心することで非結合画分を取り除き、300 μlの0.5M塩化ナトリウム/TBSで3回洗浄した。GlycOCATCHレジン結合タンパク質は、50μlの8M尿素で5min振盪することで回収された。GlycOCATCH溶出画分はアセトン沈殿による糖ペプチド画分と結合し、GL-Tip SDB精製後、Speed Vac.した。 O-type glycopeptide enrichment using GlycOCATCH was performed according to the attached protocol. The affinity resin was washed three times with 300 μl of TBS by centrifugation at 20°C and 200 × g for 1 min. The supernatant (non-glycopeptide fraction) obtained by glycopeptide concentration with acetone was redissolved in TBS-Tx after speed vac., and reacted with GlycOCATCH affinity resin together with 10 units of SialEXO at room temperature for 2 hours. Unbound fractions were removed by centrifugation at 1000×g for 1 min and washed three times with 300 μl of 0.5M sodium chloride/TBS. GlycOCATCH resin-bound protein was recovered by shaking in 50 μl of 8M urea for 5 min. The GlycOCATCH elution fraction was combined with the glycopeptide fraction by acetone precipitation, purified with GL-Tip SDB, and then subjected to Speed Vac.
2.5. Liquid chromatography/mass spectrometry
ペプチドと糖ペプチドは L-column2 ODS trapping column (0.3×5 mm, 5 μm; Chemicals Evaluation and Research Institute, Tokyo, Japan) と Nano HPLC Capillary Column (75 μm×120 mm, 3 μm, C18; Nikkyo Technos, Tokyo, Japan)とを連結したEASY-nLC 1000 (Thermo Fisher Scientific) で分離した。溶媒は、0.1% v/v ギ酸/蒸留水 (pump A) と0.1% v/v ギ酸/アセトニトリル(pump B)を用いた。流速は0.3 μL/min とし、0 to 35% B （120 min）のリニアグラジェントで分離した。質量分析データは、イオン源としてNanospray Flex Ion Source (Thermo Fisher Scientific) を装備したQ Exactive mass spectrometer (Thermo Fisher Scientific) を用い、ポジティブイオンモードで測定することで獲得した。質量分析計の制御とデータ取得には、 Xcalibur 4.4 workstation (Thermo Fisher Scientific) を用いた。 スプレー電圧は2.0 kV, キャピラリー温度 250°C, S-lens RF レベルは50とした。マススペクトルはm/z 350-2000の範囲で、分解能70,000で取得した。プロダクトイオンスペクトルは、データ依存的取得法（Top 20）を用い分解能17,500で取得し、normalized collision energy (NCE) 値は 27 と35を用いた。
2.5. Liquid chromatography/mass spectrometry
Peptides and glycopeptides were analyzed using L-column2 ODS trapping column (0.3×5 mm, 5 μm; Chemicals Evaluation and Research Institute, Tokyo, Japan) and Nano HPLC Capillary Column (75 μm×120 mm, 3 μm, C18; Nikkyo Technos, Separation was performed using an EASY-nLC 1000 (Thermo Fisher Scientific) connected to Tokyo, Japan). The solvents used were 0.1% v/v formic acid/distilled water (pump A) and 0.1% v/v formic acid/acetonitrile (pump B). The flow rate was 0.3 μL/min, and separation was performed using a linear gradient of 0 to 35% B (120 min). Mass spectrometry data were acquired by measuring in positive ion mode using a Q Exactive mass spectrometer (Thermo Fisher Scientific) equipped with a Nanospray Flex Ion Source (Thermo Fisher Scientific) as an ion source. An Xcalibur 4.4 workstation (Thermo Fisher Scientific) was used to control the mass spectrometer and acquire data. The spray voltage was 2.0 kV, the capillary temperature was 250°C, and the S-lens RF level was 50. Mass spectra were acquired in the m/z 350-2000 range with a resolution of 70,000. Product ion spectra were acquired using the data-dependent acquisition method (Top 20) at a resolution of 17,500, and normalized collision energy (NCE) values of 27 and 35 were used.
2.6. Identification of O-glycopeptides
O-型糖ペプチドのMS2データセット はProteome Discoverer ver. 2.4 (Thremo Scientific)内に格納された検索エンジンByonic (ver. 3.11: Protein Metrics) で解析された.参照データベースとして、UniprotKB HUMAN (status/2022/02)とO-glycan (in-house database)を使用した。すなわち、10 O-glycan: HexNAc(1), HexNAc(2), HexNAc(1)Hex(1), HexNAc(1), Hex(1)Fuc(1), HexNAc(2)Hex(1), HexNAc(1)Hex(1)NeuAc(1), HexNAc(1)Hex(2)Fuc(1), HexNAc(1)Hex(1)Fuc(1)NeuAc(1), HexNAc(1)Hex(1)NeuAc(2), HexNAc(1)NeuAc(1)。検索条件は以下の通りである。enzyme, trypsin; maximum number of missed cleavages, 2; static modification, carbamidomethylation (C); dynamic modifications, Gln -> pyroGlu (N-term Q) and oxidation (M); precursor mass tolerance, 5 ppm; fragment mass tolerance, 0.1 Da; and maximum number of N-glycosylations per peptide, 2. 同定されたすべての糖ペプチドはfalse discovery rate (FDR)1%以下で選別した。Parcolatorにより信頼性は評価され、Confidence “High”の糖ペプチドのみリスト化した。
2.6. Identification of O-glycopeptides
The MS2 dataset of O-type glycopeptides was analyzed with the search engine Byonic (ver. 3.11: Protein Metrics) stored in Proteome Discoverer ver. 2.4 (Thremo Scientific). UniprotKB HUMAN (status/2022 /02) and O-glycan (in-house database) were used. That is, 10 O-glycan: HexNAc(1), HexNAc(2), HexNAc(1)Hex(1), HexNAc(1), Hex(1)Fuc(1), HexNAc(2)Hex(1), HexNAc (1)Hex(1)NeuAc(1), HexNAc(1)Hex(2)Fuc(1), HexNAc(1)Hex(1)Fuc(1)NeuAc(1), HexNAc(1)Hex(1) NeuAc(2), HexNAc(1)NeuAc(1). The search conditions are as follows. enzyme, trypsin; maximum number of missed cleavages, 2; static modification, carbamidomethylation (C); dynamic modifications, Gln -> pyroGlu (N-term Q) and oxidation (M); precursor mass tolerance, 5 ppm; fragment mass tolerance, 0.1 Da; and maximum number of N-glycosylations per peptide, 2. All identified glycopeptides were selected at a false discovery rate (FDR) of 1% or less. Confidence was evaluated using Parcolator, and only glycopeptides with “High” confidence were listed.
2.7. Statistics
Byonicで同定されたO-グリコフォームの定量的なピークエリア値は、Proteome Discoverer (ver.2.4)におけるPrecursor ion Quantifier nodeを用いた解析により得られた。解析条件は以下の通りである。Peptide to Use, unique; Precursor Quantification, area; Normalization mode, none. Principle component analysis (PCA) 解析と、and ウィルコクソンの符号順位検定（Wilcoxon matched-pairs signed rank test）は進行・再発大腸がん患者血清群と健常血清群から得られたグリコフォームピーク面積値を用い、JMP 14.2.0 (SAS Institute Inc., Cary, NC)で実行した。P<0.05、P<0.01は、各々重大な、または極めて重大な変化と規定した。
2.7. Statistics
Quantitative peak area values of O-glycoforms identified by Byonic were obtained by analysis using the Precursor ion Quantifier node in Proteome Discoverer (ver.2.4). The analysis conditions are as follows. Peptide to Use, unique; Precursor Quantification, area; Normalization mode, none. Principle component analysis (PCA) analysis and Wilcoxon matched-pairs signed rank test are and glycoform peak area values obtained from the healthy serum group and were run in JMP 14.2.0 (SAS Institute Inc., Cary, NC). P<0.05 and P<0.01 were defined as significant or extremely significant changes, respectively.
2.8. Gene Expression Analysis Using the Public Databases
The University of California Santa Cruz (UCSC) Xena Browserを用いて、遺伝子発現レベルと生存率との関連性を評価した。TCGA と GTEx データベースを用いた。候補遺伝子のExpectation-Maximization（RSEM）によるRNAシーケンスの期待カウント値は、DESeq2によりノーマライズされ、XenaBrowserから抽出した。 正常結腸（n = 307）、正常肝臓（n = 110）、結腸原発腫瘍（n = 287）、および肝臓原発腫瘍（n = 369）のRSEM予想カウントのデータセットは、それぞれTCGAおよびGTExデータベースから取得された。生存分析では、カプラン・マイヤー法を使用して、全体的な生存時間と遺伝子発現の相関関係を分析した。 疾患特異的生存の統計的有意性は、ログランク検定を使用して決定した。
2.8. Gene Expression Analysis Using the Public Databases
The association between gene expression levels and survival rate was evaluated using The University of California Santa Cruz (UCSC) Xena Browser. TCGA and GTEx databases were used. Expected counts of RNA sequences by Expectation-Maximization (RSEM) of candidate genes were normalized by DESeq2 and extracted from XenaBrowser. Datasets of RSEM expected counts for normal colon (n = 307), normal liver (n = 110), primary colon tumor (n = 287), and primary liver tumor (n = 369) were obtained from the TCGA and GTEx databases, respectively. It was done. For survival analysis, the Kaplan-Meier method was used to analyze the correlation between overall survival time and gene expression. Statistical significance of disease-specific survival was determined using the log-rank test.
3. Results
Identification of O-glycopeptides
ワークフロー（図１）に示した通り, MPL, Jacalin, SNLレクチンを用いて連続的に精製されたO-型糖鎖修飾タンパク質のトリプシン消化物から、アセトン濃縮法とGlycOCATCH法によりO-型糖ペプチドを濃縮した。Combineされた濃縮物のLC/MS/MSは異なる開裂エネルギーで２回実施され、得られたスペクトルデータは糖ペプチド検索エンジンByonicで解析された。糖ペプチドプロダクトイオンスペクトルを用いたデータベース検索の結果を図２に示す。健常血清10検体から、開裂エネルギー27では1019のO-グリコフォームが同定された。そして、開裂エネルギーを27->35に変更することで、新たに437のO-グリコフォームが同定された。最終的に、207種のタンパク質に由来する688種のO-型糖ペプチド、1456種のO-型グリコフォームが同定された。一方、大腸がん患者血清10検体から、開裂エネルギー27では1104のO-グリコフォームが同定された。そして、開裂エネルギーを27->35に変更することで、新たに443のO-グリコフォームが同定された。最終的に、231種のタンパク質に由来する744種のO-型糖ペプチド、1547種のO-型グリコフォームが同定された。健常血清から最も頻繁に付加が認められたのはT抗原（459部位）とSialyl T抗原（432部位）であった。一方、大腸がん患者血清から最も頻繁に付加が認められたのはSialyl T抗原（473部位）とT抗原（465部位）、di- Sialyl T抗原（442部位）であった。興味深いことに、一般的にがん抗原として知られるSialyl Tn、Lewis、Sialyl Lewisの付加に関して、大腸がん患者血清中における優位性は認められなかった。
3.Results
Identification of O-glycopeptides
As shown in the workflow (Figure 1), O-glycopeptides were extracted from tryptic digests of O-glycosylated proteins that were sequentially purified using MPL, Jacalin, and SNL lectins using the acetone enrichment method and the GlycOCATCH method. was concentrated. LC/MS/MS of the combined concentrate was performed twice with different cleavage energies, and the resulting spectral data were analyzed with the glycopeptide search engine Byonic. Figure 2 shows the results of a database search using the glycopeptide product ion spectrum. From 10 healthy serum samples, 1019 O-glycoforms were identified at a cleavage energy of 27. By changing the cleavage energy from 27 to 35, 437 new O-glycoforms were identified. Ultimately, 688 O-glycopeptides and 1456 O-glycoforms derived from 207 proteins were identified. On the other hand, 1104 O-glycoforms were identified at a cleavage energy of 27 from 10 serum samples from colorectal cancer patients. By changing the cleavage energy from 27 to 35, 443 new O-glycoforms were identified. Ultimately, 744 O-glycopeptides and 1547 O-glycoforms derived from 231 proteins were identified. The most frequent additions from healthy serum were T antigen (459 sites) and Sialyl T antigen (432 sites). On the other hand, the most frequently added antigens from the serum of colorectal cancer patients were Sialyl T antigen (473 sites), T antigen (465 sites), and di-Sialyl T antigen (442 sites). Interestingly, no predominance was observed in the serum of colorectal cancer patients regarding the addition of Sialyl Tn, Lewis, and Sialyl Lewis, which are generally known as cancer antigens.
Comparative O-glycoproteomic analysis (Normal & Tumor)
Byonic_Quanにより算出されたO-型グリコフォームprecursorイオンのピーク面積値データを用いたPCA解析の結果を図３aに示す。多変量解析でのPC1は、基本的なO-型糖鎖認識レクチン（MPL, Jacalin, SNL）を用いたfiltrationが健常血清群と再発・進行大腸がん血清群をグループ化することを可能にした。
Comparative O-glycoproteomic analysis (Normal & Tumor)
Figure 3a shows the results of PCA analysis using the peak area value data of the O-type glycoform precursor ion calculated by Byonic_Quan. PC1 in multivariate analysis enabled filtration using basic O-glycan recognition lectins (MPL, Jacalin, SNL) to group healthy serogroups and recurrent/advanced colorectal cancer serogroups. did.
健常血清群と再発・進行大腸がん患者血清群からの各々1456及び1547のO-型グリコフォームが同定された。 図３bは、健常血清および大腸がん患者血清から同定されたO-型グリコフォーム、糖ペプチド、糖タンパク質を比較したVenn diagramである。最終的に、重複を除くと2201のO-型グリコフォーム、1051のO-型糖ペプチドと274のO-型糖タンパク質が同定された。そして、再発・進行大腸がん患者血清群からは、ユニークな745のO-型グリコフォーム、363のO-型糖ペプチド、67のO-型糖タンパク質が大腸がん患者血清群からのみ同定された。再発・進行大腸がん血清群からのみ同定されたこれら67のタンパク質は、がん特異的なO-型糖鎖修飾もしくはO-型糖鎖修飾の異常な亢進が認められたものを示す。363のO-型糖ペプチドは進行・再発大腸がんで新たにO-型糖鎖修飾されたペプチド領域、745のO-型グリコフォームは繊細な糖鎖の変化を表しているに違いない。 1456 and 1547 O-type glycoforms were identified from the healthy serogroup and from the relapsed/advanced colorectal cancer patient serogroup, respectively. Figure 3b is a Venn diagram comparing O-type glycoforms, glycopeptides, and glycoproteins identified from healthy serum and colon cancer patient serum. Ultimately, after removing duplicates, 2201 O-glycoforms, 1051 O-glycopeptides, and 274 O-glycoproteins were identified. Furthermore, 745 unique O-type glycoforms, 363 O-type glycopeptides, and 67 O-type glycoproteins were identified only from the colorectal cancer patient serogroup. Ta. These 67 proteins, which were identified only from the recurrent/advanced colorectal cancer serogroup, were found to have cancer-specific O-type glycosylation or abnormally enhanced O-type glycosylation. The O-glycopeptide of 363 must be a newly modified peptide region with O-glycosylation in advanced/recurrent colorectal cancer, and the O-glycoform of 745 must represent a delicate change in the glycan.
再発・進行大腸がん患者の特定ならびに治療法選択を支援するマーカー開発への可能性を求めて、我々は、患者血清特異的に同定された67のO-型糖鎖修飾タンパク質から、より広範囲の検体より同定されたO-型糖鎖修飾タンパク質を抽出した。上位6タンパク質と同定されたグリコフォームをTable 1に示す。FBLN2は、最もcommonly identified O-glycoproteinsであり、8検体から同定された。Byonicの結果によると、aa330-378領域に、大腸がん特異的な複数のT抗原、そして１つのdi-Sialyl T抗原の付加が認められた。ANGPTL3は、FBLN2と同じく8検体から同定されたmost commonly identified O-glycoproteinsである。Byonicの結果によると、aa206-235領域に複数のT抗原、Sialyl T抗原、di-Sialyl T抗原が様々な組み合わせで付加していることが示された。MAN2A1は6検体から同定された。aa211-221領域にはT抗原が付加しており、シアル酸の付加による不均一性を作り出していた。VCANからは６検体から合わせて4つのアミノ酸配列領域へのO-型糖鎖修飾を検出することができた。CSF1は、aa356-395領域に対し、T抗原、Sialyl T抗原、di-Sialyl T抗原が様々な組み合わせで付加していた。C1QAのaa101-121領域には２つのフコシル化Core1が付加していた。
並行して、我々は、非ラベル化定量法を用いて、Difference≧5かつFDR LogWorth≧1.3を満たすO-型糖鎖修飾タンパク質を抽出した。同定されたグリコフォームをTable 2に示す。MRC1のaa1215-1229領域へのT抗原付加（Difference値=11.34、FDR LogWorth=10.25）、FGAのaa354-367領域へのSialyl T抗原（Difference値=7.70、FDR LogWorth=2.35）、及びaa 511-527領域へのdi-Sialyl T抗原の付加（Difference値=7.67、FDR LogWorth=1.66）は、統計学的に極めて有意なグリコフォームとして同定された。
これらは全てプロダクトイオンスペクトルからも支持された（図５～１２）。図5にFBLN2のaa330-349、aa350-364、aa365-378領域へのT抗原：Hex(1)HexNAc(1)付加を示すプロダクトイオンスペクトルを示す。m/z204 (HexNAc)と366(HexNAc-Hex)は糖鎖の診断イオンである。ペプチドイオン（aa330-349; m/z1057.04+, aa350-364; m/z1438.73+/719.372+, aa365-378; m/z1363.77+）に加え、複数のペプチド由来のフラグメントイオン（b+, y+）が認められた。図６にANGPTL3のaa206-221領域へのSialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)付加（上段）、aa222-235領域内２箇所へのSialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)付加またはaa222-235領域内２箇所へのT抗原：Hex(1)HexNAc(1)とSialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)付加（下段）を示すプロダクトイオンスペクトルを示す。m/z204 (HexNAc)と274/292 (NeuAc)、366(HexNAc-Hex)は糖鎖の診断イオンである。ペプチドイオン（aa206-221; m/z1773.95+, aa222-235; m/z828.962+）に加え、複数のペプチド由来のフラグメントイオン（b+, y+）が認められた。図７にMAN2A1のaa211-221領域内２箇所へのSialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)とdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)付加を示すプロダクトイオンスペクトルを示す。m/z204 (HexNAc)と274/292 (NeuAc)、366(HexNAc-Hex)は糖鎖の診断イオンである。ペプチドイオンm/z1372.71+/686.862+に加え、ペプチド由来のフラグメントイオンb9+/y2+が認められた。図８にCSF1のaa356-369領域内１箇所へのT抗原：Hex(1)HexNAc(1)付加（上段）、aa356-369領域内２箇所へのT抗原：Hex(1)HexNAc(1)付加（中段）、及びaa370-386領域内１箇所へのT抗原：Hex(1)HexNAc(1)付加（下段）を示すプロダクトイオンスペクトルを示す。m/z204 (HexNAc)と366(HexNAc-Hex)は糖鎖の診断イオンである。ペプチドイオン（aa356-369; m/z1410.74+, aa370-386; m/z958.992+）に加え、複数のペプチド由来のフラグメントイオン（b+, y+）が認められた。図９にCSF1のaa370-386領域内１箇所へのdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)付加及びaa370-386領域内２箇所へのT抗原：Hex(1)HexNAc(1)とdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)付加を示すプロダクトイオンスペクトルを示す。m/z204 (HexNAc)と274/292 (NeuAc)は糖鎖の診断イオンである。ペプチドイオン（aa370-386; m/z959.492+, aa370-395; m/z970.133+）に加え、複数のペプチド由来のフラグメントイオン（b+, y+）が認められた。図10にC1QAのaa101-121領域内２箇所へのフコシル化T抗原：Hex(1)HexNAc(1)dHex(1)が付加を示すプロダクトイオンスペクトルを示す。m/z204 (HexNAc)と366(HexNAc-Hex)は糖鎖の診断イオンである。複数のペプチド由来のフラグメントイオン（b+, y+）が認められた。図11に、MRC1のaa1215-1229領域内1箇所へのT抗原：Hex(1)HexNAc(1)付加を示すプロダクトイオンスペクトルを示す。m/z204 (HexNAc)と366(HexNAc-Hex)は糖鎖の診断イオンである。ペプチドイオン（aa1215-1229; m/z1607.81+）に加え、複数のペプチド由来のフラグメントイオン（b+, y+）が認められた。図12に、FGAのaa354-367領域へのSialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)、及びaa511-527領域へのdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)の付加を示すプロダクトイオンスペクトルを示す。m/z204 (HexNAc)、m/z366およびm/z292(NeuAc)は糖鎖の診断イオンである。ペプチドイオン（aa354-367; m/z1432.63+, aa511-527; m/z944.442+）に加え、複数のペプチド由来のフラグメントイオン（b+, y+）が認められた。
In search of the possibility of developing markers to support the identification of patients with recurrent/advanced colorectal cancer and the selection of treatment methods, we selected a broader range of O-glycosylated proteins from the 67 O-glycosylated proteins identified specifically in patient serum. O-type glycosylated proteins identified from the samples were extracted. The top six proteins and identified glycoforms are shown in Table 1. FBLN2 was the most commonly identified O-glycoprotein and was identified in 8 samples. According to Byonic's results, the addition of multiple colorectal cancer-specific T antigens and one di-Sialyl T antigen was observed in the aa330-378 region. ANGPTL3, like FBLN2, is a most commonly identified O-glycoprotein that was identified in eight samples. Byonic's results showed that multiple T antigens, Sialyl T antigens, and di-Sialyl T antigens were added in various combinations to the aa206-235 region. MAN2A1 was identified in 6 samples. T antigen was added to the aa211-221 region, creating heterogeneity due to the addition of sialic acid. From VCAN, we were able to detect O-type sugar chain modifications in four amino acid sequence regions in total from six samples. CSF1 had various combinations of T antigen, Sialyl T antigen, and di-Sialyl T antigen added to the aa356-395 region. Two fucosylated Core1 were added to the aa101-121 region of C1QA.
In parallel, we used a non-labeling quantification method to extract O-glycosylated proteins that satisfied Difference≧5 and FDR LogWorth≧1.3. The identified glycoforms are shown in Table 2. T antigen addition to the aa1215-1229 region of MRC1 (Difference value = 11.34, FDR LogWorth = 10.25), Sialyl T antigen addition to the aa354-367 region of FGA (Difference value = 7.70, FDR LogWorth = 2.35), and aa 511- Addition of di-Sialyl T antigen to the 527 region (Difference value = 7.67, FDR LogWorth = 1.66) was identified as a highly statistically significant glycoform.
All of these were also supported by the product ion spectra (Figures 5 to 12). Figure 5 shows a product ion spectrum showing T antigen: Hex(1)HexNAc(1) addition to the aa330-349, aa350-364, and aa365-378 regions of FBLN2. m/z204 (HexNAc) and 366 (HexNAc-Hex) are diagnostic ions for sugar chains. In addition to peptide ions (aa330-349; m/z1057.04 + , aa350-364; m/z1438.73 + /719.37 2+ , aa365-378; m/z1363.77 + ), fragment ions derived from multiple peptides (b + , y + ) was observed. Figure 6 shows the addition of Sialyl T antigen: Hex(1) HexNAc(1) NeuAc(1) to the aa206-221 region of ANGPTL3 (upper row), and the addition of Sialyl T antigen: Hex(1) HexNAc to two locations within the aa222-235 region. (1) Addition of NeuAc(1) or addition of T antigen to two sites within the aa222-235 region: Hex(1) HexNAc(1) and Sialyl T antigen: Hex(1) HexNAc(1) Addition of NeuAc(1) (lower row) The product ion spectrum showing . m/z204 (HexNAc), 274/292 (NeuAc), and 366 (HexNAc-Hex) are diagnostic ions for sugar chains. In addition to peptide ions (aa206-221; m/z 1773.95 + , aa222-235; m/z 828.96 2+ ), multiple peptide-derived fragment ions (b + , y + ) were observed. Figure 7 shows the addition of Sialyl T antigen: Hex(1) HexNAc(1) NeuAc(1) and di-Sialyl T antigen: Hex(1) HexNAc(1) NeuAc(2) to two locations within the aa211-221 region of MAN2A1. The product ion spectrum is shown. m/z204 (HexNAc), 274/292 (NeuAc), and 366 (HexNAc-Hex) are diagnostic ions for sugar chains. In addition to the peptide ion m/z 1372.71 + /686.86 2+ , a peptide-derived fragment ion b 9+ /y 2+ was observed. Figure 8 shows T antigen: Hex(1) HexNAc(1) added to one site in the aa356-369 region of CSF1 (upper row), T antigen: Hex(1) HexNAc(1) to two sites in the aa356-369 region. Product ion spectra showing addition (middle row) and addition of T antigen: Hex(1)HexNAc(1) to one location within the aa370-386 region (lower row) are shown. m/z204 (HexNAc) and 366 (HexNAc-Hex) are diagnostic ions for sugar chains. In addition to peptide ions (aa356-369; m/z1410.74 + , aa370-386; m/z958.99 2+ ), multiple peptide-derived fragment ions (b + , y + ) were observed. Figure 9 shows the addition of di-Sialyl T antigen: Hex(1) HexNAc(1) NeuAc(2) to one site within the aa370-386 region of CSF1 and the addition of T antigen: Hex(1) to two sites within the aa370-386 region. HexNAc(1) and di-Sialyl T antigen: A product ion spectrum showing Hex(1)HexNAc(1)NeuAc(2) addition is shown. m/z204 (HexNAc) and 274/292 (NeuAc) are diagnostic ions for sugar chains. In addition to peptide ions (aa370-386; m/z959.49 2+ , aa370-395; m/z970.13 3+ ), multiple peptide-derived fragment ions (b + , y + ) were observed. Figure 10 shows a product ion spectrum showing the addition of fucosylated T antigen: Hex(1) HexNAc(1) and dHex(1) to two locations within the aa101-121 region of C1QA. m/z204 (HexNAc) and 366 (HexNAc-Hex) are diagnostic ions for sugar chains. Fragment ions (b + , y + ) derived from multiple peptides were observed. FIG. 11 shows a product ion spectrum showing T antigen: Hex(1) HexNAc(1) addition to one location within the aa1215-1229 region of MRC1. m/z204 (HexNAc) and 366 (HexNAc-Hex) are diagnostic ions for sugar chains. In addition to the peptide ion (aa1215-1229; m/z1607.81 + ), multiple peptide-derived fragment ions (b + , y + ) were observed. Figure 12 shows Sialyl T antigen to the aa354-367 region of FGA: Hex(1) HexNAc(1) NeuAc(1), and di-Sialyl T antigen to the aa511-527 region: Hex(1) HexNAc(1). A product ion spectrum showing the addition of NeuAc(2) is shown. m/z204 (HexNAc), m/z366 and m/z292 (NeuAc) are diagnostic ions for sugar chains. In addition to peptide ions (aa354-367; m/z1432.63 + , aa511-527; m/z944.44 2+ ), multiple peptide-derived fragment ions (b + , y + ) were observed.
Verification of marker candidate O-glycoproteins by statistical analysis
大腸がん患者血清のみでcommonly identifiedされたFBLN2とANGPTL3をはじめとする６分子、及び非ラベル化定量法によりDifference≧5かつFDR LogWorth≧1.3を満たしたMRC1及びFGAについて、マーカー候補タンパク質としての有用性をROC解析により調査した（図４）。O-型糖鎖認識レクチンと抗体との組み合わせを利用した検出系を構築するために、O-型糖鎖のタイプごとにPeak Area値を合算した時のWilcoxon matched-pairs signed rank testを実行した。FBLN2へのT抗原、di-Sialyl T抗原の付加は、大腸がん患者血清で顕著に増加しており、診断（予測）能を示すAUC値は0.9（カットオフ値：3.E+07）であった。ANGPTL3へのT抗原の付加には明確な差異は認められなかったものの、Sialyl T抗原の付加は大腸がん患者血清で顕著に増加しており、AUC値は0.8（カットオフ値：3.E+07）であった。また、MAN2A1へのSialyl T抗原とdi-Sialyl T抗原の付加、C1QAへのフコシル化Sialyl-T抗原の付加は大腸がん患者血清で増加しており、AUC値はそれぞれ0.87（カットオフ値：4.E+07）と0.85（カットオフ値：6.E+06）であった。VCANへのT抗原の付加は大腸がん患者血清で顕著に増加しており、AUC値は0.91であった（カットオフ値：2.E+06）。特筆すべきことに、CSF1へのT抗原、またSialyl T抗原とdi-Sialyl T抗原の付加は大腸がん患者血清で増加しており、AUC値はそれぞれ0.94（カットオフ値：2.E+07）と1.00（カットオフ値：2.E+06）と高い診断能を示した。MRC1のaa1215-1229領域へのT抗原の付加は、大腸がん患者血清で増加しており、AUC値は1.00（カットオフ値：3.E+06）であった。FGAのaa354-367領域へのSialyl T抗原の付加及びaa511-527領域へのdi-Sialyl T抗原の付加は、大腸がん患者血清で増加しており、AUC値はそれぞれ0.88（カットオフ値：1.E+06）及び0.86（カットオフ値：2.E+06）であった。
Verification of marker candidate O-glycoproteins by statistical analysis
Six molecules, including FBLN2 and ANGPTL3, which were commonly identified only in the serum of colorectal cancer patients, and MRC1 and FGA, which satisfied Difference≧5 and FDR LogWorth≧1.3 by non-labeling quantitative method, are useful as marker candidate proteins. The characteristics were investigated by ROC analysis (Figure 4). In order to construct a detection system using a combination of an O-glycan recognizing lectin and an antibody, we performed a Wilcoxon matched-pairs signed rank test when summing up the Peak Area values for each type of O-glycan. . The addition of T antigen and di-Sialyl T antigen to FBLN2 is significantly increased in the serum of colorectal cancer patients, and the AUC value indicating diagnostic (predictive) ability is 0.9 (cutoff value: 3.E+07) Met. Although no clear difference was observed in the addition of T antigen to ANGPTL3, the addition of Sialyl T antigen was markedly increased in the serum of colorectal cancer patients, with an AUC value of 0.8 (cutoff value: 3.E +07). In addition, the addition of Sialyl T antigen and di-Sialyl T antigen to MAN2A1 and the addition of fucosylated Sialyl-T antigen to C1QA are increased in the serum of colorectal cancer patients, and each has an AUC value of 0.87 (cutoff value: 4.E+07) and 0.85 (cutoff value: 6.E+06). Addition of T antigen to VCAN was significantly increased in serum from colorectal cancer patients, and the AUC value was 0.91 (cutoff value: 2.E+06). Notably, the addition of T antigen, as well as Sialyl T antigen and di-Sialyl T antigen to CSF1, was increased in the serum of colorectal cancer patients, with an AUC value of 0.94 (cutoff value: 2.E+), respectively. 07) and 1.00 (cutoff value: 2.E+06), which showed high diagnostic ability. Addition of T antigen to the aa1215-1229 region of MRC1 was increased in serum from colorectal cancer patients, and the AUC value was 1.00 (cutoff value: 3.E+06). The addition of Sialyl T antigen to the aa354-367 region of FGA and the addition of di-Sialyl T antigen to the aa511-527 region are increased in the serum of colorectal cancer patients, and the AUC value is 0.88 (cutoff value: 1.E+06) and 0.86 (cutoff value: 2.E+06).
Comparison of the Gene Expression Levels or Marker Candidates Using public databases
O-グライコプロテオミクスの結果では、一部のタンパク質に対するO-型糖鎖の付加が、再発・進行大腸がん患者血清中で顕著に増加する可能性を示した。我々は、複数のO型糖鎖認識レクチンを用いて濃縮したものの、再発・進行大腸がんにおけるタンパク質発現量の亢進の影響を評価することは難しい。そこで、2つの公開データベースGTExとTCGAを使用して、これらのO-型糖鎖の付加が変化したと推定されるタンパク質（血中タンパク質だけではなくECMの構成成分であるタンパク質も含めて）の遺伝子発現レベルと、高発現群が大腸がん患者の生存期間に及ぼす影響を調査した。免疫グロブリンであるγグロブリン以外の血中タンパク質は、主に肝臓で合成されることが知られている。加えて、ECMを構成するタンパク質の多くは組織線維芽細胞から分泌されることが示唆されている。したがって、これらのO-型糖鎖の付加が変化したと推定される９つのO-型糖タンパク質の遺伝子発現レベルを正常な肝臓組織および大腸組織と、それらの腫瘍組織間で比較した。結果を図１１に示す。ECMに局在する分泌型タンパク質FBLN2遺伝子の低発現群では有意な生存期間延長が認められるものの、大腸腫瘍組織におけるFBLN2遺伝子発現レベルの重大な増加は認められなかった。肝臓で合成される血中タンパク質であるANGPTL3は、（肝臓での遺伝子発現レベルと大腸がんとの関連性は不明だが）、肝臓がんでは有意な発現レベルの上昇が認められた。しかしながら、低発現群と高発現群との2群比較において、生存期間に差は認められなかった。膜貫通領域をもちGolgiまたは細胞表面に局在する糖タンパク質であるMAN2A1において、大腸腫瘍組織における遺伝子発現レベルの重大な増加が認められたものの、発現レベルと生存期間との相関は無いようである。MAN2A1と同様に、膜貫通領域をもつ細胞表面タンパク質CSFにおいて、大腸腫瘍組織における遺伝子発現レベルの重大な増加は認められないが、遺伝子高発現群における生存期間短縮は著しく顕著である。ECMタンパク質であるVCAN遺伝子の大腸腫瘍組織における発現レベルの増加は認められず、発現レベルと生存期間との相関は無いようである。分泌型タンパク質であるC1QA遺伝子の肝臓における発現レベルは、肝がんで顕著に増加するものの、低発現群における有意な生存期間延長は認めない。膜貫通領域をもつMRC1において、大腸腫瘍組織における遺伝子発現レベルの有意な増加は認められず、発現レベルと生存期間との相関は無いようである。血中分泌タンパク質であるFGAは、肝臓がんにおける有意な発現変動は認められない。また、低発現群と高発現群との2群比較において、生存期間に有意差は認められなかった。
Comparison of the Gene Expression Levels or Marker Candidates Using public databases
The results of O-glycoproteomics showed that the addition of O-glycans to some proteins may be significantly increased in the serum of patients with recurrent or advanced colorectal cancer. Although we enriched the protein using multiple O-glycan-recognizing lectins, it is difficult to evaluate the impact of increased protein expression in recurrent or advanced colorectal cancer. Therefore, we used two public databases, GTEx and TCGA, to identify proteins (including not only blood proteins but also proteins that are components of the ECM) that are presumed to have altered O-glycan additions. We investigated gene expression levels and the effects of high expression groups on survival time of colorectal cancer patients. It is known that blood proteins other than gamma globulin, which is an immunoglobulin, are mainly synthesized in the liver. In addition, it has been suggested that many of the proteins that make up the ECM are secreted from tissue fibroblasts. Therefore, we compared the gene expression levels of nine O-glycoproteins, which are presumed to have altered O-glycan addition, between normal liver and colon tissues and their tumor tissues. The results are shown in FIG. Although survival was significantly prolonged in the group with low expression of the secreted protein FBLN2 gene, which is localized in the ECM, no significant increase in FBLN2 gene expression level was observed in colorectal tumor tissue. ANGPTL3, a blood protein synthesized in the liver, was found to have significantly increased expression levels in liver cancer (although the relationship between gene expression levels in the liver and colorectal cancer is unclear). However, no difference in survival time was observed when comparing the low expression group and the high expression group. MAN2A1, a Golgi or cell surface-localized glycoprotein with a transmembrane region, showed a significant increase in gene expression levels in colorectal tumor tissues, but there appeared to be no correlation between expression levels and survival time. . Similar to MAN2A1, the cell surface protein CSF, which has a transmembrane region, does not show a significant increase in gene expression level in colon tumor tissues, but the survival time is significantly shortened in the group with high gene expression. No increase in the expression level of the VCAN gene, an ECM protein, was observed in colon tumor tissues, and there appears to be no correlation between the expression level and survival time. Although the expression level of the C1QA gene, a secreted protein, in the liver increases markedly in liver cancer, no significant survival period was observed in the low expression group. In MRC1, which has a transmembrane region, no significant increase in gene expression level was observed in colorectal tumor tissues, and there appears to be no correlation between expression level and survival time. No significant changes in expression of FGA, a protein secreted in the blood, are observed in liver cancer. Furthermore, no significant difference in survival time was observed when comparing the two groups, the low expression group and the high expression group.
[Table 1]
[Table 1]
[Table 2]
[Table 2]
4. Refernces
1. Hintze J, Ye Z, Narimatsu Y, Madsen TD, Joshi HJ, Goth CK, et al. Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines. J Biol Chem (2018) 293: 19064-19077. 10.1074/jbc.RA118.004516
2. Bennett EP, Mandel U, Clausen H, Gerken TA, Fritz TA, Tabak LA. Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family. Glycobiology (2012) 22: 736-756. 10.1093/glycob/cwr182
3. Springer GF, Taylor CR, Howard DR, Tegtmeyer H, Desai PR, Murthy SM, et al. Tn, a carcinoma-associated antigen, reacts with anti-Tn of normal human sera. Cancer (1985) 55: 561-569. 10.1002/1097-0142(19850201)55:3<561::aid-cncr2820550315>3.0.co;2-1
4. Fu C, Zhao H, Wang Y, Cai H, Xiao Y, Zeng Y, et al. Tumor-associated antigens: Tn antigen, sTn antigen, and T antigen. Hla (2016) 88: 275-286. 10.1111/tan.12900
5. Lugo R, Avila-Nava A, Garcia-Perez R, Herrera-Escalante S, De la Cruz-Acosta J, Gutierrez-Solis AL. Systematic review and meta-analysis of the clinical survival significance of Sialyl-Tn expression in histological tissues from cancer patients. Transl Cancer Res (2020) 9: 547-555. 10.21037/tcr.2019.11.53
6. Munkley J. The Role of Sialyl-Tn in Cancer. Int J Mol Sci (2016) 17: 275. 10.3390/ijms17030275
7. Peixoto A, Fernandes E, Gaiteiro C, Lima L, Azevedo R, Soares J, et al. Hypoxia enhances the malignant nature of bladder cancer cells and concomitantly antagonizes protein O-glycosylation extension. Oncotarget (2016) 7: 63138-63157. 10.18632/oncotarget.11257
8. Chia J, Goh G, Bard F. Short O-GalNAc glycans: regulation and role in tumor development and clinical perspectives. Biochim Biophys Acta (2016) 1860: 1623-1639. 10.1016/j.bbagen.2016.03.008
9. Jiang Y, Liu Z, Xu F, Dong X, Cheng Y, Hu Y, et al. Aberrant O-glycosylation contributes to tumorigenesis in human colorectal cancer. J Cell Mol Med (2018) 22: 4875-4885. 10.1111/jcmm.13752
10. Aoyagi Y, Suzuki Y, Isemura M, Nomoto M, Sekine C, Igarashi K, et al. The fucosylation index of alpha-fetoprotein and its usefulness in the early diagnosis of hepatocellular carcinoma. Cancer (1988) 61: 769-774. 10.1002/1097-0142(19880215)61:4<769::aid-cncr2820610422>3.0.co;2-m
11. Katoh H, Nakamura K, Tanaka T, Satomura S, Matsuura S. Automatic and simultaneous analysis of Lens culinaris agglutinin-reactive alpha-fetoprotein ratio and total alpha-fetoprotein concentration. Anal Chem (1998) 70: 2110-2114. 10.1021/ac971280c
12. Kagebayashi C, Yamaguchi I, Akinaga A, Kitano H, Yokoyama K, Satomura M, et al. Automated immunoassay system for AFP-L3% using on-chip electrokinetic reaction and separation by affinity electrophoresis. Anal Biochem (2009) 388: 306-311. 10.1016/j.ab.2009.02.030
13. Ito N, Yamada M, Morishita K, Nojima S, Motooka K, Sakata N, et al. Identification of fucosylated haptoglobin-producing cells in pancreatic cancer tissue and its molecular mechanism. Glycoconj J (2021) 38: 45-54. 10.1007/s10719-020-09970-8
14. Acharya A, Markar SR, Matar M, Ni M, Hanna GB. Use of Tumor Markers in Gastrointestinal Cancers: Surgeon Perceptions and Cost-Benefit Trade-Off Analysis. Ann Surg Oncol (2017) 24: 1165-1173. 10.1245/s10434-016-5717-y
15. Mysling S, Palmisano G, Hojrup P, Thaysen-Andersen M. Utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics. Anal Chem (2010) 82: 5598-5609. 10.1021/ac100530w
16. Chen Z, Zhong X, Tie C, Chen B, Zhang X, Li L. Development of a hydrophilic interaction liquid chromatography coupled with matrix-assisted laser desorption/ionization-mass spectrometric imaging platform for N-glycan relative quantitation using stable-isotope labeled hydrazide reagents. Anal Bioanal Chem (2017) 409: 4437-4447. 10.1007/s00216-017-0387-6
17. Badr HA, Alsadek DM, Darwish AA, Elsayed AI, Bekmanov BO, Khussainova EM, et al. Lectin approaches for glycoproteomics in FDA-approved cancer biomarkers. Expert Rev Proteomics (2014) 11: 227-236. 10.1586/14789450.2014.897611
18. Gbormittah FO, Lee LY, Taylor K, Hancock WS, Iliopoulos O. Comparative studies of the proteome, glycoproteome, and N-glycome of clear cell renal cell carcinoma plasma before and after curative nephrectomy. J Proteome Res (2014) 13: 4889-4900. 10.1021/pr500591e
19. Ruiz-May E, Catala C, Rose JK. N-glycoprotein enrichment by lectin affinity chromatography. Methods Mol Biol 2014; (1072) 633-643. 10.1007/978-1-62703-631-3_43
20. Takakura D, Harazono A, Hashii N, Kawasaki N. Selective glycopeptide profiling by acetone enrichment and LC/MS. J Proteomics (2014) 101: 17-30. 10.1016/j.jprot.2014.02.005
21. Turiak L, Sugar S, Acs A, Toth G, Gomory A, Telekes A, et al. Site-specific N-glycosylation of HeLa cell glycoproteins. Sci Rep (2019) 9: 14822. 10.1038/s41598-019-51428-x
4.References
1. Hintze J, Ye Z, Narimatsu Y, Madsen TD, Joshi HJ, Goth CK, et al. Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines. J Biol Chem ( 2018) 293: 19064-19077. 10.1074/jbc.RA118.004516
2. Bennett EP, Mandel U, Clausen H, Gerken TA, Fritz TA, Tabak LA. Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family. Glycobiology (2012) 22: 736-756. 10.1093/glycob/cwr182
3. Springer GF, Taylor CR, Howard DR, Tegtmeyer H, Desai PR, Murthy SM, et al. Tn, a carcinoma-associated antigen, reacts with anti-Tn of normal human sera. Cancer (1985) 55: 561-569 . 10.1002/1097-0142(19850201)55:3<561::aid-cncr2820550315>3.0.co;2-1
4. Fu C, Zhao H, Wang Y, Cai H, Xiao Y, Zeng Y, et al. Tumor-associated antigens: Tn antigen, sTn antigen, and T antigen. Hla (2016) 88: 275-286. 10.1111/ tan.12900
5. Lugo R, Avila-Nava A, Garcia-Perez R, Herrera-Escalante S, De la Cruz-Acosta J, Gutierrez-Solis AL. Systematic review and meta-analysis of the clinical survival significance of Sialyl-Tn expression in histological tissues from cancer patients. Transl Cancer Res (2020) 9: 547-555. 10.21037/tcr.2019.11.53
6. Munkley J. The Role of Sialyl-Tn in Cancer. Int J Mol Sci (2016) 17: 275. 10.3390/ijms17030275
7. Peixoto A, Fernandes E, Gaiteiro C, Lima L, Azevedo R, Soares J, et al. Hypoxia enhances the malignant nature of bladder cancer cells and concomitantly antagonizes protein O-glycosylation extension. Oncotarget (2016) 7: 63138-63157 .10.18632/oncotarget.11257
8. Chia J, Goh G, Bard F. Short O-GalNAc glycans: regulation and role in tumor development and clinical perspectives. Biochim Biophys Acta (2016) 1860: 1623-1639. 10.1016/j.bbagen.2016.03.008
9. Jiang Y, Liu Z, Xu F, Dong X, Cheng Y, Hu Y, et al. Aberrant O-glycosylation contributes to tumorigenesis in human colorectal cancer. J Cell Mol Med (2018) 22: 4875-4885. 10.1111/ jcmm.13752
10. Aoyagi Y, Suzuki Y, Isemura M, Nomoto M, Sekine C, Igarashi K, et al. The fucosylation index of alpha-fetoprotein and its usefulness in the early diagnosis of hepatocellular carcinoma. Cancer (1988) 61: 769-774 . 10.1002/1097-0142(19880215)61:4<769::aid-cncr2820610422>3.0.co;2-m
11. Katoh H, Nakamura K, Tanaka T, Satomura S, Matsuura S. Automatic and simultaneous analysis of Lens culinaris agglutinin-reactive alpha-fetoprotein ratio and total alpha-fetoprotein concentration. Anal Chem (1998) 70: 2110-2114. 10.1021 /ac971280c
12. Kagebayashi C, Yamaguchi I, Akinaga A, Kitano H, Yokoyama K, Satomura M, et al. Automated immunoassay system for AFP-L3% using on-chip electrokinetic reaction and separation by affinity electrophoresis. Anal Biochem (2009) 388: 306-311. 10.1016/j.ab.2009.02.030
13. Ito N, Yamada M, Morishita K, Nojima S, Motooka K, Sakata N, et al. Identification of fucosylated haptoglobin-producing cells in pancreatic cancer tissue and its molecular mechanism. Glycoconj J (2021) 38: 45-54. 10.1007/s10719-020-09970-8
14. Acharya A, Markar SR, Matar M, Ni M, Hanna GB. Use of Tumor Markers in Gastrointestinal Cancers: Surgeon Perceptions and Cost-Benefit Trade-Off Analysis. Ann Surg Oncol (2017) 24: 1165-1173. 10.1245/ s10434-016-5717-y
15. Mysling S, Palmisano G, Hojrup P, Thaysen-Andersen M. Utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics. Anal Chem (2010) 82: 5598-5609. 10.1021/ac100530w
16. Chen Z, Zhong X, Tie C, Chen B, Zhang X, Li L. Development of a hydrophilic interaction liquid chromatography coupled with matrix-assisted laser desorption/ionization-mass spectrometric imaging platform for N-glycan relative quantitation using stable- isotope labeled hydrazide reagents. Anal Bioanal Chem (2017) 409: 4437-4447. 10.1007/s00216-017-0387-6
17. Badr HA, Alsadek DM, Darwish AA, Elsayed AI, Bekmanov BO, Khussainova EM, et al. Lectin approaches for glycoproteomics in FDA-approved cancer biomarkers. Expert Rev Proteomics (2014) 11: 227-236. 10.1586/14789450.2014. 897611
18. Gbormittah FO, Lee LY, Taylor K, Hancock WS, Iliopoulos O. Comparative studies of the proteome, glycoproteome, and N-glycome of clear cell renal cell carcinoma plasma before and after curative nephrectomy. J Proteome Res (2014) 13: 4889-4900. 10.1021/pr500591e
19. Ruiz-May E, Catala C, Rose JK. N-glycoprotein enrichment by lectin affinity chromatography. Methods Mol Biol 2014; (1072) 633-643. 10.1007/978-1-62703-631-3_43
20. Takakura D, Harazono A, Hashii N, Kawasaki N. Selective glycopeptide profiling by acetone enrichment and LC/MS. J Proteomics (2014) 101: 17-30. 10.1016/j.jprot.2014.02.005
21. Turiak L, Sugar S, Acs A, Toth G, Gomory A, Telekes A, et al. Site-specific N-glycosylation of HeLa cell glycoproteins. Sci Rep (2019) 9: 14822. 10.1038/s41598-019-51428 -x
本発明は、がんの診断を補助することができる。本発明により、がんの治療効果、がんの再発や進行、がんの転移リスクを評価することが可能となる。 The present invention can aid in the diagnosis of cancer. The present invention makes it possible to evaluate cancer treatment effects, cancer recurrence and progression, and cancer metastasis risk.
＜配列番号１＞FBLN2のアミノ酸配列を示す。
MVLLWEPAGAWLALGLALALGPSVAAAAPRQDCTGVECPPLENCIEEALEPGACCATCVQQGCACEGYQYYDCLQGGFVRGRVPAGQSYFVDFGSTECSCPPGGGKISCQFMLCPELPPNCIEAVVVADSCPQCGQVGCVHAGHKYAAGHTVHLPPCRACHCPDAGGELICYQLPGCHGNFSDAEEGDPERHYEDPYSYDQEVAEVEAATALGGEVQAGAVQAGAGGPPAALGGGSQPLSTIQAPPWPAVLPRPTAAAALGPPAPVQAKARRVTEDSEEEEEEEEEREEMAVTEQLAAGGHRGLDGLPTTAPAGPSLPIQEERAEAGARAEAGARPEENLILDAQATSRSTGPEGVTHAPSLGKAALVPTQAVPGSPRDPVKPSPHNILSTSLPDAAWIPPTREVPRKPQVLPHSHVEEDTDPNSVHSIPRSSPEGSTKDLIETCCAAGQQWAIDNDECLEIPESGTEDNVCRTAQRHCCVSYLQEKSCMAGVLGAKEGETCGAEDNDSCGISLYKQCCDCCGLGLRVRAEGQSCESNPNLGYPCNHVMLSCCEGEEPLIVPEVRRPPEPAAAPRRVSEAEMAGREALSLGTEAELPNSLPGDDQDECLLLPGELCQHLCINTVGSYHCACFPGFSLQDDGRTCRPEGHPPQPEAPQEPALKSEFSQVASNTIPLPLPQPNTCKDNGPCKQVCSTVGGSAICSCFPGYAIMADGVSCEDINECVTDLHTCSRGEHCVNTLGSFHCYKALTCEPGYALKDGECEDVDECAMGTHTCQPGFLCQNTKGSFYCQARQRCMDGFLQDPEGNCVDINECTSLSEPCRPGFSCINTVGSYTCQRNPLICARGYHASDDGTKCVDVNECETGVHRCGEGQVCHNLPGSYRCDCKAGFQRDAFGRGCIDVNECWASPGRLCQHTCENTLGSYRCSCASGFLLAADGKRCEDVNECEAQRCSQECANIYGSYQCYCRQGYQLAEDGHTCTDIDECAQGAGILCTFRCLNVPGSYQCACPEQGYTMTANGRSCKDVDECALGTHNCSEAETCHNIQGSFRCLRFECPPNYVQVSKTKCERTTCHDFLECQNSPARITHYQLNFQTGLLVPAHIFRIGPAPAFTGDTIALNIIKGNEEGYFGTRRLNAYTGVVYLQRAVLEPRDFALDVEMKLWRQGSVTTFLAKMHIFFTTFAL
＜配列番号２＞ANGPTL3のアミノ酸配列を示す。
MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE
＜配列番号３＞MAN2A1のアミノ酸配列を示す。
MKLSRQFTVFGSAIFCVVIFSLYLMLDRGHLDYPRNPRREGSFPQGQLSMLQEKIDHLERLLAENNEIISNIRDSVINLSESVEDGPKSSQSNFSQGAGSHLLPSQLSLSVDTADCLFASQSGSHNSDVQMLDVYSLISFDNPDGGVWKQGFDITYESNEWDTEPLQVFVVPHSHNDPGWLKTFNDYFRDKTQYIFNNMVLKLKEDSRRKFIWSEISYLSKWWDIIDIQKKDAVKSLIENGQLEIVTGGWVMPDEATPHYFALIDQLIEGHQWLENNIGVKPRSGWAIDPFGHSPTMAYLLNRAGLSHMLIQRVHYAVKKHFALHKTLEFFWRQNWDLGSVTDILCHMMPFYSYDIPHTCGPDPKICCQFDFKRLPGGRFGCPWGVPPETIHPGNVQSRARMLLDQYRKKSKLFRTKVLLAPLGDDFRYCEYTEWDLQFKNYQQLFDYMNSQSKFKVKIQFGTLSDFFDALDKADETQRDKGQSMFPVLSGDFFTYADRDDHYWSGYFTSRPFYKRMDRIMESHLRAAEILYYFALRQAHKYKINKFLSSSLYTALTEARRNLGLFQHHDAITGTAKDWVVVDYGTRLFHSLMVLEKIIGNSAFLLILKDKLTYDSYSPDTFLEMDLKQKSQDSLPQKNIIRLSAEPRYLVVYNPLEQDRISLVSVYVSSPTVQVFSASGKPVEVQVSAVWDTANTISETAYEISFRAHIPPLGLKVYKILESASSNSHLADYVLYKNKVEDSGIFTIKNMINTEEGITLENSFVLLRFDQTGLMKQMMTKEDGKHHEVNVQFSWYGTTIKRDKSGAYLFLPDGNAKPYVYTTPPFVRVTHGRIYSEVTCFFDHVTHRVRLYHIQGIEGQSVEVSNIVDIRKVYNREIAMKISSDIKSQNRFYTDLNGYQIQPRMTLSKLPLQANVYPMTTMAYIQDAKHRLTLLSAQSLGVSSLNSGQIEVIMDRRLMQDDNRGLEQGIQDNKITANLFRILLEKRSAVNTEEEKKSVSYPSLLSHITSSLMNHPVIPMANKFSSPTLELQGEFSPLQSSLPCDIHLVNLRTIQSKVGNGHSNEAALILHRKGFDCRFSSKGTGLFCSTTQGKILVQKLLNKFIVESLTPSSLSLMHSPPGTQNISEINLSPMEISTFRIQLR
＜配列番号４＞CSF1のアミノ酸配列を示す。
MTAPGAAGRCPPTTWLGSLLLLVCLLASRSITEEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQDVVTKPDCNCLYPKAIPSSDPASVSPHQPLAPSMAPVAGLTWEDSEGTEGSSLLPGEQPLHTVDPGSAKQRPPRSTCQSFEPPETPVVKDSTIGGSPQPRPSVGAFNPGMEDILDSAMGTNWVPEEASGEASEIPVPQGTELSPSRPGGGSMQTEPARPSNFLSASSPLPASAKGQQPADVTGTALPRVGPVRPTGQDWNHTPQKTDHPSALLRDPPEPGSPRISSLRPQGLSNPSTLSAQPQLSRSHSSGSVLPLGELEGRRSTRDRRSPAEPEGGPASEGAARPLPRFNSVPLTDTGHERQSEGSFSPQLQESVFHLLVPSVILVLLAVGGLLFYRWRRRSHQEPQRADSPLEQPEGSPLTQDDRQVELPV
＜配列番号５＞C1QAのアミノ酸配列を示す。
MEGPRGWLVLCVLAISLASMVTEDLCRAPDGKKGEAGRPGRRGRPGLKGEQGEPGAPGIRTGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGTKGSPGNIKDQPRPAFSAIRRNPPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQWEICLSIVSSSRGQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSA
＜配列番号６＞VCANのアミノ酸配列を示す。
MFINIKSILWMCSTLIVTHALHKVKVGKSPPVRGSLSGKVSLPCHFSTMPTLPPSYNTSEFLRIKWSKIEVDKNGKDLKETTVLVAQNGNIKIGQDYKGRVSVPTHPEAVGDASLTVVKLLASDAGLYRCDVMYGIEDTQDTVSLTVDGVVFHYRAATSRYTLNFEAAQKACLDVGAVIATPEQLFAAYEDGFEQCDAGWLADQTVRYPIRAPRVGCYGDKMGKAGVRTYGFRSPQETYDVYCYVDHLDGDVFHLTVPSKFTFEEAAKECENQDARLATVGELQAAWRNGFDQCDYGWLSDASVRHPVTVARAQCGGGLLGVRTLYRFENQTGFPPPDSRFDAYCFKPKEATTIDLSILAETASPSLSKEPQMVSDRTTPIIPLVDELPVIPTEFPPVGNIVSFEQKATVQPQAITDSLATKLPTPTGSTKKPWDMDDYSPSASGPLGKLDISEIKEEVLQSTTGVSHYATDSWDGVVEDKQTQESVTQIEQIEVGPLVTSMEILKHIPSKEFPVTETPLVTARMILESKTEKKMVSTVSELVTTGHYGFTLGEEDDEDRTLTVGSDESTLIFDQIPEVITVSKTSEDTIHTHLEDLESVSASTTVSPLIMPDNNGSSMDDWEERQTSGRITEEFLGKYLSTTPFPSQHRTEIELFPYSGDKILVEGISTVIYPSLQTEMTHRRERTETLIPEMRTDTYTDEIQEEITKSPFMGKTEEEVFSGMKLSTSLSEPIHVTESSVEMTKSFDFPTLITKLSAEPTEVRDMEEDFTATPGTTKYDENITTVLLAHGTLSVEAATVSKWSWDEDNTTSKPLESTEPSASSKLPPALLTTVGMNGKDKDIPSFTEDGADEFTLIPDSTQKQLEEVTDEDIAAHGKFTIRFQPTTSTGIAEKSTLRDSTTEEKVPPITSTEGQVYATMEGSALGEVEDVDLSKPVSTVPQFAHTSEVEGLAFVSYSSTQEPTTYVDSSHTIPLSVIPKTDWGVLVPSVPSEDEVLGEPSQDILVIDQTRLEATISPETMRTTKITEGTTQEEFPWKEQTAEKPVPALSSTAWTPKEAVTPLDEQEGDGSAYTVSEDELLTGSERVPVLETTPVGKIDHSVSYPPGAVTEHKVKTDEVVTLTPRIGPKVSLSPGPEQKYETEGSSTTGFTSSLSPFSTHITQLMEETTTEKTSLEDIDLGSGLFEKPKATELIEFSTIKVTVPSDITTAFSSVDRLHTTSAFKPSSAITKKPPLIDREPGEETTSDMVIIGESTSHVPPTTLEDIVAKETETDIDREYFTTSSPPATQPTRPPTVEDKEAFGPQALSTPQPPASTKFHPDINVYIIEVRENKTGRMSDLSVIGHPIDSESKEDEPCSEETDPVHDLMAEILPEFPDIIEIDLYHSEENEEEEEECANATDVTTTPSVQYINGKHLVTTVPKDPEAAEARRGQFESVAPSQNFSDSSESDTHPFVIAKTELSTAVQPNESTETTESLEVTWKPETYPETSEHFSGGEPDVFPTVPFHEEFESGTAKKGAESVTERDTEVGHQAHEHTEPVSLFPEESSGEIAIDQESQKIAFARATEVTFGEEVEKSTSVTYTPTIVPSSASAYVSEEEAVTLIGNPWPDDLLSTKESWVEATPRQVVELSGSSSIPITEGSGEAEEDEDTMFTMVTDLSQRNTTDTLITLDTSRIITESFFEVPATTIYPVSEQPSAKVVPTKFVSETDTSEWISSTTVEEKKRKEEEGTTGTASTFEVYSSTQRSDQLILPFELESPNVATSSDSGTRKSFMSLTTPTQSEREMTDSTPVFTETNTLENLGAQTTEHSSIHQPGVQEGLTTLPRSPASVFMEQGSGEAAADPETTTVSSFSLNVEYAIQAEKEVAGTLSPHVETTFSTEPTGLVLSTVMDRVVAENITQTSREIVISERLGEPNYGAEIRGFSTGFPLEEDFSGDFREYSTVSHPIAKEETVMMEGSGDAAFRDTQTSPSTVPTSVHISHISDSEGPSSTMVSTSAFPWEEFTSSAEGSGEQLVTVSSSVVPVLPSAVQKFSGTASSIIDEGLGEVGTVNEIDRRSTILPTAEVEGTKAPVEKEEVKVSGTVSTNFPQTIEPAKLWSRQEVNPVRQEIESETTSEEQIQEEKSFESPQNSPATEQTIFDSQTFTETELKTTDYSVLTTKKTYSDDKEMKEEDTSLVNMSTPDPDANGLESYTTLPEATEKSHFFLATALVTESIPAEHVVTDSPIKKEESTKHFPKGMRPTIQESDTELLFSGLGSGEEVLPTLPTESVNFTEVEQINNTLYPHTSQVESTSSDKIEDFNRMENVAKEVGPLVSQTDIFEGSGSVTSTTLIEILSDTGAEGPTVAPLPFSTDIGHPQNQTVRWAEEIQTSRPQTITEQDSNKNSSTAEINETTTSSTDFLARAYGFEMAKEFVTSAPKPSDLYYEPSGEGSGEVDIVDSFHTSATTQATRQESSTTFVSDGSLEKHPEVPSAKAVTADGFPTVSVMLPLHSEQNKSSPDPTSTLSNTVSYERSTDGSFQDRFREFEDSTLKPNRKKPTENIIIDLDKEDKDLILTITESTILEILPELTSDKNTIIDIDHTKPVYEDILGMQTDIDTEVPSEPHDSNDESNDDSTQVQEIYEAAVNLSLTEETFEGSADVLASYTQATHDESMTYEDRSQLDHMGFHFTTGIPAPSTETELDVLLPTATSLPIPRKSATVIPEIEGIKAEAKALDDMFESSTLSDGQAIADQSEIIPTLGQFERTQEEYEDKKHAGPSFQPEFSSGAEEALVDHTPYLSIATTHLMDQSVTEVPDVMEGSNPPYYTDTTLAVSTFAKLSSQTPSSPLTIYSGSEASGHTEIPQPSALPGIDVGSSVMSPQDSFKEIHVNIEATFKPSSEEYLHITEPPSLSPDTKLEPSEDDGKPELLEEMEASPTELIAVEGTEILQDFQNKTDGQVSGEAIKMFPTIKTPEAGTVITTADEIELEGATQWPHSTSASATYGVEAGVVPWLSPQTSERPTLSSSPEINPETQAALIRGQDSTIAASEQQVAARILDSNDQATVNPVEFNTEVATPPFSLLETSNETDFLIGINEESVEGTAIYLPGPDRCKMNPCLNGGTCYPTETSYVCTCVPGYSGDQCELDFDECHSNPCRNGATCVDGFNTFRCLCLPSYVGALCEQDTETCDYGWHKFQGQCYKYFAHRRTWDAAERECRLQGAHLTSILSHEEQMFVNRVGHDYQWIGLNDKMFEHDFRWTDGSTLQYENWRPNQPDSFFSAGEDCVVIIWHENGQWNDVPCNYHLTYTCKKGTVACGQPPVVENAKTFGKMKPRYEINSLIRYHCKDGFIQRHLPTIRCLGNGRWAIPKITCMNPSAYQRTYSMKYFKNSSSAKDNSINTSKHDHRWSRRWQESRR
＜配列番号７＞MRC1のアミノ酸配列を示す。
MRLPLLLVFASVIPGAVLLLDTRQFLIYNEDHKRCVDAVSPSAVQTAACNQDAESQKFRWVSESQIMSVAFKLCLGVPSKTDWVAITLYACDSKSEFQKWECKNDTLLGIKGEDLFFNYGNRQEKNIMLYKGSGLWSRWKIYGTTDNLCSRGYEAMYTLLGNANGATCAFPFKFENKWYADCTSAGRSDGWLWCGTTTDYDTDKLFGYCPLKFEGSESLWNKDPLTSVSYQINSKSALTWHQARKSCQQQNAELLSITEIHEQTYLTGLTSSLTSGLWIGLNSLSFNSGWQWSDRSPFRYLNWLPGSPSAEPGKSCVSLNPGKNAKWENLECVQKLGYICKKGNTTLNSFVIPSESDVPTHCPSQWWPYAGHCYKIHRDEKKIQRDALTTCRKEGGDLTSIHTIEELDFIISQLGYEPNDELWIGLNDIKIQMYFEWSDGTPVTFTKWLRGEPSHENNRQEDCVVMKGKDGYWADRGCEWPLGYICKMKSRSQGPEIVEVEKGCRKGWKKHHFYCYMIGHTLSTFAEANQTCNNENAYLTTIEDRYEQAFLTSFVGLRPEKYFWTGLSDIQTKGTFQWTIEEEVRFTHWNSDMPGRKPGCVAMRTGIAGGLWDVLKCDEKAKFVCKHWAEGVTHPPKPTTTPEPKCPEDWGASSRTSLCFKLYAKGKHEKKTWFESRDFCRALGGDLASINNKEEQQTIWRLITASGSYHKLFWLGLTYGSPSEGFTWSDGSPVSYENWAYGEPNNYQNVEYCGELKGDPTMSWNDINCEHLNNWICQIQKGQTPKPEPTPAPQDNPPVTEDGWVIYKDYQYYFSKEKETMDNARAFCKRNFGDLVSIQSESEKKFLWKYVNRNDAQSAYFIGLLISLDKKFAWMDGSKVDYVSWATGEPNFANEDENCVTMYSNSGFWNDINCGYPNAFICQRHNSSINATTVMPTMPSVPSGCKEGWNFYSNKCFKIFGFMEEERKNWQEARKACIGFGGNLVSIQNEKEQAFLTYHMKDSTFSAWTGLNDVNSEHTFLWTDGRGVHYTNWGKGYPGGRRSSLSYEDADCVVIIGGASNEAGKWMDDTCDSKRGYICQTRSDPSLTNPPATIQTDGFVKYGKSSYSLMRQKFQWHEAETYCKLHNSLIASILDPYSNAFAWLQMETSNERVWIALNSNLTDNQYTWTDKWRVRYTNWAADEPKLKSACVYLDLDGYWKTAHCNESFYFLCKRSDEIPATEPPQLPGRCPESDHTAWIPFHGHCYYIESSYTRNWGQASLECLRMGSSLVSIESAAESSFLSYRVEPLKSKTNFWIGLFRNVEGTWLWINNSPVSFVNWNTGDPSGERNDCVALHASSGFWSNIHCSSYKGYICKRPKIIDAKPTHELLTTKADTRKMDPSKPSSNVAGVVIIVILLILTGAGLAAYFFYKKRRVHLPQEGAFENTLYFNSQSSPGTSDMKDLVGNIEQNEHSVI
＜配列番号８＞FGAのアミノ酸配列を示す。
MFSMRIVCLVLSVVGTAWTADSGEGDFLAEGGGVRGPRVVERHQSACKDSDWPFCSDEDWNYKCPSGCRMKGLIDEVNQDFTNRINKLKNSLFEYQKNNKDSHSLTTNIMEILRGDFSSANNRDNTYNRVSEDLRSRIEVLKRKVIEKVQHIQLLQKNVRAQLVDMKRLEVDIDIKIRSCRGSCSRALAREVDLKDYEDQQKQLEQVIAKDLLPSRDRQHLPLIKMKPVPDLVPGNFKSQLQKVPPEWKALTDMPQMRMELERPGGNEITRGGSTSYGTGSETESPRNPSSAGSWNSGSSGPGSTGNRNPGSSGTGGTATWKPGSSGPGSTGSWNSGSSGTGSTGNQNPGSPRPGSTGTWNPGSSERGSAGHWTSESSVSGSTGQWHSESGSFRPDSPGSGNARPNNPDWGTFEEVSGNVSPGTRREYHTEKLVTSKGDKELRTGKEKVTSGSTTTTRRSCSKTVTKTVIGPDGHKEVTKEVVTSEDGSDCPEAMDLGTLSGIGTLDGFRHRHPDEAAFFDTASTGKTFPGFFSPMLGEFVSETESRGSESGIFTNTKESSSHHPGIAEFPSRGKSSSYSKQFTSSTSYNRGDSTFESKSYKMADEAGSEADHEGTHSTKRGHAKSRPVRDCDDVLQTHPSGTQSGIFNIKLPGSSKIFSVYCDQETSLGGWLLIQQRMDGSLNFNRTWQDYKRGFGSLNDEGEGEFWLGNDYLHLLTQRGSVLRVELEDWAGNEAYAEYHFRVGSEAEGYALQVSSYEGTAGDALIEGSVEEGAEYTSHNNMQFSTFDRDADQWEENCAEVYGGGWWYNNCQAANLNGIYYPGGSYDPRNNSPYEIENGVVWVSFRGADYSLRAVRMKIRPLVTQ
<SEQ ID NO: 1> Shows the amino acid sequence of FBLN2.
<SEQ ID NO: 2> Shows the amino acid sequence of ANGPTL3.
MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEI ENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSK PERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE
<SEQ ID NO: 3> Shows the amino acid sequence of MAN2A1.
<SEQ ID NO: 4> Shows the amino acid sequence of CSF1.
<SEQ ID NO: 5> Shows the amino acid sequence of C1QA.
MEGPRGWLVLCVLAISLASMVTEDLCRAPDGKKGEAGRPGRRGRPGLKGEQGEPGAPGIRTGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGTKGSPGNIKDQPRPAFSAIRRNPPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQWEICLSIVSSSRGQVRRSLGFCDTTNKGLFQVVSGGMVLQL QQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSA
<SEQ ID NO: 6> Shows the amino acid sequence of VCAN.
<SEQ ID NO: 7> Shows the amino acid sequence of MRC1.
<SEQ ID NO: 8> Shows the amino acid sequence of FGA.
MFSMRIVCLVLSVVGTAWTADSGEGDFLAEGGGVRGPRVVERHQSACKDSDWPFCSDEDWNYKCPSGCRMKGLIDEVNQDFTNRINKLKNSLFEYQKNNKDSHSLTTNIMEILRGDFSSANNRDNTYNRVSEDLRSRIEVLKRKVIEKVQHIQLLQKNVRAQLVDMKRLEVDIDIKIRSCRGSCSRALAREVDLKDYEDQQKQLEQVIAKDLLPSRDR QHLPLIKMKPVPDLVPGNFKSQLQKVPPEWKALTDMPQMRMELERPGGNEITRGGSTSYGTGSETESPRNPSSAGSWNSGSSGPGSTGNRNPGSSGTGGTATWKPGSSGPGSTGSWNSGSSGTGSTGNQNPGSPRPGSTGTWNPGSSERGSAGHWTSESSVSGSTGQWHSESGSFRPDSPGSGNARPNNPDWGTFEEVSGNVSPGTRREYHTEKLVTSKGDKELRTGKE KVTSGSTTTTRRSCSKTVTKTVIGPDGHKEVTKEVVTSEDGSDCPEAMDLGTLSGIGTLDGFRHRHPDEAAFFDTASTGKTFPGFFSPMLGEFVSETESRGSESGIFTNTKESSSHPGIAEFPSRGKSSSYSKQFTSSTSYNRGDSTFESKSYKMADEAGSEADHEGTHSTKRGHAKSRPVRDCDDVLQTHPSGTQSGIFNIKLPGSSKIFSVYCDQETSLGGW LLIQQRMDGSLNFNRTWQDYKRGFGSLNDEGEGEFWLGNDYLHLLTQRGSVLRVELEDWAGNEAYAEYHFRVGSEAEGYALQVSSYEGTAGDALIEGSVEEGAEYTSHNNMQFSTFDRDADQWEENCAEVYGGGWWYNNCQAANLNGIYYPGGSYDPRNNSPYEIENGVVWVSFRGADYSLRAVRMKIRPLVTQ
Claims (10)
(i) T抗原：Hex(1)HexNAc(1)が付加されたFBLN2、
(ii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)が付加されたが付加されたANGPTL3、
(iii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)が付加されたMAN2A1、
(iv) T抗原：Hex(1)HexNAc(1)が付加されたCSF1及びdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたCSF1、
(v) T抗原：Hex(1)HexNAc(1)が付加されたVCAN、
(vi) フコシル化T抗原：Hex(1)HexNAc(1)dHex(1)が付加されたC1QA
(vii) T抗原：Hex(1)HexNAc(1)が付加されたMRC1、並びに
(viii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)が付加されたFGA
4. The method according to claim 3, wherein the protein modified with an O-type sugar chain is at least one selected from the group consisting of (i) to (v) below.
(i) T antigen: FBLN2 with Hex(1)HexNAc(1) added,
(ii) Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1) and/or di-Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(2), but ANGPTL3 added;
(iii) Sialyl T antigen: Hex(1) HexNAc(1) NeuAc(1) and/or di-Sialyl T antigen: MAN2A1 with Hex(1) HexNAc(1) NeuAc(2) added;
(iv) T antigen: CSF1 with Hex(1) HexNAc(1) added and CSF1 with di-Sialyl T antigen Hex(1) HexNAc(1) NeuAc(2) added,
(v) T antigen: VCAN with Hex(1)HexNAc(1) added;
(vi) Fucosylated T antigen: C1QA with Hex(1) HexNAc(1) dHex(1) added
(vii) T antigen: MRC1 with Hex(1)HexNAc(1) added, and
(viii) FGA with Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1) and/or di-Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(2)
(ia) aa330-349、aa350-364及びaa365-378のうち少なくとも１つの領域内の部位にT抗原：Hex(1)HexNAc(1)が付加されたFBLN2、
(iia) aa206-221及びaa222-235のうち少なくとも１つの領域内の部位にSialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたANGPTL3、
(iiia) aa211-221領域内の部位にSialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原 Hex(1)HexNAc(1)NeuAc(2)が付加されたMAN2A1、
(iva) aa356-395領域内の部位にT抗原：Hex(1)HexNAc(1)が付加されたCSF1及び370-395領域内の部位にdi-Sialyl T抗原 Hex(1)HexNAc(1)NeuAc(2)が付加されたCSF1、
(va)aa1415-1430及びaa2468-2475のうちの少なくとも１つの領域内の部位にT抗原：Hex(1)HexNAc(1)が付加されたVCAN、
(via) aa101-121領域内の部位にフコシル化T抗原：Hex(1)HexNAc(1)dHex(1)が付加されたC1QA
(viia) aa1215-1229領域内の部位にT抗原：Hex(1)HexNAc(1)が付加されたMRC1、並びに
(viiia) aa354-367及びaa511-527のうち少なくとも１つの領域内の部位にSialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原 Hex(1)HexNAc(1)NeuAc(2)が付加されたFGA
5. The method according to claim 4, wherein the protein modified with an O-type sugar chain is at least one selected from the group consisting of (ia) to (va) below.
(ia) FBLN2 with T antigen: Hex(1) HexNAc(1) added to at least one region of aa330-349, aa350-364 and aa365-378;
(iia) Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1) and/or di-Sialyl T antigen Hex(1)HexNAc( 1) ANGPTL3 with NeuAc(2) added,
(iiia) Sialyl T antigen: Hex(1) HexNAc(1) NeuAc(1) and/or di-Sialyl T antigen Hex(1) HexNAc(1) NeuAc(2) is added to the site within the aa211-221 region. MAN2A1,
(iva) T antigen in the aa356-395 region: CSF1 with Hex(1)HexNAc(1) and di-Sialyl T antigen in the region 370-395 Hex(1)HexNAc(1)NeuAc CSF1 with (2) added,
(va) VCAN with T antigen: Hex(1) HexNAc(1) added to a site within at least one region of aa1415-1430 and aa2468-2475;
(via) C1QA with fucosylated T antigen: Hex(1)HexNAc(1)dHex(1) added to the aa101-121 region
(viia) MRC1 with T antigen: Hex(1) HexNAc(1) added to the site within the aa1215-1229 region, and
(viiia) Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1) and/or di-Sialyl T antigen Hex(1)HexNAc( 1)FGA with NeuAc(2) added
(i) T抗原：Hex(1)HexNAc(1)が付加されたFBLN2、
(ii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1) 及び/又はdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたANGPTL3、
(iii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたMAN2A1、
(iv) T抗原：Hex(1)HexNAc(1)が付加されたCSF1及びdi-Sialyl T抗原Hex(1)HexNAc(1)NeuAc(2)が付加されたCSF1、
(v) T抗原：Hex(1)HexNAc(1)が付加されたVCAN、
(vi) フコシル化T抗原：Hex(1)HexNAc(1)dHex(1)が付加されたC1QA
(vii) T抗原：Hex(1)HexNAc(1)が付加されたMRC1、並びに
(viii) Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(1)及び/又はdi-Sialyl T抗原：Hex(1)HexNAc(1)NeuAc(2)が付加されたFGA A test kit comprising a reagent capable of specifically detecting at least one O-type glycosylated protein selected from the group consisting of (i) to (v) below.
(i) T antigen: FBLN2 with Hex(1)HexNAc(1) added,
(ii) Sialyl T antigen: ANGPTL3 with Hex(1)HexNAc(1)NeuAc(1) and/or di-Sialyl T antigen Hex(1)HexNAc(1)NeuAc(2) added;
(iii) Sialyl T antigen: MAN2A1 with Hex(1) HexNAc(1) NeuAc(1) and/or di-Sialyl T antigen Hex(1) HexNAc(1) NeuAc(2) added;
(iv) T antigen: CSF1 with Hex(1) HexNAc(1) added and CSF1 with di-Sialyl T antigen Hex(1) HexNAc(1) NeuAc(2) added,
(v) T antigen: VCAN with Hex(1)HexNAc(1) added;
(vi) Fucosylated T antigen: C1QA with Hex(1) HexNAc(1) dHex(1) added
(vii) T antigen: MRC1 with Hex(1)HexNAc(1) added, and
(viii) FGA with Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(1) and/or di-Sialyl T antigen: Hex(1)HexNAc(1)NeuAc(2)
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