JPH09201191A - Alpha1-6 fucosyltransferase derived from swine - Google Patents
Alpha1-6 fucosyltransferase derived from swineInfo
- Publication number
- JPH09201191A JPH09201191A JP1036596A JP1036596A JPH09201191A JP H09201191 A JPH09201191 A JP H09201191A JP 1036596 A JP1036596 A JP 1036596A JP 1036596 A JP1036596 A JP 1036596A JP H09201191 A JPH09201191 A JP H09201191A
- Authority
- JP
- Japan
- Prior art keywords
- fucosyltransferase
- enzyme
- 2manα1
- fucose
- sugar chain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000282898 Sus scrofa Species 0.000 title abstract 3
- 108010027343 Glycoprotein 6-alpha-L-fucosyltransferase Proteins 0.000 title 1
- 102000006471 Fucosyltransferases Human genes 0.000 claims abstract description 31
- 108010019236 Fucosyltransferases Proteins 0.000 claims abstract description 31
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims abstract description 13
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims abstract description 13
- 210000004556 brain Anatomy 0.000 claims abstract description 12
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 claims abstract description 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 8
- LQEBEXMHBLQMDB-UHFFFAOYSA-N GDP-L-fucose Natural products OC1C(O)C(O)C(C)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C3=C(C(N=C(N)N3)=O)N=C2)O1 LQEBEXMHBLQMDB-UHFFFAOYSA-N 0.000 claims abstract description 5
- LQEBEXMHBLQMDB-JGQUBWHWSA-N GDP-beta-L-fucose Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C3=C(C(NC(N)=N3)=O)N=C2)O1 LQEBEXMHBLQMDB-JGQUBWHWSA-N 0.000 claims abstract description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims abstract description 5
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 claims abstract description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 4
- 230000000694 effects Effects 0.000 claims description 29
- 230000009471 action Effects 0.000 claims description 9
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 claims description 8
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 claims description 8
- 229910021645 metal ion Inorganic materials 0.000 claims description 8
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 claims description 7
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 2
- 230000005764 inhibitory process Effects 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 50
- 108090000790 Enzymes Proteins 0.000 abstract description 50
- 238000000746 purification Methods 0.000 abstract description 5
- QGWNDRXFNXRZMB-UUOKFMHZSA-N GDP Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O QGWNDRXFNXRZMB-UUOKFMHZSA-N 0.000 abstract description 4
- QGWNDRXFNXRZMB-UHFFFAOYSA-N guanidine diphosphate Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O QGWNDRXFNXRZMB-UHFFFAOYSA-N 0.000 abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 239000000032 diagnostic agent Substances 0.000 abstract description 3
- 229940039227 diagnostic agent Drugs 0.000 abstract description 3
- 230000006870 function Effects 0.000 abstract description 3
- 238000012916 structural analysis Methods 0.000 abstract 2
- 239000000872 buffer Substances 0.000 description 10
- 102000051366 Glycosyltransferases Human genes 0.000 description 8
- 108700023372 Glycosyltransferases Proteins 0.000 description 8
- 239000000284 extract Substances 0.000 description 8
- 239000012528 membrane Substances 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 108700014210 glycosyltransferase activity proteins Proteins 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 4
- 239000007987 MES buffer Substances 0.000 description 4
- 229920002684 Sepharose Polymers 0.000 description 4
- 239000013504 Triton X-100 Substances 0.000 description 4
- 229920004890 Triton X-100 Polymers 0.000 description 4
- 229960001230 asparagine Drugs 0.000 description 4
- 235000009582 asparagine Nutrition 0.000 description 4
- 201000003883 Cystic fibrosis Diseases 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 210000001124 body fluid Anatomy 0.000 description 3
- 239000010839 body fluid Substances 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 230000003902 lesion Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 238000005199 ultracentrifugation Methods 0.000 description 3
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 2
- FZHXIRIBWMQPQF-UHFFFAOYSA-N Glc-NH2 Natural products O=CC(N)C(O)C(O)C(O)CO FZHXIRIBWMQPQF-UHFFFAOYSA-N 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 102000004357 Transferases Human genes 0.000 description 2
- 108090000992 Transferases Proteins 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 229910052749 magnesium Inorganic materials 0.000 description 2
- 229910052748 manganese Inorganic materials 0.000 description 2
- 239000011572 manganese Substances 0.000 description 2
- 239000008057 potassium phosphate buffer Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 210000002288 golgi apparatus Anatomy 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- RAJSAPSERUGCBT-UHFFFAOYSA-N n'-pyridin-2-ylbutane-1,4-diamine Chemical compound NCCCCNC1=CC=CC=N1 RAJSAPSERUGCBT-UHFFFAOYSA-N 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明はブタ由来のα１→６
フコシルトランスフェラーゼに関し、詳細にはアスパラ
ギン型糖鎖（Ａｓｎ型糖鎖）の根幹部のＡｓｎに結合し
たＮ−アセチルグルコサミン（ＧｌｃＮＡｃ）に、α１
→６結合でもって、ＧＤＰ（グアノシンジホスフェー
ト）−フコースからフコースを転移する酵素であって、
糖鎖の修飾や合成などの糖鎖工学および／または癌など
の疾病の診断に有用なブタ由来の新規なα１→６フコシ
ルトランスフェラーゼに関する。TECHNICAL FIELD The present invention relates to pig-derived α1 → 6.
Regarding fucosyltransferase, in detail, α1 is added to N-acetylglucosamine (GlcNAc) linked to Asn in the root of asparagine-type sugar chain (Asn-type sugar chain).
An enzyme that transfers GDP (guanosine diphosphate) -fucose from fucose with 6 bonds,
The present invention relates to a novel α1 → 6 fucosyltransferase derived from pig, which is useful for sugar chain engineering such as sugar chain modification and synthesis and / or diagnosis of diseases such as cancer.
【０００２】[0002]
【従来の技術】生体内で種々の重要な生理作用を有する
糖タンパク質、糖脂質、プロテオグリカンの糖鎖部分の
構造と機能に関する関心が高まっており、近年、その研
究が盛んに行われている。糖鎖は糖加水分解酵素および
糖転移酵素の作用により形成されるが、その中でも糖転
移酵素が寄与するところが大きい。一般に、糖転移酵素
は糖ヌクレオチドを糖供与体として、受容体となる糖鎖
に糖を転移し、糖鎖伸長を行う酵素であると定義される
が、その受容体糖鎖構造に対する特異性は厳密であり、
通常、一つのグリコシド結合は、対応する一つの糖転移
酵素によって形成される。それ故、糖転移酵素は複合糖
質の糖鎖部分の構造研究、特定の糖鎖構造の簡便な合
成、天然の糖鎖構造の修飾など様々な用途への利用、ま
た、糖鎖の人工的な改変による複合糖質あるいは細胞性
質の改変への利用が期待されている。しかしながら、現
在、利用できる糖転移酵素の種類とその供給には限りが
あり、このことが、実用上の大きな障壁となっていた。
これらのことから、基質特異性の明らかな種々の糖転移
酵素の開発と量的供給が望まれているのが実情である。2. Description of the Related Art There has been an increasing interest in the structure and function of the sugar chain portion of glycoproteins, glycolipids, and proteoglycans, which have various important physiological actions in the living body, and in recent years, research has been actively conducted. Sugar chains are formed by the action of sugar hydrolases and glycosyltransferases, of which glycosyltransferases are the major contributors. Generally, a glycosyltransferase is defined as an enzyme that transfers a sugar to a sugar chain serving as an acceptor by using a sugar nucleotide as a sugar donor and extends the sugar chain, but its specificity for the sugar chain structure of the acceptor is Strict,
Usually, one glycosidic bond is formed by one corresponding glycosyl transferase. Therefore, glycosyltransferases are used for various purposes such as structural studies of sugar chains of glycoconjugates, simple synthesis of specific sugar chains, modification of natural sugar chains, and artificial sugar chains. It is expected to be used for the modification of glycoconjugates or cellular properties by various modifications. However, there are currently limited types of glycosyltransferases that can be used and their supply, and this has been a major obstacle in practical use.
From these facts, it is the actual situation that the development and quantitative supply of various glycosyltransferases with clear substrate specificity are desired.
【０００３】α１→６フコシルトランスフェラーゼは、
細胞内小器官のゴルジ体に存在する酵素であり、アスパ
ラギン結合型糖鎖のプロセッシングを制御する酵素のう
ちの一つであると考えられる重要な酵素である。該酵素
をアスパラギン結合型糖鎖に作用させることで、その制
御機構の解明、糖鎖構造形成の制御等に役立つと考えら
れる。また、肝臓癌や嚢胞性線維症などのいくつかの疾
病におけるα１→６フコシルトランスフェラーゼ活性の
上昇、本酵素反応生成物の割合の増加が知られており、
該酵素の診断用途としての早急なる開発も望まれてい
る。[0003] α1 → 6 fucosyltransferase is
It is an enzyme that exists in the Golgi apparatus of organelles and is an important enzyme that is considered to be one of the enzymes that control the processing of asparagine-linked sugar chains. The action of the enzyme on an asparagine-linked sugar chain is thought to be useful for elucidation of its control mechanism, control of sugar chain structure formation, and the like. In addition, it is known that α1 → 6 fucosyltransferase activity is increased in some diseases such as liver cancer and cystic fibrosis, and that the ratio of this enzymatic reaction product is increased.
Urgent development of the enzyme for diagnostic use is also desired.
【０００４】従来、α１→６フコシルトランスフェラー
ゼは、各種動物の体液あるいは臓器中、各種動物の培養
細胞に活性は検出されているものの、精製された酵素標
品としては、ヒト嚢胞性線維症細胞破砕物由来の酵素
〔ジャーナル オブ バイオロジカル ケミストリー
(Journal of Biological Chemistry）、第２６６巻、第
２１５７２〜２１５７７頁（１９９１）〕以外には知ら
れてない。しかしながら、この報告では、１）至適ｐＨ
が５．６であって、生理学的ｐＨとは異なる、２）ＳＤ
Ｓ−ポリアクリルアミドゲル電気泳動による分子量が３
４，０００および３９，０００と比較的低分子量であ
る、３）ヒト細胞由来のため大量および安定供給が事実
上、困難であるなどの欠点を有していた。Conventionally, α1 → 6 fucosyltransferase has been found to have activity in the body fluids or organs of various animals, or in the cultured cells of various animals, but as a purified enzyme preparation, human cystic fibrosis cell disruption is used. Enzymes derived from things [Journal of Biological Chemistry
(Journal of Biological Chemistry), Volume 266, 215272-21577 (1991)]. However, in this report, 1) optimum pH
Is 5.6, which is different from physiological pH 2) SD
The molecular weight by S-polyacrylamide gel electrophoresis is 3
It has a relatively low molecular weight of 4,000 and 39,000, and 3) it has a drawback that it is difficult to supply in large quantities and stably because it is derived from human cells.
【０００５】一方、糖鎖の合成法としては、化学合成法
も時として採用されているが、その複雑な合成経路およ
び反応の特異性から、実用上は少糖類の合成に適用され
るに限定されている。特に、受容体基質であるアスパラ
ギン結合型糖鎖の合成は、事実上、困難であった。さら
に、フコースをα１→６結合で、アスパラギン型糖鎖の
Ａｓｎに結合したＧｌｃＮＡｃに結合させる反応は、非
常な困難性を伴う。On the other hand, as a method for synthesizing a sugar chain, a chemical synthesis method is sometimes adopted, but due to its complicated synthetic route and reaction specificity, it is practically limited to the application to the synthesis of oligosaccharides. Has been done. In particular, the synthesis of an asparagine-linked sugar chain that is a receptor substrate was practically difficult. Furthermore, the reaction of allowing fucose to bind to GlcNAc linked to Asn of an asparagine-type sugar chain through an α1 → 6 bond is extremely difficult.
【０００６】[0006]
【発明が解決しようとする課題】本発明の目的は、糖鎖
構造解析、糖鎖工学用試薬、診断薬として、大量にしか
も安定に供給可能なα１→６フコシルトランスフェラー
ゼを提供することにある。SUMMARY OF THE INVENTION An object of the present invention is to provide α1 → 6 fucosyltransferase which can be stably supplied in a large amount as a sugar chain structure analysis, sugar chain engineering reagent, and diagnostic agent.
【０００７】[0007]
【課題を解決するための手段】上記目的を達成するため
に、本発明者らは、該酵素の受容体であるアスパラギン
型糖鎖に類似した蛍光標識された基質を用いることによ
り、フコースをα１→６結合でもって、アスパラギン型
糖鎖のＡｓｎに結合したＧｌｃＮＡｃに結合させる酵素
の検討に着手し、その結果、精製出発材料として入手が
容易なブタ脳の抽出画分に、該酵素活性を検出し、該画
分から該酵素を精製し、その酵素学的および理化学的性
質を解明し、本発明に到達した。In order to achieve the above object, the present inventors have used a fluorescently labeled substrate similar to the asparagine-type sugar chain that is the receptor for the enzyme to convert fucose to α1. → We started investigation of an enzyme that binds to GlcNAc linked to Asn of asparagine-type sugar chain by 6-linkage, and as a result, detected the enzyme activity in the extract fraction of pig brain, which is easily available as a purified starting material. Then, the enzyme was purified from the fraction, the enzymatic and physicochemical properties thereof were elucidated, and the present invention was accomplished.
【０００８】すなわち、本発明は下記理化学的性質を有
するブタ由来α１→６フコシルトランスフェラーゼであ
る。 （１）作用：ＧＤＰ−フコースのフコース（Ｆｕｃ）
を、ＧｌｃＮＡｃβ１→２Ｍａｎα１→６（ＧｌｃＮＡ
ｃβ１→２Ｍａｎα１→３）Ｍａｎβ１→４ＧｌｃＮＡ
ｃβ１→４ＧｌｃＮＡｃ−Ｒ（Ｒはペプチド鎖のアスパ
ラギン残基を示す）受容体の、最もペプチド鎖に近いＧ
ｌｃＮＡｃの６位の水酸基に転移し、ＧｌｃＮＡｃβ１
→２Ｍａｎα１→６（ＧｌｃＮＡｃβ１→２Ｍａｎα１
→３）Ｍａｎβ１→４ＧｌｃＮＡｃβ１→４（Ｆｕｃα
１→６）ＧｌｃＮＡｃ−Ｒを生成する。 （２）至適ｐＨ：至適ｐＨは、７．０である。 （３）ｐＨ安定性：４℃、５時間の処理で、ｐＨ４．０
〜１０．０の範囲で安定である。 （４）至適温度：至適作用温度は、３０〜３７℃であ
る。 （５）阻害、活性化：活性の発現に、２価金属イオンを
要求せず、また、５ｍＭ ＥＤＴＡ存在下においても活
性は阻害されない。 （６）分子量：約６０，０００（ＳＤＳ−ポリアクリル
アミドゲル電気泳動）を示す。That is, the present invention is a pig-derived α1 → 6 fucosyltransferase having the following physicochemical properties. (1) Action: GDP-fucose of fucose (Fuc)
To GlcNAcβ1 → 2Manα1 → 6 (GlcNAc
cβ1 → 2Manα1 → 3) Manβ1 → 4GlcNA
cβ1 → 4GlcNAc-R (R represents an asparagine residue of the peptide chain) G of the receptor closest to the peptide chain
GlcNAcβ1 is transferred to the 6-position hydroxyl group of lcNAc.
→ 2Manα1 → 6 (GlcNAcβ1 → 2Manα1
→ 3) Manβ1 → 4GlcNAcβ1 → 4 (Fucα
1 → 6) Generate GlcNAc-R. (2) Optimum pH: The optimum pH is 7.0. (3) pH stability: pH 4.0 after treatment at 4 ° C. for 5 hours
It is stable in the range of up to 10.0. (4) Optimum temperature: The optimum working temperature is 30 to 37 ° C. (5) Inhibition and activation: Divalent metal ions are not required for expression of activity, and activity is not inhibited even in the presence of 5 mM EDTA. (6) Molecular weight: about 60,000 (SDS-polyacrylamide gel electrophoresis) is shown.
【０００９】[0009]
【発明の実施態様】本発明の酵素の精製出発材料として
は、フコシルトランスフェラーゼ活性を有するブタの臓
器または体液がある。臓器の具体例としては、例えば、
脳、精巣、膵臓、肺、腎臓等が挙げられる。また、血液
や血清などのブタ体液を用いることもできる。BEST MODE FOR CARRYING OUT THE INVENTION The starting material for purifying the enzyme of the present invention is porcine organ or body fluid having fucosyltransferase activity. Specific examples of organs include, for example,
The brain, testis, pancreas, lung, kidney and the like can be mentioned. In addition, pig body fluid such as blood or serum can also be used.
【００１０】本発明のα１→６フコシルトランスフェラ
ーゼは、例えばブタ脳の破砕物から粗酵素抽出物を調製
し、次に、この粗酵素抽出物から、目的の酵素を分離す
る。この場合、ブタ脳中のα１→６フコシルトランスフ
ェラーゼは、膜結合型酵素であるので、脳破砕物より適
当な界面活性剤を用いて粗酵素抽出液を得ることが通
常、採用される。さらに、この粗酵素抽出液から公知の
精製手段を組み合わせて、精製酵素標品を得ることがで
きる。例えば、限外ろ過膜による濃縮や脱塩、基質類似
体を固定化したアフィニティーカラムクロマトグラフィ
ー、イオン交換カラムクロマトグラフィー、疎水結合カ
ラムクロマトグラフィーなどの組み合わせにより精製を
行い、他のトランスフェラーゼの夾雑活性のない実質的
に単一の酵素標品を得ることができる。例えば、ブタ脳
をリン酸緩衝液中でワーリングブレンダーを用いて破
砕、超遠心分離により膜画分を集めた後、この膜画分よ
り界面活性剤、トリトン（Triton）Ｘ−１００を含むリ
ン酸緩衝液で目的酵素を抽出し、さらに超遠心分離によ
って上清を集めて、粗酵素抽出液を得ることができる。
ＧＤＰ−ヘキサノールアミン−セファロース、ＧｌｃＮ
Ａｃβ１→２Ｍａｎα１→６（ＧｌｃＮＡｃβ１→２Ｍ
ａｎα１→３）Ｍａｎβ１→４ＧｌｃＮＡｃβ１→４Ｇ
ｌｃＮＡｃ−アスパラギン−セファロース等のアフィニ
ティーカラムクロマトグラフィーに供し、フコシルトラ
ンスフェラーゼ活性を示す各画分を集めて、精製するこ
とができる。For the α1 → 6 fucosyltransferase of the present invention, for example, a crude enzyme extract is prepared from a crushed product of porcine brain, and then the desired enzyme is separated from the crude enzyme extract. In this case, since α1 → 6 fucosyltransferase in porcine brain is a membrane-bound enzyme, it is usually adopted to obtain a crude enzyme extract from a crushed brain product using an appropriate surfactant. Furthermore, a purified enzyme preparation can be obtained by combining known purification means from the crude enzyme extract. For example, purification by a combination of concentration and desalting with an ultrafiltration membrane, affinity column chromatography with immobilized substrate analog, ion exchange column chromatography, hydrophobic binding column chromatography, etc., and contaminating activity of other transferases can be performed. It is possible to obtain substantially no single enzyme preparation. For example, porcine brain is crushed in a phosphate buffer using a Waring blender, and a membrane fraction is collected by ultracentrifugation. Then, a phosphate containing a surfactant, Triton X-100, is collected from the membrane fraction. The target enzyme can be extracted with a buffer, and the supernatant can be collected by ultracentrifugation to obtain a crude enzyme extract.
GDP-hexanolamine-sepharose, GlcN
Acβ1 → 2Manα1 → 6 (GlcNAcβ1 → 2M
anα1 → 3) Manβ1 → 4GlcNAcβ1 → 4G
It is possible to subject it to affinity column chromatography such as lcNAc-asparagine-Sepharose and collect each fraction showing the fucosyltransferase activity for purification.
【００１１】本発明の一実施態様であるブタ脳由来のα
１→６フコシルトランスフェラーゼの理化学的性質を下
記に示す。 （１）作用：ＧＤＰ−フコースのフコース（Ｆｕｃ）
を、ＧｌｃＮＡｃβ１→２Ｍａｎα１→６（ＧｌｃＮＡ
ｃβ１→２Ｍａｎα１→３）Ｍａｎβ１→４ＧｌｃＮＡ
ｃβ１→４ＧｌｃＮＡｃ−Ｒ（Ｒはペプチド鎖のアスパ
ラギン残基を示す）受容体の、最もペプチド鎖に近いＧ
ｌｃＮＡｃの６位の水酸基に転移し、ＧｌｃＮＡｃβ１
→２Ｍａｎα１→６（ＧｌｃＮＡｃβ１→２Ｍａｎα１
→３）Ｍａｎβ１→４ＧｌｃＮＡｃβ１→４（Ｆｕｃα
１→６）ＧｌｃＮＡｃ−Ｒの生成物を与える。Α from porcine brain, which is an embodiment of the present invention
The physicochemical properties of 1 → 6 fucosyltransferase are shown below. (1) Action: GDP-fucose of fucose (Fuc)
To GlcNAcβ1 → 2Manα1 → 6 (GlcNAc
cβ1 → 2Manα1 → 3) Manβ1 → 4GlcNA
cβ1 → 4GlcNAc-R (R represents an asparagine residue of the peptide chain) G of the receptor closest to the peptide chain
GlcNAcβ1 is transferred to the 6-position hydroxyl group of lcNAc.
→ 2Manα1 → 6 (GlcNAcβ1 → 2Manα1
→ 3) Manβ1 → 4GlcNAcβ1 → 4 (Fucα
1 → 6) gives the product of GlcNAc-R.
【００１２】（２）活性測定法 本発明のα１→６フコシルトランスフェラーゼ活性の測
定は、次のようにして行った。すなわち、糖鎖末端のア
スパラギンを４−（２−ピリジルアミノ）ブチルアミン
〔ＰＡＢＡ：−ＮＨ2 （ＣＨ2 ）4 −ＮＨ−ｐｙｒｉｄ
ｉｎｅ〕で蛍光標識してある下記化１で示される化合物
を酵素活性の測定基質として用いた。該基質を用いるこ
とにより、フコースがα１→６結合で転移した酵素反応
の生成物の高速液体クロマトグラフィーによる蛍光検出
が可能となる。(2) Activity measurement method The α1 → 6 fucosyltransferase activity of the present invention was measured as follows. That is, asparagine at the sugar chain terminal is converted to 4- (2-pyridylamino) butylamine [PABA: -NH 2 (CH 2 ) 4 -NH-pyrid.
ine] was used as a substrate for measuring enzyme activity. By using the substrate, the product of the enzymatic reaction in which fucose is transferred by α1 → 6 bond can be detected by high performance liquid chromatography.
【００１３】[0013]
【化１】 〔ＰＡ：ピリジルアミノ基を示す〕Embedded image [PA: indicates a pyridylamino group]
【００１４】具体的な測定方法を以下に述べる。上記化
１で示される受容体蛍光標識基質６２．５μＭ、供与体
基質（ＧＤＰ−フコース）６２５μＭを含む２５０ｍＭ
メス（ＭＥＳ）緩衝液、ｐＨ７．０、４０μｌに、試料
液１０μｌおよび１．２５％トリトンＸ−１００を添加
混合する。３７℃で１時間反応させた後、５分間の煮沸
により反応を停止させ、反応液を高速液体クロマトグラ
フィーに供し、生成物のピークを蛍光検出器で定量す
る。酵素量１単位は、この条件下で、１分間に１ｐｍｏ
ｌｅのＧｌｃＮＡｃβ１→２Ｍａｎα１→６（ＧｌｃＮ
Ａｃβ１→２Ｍａｎα１→３）Ｍａｎβ１→４ＧｌｃＮ
Ａｃβ１→４（Ｆｕｃα１→６）ＧｌｃＮＡｃ−Ｒ〔Ｒ
はＡｓｎ−ＮＨ−（ＣＨ2 ）4 −ＰＡ〕を生じるものと
した。A specific measuring method will be described below. 250 mM containing the acceptor fluorescence-labeled substrate 62.5 μM and the donor substrate (GDP-fucose) 625 μM represented by the above chemical formula 1
10 μl of sample solution and 1.25% Triton X-100 are added to 40 μl of MES buffer, pH 7.0, and mixed. After reacting at 37 ° C. for 1 hour, the reaction is stopped by boiling for 5 minutes, the reaction solution is subjected to high performance liquid chromatography, and the peak of the product is quantified with a fluorescence detector. One unit of the enzyme amount is 1 pmo per minute under these conditions.
le GlcNAcβ1 → 2Manα1 → 6 (GlcN
Acβ1 → 2Manα1 → 3) Manβ1 → 4GlcN
Acβ1 → 4 (Fucα1 → 6) GlcNAc-R [R
Was as causing a Asn-NH- (CH 2) 4 -PA ].
【００１５】（３）至適ｐＨ 本発明のα１→６フコシルトランスフェラーゼは、ｐＨ
７．０〜７．５付近に高い活性を示す。 （４）ｐＨ安定性 本発明のα１→６フコシルトランスフェラーゼは、ｐＨ
４〜１０で比較的安定であり、特にｐＨ５〜９の間にお
いてより良好な安定性を示す。 （５）至適温度 本発明のα１→６フコシルトランスフェラーゼは、至適
温度が３７℃付近に認められ、また、２０〜４０℃の範
囲で十分な作用を保持する。 （６）２価金属イオン要求性 本発明のα１→６フコシルトランスフェラーゼは、マグ
ネシウム、マンガンなどの２価金属イオンの非存在下で
も十分な活性を示す。さらに、キレート剤であるＥＤＴ
Ａ５ｍＭの存在下でも十分な活性を示す。 （７）分子量 本発明のα１→６フコシルトランスフェラーゼの精製標
品は、ＳＤＳ−ポリアクリルアミドゲル電気泳動におい
て、分子量約６０，０００のところに単一バンドを示
す。(3) Optimum pH The α1 → 6 fucosyltransferase of the present invention has a pH of
It exhibits high activity around 7.0 to 7.5. (4) pH stability The α1 → 6 fucosyltransferase of the present invention has a pH of
It is relatively stable at 4-10, and shows better stability especially at pH 5-9. (5) Optimum temperature The α1 → 6 fucosyltransferase of the present invention has an optimum temperature of around 37 ° C., and retains a sufficient action in the range of 20 to 40 ° C. (6) Divalent Metal Ion Requirement The α1 → 6 fucosyltransferase of the present invention shows sufficient activity even in the absence of divalent metal ions such as magnesium and manganese. In addition, the chelating agent EDT
It shows sufficient activity even in the presence of A5 mM. (7) Molecular weight The purified preparation of α1 → 6 fucosyltransferase of the present invention shows a single band at a molecular weight of about 60,000 in SDS-polyacrylamide gel electrophoresis.
【００１６】以上の性質より判断して、本発明のα１→
６フコシルトランスフェラーゼは、至適ｐＨ、金属イオ
ン要求性、分子量の点で、従来のヒト嚢胞性線維症細胞
由来のα１→６フコシルトランスフェラーゼ（至適ｐＨ
５．６、分子量３４，０００および３９，０００）とは
明らかに相違する新規な酵素である。Judging from the above properties, α1 →
6-fucosyltransferase is an α1 → 6 fucosyltransferase (optimum pH) derived from conventional human cystic fibrosis cells in terms of optimum pH, metal ion requirement and molecular weight.
5.6, molecular weights 34,000 and 39,000) is a novel enzyme which is clearly different.
【００１７】本発明のα１，６−フコシルトランスフェ
ラーゼの期待される用途としては、１）糖鎖構造の改変
と機能解析：アスパラギン型糖鎖に新たにフコースを導
入し、糖鎖構造を人工的に改変することができ、細胞機
能変化や複合糖質のプロセッシング制御機構の解明およ
び糖鎖の役割を解明することができる、２）酵素活性に
よる病変の診断：本酵素活性は腫瘍化に伴う種々の病変
を反映しており、酵素活性を測定することにより癌など
の疾病の診断を行うことができる、３）特異抗体による
病変の診断：本酵素により誘導される特異抗体を用いる
ことにより種々の疾病の診断を行うことができる、など
の極めて有用な用途がある。The expected uses of the α1,6-fucosyltransferase of the present invention are as follows: 1) Modification of sugar chain structure and functional analysis: New fucose is introduced into asparagine-type sugar chain to artificially change the sugar chain structure. It can be modified to elucidate the mechanism of cell function change, processing control mechanism of glycoconjugate and role of sugar chain. 2) Diagnosis of lesions by enzyme activity: This enzyme activity is associated with various tumorigenesis. Diseases such as cancer can be diagnosed by measuring the enzyme activity, which reflects the lesions. 3) Diagnosis of lesions by specific antibody: Various diseases by using specific antibody induced by this enzyme There is an extremely useful application such as the diagnosis of
【００１８】次に、実施例を挙げて本発明を具体的に説
明する。実施例１ （１）ブタ脳の破砕物と粗抽出液の調製 ブタ脳、１００ｇを２０ｍＭ リン酸カリウム緩衝液
（ｐＨ７．０）中でワーリングブレンダーを用いて破砕
した後、超遠心分離により膜画分を集めた。この膜画分
を、濃度０．５％のトリトン Ｘ−１００を含む同緩衝
液を用いて、酵素の抽出を行った。抽出操作後、遠心分
離によって上清を集め、粗酵素抽出液とした。Next, the present invention will be specifically described with reference to examples. Example 1 (1) Preparation of crushed pig brain and crude extract 100 g of porcine brain was crushed in a 20 mM potassium phosphate buffer (pH 7.0) using a Waring blender and then subjected to ultracentrifugation to form a membrane. I collected the minutes. The membrane fraction was subjected to enzyme extraction using the same buffer containing Triton X-100 at a concentration of 0.5%. After the extraction operation, the supernatant was collected by centrifugation to obtain a crude enzyme extract.
【００１９】（２）粗抽出液からの酵素の精製 ０．０５％ トリトンＸ−１００、５０ｍＭ ＫＣｌを
含む２０ｍＭ リン酸カリウム緩衝液（ｐＨ７．０）
で、ＧｌｃＮＡｃβ１→２Ｍａｎα１→６（ＧｌｃＮＡ
ｃβ１→２Ｍａｎα１→３）Ｍａｎβ１→４ＧｌｃＮＡ
ｃβ１→４ＧｌｃＮＡｃ−アスパラギン−セファロース
のカラム（トランスフェリン由来のアシアロアガラクト
糖ペプチドカラム）を平衡化し、実施例１で調製した粗
酵素抽出液を供した。非吸着画分に蛋白質が検出されな
くなるまでカラムを同緩衝液で洗浄した後、活性画分
を、１Ｍ ＫＣｌを含む同緩衝液で溶出させた。次に、
酵素の活性画分を限外濾過膜により濃縮、脱塩を行った
後、同緩衝液で平衡化したＧＤＰ−ヘキサノールアミン
−セファロースのカラムに供した。溶出は１００ｍＭＧ
ＤＰを含む同緩衝液により行った。活性画分を集めて限
外ろ過膜で濃縮、脱塩を行うことにより、α１→６フコ
シルトランスフェラーゼを得ることができた。このよう
にして得られたα１→６フコシルトランスフェラーゼ
は、ＳＤＳポリアクリルアミドゲル電気泳動で、分子量
約６０，０００の位置に単一バンドを示し、不純物に起
因する他のバンドはみられず、また、他のトランスフェ
ラーゼ活性もなく、高純度の酵素標品であった。(2) Purification of enzyme from crude extract 20 mM potassium phosphate buffer (pH 7.0) containing 0.05% Triton X-100, 50 mM KCl
Then, GlcNAcβ1 → 2Manα1 → 6 (GlcNAc
cβ1 → 2Manα1 → 3) Manβ1 → 4GlcNA
A column of cβ1 → 4GlcNAc-asparagine-Sepharose (transferrin-derived asialogagalactoglycopeptide column) was equilibrated, and the crude enzyme extract prepared in Example 1 was provided. The column was washed with the same buffer until no protein was detected in the non-adsorbed fraction, and then the active fraction was eluted with the same buffer containing 1 M KCl. next,
The active fraction of the enzyme was concentrated and desalted by an ultrafiltration membrane, and then applied to a GDP-hexanolamine-Sepharose column equilibrated with the same buffer solution. Elution is 100 mMG
The same buffer solution containing DP was used. By collecting the active fractions, concentrating and desalting with an ultrafiltration membrane, α1 → 6 fucosyltransferase could be obtained. The α1 → 6 fucosyltransferase thus obtained showed a single band at a position of a molecular weight of about 60,000 on SDS polyacrylamide gel electrophoresis, and no other band due to impurities was observed. There was no other transferase activity and it was a highly purified enzyme preparation.
【００２０】本発明の酵素の至適ｐＨを緩衝液のｐＨを
変化させ求めた結果を図１に示す。該酵素は、ｐＨ７．
０〜７．５付近に高い活性を示した。なお、緩衝液は、
２００ｍＭ メス（ＭＥＳ）緩衝液（黒丸）を使用し
た。グラフの横軸は本発明により得られるα１→６フコ
シルトランスフェラーゼのｐＨ、縦軸は相対活性（％）
を表す。The optimum pH of the enzyme of the present invention was determined by changing the pH of the buffer solution, and the results are shown in FIG. The enzyme has a pH of 7.
It exhibited high activity around 0 to 7.5. The buffer solution is
A 200 mM female (MES) buffer solution (black circle) was used. The horizontal axis of the graph is the pH of α1 → 6 fucosyltransferase obtained by the present invention, and the vertical axis is the relative activity (%).
Represents
【００２１】本発明の酵素のｐＨ安定性についても、同
様に検討した。図２は、該酵素を各緩衝液中でそれぞれ
のｐＨにおいて、４℃、５時間処理した後の残存活性を
示しているが、該酵素はｐＨ４〜１０で比較的安定であ
り、特にｐＨ５〜９の間においてより良好な安定性を示
した。緩衝液は、ｐＨ３．５〜５．５は５０ｍＭ酢酸緩
衝液（黒三角）を、ｐＨ５．５〜７．５は５０ｍＭ メ
ス（ＭＥＳ）緩衝液（黒丸）を、ｐＨ７．５〜９．０は
５０ｍＭ トリス−塩酸緩衝液（白丸）を、ｐＨ９．０
〜１１．５は炭酸水素ナトリウム緩衝液（白三角）を、
それぞれ用いた。グラフの横軸は本発明により得られる
α１→６フコシルトランスフェラーゼのｐＨ、縦軸は残
存活性（％）を表す。Similarly, the pH stability of the enzyme of the present invention was examined. FIG. 2 shows the residual activity after treating the enzyme in each buffer at each pH for 5 hours at 4 ° C., but the enzyme is relatively stable at pH 4-10, especially pH 5-5. Better stability was exhibited between 9. As for the buffer, pH 3.5 to 5.5 is 50 mM acetate buffer (black triangle), pH 5.5 to 7.5 is 50 mM female (MES) buffer (black circle), and pH 7.5 to 9.0 is pH 9.0 to 9.0. 50 mM Tris-hydrochloric acid buffer solution (open circle), pH 9.0
~ 11.5 is sodium hydrogen carbonate buffer (white triangle),
Used respectively. The horizontal axis of the graph represents the pH of α1 → 6 fucosyltransferase obtained by the present invention, and the vertical axis represents the residual activity (%).
【００２２】本発明の酵素の至適温度は、図３に示すよ
うに３７℃付近に認められ、また、２０〜４０℃の範囲
で十分な作用を保持すると考えられた。また、凍結品は
−２０℃で少なくとも数カ月間は安定にその活性を保持
した。緩衝液は、２００ｍＭメス（ＭＥＳ）緩衝液、ｐ
Ｈ７．０（黒丸）を使用した。グラフの横軸は、処理温
度（℃）、縦軸は、本発明により得られるα１→６フコ
シルトランスフェラーゼの相対活性（％）を表す。The optimum temperature of the enzyme of the present invention was found around 37 ° C. as shown in FIG. 3, and it was considered that the enzyme had a sufficient action in the range of 20-40 ° C. The frozen product retained its activity stably at -20 ° C for at least several months. The buffer is 200 mM female (MES) buffer, p
H7.0 (black circle) was used. The horizontal axis of the graph represents the treatment temperature (° C.), and the vertical axis represents the relative activity (%) of α1 → 6 fucosyltransferase obtained by the present invention.
【００２３】多くの糖転移酵素はその活性の発現にマグ
ネシウム、マンガンなどの２価金属イオンを必要とする
が、本発明の酵素はこれらの２価金属イオン非存在下で
も十分な活性を示した。さらに、キレート剤であるＥＤ
ＴＡ５ｍＭの存在下でも十分な活性を示したことから、
２価金属イオンの要求性は示さないと結論した。Many glycosyltransferases require divalent metal ions such as magnesium and manganese for expression of their activities, but the enzyme of the present invention showed sufficient activity even in the absence of these divalent metal ions. . In addition, the chelating agent ED
Since it showed sufficient activity even in the presence of TA5mM,
It was concluded that there was no requirement for divalent metal ions.
【００２４】[0024]
【発明の効果】本発明のブタ由来α１→６フコシルトラ
ンスフェラーゼは、公知のヒトα１→６フコシルトラン
スフェラーゼとは、理化学的性質が種々の点で大きく異
なり、より生理学的条件に近い反応至適条件で作用を示
す。したがって、本発明により、糖鎖の修飾や合成など
の糖鎖工学および／または癌などの疾病の診断に、有用
な新規α１→６フコシルトランスフェラーゼを提供する
ことができる。INDUSTRIAL APPLICABILITY The porcine α1 → 6 fucosyltransferase of the present invention differs greatly from the known human α1 → 6 fucosyltransferase in various points in terms of physicochemical properties, and is suitable for reaction conditions closer to physiological conditions. Shows the action. Therefore, the present invention can provide a novel α1 → 6 fucosyltransferase useful for sugar chain engineering such as modification and synthesis of sugar chains and / or diagnosis of diseases such as cancer.
【図１】本発明の酵素の至適ｐＨを示す。FIG. 1 shows the optimum pH of the enzyme of the present invention.
【図２】本発明の酵素の４℃におけるｐＨ安定性を示
す。FIG. 2 shows pH stability of the enzyme of the present invention at 4 ° C.
【図３】本発明の酵素の至適温度を示す。FIG. 3 shows the optimum temperature of the enzyme of the present invention.
Claims (2)
→６フコシルトランスフェラーゼ。 （１）作用：ＧＤＰ−フコースのフコース（Ｆｕｃ）
を、ＧｌｃＮＡｃβ１→２Ｍａｎα１→６（ＧｌｃＮＡ
ｃβ１→２Ｍａｎα１→３）Ｍａｎβ１→４ＧｌｃＮＡ
ｃβ１→４ＧｌｃＮＡｃ−Ｒ（Ｒはペプチド鎖のアスパ
ラギン残基を示す）受容体の、最もペプチド鎖に近いＧ
ｌｃＮＡｃの６位の水酸基に転移し、ＧｌｃＮＡｃβ１
→２Ｍａｎα１→６（ＧｌｃＮＡｃβ１→２Ｍａｎα１
→３）Ｍａｎβ１→４ＧｌｃＮＡｃβ１→４（Ｆｕｃα
１→６）ＧｌｃＮＡｃ−Ｒを生成する。 （２）至適ｐＨ：至適ｐＨは、７．０である。 （３）ｐＨ安定性：４℃、５時間の処理で、ｐＨ４．０
〜１０．０の範囲で安定である。 （４）至適温度：至適作用温度は、３０〜３７℃であ
る。 （５）阻害、活性化：活性の発現に、２価金属イオンを
要求せず、また、５ｍＭ ＥＤＴＡ存在下においても活
性は阻害されない。 （６）分子量：約６０，０００（ＳＤＳ−ポリアクリル
アミドゲル電気泳動）を示す。1. Pig-derived α1 having the following physicochemical properties:
→ 6 Fucosyltransferase. (1) Action: GDP-fucose of fucose (Fuc)
To GlcNAcβ1 → 2Manα1 → 6 (GlcNAc
cβ1 → 2Manα1 → 3) Manβ1 → 4GlcNA
cβ1 → 4GlcNAc-R (R represents an asparagine residue of the peptide chain) G of the receptor closest to the peptide chain
GlcNAcβ1 is transferred to the 6-position hydroxyl group of lcNAc.
→ 2Manα1 → 6 (GlcNAcβ1 → 2Manα1
→ 3) Manβ1 → 4GlcNAcβ1 → 4 (Fucα
1 → 6) Generate GlcNAc-R. (2) Optimum pH: The optimum pH is 7.0. (3) pH stability: pH 4.0 after treatment at 4 ° C. for 5 hours
It is stable in the range of up to 10.0. (4) Optimum temperature: The optimum working temperature is 30 to 37 ° C. (5) Inhibition and activation: Divalent metal ions are not required for expression of activity, and activity is not inhibited even in the presence of 5 mM EDTA. (6) Molecular weight: about 60,000 (SDS-polyacrylamide gel electrophoresis) is shown.
ブタ由来α１→６フコシルトランスフェラーゼ。2. The porcine α1 → 6 fucosyltransferase according to claim 1, which is purified from porcine brain.
Priority Applications (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP01036596A JP3642361B2 (en) | 1996-01-24 | 1996-01-24 | Α1- → 6 fucosyltransferase from pig |
| US08/913,805 US6054304A (en) | 1996-01-24 | 1997-01-23 | α1-6 fucosyltransferase |
| PCT/JP1997/000171 WO1997027303A1 (en) | 1996-01-24 | 1997-01-23 | Alpha-1-6 fucosyltransferases |
| EP97900780A EP0816503B1 (en) | 1996-01-24 | 1997-01-23 | Alpha-1-6 fucosyltransferases |
| DE69736261T DE69736261T2 (en) | 1996-01-24 | 1997-01-23 | ALPHA-1-6-fucosyltransferases |
| US09/442,629 US6291219B1 (en) | 1996-01-24 | 1999-11-18 | α1-6 fucosyltransferase |
| US09/839,136 US20020081694A1 (en) | 1996-01-24 | 2001-04-23 | Alpha 1-6 fucosyltransferase |
| US10/844,432 US7264955B2 (en) | 1996-01-24 | 2004-05-13 | α1-6 fucosyltransferase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP01036596A JP3642361B2 (en) | 1996-01-24 | 1996-01-24 | Α1- → 6 fucosyltransferase from pig |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH09201191A true JPH09201191A (en) | 1997-08-05 |
| JP3642361B2 JP3642361B2 (en) | 2005-04-27 |
Family
ID=11748146
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP01036596A Expired - Fee Related JP3642361B2 (en) | 1996-01-24 | 1996-01-24 | Α1- → 6 fucosyltransferase from pig |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3642361B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2078455A1 (en) | 2001-03-06 | 2009-07-15 | The Dow Chemical Company | Plant cell having animal-type sugar chain adding function |
| JP2009539350A (en) * | 2006-06-02 | 2009-11-19 | ロバート サックステイン， | Compositions and methods for modifying cell surface glycans |
-
1996
- 1996-01-24 JP JP01036596A patent/JP3642361B2/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2078455A1 (en) | 2001-03-06 | 2009-07-15 | The Dow Chemical Company | Plant cell having animal-type sugar chain adding function |
| JP2009539350A (en) * | 2006-06-02 | 2009-11-19 | ロバート サックステイン， | Compositions and methods for modifying cell surface glycans |
| JP2016188242A (en) * | 2006-06-02 | 2016-11-04 | サックステイン， ロバート | Compositions and methods for modifying cell surface glycans |
| JP2018153192A (en) * | 2006-06-02 | 2018-10-04 | サックステイン， ロバート | Compositions and methods for modifying cell surface glycans |
| JP2020137523A (en) * | 2006-06-02 | 2020-09-03 | サックステイン， ロバート | Compositions and Methods for Modifying Cell Surface Glycans |
| JP2022033352A (en) * | 2006-06-02 | 2022-02-28 | サックステイン， ロバート | Compositions and Methods for Modifying Cell Surface Glycans |
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| Publication number | Publication date |
|---|---|
| JP3642361B2 (en) | 2005-04-27 |
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