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ENZYME HAVING ACTIVITY FOR RELEASING GLYCOPROTEIN SUGAR CHAINS AND METHOD FOR PRODUCING SAME, AND METHOD FOR RELEASING SUGAR CHAINS USING SAID ENZYME commons

Foreign code F140008045
File No. S2013-0214-C0
Posted date Dec 4, 2014
Country WIPO
International application number 2013JP081422
International publication number WO 2014080991
Date of international filing Nov 21, 2013
Date of international publication May 30, 2014
Priority data
  • P2012-255631 (Nov 21, 2012) JP
  • P2013-108153 (May 22, 2013) JP
Title ENZYME HAVING ACTIVITY FOR RELEASING GLYCOPROTEIN SUGAR CHAINS AND METHOD FOR PRODUCING SAME, AND METHOD FOR RELEASING SUGAR CHAINS USING SAID ENZYME commons
Abstract The problem is to provide an enzyme that acts on complex sugar chains and has activity for directly releasing sugar chains from glycoproteins themselves. The problem is solved by an enzyme derived from microorganisms selected from the genus Lactobacillus and the genus Prevotella, or having an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11 or an amino acid sequence having at least 70% homology with at least one of these sequences, and having activity for releasing glycoprotein sugar chains.
Outline of related art and contending technology BACKGROUND ART
Sugar chain, protein, deoxyribonucleic acid (DNA) 3 followed by the second biometric information molecule referred to as biopolymers. In humans, such as about 10 glucose sugar chain is composed of the monosaccharide type dendritic molecules, most of the proteins bound to glycoproteins and glycolipids present as lipid. Sugar chain, or present in a body fluid as a secreted protein, or membrane protein or glycolipid as often covers the surface of the cells.
A recent study, a wider variety of functions of the sugar chains are revealed. Specifically, sugar chain, cancer (metastasis, such as a tumor marker), immune (immune receptor modulators, immune cell differentiation, such as antibody pharmaceuticals), fertilization, generation, differentiation (such as regenerative medicine), infection (influenza, H. pylori, such as cholera toxin) such as in, play an important role in the currently known. The current, the structure and function of sugar chain by analysis, the diagnosis of cancer or immune disease, development of methods for prophylaxis or treatment, such as the construction of drug delivery system, various fields are expected to be applied to.
Glycoproteins of the sugar chain structure and functional analysis of order, it is necessary to release the sugar chain. Patent Document 1 is, of an aldehyde group on a sugar chain that specifically binds to substances with hydrazide groups, free sugar chains from glycoprotein disclosed is a method. However, in this method, due to the decomposition of the protein moiety. In addition, free of the sugar chain structure is partially changed.
And the protein portion of the carbohydrate moieties without change both the free sugar chains from glycoprotein as a method, also known the method of using a. Specifically, asparagine-type sugar chain releasing enzyme, used worldwide end - β-N - acetylglucosaminidase (hereinafter, simply also referred to as' end ') as, Streptomyces, and derived from end H (Streptomyces plicatus) . However, the end H, yeast and filamentous fungi of the characteristic of a glycoprotein sugar chain asparagine -type and hybrid type N-linked carbohydrate chain acting, higher animals such as a human glycoprotein which is characteristic of a complex-type sugar chain does not act on.
Recent has become commercially available, is derived from end M (Mucor hiemalis), in the same manner as end H, and hybrid-type sugar chain to well in the mold. End M is, complex-type sugar chain acting on the chain 2 by its nature, glycosides are used in the synthesis. However, their action is, lower than -type effect. In addition, carbohydrate moieties and between the stopper and the fucose glycoprotein comprising 3-5 of the sugar chain and complex-type sugar chain is present chain branching does not easily act. Further, in order to free sugar chains, digested with a protein moiety, a sugar or sugar asparagine is necessary. Therefore, the end M may be used, directly free sugar chains from glycoprotein itself is difficult.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 ラクトバシラス属およびプレボテラ属から選択される微生物に由来するか、または配列番号1、配列番号3、配列番号5、配列番号7、配列番号9もしくは配列番号11のアミノ酸配列またはこれらの配列のいずれか一つと少なくとも70%の相同性を有するアミノ酸配列を有し、かつ、糖タンパク質の糖鎖を遊離させる活性を有する酵素。

[請求項2]
 前記微生物が、ラクトバシラス・ファーメンタム、プレボテラ・ビビアおよびプレボテラ・メラニノゲニカから選択され、かつ、糖タンパク質の糖鎖を遊離させる活性を有する請求項1に記載の酵素。

[請求項3]
 前記酵素が、膜貫通領域を除去されている請求項1に記載の酵素。

[請求項4]
 前記糖タンパク質が、脊椎動物を含む動物に由来する糖タンパク質である請求項1に記載の酵素。

[請求項5]
 前記糖鎖が、前記糖タンパク質のタンパク質部分のアスパラギンのアミノ基にジアセチルキトビオース部分を介して結合している糖鎖である請求項1に記載の酵素。

[請求項6]
 前記糖鎖が、2~5本鎖分岐のコンプレックス型の糖鎖である請求項5に記載の酵素。

[請求項7]
 前記糖タンパク質が、糖鎖部分の根元の糖とジアセチルキトビオース部分との間に、フコースを更に含む請求項1に記載の酵素。

[請求項8]
 前記糖鎖が、その末端側にシアル酸を含む請求項1に記載の酵素。

[請求項9]
 請求項1に記載の酵素をコードする遺伝子を含む組換えベクター。

[請求項10]
 前記遺伝子が、膜貫通領域をコードする領域を除去されている請求項9に記載の組換えベクター。

[請求項11]
 請求項9に記載の組換えベクターにより形質転換された宿主細胞。

[請求項12]
 請求項11に記載の細胞を培養することにより、培養物を得る工程と、
 得られた培養物を精製することにより、請求項1に記載の酵素を得る工程と
を含む酵素の製造方法。

[請求項13]
 請求項1に記載の酵素と、糖タンパク質を含む試料とを接触させることにより、前記糖タンパク質の糖鎖を遊離させる糖鎖の遊離方法。

[請求項14]
 請求項11に記載の宿主細胞を含む培養物。

[請求項15]
 請求項14に記載の培養物の処理物。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • PUBLIC UNIVERSITY CORPORATION OSAKA CITY UNIVERSITY
  • Inventor
  • ITO, Kazuo
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN TD TG

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