Modified lectin derived from Wisteria floribunda

Patent metadata

Field	Value
Publication number	US-9796765-B2
Application number	US-201314654223-A
Country	US
Kind code	B2
Filing date	Dec 18, 2013
Priority date	Dec 21, 2012
Publication date	Oct 24, 2017
Grant date	Oct 24, 2017

Abstract

[Problem] The purpose of the present invention is to stably supply high-quality and highly uniform Wisteria floribunda agglutinin (WFA) that recognizes biologically important sugar-chain markers, to elucidate the sugar-chain recognition activity in detail, and to furthermore increase the specificity of the sugar-chain recognition activity.
[Solution] The present invention involves the development of a technique for cloning genes for coding Wisteria floribunda agglutinin (WFA) and producing recombinant WFA having the same sugar-chain recognition activity as natural WFA from transformed bacteria. Natural WFA is reduced to thereby manufacture a reduced WFA monomer for specifically recognizing terminal GalNAc residue. A recombinant monomer WFA for recognizing LDN (GalNAcβ1, 4GlcNAc) sugar chain, which is important as a diagnostic marker among sugar chains having a terminal GalNAc residue, is manufactured by introducing cysteine mutation to recombinant WFA or by C-terminal-side amino acid deletion.

First claim

The invention claimed is:

1. A Wisteria floribunda monomeric lectin polypeptide comprising any one of the amino acid sequences selected from the group consisting of:
(1) the amino acid sequence represented by SEQ ID NO: 2 except that Cys272 is substituted with an amino acid other than Cys;
(2) the amino acid sequence defined in (1) above, except that one to 20 amino acids at positions other than Cys272 is/are deleted, substituted, inserted, or added;
(3) the amino acid sequence identical to the amino acid sequence represented by SEQ ID NO: 2 except that 13 to 15 amino acids are deleted from the C-terminus of SEQ ID NO: 2; and
(4) the amino acid sequence defined in (1) to (3) above, further having an N-terminus deletion of one to 30 amino acids,
wherein the polypeptide is capable of specifically binding to LDN sugar chain.

2. A reagent composition for detecting a LDN sugar chain marker, wherein the reagent comprises the polypeptide of claim 1 .

3. A cDNA molecule encoding a Wisteria floribunda monomeric lectin polypeptide comprising any one of the amino acid sequences selected from the group consisting of:
(1) the amino acid sequence represented by SEQ ID NO: 2 except that Cys272 is substituted with an amino acid other than Cys;
(2) the amino acid sequence defined in (1) above, except that one to 20 amino acids at positions other than Cys272 position is/are deleted, substituted, inserted, or added;
(3) the amino acid sequence represented by SEQ ID NO: 2 except that 13 to 15 amino acids are deleted from the C-terminus of SEQ ID NO: 2; and
(4) the amino acid sequence defined in (1) to (3) above, further having an N-terminus deletion of one to 30 amino acids,
wherein the polypeptide is capable of specifically binding to LDN sugar chain.

4. An expression vector comprising the cDNA of claim 3 .

5. A transformed cell transformed by using the cDNA of claim 3 .

6. A method for preparing a recombinant polypeptide having LDN sugar-chain-specific binding activity, the method comprising the steps of:
culturing the transformed cell of claim 5 ; and
collecting an expression product from a culture product obtained by culturing the transformed cell of claim 5 .

7. A method for diagnosing development of a disease defined by high expression of LDN sugar chain in a subject, the method comprising the step of detecting a LDN sugar chain marker by binding the reagent composition of claim 2 to cells having high expression of LDN sugar chain in said subject and diagnosing the development of the disease defined by high expression of LDN sugar chain in said subject.

8. The method of claim 7 , wherein the disease defined by high expression of LDN sugar chain is an endocrine tumor or a lung small cell carcinoma.

Assignees

• Nat Inst Advanced Ind Science & Tech

Inventors

• Sato Takashi
• Chiba Yasunori
• Tateno Hiroaki
• Kaji Hiroyuki
• Goto Masanori
• Narimatsu Hisashi

Classifications

• G01N2333/42

  Lectins, e.g. concanavalin, phytohaemagglutinin · CPC title

• C07K14/42 Primary

  Lectins, e.g. concanavalin, phytohaemagglutinin · CPC title

• G01N33/5308

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• G01N2400/00

  Assays, e.g. immunoassays or enzyme assays, involving carbohydrates · CPC title