JP5233347B2 - Recombinant willow matsutake galectin - Google Patents

Description

  The present invention relates to recombinant willow matsutake galectin molecules and variants thereof. The present invention also relates to a nucleic acid molecule encoding the recombinant willow matsutake galectin molecule or a variant thereof, and an expression vector containing the nucleic acid molecule. Furthermore, the present invention relates to a sugar chain analysis method using a recombinant willow matsutake galectin molecule or a variant thereof, and a lectin library and a lectin chip used in the method.

  Agrocybe cylindracea galectin (hereinafter, also referred to as “ACG”) is a kind of lectin that was first isolated from the fruit body of Willow matsutake (Non-patent Document 1). In the same document, Yagi et al. Have a high affinity for ACG to sialyl α2,3-galactose structure (NeuAcα2-3Galβ-), particularly sialyl α2,3-lactose (NeuAcα2-3Galβ1-4Glc) and It has been reported that it exhibits high binding affinity / specificity for sialyl α2,3-N-acetyllactosamine (NeuAcα2-3Galβ1-4GlcNAc).

  However, ACG is regarded as a lectin molecular species (galectin) specific to a galactosyl sugar chain rather than specific to sialic acid (for example, Non-Patent Document 2 and Patent Document 1). That is, in Non-Patent Document 1, although the affinity of ACG for sialyl α2,3-lactose is nearly 100 times that of lactose, the ACG is low for the sialic acid residue itself. It is also known that only affinity is shown (Non-patent Document 2).

  Further, in relation to the sugar chain recognition mechanism of ACG, Non-Patent Document 2 describes X-ray diffraction experiments on each crystal of ACG / lactose, ACG / 3′-lactose and ACG / sialyl α2,3-lactose. As a result, in the sugar chain binding site of ACG, asparagine at position 46, histidine at position 62, arginine at position 66, asparagine at position 75 and glutamic acid at position 86 contribute to hydrogen bonding with galactose and glucose units in lactose. Have reported that. On the other hand, regarding the binding between ACG and sialyl α2,3-lactose, serine at position 44, arginine at position 77 and tryptophan at position 83 form a hydrogen bond with the sialic acid unit in sialyl α2,3-lactose. Make sure you do. Non-Patent Document 2 discloses that in ACG, in addition to the individual amino acids involved in the hydrogen bond, a unique loop structure containing 44th serine-45th proline-46th asparagine has lactose and sialyl of the galectin molecule. It concludes that it is essential for expressing the specific binding specificity for α2,3-lactose. (Incidentally, the amino acid sequence of natural ACG is published as NCBI Accession Code: 1WW4A, and the sequence consists of 160 amino acid residues starting from threonine. Since an amino acid number is assigned based on a sequence in which one Ac-serine is added to the N-terminus, according to the sequence described in NCBI Access Code: 1WW4A, for example, asparagine at position 46 in Non-Patent Document 2 is NCBI. (The asparagine at position 45 in the sequence and the glutamic acid at position 86 correspond to the glutamic acid at position 85 in the NCBI sequence. In the present specification, the amino acid numbers are referred to hereinafter according to the NCBI sequence.)

  By the way, in recent years, it has become clear that sugar chains having sialic acid have biologically important functions. For example, Non-Patent Document 3 discloses that the sugar chain structure on bovine peripheral nerve α-dystroglycan is Neu5Acα2-3Galβ1-4GlcNAcβ1-2Man-Ser / Thr (: sialyl α2,3-N-acetyllactosamine β1,2-mannose- Ser / Thr). α-dystroglycan is a glycoprotein that cross-links the transmembrane protein β-dystroglycan and laminin in the extracellular matrix. Since the dystrophin binds to dystrophin and then binds to the actin cytoskeleton, an association between the abnormality in these complexes and muscular dystrophy has been suggested (Non-patent Document 4). Patent Document 1 also points out the relationship between Congenital Disorders of Glycosylation (CDG) and sialic acid-containing sugar chains.

  However, the precise identification and analysis of sialic acid-containing glycans is an interesting biochemical and clinical target, and the importance of searching for sialic acid-specific lectin species that can meet such objectives has also been recognized (non- Patent Document 1).

  Patent Document 2 describes modification of sugar chain specificity of a natural lectin molecule by gene recombination technology, and identification of a sugar chain using the gene recombination lectin molecule. However, this document also does not disclose changes in binding properties of ACG to sialic acid / lactose and N-acetyllactosamine-containing sugar chains.

JP 2008-32520 A International Publication No. WO2002 / 066664 Pamphlet Glycoconjugate Journal, Vol. 14, pp. 281-288 (1997) J. et al. Mol. Biol. , Vol. 351, pp. 695-706 (2005) J. et al. Biol. Chem. , Vol. 272, no. 4, pp. 2156-2162 (1997) J. et al. Biol. Chem. , Vol. 278, No. 18, pp. 15457-15460 (2003)

  Accordingly, an object of the present invention is to provide a lectin molecule with improved specificity for sialic acid-containing sugar chains. More specifically, recombination that is specific for the sialyl α2,3-lactose and sialyl α2,3-N-acetyllactosamine structures of the sugar chain and has limited binding affinity with the corresponding non-sialylated sugar chain The object is to provide lectin molecules.

It was found that substitution of glutamic acid at position 85 in the amino acid sequence of the natural ACG molecule with a specific amino acid limits the binding with the corresponding non-sialylated sugar chain. Although this finding clarifies the existence of a hydrogen bond between 85-position (86-position) glutamic acid of ACG and lactose of the sugar chain, Non-Patent Document 2 shows, at the same time, important involvement of other amino acids. However, it was surprising that only the substitution of glutamic acid at position 85 showed that the sugar chain binding property of ACG could be substantially changed. In any known literature, the amino acid to be substituted for glutamic acid at position 85 of the natural ACG molecule is one amino acid selected from the group consisting of alanine, arginine, lysine, aspartic acid, cysteine, glycine, serine and threonine. It does not suggest that there is. Therefore, in the first aspect of the present invention,
(1) The 85th-position glutamic acid residue of the amino acid sequence of a galectin molecule derived from a natural type of willow matsutake (Agrocybe cylindracea) is selected from the group consisting of alanine, arginine, lysine, aspartic acid, cysteine, glycine, serine and threonine Provided is a recombinant willow matsutake galectin molecule characterized in that it is substituted with an amino acid residue.

The above-mentioned recombinant willow matsutake galectin molecules typically have the following:
ThrThrSerAlaValAsnIleTyrAsnIleSerAlaGlyAlaSerValAspLeuAlaAlaProValThrThrGlyAspIleValThrPhePheSerSerAlaLeuAsnLeuSerAlaGlyAlaGlySerProAsnAsnThrAlaLeuAsnLeuLeuSerGluAsnGlyAlaTyrLeuLeuHisIleAlaPheArgLeuGlnGluAsnValIleValPheAsnSerArgGlnProAsnAlaProTrpLeuVal Ala GlnArgValSerAsnValAlaAsnGlnPheIleGlySerGlyGlyLysAlaMetValThrValPheAspHisGlyAs LysTyrGlnValValIleAsnGluLysThrValIleGlnTyrThrLysGlnIleSerGlyThrThrSerSerLeuSerTyrAsnSerThrGluGlyThrSerIlePheSerThrValValGluAlaValThrTyrThrGlyLeuAla (SEQ ID NO: 4),
ThrThrSerAlaValAsnIleTyrAsnIleSerAlaGlyAlaSerValAspLeuAlaAlaProValThrThrGlyAspIleValThrPhePheSerSerAlaLeuAsnLeuSerAlaGlyAlaGlySerProAsnAsnThrAlaLeuAsnLeuLeuSerGluAsnGlyAlaTyrLeuLeuHisIleAlaPheArgLeuGlnGluAsnValIleValPheAsnSerArgGlnProAsnAlaProTrpLeuVal Arg GlnArgValSerAsnValAlaAsnGlnPheIleGlySerGlyGlyLysAlaMetValThrValPheAspHisGlyAs LysTyrGlnValValIleAsnGluLysThrValIleGlnTyrThrLysGlnIleSerGlyThrThrSerSerLeuSerTyrAsnSerThrGluGlyThrSerIlePheSerThrValValGluAlaValThrTyrThrGlyLeuAla (SEQ ID NO: 6),
ThrThrSerAlaValAsnIleTyrAsnIleSerAlaGlyAlaSerValAspLeuAlaAlaProValThrThrGlyAspIleValThrPhePheSerSerAlaLeuAsnLeuSerAlaGlyAlaGlySerProAsnAsnThrAlaLeuAsnLeuLeuSerGluAsnGlyAlaTyrLeuLeuHisIleAlaPheArgLeuGlnGluAsnValIleValPheAsnSerArgGlnProAsnAlaProTrpLeuVal Lys GlnArgValSerAsnValAlaAsnGlnPheIleGlySerGlyGlyLysAlaMetValThrValPheAspHisGlyAs LysTyrGlnValValIleAsnGluLysThrValIleGlnTyrThrLysGlnIleSerGlyThrThrSerSerLeuSerTyrAsnSerThrGluGlyThrSerIlePheSerThrValValGluAlaValThrTyrThrGlyLeuAla (SEQ ID NO: 8),
ThrThrSerAlaValAsnIleTyrAsnIleSerAlaGlyAlaSerValAspLeuAlaAlaProValThrThrGlyAspIleValThrPhePheSerSerAlaLeuAsnLeuSerAlaGlyAlaGlySerProAsnAsnThrAlaLeuAsnLeuLeuSerGluAsnGlyAlaTyrLeuLeuHisIleAlaPheArgLeuGlnGluAsnValIleValPheAsnSerArgGlnProAsnAlaProTrpLeuVal Asp GlnArgValSerAsnValAlaAsnGlnPheIleGlySerGlyGlyLysAlaMetValThrValPheAspHisGlyAs LysTyrGlnValValIleAsnGluLysThrValIleGlnTyrThrLysGlnIleSerGlyThrThrSerSerLeuSerTyrAsnSerThrGluGlyThrSerIlePheSerThrValValGluAlaValThrTyrThrGlyLeuAla (SEQ ID NO: 10),
ThrThrSerAlaValAsnIleTyrAsnIleSerAlaGlyAlaSerValAspLeuAlaAlaProValThrThrGlyAspIleValThrPhePheSerSerAlaLeuAsnLeuSerAlaGlyAlaGlySerProAsnAsnThrAlaLeuAsnLeuLeuSerGluAsnGlyAlaTyrLeuLeuHisIleAlaPheArgLeuGlnGluAsnValIleValPheAsnSerArgGlnProAsnAlaProTrpLeuVal Cys GlnArgValSerAsnValAlaAsnGlnPheIleGlySerGlyGlyLysAlaMetValThrValPheAspHisGlyAs LysTyrGlnValValIleAsnGluLysThrValIleGlnTyrThrLysGlnIleSerGlyThrThrSerSerLeuSerTyrAsnSerThrGluGlyThrSerIlePheSerThrValValGluAlaValThrTyrThrGlyLeuAla (SEQ ID NO: 12),
ThrThrSerAlaValAsnIleTyrAsnIleSerAlaGlyAlaSerValAspLeuAlaAlaProValThrThrGlyAspIleValThrPhePheSerSerAlaLeuAsnLeuSerAlaGlyAlaGlySerProAsnAsnThrAlaLeuAsnLeuLeuSerGluAsnGlyAlaTyrLeuLeuHisIleAlaPheArgLeuGlnGluAsnValIleValPheAsnSerArgGlnProAsnAlaProTrpLeuVal Gly GlnArgValSerAsnValAlaAsnGlnPheIleGlySerGlyGlyLysAlaMetValThrValPheAspHisGlyAs LysTyrGlnValValIleAsnGluLysThrValIleGlnTyrThrLysGlnIleSerGlyThrThrSerSerLeuSerTyrAsnSerThrGluGlyThrSerIlePheSerThrValValGluAlaValThrTyrThrGlyLeuAla (SEQ ID NO: 14),
ThrThrSerAlaValAsnIleTyrAsnIleSerAlaGlyAlaSerValAspLeuAlaAlaProValThrThrGlyAspIleValThrPhePheSerSerAlaLeuAsnLeuSerAlaGlyAlaGlySerProAsnAsnThrAlaLeuAsnLeuLeuSerGluAsnGlyAlaTyrLeuLeuHisIleAlaPheArgLeuGlnGluAsnValIleValPheAsnSerArgGlnProAsnAlaProTrpLeuVal Ser GlnArgValSerAsnValAlaAsnGlnPheIleGlySerGlyGlyLysAlaMetValThrValPheAspHisGlyAs LysTyrGlnValValIleAsnGluLysThrValIleGlnTyrThrLysGlnIleSerGlyThrThrSerSerLeuSerTyrAsnSerThrGluGlyThrSerIlePheSerThrValValGluAlaValThrTyrThrGlyLeuAla (SEQ ID NO: 16), or ThrThrSerAlaValAsnIleTyrAsnIleSerAlaGlyAlaSerValAspLeuAlaAlaProValThrThrGlyAspIleValThrPhePheSerSerAlaLeuAsnLeuSerAlaGlyAlaGlySerProAsnAsnThrAlaLeuAsnLeuLeuSerGluAsnGlyAlaTy rLeuLeuHisIleAlaPheArgLeuGlnGluAsnValIleValPheAsnSerArgGlnProAsnAlaProTrpLeuVal Thr GlnArgValSerAsnValAlaAsnGlnPheIleGlySerGlyGlyLysAlaMetValThrValPheAspHisGlyAspLysTyrGlnValValIleAsnGluLysThrValIleGlnTyrThrLysGlnIleSerGlyThrThrSerSerLeuSerTyrAsnSerThrGluGlyThrSerIlePheSerThrValValGluAlaValThrTyrThrGlyLeuAla (SEQ ID NO: 18)
Have Therefore, in the second aspect of the present invention,
(2) consisting of the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18. The recombinant willow matsutake galectin molecule described in (1) above is provided.

Furthermore, the present invention also contemplates a modified form of the above-mentioned recombinant willow matsutake galectin molecule (herein, including the above-mentioned recombinant willow matsutake galectin molecule, also referred to as “recombinant galectin molecule”). The modified product is composed of alanine, arginine, lysine, aspartic acid, cysteine, glycine, serine, and threonine, in which the amino acid residue corresponding to the 85th glutamic acid residue of the amino acid sequence of the galectin molecule derived from natural type willow matsutake (Agrocybe cylindracea) Provided that the amino acid sequence is substantially homologous to the amino acid sequence of each recombinant willow matsutake galectin molecule, provided that the amino acid sequence is one amino acid residue selected from the group, It may exhibit substantially the same function as the above-mentioned recombinant willow matsutake galectin molecule, particularly limited binding to non-sialylated sugar chains. Therefore, in the third aspect of the present invention,
(3) From the group consisting of alanine, arginine, lysine, aspartic acid, cystine, glycine, serine and threonine, the amino acid residue corresponding to the 85th glutamic acid residue of the amino acid sequence of the galectin molecule derived from natural type Agaricus cylindracea An amino acid having one or several amino acid substitutions, deletions, insertions or additions to any one of the amino acid sequences described in (2) above, provided that it is one selected amino acid residue Recombinant galectin comprising a sequence and characterized in that the binding affinity for sialyl α2,3-N-acetyllactosamine and sialyl α2,3-lactose is higher than the binding affinity for N-acetylactosamine and T antigen A molecule is provided.

The present invention also relates to a nucleic acid molecule encoding the recombinant galectin molecule of the present invention and a vector for expressing the nucleic acid molecule. These nucleic acid molecules and vectors can be suitably used for producing the recombinant galectin molecule of the present invention. That is, in the fourth and fifth aspects of the present invention,
(4) a nucleic acid molecule encoding the recombinant galectin molecule according to any one of (1) to (3) above, and (5) a nucleic acid molecule according to (4) functionally linked to a transcription control element. An expression vector is provided.

Furthermore, the present invention provides a more precise and simple method for analyzing a sugar chain containing sialic acid. In view of the biological importance of sialic acid-containing sugar chains, the advantages of the recombinant galectin molecules of the present invention that can clearly distinguish sialic acid-containing sugar chains from the corresponding non-sialylated sugar chains are apparent. Therefore, the sixth to ninth aspects of the present invention are respectively
(6) A method for analyzing a sugar chain containing sialic acid, comprising:
(I) A sample in which a test substance having a sugar chain containing sialic acid is expected to exist and the recombinant galectin molecule according to any one of (1) to (3) above are combined with the test substance. Contacting under conditions sufficient for the recombinant galectin molecule to bind, and (ii) observing the binding or non-binding of the recombinant galectin with the test substance,
A method for analyzing a sugar chain containing sialic acid, comprising:
(7) The method according to (6) above, wherein the recombinant galectin molecule is the recombinant galectin molecule according to (2) above;
(8) In the above (6) or (7), the test substance is a sugar chain itself, glycoprotein or glycolipid, or a cell, pseudo cell or cell membrane containing the sugar chain itself, glycoprotein or glycolipid. (9) The method according to (8) above, wherein the sample containing the test substance is a biological fluid.

In the sugar chain analysis method described above, it is also a preferable aspect to simultaneously observe the binding property of the test substance to other known lectin molecules having a sugar chain binding specificity different from the recombinant galectin molecule of the present invention. Such analysis, coupled with the use of the recombinant galectin molecules of the present invention, allows for more detailed glycan structure profiling on the analyte. Thus, for such analysis, it is advantageous to utilize a lectin library containing the recombinant galectin molecules of the present invention and other known lectin molecules. Other known lectin molecules include, for example, yellow slug / agglutinin (LFA), dog endangered bean haemagglutinin (MAH) or elderberry agglutinin (SNA) mentioned in Non-Patent Document 1. These natural lectin molecules are also known to be specific for sialic acid-related sugar chains, and by using these lectin molecules, it is possible to grasp structural differences other than the sialic acid part of the sugar chain. You will get. Accordingly, in the tenth and eleventh aspects of the present invention,
(10) A lectin library comprising as a member the recombinant galectin molecule according to any one of (1) to (3) above, and (11) yellow slug agglutinin (LFA), cane lentil hemagglutinin (MAH) Or the lectin library as described in said (8) which further contains elderberry agglutinin (SNA) is provided.

The advantages of constructing the above lectin library in the form of a so-called lectin chip will be apparent to those skilled in the art. In short, the sugar chain analysis method of the present invention can be achieved more easily by using the lectin chip. Thus, a further aspect of the present invention is
(12) A lectin chip in which each member of the lectin library according to (10) or (11) is immobilized at a predetermined position on a substrate.

  By utilizing the recombinant galectin molecule of the present invention, a more precise and simple analysis of biologically important sialic acid-containing sugar chains can be achieved.

Recombinant willow matsutake galectin molecule The recombinant willow matsutake galectin molecule of the present invention has a glutamic acid residue at position 85 in the amino acid sequence of a natural rapeseed matsutake galectin molecule, alanine, arginine, lysine, aspartic acid, It is characterized by being substituted with one amino acid residue selected from the group consisting of cysteine, glycine, serine and threonine. The amino acid sequence of the natural type willow matsutake galectin molecule has been published as NCBI Accession Code: 1WW4A. Specifically,
ThrThrSerAlaValAsnIleTyrAsnIleSerAlaGlyAlaSerValAspLeuAlaAlaProValThrThrGlyAspIleValThrPhePheSerSerAlaLeuAsnLeuSerAlaGlyAlaGlySerProAsnAsnThrAlaLeuAsnLeuLeuSerGluAsnGlyAlaTyrLeuLeuHisIleAlaPheArgLeuGlnGluAsnValIleValPheAsnSerArgGlnProAsnAlaProTrpLeuVal Glu GlnArgValSerAsnValAlaAsnGlnPheIleGlySerGlyGlyLysAlaMetValThrValPheAspHisGlyAs LysTyrGlnValValIleAsnGluLysThrValIleGlnTyrThrLysGlnIleSerGlyThrThrSerSerLeuSerTyrAsnSerThrGluGlyThrSerIlePheSerThrValValGluAlaValThrTyrThrGlyLeuAla (SEQ ID NO: 2)
Has been identified as having In Non-Patent Document 2, since the amino acid number is assigned based on the sequence in which Ac-serine is added to the N-terminus of the amino acid sequence, the 85th-position glutamic acid in the present invention is the 86th position in Non-Patent Document 2. Same as glutamic acid.

  As described above, this natural ACG does not necessarily specifically bind only to sialic acid-containing sugar chains. For example, as shown in the Examples below, natural ACG is more effective against N-acetyllactosamine (Galβ1-4GlcNAcβ) than sialyl α2,3-N-acetyllactosamine (Neu5Acα2-3Galβ1-4GlcNAcβ). Can have a binding affinity equal to or greater than. Similarly, natural ACG has a binding affinity for T antigen (Galβ1-3GalNAcα) that is equal to or higher than that for sialyl α2,3-T antigen (Neu5Acα2-3Galβ1-3GalNAcα). Therefore, when a sialic acid-containing sugar chain on a test substance such as sialyl α2,3-N-acetyllactosamine or sialyl α2,3-T antigen is analyzed using a natural ACG, it is mixed in the sample. N-acetyllactosamine or T antigen can interfere with the analysis.

  Thus, the present invention limits the specificity of glycan binding of natural ACG, in other words, the specificity for sialic acid-containing glycans by limiting the binding affinity with the corresponding non-sialylated glycans. As described above, the purpose is to change the glutamic acid residue at position 85 in the amino acid sequence of natural ACG from the group consisting of alanine, arginine, lysine, aspartic acid, cysteine, glycine, serine and threonine. It can be achieved by substitution with one selected amino acid residue. Here, as also shown in the examples described later, a substitution of a 85-position glutamic acid residue of natural ACG with alanine (clone 1), a substitution with arginine (clone 2), a substitution with lysine (clone 3) Aspartic acid substitute (clone 4), cysteine substitute (clone 5), glycine substitute (clone 6), serine substitute (clone 7), and threonine substitute (clone 8) Retains substantial binding affinity for sialyl α2,3-N-acetyllactosamine and / or sialyl α2,3-lactose, but their substitutions bind to N-acetyllactosamine and T antigen The affinity is markedly limited. Moreover, in the substitution product (clone 9) by glutamine, in addition to sialyl α2,3-N-acetyllactosamine and sialyl α2,3-lactose, substantial binding affinity for sialyl α2,3-T antigen is also retained. Yes.

  In addition, in the histidine substitute (clone 10), only the binding affinity for sialyl α2,3-lactose is significantly preserved, but also the substantial binding affinity for N-acetyllactosamine and T antigen. Sex is not observed.

  On the other hand, in the substitution product (clone 14) by methionine, the binding affinity for both sialyl α2,3-N-acetyllactosamine and N-acetyllactosamine remains. In the case of a substitute by proline (clone 16), a substitute by tryptophan (clone 17) and a substitute by tyrosine (clone 18), the binding affinity for sialyl α2,3-N-acetyllactosamine and sialyl α2,3-lactose Even sex is impaired.

  Therefore, in the present invention, the above-mentioned alanine substitute (clone 1), arginine substitute (clone 2), lysine substitute (clone 3), aspartate substitute (clone 4), cysteine substitute (clone 5) ), A glycine substitute (clone 6), a serine substitute (clone 7) and a threonine substitute (clone 8), which are specifically shown in SEQ ID NO: 4 and SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18.

Recombinant willow matsutake galectin molecule variants The scope of the present invention includes functional variants of the above-mentioned recombinant willow matsutake galectin molecules. Here, the variant is a compound having a biological activity (functional or structural) substantially similar to the biological activity of the recombinant willow matsutake galectin molecule of the present invention. That is, the term “variant” of the present invention includes any fragment, mutant, analog and homologue, or chemical derivative having a sequence derived from the amino acid sequence of the recombinant willow matsutake galectin molecule. . The term “fragment” is intended to refer to any polypeptide subset of the recombinant willow matsutake galectin molecule of the present invention. The term “variant” is intended to refer to a molecule that is structurally, functionally and substantially similar to the intact recombinant willow matsutake galectin molecule of the present invention or a fragment thereof. If both molecules have a substantially similar structure, or if both molecules have similar biological activity, the molecules are substantially similar. Thus, if the two molecules have substantially similar activity, the molecules are mutants, even if one structure of the molecules is not in the other or the two amino acid sequences are not completely identical. it is conceivable that. For example, substitution of leucine for valine, lysine for arginine, and glutamine for asparagine may not change the function of the polypeptide (however, an amino acid corresponding to the glutamic acid residue at position 85 in the amino acid sequence of natural ACG) The residue is one amino acid residue selected from the group consisting of alanine, arginine, lysine, aspartic acid, cysteine, glycine, serine and threonine). The term “analog” refers to a molecule that is functionally substantially similar to a fully recombinant willow matsutake galectin molecule or fragment thereof.

  Examples of preferable variants are those showing 70% or more homology, more preferably 80%, particularly preferably 90% or more amino acid sequence homology to the amino acid sequence of the recombinant willow matsutake galectin molecule of the present invention. It is. The amino acid sequence homology referred to here is obtained by searching using the default parameters (matrix = Blosum62; gap existence cost = 11, gap extension cost = 1) of the Internet site http: // www. ncbi. n / m. nih. It can be defined as the percentage of positives indicated by the BLASTP algorithm, which can be implemented with gov / egi-gin / BLAST.

  In addition, a variant having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of the recombinant willow matsutake galectin molecule of the present invention is also suitable. For example, it is a preferred embodiment to add a His-tag sequence or the like that can be suitably used for purification of the above-mentioned recombinant willow matsutake galectin molecule. In such a case, an endopeptidase recognition site such as a thrombin recognition site or a caspase 3 recognition sequence (DEVD) is further added between the additional sequence and the target sequence in order to remove the His-tag sequence. Can do. Alternatively, in order to improve the stability of the recombinant willow matsutake galectin molecule of the present invention, it may be possible to block, for example, the N-terminus thereof with proline or Ac-serine. In short, for the amino acid sequence of the recombinant willow matsutake galectin molecule of the present invention, as long as the function of the molecule is not impaired, any modification (amino acid substitution) that has established utility in the recombinant protein technology. , Deletions, insertions and / or additions) can be introduced, and those skilled in the art will readily understand that the advantages of those techniques can also be enjoyed in the variants.

Nucleic acid molecules and expression vectors Recombinant willow matsutake galectin molecules of the present invention and variants thereof (ie, recombinant galectin molecules of the present invention) are transformed into appropriate hosts using nucleic acid molecules such as cDNA encoding them. However, it can be suitably produced by expressing the nucleic acid molecule in the host. Such techniques are well known to those skilled in the art (see, for example, J. Sambrook et al., Molecular Cloning, a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory, New York (1989)).

  Nucleic acid molecules such as cDNA that can be used in the above technique are SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, as described herein. It can be designed based on the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 18. Suitably, the nucleic acid molecule may consist of a nucleotide sequence designed with the codons appropriate for the host in which it is expressed. That is, since the genetic code is degenerate, a plurality of codons can be used to encode a specific amino acid. In this case, it is preferable to use a codon that is frequently used in the host cell. For example, when E. coli is used as a host, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and The nucleic acid molecule can be designed based on the nucleotide sequence shown in SEQ ID NO: 17.

  In preparing the nucleic acid molecule, a chemical synthesis method such as a phosphoramidite method can be used. Alternatively, a nucleic acid molecule encoding the recombinant galectin molecule of the present invention can be prepared by introducing a predetermined mutation into a nucleic acid molecule encoding natural ACG, and as such a technique, For example, it is easy to use a site-directed mutagenesis technique based on the Inverse PCR method. The site-directed mutagenesis based on the Inverse PCR method is a technique well known to those skilled in the art, and kits therefor are also commercially available (for example, “KOD-Plus-Mutageness Kit” (trade name from Toyobo Co., Ltd.) ) Is available.) In general, the technique involves amplifying the entire circumference of the plasmid by performing PCR with two types of primers set in opposite directions with respect to the template circular plasmid, and including the mutation to be introduced into the primer used at that time. It should be noted that specific embodiments of the technique are illustrated in the examples herein.

  Thus, the scope of the present invention also includes an expression vector containing the nucleic acid molecule described above. That is, the above-described cDNA encoding the recombinant galectin molecule according to the present invention can be expressed recombinantly by molecular cloning into an expression vector containing regulatory sequences, that is, an appropriate promoter and other appropriate transcription control elements. Can be transferred to prokaryotic or eukaryotic host cells to produce the recombinant galectin molecules of the invention. Such operation techniques are also described in J. Org. Fully described in Sambrook et al. (Supra) and well known in the art.

  That is, the expression vector contains the DNA sequences necessary for transcription of the cloned copy of the gene in a suitable host and translation of the resulting mRNA, and using such vectors, bacteria, cyanobacteria, plant cells, Eukaryotic genes can be expressed in various hosts such as insect cells, fungal cells, and animal cells. Specially designed vectors can shut down DNA between hosts, eg, bacteria-yeast, bacteria-animal cells, bacteria-fungal cells, or bacteria-invertebrate cells.

  A properly constructed expression vector should contain an origin of replication for autonomous replication in the host cell, a selectable marker, a limited number of useful restriction enzyme sites, possible elements for high copy number, an active promoter It is. A promoter, whether a structural promoter or a regulated promoter, is defined as a DNA sequence that binds RNA polymerase to DNA and initiates RNA synthesis. A strong promoter is a promoter that initiates mRNA at a high frequency. In addition, other regulatory sequences in the expression vector include transcription enhancers and terminators, and other sequences that regulate protein expression by known mechanisms such as binding, attenuation or anti-attenuation of transcription repressors. It is. SV40 or adenovirus early and late promoters, lac system, trp system, TAC or TRC system, lambda phage major operator and promoter region, fd coat protein regulatory region, glycolytic enzymes (eg, 3-phosphoglycerate kinase ) Promoter, Pho5 promoter, yeast α-junction promoter, etc. can be used depending on the properties of the host cell. Expression vectors include, but are not limited to, cloning vectors, modified cloning vectors, specially designed plasmids or viruses.

  A variety of bacterial expression vectors can be used to express recombinant galectin DNA in bacterial host cells. In addition, various mammalian expression vectors can be used to express the recombinant galectin DNA of the present invention in mammalian host cells. Similarly, recombinant galectin DNA can be expressed in fungal cells using a variety of fungal host cell expression vectors, and further, recombinant galectin DNA can be expressed in insect host cells using a variety of insect cell expression vectors. be able to. Various host / vector systems for expression are known. For example, for bacterial hosts, in addition to pBR322 and pUC vectors having a colicin E1-type replication initiation mechanism, His-tag / thrombin / T7-tag / Examples include pET-28a (+) vector having MCS (BamHI, EcoRI, SacI, SalI, HindIII, EagI, NotI, XhoI) / His-tag. In addition, lambda phage derivatives such as M13, 2μ plasmid in yeast cells, SV40 for eukaryotic hosts, vectors consisting of expression control sequences obtained from bovine papilloma virus, adenovirus and cytomegalovirus, pVL941 for insects, etc. Is mentioned.

  Examples of host cells include E. coli, Pseudomonas and Bacillus bacteria, Saccharomyces and Pichia yeasts, insect cells such as SF9, and animal cells such as CHO and COS7. Particularly preferred hosts are prokaryotic cells, more preferably E. coli derived host cells. Methods for introducing a vector into a host cell include electroporation methods, protoplast methods, alkali metal methods, calcium phosphate precipitation methods, DEAE dextran methods, microinjection methods, methods using virus particles, etc. There is a genetic engineering handbook published on March 20, 1991, Yodosha), but any method may be used.

  In addition, as described above, the recombinant willow matsutake galectin molecule of the present invention can be expressed as a fusion protein with a protein or tag other than His-tag, such as glutathione S transferase, protein A, or FLAG tag. It is. The expressed fusion form can be excised using an appropriate protease (such as thrombin).

  After expression of the recombinant galectin molecule in the host cell, the protein can be recovered and obtained in a purified form. Several purification methods are available, such as cell lysates, by salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxyapatite adsorption chromatography, various combinations of hydrophobic interaction chromatography, or individual applications. And the recombinant galectin molecule of the present invention can be purified from the cell extract.

Sugar chain analysis method The recombinant galectin molecule of the present invention can be suitably used for sugar chain analysis, that is, analysis of sugar chain structure, profiling, identification, etc., and particularly sugar chains containing sialic acid (sialic acid-containing sugar chains). It is useful in the analysis of A sialic acid-containing sugar chain typically has a structure in which sialic acid is bound to the non-reducing end of the sugar chain. In the present invention, sialic acid is bound to a galactosyl sugar chain, such as sialyl α2,3- Examples thereof include N-acetyllactosamine structural unit, sialyl α2,3-lactose structural unit, or sialyl α2,3-T antigen (Neu5Acα2-3Galβ1-3GalNAcα) structural unit. Neu5Acα2-3Galβ1-4GlcNAcβ1-2Man-sugar chain and the like described in the above relationship are interesting analysis objects.

  In the method for analyzing a sugar chain of the present invention, a sample in which a test substance having a sugar chain containing sialic acid is predicted and the recombinant galectin molecule of the present invention are bound to the test substance and the recombinant galectin molecule. Including a step of contacting under conditions sufficient to do so. Examples of the test substance include, but are not limited to, a conjugate of a sugar chain and another chemical substance other than the sugar chain itself, such as glycoprotein and glycolipid. In addition, cells, pseudocells, cell membranes, tissues, and the like containing the sugar chains, glycoproteins, glycolipids, and the like can be included in the test substance in a broad sense. Therefore, the sample predicted to contain the test substance referred to in the present invention includes the sugar chain, glycoprotein or glycolipid, or a solution containing cells, pseudocells or cell membranes having them. possible. That is, the sample to be subjected to the analysis method of the present invention may include a suspension or extract of the cells, pseudo-cells or cell membranes, or an extract of the tissue. In the application of glycan analysis, various biological fluids such as blood, lymph, saliva, urine, mucosal fluid and the like can be directly or in the form of a diluted solution as an analysis sample.

  The sample and the recombinant galectin molecule of the present invention must be brought into contact under conditions sufficient for the test substance to bind to the recombinant galectin molecule. Such conditions vary depending on the specific measurement technique or protocol in which the specific analysis is performed, in addition to the origin and state of the sample, but the conditions when a conventional measurement technique or protocol is applied. Again, those skilled in the art can easily select or make appropriate changes.

  For example, the sugar chain analysis method of the present invention can be adapted to a so-called sandwich assay protocol simulating ELISA (see Non-Patent Document 1). Briefly, first, the recombinant galectin molecule of the present invention is immobilized on a solid support. Solid-phase immobilization can be achieved, for example, by dissolving recombinant galectin molecules in phosphate buffered saline (PBS), adding the solution to wells of a microplate, and incubating for a predetermined time. Thereafter, the well may be blocked with bovine serum albumin (BSA) or the like. After wells are thoroughly washed with PBS containing 0.05% Tween 20, etc., a sample is added to the wells and incubated at room temperature for 10 minutes to several hours, preferably 30 minutes to around 2 hours. After the incubation is complete, remove the sample from the well and wash the well thoroughly as described above. After washing, a detection probe, typically an antibody against the test substance, is added to the well to form a recombinant galectin-test substance-antibody sandwich complex. In order to react the antibody with the test substance, for example, incubation may be performed at room temperature for about 30 minutes to 3 hours. Thereafter, excess antibody is removed, the wells are washed, and the presence or amount of the formed sandwich complex is detected to observe the binding or non-binding of the test substance and the recombinant galectin.

  When used in the above sandwich assay protocol, it is advantageous in detecting the sandwich complex formed to label the antibody against the test substance. For example, the antibody can be labeled with a radioactive substance, colored particles such as gold colloid, a fluorescent or chemiluminescent label, or an enzyme (ELISA method). Any method known to those skilled in the art can be used as a method for labeling an antibody. Biochem. vol. 11, pages 395-399 (1979), J. MoI. Biochem. vol. 14, 41-57 (1982), Immunofluorescence and Related Technologies, Elsevier / North Holland Biomedical Press, pages 215-225 (1978). Detection depends on the nature of the labeling substance labeled on the antibody. If radioactive labeling is used, the radiation dose will be higher. If colored particle labeling, the amount of color development and absorbance will be appropriate. If it is enzyme labeling (ELISA), it will be more appropriate An appropriate substrate is added to the well, and the absorbance after a predetermined incubation is detected. In the example of the ELISA method, the enzyme used is not particularly limited, and for example, an enzyme such as horseradish peroxidase or alkaline phosphatase is advantageously used. When labeling with horseradish peroxidase, 3,3 ', 5,5'-tetramethylbenzidine or the like can be used as a substrate for the enzyme. When alkaline phosphatase is used, p-nitrophenyl phosphate is used as a substrate.

Lectin library and lectin chip As already described, in the sugar chain analysis method described above, it is advantageous to use a lectin library containing the recombinant galectin molecule of the present invention and other known lectin molecules. is there. Simultaneously observing the binding of a test substance to other known lectin molecules having a sugar chain binding specificity different from that of the recombinant galectin molecule of the present invention is combined with the use of the recombinant galectin molecule of the present invention. Thus, it becomes possible to profile the sugar chain structure on the test substance in more detail. In addition, the advantage of configuring the lectin library in the form of a so-called lectin chip in such profiling will be further apparent to those skilled in the art. The lectin library and lectin chip are described in detail in Patent Document 2, and the contents of the document are incorporated herein by reference.

  Without further explanation, those skilled in the art given the above explanation can make full use of the present invention. The following examples are given for illustrative purposes only. In the present specification, unless otherwise specified, the amino acid sequence is described from the N-terminal to the C-terminal, and the nucleotide sequence is described from the 5'-terminal to the 3'-terminal.

Preparation of Recombinant Galectin Molecule-Coding Sequence In order to replace the glutamic acid at position 85 in the amino acid sequence of natural ACG, first, a plasmid (pET having a natural ACG cDNA inserted into the multiple cloning site of pET-28a (+) vector -28a (+)-ACG) was prepared, and the plasmid was used to transform E. coli JM109 (TaKaRa; 9052) to methylate the pET-28a (+)-ACG. Next, using the methylated pET-28a (+)-ACG as a template, site-directed mutagenesis by Inverse PCR was performed according to the protocol of KOD-Plus-Mutageness Kit (Toyobo; code No. SMK-101). did. The methylation treatment of the above-described template plasmid (pET-28a (+)-ACG) with E. coli JM109 was performed by PCR according to the protocol of the Inverse PCR method kit, and then only the methylated template was converted to the restriction enzyme DpnI ( For degradation with methylated DNA specific).

  The natural ACG cDNA used above was prepared by nucleotide synthesis (requested from IDT). Specifically, the natural type ACG cDNA was synthesized as a form inserted into the vector pZEr0-2 (pZEr0-2: ACG). Subsequently, in order to transfer the natural ACG cDNA in the synthetic vector to the expression vector pET-28a (+), it was PCR amplified using the pZEr0-2: ACG as a template. In this amplification, a restriction enzyme recognition sequence and a primer capable of introducing a caspase 3 recognition site (DEVD) coding sequence into the 5 'end of the native ACG cDNA were designed and used.

  The PCR product amplified above and pET-28a (+) are both digested with restriction enzymes (NheI and XhoI, manufactured by Toyobo Co., Ltd.), and ligated between target sequences, followed by transformation of E. coli JM109 (TaKaRa; 9052). Used for. Transformants were selected and the cDNA sequence from the selected clone was confirmed to obtain the above methylated pET-28a (+)-ACG. The pET-28a (+) vector used here has a kanamycin resistance marker, and His-tag / thrombin / T7-tag / MCS (BamHI, EcoRI, SacI, SalI, HindIII, EagI, NotI, N-terminal). XhoI) / His-tag expression vector for E. coli, which can be purchased from Novagen (code No. 69864-3). The natural ACG cDNA was inserted into the expression vector at the NheI site in the T7-tag sequence of pET-28a (+) and the XhoI site in the MCS sequence.

を使用し、またプライマーとしては：
プライマー１:5’-nnncagcgtgtttctaacgtagcaaaccagttcattgg-3’
（配列番号：４１）
プライマー２:5’-gaccagccaaggagcgttcggctgacgggagttgaacacgat-3’
（配列番号：４２）
を用いた。なお、上記プライマー１の５‘末端ｎｎｎ部分にランダムな配列が導入された。また、このＰＣＲの反応サイクルは、９４℃（２分）の後、９８℃（１０秒）→６８℃（５分４５秒）を３５サイクル行い、４℃でホールドするものであった。 As described above, site-directed mutagenesis by the subsequent Inverse PCR method was performed according to the protocol of KOD-Plus-Mutageness Kit (Toyobo; code No. SMK-101). Specifically, the reaction solutions in Table 1:

And as a primer:
Primer 1: 5'-nnncagcgtgtttctaacgtagcaaaccagttcattgg-3 '
(SEQ ID NO: 41)
Primer 2: 5'-gaccagccaaggagcgttcggctgacgggagttgaacacgat-3 '
(SEQ ID NO: 42)
Was used. In addition, a random sequence was introduced into the 5 ′ end nnn portion of the primer 1. The PCR reaction cycle was 94 ° C. (2 minutes) followed by 35 cycles of 98 ° C. (10 seconds) → 68 ° C. (5 minutes 45 seconds) and held at 4 ° C.

After completion of the above PCR reaction, the target PCR product was confirmed using 1% agarose (Wako Pure Chemical; code No. 312-1193, Agarose S). Next, in order to digest the template Plasmid, 2 μl of DpnI was added to the PCR reaction solution and incubated at 37 ° C. for 1 hour. After incubation, reaction solutions shown in Table 2 were prepared for PCR product self-ligation, and incubated at 16 ° C. in the reaction solution.

  Using the reaction solution after the ligation reaction as it was, E. coli JM109 (TaKaRa; 9052) was transformed, and the E. coli was plated on an LB agar medium containing kanamycin (final concentration 20 μg / ml). After overnight culture at 37 ° C., 48 single colonies were picked up, liquid-cultured, and ACG cDNA into which site-specific mutation was introduced was introduced using Mini Plus ™ Plasmid DNA Extraction System (VIOGENE; Code No. GF2002). The contained plasmid DNA was extracted. The obtained ACG cDNA was confirmed, and 19 clones in which mutations were introduced at the target site, that is, clone 1 to clone 19, were selected.

  Each of the above clones is alanine-substituted (clone 1), arginine-substituted (clone 2), lysine-substituted (clone 3), aspartic acid-substituted (clone) with respect to glutamic acid 85 in the amino acid sequence of natural ACG. 4), cysteine substitute (clone 5), glycine substitute (clone 6), serine substitute (clone 7), threonine substitute (clone 8), glutamine substitute (clone 9), histidine substitute (clone 10) , Isoleucine substitute (clone 11), leucine substitute (clone 12), asparagine substitute (clone 13), methionine substitute (clone 14), fail alanine substitute (clone 15), proline substitute (clone 16), Tryptophan substitute (clone 17), tyrosine substitute (clone 18) And it was in relation valine substitutions (clone 19). The protein (recombinant galectin molecule) -encoding nucleotide sequence in each clone is SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, respectively. , SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75 SEQ ID NO: 77, SEQ ID NO: 79 and SEQ ID NO: 81. The coding sequence for the corresponding native ACG is shown in SEQ ID NO: 43.

Expression and Purification of Histag Fusion Protein (Recombinant Galectin Molecule) E. coli Rosetta2 DE2 (TaKaRa; code No. 71405-5) was transformed with the natural plasmid and the above expression plasmid from clones 1-19. A single colony of each transformant was inoculated into 5 ml of LB medium containing kanamycin (final concentration 20 μg / ml) and pre-cultured overnight at 37 ° C. 5 ml of the preculture was added to 150 ml of LB medium containing kanamycin (final concentration 20 μg / ml), and cultured at 37 ° C. and 100 rpm to OD600 = 0.7, and isopropyl β-D-thiogalactoside (IPTG; final concentration) Expression was induced at 1 mM). After induction of expression, the cells were continuously cultured at 30 ° C. and 100 rpm for 5 hours. Bacterial cell pellets were collected at 11000 rpm for 10 min. 10 ml of buffer I (2% Triton X100, 50 mM Tris-HCl, 150 mM NaCl: pH 7.4) was added to the collected bacterial cell pellet, and the pellet was suspended. To the resulting bacterial cell pellet suspension, a protease inhibitor cocktail (Nacalai Tesque: code No. 25955-11) was added at a 100-fold dilution, and lysozyme (final concentration 1 mg / ml) was further added. And incubated for 30 minutes on ice. After incubation, the bacterial cells in the bacterial cell pellet suspension were crushed using an ultrasonic crusher. The disrupted bacterial cell was centrifuged at 11000 rpm for 10 minutes with a cooling centrifuge, and the soluble fraction was collected. The collected soluble fraction was filtered through a 0.45 μm filter. Next, in order to purify the obtained histag fusion protein, 10 ml of Buffer I was added to His GraviTrap (manufactured by GE healthcare: code No. 11-0033-99) and equilibrated. To the equilibrated His GraviTrap, the soluble fraction filtered above was added. After the addition, washing was performed with buffer II (50 mM Tris-HCl, 500 mM NaCl: pH 7.0). Subsequently, a second wash was performed with Buffer III (50 mM Tris-HCl, 500 mM NaCl, 75 mM Imidazole: pH 7.0). After washing, 5 ml of Buffer IV (50 mM Tris-HCl, 500 mM NaCl, 500 mM Imidazole: pH 7.0) was added to elute the desired histag fusion protein. The eluted histag fusion protein was dialyzed with PBS to exchange the buffer in the sample. For the dialyzed histag fusion protein, the concentration of purified histag fusion protein was determined using the BCATM Protein Assay kit. In addition, the purity of the purified histag fusion protein was confirmed by SDS-PAGE. The results are shown in FIG.

Each multivalent biotinylated glycopolymer purchased from Glycotech Inc. indicating the sugar chain specificity of each recombinant galectin molecule by BIAcore :
Galβ1-4GlcNAcβ-PAA-biotin (N-acetyllactosamine-PAA-biotin),
Galβ1-3GalNAcα-PAA-biotin (T antigen-PAA-biotin),
Neu5Acα2-6Galβ-PAA-biotin (sialyl α2,6-galactose-PAA-biotin),
Neu5Acα2-3Galβ-PAA-biotin (sialyl α2,3-galactose-PAA-biotin),
3′-Sialylactose-PAA-biotin (sialyl α2,3-lactose-PAA-biotin),
Neu5Acα2-3Galβ1-4GlcNAcβ-PAA-biotin (sialyl α2,3-N-acetyllactosamine-PAA-biotin),
Neu5Acα2-3Galβ1-3GalNAc? -PAA-biotin (sialyl α2,3-T antigen-PAA-biotin)
Were prepared in PBS to a final concentration of 2.5 μg / ml. The prepared sugar polymer was solidified under the following conditions.

[Solidification conditions]
Measuring apparatus: BIAcore 3000 (manufactured by GE Healthcare (BIAcore)),
Sensor chip: sensor chip SA (code No. BR-1000-32),
Inside temperature: 25 ° C setting,
Flow rate: 5 μl / min,
Injection: 100 μl (PBS solution with sugar polymer adjusted to a final concentration of 2.5 μg / ml),
Running buffer: PBS

  Next, using the sensor chip in which the multivalent biotinylated sugar polymer was solidified, the sugar chain specificity of each recombinant galectin molecule was pointed out.

[BIAcore measurement conditions]
Measuring apparatus: BIAcore 3000 (manufactured by GE Healthcare (BIAcore)),
Sensor chip: The above sensor chip in which each multivalent biotinylated sugar polymer is solidified,
Inside temperature: 25 ° C setting,
Flow rate: 20 μl / min,
Injection: 100 μl (each recombinant galectin molecule in 100 μg / ml PBS),
Running buffer: PBS,
Regeneration solution: 50 mM NaOH

  The results of the above BIAcore measurement are shown in FIG. FIG. 2 shows that the 85-position glutamic acid residue of natural ACG is replaced with alanine (clone 1), arginine (clone 2), lysine (clone 3), aspartic acid (clone 4). ), Cysteine substituted (clone 5), glycine substituted (clone 6), serine substituted (clone 7) and threonine substituted (clone 8) are all sialyl α2,3-N-acetyllacto Retains substantial binding affinity for samine and / or sialyl α2,3-lactose, but their substitutions have significantly limited binding affinity for N-acetyllactosamine and T antigen I understand that. Moreover, in the substitution product (clone 9) by glutamine, in addition to sialyl α2,3-N-acetyllactosamine and sialyl α2,3-lactose, substantial binding affinity for sialyl α2,3-T antigen is also retained. In the histidine substitute (clone 10), only the binding affinity for sialyl α2,3-lactose is significantly conserved, but again the substantial binding affinity for N-acetyllactosamine and T antigen. Sex has not been observed. On the other hand, in the substitution product (clone 14) with methionine, it can be seen that the binding affinity for both sialyl α2,3-N-acetyllactosamine and N-acetyllactosamine remains. In the case of a substitute by proline (clone 16), a substitute by tryptophan (clone 17) and a substitute by tyrosine (clone 18), the binding affinity for sialyl α2,3-N-acetyllactosamine and sialyl α2,3-lactose It was found that even sex was impaired.

  As is clear from the above, the recombinant galectin molecule of the present invention is specific to the sialyl α2,3-lactose and sialyl α2,3-N-acetyllactosamine structures of the sugar chain and the corresponding non-sialylated sugar chain Since its binding affinity is limited, it can be suitably used for precise and simple analysis of biologically important sialic acid-containing sugar chains that are attracting attention in biochemical research and clinical tests. Therefore, it can be used especially in the medical industry.

It is a figure which shows the result of the purity confirmation of the recombinant galectin molecule | numerator (as a refinement | purification histag fusion protein) of this invention by SDS-PAGE. It is a figure which shows the sugar_chain | carbohydrate specificity analysis result of the recombinant galectin molecule | numerator of this invention by BIAcore. The unit on the front side (RU) is a resonance unit in BIAcore measurement, which corresponds to a change of 1 ng per 1000 RU = 1 mm ² . The label below the table indicates the clone number (substituted amino acid residue). AGC represents a natural willow matsutake galectin molecule. It is a figure which shows the sugar_chain | carbohydrate specificity analysis result of the recombinant galectin molecule | numerator of this invention by BIAcore. The unit on the front side (RU) is a resonance unit in BIAcore measurement, which corresponds to a change of 1 ng per 1000 RU = 1 mm ² . The label below the table indicates the clone number (substituted amino acid residue). AGC represents a natural willow matsutake galectin molecule. MALI stands for canine endangered bean leucogglutinin, MALII stands for canine endangered bean hemagglutinin, ECA stands for dei legume agglutinin, and SNA stands for elderberry agglutinin molecule (both natural forms).

Claims (11)

A recombinant willow matsutake galectin molecule in which the 85-position glutamic acid residue of the amino acid sequence of a galectin molecule derived from a natural type of willow matsutake mushroom (Agrocybe cylindracea) is substituted, comprising SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: A recombinant willow matsutake galectin molecule comprising the amino acid sequence represented by SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18.   The amino acid residue corresponding to the glutamic acid residue at position 85 in the amino acid sequence of the galectin molecule derived from natural type Agaricus cylindracea is selected from the group consisting of alanine, arginine, lysine, aspartic acid, cysteine, glycine, serine and threonine Consisting of an amino acid sequence having one or several amino acid substitutions, deletions, insertions or additions to any one of the amino acid sequences of claim 1, provided that it is one amino acid residue, A recombinant galectin molecule characterized in that the binding affinity for sialyl α2,3-N-acetyllactosamine and sialyl α2,3-lactose is higher than the binding affinity for N-acetylactosamine and T antigen.   A nucleic acid molecule encoding the recombinant galectin molecule according to claim 1 or 2.   An expression vector comprising the nucleic acid molecule of claim 3 operably linked to a transcriptional regulatory element. A method for analyzing a sugar chain containing sialic acid, comprising:
(1) A test substance having a sugar chain containing sialic acid is predicted to bind to the recombinant galectin molecule according to claim 1 or 2 and the test substance and the recombinant galectin molecule bind to each other. And (2) observing the binding or non-binding of the recombinant galectin with the test substance,
A method for analyzing a sugar chain containing sialic acid, comprising:   6. The method of claim 5, wherein the recombinant galectin molecule is the recombinant galectin molecule of claim 1.   The method according to claim 5 or 6, wherein the test substance is a sugar chain itself, glycoprotein or glycolipid, or a cell, pseudo cell or cell membrane containing the sugar chain itself, glycoprotein or glycolipid.   The method according to claim 7, wherein the sample containing the test substance is a biological fluid.   A lectin library comprising the recombinant galectin molecule according to claim 1 or 2 as a member. The lectin library according to claim 9 , further comprising yellow slug agglutinin (LFA), dog endangered hemagglutinin (MAH) or elderberry agglutinin (SNA). A lectin chip in which each member of the lectin library according to claim 9 or 10 is immobilized at a predetermined position on a substrate.

Images