JP5480644B2 - peptide - Google Patents

Description

  The present invention relates to a novel peptide, and more particularly to a novel peptide having sugar-binding activity.

  The present inventor newly isolated Tsutsugitake lectin (hereinafter referred to as PTL) from Tsutsugitake, which is a kind of basidiomycete (Patent Document 1). PTL has the property of specifically recognizing fucose α1 → 6 sugar chains. As conventional lectins having affinity for fucose α1 → 6 sugar chains, lentil lectin (LCA), pea lectin (PSA), yellow bamboo lectin (AAL), daffodil lectin (NPA), broad bean lectin (VFA), gonococcus Lectins (AOL) and the like are known, but these lectins also show affinity for glycolipid-based sugar chains having fucose other than α1 → 6 bonds and high mannose sugar chains not having fucose. On the other hand, PTL is superior to conventional lectins as a lectin that specifically recognizes fucose α1 → 6 sugar chain.

  Due to its sugar recognition properties, PTL is expected to be widely used for diagnostic agents for diseases involving fucose α1 → 6 sugar chains, methods for detecting fucose α1 → 6 sugar chains, and methods for separating fucose α1 → 6 sugar chains. Is done. For example, the fucose α1 → 6 transferase (α1 → 6FucT) gene, which transfers fucose as an α1 → 6 bond to N-acetylglucosamine at the reducing end of an N-linked sugar chain, is expressed as hepatocytes become cancerous. It is known. The presence or absence of these cancerous hepatocytes can be detected by lectin affinity electrophoresis using PTL.

PCT / JP2009 / 003346

  According to the method described in Patent Document 1, it is easy to isolate and extract PTL from tsujisugitake, but if PTL can be chemically synthesized, there are advantages in various points such as stable supply, production cost reduction, and purity improvement. Furthermore, if physical properties such as isoelectric point, solubility, and sugar binding activity can be freely changed, the utility value is further enhanced.

  Therefore, an object of the present invention is to provide a peptide having sugar recognition characteristics such as PTL and further changing or improving physical properties.

  As a result of intensive studies on the above problems, the present inventors have found that the amino acid sequence of SEQ ID NO: 1 can be solved by substituting, adding, or deleting a specific amino acid. That is, the present invention provides a peptide in which one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 are substituted with lysine and / or arginine and have sugar binding activity.

  In the peptide, the amino acid substituted by lysine and / or arginine is preferably at least one selected from the group consisting of alanine, proline, valine, threonine and glycine. The amino acid substituted with lysine or arginine is preferably threonine and glycine at the amino acid sequence C-terminal.

  The peptide may further be added with one or several lysine and / or arginine as long as it has sugar binding activity.

  The present invention also provides a peptide having one or several lysine and / or arginine added to the amino acid sequence shown in SEQ ID NO: 1 and having a sugar binding activity.

  As long as the peptide has sugar-binding activity, amino acids other than lysine and arginine may be deleted.

  The amino acid to be deleted is preferably at least one of threonine and glycine.

  The sugar-binding activity of the peptide is, for example, the binding activity to fucose α1 → 6 sugar chain.

  The isoelectric point of the peptide is preferably higher than 4.67.

  The peptide of the present invention is preferably labeled.

  The present invention also provides a peptide in which a plurality of the same or different peptides are associated with each other and have a sugar binding activity.

  The present invention is a peptide represented by any of SEQ ID NOs: 2 to 14, for example.

  The present invention provides a diagnostic agent or kit containing the peptide.

  The diagnostic agent or kit is useful for detecting, for example, fucose α1 → 6 sugar chain.

  The present invention provides a method for modifying a peptide having sugar-binding activity, comprising substituting one or several amino acids with lysine and / or arginine in the amino acid sequence shown in SEQ ID NO: 1.

  The method may further add one or several lysines and / or arginines.

  The present invention also provides a method for modifying a peptide having sugar-binding activity, which comprises adding one or several amino acids with lysine and / or arginine in the amino acid sequence shown in SEQ ID NO: 1.

  In the above method, one or several amino acids other than lysine and / or arginine may be deleted.

  According to the present invention, a novel peptide having properties similar to PTL can be provided inexpensively and easily. The peptide of the present invention can freely change physical properties such as sugar-binding activity, hemagglutination activity, isoelectric point, solubility, and heat stability with respect to PTL.

  Since this peptide is particularly highly specific for fucose α1 → 6 sugar chain, a specific binding agent for fucose α1 → 6 sugar chain, a reagent for sugar chain research, and a tumor marker related to fucose α1 → 6 sugar chain are accurate. Use for detection, prognosis determination, therapeutic effect determination, and search for new tumor markers is expected.

The result of having performed the separation test of AFP-L1 and AFP-L3 using the gel which added conventional LCA is shown. In the figure, the real gland arrow indicates AFP-L3, and the wavy arrow indicates AFP-L1 (hereinafter the same). The result of having performed the separation test of AFP-L1 and AFP-L3 using the control peptide (SEQ ID NO: 1) is shown. The result of having performed the isolation | separation test of AFP-L1 and AFP-L3 using the peptide (sequence number 10) of Example 9 is shown. The result of having performed the isolation | separation test of AFP-L1 and AFP-L3 using the peptide (sequence number 7) of Example 6 is shown. The AFP analysis result using the peptide of Example 6 is shown about serum (3 specimens) of cancer patients.

  Below, the peptide of this invention is demonstrated in detail. The peptide of the present invention is based on PTL contained in the mussel, which is a kind of basidiomycete. One type of PTL amino acid sequence is shown in SEQ ID NO: 1. PTL can be obtained by the production method described in Patent Document 1, chemical synthesis based on amino acid sequences, genetic engineering techniques, and the like.

  One embodiment of the peptide of the present invention is a peptide in which the amino acid sequence shown in SEQ ID NO: 1 is substituted with one or several lysines and has sugar-binding activity. It has been found that substitution with lysine, which is a basic amino acid, can enhance the sugar binding activity and increase the isoelectric point of the peptide while maintaining the sugar binding activity. Examples of such are SEQ ID NOs: 2, 3, 4, 5 and 10. An increase in isoelectric point contributes to an improvement in resolution of affinity electrophoresis analysis.

  Another embodiment of the present invention provides a peptide in which the amino acid sequence shown in SEQ ID NO: 1 is substituted with one or several arginines and has sugar-binding activity. It has been found that substitution with arginine, a basic amino acid, can increase the isoelectric point of the peptide while maintaining sugar binding activity. An example of such is SEQ ID NO: 12.

  Depending on the place of substitution with lysine or arginine, the strength of the sugar binding activity can vary. The substituted amino acid is preferably at least one selected from the group consisting of alanine, proline, valine, threonine and glycine.

  When substituted with lysine, amino acid N-terminal alanine (SEQ ID NO: 2-5), proline (SEQ ID NO: 3-5), valine (SEQ ID NO: 4-5), or amino acid sequence C-terminal threonine and glycine are 2 Preferably substituted with 1 lysine. An example of such is SEQ ID NO: 10.

  In the case of substitution with arginine, it is particularly preferred that threonine and glycine at the C-terminal of the amino acid sequence are substituted with two arginines. An example of such is SEQ ID NO: 12.

  The number of amino acids to be substituted is 1 or several, preferably 1 to 4.

  One or several lysine and / or arginine may be added to the amino acid sequence substituted with lysine and / or arginine. An example of such is SEQ ID NO: 9.

  Another embodiment of the present invention provides a peptide having lysine and / or arginine added to the amino acid sequence shown in SEQ ID NO: 1 and having sugar-binding activity. An example of such is SEQ ID NO: 14.

  The number of amino acids to be added is one or several, preferably 1-2.

  When substituted and / or added with the lysine and / or arginine, it is possible to further delete amino acids other than lysine or arginine as long as they have sugar-binding activity. Examples of such include SEQ ID NOs: 6-8, 11, and 13.

  When substituted or added with lysine, the amino acid that may be deleted is preferably at least one of threonine and glycine. Examples of threonine deletion include SEQ ID NOs: 8 and 13, and examples of glycine deletion include SEQ ID NOs: 6, 7, 8, 11, and 13.

  Amino acids that may be substituted with lysine when accompanied by amino acid deletion include threonine (SEQ ID NO: 6, 7 and 11), alanine (SEQ ID NO: 7), tyrosine (SEQ ID NO: 7 and 8), and histidine (SEQ ID NO: It is preferably at least one selected from the group consisting of 13).

  The number of amino acids to be deleted is one or several, preferably 1-2.

  The number of amino acids changed by at least one of substitution, deletion, and addition to the amino acid sequence of SEQ ID NO: 1 is 1 to several, preferably 1 to 4.

  The specificity of the peptide of the present invention is not limited as long as it has sugar binding activity. In particular, it is preferable to have sugar binding specificity to fucose α1 → 6 sugar chain like PTL. The measurement of sugar bond specificity can be performed by a conventional method. For example, a capillary electrophoresis apparatus is filled with a solution to which a peptide is added, and a labeled standard sugar chain is developed. After electrophoresis, detection is performed by a detection means based on the label.

  The isoelectric point of the peptide of the present invention is preferably higher than the isoelectric point (4.67) of PTL, and is more preferably in the range of 4.69 to 9.00.

  The peptide of the present invention is preferably labeled. As the labeling means, a known labeling method can be applied without particular limitation. For example, labeling with a radioisotope, binding of a labeled compound, and the like can be mentioned. Examples of the labeling compound include direct or indirect labeling compounds, enzymes, fluorescent compounds, and the like. Specific examples include biotin (Examples 13 and 14), digoxigenin, horseradish-derived peroxidase, fluorescein isothiocyanate, CyDye, and the like. These labeling compounds can be bound to peptides by a conventional method.

  The peptide of the present invention may be a peptide having the same or different peptides as subunits and multiple associations and having sugar-binding activity. By association, sugar binding activity and hemagglutination activity can be improved.

  The number of peptides associated is usually 2 or more, preferably 2 to 10, and particularly preferably 2 to 4. The method for associating a plurality of peptides is not particularly limited as long as the sugar binding activity is not lost. Examples thereof include a reaction with a condensing agent such as avidin-biotin reaction (Example 14) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.

  The present invention also provides a diagnostic agent or kit containing the peptide. This diagnostic agent or kit is useful for detecting fucose α1 → 6 sugar chain. This detection includes grasping the tendency of increase or decrease in fucose α1 → 6 sugar chain. Examples of diseases associated with fucose α1 → 6 sugar chain include hepatocellular carcinoma, colon cancer, pancreatic cancer, prostate cancer, breast cancer, gastric cancer, small intestine cancer, colorectal cancer, renal cell cancer, pancreatic cancer, small cell lung cancer, non Small cell carcinoma, uterine cancer, ovarian cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, melanoma, glioblastoma, astrocytoma, medulloblastoma, acute lymphoma, malignant lymphoma, Hodgkin's disease, non-Hodgkin's disease, acute bone marrow It is expected to be used for diagnosis of malignant tumors such as sex leukemia and chronic lymphocytic leukemia; pancreatitis; allergic diseases; autoimmune diseases; circulatory diseases such as emphysema.

  In the diagnostic agent or kit of the present invention, in addition to the peptide of the present invention, a reagent (buffer or the like) generally used in the diagnostic drug field can be included. In addition, the diagnostic agent or kit of the present invention may include those known (for example, antibodies and known lectins) as diagnostic agents for tumor markers.

  The present invention also provides a method for modifying a peptide comprising substituting one or several amino acids with lysine and / or arginine in the amino acid sequence shown in SEQ ID NO: 1.

  The method may further add one or several lysines and / or arginines.

  In the above method, one or several amino acids other than lysine and arginine may be deleted or substituted.

  The present invention also provides a method for modifying a peptide having sugar-binding activity, which comprises adding one or several amino acids with lysine and / or arginine in the amino acid sequence shown in SEQ ID NO: 1.

  In the above method, one or several amino acids other than lysine and arginine may be deleted or substituted.

  The actual method for obtaining the peptide is not particularly limited. For example, there are peptide solid phase synthesis method and liquid layer synthesis method, and organic synthetic chemical methods such as Fmoc (Fluorenyl-Methoxy-Carbonyl) method, Boc (tert-Butyl Oxy Carbon) method, Cbz (Benzyloxycarbon Chloride) method, etc. Engineering methods are possible.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. However, the present invention is not limited to the following examples.
[Examples 1-14, Comparative Examples 1-6]
(Synthesis of peptides)
Peptides having the amino acid sequences shown in Table 1 were actually synthesized by the Fluorenyl-Methoxyxy Carbonyl (Fmoc) method. The number of amino acids, solubility, isoelectric point (Theoretical PI), and molecular weight of the synthesized peptides are also shown in Table 1. The underlined portion of the amino acid sequence is substituted, deleted or added as compared to the control (SEQ ID NO: 1).

(Synthesis of labeled peptide)
In Example 13 (Table 1), peptides having amino acid sequences were synthesized by the Fmoc method and labeled with biotin by a conventional method. Table 1 shows the solubility of the obtained labeled peptide.

(Production of multivalent peptide)
In Example 14, a multivalent peptide was prepared by reacting the labeled peptide of Example 13 with biotin-avidin reaction. Specifically, the biotin-labeled peptide of Example 13 and streptavidin (manufactured by Vector) were each weighed in 1 mg tubes and adjusted to 1 mg / ml with phosphate buffered saline. A streptavidin solution (68 μl) and a biotin-labeled peptide solution (45 μl) were mixed and reacted at room temperature for 30 minutes. Then, it refine | purified by the gel filtration using Sephadex G25 (GE health care bioscience). Table 1 shows the solubility of the prepared multivalent peptide.

(Preparation of lectin)
For comparison, as a commercially available lectin said to be a fucose-specific lectin,
Comparative Example 1: Harie Nishida Lectin (UEA-I, Biochemical Biobusiness Co., Ltd.-J-Oil Mills),
Comparative Example 2: Miyakogusa lectin (Lotus, Biochemical Biobusiness Co., Ltd.-J-Oil Mills),
Comparative Example 3: Hilochawantake lectin (AAL, Biochemical Biobusiness Co., Ltd.-J-Oil Mills),
Comparative Example 4: Aspergillus lectin (AOL, Tokyo Chemical Industry Co., Ltd.-Laurel Wreath Co., Ltd.)
Comparative Example 5: Lentil lectin (LCA, Biochemical Biobusiness Co., Ltd.-J-Oil Mills),
Comparative Example 6: Pea lectin (PSA, Biochemical Biobusiness Co., Ltd.-J-Oil Mills)
Prepared.

(Measurement of sugar binding specificity)
P / ACE-MDQ (manufactured by Beckman Coulter, Inc.) was used for the capillary electrophoresis apparatus. The sugar chains shown in Tables 2A to C were labeled with trisodium 8-aminopyrene-1,3,6-trisulfonate (APTS). Laser fluorescence detection (ex 488 nm / em 520 nm) was used for the detection (THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 34, Issue of August 22, pp. 32439-32447). The peptide, labeled peptide, multivalent peptide and lectin were prepared in a concentration of 0.01 to 1 mg / ml with 0.1 M Tris-acetate buffer.

  Judgment of the binding was judged to be binding when the labeled sugar chain migration time under the condition without peptide was delayed by 0.06 minutes or more. ◎ when binding was observed at a peptide or lectin concentration of 0.01 mg / ml, ○ when binding was observed at 0.05 mg / ml, and □ when binding was observed at 0.1 mg / ml When the binding was observed at 1 mg / ml, the evaluation was written as Δ. Those not bound at 1 mg / ml were marked with x. The results are shown in Tables 3 and 4.

  From the comparison between Table 3 and Table 4, unlike the known lectin, the peptide of the present invention binds only to fucose α1-6 sugar chain, fucose α1-2 sugar chain, fucose α1-3 sugar chain, fucose α1- It turns out that it did not couple | bond with 4 sugar chains.

(Hemagglutination activity test)
20 μl of PBS was placed in one stage of a 96-well U-bottom titer plate, and 20 μl of sample solution (0.5 mg / ml) was placed in the well to prepare a serial 1/2 dilution series. 40 μl of 2% rabbit erythrocyte solution was added to each of the above series, and after standing at room temperature for about 60 minutes, the hemagglutination activity was visually observed. The reciprocal of the maximum dilution at which aggregation was observed was defined as the aggregation titer of the stock solution, and the logarithm with the aggregation titer of 2 as the base was defined as the hemagglutination activity.

  The peptides of Examples 1 to 13 did not have hemagglutination activity. On the other hand, in the multivalent peptide of Example 14, as shown in Table 5, hemagglutination activity was confirmed.

(Thermal stability test)
The control peptide, Examples 6 and 9, and Comparative Examples 3-5 were dissolved in PBS to 1 mg / ml. 50 μl of this solution was added to a microtube and incubated at 30-100 ° C. for 30 minutes. Those that were not kept warm were stored at 4 ° C. until analysis. Immediately after the incubation, the mixture was ice-cooled, diluted 4-fold, and the presence or absence of binding to the fucose sugar chain (NG2AF) (sugar chain 23) was measured using the capillary electrophoresis. The results are shown in Table 6.

○: With connection ×: Without connection

  The control peptide and Examples 6 and 9 all retained sugar-binding activity even when incubated at 100 ° C. On the other hand, the lectins of Comparative Examples 3 to 5 lost sugar binding activity at 70 ° C. or higher.

(Solubility test)
The peptides and lectins shown in Table 7 were prepared in the following five types of buffers so as to be 0.1 mg / ml:
A: Phosphate buffered saline (PBS)
B: 10 mM Tris-HCl buffer (pH 7.4)
C: 10 mM sodium phosphate buffer (pH 7.4)
D: 10 mM potassium phosphate buffer (pH 7.4)
E: 10 mM sodium citrate buffer (pH 7.4)
And dissolved. Then, it centrifuged for 2 minutes with the desktop centrifuge, and the presence or absence of the deposit was evaluated. The evaluation criteria are as follows.
○: no insoluble matter (good dissolution)
Δ: Slightly insoluble material ×: Many insoluble materials (dissolved poorly)
The results are shown in Table 7.

  The peptide of the present invention dissolved well in any buffer solution, but there was a buffer solution that was not sufficiently dissolved in LCA (Comparative Example 5) and PSA (Comparative Example 6).

(AFP-L3 analysis by affinity electrophoresis)
α-fetoprotein (AFP) is a marker for hepatocellular carcinoma. However, since it increases even in patients with non-hepatic cancer such as chronic hepatitis and cirrhosis, it is difficult to differentiate in mild (˜100 ng / ml) to moderate (˜400 ng / ml) cases. Among the AFP lectin fractions, many non-hepatic cancer patients appear in the L1 fraction, and the L3 fraction increases in hepatocellular carcinoma patients. The AFP lectin fraction is useful for differential diagnosis between hepatocellular carcinoma and hepatic benign disease.

  As the L3 fraction (AFP-L3 type), the structure of the sugar chain of serum AFP derived from a cancer patient (hepatocellular carcinoma) is shown below.

As the L1 fraction (AFP-L1 type), the structure of the sugar chain of serum AFP derived from benign diseases (acute hepatitis, chronic hepatitis, cirrhosis, congenital biliary atresia) in the embryonic period is shown below.

(Preparation of gel for affinity electrophoresis)
Lectin affinity electrophoresis was performed with reference to the following literature (Taketa, K., Clinical Laboratory, 39 (1), 66 (1995), Shimizu, K., et al., Clinica. Chimica. Acta, 214, 3 (1993), Yamashita, K., et al., Cancer Res., 53, 2970 (1993), Taketa, K., J. Chromatogr., 569, 229 (1991), Taketa, K., et al. , Gastroenterology, 99, 508 (1990), Taketa, K., et al., Electrophoresis, 10, 562 (1989)).

  Peptide (control, Example 9, Example 6) and LCA were each dissolved in pure water to 3 mg / ml. To 4.2 ml of the agarose solution kept warm, 0.3 ml of the peptide solution or LCA solution was added and gently mixed. Poured into a gel mold and left at room temperature for 10-20 minutes. The gel was removed from the gel and used as an affinity electrophoresis gel.

(Analytical sample)
α-fetoprotein (AFP) (manufactured by Fitzgerald) and fucosylated AFP (AFP-L3) (manufactured by Wako Pure Chemical Industries, Ltd.) were used. AFP and AFP-L3 were prepared at 4 μg / ml and 0.1 μg / ml, respectively, and used as analysis samples.

(Electrophoresis)
An electrophoresis apparatus (manufactured by Kayaki) was filled with 50 mM veronal (pH 8.6) (buffer A) in a buffer layer, and an affinity electrophoresis gel was installed. A solution obtained by adding 1 μl of 2% bromophenol blue to 2 μl of an analytical sample was added to the well of the gel, and electrophoresis was performed.

(Transcription)
The gel was placed on an anti-AFP membrane soaked in buffer A in advance, and a filter paper, an acrylic plate, and a weight were placed thereon, and left for 30 minutes to perform transfer.

(Immunostaining)
After the transferred anti-AFP membrane was washed, the anti-AFP membrane was immersed in an anti-human AFP polyclonal antibody solution and allowed to stand at room temperature for 30 minutes. It was immersed in a cleaning solution and cleaned. The anti-AFP membrane was immersed in a horseradish peroxidase-labeled anti-rabbit IgG antibody solution. After washing, color was developed with a POD immunostain set (manufactured by Wako Pure Chemical Industries, Ltd.), thoroughly washed with distilled water, and dried.

  FIG. 1 shows the LCA gel, FIG. 2 shows the control peptide, FIG. 3 shows the peptide of Example 9, and FIG. 4 shows the results of AFP separation using the peptide of Example 6.

  In the control peptide shown in FIG. 2, since the isoelectric point is as low as 4.67, the separation between AFP-L1 and AFP-L3 is slightly insufficient. This is difficult to apply to liver cancer diagnostic methods. On the other hand, the peptide of Example 9 (isoelectric point 6.77) and the peptide of Example 6 (isoelectric point 8.01) according to the present invention obtain very good separation as shown in FIGS. I was able to. From these results, it was shown that the sugar chain change of AFP which is a tumor marker can be separated and detected by the peptide of the present invention.

  In the case of the peptides of Example 9 and Example 6, the thermostability is superior to that of LCA, and therefore the risk of a decrease in activity due to heating during preparation of the gel for affinity electrophoresis is extremely low. The ability to produce agarose with different isoelectric points as in the present invention is expected to be effective for the separation and detection of other tumor markers.

(Detection test of AFP of patient specimens by affinity electrophoresis)
Using the peptide of Example 6, human serum was analyzed by lectin affinity electrophoresis. Healthy samples and liver cancer patient sera (purchased from ProteoGenex) were used as analysis samples. The clinical data of the patients used are shown in Table 8 below.

  FIG. 5 shows the results of AFP analysis using the peptide of Example 6 for cancer patient sera (3 samples). Patients 1 and 3 have an AFP value of 5 ng / ml or more and can clearly analyze the appearance of AFP-L3, which is an indicator of canceration. Further, when AFP analysis was performed on serum (3 samples) of healthy subjects using the same method, AFP was not detected.

Claims (18)

  A peptide in which one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 are substituted with lysine and / or arginine and have sugar-binding activity.   The peptide according to claim 1, wherein the amino acid substituted by lysine and / or arginine is at least one selected from the group consisting of alanine, proline, valine, threonine and glycine.   The peptide according to claim 1 or 2, wherein the amino acid substituted with lysine or arginine is threonine and glycine at the C-terminal of the amino acid sequence.   Furthermore, the peptide in any one of Claims 1-3 to which 1 or several lysine and / or arginine are added, and have saccharide | sugar binding activity.   A peptide having one or several lysine and / or arginine added to the amino acid sequence shown in SEQ ID NO: 1 and having a sugar-binding activity.   Furthermore, the peptide in any one of Claims 1-5 from which amino acids other than a lysine and arginine are deleted, and have sugar-binding activity.   The peptide according to claim 6, wherein the amino acid to be deleted is at least one of threonine and glycine.   The peptide according to any one of claims 1 to 7, wherein the sugar binding activity is a binding activity to a fucose α1 → 6 sugar chain.   The peptide according to any one of claims 1 to 8, wherein the isoelectric point is higher than 4.67.   The peptide according to any one of claims 1 to 9, which is labeled.   A peptide having the same or different peptides according to any one of claims 1 to 10 and having a sugar-binding activity.   The peptide shown by either SEQ ID NO: 2-14.   A diagnostic or kit comprising the peptide according to claim 1.   The diagnostic or kit according to claim 13 for detecting fucose α1 → 6 sugar chain.   A method for modifying a peptide having sugar-binding activity, comprising substituting one or several amino acids with lysine and / or arginine in the amino acid sequence shown in SEQ ID NO: 1.   The method for modifying a peptide having a sugar binding activity according to claim 15, further comprising adding one or several lysine and / or arginine.   A method for modifying a peptide having sugar-binding activity, which comprises adding one or several amino acids with lysine and / or arginine in the amino acid sequence shown in SEQ ID NO: 1.   The method for modifying a peptide having a sugar binding activity according to claim 15, further comprising deleting one or several amino acids other than lysine and / or arginine.