KR20040108112A - Sialic acid derivative-specific lectin protein isolated from Cordyceps militaris, process for isolating the same and use thereof - Google Patents

Abstract

PURPOSE: A sialic acid derivative-specific lectin protein from Cordyceps militaris, an isolation process of the same and use thereof are provided. The protein is useful for diagnosis of disease associated with N-acetylneuramic acid, transfers a selective anticancer drug to a target local region with reduced adverse side-effects, and inhibits various cancers. CONSTITUTION: The sialic acid derivative-specific lectin protein from Cordyceps militaris is Cordyceps militaris agglutinin(CMA) protein having molecular weight of 45 kDa, and specifically binds to N-acetylneuramic acid or a glycoprotein containing N-acetylneuramic acid via red cell agglutination and red cell agglutination inhibition, wherein the N-terminal of the CMA comprises the amino acid sequence of X1-X2-X3-Ala-Val-Glu-Gln-Pro-X9-X10 in which X1 is any one selected from His and Arg, X2 is any one selected from Gln, Leu, Pro, Ile and Try; X3 is any one selected from Pro and Lys; and X9 and X10 are independently Cys or an modified amino acid which is not detected by a sequencer. The process for isolating the CMA comprises the steps of: finely cutting Cordyceps militaris; adding NaCl solution into the cut pieces of Cordyceps militaris and mixing them; filtering the Cordyceps militaris solution; and subjecting the filtered solution to fetuin-affinity chromatography.

Description

Sialic acid derivative-specific lectin protein isolated from Cordyceps militaris, process for isolating the same and use approximately}

The present invention relates to a lectin protein isolated and purified from a Millitaris cordyceps fungus (Cordyceps militaris) mushroom, a method and its use, and more specifically, to N-acetylneuraminic acid isolated and purified from a millitaris cordyceps fungus. Lectin CMA, which is a protein component that binds to a protein, methods for isolation thereof and various uses based on the biological activity of the isolated CMA.

Oligosaccharides, one of the important constituents of the living body, form complex molecules such as glycoproteins or glycolipids that bind to proteins or lipids, which are indispensable for maintaining the biological activity of an individual such as cell adhesion, information transfer between cells, and morphology of tissues. It is in charge of function.

The functions of oligosaccharides known to date are largely three. First, oligosaccharides bind to other proteins, and their interactions play an important role in cell-specific recognition. Second, when the oligosaccharides bound to the protein itself significantly change its specific function. For example, N-CAM, which is a cell adhesion molecule of oligosaccharides specifically expressed in the brain, such as polysialic acid. Third, most proteins are glycoproteins, and oligosaccharides are bound through Asn or Ser / Thr of the protein itself to activate the function of the protein. In other words, oligosaccharides can be thought of as important substances that can cause changes in their function not only by themselves but also by binding to other biological substances.

Intercellular recognition occurs mainly through sugars on the cell surface, and in order to mediate this smoothly, molecules that recognize sugar must exist on the cell surface. One example is the presence of carbohydrate-binding proteins such as lectins on the cell surface.

Militaris cordyceps (Cordyceps militaris) belongs to Cordyceps sp., Which live in insect bodies in winter, absorb nutrients to kill insects, and in summer they become mushrooms in dead insect bodies. It forms a dark orange color and the total length is about 2-7cm. To date, the effects of cordycepin, cordyceptic polysaccharides, cordyceptic acid, etc. have been isolated from the Militaris cordyceps, but lectins have specific functions. There is no example of pure separation.

On the other hand, the fact that the lectin is present in the high molecular protein and other fungi other than the fungus is was already known, to have a sugar binding characteristics of these known is Agaricus bisporus lectin derived βGal1-3GalNAC bond, Agrocybe aergerita derived galactose binding lectin, Alueuria derived aurantia Fucose binding lectins, N-acetyl-D-galactosamine binding lectins derived from Fomes fomentarius , N-acetyl-D-glucosamine binding lectins derived from Psathyrella velutina are known (Kawagishi H. Food Reviews International 11, 63-68, 1995). However, lectins that recognize sialic acid are rarely present in mushrooms. Especially, the lectins that have confirmed the sugar-binding properties are isolated and purified from Militaris cordyceps to obtain a single substance, and the molecular properties of the lectins, the binding sugars, There are no examples of information on anticancer effects.

The present inventors have isolated and purified a protein lectin CMA, which has not been known for some time, from the Militaris cordyceps below, to characterize this substance and to test its biological activity, which is specific for N-acetylneuraminic acid. The present invention has been completed by confirming that it can be used for various glycoprotein-related research reagents and cancer cell proliferation inhibitors by having various beneficial biochemical properties such as binding.

Therefore, in one aspect, an object of the present invention is to provide a CMA, which is a lectin component, which is a kind of protein that recognizes N-acetylneuraminic acid, extracted from Millyris cordyceps militaris.

In another aspect, an object of the present invention is to provide a method for separating the lectin CMA.

In a further aspect, an object of the present invention is to provide a diagnostic reagent capable of detecting when the structure of the sugar in the cell containing the lectin CMA contains N-acetylneuraminic acid and its distribution changes during the onset .

In another aspect, an object of the present invention is to provide a use of the lectin CMA as an anticancer agent that exhibits a proliferation inhibitory effect on cancer cells.

In still another aspect, an object of the present invention is to provide a use of the lectin CMA in immunobiochemical research materials or in aggregation of cancer cells and the like.

1 is a graphical representation of UV (280 nm) spectrophotometric chromatograms using a Sephadex G-75 column of an extract comprising lectin CMA isolated according to the method of the present invention.

FIG. 2 is a graphical representation of UV (280 nm) spectroscopy chromatograms with a petuin-affinity column of lectin CMA isolated according to the method of the present invention. FIG.

Figure 3 is a photograph showing the results of electrophoresis of lectin CMA isolated according to the method of the present invention. Lane 1: molecular marker (66, 45, 36, 29, 24, 20.1, 14.2 kDa); Lane 2: lectin CMA.

Hereinafter, the present invention will be described in more detail.

Lectin is a protein that selectively binds to carbohydrates. It is isolated and purified from plants, microorganisms, viruses, invertebrates, vertebrates, etc., and is widely used as a material for research on various diseases. To date, about 300 species have been reported. Lectin has a variety of functions, such as agglutination of erythrocytes and sedimentation is well known, classifying blood types, and recently, the ability to specifically aggregate cancer cells. In other words, lectins bind to sugars with specific structures and allow selective exchange of information between cells with lectin substances and cells with sugars that bind to them.

Lectin research can be considered as an important field for elucidating the mechanism of information transfer between cells through molecular recognition.Neutral system, immune system, inflammatory response, etc. In terms of research and the relationship between infections, diseases (cancer, rheumatism) and sugar, research is being actively conducted.

In particular, the sugar chains on the cell surface have been found to be an important factor in determining the metastasis of cancer to experimental models in connection with cancer. Metastasis is different in cancer cells selected in vitro, and the possibility of changing metastasis by modifying the sugar chains of cancer cells has been confirmed. These properties are known to be common to all types of cancer cells and to organs that target metastasis. Metastatic tumors appear frequently over time even after the primary tumor is completely removed by surgical treatment such as surgery, which is one of the biggest causes of difficulty in treating malignant tumors. In view of this, recently, the use of asialoglycoprotein as a carrier, the method using sugar chain-modified polymers, the targeting of protein-modified sugars by sugar modification, and the introduction of sugar chains by particulate carriers have been associated with cancer. It has been studied. In particular, glucose recognition mechanisms frequently used for systemic drug targeting include galactose receptors on the surface of hepatic parenchymal cells, mannose receptors on various macrophages, including cooper cells, and the like. Since these receptors are highly binding and express each organ and cell-specific, cell-specific drug targeting using this apparatus is expected.

In recent years, structural changes in oligosaccharides of proteins in the body during the onset of rheumatism, cancer, and AIDS have been reported, and attempts have been made to interpret the results of these diseases from structural changes in oligosaccharides. In particular, the significant change of sialyl epitope in the sialoconjugate on the cell surface of pathogens or tumors is related to the type of sialic acid in the oligosaccharides and the sugar binding method to adjacent sugars. It is believed to be attributable. Therefore, lectins that specifically recognize only various sialic acids and their sugar binding methods can be used as useful tools in identifying various sialyl epitopes in pathogens and malignant tumor biopsies.

Sialic acid refers to about 30 derivatives such as N-acetylneuraminic acid or N-glycolylneuraminic acid, an acyl substituent of the C-5 amino group. Compound. Most other sialic acids have one or more O-acetyl substituents on one or more C-4, C-7, C-8, C-9 hydroxyl groups. Such substituted sialic acid may cause transformation or other changes in the cellular environment, and by inhibiting or weakening sialic acid hydrolase of bacteria or virus, altering their immune capacity, affecting the enzymatic ability of the complex sugars. It is known to give.

Most of the lectins reported so far have been reported to specifically recognize mannose (D-mannose) and galactose (D-galactose), and specific sialic acid that plays a structural and functional role in the non-reducing end of sugar. Lectin is recognized as a species. Sialic acid binding lectins are mainly found in invertebrates, and isolated lectins are known to recognize part or all of sugar, similar to lectins isolated from other sources such as plants. Among them, molluscs and arthropods are reported to produce lectins that have specificity for sialic acid, and crustaceans do not synthesize sialic acid but use sialic acid-binding lectin as a defense against bacteria that express various sialoconjugates. It is known to produce. Lectins that bind to N-acetylneuraminic acid include Wheat germ agglutinin (WGA), Maackia amurensis agglutinin (MAA), and Limulus polyphemus lectin (limulin), Sambucus niger agglutinin (SNA) Tree-derived).

There are many reports on the anti-tumor effect of plant-derived lectins that inhibit cancer growth and the inhibition of cancer by carcinogens. WGA and MAA are known to have cytotoxic effects on liver cancer, melanoma, chorionic cancer, and osteosarcoma, and in vivo, murine ascitic tumors and murine ascitic murine lymphomas. In lymphoma tumors, in vitro, the effects of inhibiting human nasal septum tumor, human colon adenocarcinoma, and mouse embryonal carcinoma are known. MAA and SNA are reported to be selective for the receptor of influenza A virus. WGA has also been used to analyze sugar structure in HIV and SNA has been used to identify N-acetylneuraminic acid in liver cancer cells (Abdullaev F. et al. Natural Toxin 5, 157-163, 1997).

In addition, many cancer-related experimental models have been reported to have a significant relationship with the increase or dramatic change in sugar chains containing N-acetylneuraminic acid and the corresponding increase in lectin binding. This increase in N-acetylneuraminic acid is simultaneously observed in N- or O-linked oligosaccharides of glycoproteins. Inhibition of the formation of sugar chains by sugar chain synthesis inhibitors such as tunicamycin shows a decrease in metastatic properties. In addition, the glycolipids also show an increase in gangliosides and an increase in sugar chains containing many neuramic acids. Recently, IGR39 from primary tumors and IGR37 from metastatic lymph nodes in human melanoma cell lines from the same cancer patients have been reported to bind strongly with lectins. HM7 melanoma Affinity columns that combine lectins with sepharose have also been used to separate and purify mucin-type glycoproteins from plasma membranes of cells. In addition, a substance combining fluorochrome tetramethyltamine isothiocyanate with lectin is used for effective detection in B16F10 melanoma and Lewis lung carcinoma cells. On the other hand, there have been reports of the possibility of a carrier protein using acid-labile chemotherapy after binding doxorubicin to lectin for colon carcinoma.

The present inventors conducted a variety of experiments by selecting Militaris Cordyceps sinensis as a material to search for useful materials from natural organisms.

In one embodiment, the present invention provides a CMA, which is a lectin component, which is a protein that recognizes N-acetylneuraminic acid, extracted from Militaris cordyceps.

The lectin component was found to bind to glycoproteins containing N-acetylneuraminic acid or N-acetylneuraminic acid by electrophoresis and mass spectrometry at 45 kDa and by hemagglutination and hemagglutination inhibition. .

In another aspect, the present invention provides a method for separating lectin CMA from Militaris cordyceps.

Specifically, the lectin CMA was finely cut into Cordyceps sinensis and stirred at 4 ° C. for 24 hours by adding 0.15 M NaCl aqueous solution, and then filtering the precipitate with a filter to separate the protein component through a petuin-affinity column. It can be obtained by.

In a further aspect, the present invention provides a use as a diagnostic reagent for diagnosing a disease in which the structure of a sugar in a cell, including the lectin CMA, contains N-acetylneuraminic acid and the amount thereof changes. Examples of such diseases include, but are not limited to, colorectal cancer, stomach cancer, lung cancer, cervical cancer, ovarian cancer, liver cancer, pancreatic cancer, and the like.

As a result of testing the biological activity of the CMA, it specifically binds to N-acetylneuraminic acid as monosaccharide and specifically to petuin including N-acetyl acid as glycoprotein.

In another aspect, the present invention provides a use of the lectin CMA as an anticancer agent that exhibits a proliferation inhibitory effect on cancer cells.

The lectin CMA material of the present invention selectively recognizes and binds to a glycoprotein comprising N-acetylneuraminic acid or N-acetylneuraminic acid as confirmed by hemagglutination and hemagglutination inhibition. Not only can be used, but also has an anticancer effect as evidenced in the examples described below. Therefore, the lectin CMA of the present invention can be effectively used as an active ingredient of cancer cell proliferation inhibitors (or anticancer agents) in diseases in which N-acetylneuraminic acid is present. Examples of the target diseases include, but are not limited to, various malignant tumors including colon cancer, stomach cancer, lung cancer, cervical cancer, ovarian cancer, liver cancer, pancreatic cancer, and the like.

The pharmaceutical composition of the cancer cell proliferation inhibitor according to the present invention can be formulated in combination with a known pharmaceutical carrier. Generally, the active ingredient is blended with a pharmaceutically acceptable liquid or solid carrier, and a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant and the like are added as necessary and purified. And solid solutions such as granules, powders, powders, and capsules, liquids such as ordinary liquids, suspensions, and emulsions. Moreover, it can also be made into the dry goods which can be made into a liquid state by addition of a suitable carrier before use.

The pharmaceutical composition of the present invention can be administered by any of parenteral agents such as oral preparations, injections, and drops.

A medical carrier can be selected according to the said dosage form and formulation, and, in the case of an oral preparation, starch, lactose, white sugar, mannite, carboxymethylcellulose, corn starch, an inorganic salt, etc. are used, for example. Moreover, in preparation of an oral preparation, a binder, a disintegrating agent, surfactant, a lubricating agent, a fluidity promoter, a mating agent, a coloring agent, a fragrance | flavor, etc. can also be mix | blended further.

On the other hand, in the case of parenteral preparations, a distilled water for injection as a diluent, physiological saline, aqueous glucose solution, injectable vegetable oil, sesame oil, peanut oil, soybean oil, corn oil according to a conventional method It is prepared by dissolving or suspending in propylene glycol, polyethylene glycol or the like and adding a fungicide, stabilizer, isotonicity agent, analgesic agent and the like as necessary.

The pharmaceutical composition of the present invention is administered by a suitable route of administration according to the formulation form. The administration method need not be particularly limited and can be administered by internal content, external application, and injection. Injections can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally or the like.

The dosage of the pharmaceutical composition of the present invention is appropriately set depending on the form of the preparation, the method of administration, the purpose of use, and the age, weight and symptoms of the patient to be applied thereto. Amounts are, for example, 10 μg-200 mg / kg body weight per adult. Of course, since the dose varies depending on various conditions, an amount smaller than the dose may be sufficient, or it may be necessary beyond the range.

In a still further aspect, the present invention provides a drug conjugate in which the lectin CMA is combined as a carrier protein for topical delivery and the selective drug is used as an anticancer agent. Examples of diseases targeted by such anticancer agents include, but are not limited to, various tumors including colon cancer, stomach cancer, lung cancer, cervical cancer, ovarian cancer, liver cancer, pancreatic cancer, and the like.

Hereinafter, the present invention is described in more detail by the following representative examples, but the present invention is not limited in any way by these examples.

<Example>

Example 1 Isolation of Protein Solution Containing Lectin CMA from Militaris Cordyceps Sinensis

After removing the pupa part, 4.7 g of Militaris cordyceps was collected, washed with tertiary distilled water, and then rinsed with 0.15 M NaCl solution using a grinder and mixed well at 4 ° C. for 24 hours. The mixed solution was subjected to a primary filter with a 6 μm membrane filter and secondary filtration with a 0.45 μm membrane filter. The filtrate was subjected to Sephadex G-75 column chromatography. In this case, 1 g of Sephadex G-75 was dissolved in 20 ml of 10 mM Tris-HCl, 150 mM NaCl (pH 8.0), and 12.5 ml of gel was poured into the column (Econo Pac column, Bio-Rad) and 10 mM Tris-HCl, 150 mM NaCl (pH 8.0). Equilibrate by washing with. Here Chromatogram was prepared by measuring the absorbance at 280 nm of 30 fractions obtained by flowing 1 ml of Cordyceps sinensis filtrate in 1 ml each, and the results are shown in FIG. 1. In the chromatogram using a Sephadex G-75 column, fractions with hemagglutination activity described below (No. 7-10) were collected to separate the protein solution containing CMA.

Example 2: Isolation of Lectin CMA from Millitaris Cordyceps Sinensis Protein Solution

The protein solution obtained in Example 1 was applied to a pettuin-affinity column, and chromatograms were prepared by UV / VIS spectroscopy at 50 fractions of 280 nm. The results are shown in FIG. 2. The fractions were placed on a petuniin affinity column and allowed to stand at 4 ° C. for 12 hours, and then aliquoted with 1 ml while slowly flowing 10 mM Tris-HCl and 150 mM NaCl (pH 8.0). Subsequently, 10 mM Tris-HCl and 150 mM NaCl (pH 8.0) were sent until the absorbance value became 0.0 at 280 nm. Received fractions 2-4 with absorbance values greater than 0.1, and these were confirmed by erythrocyte agglutination reactions, which are not lectin components, and 100mM glycine buffer (pH 3.0) was rapidly obtained from fraction 28, where the absorbance values were low. 1 ml aliquots were flushed and immediately neutralized with 1.5 M Tris-HCl (pH 8.5). As a result of measuring absorbance at 280 nm for these fractions, the fractions 31-35 showed high absorbance. In addition, the results of the hemagglutination reaction experiments described below with respect to these fractions, it was confirmed that the lectin having a high activity.

The results of the total protein amount and the recovery rate of the lectin CMA obtained by Example 1 and Example 2 are shown in Table 1 below.

Total protein amount (mg) CMA recovery (%) Protein extracts 407.7 100.0 Sephadex G-75 Columns 7-10 133.2 32.7 Thyroglobulin-affinity Columns 31-35 Fractions (CMA) 58.7 14.4

Example 3: Confirmation of Purity and Molecular Weight of Lectin CMA

Electrophoresis was applied to confirm the molecular weight and purity of the lectins obtained in Example 2. First, electrophoresis was performed on a 12% polyacrylamide gel containing 0.1% SDS to compare with a molecular weight standard. After completion of development, the protein bands were stained with Coomassie blue staining reagent. This experimental result is shown in FIG. In Figure 2, lane 1 is molecular marker (66, 45, 36, 29, 24, 20.1, 14.2 kDa); Lane 2 is lectin CMA.

As confirmed from the electrophoretic results of FIG. 3, a single band having a molecular weight of 45 kDa was identified. From this result, it was confirmed that CMA was separated into a single substance and its molecular weight was 45 kDa.

Example 4: N-terminal amino acid analysis of lectin CMA

Amino acid sequence analyzer the N- terminal amino acid of the lectin CMA (manufacturer: AppliedBiosystems; product name: Automatic Protein Sequencer, 476A-01-120 ) results using the analysis performed according to the manufacturer's instructions The sequence X ₁ in the N- terminal -X ₂ -X ₃ -Ala-Val-Glu-Gln-Pro-X ₉ -X ₁₀ . Wherein X ₁ is any one selected from His and Arg, X ₂ is any one selected from Gln, Leu, Pro, Ile and Try, and X ₃ is any one selected from Pro and Lys , X ₉ and X ₁₀ are each Cys or one of the modified amino acids and represents an amino acid not detected by the amino acid sequencer. In amino acid sequencing, the substance having an amino acid sequence corresponding to residues 4 to 8 has not been reported yet, and it was confirmed that it is a novel substance.

Example 5 Hemagglutination of Lectin CMA

Using the lectin CMA obtained in Example 2, serum of humans, rats, and rabbits was diluted with 10 mM phosphate buffer, 0.15 M NaCl (pH 7.2) to 6.25%, and then hemagglutination was performed. Fractions positive in this reaction were collected and subjected to electrophoresis as shown in Example 3 to confirm that it was a single substance of a single band, and this portion was named CMA (Cordyceps militaris agglutinin). Erythrocytes of human A, B, O, rats, and rabbits were diluted in the same manner as above, and 50 μl was left at room temperature with 50 μl of lectin CMA to check for hemagglutination. As a result, lectin CMA did not react to human erythrocytes, and thus erythrocytes aggregated normally, inhibiting aggregation until 8-fold dilutions of rat erythrocytes and 2-fold red rabbit cells, and the results are shown in Table 2 below. As shown.

Red blood cells Present sialic acid class Minimal hemagglutination inhibitory activity Person A type Neuac 0 Person B type Neuac 0 Person O type Neuac 0 Rat Neu, Gc, NeuAc, O-acetylsialic acid 8 rabbit Neu, Gc, NeuAc, O-acetylsialic acid 2 Note: The minimum hemagglutination inhibitory activity is the minimum erythrocyte aggregation inhibition dilution factor obtained by diluting lectin CMA by 2 times for various red blood cells diluted to 6.25%, NeuGc is N-glycolylneuraminic acid, and NeuAc is N-acetyl. Represents neuramic acid.

Example 6 Hemagglutination Inhibition of Lectin CMA

In order to confirm the sugar binding selectivity of the lectin CMA of the present invention, hemagglutination inhibition reaction using monosaccharide, disaccharide, and glycoprotein was performed. 50 μl of CMA lectin solution dissolved in 0.15 M NaCl in each well of a 96 well microplate and various monosaccharides (β-D-glucose, D-galactose, L-fucose, D-mannose, N-acetylneuraminic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine), disaccharides (α-lactose, β-lactose, D-lactose, maltose) and glycoproteins (bovine submaxillary mucin), 50 μl of a solution in which Petuin and asialofetuin) were dissolved in 0.15 M NaCl was added, and reacted at room temperature for at least 1 hour. Then, 50 μl of red blood cells suspended in 0.15 M NaCl was added to each well, and for 1 hour. The reaction was carried out at room temperature. As a result, N-acetylneuraminic acid inhibited aggregation at 3.3mM as a monosaccharide, and 0.07mM for petuine containing N-acetylneuraminic acid as a glycoprotein, and at a low concentration of 0.01mM for bovine maxillary mucin. And other monosaccharides, disaccharides, and glycoproteins did not inhibit aggregation. The results are as shown in Table 3 below.

Classification carbohydrate Sialic acid type Minimal hemagglutination inhibition concentration Monosaccharides β-D-glucose - － D-galactose - － L-fucose - － D-Mannose - － N-acetylneuraminic acid Neuac 3.3mM N-acetyl-D-galactosamine - － N-acetyl-D-glucosamine - － saccharose α-lactose - － β-lactose - － D-lactose - － maltose - － N-acetyllactozamin - － Glycoprotein Cow maxillary mucin NeuGc / NeuAc / O-acetylsialic acid 0.01mM Petuin Neuac 0.07mM Asialopetuin - －

In Table 3, the minimum concentration of erythrocyte aggregation inhibition was diluted by 8-fold with the minimum aggregation of CMA in 6.25% erythrocytes of rats showing minimal hemagglutination inhibition and 2-fold after preparation of monosaccharides, disaccharides and glycoproteins at each concentration. Dilution represents the minimum concentration that indicates aggregation inhibition,-indicates no aggregation inhibition. Monosaccharides β-D-glucose, D-galactose, L-fucose, D-mannose and disaccharides α-lactose, β-lactose, D-lactose and maltose are 100 mM, monosaccharide N-acetyl-D-glucosamine, N-acetyl-D-galactosamine showed no aggregation inhibition even at 10 mM. In addition, asialopettuin, a glycoprotein to which sialic acid was not bound, showed no aggregation inhibition even at 0.1 mM. NeuAc stands for N-acetylneuraminic acid, NeuGc stands for N-glycolylneuraminic acid.

Example 7 Effect of Temperature of Lectin CMA

In order to confirm the activity according to the temperature of Cordyceps lectin PJA, the activity was confirmed after 1 hour at 4 ° C, 25 ° C, 37 ° C, 50 ° C and 60 ° C. Indicated.

Temperature activation 4 ?? 25 ?? 37 ?? 50 ?? 60 ?? 100% 100% 100% 100% 0%

Example 8 Inhibition of Cancer Cell Proliferation by Lectin CMA

Proliferation inhibitory effect on cancer cells was carried out using MTT (3- [4,5-dimethyldiazol-2-yl] -2,5-diphenyl tetrazolium bromide) assay (Mosmann T et al. J. Immunol. Methods 65, pp 55-63 (1983). After culturing each cancer cell in RPMI 1640 90%, FBS (fetal bovine serum) 10% medium and using a hematocytometer to confirm that the number is about 1x10 ^{4 in} 100ul, put into a 96 well plate 1, 0.1 μM lectin CMA 100 Μl was added and it was left for 72 hours in a 37 ° C. incubator maintaining 5% CO ₂ . 50 μl of MTT reagent was added to each well, and the mixture was left in a CO ₂ incubator for 4 hours. The supernatant was discarded, 150 μl of dimethyl sulfoxide was added, mixed for 10 minutes, and the absorbance was measured at 540 nm using a microplate. When the experiment was conducted in the same manner without adding lectin CMA under the experimental conditions, the reduction ratio was confirmed by comparing with the case where lectin CMA was added at 100%. As a result, as shown in Table 6, lectin CMA showed a cell proliferation inhibitory effect on human human colorectal cancer, human gastric cancer, and human lung cancer, and showed the strongest cancer cell proliferation inhibitory effect on human colorectal cancer.

Cancer cell origin Cancer cell common name Badge composition Lactin PJA Concentration Cancer cell growth inhibition% Human colon cancer SNU-1 RPMI 1640 90%, FBS 10% 1 μM0.1 μM 98.1% 58.6% Human stomach cancer A-549 RPMI 1640 90%, FBS 10% 1 μM0.1 μM 34.8% 17.8% Human lung cancer HCT-116 RPMI 1640 90%, FBS 10% 1 μM0.1 μM 20.4% 8.1%

Example 7: Pharmaceutical Formulations

Form of the formulation (eg tablet)

Active ingredient (Lectin CMA) 10 mg

Lactose 80 mg

Starch 17 mg

Magnesium Stearate 3 mg

10 mg of crystalline cellulose

Tablets were prepared by conventional methods and contain the above ingredients as raw material in one tablet. Tablets may be added as usual, enteric coat (eg hydroxypropylmethylcellulose phthalate), sugar coating or film (ethyl cellulose).

According to the present invention, a lectin CMA that specifically recognizes N-acetylneuraminic acid can be used as a reagent for carbohydrate-related research based on the principle of recognizing N-acetylneuraminic acid and the distribution of N-acetylneuraminic acid. It can be used for diagnosis of changing diseases, and it can be applied to humans because it does not aggregate human erythrocytes while maintaining 100% activity at human body temperature of 37 ℃, and it is a selective drug that minimizes side effects by combining anticancer drugs with lectins. It can be used as a cancer cell proliferation inhibitor for colorectal cancer, gastric cancer, lung cancer as well as carrier protein for delivery to the local site.

Claims (9)

Molecular weight of 45 kDa by electrophoresis and mass spectrometry. CMA (Cordyceps militaris agglutinin protein), a lectin component extracted from Cordyceps sinensis. The CMA of claim 1, wherein the CMA specifically binds to a glycoprotein comprising N-acetylneuraminic acid or N-acetylneuraminic acid by erythrocyte agglutination and erythrocyte agglutination. The CMA of claim 1, wherein the N-terminus of the protein comprises the following nucleotide sequence: X ₁ -X ₂ -X ₃ -Ala-Val-Glu-Gln-Pro-X ₉ -X ₁₀ Wherein X ₁ is any one selected from His and Arg, X ₂ is any one selected from Gln, Leu, Pro, Ile and Try, and X ₃ is any one selected from Pro and Lys , X ₉ and X ₁₀ are each Cys or one of the modified amino acids and represents an amino acid not detected by the amino acid sequencer. Chopping the cordyceps finely, adding NaCl aqueous solution to the mixture, filtering the mixture with a filter, and applying the fetuin-affinity chromatography to the filtrate. How to detach a CMA. A diagnostic reagent comprising a lectin CMA according to claim 1 for diagnosing a disease associated with a change in the distribution of cells comprising N-acetylneuraminic acid in the intracellular sugar structure. A drug conjugate wherein the lectin CMA according to claim 1 as a carrier protein for topical delivery agent is combined with an optional drug as an optional agonist. 7. The drug conjugate of claim 6, wherein said agonist is an anticancer agent. Cancer cell proliferation inhibitor comprising the lectin CMA according to claim 1 as an active ingredient. The cancer cell proliferation inhibitor according to claim 8, wherein the cancer is selected from the group consisting of colon cancer, gastric cancer, lung cancer, cervical cancer, ovarian cancer, liver cancer, pancreatic cancer and various tumors.