JP2587863B2 - Novel glycoprotein - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel glycoprotein having an action of suppressing complement-induced cell membrane damage.

[Problems to be solved by conventional technology and invention]

Complement is a reaction system that includes a group of proteases present in blood and body fluids. When the foreign body invades the living body, the complement system is activated, and some of the complement components deposited in response to the foreign body promote poor phagocytosis by the phagocytic cells or cause dissolution of the foreign body, and actively remove the foreign body. work. In addition, various peptide fragments of complement components generated in the process of complement activation are used for various cells such as activating lymphocytes, migrating neutrophils, and promoting degranulation of mast cells. Are involved in a variety of immune and inflammatory responses.

Although complement responds rapidly to foreign bodies, complement activation does not proceed on its own normal cells. However, for example, in inflammation sites such as rheumatoid arthritis, systemic lupus erythematosus, and glomerulonephritis, complement recognizes its own tissue as a foreign body, and induces an inflammatory reaction in response to the cell membrane of the tissue, It is thought that they promote phagocytosis by poor phagocytes or cause cell damage such as dissolution of the cell membrane, thereby exacerbating the inflammatory response.

In recent years, the presence of a protein that suppresses the activation of autologous complement on the autologous cell membrane has been shown [Mol. Immunol., 20 , 1233-1236.
(1983)], and some have been identified as proteins having that function. For example, Decay acceler-ating with a molecular weight of 68 Kd
factor (DAF) [J. Exp. Med., 160 , 1558-1578 (198
4)] and a homologous restriction factor with a molecular weight of 65Kd
(HRF) [PNAS, 83 , 6975-6979 (1986)] has been obtained as a complement inhibitory factor present on human cell membranes. However, the detailed functions of these DAFs and HRFs in living bodies have not yet been elucidated.

The present inventors have studied to obtain a membrane protein which has a function of suppressing complement activation on an autologous cell membrane in addition to these factors. First, the present inventors immunized a mouse with human erythrocytes, removed the spleen of the mouse, and fused the spleen cells with mouse myeloma cells (P3U1) using polyethylene glycol to obtain a large number of antibody-producing hybridomas. From among the antibodies (culture supernatants) produced by these hybridomas, cells that produce antibodies that react with human erythrocytes that have been treated with neuraminidase in advance and also cause hemolysis by human complement are repeatedly screened. to obtain a hybridoma producing the antibody, to obtain a single monoclonal antibody 1F5 antibody [the Japanese Society for Immunology meeting article, 1
7 , 498 (1987), Complement Symbolic Abstracts, 24 , 180 (198
7)].

[Means for solving the problem]

Recently, the present inventors have conducted intensive studies to obtain an antigen of the 1F5 antibody, and as a result, have obtained a novel glycoprotein having a molecular weight of 20 to 25 Kd, which is different from the conventional complement inhibitory factor, and completed the present invention. did.

That is, the gist of the present invention is a glycoprotein derived from human cell membrane, which is obtained by SDS-polyacrylamide gel electrophoresis (hereinafter referred to as “S
(Abbreviated as "DS-PAGE") having a molecular weight of 20 to 25 Kd, containing an N-glycoside type sugar chain and phosphatidylinositol, and having an action of suppressing cell membrane damage caused by complement.

In the following, the present invention will be described. The glycoprotein of the present invention is obtained from human cell membranes such as erythrocytes, lymphocytes, vascular endothelial cells or tumor cells and normal cell lines derived therefrom. For example, human cells such as human red blood cells are centrifuged (500 × g, 1
After washing with Dulbecco's PBS (manufactured by Nissui Pharmaceutical Co., Ltd., hereinafter referred to as “PBS”) or the like, the solution is transferred to a hypotonic solution and crushed by osmotic pressure or crushed by nitrogen gas pressure. A hypotonic solution such as 1 mA phenylmethylsulfonyl fluoride (PMSF) (manufactured by Sigma) and a 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA are added to the obtained crushed solution.
And centrifuge (5000-1000 xg, about 30 minutes). This operation is repeated to obtain a cell membrane fraction.

Then, a surfactant such as 1%
Glycoprotein is extracted using the above buffer containing about β-N-octylglucoside (manufactured by Dojinsha) or an organic solvent, for example, about 20% (v / v) butanol, and ammonium sulfate is added to the glycoprotein extract. In addition, a protein precipitate with a saturated concentration of ammonium sulfate of 30 to 60% is obtained by centrifugation.

The above ammonium sulfate precipitate fraction was dissolved in a small amount of the above buffer solution, and DE was dissolved.
AE-Sepharose column chromatography, MonoS-
A suitable combination of column chromatography, hydroxyapatite column chromatography, phenyl sepharose chromatography, and column chromatography such as TSK2000SW (manufactured by Tosoh Corporation), and the glycoprotein of the present invention is used as an index based on the activity of inhibiting the hemolysis of human guinea pig erythrocytes by human complement. Can be purified.

Alternatively, 1F5 antibody affinity sepharose is prepared by a conventional method using the 1F5 antibody obtained by the method described in the Japanese Society of Immunology Society general meeting article 17 , 498 (1987) and complement symposium abstract 24 , 180 (1987), and described above. The ammonium sulfate precipitate fraction of the glycoprotein is provided, and the glycoprotein of the present invention is purified by elution with a 0.1 M glycine-HCl buffer (pH 3.0) containing 0.15 M sodium chloride and 0.1% CHAPS (manufactured by Sigma). be able to. The 1F5 antibody can be freely obtained without limitation from the Department of Microbiology, School of Medicine, Fukuoka University. Alternatively, a peptide having the amino acid sequence of the following portion of the glycoprotein obtained by the above method is synthesized, and immunized to a rabbit according to a conventional method to obtain an antiserum, from which an IgG fraction is purified. IgG was bound to Sepharose, antibody affinity sepharose was prepared, and the above ammonium sulfate precipitated fraction was provided, and 0.1 M glycine-
The glycoprotein of the present invention can be purified by elution with a hydrochloric acid buffer (pH 2.8).

(A) Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pr
o (b) Thr-Ala-Asp-Cys-Lys-Thr-Ala-Val The glycoprotein of the present invention thus obtained has the following properties.

(1) The molecular weight by electrophoresis (12.5% SDS-PAGE) is 2
0 to 25 Kd (2) The sugar chain contains an N-glycoside type sugar chain (3) It contains phosphatidylinositol (4) It has an action of suppressing cell damage caused by complement.
This is confirmed, for example, by the fact that guinea pig erythrocytes are readily taken up by guinea pig erythrocytes and suppress the hemolysis of guinea pig erythrocytes by human complement.

(5) The isoelectric point was determined by isoelectric focusing using Ampholine (manufactured by Pharmacia) pI 3.5-10 in the first dimension, and performing SDS-PAGE (12.5%) under non-reducing in the second dimension. As a result of the analysis, bands were obtained at a pI of about 5.0 (main band) and a pI of about 6.5.

(6) Asp having the following amino acid composition: 20.2%, Thr: 8.9%, Ser; 3.1%, Glu; 10.6%, Gly;
2%, Ala; 6.2%, Cys; 0.6%, Val; 4.5%, Ile; 1.4%, Leu; 1
0.1%, Tyr; 8.1%, Phe; 6.4%, Lys; 9.1%, His; 2.1%, Arg;
3.0%, Pro; 3.5% (7) Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pro-Thr having the following partial amino acid sequence
-Ala-Asp-Cys-Lys-Thr-Ala-Val-XX-Ser-
Ser-Asp-Phe-Asp-Ala-X-Leu-X-Thr-Lys-Al
a-Gly-X-Gln-Val-Tyr-Asn-X represents Cys or another amino acid residue.

〔Example〕

Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1 (1) Preparation of 1F5 antibody affinity sepharose CNBr-activated Sepharose 4B (Pharmacia)
Was swelled in 1 mM hydrochloric acid for 15-30 minutes. This gel solution was suctioned on a glass filter, 200 mM of 1 mM hydrochloric acid was added per 1 g of the dried gel, and the suction washing was repeated.

The gel was then washed with coupling buffer (0.2 M sodium bicarbonate containing 0.5 M sodium chloride, pH 8.5-8.7) at 10 ml per dry gel. Immediately transfer the washed gel to the 1F5 antibody solution dissolved in the coupling buffer,
The coupling reaction was started. At this time, the amount of 1F5 antibody is 1 ml
The amount was 5 to 10 mg for the swollen gel.

The coupling reaction was performed at 4 ° C. overnight with gentle stirring. After the reaction, transfer the gel solution onto a glass funnel,
Unreacted antibody solution was recovered. The gel was then transferred to a 1 M ethanolamine solution and left at 4 ° C. for 16 hours.

The gel after the reaction was transferred to a glass funnel and aspirated. Next, washing and suction were repeated 10 times with a coupling buffer, and further, washing and suction were repeated 10 times with a 0.1 M acetate buffer (pH 4.0) containing 0.5 M sodium chloride. Lastly, gel P
After washing and suctioning three times with BS, 1F5 antibody affinity sepharose was prepared.

(2) Preparation of human erythrocyte membrane (a) Washing of human erythrocytes 300 to 400 ml of human blood was centrifuged at 400 to 500 × g for 15 minutes, and the supernatant was removed to obtain a red blood cell fraction as a precipitate. PBS containing 10 mM EDTA and 1 mM PMSF was added to the precipitate, and 400 to 500 × g
For 15 minutes. Next, 1 mM EDTA, 1 mM PMM
PBS containing SF was added and centrifuged at 400-500 × g for 15 minutes.
The supernatant was removed again to obtain a precipitate. This operation was repeated until the supernatant became transparent.

(B) Hemolysis of erythrocytes and preparation of erythrocyte membrane The washed erythrocytes obtained in (a) above were subjected to 1 mM EDTA and 1 mM PMSF.
Was transferred to 3 to 4 of a 10 mM Tris-HCl buffer (pH 7.5) (hereinafter, abbreviated as "buffer A") containing, and hemolyzed while stirring at 4 ° C overnight.

Then, the mixture was centrifuged at 25,000 × g for 30 minutes, and the erythrocyte membrane fraction was collected as a precipitate. This precipitate was suspended in buffer A and centrifuged at 25,000 × g for 30 minutes. This operation was performed until the color of the erythrocyte membrane obtained as a precipitate became pale pink.

(C) Solubilization of erythrocyte membrane protein The erythrocyte membrane fraction obtained in the above (b) was treated with 1% n-octyl-β-D-glucopyranoside (Sigma, hereinafter referred to as “NO
G ") and stirred at 500C overnight.

The solution was then centrifuged at 105,000 xg for 30 minutes,
The supernatant was obtained. While stirring the solution at 4 ° C., solid ammonium sulfate was added little by little so as to become 30% saturated. After stirring was continued for 2 to 3 hours, the mixture was centrifuged at 10,000 × g for 15 minutes to obtain a supernatant.
Solid ammonium sulfate was added to this supernatant so as to be 60% saturated, and 4 ° C.
After stirring for 2 to 3 hours, the mixture was centrifuged at 10,000 × g for 15 minutes to obtain a precipitate. This precipitate was dissolved in a small amount of buffer A and the final concentration was 0.1
% NOG was added. This solution was added to buffer A containing 0.1% NOG and 0.15 M sodium chloride at 4 ° C.
3500 dialysis membrane (Made by Spectrum) overnight
Was dialyzed against

(D) Affinity column chromatography A column of the 1F5 antibody affinity sepharose gel (5 ml capacity) prepared in the above (1) was prepared and equilibrated with buffer A containing 0.15 M sodium chloride and 0.1% NOG. The solubilized solution of the glycoprotein obtained in the above (c) was adsorbed on this column. After the adsorption, buffer A containing 0.5 M sodium chloride and 0.1% NOG was washed with one sulfuric acid. After washing, 100 ml of buffer A containing 0.15 M sodium chloride and 0.1% CHAPS was flowed. Next, elution was performed with a 0.1 M glycine-HCl buffer (pH 3.0) containing 0.1% CHAPS and 0.15 M sodium chloride. The eluate was fractionated in 1 ml portions, and quickly neutralized by adding 1 M glycine-sodium hydroxide buffer (pH 9.5). Concentrated by ultrafiltration collected elution fractions protein was eluted as monitored by A ₂₈₀ (emission molecular weight of 10,000 or less), PB containing 0.1% CHAPS
Dialyze against S using the above dialysis membrane and 100 ~ 150μg
Of the present invention was obtained.

(E) Properties of the glycoprotein of the present invention (1) Molecular weight: 20 to 25 Kd (12.5% reduced, non-reduced SDS-
(PAGE) (2) The sugar chain contains an N-glycoside type sugar chain. 50% of the 2% SDS solution is added to 50 μm of the glycoprotein of the present invention (mg / ml).
Add μ and boil for 1 minute. Next, 1 unit of endoglycosidase F (manufactured by Boehringer), 100 µM of 1 M acetate buffer (pH 6.0), and 100 µ of 0.25 MEDTA (pH 7.5) were added to this solution.
μ, 10% Triton X-100 (manufactured by Sigma) 50μ, 2
-10 µ of mercaptoethanol and 619 µ of distilled water were added, and reacted while stirring at 37 ° C for 16 hours. After the reaction is completed, add 200 μl of a 50% trichloroacetic acid solution, and place on ice for 10 minutes.
The mixture was left standing for 10 minutes, centrifuged (10,000 × g for 10 minutes), and the supernatant was discarded. 1 ml of cold acetone was added to the precipitate, and the mixture was stirred.
After standing for 1 minute, the mixture was centrifuged (10,000 × g, 10 minutes), the supernatant was discarded, and this operation was repeated once. This precipitate was subjected to SDS-P
After dissolving in AGE sample solution and boiling for 3 minutes, SDS-PAGE1
2.5%. As a control, a reaction solution not containing only endoglycosidase F was added to the glycoprotein of the present invention, and the same treatment was carried out for the same time. Those treated with endoglycosidase F showed about a band on SDS-PAGE as compared to the control.
It shifted to the low molecular side of 0.5 Kd (FIG. 1).

From the above results, it was shown that the glycoprotein of the present invention contains an N-glycoside type sugar chain.

(3) MT2 containing phosphatidylinositol (Human T-cell leukemia virus-1 (HTLV-1) infected human T cell line) having the glycoprotein of the present invention on the cell surface
Was used. Centrifuge the culture solution containing MT21 × 10 ⁶ cells (100 to 40
0 × g for 5 minutes) to collect the cells. After PBS was added thereto and the mixture was gently stirred, the mixture was centrifuged in the same manner, and the supernatant was discarded. This operation was repeated twice. 1 × 10 ⁶ MT2 cells (cells) were phosphatidylinositol-specific phospholipase C15 unit (hereinafter abbreviated as “PI-PLC”; J. Biochem., 93 , 1717).
(1983), B. thruringeinsis IAM 12077
Purified), 10 mM Tris-HCl (pH 7.5) buffer containing 0.25 M sucrose, 10 mM EDTA and 0.1% bovine serum albumin.
μ was added, and the mixture was left at 37 ° C. for 90 minutes with gentle stirring. After the reaction, 2 ml of PBS was added and centrifuged (100-400 × g10
Minutes) and the supernatant was discarded. This operation was repeated twice more. Sheep IgG (Capel) (1
(0 mg / ml), and the mixture was stirred and allowed to stand at room temperature for 10 minutes, and PBS was added as described above, and washing by centrifugation was repeated three times. 20 μl of 1F5 antibody (A ₂₈₀ 0.1 concentration) was added to this precipitate,
After stirring, the mixture was allowed to stand at room temperature for 20 minutes. The PBS was added again, and washing by centrifugation was repeated three times, and the precipitate was subjected to FITC-labeled anti-mouse IgG.
After adding 20 μl (manufactured by Capel), stirring, the mixture was allowed to stand at room temperature for 20 minutes, and PBS was added thereto, followed by washing by centrifugation three times. The precipitate was added with 200 μl of sheath solution (manufactured by Fujisawa Pharmaceutical Co., Ltd.) to obtain a cell suspension, and the filtrate filtered through a nylon mesh was further added to the filtrate.
0 μ sheath solution was added, and subjected to a FACS analyzer (flow cytometry). As a control, MT2 cells treated with a reaction solution containing no PI-PLC as described above were used.

As a result, MT2 cells treated with PI-PLC were 95% glycoprotein-negative cells of the present invention as compared to the control (FIG. 2).

From the above results, it was presumed that the glycoprotein of the present invention had phosphatidylinositol and was bound to the membrane via this.

(4) It has the effect of suppressing cell damage caused by human complement.

For example, 25μ of 2% guinea pig erythrocytes thoroughly washed with PBS
12.5 μg of the glycoprotein of the present invention (arbitrary protein concentration)
In addition, 462.5 µ of PBS was further added, and the mixture was allowed to stand with gentle stirring at 37 ° C for 1 hour. After completion of the reaction, 2 ml of veronal buffer (manufactured by Wako Pure Chemical Industries, Ltd.) containing 5 mM magnesium, 10 mM ETA and 0.1% gelatin (abbreviated as “Mg-EGTA-GVB”) was used.
In addition, the mixture was centrifuged (100-400 × g, 10 minutes), and the supernatant was discarded.
Again, 2 ml of Mg-EGTA-GVB was added, centrifuged in the same manner as above, and the supernatant was discarded. 100 μg of Mg-EGTA-GVB and 50 μm of normal human serum were added to the precipitate, and the mixture was slowly stirred at 37 ° C. for 60 minutes.
Next, 1.0 ml of veronal buffer containing 40 mM EDTA and 0.1% gelatin was added, and centrifugation was performed (100 to 400 × g, 10 minutes). The supernatant was measured by A _414, to measure the hemolysis of guinea pig erythrocytes by human complement. As a control, the same concentration of bovine serum albumin was used instead of the glycoprotein of the present invention.

As a result, when the glycoprotein of the present invention was added to a final concentration of 1 μg / ml, the hemolysis rate of guinea pig erythrocytes was 90%.
% Was suppressed. Therefore, it was shown that the glycoprotein of the present invention was adsorbed to the erythrocyte membrane and had an effect of suppressing cell damage caused by human complement (FIG. 3).

(5) Isoelectric point: Isoelectric point electrophoresis using Ampholine (manufactured by Pharmacia) pI 3.5 to 10 in the first dimension was performed.
As a result of analysis using SDS-PAGE (12.5%) under the non-reducing condition, bands having a pI of about 5.0 (main band) and a pI of about 6.5 were obtained.

(6) Amino acid composition (automatic amino acid analyzer) Asp; 20.2%, Thr; 8.9%, Ser; 3.1%, Glu: 10.6%, Gly; 2.2
%, Ala; 6.2%, Cys; 0.6%, Val; 4.5%, Ile; 1.4%, Leu; 1
0.1%, Tyr; 8.1%, Phe; 6.4%, Lys; 9.1%, His; 2.1%, Arg;
3.0%, Pro; 3.5% (7) Partial amino acid sequence Approximately 20 μg of the glycoprotein obtained in the above (d) was separated by reversed-phase high-performance liquid chromatography using a “Bakerbond Butyl” (0.46 × 25 cm) column manufactured by JTBaker. did. The elution condition was a linear gradient of 40% from 0.1% trifluoroacetic acid aqueous solution to 0.1% trifluoroacetic acid aqueous solution containing 80% acetonitrile / isopropyl alcohol (3/7: v / v) mixture at a flow rate of 1 ml / min. went. When the protein was detected by the absorbance at 215 nm, about 13 fractions were obtained as shown in FIG.

Among them, the fraction eluted in about 36 minutes was dried under vacuum, and then dried in Tris buffer (1M Tris-HCl buffer (pH 8.5),
After dissolving in 100 μm of 5M guanidine hydrochloride), adding 0.5 μl of 2-mercaptoethanol and reducing at 40 ° C. for 2 hours under nitrogen gas, adding 1 mg of monoiodoacetic acid and reacting under nitrogen gas at room temperature under common light for 1 hour. Thus, a reduced carboxymethylated product of the protein was obtained.

The reduced carboxymethylated protein was subjected to the above-mentioned reversed-phase high performance liquid chromatography under a linear concentration gradient of 30% to 90% for 30 minutes. When the same detection was performed as described above, about three fractions were obtained (FIG. 5).

Among them, the fraction eluted in about 21 minutes was dried under vacuum, dissolved in 60% 50% trifluoroacetic acid, added to a polybrene-treated glass filter, and applied to an Applied Biosyste.
The product was digested by Edman using an MS type 470A sequencer.

The identity of PTH-amino acid was determined by Mitsubishi Kasei's “MCI gel ODS I
Using a HU "(0.46 x 15 cm) column, a single solvent elution method using an acetate buffer (containing 10 mM acetate buffer (pH 4.7), 0.01% SDS and 38% acetonitrile) was performed at a flow rate of 1.2 ml / min and a temperature of 43.
C., and the detection of PTH-amino acid was performed at an absorbance of 269 nm.

As a result, it was revealed that the amino terminal sequence of this protein is as follows.

Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pro-Thr
-Ala-Asp-Cys-Lys-Thr-Ala-Val-XX-Ser-
Ser-Asp-Phe-Asp-Ala-X-Leu-X-Thr-Lys-Al
a-Gly-X-Gln-Val-Tyr-Asn-X represents Cys or another amino acid residue.

Example 2 (1) Preparation of polyclonal antibody against synthetic peptide (a) Preparation of antigen Approximately 30 mg of keyhole limpet hemocyan (hereinafter abbreviated as “KLH”) (manufactured by Calbiochem) against PBS at 4 ° C. overnight. Dialyzed. After dialysis, the protein was diluted with 50 mM sodium borate-hydrochloric acid buffer (pH 9.0) to adjust the protein concentration to 30 mg / ml.

Next, 7.5 mg of each of the following synthetic peptides was dissolved in 375 μ of 0.1 M sodium phosphate buffer (pH 8.0),
125 μl of the solution was added to each.

(A) Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pr
o (b) Thr-Ala-Asp-Cys-Lys-Thr-Ala-Val 25% glutaraldehyde (manufactured by Sigma) is added to this solution.
5 μl of the solution was added to each and left at room temperature for 15 minutes. again
5 μ of a 25% glutaraldehyde solution was added to each, and the mixture was allowed to stand at room temperature for 15 minutes.

Next, 100 μM of a 1 M glycine-hydrochloric acid (pH 6.0) buffer was added, and the mixture was allowed to stand at room temperature for 10 minutes. This solution was dialyzed against PBS at 4 ° C. for 24 hours. After dialysis, centrifuge at high speed.
It was sterilized, aliquoted and stored at 4 ° C.

(B) Acquisition of Antibody Two equal amounts of the KLH-peptide conjugate obtained in (a) above were mixed, and PBS was added to adjust the total protein amount to about 2 mg / ml. An equal amount of adjuvant (manufactured by Difco) was added and mixed to obtain an emulsion. 0.1 ml each at four subcutaneous sites on the back of the rabbit and 0.1 ml on each footpad
Immunized one by one. Four weeks later, a booster immunization was performed at four subcutaneous sites on the back. Two weeks later, 1 ml of an emulsion of the same KLH-peptide conjugate (1 mg / ml) was boosted subcutaneously at 6 sites on the back. Two to three weeks after injection, whole blood was collected to obtain antiserum. Solid ammonium sulfate was added to the obtained antiserum while stirring so as to be 40% saturated, and stirring was further continued at 4 ° C for 2 to 3 hours. After stirring, the mixture was centrifuged at 10,000 × g for 15 minutes to obtain a precipitate, which was dissolved in a small amount of PBS. This solution was applied to Protein A Cellulofine (manufactured by Seikagaku Corporation) which had been equilibrated with PBS in advance, washed thoroughly with PBS, and eluted with 0.1 M glycine-hydrochloric acid (pB2.8). The fractions from which the protein was eluted were collected, immediately neutralized by adding a 1 M Tris solution, and concentrated using an ultrafiltration device (discharge molecular weight: 30,000). The antibody concentrate was dialyzed against PBS overnight at 4 ° C. to obtain a purified polyclonal antibody.

(2) Purification of Glycoprotein An affinity sepharose gel was prepared in the same manner as in Example 1 (2) (d) except that the polyclonal antibody obtained in (2) above was used instead of the 1F5 antibody. Using this polyclonal antibody Sepharose column, the lysate of the glycoprotein obtained in (2) of Example 1 (a) to (c) was used, and purification was performed in the same manner.
A glycoprotein of the present invention having the same properties as in Example 1 was obtained.

〔The invention's effect〕

The glycoprotein of the present invention obtained as described above can be used, for example, for the following purposes.

(1) By immunizing a rabbit, a mouse, or the like with the glycoprotein of the present invention, a stable polyclonal antibody or monoclonal antibody against a membrane protein that plays an important role in regulating complement activity can be obtained.

(2) By quantifying the glycoprotein on cells such as erythrocytes and lymphocytes using the antibody against the glycoprotein of the present invention, various types of diseases such as pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis and the like can be obtained. Can be diagnosed.

(3) The glycoprotein of the present invention can be used as a therapeutic agent for various diseases involving activation of complement.

(4) The antibody can be used for treatment such as lysis of cancer cells and removal of malignant cells.

[Brief description of the drawings]

FIG. 1 shows 12.5% SDS-P of the glycoprotein of the present invention treated with endoglycosidase F (1 in the figure) and untreated (2 in the figure).
It is a drawing showing the electrophoresis pattern of AGE in a photograph. Figure 2 shows PI-PLC processing And no processing 1 is a drawing showing the released state of the glycoprotein of the present invention from the cell surface subjected to the above. In the figure, Indicates a negative control (non-specific adsorption of FITC-labeled anti-mouse IgG to MT2). FIG. 3 shows the glycoprotein of the present invention. And bovine serum albumin as a control 3 is a drawing showing the effect of human complement on the lysis of guinea pig erythrocytes by addition of guinea pig erythrocytes. 4 and 5 are diagrams showing elution patterns of reversed-phase high performance liquid chromatography, in which the horizontal axis shows elution time (minutes).

Claims (1)

(57) [Claims] 1. A glycoprotein derived from human cell membrane, comprising SDS
-Molecular weight of 20 to 20 by polyacrylamide gel electrophoresis
At 25 Kd, Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pr
o-Thr-Ala-Asp-Cys-Lys-Thr-Ala-Val-XX
-Ser-Ser-Asp-Phe-Asp-Ala-X-Leu-X-Thr-
Lys-Ala-Gly-X-Gln-Val-Tyr-Asn- (X in the sequence
Has an amino-terminal sequence represented by Cys or another amino acid residue), contains an N-glycoside type sugar chain and phosphatidylinositol, and has an action of suppressing cell membrane damage caused by complement. Glycoprotein to do.