JPH03287600A - Protein inhibiting phospholipase a2 originated from affected part of human inflammation, its production and gene - Google Patents

Abstract

NEW MATERIAL:Protein inhibiting phospholipase A2 originated from affected part of human inflammation and containing the amino acid sequence of formula or a physiologically equivalent amino acid sequence. USE:Remedy for inflammatory diseases. PREPARATION:The objective protein of formula can be produced e.g. by adding NaN3 to human serum, treating at 37 deg.C for 10 days, adding a protease inhibitor, dialyzing by using a tris-HCl buffer solution of pH7.5, removing insoluble material by centrifugal separation, fractionating by anion-exchange high-performance liquid chromatography, collecting the fraction exhibiting an ultraviolet absorption spectrum having an absorption band at 280nm, subjecting the fraction to SDS-polyacrylamide gel electrophoresis, collecting fractions exhibiting 39kDa band, subjecting to anion-exchange high-performance liquid chromatography and separating a fraction active to inhibit phospholipase A2 originated from affected part of human inflammation.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a protein that is expected to be useful in the treatment of inflammatory diseases. More specifically, the present invention relates to a human-derived protein that inhibits phospholipase A2 derived from human inflamed areas, a method for producing the same, and a gene thereof.

<Prior Art> Steroids have been used as powerful anti-inflammatory drugs to treat many inflammatory diseases. However, steroids have various side effects due to their hormonal effects, so their use must be limited to relatively seriously ill patients. Therefore, the development of new drugs that have anti-inflammatory effects equivalent to those of steroids and have fewer side effects is the most important requirement for doctors treating inflammatory diseases.

One of the mechanisms by which steroids exert their anti-inflammatory effects is that by inducing the synthesis of a protein that inhibits phospholipase A2 in the inflamed area, they suppress the release of arachidonic acid by this enzyme, resulting in an inflammatory effect. It is thought to reduce the production of arachidonic acid metabolites (Floweret at Nature 27
8.456.1979), this phospholipase A2 inhibitory protein is expected to have an activity that satisfies the above requirements, and many researchers have attempted to isolate and purify it and evaluate its anti-inflammatory effect. It has been.

However, so far no substance has been found that exhibits a strong anti-inflammatory effect comparable to steroids. There are two possible reasons for this.

(1) In previous studies, enzymes derived from the pancreas were used in the evaluation system for inhibitory activity, but phospholipase A2 derived from inflamed local areas
has a structure and enzymatic activity different from pancreatic-derived phospholipase A2.

(2) It did not act directly on phospholipase A2, but detected an apparent inhibitory activity due to interaction with the substrate side.

The present inventors focused on these problems and first purified phospholipase A2 from the peritoneal exudate of rats that had developed peritonitis due to casein administration. We then developed a protein that specifically inhibits this enzyme.

Discovered in the peritoneal lavage fluid of rats administered Samethazone, we sought to successfully purify it and determine its N-terminal amino acid sequence.
-246397 publication>. This inhibitory protein showed no inhibitory activity against pancreatic phospholipase A2. In other words, this inhibitory protein is completely unique in that it has an activity that overcomes the two problems mentioned above in previous research, and it has a much stronger anti-inflammatory effect than conventional phospholipase A2 inhibitory proteins. It is expected that the results will be shown.

The N-terminal amino acid sequence of this inhibitory protein showed extremely high homology with part of the amino acid sequence of the human C3 chain. Therefore, this inhibitory protein was considered to be a degradation product of C3. C3 is gradually degraded by proteases in serum to form C3a.

It is known that various fragments such as C3b are generated. The inflammatory local phospholipase A2 inhibitory protein that the present inventors purified from rat peritoneal lavage fluid was thought to be similar to C3ag or C3d, which are the final degradation products of human C3 (Suwaet et al., Pro.
, Natl, Acad, Sci, LISA
, in press.

1990+. The reason is that the human C3 site corresponding to the N-terminal amino acid sequence of this inhibitory protein is C3ag or C3
It is very similar to the N-terminal site of d, and has a molecular weight of 37 kDa or 33 kDa.

Therefore, the present inventors separately purified rat serum C3ag, and found that rat C3dg was an inflamed local-derived phospholipase A.
It was discovered that this compound has the activity of specifically inhibiting 2. Additionally, the entire base sequence and amino acid sequence of the gene encoding rat C3dg was determined by gene cloning (Japanese Patent Application No. 1-200246).

<Problems to be Solved by the Invention> However, the rat-derived phospholipase A2 inhibitory protein has problems in terms of antigenicity and the like for clinical evaluation as an anti-inflammatory drug.

The present invention determines the structure of a human-derived protein that inhibits human inflammatory topical phospholipase A2, and at the same time provides a method for producing this protein and a protein gene for the protein that can be used to produce this protein, and provides an anti-inflammatory drug. The purpose is to enable development as a.

Means for Solving the Problems> That is, the present invention is a human inflammatory topical phospholipase A2 inhibitory protein having the amino acid sequence shown in FIG. 1 or an amino acid sequence physiologically equivalent thereto.

The present invention also provides a method for producing a phospholipase A2 inhibitory protein derived from a human inflamed local area having the amino acid sequence shown in FIG. 1 or an amino acid sequence physiologically equivalent thereto, which comprises enzymatically treating human serum.

Furthermore, the present invention is a gene encoding a human inflammatory topical phospholipase A2 inhibitory protein having the amino acid sequence shown in FIG. 1 or an amino acid sequence physiologically equivalent thereto.

The human inflammatory topical phospholipase A2 inhibitory protein of the present invention is a polypeptide consisting of the 349 amino acid sequence shown in FIG. Polypeptides composed of partial substitutions, deletions, insertions, etc. are also included in the human inflammatory topical phospholipase A2 inhibitory protein of the present invention.

In the production method of the present invention, human serum is enzymatically treated to produce inflammatory phospholipase A2 having the above amino acid sequence.
Inhibitory proteins can be produced, but enzyme treatment in such production methods means, for example, directly heating human serum at 37°C for 6 to 10 days.
03 is purified from the same serum and subjected to normal enzyme treatment using Facto®.

Furthermore, the human inflammatory topical phospholipase A of the present invention
An example of the gene for the 2-inhibitory protein consists of 1047 DNA base sequences shown in Figure 2.
Any double-stranded DNA consisting of heavy chain DNA and complementary DNA is included in this category.

Furthermore, if it encodes the amino acid sequence of the above-mentioned inhibitory protein, DNA formed by partial substitution, deletion, or insertion of the above-mentioned base sequence, DNA containing the above-mentioned base sequence,
Naturally, a DNA having only a part of the above base sequence is also included in the gene for the human inflammatory topical phospholipase A2 inhibitory protein of the present invention.

Next, the entire amino acid sequence of the human inflammatory locally derived phospholipase A2 inhibitory protein of the present invention and the method of the present invention leading to the gene of this inhibitory protein will be explained.

As mentioned above, the present inventors found that rat C3dg has the activity of specifically inhibiting rat inflammatory localized phospholipase A2.

In addition, the present inventors have successfully purified phospholipase A2 from human inflammatory sites, that is, synovial fluid of patients with chronic rheumatoid arthritis.
a et al., J. Biochem, 104.
326-3281988), human inflammatory topical phospholipase A2 is rat inflammatory topical phospholipase A2
It was revealed that they are extremely similar in structure and enzyme activity. In other words, in terms of enzyme activity,
Both enzymes had high specificity for phosphatidylethanolamine. Pancreatic-derived phospholipase A
2 does not have such substrate specificity. Regarding the structure, as a result of comparing the N-terminal 34 amino acid sequences,
It has been revealed that the homology between phospholipase A2 derived from rat inflamed areas and phospholipase A2 derived from human inflamed areas is 67%, while the homology between phospholipase A2 derived from human inflamed areas and human pancreatic phospholipase A2 is only 45%. Ta.

On the other hand, it has already been reported that high CBdg values can be detected by immunochemical methods in the synovial fluid of patients with rheumatoid arthritis, and that the values show a correlation with the severity of the disease condition (Nydegger et al., C11
n, Invest, 59862-8681977
).

Based on the above findings, the present inventors found that C
They were the first to realize that 3dg has a high possibility of specifically inhibiting phospholipase A2 derived from inflamed local areas, and as a result of intensive study on this point, they arrived at the present invention.

The human inflammatory topical phospholipase A2 inhibitory protein of the present invention is obtained by anion-exchange HPLC after treating human serum at 37°C.
, isolated and purified by gel filtration HPLC (1 reverse phase HPLC), or after enzyme treatment of C3 purified from human serum, isolated and purified by gel filtration HPLC (1 reverse phase) IPLC, etc.

Furthermore, since the human inflammatory local phospholipase A2 inhibitory protein of the present invention is a degradation product of C3, the gene encoding this protein is the previously reported human 03 gene (de Bruijn et al., Proc.

Natl, Acad, Sci, t! SA, 82
．． 708-712.1985). Therefore, the N-terminal amino acid sequence of the human inflammatory topical phospholipase A2 inhibitory protein of the present invention purified as described above was determined, and using this as an index, the entire base sequence of the gene of this inhibitory protein was determined.

Furthermore, the entire amino acid sequence of this inhibitory protein was determined from the entire base sequence of this inhibitory protein gene determined as described above.

Various tests and measurement methods used in the present invention are as follows.

(1) Method for measuring human inflammatory locally derived phospholipase A2 inhibitory activity.

Phospholipase A2 isolated and purified from synovial fluid of patients with rheumatoid arthritis was used as the enzyme, and 14C- as the substrate.
14C-phosphatidylethanolamine extracted and purified from E, Co11 cultured with acetic acid <approximately 2,000
0dpm/mmo1.1mM) was used.

Enzyme reactions were carried out using 50 μm of 0.5 M Tris-CI.
(pH9,0), 25. tz, 40mM Ca in Q
Add the sample and water to the Cl2.25AIQ substrate, mix to make a total volume of 240 μg, and finally add 10 μg of enzyme solution (0
, 1 ng/μ2) and reacted for 10 minutes at 37°C, the reaction was stopped by adding Dole's reagent (1.5 ml), and the 14C-fatty acid produced was extracted according to Dole's method, followed by liquid scintillation.・Measured with a counter.

<2) SDS-polyacrylamide electrophoresis and Western blotting Samples obtained by enzyme treatment or HPLC were
10 volumes of dye solution <0.1% BPB, XC.

10% 5DS) was added, and after heat treatment at 100°C for 5 minutes,
．． Apply 1% SDS to 5% polyacrylamide gel.
Electrophoresis was performed for 100 hours at 15-25 mA in the presence of 2-Mercaptoethanol (2MB) was added to the samples for analysis under reduced conditions.

After electrophoresis, the cells were stained with CBB for protein detection.

After electrophoresis in the same manner, the protein was transferred from the gel to a nitrocellulose filter using a Millibore electroblotting device. Then, using anti-human C3d sheep serum (Dako), horseradish peroxidase-conjugated anti-sheep IgG antibody (Cappell) and 4-chloro-1 naphthol (Bio-Rad) as a substrate,
C3d bands were detected by enzyme antibody staining.

(3) Deterministic gas phase protein sequencer for protein N-terminal amino acid sequence (Applied Biosystems 477
A) and HPLC (Applied Biosystems 12
0A) was used.

That is, a 100 μm sample dissolved in 0.1% TFA was applied to the above gas phase protein sequencer.

The automatically Edman-decomposed PTH (phenylthiohydantoin) amino acid derivative was then analyzed as each amino acid by high performance liquid chromatography.

The human inflammatory topical phospholipase A2 inhibitory protein of the present invention, whose entire amino acid sequence has been clarified by the present invention, can be produced by known chemical synthesis methods, but it can also be produced easily and in large quantities by genetic recombination methods. can be manufactured.

<Example> The present invention will be explained in more detail with reference to Examples below.

Example 1 Phospholipase A2 inhibitory protein derived from human inflammatory local area (hereinafter referred to as
(abbreviated as “inhibitory protein”) and activity measurement (A>Preparation of inhibitory protein (1) Add 0.1% NaN3 to 30ml of human serum,
It was treated at 7°C for 10 days.

The treated and untreated samples were each diluted 50 times and transferred to a filter by 5DS-PAGE and Western blotting according to a conventional method. As a result of enzyme antibody staining using anti-human C3d serum on this filter, only a band of 100 kDa or more was detected in untreated serum, and only a 39 kDa band was detected in serum treated at 37°C (Fig. 3). The results show that treatment at 37°C causes C3 to be cleaved by enzymes in serum, resulting in C3dg.
was confirmed to be generated.

Therefore, this serum contains protease inhibitor <2 (b
cg/ml aprotinin, 1 (ltct/ml soybean trypsin inhibitor, 0.5mM EDTA) followed by 3fJ of 2QmM Tris-HCp)17.
Dialysis was performed twice against 5.

The sample after dialysis obtained at +21 <1+ (protein concentration 30 μ/ml) was diluted 20 times, and after removing insoluble matter by centrifugation, the target protein was isolated and purified as follows. .

First, the sample was anion-exchanged to a size for collection.
C (TSKge IDEAE-5PW) (
Figure 4. In FIG. 4a, the solid line shows the UV absorption at 280 nm, and the broken line shows the NaCI concentration gradient. Elution is 2
0mM Tris-HCl, pH 7,5-NaCl
(0→Oj5M), 2°5 ml/nin, 2m
It was performed at 1n/1ube. Column size is 21.5m
The number is mφ×30 pieces.

Then, 5DS- in the fraction shown in FIG.
Fraction Nr, 52-54, in which a 39 kDa band was detected by PAGE, was collected and diluted 4 times, and then subjected to anion-exchange HPLC (TSKge
IDEAE-5PW) (Fig. 5). In FIG. 5a, the solid line shows the absorption at UV 280 nm, the broken line shows the NaCl concentration gradient, and the bar graph shows the inhibitory activity of human inflammatory local phospholipase A2.

Purification was performed using 20mM Tris-HCl, pH 7.5.
-NaC1 (0→1.OM) + 1.0 ml/mi
n, 2m1n/1ube.

Next, a 39 kDa band was detected by 5DS-PAGE in Figure 5, and the peak portion of fraction Nr.
I would like to further fractionate with G300O3Wl (Figure 6). Figure 6a
In the figure, the solid line shows the absorption at UV 280 nm, and the bar graph shows the inhibitory activity of human inflammatory local phospholipase A2.

Elution conditions were 20mM Tris-HCl ptl 7
．． 5-IMNaCI 0.5 ml/min, 2
It was performed with m1n/1ube.

Finally, a 39kDa band was detected by 5DS-PAGE in Figure 6, indicating that phospholipase A2 derived from human inflammation local.
Fraction Nr with the strongest inhibitory activity, 20.2
1 was further subjected to reverse phase HPLC (Bio-Rad RP304)
(Figure 7). In Figure 7a, the solid line is U V
Absorption at 210 nm, dashed line indicates CHaCN concentration gradient. Purification was performed using 0.1% TFA/CH3CN0-80
%, 1ml/min, 2m1n/1ube.

As shown in 5DS-PAGE in Figure 7, the molecular weight was 3.
Although the 9 kDa inhibitory protein was eluted in fractions Nr and 30, it was found that a protein that appeared to be albumin, which was mainly eluted in fractions Nr and 23, was slightly mixed therein. However, albumin is a strong phospholipase A2
Having no inhibitory activity, and fraction Nr, 2
Since No. 3 also showed no phospholipase A2 inhibitory activity, it was considered that No. 3 had no effect on the measurement of the inhibitory protein activity.

(B) Phospholipase A2 inhibitory activity of the inhibitory protein (A) Inhibition of the inhibitory protein of the present invention (reverse phase HPLC fraction NrjO1 protein concentration approximately 15 ng, /ufJ) against human inflammatory locally derived phospholipase A2 and other phospholipases A2 Activity was determined using phospholipase A2 as previously described.
It was measured by the method of measuring inhibitory activity.

The results are shown in FIG. 8 and Table 1 below.

Table 1 Comparison of activities of inhibitory proteins of the present invention against various phospholipase A2 Types of phospholipase A2 Phospholipase A2 inhibitory activity (%).

Rheumatoid arthritis patient joint fluid 47.4 (derived from human inflamed area) Rat platelets 22.0 (derived from rat inflamed area) Porcine pancreas 6.5 Inhibitory activity when 15 ng of inhibitory protein was added to 21 ng of phospholipase A From FIG. 8, it is clear that this inhibitory protein inhibits human inflammatory local phospholipase A2 in a dose-dependent manner. Furthermore, the amount of inhibitory protein required to inhibit the human inflammatory local phospholipase A2 activity of lng was about 50 ng. Furthermore, as is clear from Table 1, this inhibitory protein inhibited rat inflammatory locally derived phospholipase A2, but did not exhibit significant inhibitory activity against pig pancreatic phospholipase A2.

From the above results, it is clear that this inhibitory protein has the activity of specifically inhibiting phospholipase A2 derived from inflamed local areas.

Example 2 Determination of the entire nucleotide sequence of the human inflammatory locally derived phospholipase A2 inhibitory protein gene and the entire amino acid sequence of this inhibitory protein + A) Identification of the inhibitory protein (1) Determination of the N-terminal amino acid sequence of the inhibitory protein Example 1 (
The inhibitory protein (with a molecular weight of 39 kDa) isolated and purified in A)
The N-terminal amino acid sequence of the reversed phase H, PLC fraction Nr, 30) was determined by the method described above.

As a result, it was found that the sequence was as shown below.

N) 12-Gln-Gly-Val-Asn-Lys-
Glt+-Agp-11e-Pro-Pro- This completely matched the already known N-terminal amino acid sequence of human C3dg.

(2) Immunochemical analysis of inhibitory protein The inhibitory protein with a molecular weight of 39 kDa isolated and purified in Example 1 (A) was subjected to 5DS-PAGE using the method described above, followed by Western analysis. Plotting was performed, transferred to a filter, and detected by enzyme antibody staining using anti-human C3d serum. As a result, this inhibitory protein specifically reacted with anti-human C3d serum. Figure 9a shows the results of 5DS-PAGE. 9th
The figure shows the results of Western plotting.

From the above results (1) and <2), it is clear that this inhibitory protein is human C3dg.

(B) Phospholipase A2 inhibitory protein derived from human inflammatory local area (
The complete nucleotide sequence of the CBdg) gene and the entire amino acid sequence of this inhibitory protein cDNA cloning has already been carried out for human C3, and its entire nucleotide sequence and the entire amino acid sequence deduced from it have been determined.

Additionally, the part of the 03 gene that encodes C3dg has been determined (de Brutjn et al.
Proc.

Natl, Aead, Sci, USA, 82.7
08-712.1985).

From the above results, it is clear that the human inflammatory local phospholipase A2 inhibitory protein of the present invention is human C3dg. Therefore, the base sequence of this inhibitory protein gene is human C.
It is thought that it completely matches the portion encoding C3dg among the entire base sequences of the three genes. Furthermore, the entire amino acid sequence of this inhibitory protein is considered to completely match the portion corresponding to C3dg of the entire amino acid sequence of human C3 deduced from the entire nucleotide sequence of the human C3 gene.

Based on the above, it was determined that the entire amino acid sequence of the phospholipase A2 inhibitory protein derived from human inflamed areas of the present invention and the entire base sequence of the phospholipase A2 inhibitory protein gene derived from human inflamed areas of the present invention are those in FIGS. 1 and 2. The human inflammatory topical phospholipase A2 inhibitory protein was encoded by 1047 bases and consisted of 349 amino acids.

[Brief explanation of the drawing]

Figure 1 shows the human inflammatory topical phospholipase A of the present invention.
2 shows the amino acid sequence of the 2 inhibitory protein. Figure 2 shows the human inflammatory local phospholipase A of the present invention.
An example of the DNA base sequence of the 2 inhibitory protein gene is shown. Figure 3 shows the results of Western blotting of untreated and 37°C treated human serum. Figure 4 shows the elution pattern of phospholipase A2 inhibitor protein derived from human inflamed local areas using preparative size anion exchange (PLC) (Figure 4a) and 5DS-PAGE (
Same b) is shown. Figure 5 shows the elution pattern of inflammatory phospholipase A2 inhibitory protein derived from human inflammatory local area using analytical size anion exchange HPLC (Figure 5a) and 5DS-PAGE.
(Figure 6 shows the elution pattern of inflammatory phospholipase A2 inhibitory protein derived from human inflamed local area using gel filtration HPLC (Figure 6 a) and SDS-PAGE (Figure 6 b). Figure 7 shows the elution pattern of rat proinflammatory phospholipase A2 inhibitory protein using reversed phase HPLC (Figure 7a).
and 5DS-PAGE (same b). Figure 8 shows the inhibitory activity of the inhibitory protein of the present invention. Figure 9 shows inflammatory phospholipase A2 derived from human inflamed local area.
The results of 5DS-PAGE <Figure 9 a) and Western blotting (Figure 9 b) of inhibitory proteins are shown.

Claims (1)

[Claims] 1. A human inflammatory local phospholipase A_2 inhibitory protein having the amino acid sequence shown in FIG. 1 or a physiologically equivalent amino acid sequence. 2. The human inflammatory topical phospholipase A_2 inhibitory protein according to claim 1, which has a part of the amino acid sequence shown in FIG. 1 or a physiologically equivalent amino acid sequence. 3. Claim 1, characterized in that human serum is treated with an enzyme.
A method for producing the human inflammatory local phospholipase A_2 inhibitory protein described above. 4. Phospholipase A derived from human inflammation topic according to claim 1
_2 inhibitory protein gene. 5. The gene for the human inflammatory topical phospholipase A_2 inhibitory protein according to claim 4, which comprises the DNA base sequence shown in FIG.