WO1992015615A1

WO1992015615A1 - Serum calcium depressing factor

Info

Publication number: WO1992015615A1
Application number: PCT/JP1992/000251
Authority: WO
Inventors: Akito Tomomura; Takeyori Saheki; Yoshito Takaoka
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 1991-03-06
Filing date: 1992-03-03
Publication date: 1992-09-17
Also published as: AU1338292A; JPH04279598A; OA09712A

Abstract

A novel serum calcium depressing factor derived from swine pancreas, which has a molecular weight of 26,500 to 28,000 D as determined by polyacrylamide gel electrophoresis, an isoelectric point of about 4.5 as determined by isoelectric electrophoresis, and an amino acid sequence of Val-Val-Gly-Gly-Gln-Asn-Ala-Ile-Pro-His-Ser-Trp-Pro-Trp-Gln-Ile-Arg-Leu- at or near the N-terminus. It has a protease activity and is inhibited by PMSF, chymostatin and trypsin inhibitor but not by APMSF and E-64. The novel depressing factor is expected to be applicable as a medicine for treating and preventing bone diseases such as osteoporosis.

Description

Description Serum lucidum-lowering factor

Minutes g

The present invention relates to a serum calcium-lowering factor derived from pig, a method for producing the factor, and a medicament containing the factor. Background technology

Takaoka et al. [The existence of serum calcium lowering factor

9 5 1, 1 5-2 0 (1 9 8 0)] has been suggested by - not yet purified, nor is elucidated characteristics as the material ₀

Accordingly, an object of the present invention is to provide a novel isolated serum calcium-lowering factor derived from pig.

Disclosure of the invention

The present inventors have succeeded in isolating and purifying the desired serum calcium-lowering factor from swine spleen, elucidating its characteristics as a substance, and further clarifying some biological characteristics. Thus, the present invention has been completed. That is, the present invention has the following properties:

(1) dose-dependently lower serum calcium levels in mice;

(2) SDS—Polyacrylamide gel electrophoresis has a molecular weight of about 26,500-28,000 D; (3) the isoelectric point measured by isoelectric focusing is about 4.5;

() N—The following amino acid sequence at or near the terminal (SEQ ID NO: 1): V al — V al — G ly — G ly— G in— A sn — A la — I 1 e — P ro — H having is-Ser-Trp-Pro-Trp-Gln-I1e-Arg-Leu-;

(5) Protease activity is measured by the protease inhibitor phenylmethansulfonyl fluoride (PMSF) (1 mM), chymostatin (100 μM) and trypsin inhibitor (100 g / ml). Harmful, but (4-amidinofenyl) methanesulfonyl fluoride (APMSF) (50 μΜ) and Ν—CΝ- (L-13—trans-carboxyxylane-2-carbonyl) ) 1 L 1 bit isl] Agmatine (Ε-64) (2.5 g ml) is not inhibited;

And a novel serum calcium-lowering factor derived from a pig having the following. .

The present invention further provides a method for producing the serum calcium-lowering factor, which comprises isolating the serum calcium-lowering factor from porcine excreta. .

The present invention further provides a medicament comprising the serum calcium lowering factor. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the elution profile of Q-Sepharose Fast Fast Flow Chromatography. Only Beak III Has serum calcium lowering effect.

In FIG. 2, A shows the state of elution in gel filtration chromatography using Superdex 75 HR. A shows inhibition of calcium release (by PTH) in Lewis bone culture system by several aspects corresponding to B. Only the main peak had an inhibitory effect on calcium release.

Figure 3 shows the elution of ion-exchanged chromatographic liquid with a Mono Q column. Only the main beak has a serum calcium lowering effect.

Figure 4 shows the appearance of elution in reverse-phase liquid chromatography using Wakoshi15C-18 columns.

FIG. 5 is a graph showing that serum calcium-lowering factor (open circles) and serum calcium-lowering factor (black circles) treated with protease inhibitor PMSF lower serum calcium in a dose-dependent manner. BEST MODE FOR CARRYING OUT THE INVENTION

Production method

The serum calcium-lowering factor of the present invention can be isolated and purified from pig kidney as follows. First, a pig's sarcoma is excised, crushed according to a conventional method, and dehydrated with acetate to prepare aceton powder. Next, an extract is obtained by extracting the acetate buffer with a suitable buffer, for example, 0.1% squirrel hydrochloric acid (PH = 8) containing 2% NaCl. Next, acetate was added to this extract at a concentration of 30% to form a precipitate. After removing the solid, add acetone to the supernatant to a concentration of 60% to obtain the formed solid.

The 30-60% acetone fraction thus obtained was filtered through a suitable buffer solution, for example, deionized water, to remove the acetate, and then added with ammonium sulfate. 4 Remove the deposits generated after 5% saturation. Next, ammonium sulfate is added to the supernatant to make it 60% saturated, and the resulting precipitate is obtained. This solution is dissolved in a suitable buffer, for example, 5 mM acetate buffer (pH 5.5), and the solution is dialyzed to remove ammonium sulfate to obtain a crude serum calcium-lowering factor extract.

The serum calcium-lowering factor of the present invention can be obtained from this extract by the method described in detail in Example 1. Briefly, the crude extract was first applied to a Q-Sepharose Fast Flow column, and a 5 OmM acetate buffer (pH 5.5) and 0.1 M Na After passing the same acetate buffer containing C 1 through the column, 0.2 M

Elution with the same acetate buffer containing Na C 1 yields the 3 橱 peak shown in Figure 1. Of these beaks, only Beak III shows a serum calcium-lowering effect in mice in in vivo measurements (Example 1). The results are shown in Table 1 of Example 1.

Next, if the above peak 111 was subjected to gel filtration using Superdex 75 HR power ram, one sharp main peak and three minor peaks were observed as shown in Fig. 2B. Only this main beak is used for in vivo measurements. Calcium-lowering effect in rats and parathyroid hormone in the Louis bone culture system [Raisz L.G., J. Clin. Invest., 4401-03-116 (19665)) (PTH) has an inhibitory effect on mono-induced calcium release. The results are shown in FIG. Next, the main beak obtained by the gel filtration chromatography was subjected to ion exchange chromatography using a Mono QHR column, and N in 50% sodium acetate buffer (PH 5.5) was added. a Elute with C1 concentration linear drug. As a result, as shown in FIG. 3, the main beak elutes at about 0.2 MNaCl, and this beak has a serum calcium-lowering effect in in vivo measurement.

Next, the main beak obtained by the gel filtration chromatography was subjected to reverse-phase liquid chromatography using Wakosi 15 C-18 column to obtain a solution in 0.1% trifluoroacetic acid (TFA). The elution profile shown in Fig. 4 was obtained by elution with a linear gradient of tonitrile concentration, indicating that the serum calcium-lowering factor of the present invention was purified as a single protein by purification with a M0 no QHR column. It is confirmed.

Although the serum calcium-lowering factor of the present invention was isolated and purified as described above, it was once isolated and purified, and after its properties were elucidated as described below, the properties of the protein It is clear that the serum calcium-lowering factor of the present invention can be isolated and purified using any conventional method used for isolation and purification. You.

Further, the serum calcium lowering factor of the present invention can be produced by genetic engineering means. For example, mRNA can be isolated from pig kidney according to a conventional method, and a cDNA library can be prepared based on the mRNA according to a conventional method. A DNA probe for screening this cDNA library can be designed, for example, based on the amino acid sequence at or near the N-terminus of serum calcium-lowering factor identified according to the present invention. . Alternatively, after the serum calcium-lowering factor of the present invention is enzymatically or chemically cleaved and the amino acid sequence of the fragment is determined, a DNA probe can be designed based on the amino acid sequence. .

Next, the cDNA encoding the serum calcium-lowering factor thus obtained is inserted into an appropriate expression vector, and then a host is transformed with the expression vector and the transformant is cultured. Thus, the serum calcium-lowering factor of the present invention can be produced. Conventional hosts such as prokaryotic cells such as Escherichia coli, lower eukaryotic cells such as yeast, and higher eukaryotic cells such as cultured mammalian cells can be used.

Properties of serum calcium lowering factor

(1) Molecular weight

When the molecular weight of the serum calcium-lowering factor of the present invention is measured by SDS-acrylamide gel electrophoresis, it shows a single molecular weight of about 26,500 to 28,000 D. (2) m ^

When measured by isoelectric focusing, the serum calcium-lowering factor of the present invention exhibits a single isoelectric point of about 4.5.

(3) Partial amino acid sequence

When the purified serum calcium-lowering factor of the present invention is subjected to Edman degradation according to a conventional method, the following amino acid sequence (SEQ ID NO: 1) appears at or near the N-terminus:

V a 1-V a 1-G 1-G 1 y-G 1 n— A sn— A la — I 1 e — P ro — H is — Ser — T rp — P rp — T rp — G in— It has I le —A rg—L eu—. This amino acid sequence is composed of porcine plasminogen, human apoprotein 8, porcine elastase 2, ゥ一トモププ A キト A, ゥモモモブブ B B B, and プラズマThe serum calcium-lowering factor of the present invention is a novel protein that is homologous to, but not identical to, the amino acid sequence of human coagulation factor (Pre) and human coagulation factor XI (Pre). is there.

(4) Action by protease inhibitors

The serum calcium-lowering factor of the present invention has protease activity, and the behavior of the serum calcium-lowering factor of the present invention with respect to a protease inhibitor is described in detail in Example 3 and Table 2.

(Effect)

Since the serum calcium-lowering factor of the present invention has a serum calcium-lowering effect in vivo, it has various bone diseases, for example, osteoporosis, primary hyperparathyroidism, hypercalcemia associated with malignant tumors. Active ingredients of medicines for treatment and prevention of etc. It is expected to be useful as. The serum calcium-lowering factor of the present invention can be formulated parenterally with a conventional pharmaceutical carrier and administered parenterally, for example, by intravenous administration, subcutaneous administration, intramuscular administration, or enteral or oral administration. it can.

Example 1. Purification of serum calcium-lowering porcine derived pig swine Acetone powder of swine spleen was prepared and extracted with 0.1 M Tris-HCl buffer containing 2% NaC1. Then, an acetone sedimentation fraction was obtained at an acetate concentration of 30 to 60%. This fraction was filtered through deionized water, and then subjected to ammonium sulfate salt to obtain a precipitate fraction at 45 to 60% saturation with ammonium sulfate.

This ammonium sulfate precipitated fraction was dissolved in 50 i »M acetate buffer (ρΗ5.5), and then filtered through the same buffer to remove the ammonium sulfate. I got This extract was subjected to ion-exchange chromatography using a Q-Sepharose Fast Flow (Q-SepharoseFastFlow) column. Most of the proteins were passed through 50 acetate buffer (pH 5.5). next,

After stepwise elution with the same buffer containing 0.1 M NaC1, it was stepwise eluted with the same buffer containing 0.2 M NaC1. As a result, three beaks shown in FIG. 1 were obtained.

Next, these beaks were examined for serum calcium lowering effect in mice by the following method (in vivo measurement).

4 to 6-week-old male BAL BZC mice were fasted for 18 hours, and 0.1 M Tris-HCl buffer (pH = 7.5) alone or a sample prepared by dissolving each of the above beaks in the same buffer was used. Weight 20 g The dose should be administered via the tail vein of the mouse at a rate of 100 0 //〗. Four hours later, blood is collected from the heart under ether anesthesia to obtain serum. Serum calcium concentration was measured according to the orthocresol phthalein complexone method.

The results are shown in Table 1 below.

table 1

Serum calcium-lowering activity of Q-Sepharose FF column elution beak Serum Ca concentration Serum Ca concentration Significance of decrease in test sample (mg / dl) 8.9 1 ± 0.15

Beak I

1.98 mgZkg body weight8.90 ± 0.20

Peak 11

2 7 3 mgZkg body weight 8.8 9 ± 0.1 2

Peak 111

5.3 0 Big / kg body weight 8 2 3 P <0.01 ⁽ * ⁾ 0.5 3 mgZkg body weight 8

2 0 P <0. 0 5

(*) By t-test.

As is evident from the results in Table 1, the serum potency lowering activity was present in beak III, and a significant serum calcium lowering activity was observed even when this beak was diluted to a concentration of 1Z10.

Next, the above-mentioned beak I II was applied to gel filtration chromatography. That is, Beak III was applied to a column of Superdex 75 HR 10/30 in 0.2 M ammonium acetate buffer (PH 6.8), and eluted with 0.5 mlZ. , And fractionated by 1 ml. The results are shown in FIG. This As a result, one sharp main peak and three minor peaks were observed. This main beak corresponds to a molecular weight of about 22,000 D, and this beak was added to serum by in vivo measurement. Calcium lowering effect was observed. Next, the activity of each of the fractions that inhibit the release of calcium by parathyroid hormone (PTH) was determined using a Lewis bone culture system for several fractions including the fractions corresponding to the four beaks. Examined. This test was performed as follows.

To the ICR system pregnancy 1 day 5 mouse, 1 dispensed subcutaneous the ⁴⁵ C a of 0~ 2 0 ^ C i, the next day, cut out before humerus of the fetus, one day in or- ME M medium, performing a pre-culture. Replace the medium with the same medium containing parathyroid hormone (PTH) and culture for 3 days. Bone C a is determined by acid decalcification. The power release effect of parathyroid hormone (PTH) is expressed as the ratio of ^<5 Ca in medium to the sum of ⁴⁵ Ca in bone and medium. The results are shown in FIG. In FIG. 2, the position of the bar in A corresponds to the fraction number in B. As shown in ^{FIG, PTH (1 0 - 7 M} ) is to promote about 80% the release of calcium, fractions corresponding to the main Lee Nbiku completely inhibited the calcium release promoting. On the other hand, the fractions corresponding to the three minor beaks and the areas not corresponding to any of the peaks did not show any inhibitory action.

This main beak was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of sodium dodecyl sulfate, and the molecular weight standards (albumin 67 KD, ovalbumin 43 KD, force-carbonic anhydrase 30%) were determined. KD, trypsin inhibitor The main band showed a molecular weight of about 26,500 D when compared with 20 KD and 14 KD of n-lactalbumin. Next, the main beak obtained by the gel filtration chromatography was subjected to ion exchange chromatography using a M0n0QHR5Z5 column. Buffer A [50 mM sodium acetate (PH5.5)] and Buffer B [50 mM sodium acetate (pH 5.5) containing 0.5 MNaCl] The mixture was eluted at a flow rate of 0.5 mlZ with a linear gradient of NaCl concentration using, and fractionated 1 ml each. Figure 3 shows the results. A main beak was obtained at a NaC1 concentration of about 0.2 M, and this beak had a serum calcium lowering effect in in vivo measurement. Next, the purity of the active main beak obtained by ion exchange chromatography using the M0n0Q column was examined by reversed-phase liquid chromatography. W akosi 1 5 C

— Using a 1802 column, solvent A (0.1% trifluoroacetic acid (TFA) in 20% acetonitrile) and solvent B (80% acetonitrile) Elution was carried out at a flow rate of 1 mlZ by linear gradient of acetonitrile concentration using 0.1% TFA)-. Elution was performed at 100% A _: 5 minutes; 0 to 50% B _: 25 minutes; 50 to 100% B _: 5 minutes; 100% B: 5 minutes. Fig. 4 shows the results. A single peak was obtained at an acetonitrile concentration of about 50%, confirming that the serum calcium-lowering factor of the present invention was purified to a single protein.

Next, the active beak obtained by the Mono Q ion exchange chromatography and Wak 0 si 15 C-18 reverse When the molecular weight of the peak obtained by phase liquid chromatography was determined by non-reducing SDS-PAGE according to the method described above, the molecular weight of serum calcium-lowering factor was calculated to be about 28,000 D. The isoelectric point (PI) of the fraction from the M0n0Q ion-exchange chromatography was examined by isoelectric focusing, and the isoelectric point standard (human carbonic anhydrase B: p1 = 6.55, ゥ carbonic anhydrase B: p1 = 5.85, グロ A-globulin A: PI = 5.2, soybean trypsin inhibitor, pl = 4.55) The serum calcium-lowering factor of the present invention showed a single beak at about PI = 4.5.

Example 2. Determination of partial amino acid sequence

For the main active fraction obtained by gel filtration chromatography using the Mono Q column and the area obtained by reverse phase liquid chromatography using Wak0si15C-18, protein The amino acid sequence at the N-terminus was examined by a conventional method using the Edman degradation method. As a result, the same results were obtained for the sample of 2) above, and it is estimated that the serum calcium lowering factor of the present invention has the following amino acid sequence (SEQ ID NO: 1) near its N-terminal or N-terminal. Was done.

V a 1-V a 1-G 1 y— G l -G ln -A sn -A la-I 1 e— P ro— H is— S er— T rp— P rp— T rp— G 1 n— I 1 e— A rg— L eu—

Next, a homology search was performed based on the amino acid sequence at positions 10 to 18 to find that pig-plasminogen and human Pop mouth tin A, porcine elastase 2, ゥキトトブブノー、ゥ, ゥキゥモヒヒヒヒヒヒヒヒ) And a homologous amino acid sequence were found to be present, but they were not the same, confirming that the serum calcium-lowering factor of the present invention was a novel protein.

Spiritual example 3.

In the above-mentioned purification step, the formation of a fragment presumed to be a degraded fragment of the serum calcium-lowering factor protein was observed, and it was estimated that the serum calcium-lowering factor itself had protease activity. The effect of various protease inhibitors on the protease activity of calcium-lowering factor was examined as follows. Specifically, lmg / ml azocasein was used as a substrate, incubation was performed for 37 minutes and 30 minutes in a 0.25 ml system, and 1 ml of 5% trifluoroacetic acid was added. Was measured at 0 D ₃₄₀ nm, and this was defined as the protease activity. The effect of each protease inhibitor is shown as% when the protease activity of a control (no inhibitor) is defined as 100.

Phenylmethansulfonyl fluoride (PMSF), which is a serine protease inhibitor; and (41-amidinophenyl) methansulfonyl fluoride (APMSF), which is a triscine-type serine protease inhibitor; Leupeptin, a tribosine-type serine proteinase thiol protease inhibitor; chymostatin, a chymotribine-type serine proteinase inhibitor (chymostatin), a thiol protease inhibitor N-C-(L 13-trans-lipoxyxanlan 2-carbonyl) 1 L-lysyl] agmatine (E-64), an acidic protease inhibitor Pepstatin; trypsin inhibitor, an inhibitor of trypsin, factor Xa, plasmin and plasmacaline

(trypsininhiibitor); and aprotung (apr0tinin), which is an inhibitor of plasmin and force grains.

As a result, as shown in Table 2, it was inhibited by PMSF (lmM), chymotrypsin (100 µM), and trypsin inhibitor (100 fig / ml), and APMSF (5 0 M) and E-64 (2.5 μg / ml), and are not inhibited by oral piptin (100 M), pipstatin (100 μM) and ab mouth (S iiigZml).

Table 2

Inhibition of serum calcium-lowering factor protease activity by various protease inhibitors Protease activity inhibitor Inhibitor concentration (% of control) Control (no addition) 0 1 0 0

PSF 1 mM 8 8 APMSF 5 0 ^ M 1 0 7 0 Loupin 1 0 0 M 8 6 8 Chymostatin 1 0 0 M 1 6 1 E-6 4 2.5 g / ml 1 0 7 9 ぺBusatin 100 / M67 9 Trypsin inhibitor 100 g / ml 44 aprotin 2 Big / ml 7 8 7 Example 4. Study of the mechanism of action of serum calcium-lowering factor Serum calcium-lowering Since the factor has protease activity, it may be that it degrades parathyroid hormone (PTH), which regulates serum calcium. Therefore, after the serum calcium-lowering factor and PTH were incubated together, the reaction product was examined by reversed-phase liquid chromatography, and PTH was degraded by serum calcium-lowering factor to reduce the molecular weight. The coexistence of the protease inhibitor PMSF in this reaction inhibited PTH degradation.

Therefore, in vivo vaccines containing high concentrations of serum proteins It was examined whether serum calcium-lowering factor degrades PTH in a chair bone culture system (described above).

We investigated whether serum calcium-lowering factor inhibits the release of calcium by PTH or vitamin D in a Louis bone culture system. As a result, serum calcium-lowering factor inhibited the effect of PTH at a concentration as low as 1 ng / ml, but did not inhibit the effect of vitamin D at a concentration of 10 ng / ml.

Therefore, to investigate whether the inhibition of PTH effect was due to the protease activity of serum calcium-lowering factor, serum calcium-lowering pretreated with PMSF, a protease inhibitor that irreversibly binds to proteases, was investigated. Whether or not the factor inhibited the calcium releasing effect of PTH and vitamin D was examined. As a result, serum calcium-lowering factor pretreated with the protease inhibitor PMSF inhibited the action of PTH, but did not inhibit the action of vitamin D, like the untreated lowering factor. Next, the decrease in serum calcium concentration due to serum calcium-lowering factor in the in vivo measurement in mice was examined using PMSF-treated serum calcium-lowering factor and untreated factor. Fig. 5 shows the results. In the figure, open circles show the results for untreated serum calcium-lowering factor, black circles show the results for PMSF-treated serum calcium-lowering factor, and black squares show serum calcium-lowering factor only as shown on the horizontal axis. Results when only PMSF is administered in the concomitant dose when is administered

(Control) is shown.

PMSF does not lower serum calcium, but untreated serum Calcium-lowering factors reduced serum calcium in a dose-dependent manner. PMSF-treated serum calcium-lowering factor also reduced serum calcium in a dose-dependent manner, with the curve shifting to a much lower concentration. This result suggests that serum calcium-lowering factor, whose protease activity was inhibited, may have a serum calcium-lowering effect. Industrial applicability

Since the serum calcium-lowering factor of the present invention has a serum calcium-lowering effect in vivo, it is used for treatment of various bone diseases, for example, osteoporosis, primary hyperparathyroidism, hypercalcemia associated with malignant tumors, and the like. And it is expected to be useful as an active ingredient of a medicine for prevention.

Sequence listing

SEQ ID NO: 1

Array length: 1 8

Sequence type: amino acid

Topology: Direct letter

Sequence type: peptide

Array

Val Val Gly Gly Gin Asn Ala lie Pro His Ser Trp Pro Trp Gin lie

1 5 10 15

Arg Leu

Claims

The scope of the claims

1. Serum calcium-lowering factor from pigs with the following properties: (1) Dose-dependently lower serum calcium levels in mice;

(2) the molecular weight measured by SDS-polyacrylamide gel electrophoresis is about 26,500 to 28,000 D;

(3) the isoelectric point measured by isoelectric focusing is about 4.5;

(4) N-terminal or near the following amino acid sequence (SEQ ID NO: 1): V al — V al — G ly — G ly— G in— A sn-A 1 a — I 1 e — P ro — H is-Ser-T rp-Pro — T rp — G in — I le — A rg — L eu —

(5) Phenylmethanesulfonyl fluoride (PMSF) (1 wM), chymostatin (100 M), and trypsin inhibitor (100), which have protease activity and are protease inhibitors. g / ml), but (4-amidinophenyl) methansulfonyl fluoride (APMSF) (50M) and N— (N- (L-13—trans-carboxy) It is not inhibited by l- (2-carbonyl) -l-l-isyl] agmatine (E-64) (2.5 # g / ml).

2. A method for producing a serum calcium-lowering factor, comprising isolating the protein of claim 1 from pig pig.

3. A medicament comprising the serum calcium-lowering factor according to claim 1.