JP2001103992A - Papain treated collagen modified substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain both a useful collagen modified substance provided with a new pathological activity without changing and losing characteristics of collagen and an MMP-2 activator containing the collagen modified substance. SOLUTION: This papain treated collagen modified substance is essentially constituted of a mixture of a collagen molecule comprising two α1 chain N end amino acid residues which are 13 isoleucine residues counted from N end glutamine residues of acetic acid-extracted collagen and α2 chain N end amino acid residue which is 14 leucine residue counted from the N end glutamine residue of the acetic acid-extracted collagen and a collagen molecule comprising two α1 chain N end amino acid residues which are 13 isoleucine residues counted from the N end glutamine residues of the acetic acid-extracted collagen and α2 chain N end amino acid residue which is 6 glycine residue counted from the N end glutamine residue of the acetic acid-extracted collagen. The MMP-2 activator containing the papain treated collagen modified substance is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for treating collagen with a specific proteolytic enzyme.
The present invention relates to a novel collagen modified product obtained by imparting a new physiological activity to collagen. More specifically, the present invention provides a method for treating MMP- by treating collagen with papain.
(2) Useful modified collagen having an activating effect. Further, the present invention relates to an MMP-2 activator containing the above modified collagen.

[0002]

2. Description of the Related Art Collagen is a hard protein that constitutes the connective tissue of animals. It is composed of three polypeptide chains and has a triple helix structure in which the GXY sequence of amino acids is repeated about 330 times. Is a molecule. At its N-terminus and C-terminus, there is a non-triple helix structural part called telopeptide. Collagen that maintains the triple helical structure can polymerize in vivo to form fibers,
Provides a scaffold for cell attachment and migration. The telopeptide region crosslinks between the molecules of collagen that has formed fibrils, and as a result, the collagen is stabilized and hardly extracted from the living body. Therefore, in order to extract collagen, it is necessary to cleave the cross-links formed in the telopeptide or to decompose the telopeptide itself. The general method is to treat the connective tissue with an acid, alkali, or various proteolytic enzymes.

[0003] In the acid treatment, collagen (tropocollagen) retaining the telopeptide can be extracted by cleaving the cross-link through the telopeptide. on the other hand,
In the treatment with an alkali or proteolytic enzyme, the telopeptide is decomposed to extract collagen (atelocollagen) in which the telopeptide is degraded to various degrees. In the extraction using a protease, the cleavage site of the telopeptide varies depending on the substrate specificity of the protease used. In addition, in the alkali treatment, not only cleavage of the telopeptide but also chemical modification of the amino acid side chain occurs, so that the isoelectric point of the triple helix region changes, resulting in a decrease in denaturation temperature and loss of fibril-forming ability.

[0004] Collagen extracted with acids and proteolytic enzymes has almost no difference in chemical properties of amino acid side chains, molecular weight, isoelectric point, denaturation temperature, etc., and maintains a triple helical structure. All have the ability to form fibrils. That is, by maintaining the temperature at 20 ° C. to 37 ° C. in a neutral solution under physiological conditions, a fiber having the same structure as that in a living body can be formed (Reference 1:
Prockop, DJ, Hulmes, DJ: Assembly of collagen fibri
ls de nove from soluble precursors: polymerization
and copolymerization of procollagen, pN-collagen,
and mutated collages.In: Extracellular matrix as
sembly and structure (Yurchenco, PD., Birk, DE.an
d Mecham, RP.eds.) pp47-90, Academic Press, San D
iego, 1994).

[0005] Collagen is generally known as a protein having extremely low immunogenicity, and is widely used in the fields of food, cosmetics and pharmaceuticals. In particular, in the field of medicine, it is widely used as a wound dressing or a base material for artificial organs, or as an injection for wrinkle expansion or injection into a connective tissue defect, etc., for diseases involving remodeling of connective tissue. (Reference 2:
Toshihiko Hayashi, "Can the human body be regenerated-a mechanism for designing and assembling cells from collagen", see McGraw-Hill Publishing (1991)). Collagen is known to three-dimensionally fill tissue defects, provide a scaffold for cell adhesion, and regulate cell growth and activation. There are many unknown parts about the mechanism (Reference 3: Rubin K, Gullberg D, Tomas
ini-Johansson B, Reed RK, Ryden C, Borg TK: Molecu
lar recognition of the extracellular matrix by cel
l surface receptors.In: Extracellular matrix (Com
per WD ed.) pp262-309, Harwood Academic Publisher
s, Amsterdam, 1996).

[0006] The telopeptide region of collagen contributes to stabilization of the fiber by cross-link formation, but recently it has become clear that the difference in structure greatly changes the physiological activity of the collagen fiber. For example, fibroblasts cultured in tropocollagen with all telopeptide regions have an elongated morphology. In contrast, fibroblasts cultured in collagen treated with proctase have elongated dendrites, which is a result of the loss of the telopeptide region and part of the triple helix region by proctase treatment. (Reference 4:
Properties of bovine hide collagen with different N-terminal cleavage sites depending on the extraction method. Kaori Sato, Tetsuya Ebihara, Katsuaki Iijima, Shunji Hattori, Shinkichi Irie. The 45th Matrix Study Group (1998)).

[0007] In the process of reconstructing connective tissue accompanying wound healing, various matrix metalloproteases (MMPs) are released, and this degrades extracellular matrix molecules. Is believed to be essential for the reconstruction of
-Hansen H: Proteolytic remodeling of extracellular
matrix.Current Oponion in cell biology 7: 728-735,
1995). MMP-1, MMP-8, MMP-13, M
A matrix metalloprotease such as MP-14 is activated at an early stage of wound healing, and degrades collagen at three-quarters from the N-terminal side (cleavage site A). In particular, it has been reported that MMP-13 is not induced in acute wound healing but is induced in chronic wound healing, and its action has been noted in relation to chronic disease (Reference 6: Vaalamo M, Mattila L, Johansson N, Karin
iemi AL, Karjalainen-Lindsberg ML, Kahari VM, Saar
ialho-Kere U: Distinct populations of stromal cell
s express collagenase-3 (MMP-13) and collagena
se-1 (MMP-1) in chronic ulcers but not in norm
ally healing wounds.J Invest Dermatol 109: 96-101,
1997). MMP-13 not only degrades collagen at cleavage site A, but also in the N-terminal telopeptide region (cleavage site B)
But it breaks down. It is unknown how the cleavage site affects the physiological activity of collagen. However, collagen degraded at cleavage site A gelatinizes due to body temperature because it cannot maintain a triple helical structure. Therefore, the physiological activity of the telopeptide region cannot be analyzed while maintaining the fibrous structure under physiological conditions. This also means that it is difficult to use the collagen treated with MMP-13 in vivo while maintaining the fibrous structure.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to provide a new physiological activity without altering or losing the properties of collagen such as the amino acid sequence, molecular weight, isoelectric point, triple helix structure and fibril-forming ability of collagen. Another object of the present invention is to provide a useful collagen modified product provided with the above.

It is a further object of the present invention to provide an MMP-2 activator containing the modified collagen.

[0010]

SUMMARY OF THE INVENTION Generally, the triple helical region of collagen is resistant to many proteolytic enzymes, while the telopeptide region is susceptible to limited degradation. The present inventors have focused on the difference in the biological activities of various telopeptide regions and searched for a method for preparing collagen having a different telopeptide structure while maintaining a triple helix structure. As a result, by treating collagen with papain, a proteolytic enzyme that is inexpensive and can be used in large quantities, we succeeded in imparting a new physiological activity to collagen while maintaining fibril-forming ability. completed.

[0011] Accordingly, the present invention provides an
The terminal amino acid residue is the 13th isoleucine residue counted from the N-terminal glutamine residue of the acetate-extracted collagen, and the N-terminal amino acid residue of the α2 chain is the 14th position counted from the N-terminal glutamine residue of the acetate-extracted collagen. The collagen molecule that is a leucine residue and the N-terminal amino acid residue of the two α1 chains are the 13th isoleucine residue counted from the N-terminal glutamine residue of acetic acid-extracted collagen, and α2
A papain-treated collagen modification consisting essentially of a mixture with a collagen molecule in which the N-terminal amino acid residue of the chain is the sixth glycine residue counted from the N-terminal glutamine residue of acetic acid-extracted collagen.

Further, the present invention relates to a modified papain-treated collagen having an MMP-2 activating effect. The present invention also relates to a modified papain-treated collagen, wherein the collagen is treated with papain under conditions where the collagen is not denatured.

Further, the present invention resides in an MMP-2 activator containing the above-mentioned modified papain-treated collagen. That is, the present invention is to appropriately treat purified collagen or connective tissue containing collagen with papain to produce collagen having a new physiological activity while maintaining fibril forming ability under physiological conditions. About.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described below in more detail. Papain is a widely used plant-derived proteolytic enzyme that is neutral and active.

[0015] Collagen for treatment with papain is:
For example, not only collagen extracted by using acid or pepsin from connective tissues such as skin and collagen produced by genetic recombination, but also pretreatment such as defatting, which is usually performed to extract collagen with acid, pepsin, etc. It can be connective tissue performed. This collagen, including humans,
It may be derived from any animal such as cow, pig, sheep, goat, horse, rabbit, mouse, rat, chicken and the like.

The above-mentioned method of treating collagen with papain is characterized in that collagen or connective tissue is maintained at a pH of 4 to 8.5, preferably pH 6 to 8 at which papain has an activity, and the amount of collagen or connective tissue is based on the weight of collagen or connective tissue as a substrate. 1/10000 weight or more, desirably 1/100 to 1 /
Add 10 weight of papain and the enzyme is fully active,
And a temperature at which collagen is not denatured, for example, 4 ° C.
Above 37 ° C, preferably between 10 ° C and 30 ° C.
° C, more preferably 20 ° C to 25 ° C. The papain reaction can be stopped by adding an enzyme inhibitor of papain, adding iodoacetic acid or the like which is a reagent for deactivating papain, or changing the pH. However, it is not particularly necessary to perform a deactivation operation, and it can be easily removed by performing a method similar to a commonly used collagen purification method such as salting out, isoelectric point precipitation, and column operation. .

The fact that the physiological activity of the papain-treated collagen modified product is different from that of conventional collagen was proved by, for example, the following method. The papain-treated collagen was adsorbed on a culture dish under conditions capable of forming fibrils, and fibroblasts derived from human skin were cultured thereon. The activation of MMP-2 in the culture was analyzed by gelatin zymography. The amount of active MMP-2 therein was significantly increased as compared with untreated collagen, pepsin-treated collagen or proctase-treated collagen. Activated MMP-2 was not induced when collagen was adsorbed under conditions that could not form fibers. In addition, collagen gel contraction experiments are used as a wound healing model, but as a result of culturing human skin-derived fibroblasts in various collagen gels,
When cultured in papain-treated collagen, MMP-
2 was significantly activated, but not activated in untreated collagen, pepsin-treated collagen or proctase-treated collagen. MMP-2 has an activity of degrading denatured collagen and the like, and is used for wound healing (Reference 7: Agre
n MS: Gelatinase activity during wound healing. Br
J Dermatol 131: 634-640, 1994; Reference 8: Moses MA, M
arikovsky M, Harper JW, Vogt P, Eriksson E, Klagsb
run M, Langer, R: Temporal study of theactivity of
matrix metalloproteinases and their endogenous in
hibitors durign wound healing.J Cell Biochem 60: 3
79-386, 1996) and infiltration of T lymphocytes (Reference 9: Madri)
JA, Graesser D, Haas T: The roles of adhesion mol
ecules and proteinases in lymphocyte transendothel
ial migration. Biochem Cell Biol 74: 749-757, 1996), and MMPs that are thought to play an important role in metastasis of cancer cells. Therefore, a new physiological activity is expected in the use of collagen as a drug material in wound dressings and artificial organ substrates, as well as in the use of injections to expand wrinkles and to inject into connective tissue defects. You can do it.

The modified papain-treated collagen has no difference in physical properties such as molecular weight, isoelectric point, denaturation temperature and fibril-forming ability as compared with conventionally used acid-soluble collagen and pepsin-treated collagen. Since it can be handled in the same manner as conventional collagen, it is easy to replace it with conventional collagen or to mix it with them.

As described above, the present invention has been completed in which collagen is treated with papain to produce collagen having a new physiological activity, which is not available in conventional collagen.

[0020]

The present invention will be described in more detail with reference to the following Examples, which do not limit the present invention. (Example 1) Acid soluble type I collagen derived from bovine skin was added to 0.1%.
It is dissolved in a 0.05 M Tris-HCl buffer (pH 7.5) containing 15 M sodium chloride to a final concentration of 2 mg / ml. The final concentration of this collagen solution is 0.02 m
After adding papain (manufactured by Sigma) to a concentration of g / ml, the mixture is kept at 20 ° C. overnight. Collagen is precipitated by adding 0.5 M acetic acid containing an equal volume of 2 M sodium chloride. After re-dissolving the precipitated collagen in 0.05 M acetic acid, the collagen is precipitated by dialyzing against 0.05 M Tris-HCl buffer (pH 7.5). The precipitate is collected and dissolved in 0.005M acetic acid. For comparison, collagen treated with pepsin and proctase, which are widely used for collagen preparation, was similarly prepared. The pepsin was treated with a 0.05 M acetic acid solution, and the treatment with proctase was performed using a 0.05 M tartaric acid-sodium tartrate buffer (pH 2.8). Purification was performed in the same manner as described above. . Α1 chain and α2 of the obtained collagen
The analysis result of the N-terminal of the chain is shown in FIG. In FIG. 1, the upper two chains of the three chains of each collagen shown are the α1 chain, and the lowermost chain is the α2 chain. In the modified papain-treated collagen obtained in this example, the N-terminal amino acid residues of the two α1 chains are the 13th isoleucine residue counted from the N-terminal glutamine residue of the acetic acid-extracted collagen, and the α2 chain A collagen molecule in which the N-terminal amino acid residue is the 14th leucine residue counted from the N-terminal glutamine residue of acetic acid-extracted collagen, and the N-terminal glutamine of acetic acid-extracted collagen is the N-terminal amino acid residue of two α1 chains A 13th isoleucine residue counted from the residue, and a collagen molecule in which the N-terminal amino acid residue of the α2 chain is the sixth glycine residue counted from the N-terminal glutamine residue of acetic acid-extracted collagen. Was.

FIG. 2 shows the results of analyzing the molecular weight of each collagen by polyacrylamide gel electrophoresis. From left to right, acetic acid-extracted collagen (AcOH), pepsin-treated collagen (pepsin), proctase-treated collagen (proctase), and the papain-treated collagen (papain) of the present invention are shown. Single-chain α1, α
The mobility of No. 2 is the same, and it can be seen that there is no difference in the molecular weight of these collagens.

The isoelectric point of each collagen is 0.5 ml of 1
% Collagen solution was heat denatured by heating at 100 ° C. for 3 minutes, and then Amberl was added to the resulting collagen denatured solution.
An equal amount (0.5 ml) of an ion-exchange resin mixture obtained by mixing iteIRA400 and Amberlite IR120B at a ratio of 2: 1 was added, and the mixture was heated at 40 °
The pH of the solution when it was kept for 2 hours was measured. As a result, the pH of each collagen was 9.3, and there was no difference.

The maintenance of the triple helix structure and the denaturation temperature were analyzed using circular dichroism. In collagen, the molecular ellipticity at 221 nm decreases as the triple helix structure is destroyed. Then, the temperature of the collagen was raised from 20 ° C. to 50 ° C., and the change in the molecular ellipticity at 221 nm was measured (FIG. 3). As a result, all collagens were at 37 ° C.
At ~ 45 ° C, the molecular ellipticity at 221 nm sharply decreased, indicating that there was no difference between the denaturation temperature and the ability to maintain the triple helical structure.

Generally, when a collagen solution dissolved in an ice-cooled neutral saline solution is kept at 37 ° C., the collagen solution becomes cloudy. This is a form in which collagen is self-associated and exists in a living body. This is because fibers are formed. Then, after each collagen was dissolved in a neutral physiological saline solution under ice-cooling and kept at 37 ° C. for 1 hour, it was shown that all collagens became cloudy and formed fibers. When this was observed with an electron microscope, fibers having a stripe structure with a period of 67 nm were formed for all collagens (FIG. 4).

From the above results, it was confirmed that the general properties of collagen were not lost by the papain treatment. (Example 2) The collagen prepared in Example 1 was replaced with 0.0
0.1 mg / m 2 in Dulbecco's modified Eagle medium (pH 7.5) containing 05 M acetic acid or 0.01 M Hepes-HCl.
After dissolving so as to be 1 l, the mixture was adsorbed to a culture dish. The former is a condition in which collagen molecules are adsorbed in a random state and thus fiber formation is not possible, and the latter is adsorbed in a state where collagen fibers are formed. Human skin-derived fibroblasts were cultured on each collagen for 24 hours at 37 ° C. under 5% carbon dioxide using Dulbecco's modified Eagle's medium. Activation of MMP-2 in the culture supernatant was analyzed by gelatin zymography.

As a result, when each collagen was adsorbed with an acetic acid solution, no expression of active MMP-2 was observed in any case (FIG. 5). When collagen was adsorbed under conditions capable of forming fibrils, expression of active MMP-2 was not observed in acid-soluble collagen, pepsin-treated collagen or proctase-treated collagen, but in the case of papain-treated collagen, active MMP- 2 was strongly expressed (FIG. 6). (Example 3) The collagen prepared in Example 1 was replaced with 0.0
After dissolving in Dulbecco's modified Eagle's medium (pH 7.5) containing 1 M Hepes-HCl to a concentration of 0.8 mg / ml, 1 × 10 ⁵ / ml of human skin-derived fibroblasts were mixed, and cultured in a culture dish. The in-gel culture was performed at 37 ° C. for 24 hours under 5% carbon dioxide. Under these conditions, collagen fibrils within 30 minutes of culture (this is referred to as three-dimensional culture in collagen gel). After 24 hours, the collagen gel was removed from the culture dish, and the culture supernatant was separated from the cell-containing collagen gel by centrifugation. M in culture supernatant
Activation of MP-2 was analyzed by gelatin zymography.

As a result, no expression of active MMP-2 was observed in the case of acid-soluble collagen, collagen treated with pepsin or collagen treated with proctase, but in the case of collagen treated with papain, active MMP-2 was particularly strongly expressed. (FIG. 7). As compared with the results of FIG. 6, the amount of the activated MMP-2 detected was more remarkably increased.

[0028]

The papain-treated collagen modified product of the present invention has a remarkable MMP-2 activating effect. Therefore,
The composition containing the modified collagen can be used as an MMP-2 activator, and collagen is injected into a wound dressing or artificial organ base used as a pharmaceutical material, or to a wrinkle-extending or connective tissue defect. It can be expected to be useful for use in injections and the like for the purpose.

Furthermore, the papain-treated collagen modified product has no difference in physical properties such as molecular weight, isoelectric point, denaturation temperature and fibril-forming ability as compared with conventionally used acid-soluble collagen and pepsin-treated collagen. Because it can be treated in the same way as conventional collagen,
It is easy to replace with conventional collagen or to mix with them.

[Brief description of the drawings]

FIG. 1 shows amino acid sequences near the N-terminus of various collagens.

FIG. 2 is a photograph showing the results of electrophoresis of various collagens. From left to right, acetic acid-extracted collagen (AcOH), pepsin-treated collagen (pepsin), proctase-treated collagen (proctase), and papain-treated collagen (papain) are shown.

FIG. 3 is a graph showing analysis results of circular dichroism of various collagens.

FIG. 4 is an electron micrograph of various collagen fibers.

FIG. 5 is a photograph showing MMP-2 activation by fibroblasts cultured on non-fibrotic collagen. The results of analysis of the culture supernatant by gelatin zymography are shown. Here, lane 1: bovine serum albumin (negative control); lane 2: acetate-extracted collagen; lane 3: pepsin-treated collagen; lane 4: papain-treated collagen; and lane 5: proctase-treated collagen.

FIG. 6 is a photograph showing the activation of MMP-2 by fibroblasts cultured on collagen forming fibrils.
The results of analysis of the culture supernatant by gelatin zymography are shown. Here, lane 1: bovine serum albumin (negative control); lane 2: acetate-extracted collagen; lane 3: pepsin-treated collagen; lane 4: papain-treated collagen; and lane 5: proctase-treated collagen.

FIG. 7 is a photograph showing activation of MMP-2 by fibroblasts cultured in a collagen gel. The results of analysis of the culture supernatant by gelatin zymography are shown. Here, lane 1: bovine serum albumin (negative control); lane 2: acetate-extracted collagen; lane 3: pepsin-treated collagen; lane 4: papain-treated collagen; and lane 5: proctase-treated collagen.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C08H 1/00 A61K 37/18 (72) Inventor Shunji Hattori 1-chome, Senju-Midoricho, Adachi-ku, Tokyo Stock Nippi Biomatrix Research Laboratories (72) Inventor Shinkichi Irie 1-1-1, Senju-Midori-cho, Adachi-ku, Tokyo F-term (reference) 4B064 AG01 CA10 CA11 CB02 DA03 4C084 AA02 AA07 BA01 BA08 BA20 BA21 DA40 MA01 NA14 ZB222 ZC192

Claims (4)

[Claims] 1. The N-terminal amino acid residues of two α1 chains are 1 as counted from the N-terminal glutamine residue of acetic acid-extracted collagen.
A third isoleucine residue, a collagen molecule in which the N-terminal amino acid residue of the α2 chain is the 14th leucine residue counted from the N-terminal glutamine residue of acetic acid-extracted collagen, and an N-terminal of two α1 chains The amino acid residue is the 13th isoleucine residue counted from the N-terminal glutamine residue of the acetate-extracted collagen, and the N-terminal amino acid residue of the α2 chain is the 6th glycine residue counted from the N-terminal glutamine residue of the acetate-extracted collagen. A papain-treated collagen modification consisting essentially of a mixture with residues of collagen molecules. 2. A modified papain-treated collagen having an MMP-2 activating action. 3. A modified papain-treated collagen, wherein the collagen is treated with papain under conditions where the collagen is not denatured. 4. An MMP-2 activator containing the modified papain-treated collagen according to claim 1.