WO1987000176A1

WO1987000176A1 - Unduline and its fragmentation products, production process and utilization thereof

Info

Publication number: WO1987000176A1
Application number: PCT/EP1986/000404
Authority: WO
Inventors: Detlev Schuppan; Eckhart G. Hahn
Original assignee: Heyl Chemisch-Pharmazeutische Fabrik Gmbh + Co. Kg
Priority date: 1985-07-10
Filing date: 1986-07-09
Publication date: 1987-01-15
Also published as: DE3524644A1; EP0227827A1; JPS63500245A

Abstract

The unduline is a macromolecular glycoproteine of the interstitial conjunctive tissue, which is interleaved in large quantity between the collagen (I)-fibrils. The unduline and its fragmentation products are useful for the production of antibodies, the immunologic detection methods as well as the diagnostic of inflammatory, fibrotic and tumoral affections.

Description

Undulin and its fragmentation products,

Process for their preparation and their use

The invention relates to undulin and its peptide fragments, processes for their preparation and their use for the production of specific antibodies and for the diagnosis, prognosis and therapy of diseases and tumor diseases affecting the connective tissue.

Interstitial connective tissue mainly consists of collagen I fibrils. Suggest ultrastructural studies convey that macromolecules of collagen fibrils cohesion ^". It is believed that nektin next fibro- and proteoglycan another structural component of the supramolecular organization of collagen fibrils I ist.- responsible Until now, attempts to this component to isolate failed. ^"

Surprisingly, it has now been found that the extraction of skins from fetal or newborn mammals gives the new protein undulin, which plays an essential role in the supramolecular organization of collagen I fibrils and which is used in medicine and cosmetics leaves.

The invention relates to undulin and its peptide fragments, as well as a process for their production and their use.

Among the interstitial connective tissue proteins, undulin appears to be a major component alongside collagen I and III. Undulin is a highly aggregated, macro-molecular glycoprotein, which is mainly present as a cross-linked complex with a molecular weight of >> 1,000,000, determined using SDS-polyacrylamide gel electrophoresis under non-reducing l conditions. Using a collagen I standard, a characteristic band at 270,000 (A chain) and a doublet at 175,000/180,000 (Bl or B2 chain) can be stained after reduction. The mobility of the doublet bands is clear larger than that of reduced fibronectin.

Figure 1 shows undulin before (1) and after (2) reduction in SDS-polyacrylamide gel electrophoresis. The position of collagen α, ß and γ components is marked by arrows (standards).

After lyophilization, undulin is practically insoluble in neutral, non-denaturing buffer systems, e.g. Tris • HC1 phosphate buffer or dilute acetic acid, g In contrast, undulin is readily soluble in 6 M guanidine HCl, pH 7 to 9, in the presence of protease inhibitors. In association with collagen-I, undulin can be dissolved in 0.5 M acetic acid / 1% Triton X-100.

Such preparations can be examined by electron microscopy after cone evaporation. The molecule appears in the form of a hitherto unknown structure, consisting of a globular head portion with a diameter 5 of approx. 18 nm, an approx. 70 nm long tail part and at the other end, sometimes a smaller globular domain (diameter approx. 6 nm) consists. Aggregate forms composed of two, four or more of these structures and which are often associated with collagen I molecules are frequently observed. An electron micrograph after cone evaporation, on which undulin in isolated (arrows) and aggregated (double arrows) form next to collagen I can be seen, is shown in FIG. 2. 5 In amino acid analysis (6 M HC1, 24 hours, under nitrogen at 110 ° C.), undulin gives a typical but uncharacteristic amino acid composition for large glycoproteins with a high proportion of hydrophobic and acidic amino acids.

The amino acid composition, which differs significantly from that of the basal branglycoprotein laminin or from that of fibronectin, can be seen from the table below.

Table 1: Amino acid analysis of And

4-Hyp - Met 22

Asx 98 Ile 54

Thr 65 Leu 89

Ser ^* • 88 Tyr 33

Glx 93 Phe 39

Per 55 His 26

Gly 86 Hyl

Ala. 59 Lys 52

Cys 27 Arg 49

Val 65

The carbohydrate content of undulin, determined by gas chromatography of the alditol acetates and by mass spectrometry, is approximately 2% (weight per 100 g undulin), significantly lower than that of laminin (approx. 12) and fibronectin (approx. 5%) . The table below shows the carbohydrate analysis. Table 2: Carbohydrate analysis of undulin

g carbohydrate per 100 g protein

N-acetylglucosamine 0.94

N-acetylgalactosamine 0.22

Mannose 0.26

10 galactose 0.59

Fucose 0.05

Another property of undulin is shown by

, g indulin antibodies, the production of which will be described later, clearly:

Undulin antibodies - in indirect immunofluorescence - show a novel pattern on unfixed 4 μm cryostat sections of the skin or liver that differs from all

20 known patterns for connective tissue proteins. As shown in Fig. ^'3, the Undulinmuster of wavy parallel arranged fibrils having a uniform diameter (approximately 1 micron). These typical wavy fibril bundles run through it

«Interstitium and are not present in basement membranes. Undulin is found predominantly in the dense connective tissue, where mature collagen (type I) fibrils can also be expected, for example in the deeper layers of the dermis (FIG. 3), in the adventitia larger blood vessels or in the portal field

" _{Q of} the liver (Fig. 4), comparable distribution patterns can be found not only in humans, but also in mice, rats and cattle. etc. find. Interestingly, in the area of tumors, undulin is broken down faster than other connective tissue proteins.

" _P Decorate in immunoelectron microscopy. Antibodies against undulin (with ferritin-conjugated 2nd anti- body) _. globular material on the surface of and between collagen fibrils as shown in FIG. 5. (E = elastic fiber; bar = 500 nm).

The process for the production of undulin is characterized in that in the presence of protease inhibitors

A) subjecting the skins of fetal or newborn mammals to a neutral salt extraction,

B) subjecting the extract to fractional salt precipitation,

C) dissolve the precipitate and purify by DEAE chromatography to give undulin as a crude product,

D) ^* the raw product is further purified by extraction after dialysis and lyophilization,

E) cleaves the undulin to produce the fragmentation products with neutral or acidic proteases until fragments with a molecular weight are obtained

<400,000 receives, and

F) the product obtained according to stage E is purified by means of CM and DEAE cellulose chromatography, molecular sieves and / or HPLC in a buffer of pH 2.5-9.0.

The extraction according to stage A) is preferably carried out by first performing a pre-extraction with neutral buffer to remove serum proteins and lipids. A 10-200 mM neutral buffer base, for example 50-200 mM Tris.HCl, 4-5 M NaCl from pH 7-8, 20-200 M HEPES- (2- [4- (2-hydroxyethyl) -l-piperazinyll-ethanesulfonic acid) - or PIPES (piperazine-N, N '~ bis (2-ethanesulfonic acid) buffer together with 0.1-2% of a non-ionic detergent, for example Triton X-100, about 2-4 l of buffer per kg of skin (wet weight) are used, followed by the actual extraction, which is preferably carried out in the same buffer system and is carried out with the same amount, but is carried out at a lower ionic strength.

The extract is then subjected to fractional salt precipitation in accordance with stage B). In the first step, the extract is precipitated by adding ammonium sulfate until a degree of saturation of 20 to 50% is reached. The precipitate thus obtained is then redissolved in 10-200 mM neutral buffer base and subjected to fractional salt precipitation with an alkali metal halide, for example NaCl, KCl. Most of the undulin is precipitated between 1.5M and 4.5M.

For cleaning, the precipitate is dissolved in neutral buffer / 1-4 M urea / 0.25-0.5 M NaCl and treated with DEAE cellulose. The unbound fraction is dialyzed against the same buffer, which however only contains 0.01-0.05 M NaCl. The precipitate thus obtained consists of approximately 70% undulin. The supernatant contains further undulin, which one can gain in more than 90% purity by chromatography on DEAE-cellulose, thereby obtaining a ^'salt gradient det verwen¬ as eluent.

The preparations are freed from salts by dialysis, for example against acetic acid, and lyophilized. The 70% preparation can be extracted by extraction with neutral buffer, optionally in the presence of a non-ionic detergent, are freed of impurities. The product obtained in this way with a purity of more than 80% can be dissolved in 10-100 mM Tris-HCl, 5-6 M guanidine, pH 8-9, and on a molecular sieve column (for example Sephacryl 2B, Cl 4B or 5- 1000) separate from low molecular weight impurities. The purity obtained is now almost 100%.

The yield is approximately 100 mg / kg skin (wet weight). Undulin can also be cleaned, as described below for the fragments of undulin.

The entire process is expediently carried out at cold room temperatures (4-10 ° C.) and in the presence of protease inhibitors, for example phenylmethylsulfonyl fluoride (PMSF), N-ethylmaleimide (NEM), ethylenediaminetetraacetic acid (EDTA), soybean type inhibitor ^' ( SBTI), Trasylol and Benza idinhydrochlorid carried out.

Undulin is very sensitive to neutral or acidic proteases. Suitable neutral proteases are e.g. Trypsin, thrombin, plasmin, elastase, cathepsin, V8 staphylococcal protease, chymotrypsin, papain and thermolysin. Examples of suitable acidic proteases are pepsin, acidic cathepsin, chymosin etc.

Preferred proteases are trypsin, elastase, plasmin, thrombin, V8 staphylococcal protease and chymotrypsin, and pepsin.

Undulin protease fragments with a molecular weight of <400,000, preferably <100,000, are partially soluble in neutral buffers, while maintaining some of the antigenic properties of undulin. So for example, provides the breakdown of the Undulins with trypsin at 30 ° C for intermediates with a molecular ₅ weight from 300,000 to 400,000, the main products with a molecular weight of 30,000 to 50,000, from 60,000 to 70,000 and 80 000. The final product of Pepsin degradation at 4 ° C has a molecular weight between 25,000 and 50,000.

10 The fragments can be purified by means of CM and DEAE cellulose chromatography, molecular sieves and / or HPLC in customary buffers with a pH of 2.5 - 9.0. Suitable acidic buffers are, for example, acetate and citrate buffers. Suitable neutral buffers were already mentioned above and, _c suitable alkaline buffers are A moniumbicarbonat, sodium acetate.

Undulin and its fragments are highly antigenic. For example, a triple application of 0.3 mg undulin or its fragments is sufficient to produce ^"

20 of high-titre antisera, especially in rabbits and mice, specific antibodies can be obtained with the help of known ones

Processes, e.g. Affinity chromatography. These reagents react in the Western blot, in the ELISA ©

25 in the radioimmunoassay in no way cross with other connective tissue proteins. Monoclonal technology can also be used to produce high-affinity antibodies against undulin and its fragments. Such immunoreagents can be used in immunohistology, for cell culture experiments, in genetic engineering and for the analytical detection of

30 Use undulin and its fragments. It was shown that monoclonal antibodies to undulin in immunohistology have the same characteristic pattern as polyclonal antibodies.

35

1)

ELISA = Enzyme - Linked Immuno Sorbent Assay In addition, the above-mentioned antibodies and antigens are used for the quantitative determination of undulin and / or g of its fragments in body fluids, cell media and extracts by means of radio-immunoassay, in ELISA, fluorescence immunoassay (FIA), luminescence immunoassay. Assay and the like. It has in fact by means of a hoch¬ specific and sensitive radioimmunoassay shown ₀ are that undulin antigenicity circulates in body fluids "and relate to disease states, such as the interstitial tielle connective tissue, such as inflammation, fibrotic's diseases and neoplastic diseases, elevated in the form of mirror correlated Undulin and its fragments can also be used in cosmetic preparations, such as lotions, gels, creams, to improve the appearance and condition of the skin.

The following examples illustrate the invention. 0

Example 1

_κ Undulin production

The skin of fetal or newborn monkeys, calves or rats is soaked, homogenized and deep-frozen in 100 M Tris 4.5 M NaCl, pH 7.4, which contains 2 M PMSF, 4 mM NEM and 10 M EDTA as protease inhibitors . All work is carried out at 4 ° C. and in the presence of protease inhibitors (PI) if buffers are used whose pH is above 4.0. The homogenate is first extracted with 50 mM Tris / PI / 4.5 M NaCl, pH 7.4 and 1% Triton X-100 (31 / kg wet weight). The residue is then extracted with 50 mM Tris / PI / 0.5 M NaCl, pH 7.4 (31 / kg wet weight). From the Supernatant liquid is obtained by adding ammonium sulfate to a degree of saturation of 30% (168 g / 1). This is redissolved in Tris / PI / 0.5 M NaCl (31 / kg wet weight). The solution is subjected to fractional salt precipitation with NaCl up to final concentrations of 1.7, 2.6 and 4.5 M. The fraction at 2.6 M NaCl is dissolved in 50 mM Tris, PI, .0.3 M NaCl , 1 - 4 M, pH 7.4 dissolved urea and dialyzed against this mixture. A precipitate (collagen I aggregate) is centrifuged off and the supernatant is stirred with DEAE cellulose. The unbound fraction is dialyzed against Tris, PI, 0.02 M NaCl, pH 7.4, whereby a precipitate is obtained which consists of undulin, collagen I and a protein with a molecular weight of 35,000. The soluble material is chroographed on DEAE cellulose, and after elution with a salt mixture of approximately 0.25 M NaCl undulin is obtained with a purity of> 90%.

The fractions containing undulin are dialyzed against 0.1 M acetic acid and lyophilized. For further purification, the lyophilisate is extracted with 50 M Tris-HCl, 2 M urea, protease inhibitors, 1% Triton X-100, pH 8.2, at 4 ° C., whereby contaminating proteins are removed. The undissolved approximately 80% pure undulin is then dissolved in 50 M Tris-HCl, 6 M guanidine, pH 8.2 and freed of low molecular weight impurities in the same buffer of Sepharose Cl 4B. Undulin produced in this way is homogeneous and pure, the yield is 100 mg / kg of skin used (wet weight). 1

Example: 2

5 Undulin trypsin breakdown

The lyophilized protein is suspended at 0.5 mg / ml in 0.2 M Na.HCO- / acetic acid, pH 7.0 and with trypsin-TPCK (Serva, Heidelberg) in the enzyme: substrate ratio 0 1:50 Degraded at 25 ° C with shaking. Aliquots are removed after 10, 30, 60 minutes, 4 and 16 hours, mixed with trypsin inhibitor (Soybean trypsin inhibitor, Serva, Heidelberg) and lyophilized. Degradation after 16 hours mainly contains peptides of the molecular

₁₅ weights less than or equal to 80,000.

. Example 3

0 Pepsin breakdown of undulin

For pepsin degradation, undulin is suspended at 1 mg / 1 ml in 0.5 M acetic acid and at 4 - 8 ° C in a ratio of 1:50 (enzyme to substrate) with shaking with pepsin

25 (Sigma, baptized Irish) dismantled. After 6 to 10 hours, the main product obtained is a fragment with a molecular weight between 25,000 and 50,000.

₃ 0 Example 4

Production of polyvalent antisera and polyclonal

antibody

“High-titre antisera against undulin and its fragments (from monkeys, calves or rats) can be prepared as follows: g 0.2 mg of antigen which is suspended in 0.5 ml of neutral buffer and emulsified in 0.5 ml of complete Freund's ad uvans is administered subcutaneously. This is followed by two further injections at 3-week intervals using incomplete adjuvant. The _1Q antiserum is administered via column-immobilized connective tissue antigens. After extensive rinsing with buffer, specific antibodies of high affinity can finally be eluted from immobilized undulin.

15

Example 5

Production of monoclonal antibodies

_ _N 8-12 week old Balb / c mice are given sc 3 - 4 In¬ injections of 100 ug undulin or Undulinfragmenten at intervals of 2 weeks. The first is mixed 1: 1 with Freund'sehen adjuvant (complete), the next are in a ratio 1: 1 with Freund'sehen adjuvant

₂₅ (incomplete) mixed.

After a positive antibody titer in the mouse, its spleen is fused with isogenic myelo cells (NS1) and brought into tissue culture. Cell clones producing positive antibodies are subcloned in order to obtain individual antibody-producing cell lines. The antibody can be purified using known methods from the tissue culture supernatant or from the ascites of mice serving as cell culture carriers.

5 Example 6

Radioimmunoassay

The undulin purified in Example 1 after chromatography on Sepharose C14B can be added to 10 μg / 50 μl 50 mM Tris-HCl, 6M guanidine, 0.05 X Triton, pH 8.2 using the

Radioactively label 125 chloramine-T method with 1 mCi I.

(Marking time 0.5 - 3 min.). Low-molecular activity is removed by dialysis against 50 mM Tris-HCl, 8 M urea, 0.05% Triton x-100, pH 8.2 ^ at room temperature. 125I-undulin (3,000-10,000 cpm / ng protein) shows 90-100 * binding to its antiserum and allows a sensitive liquid-phase radio-immuno-inhibition assay to be carried out, for example for the determination of circulating antigens in serum or in plasma ..

Claims

"4P claims

1. Undulin and its fragmentation products, obtainable in that in the presence of protease inhibitors

B) subjecting the extract to fractional salt precipitation,

D) the raw product is further purified by extraction after dialysis and lyophilization,

E) cleaving the undulin to produce the fragmentation products with neutral or acidic proteases until fragments with a molecular weight <400,000 are obtained, and

F) the product obtained according to stages D and E is purified by means of CM and DEAE cellulose chromatography, molecular sieve and / or HPLC in a buffer of pH 2.5-9.0.

2. A process for the preparation of the compounds according to claim 1, d a d u r c h g e k e n n z e i c h n e t,

that one in the presence of protease inhibitors

B) subjecting the extract to fractional salt precipitation,

C) the precipitate dissolves and is purified by DEAE chromatography, giving undulin as the crude product,

D) ^" further purifies the crude product by extraction after dialysis and lyophilization,

E) cleaving the undulin to produce the fragmentation products with neutral or acidic proteases until fragments with a molecular weight of <400,000 are obtained, and

F) the product obtained according to stages D and E by means of CM and DEAE cellulose chromatography, molecular sieve and / or HPLC in a buffer of pH 2.5

9.0 cleans.

3. The method according to claim 2, characterized in that the neutral salt extraction according to stage A is carried out in two stages, the first stage being carried out with a 10-200 mM neutral buffer base and the second stage being carried out with a neutral buffer having a lower ionic strength.

4. The method according to claim 3, characterized in that one carries out the pre-extraction in the presence of 0.1 - 2% g of a nonionic detergent.

5. The method according to any one of claims 2-4, characterized in that one carries out the fractional salt precipitation according to stage B by adding ammonium sulfate to the extract obtained from _Q stage A until a degree of saturation of 20-50% is reached, so the precipitate obtained is dissolved in a 10-200 mM neutral buffer base and the solution is subjected to fractional salt precipitation with an alkali metal halide. 5

Process according to one of claims 2-6, characterized in that the purification according to stage C is carried out by the precipitate obtained from stage B in 10-200 mM neutral buffer base / 1-4M urea / 0.25-0.5M Dissolve NaCl and treat with DEAE-0 cellulose, and dialyze the unbound fraction against the same buffer containing 0.01-0.05 M NaCl to give a 70% undulin precipitate. 5

Process according to Claim 6, characterized in that the liquid supernatant obtained after dialysis is purified by chromatography on DEAE cellulose using a salt gradient as the eluent, giving undulin of more than 0 90% purity.

Process according to one of claims 2-7, characterized in that the purification according to stage D is carried out by dialyzing against acetic acid, lyophilizing 5 and extracting the lyophilisate with 10-200 mM neutral buffer base or dilute acetic acid, if necessary several times. 1

9. The method according to claim 8, characterized in that one carries out the extraction in the presence of 0.1 - 2% g of a nonionic detergent.

10. The method according to claim 2, characterized in that kokkenprotease as the protease to cleave the Undulins trypsin, elastase, plasmin, thrombin, staphylococcus _V8-0, turns chymotrypsin and / or pepsin ver¬.

11. The method according to claim 10, characterized in that trypsin or pepsin is used. 5

12. Use of the products according to claim 1 for the production of antibodies, for immunological detection methods and for diagnosis, prognosis and course control of fibrotic, inflammatory and tumorous diseases. 0

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