WO1991009054A1 - Polypeptide au collagene de type iv - Google Patents
Polypeptide au collagene de type iv Download PDFInfo
- Publication number
- WO1991009054A1 WO1991009054A1 PCT/US1990/007164 US9007164W WO9109054A1 WO 1991009054 A1 WO1991009054 A1 WO 1991009054A1 US 9007164 W US9007164 W US 9007164W WO 9109054 A1 WO9109054 A1 WO 9109054A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- iii
- peptide
- cells
- collagen
- type
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
Definitions
- Type IV collagen is a distinctive glycoprotein which occurs almost exclusively in basement membranes, structures which are found in the basal surface of many- cell types, including vascular endothelial cells, epithelial cells, etc.
- Type IV collagen is a major component of basement membranes. It differs from interstitial collagens. See New Trends in Basement Membrane Research, K. Kuehn et al., eds., Raven Press, NY, at pp. 57-67 (1982).
- Type IV collagen has a molecular weight (M ) of about 500,000 and consists of three polypeptide chains: two ⁇ l_ (MW 185,000) chains and one a2_ (MW 170,000) chain.
- Type IV collagen has two major proteolytic domains: a large, globular, non- collagenous, NCI domain and another major triple-helical collagenous domain. The latter domain is often interrupted by non-collagenous sequences of variable length.
- a diagrammatic representation of the type IV collagen molecule is shown in Figure 1. It is a complex and irtultidomain protein with different biological activities residing in different domains.
- Type IV collagen self-assembles to polymeric structures which constitute the supportive frame of basement membranes.
- Various other macromolecular components bind to type IV collagen, such as: laminin, entactin/nidogen and heparan sulfate proteoglycan.
- An additional function of type IV collagen is to mediate cell binding.
- the present invention provides a polypeptide (hereinafter designated "Hep-III") which represents a fragment of the al_ chain of type IV collagen.
- Hep-III a polypeptide which represents a fragment of the al_ chain of type IV collagen.
- This polypeptide can be prepared by conventional solid phase synthesis.
- the formula of the polypeptide is:
- Polypeptide Hep-III formally represents isolated type IV collagen residues 531-543 from the amino-ter inus of the al chain of type IV collagen.
- the single letter amino acid code for this polypeptide is GEFYFDLRLKGDK.
- polypeptide Hep-III was assayed for biological activity and found to be an extremely potent promoter of heparin-binding to synthetic substrates.
- Polypeptide Hep-III was also a potent promoter of cell adhesion and spreading of many cell types, including melanoma and endothelial cells. Therefore, it is believed that polypeptide Hep-III may be useful to (a) promote cellular attachment to culture substrata, (b) inhibit the metastasis and invasion of malignant cells, and (c) promote wound healing and implant acceptance. Since other cell types have been shown or are expected to have similar behavior in response to Hep-III, other uses of peptide Hep-III could be envisioned, such as assistance in nerve regeneration. Furthermore, since it is expected that further digestion/hydrolysis of peptide Hep-III in vitro or in vivo will yield some fragments of substantially equivalent bioactivity, such lower molecular weight peptides are also considered to be within the scope of the present invention.
- Figure 1 is, a diagrammatic representation of type IV collagen, indicating the structure of the l(IV) and ⁇ 2_(IV) chains, each with a major non-collagenous, NCI domain and the triple helix-rich domain containing interruption of the gly-X-Y triple helical motif.
- Figure 2 depicts the primary amino acid sequence of the human and murine ⁇ l chain of type IV collagen in comparison. The sequence is highly conserved between these two species.
- Figures 3 through 3F are composites showing 10 heparin molecules bound to type IV collagen molecules by the method of rotary shadowing.
- Figure 4 is a histogram showing the distribution of heparin molecules to three distinctive sites along the length of type IV collagen, at the NCI 15. domain, and at 100 nm and 300 nm from the NCI domain.
- Figure 5A is a graph showing the direct binding of increasing concentrations of heparin to peptide Hep- III coated on plastic substrates.
- Figure 5B is a graph showing the direct binding 20 of increasing concentrations of heparin to peptide Hep-I (TS-2 in U.S. Patent No. 4,876,332) coated on plastic substrates.
- Figure 5C is a graph showing the direct binding of increasing concentrations of heparin to type IV 25 collagen coated on plastic substrates.
- Figure 6 is a graph showing the inhibition of the binding of heparin to the triple helix-rich domain of type IV collagen, by increasing concentrations of peptide Hep-III ( ⁇ ) [or control peptides 15 (m) and 17 30 ( ⁇ )] present in solution.
- Figure 7 is a graph depicting the competition of the binding of heparin to peptide Hep-III coated on plastic by various glycosoaminoglycans [heparin (o), dextran (A) and chondroitin ( ⁇ )] at increasing 35 concentrations.
- Figure 8A is a graph depicting the direct binding of melanoma cells to peptide Hep-III (M) , or control peptide ET-2 (•), coated onto plastic at increasing concentrations.
- Figure 8B is a graph depicting the direct binding of aortic endothelial cells to peptide Hep-III ( ⁇ ) , control peptide ET-2 ( ⁇ ) and BSA (Q), coated onto plastic at increasing concentrations.
- Figure 9A is a graph depicting the competition of the binding of melanoma cells to type IV collagen coated substrata in the presence of peptide Hep-III in solution at increasing concentrations.
- Figure 9B is a graph depicting the competition of the binding of endothelial cells, to type IV collagen coated substrata in the presence of peptide Hep-III in " solution at increasing concentrations.
- Figure 10A is a graph depicting the direct binding of increasing concentrations of iodinated ( 125 I- labeled) peptide Hep-III to the surface of melanoma cells.
- Figure 10B is a graph depicting the direct binding of increasing concentrations of iodinated ( 125 I- labeled) peptide Hep-III to the surface of endothelial cells.
- Figure 11A is a graph depicting the competition of the binding of iodinated peptide Hep-III (B), and control peptide 15 ( ⁇ ) , to melanoma cells in the presence of increasing concentrations of unlabeled Hep- III.
- Figure 11B is a graph depicting the competition of the binding of iodinated peptide Hep-III (a), and control peptide ET-2 ( ⁇ ) to endothelial cells in the presence of increasing concentrations of unlabeled Hep- III.
- the sequence of the al_ chain is shown in Figure 2. Two copies of the ⁇ l_ chain and one copy of the ⁇ 2. chain are put together to make up the type IV collagen molecule. The total number of amino acids per collagen molecule is approximately 4,550. The ⁇ l.(IV) chain contains about 1,645 amino acids.
- Binding sites for heparin are of special interest since heparin-related macromolecules such as heparan sulfate proteoglycans are present in basement membranes and cell surfaces as well. Therefore, the association of these heparin-related molecules with type IV collagen may affect basement membrane structure and various cellular functions (such as adhesion, motility/migration, spreading, etc.).
- Hep-III was synthesized, which corresponds to residues 531-543 from the NH 2 end of ⁇ l_(lV) located in the binding site 300 nm from the NCI domain. This peptide was found to exhibit strong binding to heparin.
- polypeptide of the invention was synthesized using the Merrifield solid phase method. This is the method most commonly ' used for peptide synthesis, and it is extensively described by J. M. Stewart and J. D. Young in Solid Phase Peptide Synthesis , Pierce Chemical Company, pub., Rockford, IL (2nd ed. , 1984), the disclosure of which is incorporated by reference herein.
- the Merrifield system of peptide synthesis uses a 1% crosslinked polystyrene resin functionalized with benzyl chloride groups.
- the halogens when reacted with the salt of a protected amino acid will form an ester, linking it covalently to the resin.
- the benzyloxycarbonyl (BOC) group is used to protect the free amino group of the amino acid.
- This protecting group is removed with 25% trifluoroacetic acid (TFA) in dichloromethane (DCM) .
- TAA triethylamine
- the next BOC-protected amino acid is then coupled to the free amino of the previous amino acid by the use of dicyclohexylcarbodiimide (DCC) .
- DCC dicyclohexylcarbodiimide
- Side chain functional groups of the amino acids are protected during synthesis by TFA stable benzyl derivatives. All of these repetitive reactions can be automated, and the peptides of the present invention were synthesized at the University of Minnesota Microchemical facility by the use of a Beckman System 990 Peptide synthesizer.
- the polypeptide resin is treated with anhydrous hydrofluoric acid (HF) to cleave the benzyl ester linkage to the resin and thus to release the free polypeptide.
- HF anhydrous hydrofluoric acid
- the benzyl-derived side chain protecting groups are also removed by the HF treatment.
- the polypeptide is then extracted from the resin, using a 1.0 M acetic acid, followed by lyophilization of the extract. Lyophilized crude polypeptides are purified by preparative high performance liquid chromatography
- HPLC reverse phase technique by reverse phase technique on a C-18 column.
- a typical elution gradient is 0% to 60% acetonitrile with 0.1% TFA in H 2 0.
- Absorbance of the eluant is monitored at 220 nm, and fractions are collected and lyophilized.
- Characterization of the purified polypeptide is by amino acid analysis.
- the polypeptides are first hydrolyzed anaerobically for 24 hours at 110°C in 6 M HC1 (constant boiling) or in 4 N methanesulfonic acid, when cysteine or tryptophane are present.
- the hydrolyzed airiino acids are separated by ion exchange chr ⁇ atography using a Beckman System 6300 amino acid analyzer, using citrate buffers supplied by Beckman. Quantitation is by absorbance at 440 and 570 nm, and comparison with standard curves.
- the polypeptides may be further characterized by sequence determination. This approach is especially useful for longer polypeptides, where amino acid composition data are inherently less informative.
- Sequence determination is carried out by sequential Edman degradation from the amino terminus, automated on a Model 470A gas-phase sequenator (Applied Biosysterns, Inc.), by the methodology of R. M. Hewick et al., J. Biol. Chem. , 256, 7990 (1981).
- Peptide Hep-III in solution was screened for the ability to inhibit the binding of heparin to intact, native type IV collagen coated on plastic.
- This experimental approach avoids problems due to differential coating of peptides in heparin binding assays.
- Type IV collagen at 60 ⁇ g/ml in PBS was coated on 96-well plates, using 50 ⁇ l per well and dried overnight at 28°C. The wells were then treated for two hours with 2 mg/ml BSA in wash buffer (described above in Example 1) .
- Peptide Hep-III at various dilutions ranging from 0.5 mg/ml to 5 ⁇ g/ml in PBS and CHAPS (cholamido-propyl-dimethyl-ammonio- propane-sulfonate) (a detergent used to avoid non ⁇ specific sticking) was co-incubated with a standard amount of 3 H-heparin (500 ng per well 50 ⁇ g/ml final concentration) for two hours at 37°C and the mixture was then transferred to the collagen coated plate (50 ⁇ l) and allowed to incubate for another two. hours at 37°C. The wells were then washed and radioactivity was counted as described above.
- heparin along with other sulfated glucosaminoglycans, dextran and chondroitin sulfate were used in competition experiments.
- K- 1735-M4 Highly metastatic murmine melanoma cells, K- 1735-M4 were originally provided by Dr. I. J. Fidler of Anderson Hospital, University of Texas Health Sciences Center, Houston, Texas. When the cells were received, a large number of early passage cells were propagated and frozen in liquid nitrogen. The tumor cells are usually cultured in vitro for no longer than six weeks.
- the cells are discarded and new cells withdrawn from storage for use in further in vitro or in vivo experiments. This precaution is taken to minimize phenotypic drift that can occur as a result of continuous in vitro passage.
- the cells were cultured in Dulbecco's Modified Eagle's Medium containing 5% heat inactivated fetal calf serum. The cultures were grown in 37°C incubators with a humidified atmosphere containing ' 5% C0 2 . Cells were subcultured twice weekly by releasing cells gently from the flask, using 0.05% trypsin and 1 mM EDTA.
- the assay mixture was then incubated at 37°C for 120 minutes. At the end of the incubation, the wells were washed with warm PBS containing 10 mM Ca ++ , and the adherent population was solubilized with 0.5 N NaOH containing 1% sodium dodecyl sulfate. The solubilized cells were then quantitated using a liquid scintillation counter. As shown in Figure 8A, increasing peptide concentrations produced a higher percentage of cell adhesion. Melanoma cell adhesion reached 80% of input at peptide concentrations of 0.5 ⁇ g/well.
- EXAMPLE 5 Inhibition of Adhesion of Cancer Cells in the Presence of Peptide Hep-III in Solution
- the melanoma cell line described above M4 was used. Cells were grown, labeled and harvested as described in Example 4, but after detachment and washing they were coincubated for 20 min. in the presence of various concentrations of peptide Hep-III in solution. They were then applied for another 20 min. on type IV collagen-coated plastic wells. At the end of the incubation the same treatment described in Example 4 was used. As shown in Figure 9A, increasing concentrations of peptide Hep-III were able to dramatically decrease the binding of melanoma cells to type IV collagen-coated substrata.
- Bovine aortic endothelial cells were isolated according to the following protocol. Aortas were obtained from a local slaughterhouse, washed in cold phosphate buffered saline (PBS) (136 mM NaCl, 2.6 mM KC1, 15.2 mM Na 2 HP0 4 , pH 7.2) and processed within 2 hours. Crude collagenase (CLS III, 125-145 units per mg dry weight, Cooper Biomedical) was used at 2 mg/ml in Dulbecco's modified Eagle's medium (DMEM) (GIBCO) . The vessel was clamped at the distal end, filled with the collagenase-PBS solution and digestion was carried out for 10 minutes.
- PBS cold phosphate buffered saline
- DMEM Dulbecco's modified Eagle's medium
- the lu enal contents were harvested, followed by the addition of fresh collagenase for two additional 10-minute periods.
- the enzyme-cell suspensions were added to an equal volume of DMEM containing 10% fetal bovine serum (FBS) to inhibit the enzyme and spun in a centrifuge at 400 x g for 10 minutes.
- the resulting cell pellet was resuspended in DMEM containing 10% FBS, 100 units/ml of penicillin G, 100 ⁇ g/ml of streptomycin and 100 ⁇ g/ml of crude fibroblast growth factor.
- Cells are cultured in 75 cm 2 flasks in a humidified 5% C0 2 atmosphere at 37°C.
- This method routinely gives a high yield of endothelial cells with little contamination (less than 5%) by smooth muscle cells, pericytes or fibroblasts as judged by phase contrast microscopy as well as by immunostaining.
- Direct adhesion of endothelial cells was performed as discussed in Example 4.
- Plastic substrates were coated with increasing concentrations of peptide Hep-III and a constant number of 35 S-labeled cells were added per well and they were incubated for 120 min. at 37°C. Subsequently, the wells were treated as discussed in Example 4.
- Peptide Hep-III promotes substantial adhesion of endothelial cells even at very low plating concentrations (20 ⁇ g/ml) (Fig. 8B) .
- Inhibition of adhesion was measured using 96- well microtiter plates. In each well 50 ⁇ l of a type IV collagen solution at 60 ⁇ g/ml were absorbed by incubating overnight at 29°C.
- Endothelial and melanoma cells were grown in culture as described in Examples 4, 5, and 6 (supra). Cells used for this type of experiment were not labeled 40 with radioactivity. Unlabeled cells were harvested by trypsinization (supra) on the day of the experiment. About 5,000 cells were mixed with 50 ⁇ l of a given concentration of peptide Hep-III in solution. Increasing concentrations of peptide Hep-III were used. The cells were incubated with the iodinated peptide for 15 min. at 4°C and they were then pelleted by centrifugation. ' The cells were then resuspended and washed 3 times with DMEM containing 2 mg/ml BSA and 50 mM Hepes.
- FIG 11A shows that the binding of radiolabeled Hep-III to the surface.of melanoma cells can be competed only by an excess of unlabeled peptide Hep-III, whereas control (negative) peptide 15 (formula GHATEGPK) failed to compete; a similar competition can be observed for the binding of labeled peptide Hep-III to the surface of endothelial cells (Fig. 11B) .
- an excess of unlabeled peptide Hep-III could efficiently compete for the binding of radiolabeled Hep-III, but control peptide ET-2 (formula GDSRTITTKGERGQP) failed to compete.
- peptide Hep-III is a major participant in the process of endothelial cell adhesion.
- a number of practical applications for the polypeptides of the present invention can be envisioned. Such applications include the promotion of the healing of wounds caused by the placement of synthetic substrata within the body.
- Such synthetic substrata can include artificial vessels, ' intraocular contact lenses, hip replacement implants and the like, where cell adhesion is an important factor in the acceptance of the synthetic implant by normal host tissue.
- medical devices can be designed making use of these polypeptides to attract cells to the surface in vivo or even to promote the growing of a desired cell type on a particular surface prior to grafting.
- An example of such an approach is the inducation of endothelial cell growth on a prosthetic device such as a blood vessel, heart valve or vascular graft, which is generally woven or knitted from nitrocellulose or polyester fiber, particularly DacronTM (polyethylene terephthalate) fiber.
- DacronTM polyethylene terephthalate
- the latter point indicates the potential usefulness of these defined polypeptides in coating a patch graft or the like for aiding wound closure and healing following an accident or surgery.
- the coating and implantation of synthetic polymers may also assist in the regeneration of nerves following crush traumas, e.g., spinal cord injuries.
- Such devices include controlled drug delivery reservoirs or infusion pumps.
- the polypeptides of the present invention can be used to promote cell adhesion of various cell types to naturally occurring or artificial substrata intended for use in vitro.
- a culture substrate such as the wells of a microtiter plate or the medium contacting surface of microporous fibers or beads, can be coated with the cell-attachment polypeptides. This can obviate the use of type IV collagen in the medium, thus providing better defined conditions for the culture as well as better reproducibility.
- Cytodex particles manufactured by Pharmacia, are coated with gelatin, making it possible to grow the same number of adherent cells in a much smaller volume of medium than" would be possible in dishes.
- the activity of these beads is generally dependent upon the use of coating protein in the growth medium and the present polypeptides are expected to provide an improved, chemically defined coating for such purposes.
- Other surfaces or materials may be coated to enhance attachment, such as glass, agarose, synthetic resins or long-chain polysaccharides.
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Abstract
Est décrit un polypeptide répondant à la formule suivante: gly-glu-phe-tyr-phe-asp-leu-arg-leu-lys-gly-asp-lys qui peut lier l'héparine et stimuler l'adhérence des cellules. Sont également décrits des dispositifs utilisés en médecine comme par exemple des implants prosthétiques, des dispositifs percutanés et des substrats pour la culture cellulaire revêtus d'une composition comprenant le polypeptide.
Applications Claiming Priority (2)
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US45086189A | 1989-12-14 | 1989-12-14 | |
US450,861 | 1989-12-14 |
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WO1991009054A1 true WO1991009054A1 (fr) | 1991-06-27 |
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PCT/US1990/007164 WO1991009054A1 (fr) | 1989-12-14 | 1990-12-06 | Polypeptide au collagene de type iv |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6274134B1 (en) | 1992-01-29 | 2001-08-14 | National Institutes Of Health | Human cell adhesion protein AAMP-1 and uses thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4578079A (en) * | 1982-08-04 | 1986-03-25 | La Jolla Cancer Research Foundation | Tetrapeptide |
US4876332A (en) * | 1987-10-08 | 1989-10-24 | Regents Of The Univeristy Of Minnesota | Polypeptides with type IV collagen activity |
-
1990
- 1990-12-06 WO PCT/US1990/007164 patent/WO1991009054A1/fr unknown
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4578079A (en) * | 1982-08-04 | 1986-03-25 | La Jolla Cancer Research Foundation | Tetrapeptide |
US4876332A (en) * | 1987-10-08 | 1989-10-24 | Regents Of The Univeristy Of Minnesota | Polypeptides with type IV collagen activity |
Non-Patent Citations (3)
Title |
---|
EUROPEAN JOURNAL OF BIOCHEMISTRY, Vol. 168, issued 1986, BRAZEL et al., "Completion of the Amino Acid Sequence of the 1 Chain of Human Basement Membrane Collagen (type IV), Reveals 21-Non-Triplet Located within the Collagenous Domain", pages 529-536. * |
JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 263, No. 35, issued 15 December 1988, TSILIBARY et al., "Heparin Type IV Collagen Interactions Equilibrium Binding and Inhibition of Type IV Collagen Selfassembly", pages 19112-19118. * |
JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 264, No. 4, issued 05 February 1989, KOLIAKOS et al., "The Binding of Heparin to Type IV Collagen Domain Specificity with Identification of Peptide Sequence from the 1(IV) and 2(IV) which Preferentially Bind Heparin", pages 2313-2323. * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6274134B1 (en) | 1992-01-29 | 2001-08-14 | National Institutes Of Health | Human cell adhesion protein AAMP-1 and uses thereof |
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