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WO1992006112A1 - Peptides activateurs de tissus connectifs actives, leurs analogues et leur utilisation - Google Patents

Peptides activateurs de tissus connectifs actives, leurs analogues et leur utilisation Download PDF

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Publication number
WO1992006112A1
WO1992006112A1 PCT/US1991/007556 US9107556W WO9206112A1 WO 1992006112 A1 WO1992006112 A1 WO 1992006112A1 US 9107556 W US9107556 W US 9107556W WO 9206112 A1 WO9206112 A1 WO 9206112A1
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iii
ctap
peptide
des
connective tissue
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PCT/US1991/007556
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English (en)
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Paul H. Johnson
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Sri International
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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
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to peptides. More particularly, this invention concerns analogs of human connective tissue-activating peptides (CTAPs) , in particular analogs of the human connective tissue- activating peptide CTAP-III.
  • CTAPs connective tissue-activating peptides
  • CTAPs- are a group of naturally occurring polypeptides that are capable of activating connective tissue cells. These peptides are present in platelets and leukocytes and stimulate mitogenesis, glycosaminoglycan and hyaluronic acid synthesis, prostaglandin E 2 and cyclic AMP formation, plasminogen activator secretion, fibroblast chemotaxis, glucose transport and glycolysis. CTAPs are being investigated as pharmaceuticals for regenerating connective tissue (e.g., would healing). Castor, C. ., et al., PNAS (USA) (1983) 80.765-769 reports the amino acid sequence of o,ne CTAP, known as CTAP-III, and the biological characteristics of CTAP- III.
  • CTAP- III Connective tissue activating peptide III is a human platelet granule-derived growth factor found in 1000 times the quantity of other growth factors presently known to be in platelets.
  • CTAP-III stimulates synthesis of DNA, hyaluronic acid (HA) , sulfated glycosaminoglycan (GAG) chains, proteoglycan monomer and proteoglycan core protein in human synovial fibroblast cultures (1-6) . These references are listed below under “References.”
  • HA hyaluronic acid
  • GAG sulfated glycosaminoglycan
  • proteoglycan monomer proteoglycan monomer
  • proteoglycan core protein in human synovial fibroblast cultures
  • CTAP-III isolated by immunoaffinity chromatography showed significant molecular size heterogeneity by SDS PAGE when visualized by silver. stains and/or Western blotting (11) .
  • CTAP-III purified by immunoaffinity methods appeared heterogeneous by analytical isoelectric focusing (IEF) (12) .
  • This heterogeneity was detectable immediately after extraction from platelet -granules freshly obtained from individual donors as well as in pooled outdated blood bank platelets.
  • Such microheterogeneity was thought likely to have biologic significance since fractions of CTAP-III isolated by preparative IEF had varying specific activities in stimulating DNA and glycosaminoglycan synthesis in human synovial cell cultures (13) .
  • CTAP-III Two NH 2 - terminal cleavage products were identified: CTAP-III (des 1-13) and CTAP-III (des 1-15).
  • CTAP-III (des 1-13) had a pi of 8.6 and was a stable proteolytic cleavage product that retained the capacity to stimulate [ C]GAG synthesis in human synovial cell cultures.
  • CTAP-III (de 1-15) was an elastase and chymotrypsin cleavage product identical to NAP-2, an entity thought to have neutrophil activating properties (14) .
  • CTAP-III connective tissue activating peptide
  • CTAP-I lymphocyte
  • CTAP- III platelet
  • CTAP-III connective tissue activating peptide III
  • CTAP-III connective tissue activating peptide III
  • CTAP-I lymphocyte
  • CTAP- III platelet
  • CTAP-III connective tissue activating peptide-III
  • this invention concerns a peptide having human connective tissue-activating activity and the peptide sequence of human connective tissue-activating peptide III but with from 10 to 19 amino acids deleted from the NH- end.
  • this invention provides these 1-10 des through 1-19 des CTAP-IIIs with the 21- position methionine replaced with an acyclic side-chain hydrophobic amino acid such as leucine.
  • this invention provides pharmaceutical compositions for connective tissue activation made up of one or more of these peptides and the use of these compositions to bring about connective tissue activation.
  • Figure 1 is a graph showing the results of preparative IEF over a range of pH 3-10 separated CTAP-III into four major peaks, each possessing the capacity to stimulate [ 3H]DNA synthesis.
  • Figure 2 is a silver stained SDS-PAGE gel which shows two CTAP-III preparations.
  • Lane 1 contains a typical CTAP-III preparation eluted from a heparin affinity column with 0.3M NaCl.
  • Band A shows the CTAP- III sequence on icrosequencing
  • band B contains variable mixtures of small isoforms (see text) .
  • Lane 2 contains molecular weight markers
  • Lane 3 shows CTAP-III forms which on microsequencing of Immobilon blots revealed the structural alterations (CTAP-III [Asp-1] , CTAP-III [des 1-14]) as labeled in the figure.
  • Figure 3 is a pair of graphs.
  • the top panel plots the mitogenic activity (mean ⁇ S.E.M.) of the CTAP- III (des 1-14) isofor and CTAP-III at different concentrations; the lower panel records the GAG stimulating activity of the two forms.
  • the mitogenic activity of the des 1-14 isoform is not significantly different from CTAP-III.
  • the apparent reduction of GAG stimulating activity of the des 1-14 isoform was significant (P ⁇ 0.01).
  • Figure 4 is a pair of graphs in which immunoaffinity purified CTAP-III is compared to an aliquot cleaved to the des 1-15 form with porcine elastase.
  • the heparin affinity column used to separate elastase from the isoforms yielded two affinity forms of CTAP-III (des l-15)/NAP-2, (0.3M and 0.5M) .
  • Biologic activity (mean ⁇ SEM) is plotted versus increasing concentrations of the peptides. Both affinity forms had mitogenic activity similar to the parental form (P ⁇ 0.15).
  • the 0.3M form showed a significant increase in GAG stimulating activity (P ⁇ 0.01); the 0.5M form appeared similar to the parental CTAP-III in activity.
  • FIG. 5 is a pair of graphs which show the biologic activities (mean ⁇ S.E.M.) of rCTAP-III-Leu-21 and its elastase cleavage products, des 1-15, 0.3M and 0.5M heparin affinity forms plotted as a function of concentration.
  • the upper panel shows that rCTAP-III- Leu-21 had no mitogenic activity for human synovial cells; the lower panel shows that the intact recombinant molecule had very little GAG stimulating activity.
  • the GAG stimulating capacity of the des 1-15 forms was significantly enhanced compared to the intact molecule (P ⁇ 0.001). In the mitogenic assay both des 1-15 forms were significantly more active than the parent molecule (for the 0.3M isoform, P ⁇ 0.02; for the 0.5M isoform, P ⁇ 0.001) .
  • Figure 6 is a schematic representation of CTAP- III.
  • the single letter code identifies the amino acid residues in this schematic representation of CTAP-III; cleavage sites giving rise to the isoforms studied in this report are indicated.
  • CTAP-III is a peptide material of known sequence.
  • Figure 6 shows its structure.
  • the peptides of interest herein differ from CTAP-III by having at least their first 10 amino acids and at most the first 19 amino acids deleted from the NH 2 end. These materials are known as des 1-10 CTAP-III through des 1-19 CTAP-III.
  • the peptides can also have their "21" position methionine replaced with a leucine or with another acyclic side-chain, hydrophobic amino acid.
  • the parent CTAP-III material can be obtained from natural sources as known in the art or produced recombinantly.
  • the recombinant method and its application to produce native or 21-substituted materials are fully set forth in incorporated U.S. patent no. 4,897,348.
  • the products of the invention can be attained by incubating the CTAP-III or 21-substituted CTAP-III with a suitable proteolytic enzyme such as chymotrypsin or porcine elastase, or the like. Thereafter the cleavage products can be separated and isolated.
  • a suitable proteolytic enzyme such as chymotrypsin or porcine elastase, or the like.
  • the peptides of the invention have CTAP-III activity and thus can be administered, typically by injection.
  • a peptide of this invention or a pharmaceutical composition containing the same is administered to the subject in need of such treatment.
  • peptide compositions may be administered by any of a variety of routes depending upon the specific end use, including particularly parenterally (including subcutaneous, intramuscular, and intravenous administration) .
  • the materials can be administered to mammals such as humans, monkeys, dogs, rodents, and the like.
  • the compositions generally include a pharmaceutical diluent such as injectable saline, mineral oil or the like.
  • the compound or composition may also be administered by means of slow-release, depot, or implant formulations, as is well known in the art.
  • the polypeptides described herein are usually administered in amounts of 0.001 to 1000 micrograms per kg of body weight, particularly in amounts of 1-500 micrograms per kg of body weight, although higher or lower amounts may be used.
  • CTAP-III was isolated from platelet pellets by extraction into acid/ethanol and precipitation with cold acetone (1,2,4).
  • 50g of pelleted platelets were added to 500 ml of acid ethanol (5 ml 1.25 N HCl/95 ml ethanol); this was stirred slowly at 4°C for 16 hr, centrifuged (15,000g, 10 min) , and the supernatant fluid was added to 1500 ml cold (4°C) acetone.
  • Glycosaminoglycan polysulfate (Arteparon, Luitpold Werke, Jupiter, Fla.), a proteinase inhibitor, was then added at a concentration of 100 ⁇ g/ l. This resulted in prompt flocculation of several protein species including essentially all of the CTAP-III.
  • the turbid preparation was allowed to settle for 2 hr at 4H°C and then centrifuged at 12,000g; the resultant pellet was dispersed in PBS, pH 7.5, allowed to stand overnight at 4°C and then centrifuged at 12,003g and the CTAP-III rich supernatant fluid was recovered for further processing.
  • the isolation process was continued by heparin affinity and/or immunoaffinity chromatography.
  • Heparin affinity columns were made by coupling heparin (Sigma crude unbleached heparin, Sigma Chemical Co., St. Louis, MO) to Affigel 15 (Bio-Rad Laboratories, Richmond, CA) as directed by the manufacturer (15) .
  • Partially purified CTAP-III was applied to a heparin affinity column in phosphate-buffered saline, pH 7.0; CTAP-III was eluted with 0.3M sodium chloride and dialyzed against PBS.
  • Monospecific immunoaffinity isolated anti-CTAP-III IgG was prepared by passing polyclonal rabbit anti-CTAP-III antisera over an antigen column of highly purified CTAP- .
  • Protein measurement and antisera development Protein was measured by a colorimetric method (18) and/or UV absorption (19) . Antisera to both platelet-derived and recombinant CTAP-III-Leu-21 were raised in rabbits. Eight- to 10-week old male New Zealand white rabbits were immunized with 50 ⁇ g of CTAP-III or rCTAP-III in 0.15M NaCl in Freund's complete adjuvant. Booster injections were given at 6 and 12 weeks with antigens in incomplete Freund's adjuvant. Animals were bled at 4 weeks and then at biweekly intervals after initial immunization. Measurement of CTAP-III by radial immunodiffusion (RID) utilized filtered, heat inactivated rabbit anti-human CTAP-III (20) .
  • RID radial immunodiffusion
  • Analytic polyacrylamide gel electrophoresis Highly purified CTAP-III and its isoforms were analyzed by sodium dodecyl sulfate-polyacryia-iti ⁇ e gel electrophoresis (SDS-PAGE) in 8M urea/8% total acrylamide and by analytical IEF in ampholyte gradients pH 3-10 (15,21). Proteins separated by SDS-PAGE were detected by silver stain and on IEF by both silver and Coomassie Brilliant Blue R-250 staining (15,22,23).
  • Proteins were prepared for sequencing by blotting onto Immobilon-P using a semi-dry blotter (Polyblot, American Bionetics, Inc., Hayward, CA 94545) and identified by Coomassie Brilliant Blue R-250 or immunostaining with antisera to recombinant CTAP-III (15,20,24) .
  • Western blots of proteins following electrophoretic separation and immobilization in a nitrocellulose membrane were accomplished as described (15) .
  • Membrane-bound antigens were probed with antisera to CTAP-III or rCTAP-III (1:500) and the complexes detected with a Bio-Rad Immunoblot (GAR-HRP) assay kit (BioRad Laboratories, Richmond, CA) .
  • GAR-HRP Bio-Rad Immunoblot
  • Preparative isoelectric focusing of CTAP-III Platelet preparations were fractionated using the Pharmacia Flat Bed Apparatus FBE 3000 and the Electrophoresis Constant Power Supply ECPS 3000/150. Sephadex G-75M was washed with 2 x deionized water and dried. A stable pH gradient was achieved using a system of amphoteric and nonamphoteric buffers according to the method of Prestidge and Hearn (25) . The gel bed was then cut into 26 separate segments and the pH of each was measured. The protein focused in individual segments was eluted with PBS pH 7.0, concentrated, dialyzed against PBS, and stored frozen until assay.
  • Carbohydrate analysis Carbohydrate analyses were performed by methods previously described in detail (26,27). Three different acid hydrolyses were used for each sample aliquot. Peptides were incubated 1 hr at 80°C in 0.1N sulfuric acid to release sialic acid residues for assay. For analyses of neutral sugars, samples were hydrolyzed in 4N trifluoroacetic acid at 100°C for 2 hr. Analysis of amino sugars required hydrolyses in 6N HC1 at 100°C for 3 hr.
  • Sialic acid was measured by the thiobarbituric acid method; neutral sugars were converted to the corresponding glycamines by reductive amination with 0.2M sodium cyanoborohydride in 1M ammonium sulfate (100°C, 90 min) . Amino sugars and neutral sugar glycamines were separated and quantitated as described for amino acids, by cation exchange on a Kratos automated amino acid analyzer using post-column o- phthalaldehyde derivatization and fluorometric detection.
  • fibroblasts Normal human fibroblastic cells (synovium and cartilage) were developed from explants obtained at amputation or arthrotomy; fibroblasts from dermis were obtained • following reduction mammoplasty as described earlier (1,2). Cells were grown as monolayer cultures in T-75 flasks in CMRL 1066 (Gibco, Grand Island, NY) medium supplemented with 5% human serum and 15% fetal calf serum (FCS) , sodium bicarbonate, L-glutamine, 0.02M Hepes buffer, penicillin, streptomycin and genta icin. Trypsin dispersal was performed to facilitate cell propagation, study, and preparation for cold storage.
  • FCS fetal calf serum
  • rCTAP-III Recombinant CTAP- III-Leu-21, an analogue containing a leucine substitution for methi'onine at position 21, was produced from a synthetic gene using an E. coli expression system, pNP6, based on the genetic regulatory elements of the colicin El operon (30,31) . .
  • Six liter shake-flask cultures were grown to an optical density (650 nm) of 0.4 and gene expression was induced by addition of mitomycin C to the culture at a final concentration of 0.5 ⁇ g/ml for 4 hr.
  • the protein reaction mixture was dialyzed against 0.1N acetic acid and partially purified by gel filtration chromatography using Sephacryl-200 (Pharmacia) . Fractions containing rCTAP-III-Leu-21 were identified by polyacrylamide gel electrophoresis, pooled and lyophilized.
  • the protein was dissolved in 50 mM Tris buffer, pH 8.5 containing 6M guanidinium hydrochloride at a concentration of 0.2 mg per ml. Protein folding was initiated by the addition of a redox agent to a final concentration of 2 mM oxidized glutathione and 1 mM reduced glutathionine and dialyzed against 100 volumes of buffer without GndHCl for 12 hr at room temperature.
  • rCTAP-III- Leu-21 refolded to the native conformation as verified by analysis using analytical reverse-phase HPLC and purified platelet-derived CTAP-III as a control. Finally, the rCTAP-III-Leu-21 was purified to greater than 95% purity by heparin affinity chromatography using sodium chloride gradient elution.
  • Isoelectric point (pi) microheterogeneity Highly purified, biologically active CTAP-III was separated by preparative IEF. Fractions were examined for total protein content, CTAP-III content by RID, purity by SDS-PAGE and biologic activity was measured in human synovial cell cultures. Mitogenic activity of the IEP (isoelectric point) variants of CTAP-III is shown in Figure 1; previously we showed a similar biologic activity profile by measuring the incorporation of [ C]glucosamine into [ C]HA (12) . An analytical IEF gel (inset, Figure 1) suggests that the preparative fractions are mixtures of charge isomers. Data summarized in Figure 1 suggest that the specific biologic activity of CTAP-III varies with the IEP of the different fractions. Three additional preparative IEF experiments generated similar data. The relative magnitude of two CTAP-III anabolic activities, measured as enhanced GAG and DNA synthesis, were concordant in the different fractions.
  • CTAP-III Glycosylation of CTAP-III: To explain some of the IEP heterogeneity we examined immunoaffinity isolated CTAP-III for covalently linked carbohydrate before and following acid hydrolysis. A mixture of CTAP-III variants not separated by IEF was studied. Unhydrolyzed CTAP-III (4.76 nmole based on amino acid composition) contained 0.6 nmole glucose; acid hydrolyzed CTAP-III had 10.0 nmole glucose/4.76 nmole CTAP-III. Therefore, this mixture of CTAP-III IEP variants contained 1.97 nmole of covalently linked glucose/nmole CTAP-III. Only glucose was detected; no galactose or amino sugars were found.
  • CTAP-III Seven additional biologically active samples, including four IEP species of CTAP-III (from two separate preparative IEF studies) , were examined by the same procedures (see Table 1) . Glucose and lysine content of the four separate CTAP-III charge-isomers is shown. The amino acid composition of CTAP-III charge-isomers was examined prior to and following treatment with sodium borohydride to stabilize sugar-lysyl residue Schiff base adducts.
  • Lysyl residues not determined by virtue of borohydride treatment are an estimate of sugar-lysyl adducts and are labeled "% lysine glycosylated.”
  • bound glucose ranged from 1.97 to 2.81 nmole glucose/nmole CTAP-III.
  • Glycosylation of CTAP-III occurs commonly, varies with the preparation and was not clearly related to pi or biologic activity. Glycosylation was not required for biologic activity; one nonglycosylated preparation (PI861 S 5-I M) stimulated [ C]HA synthesis by over 900%.
  • CTAP-III Amino-terminal Deamidation, (CTAP-III [Asp- 1]): CTAP-III (Asp-1) was detected by microsequencing the band from an Immobilon blot of an analytical IEF gel at the pi 7.0 locus, as well as in the SDS-PAGE gel shown in Figure 2.
  • CTAP-III was detected by microsequencing the band from an Immobilon blot of an analytical IEF gel at the pi 7.0 locus, as well as in the SDS-PAGE gel shown in Figure 2.
  • CTAP-III was detected by microsequencing the band from an Immobilon blot of an analytical IEF gel at the pi 7.0 locus, as well as in the SDS-PAGE gel shown in Figure 2.
  • Our earlier studies showed asparagine to be the amino terminal residue of CTAP-III (3,4); further, the gene for human CTAP-III was recently shown to code for an amino terminal asparagine residue (32) .
  • CTAP-III (des 1-13) was identified in purified preparations of CTAP-III recovered from prolonged cold storage (11) . SDS-PAGE gels showed a single silver stained band which immunostained with anti- rCTAP-III in a Western blot. The apparent molecular weight of the isoform was 6200 Da, the pi was 8.6 and NH 2 -terminal sequencing showed:
  • CTAP-III (des 1-13) was the same as that for intact CTAP-III.
  • CTAP-III (des 1-13) stimulated synthesis of [ 14C]HA in human synovial cell cultures with a specific activity similar to that attributed to the parent molecule; interestingly, no mitogenic activity was detected.
  • CTAP-III (des 1-14) was identified for the first time during the present study as an isoform which failed to bind to a heparin column. After isolation from an immunoaffinity column, an aliquot of this material was separated by SDS-PAGe. A Western blot of CTAP-III antigen using antik-rCTAP-III antibody then showed the major reactive species to have a molecular weight of 6500-7000 Da; a minor fraction had a molecular weight of 9300 Da ( Figure 2) . Amino terminal sequencing of the bands from an Immobilon blot showed that the larger protein, representing 25% of the total, was CTAP-III (Asp-1) . The 6500 Da fragment was sequenced through 10 cycles which showed:
  • CTAP-III Asp-l
  • CTAP-III Asp-l
  • CTAP-III (des 1-14) stimulated by 50-60 percent the incorporation of [ 35 S0 4 ] into [ 35 S]GAG formed in human chondrocyte cultures.
  • the 75%/25% mixture of CTAP- III (des 1-14) /CTAP-III (Asp-l) contributes some degree of ambiguity to the activity studies reported here.
  • CTAP-III (Asp-l) to be responsible for the observed activity, its specific activity would have to be much greater than the parent (control) molecule.
  • CTAP-III (Asp-l) appears to have "normal” specific biologic activity; therefore it seems unlikely that this minor component could account for all of the biologic activity shown in Figure 3. Consequently, we attribute the major portion of the biologic activity of this preparation to CTAP-III (des 1-14) .
  • CTAP-III (des 1-15) was detected in platelet- derived CTAP-III preparations as a small isoform (11) .
  • NH 2 -terminal sequencing of the electrophoretically- separated band blotted onto Immobilon showed:
  • CTAP-III (des 1-10) was detected when microsequencing a small isoform band blotted onto Immobilon. This band contained approximately 20 picomoles of CTAP-III (des 1-14) , 20 pico oles of CTAP- III (des 1-15) and 10 picomoles of CTAP-III (des 1-10) .
  • the (des 1-10) variant should probably be considered a naturally-occurring form in platelet extracts. it has not yet been possible to test the biologic activity of this form as a single entity.
  • CTAP-III node 1-14
  • des 1-10 the present application provides evidence for several naturally-occurring isoforms of CTAP-III not previously known, including CTAP-III (des 1-14) , and des 1-10. Further, these studies show that CTAP-III (des 1- 14) and CTAP-III (des 1-15) retain or enhance the anabolic biologic properties of uncleaved CTAP-III. Recombinant CTAP-III-Leu-21 clearly acquired increased specific activity with respect to stimulating DNA and GAG synthesis after cleavage to the des 1-15 form.
  • the structural relationships of the CTAP-III cleavage isoforms to CTAP-III and ⁇ -TG are illustrated in Figure 6 (4,33). The carboxyterminus of these CTAP-III isoforms described in detail was found to be intact.
  • CTAP-III and its isoforms cannot be avoided during organic extraction and conventional molecular sieve chromatography. Further, most of the isoforms bind to heparin affinity columns and all bind to immunoaffinity columns. The fact that all of the isoforms discussed in this report react with polyvalent antisera raised against CTAP-III has one unfortunate consequence: published studies of plasma CTAP-III/ ⁇ -TG antigen levels measured by RIA or ELISA have much less specificity than once thought.
  • CTAP-III and CTAP-III (des l-15)/NAP-2 have recently been separated by reverse-phase HPLC with a gradient of acetonitrile in 0.1% trifluoroacetic acid (34) .
  • HPLC offers an attractive approach to separating CTAP-III from its isoforms if it is accomplished without modifying the biologic activities of the proteins. This might allow separate testing of the platelet-derived CTAP-III and its isoforms and permit the "activation by cleavage experiment" described above for rCTAP-III-Leu- 21.
  • Immunoaffinity isolated CTAP-III exhibits significant microheterogeneity as determined by SDS-PAGE and IEF analytical methods.
  • Table 3 shows that the calculated IEPs of known and hypothetical members of the CTAP-III family range from about 7 to 9.3; measured values agree reasonably well with the predicted IEPs. These data account for a substantial portion of the observed IEP heterogeneity and identify some of the cationic forms possessing increased specific biologic activity. Nonenzymatic glycosylation of lysine in CTAP- III was considered as a cause for IEP heterogeneity.
  • Table 3 shows that pi values computed for hypothetical glycosylated CTAP-III isoforms appear to support this hypothesis. Experimentally we found that six of eight CTAP-III preparations contained modest amounts of covalently-bound glucose.
  • PF-4 platelet ⁇ -granule protein
  • PF-4 has extensive homology and different actions; it is noted for its heparin neutralizing properties, chemotactic activity, and has been shown to be an immunoregulator which reverses immunosuppression in mice (35) . It is pertinent that the immunoregulatory activity of PF-4 is protease-induced immediately after platelet aggregation.
  • a monocyte-derived protein (MDNCF/NAP-1/I1-8) has extensive homology with CTAP-III and is a potent neutrophil chemotactic agent (36) .
  • Chick fibroblasts, activated by serum or Rous sarcoma virus (RSV) are induced to form the 9E3 protein which is homologous to CTAP-III (37,38) .
  • RSV-transformed cells form copious amounts of hyaluronic acid, as do human fibroblasts stimulated by CTAP-III.
  • a tumorigenic hamster cell line produces increased amounts of mRNA coding for a protein (CHEF-GRO) with homology to CTAP-III (39) .
  • Recombinant gamma-interferon treated human fibroblasts, monocytes and endothelial cells exhibit induction of a gene which codes for a protein, gamma-IP-10, that also shows homology to CTAP-III and PF-4 (40) .
  • MGSA Melanoma growth stimulatory activity isolated from Hs294T melanoma cells shows striking homology to CTAP-III and is a potent mitogen which exists in two molecular weight forms (41) .
  • These 10 to 19-des CTAP-III amino acid sequence homologies reflect a family of materials which may play roles in inflammation, wound healing and growth (37) , and may be derived from a common ancestral gene.
  • Numbering of residues is from (4) .

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Abstract

On décrit l'activité de peptides activiateurs de tissus connectifs (CTAP en anglais) humains. Ces peptides se différencient des CTAP-III natifs en ce qu'ils ont les premiers acides aminés de 10 à 19 supprimés de leur extrémité HN2. De plus, ils peuvent éventuellement avoir une substitution à la position correspondant à la 21-méthionine de CTAP-III. Ces peptides sont plus actifs que les CTAP-III.
PCT/US1991/007556 1990-10-09 1991-10-09 Peptides activateurs de tissus connectifs actives, leurs analogues et leur utilisation WO1992006112A1 (fr)

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Cited By (1)

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 159, No. 3, issued 31 March 1989, A. WALZ et al., "A Novel Cleavage Product of B-Thromboglobulin Formed in Cultures of Stimulated Mononuclear Cells Activates Human Neutrophils", pages 969-975. *
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 163, No. 2, issued 15 September 1989, C.W. CASTOR et al., "Connective Tissue Activation XXXIII. Biologically Active Cleavage Products of CTAP-III From Human Platelets", pages 1071-1078. *
EUROPEAN JOURNAL OF IMMUNOLOGY, Volume 20, issued September 1990, J. VAN DAMME et al., "The Neutrophil-activating proteins interleukin 8 and B-thromboglobulin: in vitro and in vivo comparison of NH2 -terminally processed forms", pages 2113-2118. *

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US5705360A (en) * 1993-11-12 1998-01-06 Dana-Farber Cancer Institute Chemokine N-terminal deletion mutations
US5739103A (en) * 1993-11-12 1998-04-14 Dana-Farber Cancer Institute Chemokine N-terminal deletion mutations
US5854412A (en) * 1993-11-12 1998-12-29 Dana-Farber Cancer Institute Chemokine N-terminal deletion mutations
WO1996038559A1 (fr) * 1995-05-31 1996-12-05 Dana Farber Cancer Institute Mutations de deletion de l'extremite n de chemokine
EP1647597A1 (fr) * 1995-05-31 2006-04-19 Dana Farber Cancer Institute Mutations de deletion de l'extremite N de chemokine

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