WO1995028955A1 - PEPTIDES SE LIANT A LA PROTEINE ASSOCIEE AU RECEPTEUR DE MACROGLOBULINE-α2/RECEPTEUR DE LIPOPROTEINE DE FAIBLE DENSITE - Google Patents

PEPTIDES SE LIANT A LA PROTEINE ASSOCIEE AU RECEPTEUR DE MACROGLOBULINE-α2/RECEPTEUR DE LIPOPROTEINE DE FAIBLE DENSITE Download PDF

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Publication number
WO1995028955A1
WO1995028955A1 PCT/DK1995/000161 DK9500161W WO9528955A1 WO 1995028955 A1 WO1995028955 A1 WO 1995028955A1 DK 9500161 W DK9500161 W DK 9500161W WO 9528955 A1 WO9528955 A1 WO 9528955A1
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Prior art keywords
lipoprotein
lpl
peptide
lrp
interaction
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PCT/DK1995/000161
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English (en)
Inventor
Jørgen GLIEMANN
Anders Nykjaer
Morten Schallburg Nielsen
Gunilla Olivecrona
Original Assignee
Gliemann Joergen
Anders Nykjaer
Morten Schallburg Nielsen
Gunilla Olivecrona
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Application filed by Gliemann Joergen, Anders Nykjaer, Morten Schallburg Nielsen, Gunilla Olivecrona filed Critical Gliemann Joergen
Priority to AU23429/95A priority Critical patent/AU2342995A/en
Publication of WO1995028955A1 publication Critical patent/WO1995028955A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to pharmaceutical compositions containing peptides capable of binding to the ⁇ 2-macroglobulin receptor/low density lipoprotein receptor-related protein, as well as a method of using such peptides in therapy.
  • LPL Lipoprotein lipase
  • VLDL very low density lipoprotein
  • C.2-MR/LRP ⁇ 2-macroglobulin receptor/low density lipoprotein receptor-related protein
  • Lipoprotein lipase is a non-covalent homodimer of 450 (bovine) or 448 (human) amino acid residues, the monomers each containing an N-terminal folding domain including the catalytic site, and a C-terminal folding domain (for a review, see 1) .
  • the 2-macroglobulin receptor/low density lipoprotein receptor- related protein c.2-__R/LRP
  • c.2-___R/LRP consists of a membrane-spanning 85 kD /3-chain and a 500 kD extracellular and ligand-binding ⁇ - chain (2,3) .
  • C.2-MR/LRP binds and mediates the uptake of several unrelated ligands including ⁇ 2 -macroglobulin/proteinase complexes through receptor-mediated endocytosis (3,4,5).
  • Three established ligands are bound to other molecules at the cell surface before uptake via LRP: complex of plasminogen activator inhibitor-type 1 and urokinase-type plasminogen activator binds to the urokinase receptor (6) , and LPL and apoE bind to proteoglycans (7,8,9) .
  • chylomicron remnants and VLDL are taken up through the C.2-MR/LRP when associated with multiple LPL molecules (7,11) or when activated by apolipoprotein E (apoE) (12,13) .
  • apoE apolipoprotein E
  • Earlier studies have suggested LRP binding to the C-terminal domain of LPL (7) .
  • Recent results suggest that the LPL-mediated uptake through the C-2- R/LRP in cells of the arterial wall may be important for the pathogenesis of atherosclerosis.
  • LPL is expressed in subsets of smooth muscle cells and macrophages of human atherosclerotic lesions (14) .
  • uptake of lipoprotein cholesteryl ester in arterial smooth muscle cells is markedly increased by the addition of LPL, but not apoE (15) , and inbred mice exhibiting increased macrophage LPL secretion develop atherosclerosis (16) .
  • ⁇ 2 - R/LPL is expressed in the two cell types of the human arterial wall which may develop into foam cells; macrophages in atherosclerotic lesions express ⁇ 2- MR/LRP and the scavenger receptor mediating uptake of oxidised LDL; smooth muscle cells in atherosclerotic lesions express ⁇ 2- MR/LRP, but neither the scavenger receptor nor the LDL receptor.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a peptide capable of binding to the ⁇ 2- macroglobulin receptor/low density lipoprotein receptor-related protein ( ⁇ 2-MR/LRP) such as to inhibit any interaction between the C.2-MR/LRP and a lipoprotein or lipoprotein lipase or a complex of a lipoprotein and a lipoprotein lipase, said interaction resulting in the uptake of lipoprotein in mammalian cells, together with a pharmaceutically acceptable diluent or carrier.
  • ⁇ 2-MR/LRP macroglobulin receptor/low density lipoprotein receptor-related protein
  • the present invention relates to a method for the prevention or treatment of diseases or conditions involving interaction between the ⁇ 2-MR/LRP and a lipoprotein or a lipoprotein lipase or a complex of a lipoprotein and a lipoprotein lipase, said interaction resulting in the uptake of lipoprotein in mammalian cells, the method comprising administering, to a patient in need thereof, an effective amount of a peptide capable of binding to the C.2-MR/LRP so as to substantially inhibit said interaction between the ⁇ 2-MR/LRP and the lipoprotein or the lipoprotein lipase or the lipoprotein/lipoprotein lipase complex.
  • the present invention relates to the use of a peptide capable of binding to the c.2-macroglobulin receptor/low density lipoprotein receptor-related protein ( ⁇ 2- MR/LRP) such as to inhibit any interaction between the ⁇ 2- MR/LRP and a lipoprotein or lipoprotein lipase or a complex of a lipoprotein and a lipoprotein lipase, for the preparation of a medicament for the prevention or treatment of diseases or conditions involving interaction between the C.2-MR/LRP lipoprotein or lipoprotein lipase or a complex of a lipoprotein and a lipoprotein lipase, said interaction resulting in the uptake of lipoprotein in mammalian cells.
  • ⁇ 2- MR/LRP c.2-macroglobulin receptor/low density lipoprotein receptor-related protein
  • the peptide is preferably one which is capable of binding to ⁇ 2-MR/LRP such as to inhibit any interaction between the ⁇ 2- MR/LRP and a lipoprotein or lipoprotein lipase or a complex of a lipoprotein and a lipoprotein lipase, said interaction resulting in the uptake of lipoprotein in cells expressing ⁇ 2- MR/LRP, such as smooth muscle cells or macrophages.
  • the peptide is preferably a fragment of a lipoprotein lipase (LPL) or a functional homologue thereof.
  • the term "functional homologue” is intended to indicate a peptide derived from a homologous lipase, e.g. hepatic lipase, or a multiplicity of identical peptides coupled to a suitable carrier protein, or a peptide in which one or more a ino acid residues have been substituted at one or more sites in the peptide sequence, or one or more amino acid residues have been deleted at one or more sites in the peptide sequence, or one or more amino acid residues have been inserted at one or more sites of the peptide sequence, provided that the peptide retains the ability to bind to the C.2-MR/LRP and inhibit the uptake of lipoproteins into cells through this receptor.
  • the peptide may also be a chemical derivative of an LPL fragment.
  • An example of a suitable chemically derivatized peptide is hLPL 378-448 wherein SH groups (Cys 418 and Cys 438 ) have been chemically blocked, e.g. by means of iodoacetamide. This derivative is active in terms of inhibiting LpL binding to ⁇ 2 -MR/LRP.
  • a fragment of LPL capable of binding to the ⁇ 2-MR/LRP according to the invention has been found in the C-terminal domain of LPL.
  • the parent LPL may suitably be derived from a variety of sources, such as from bovine, porcine, murine or human LPL. However, when the composition of the invention is intended for administration to human beings, the parent LPL is preferably of human origin. Examples of suitable peptides derived from human LPL are a peptide comprising amino acids 378-448, or amino acids 378-423 of human LPL.
  • the peptide included in the composition of the invention may for instance be prepared by peptide synthesis or by recombinant DNA techniques in a manner known per se.
  • the peptide may be prepared by conventional methods of solution or solid phase peptide synthesis.
  • solid phase synthesis may be carried out substantially as described by Stewart and Young, Solid Phase Peptide Synthesis. 2nd. Ed., Rockford, Illinois, USA, 1976.
  • Solution peptide synthesis may for instance be carried out substantially as described by Bodansky et al., Peptide Synthesis. 2nd. Ed., New York, New York, USA, 1976.
  • Peptide synthesis is particularly advantageous for the preparation of shorter peptides.
  • the peptide may be prepared by recombinant DNA techniques involving insertion of a DNA construct coding for the peptide into a suitable expression vector, transformation of a suitable host cell with the vector and cultivation of the transformed host cell under conditions permitting production of the peptide.
  • the DNA construct encoding the present peptide may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22. 1981, pp. 1859-1869, or the method described by Matthes et al., EMBO Journal 3. 1984, pp. 801-805.
  • phosphoamidite method oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
  • the DNA construct may also be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the LPL protein by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al.. Molecular Clonin ⁇ : A Laboratory Manual. 2nd Ed., Cold Spring Harbor, 1989) .
  • a genomic or cDNA sequence encoding the entire LPL protein may be digested with one or more suitable restriction endonucleases, and a DNA fragment encoding the desired peptide may be identified in a assay for binding to ⁇ 2-MR/LRP as described in (7) .
  • the DNA sequence encoding the peptide may be further modified at a site corresponding to the site(s) at which it is desired to introduce amino acid substitutions, e.g. by site-directed mutagenesis using synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination in accordance with well-known procedures.
  • suitable mo ⁇ difications of the DNA sequence are nucleotide substitutions which do not give rise to another amino acid sequence of the present peptide, but which correspond to the codon usage of the host organism into which the DNA construct is introduced or nucleotide substitutions which do give rise to a different amino acid sequence and therefore, possibly, a different pro- tein structure without, however, impairing the properties of the native variant.
  • Other examples of possible modifications are insertion of one or more nucleotides into the sequence, or deletion of one or more nucleotides at either end or within the sequence.
  • the DNA construct encoding the present peptide may then be inserted into a suitable expression vector which may be any vector conveniently subjected to recombinant DNA procedures.
  • a suitable expression vector which may be any vector conveniently subjected to recombinant DNA procedures.
  • the choice of vector will depend on the kind of host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the DNA sequence encoding the present peptide should be operably connected to a suitable promoter sequence (i.e. operably linked to the promoter sequence in the proper reading frame) .
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • Suitable promoters for use in prokaryotic host cells include, e.g. the promoter of the Bacillus stearothermophilus maltogenic amylase gene, Bacillus licheniformis ⁇ -amylase gene, Bacillus amyloli ⁇ uefaciens BAN amylase gene, Bacillus subtilis alcaline protease gene, or Bacillus pumilus xylosidase gene, or by the phage Lambda P R or P L promoters, or the E__. coli lac promoter, the ⁇ -lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.
  • Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Che . 255. 1980, pp. 12073-12080; Alber and Kawasaki, J. Mol. APPI. Gen. 1 , 1982, pp. 419-434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microor ⁇ ani ⁇ - . for Chemicals (Hollaender et al, eds.). Plenum Press, New York, 1982), or the TPI1 (US 4, 599, 311) or ADH2-4c (Russell et al.. Nature 304. 1983, pp. 652-654) promoters.
  • Suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al.. The EMBO J. 1, 1985, pp. 2093- 2099) or the tpiA promoter.
  • suitable promoters for directing the transcription of the DNA encoding the peptide of the invention in mammalian cells are the SV 40 promoter (Subramani et al., Mol. Cell Biol. 1 , 1981, pp. 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al.. Science 222. 1983, pp. 809-814) or the adenovirus 2 major late promoter.
  • the expression vector may also include other control sequences such as an operator, riboso e binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
  • the DNA construct encoding the present peptide may be preceded by a ribosome binding site of of the Bacillus stearothermophilus altogenic amylase gene, Bacillus lichenifor is ⁇ -amylase gene.
  • the DNA sequence encoding the peptide of the invention may also be operably connected to a suitable terminator, such as (for mammalian cells) the human growth hormone terminator (Palmiter et al., op. cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) promoters.
  • the vector may further comprise elements such as polyadenylation signals (e.g. from SV 40 or the adenovirus 5 Elb region) , transcriptional enhancer sequences (e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs) .
  • a DNA sequence encoding a signal peptide may be inserted prior to the peptide- encoding sequence.
  • the recombinant expression vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a sequence when the host cell is a mammalian cell expressing the SV40 T-antigen, e.g. COS-1 or COS-7 cells
  • the SV 40 origin of replication or (when the host cell is a yeast cell) the yeast plasmid 2 ⁇ replication genes REP 1-3 and origin of replication.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, e.g. the Schizosaccharomvces pombe TPI gene (described by P.R.. Russell, Gene 40. 1985, pp.
  • a gene which confers resistance to a drug e.g. methotrexate, neomycin, hygromycin, ampicillin, kana ycin, chloramphenicol or tetracyclin.
  • a drug e.g. methotrexate, neomycin, hygromycin, ampicillin, kana ycin, chloramphenicol or tetracyclin.
  • the host cell into which the expression vector of the invention is introduced may be any cell which is capable of producing the present peptide and is preferably a bacterial, yeast, fungal or mammalian cell.
  • the host cell used in the process of the invention may be any suitable bacterium which, on cultivation, produces large amounts of the desired peptide.
  • suitable bacteria may be grampositive bacteria such as Bacillus subtilis. Bacillus licheniformis. Bacillus lentus. Bacillus brevis. Bacillus stearothermophilus. Bacillus alkalophilus. Bacillus amylolicruefaciens. Bacillus coagulans. Bacillus circulans. Bacillus lautus or Streptomvces lividans. or gramnegative bacteria such as Escherichia coli. In JE__ coli. the peptide is typically produced in the form of inclusion bodies.
  • the trans ⁇ formation of the bacteria may for instance be effected by protoplast transformation or by using competent cells in a manner known per se (cf. Sambrook et al.. Molecular Cloning. A Laboratory Manual. 2nd Ed. , Cold Spring Harbor, NY, 1989) .
  • the yeast organism used as the host cell according to the invention may be any yeast organism which, on cultivation, produces large quantities of the present peptide.
  • suitable yeast organisms are strains of the yeast species Saccharomvces cerevisiae. Saccharo yces retevveri. Schizosaccharomvces pombe or Saccharomyces uvaru .
  • the transformation of yest cells may for instance be effected by protoplast formation followed by transformation in a manner known per se.
  • fungal cells may be used as host cells of the invention.
  • suitable fungal cells are cells of filamentous fungi, e.g. Aspergillus spp.
  • suitable mammalian cell lines are the COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATCC CCL 10) or CHO (ATCC CCL 61) cell lines.
  • Methods of transfecting mammalian cells and expressing DNA sequences introduced into the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159. 1982, pp. 601-621; Southern and Berg, J. Mol. Appl. Genet, i, 1982, pp. 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79. 1982, pp. 422-426; igler et al., Cell 14, 1978, p.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing bacterial, yeast, fungal or mammalian cells, depending on the choice of host cell.
  • the peptide may be recovered from the culture medium by conventional procedures including separating the cells from the medium by centrifugation or filtration, if necessary after disrupting the cells to release an intracellular component (such as inclusion bodies) , precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulfate, purification by a variety of chro atographic procedures, e.g. ion exchange chro atography or affinity chromatography, or the like.
  • the peptide may be formulated by any of the established methods of formulating pharmaceutical compositions, e.g. as described in Remington's Pharmaceutical Sciences r 1985.
  • the composition may typically be in a form suited for systemic injection or infusion and may, as such, be formulated with sterile water or an isotonic saline or glucose solution.
  • the compositions may be sterilized by conventional sterilization techniques which are well known in the art.
  • the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration.
  • the composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • concentration of the peptide in the composition may vary widely, i.e. from less than about 0.5%, such as from 1%, to as much as 15-20% by weight.
  • a unit dosage of the composition may typically contain from about 0.1 to about 500 mg of the present peptide.
  • the present peptide is contemplated to be advantageous to use for therapeutic applications where inhibition of the interaction between LPL and C.2-MR/LRP is desired.
  • the peptide is suggested for the prevention or treatment of atherosclerosis, in that the peptide has been found capable of inhibiting the uptake of lipoprotein into cells.
  • the dosage of the peptide administered to a patient will vary with the type and severity of the condition to be treated, but is generally in the range of 1-100 mg/kg body weight.
  • Fig. 1 top panel, is a proposed model for the interaction of LPL with the C.2-MR/LRP c_-chain, proteoglycans (pg) and triglyceride-rich lipoproteins (VLDL) .
  • the C-terminal domains of the LPL dimers are drawn in black.
  • the clusters of 5 complement- type repeats of the C.2-MR/LRP ⁇ -chain are highlighted and the NPxY sequences of the .-chain, important for endocytosis via coated pits, are indicated.
  • the LPL fragment (filled circle) competes with with LPL for binding to C.2-MR/LRP (left, arrow) and for the heparan 10 sulfate part of binding thought to occur via the C-terminal (middle, small arrow) , whereas association of LPL with VLDL remains unperturbed (right) .
  • Fig. 2 shows the binding of 125 I- 2-MR/LRP to fusion proteins containing the present peptides slot-blotted onto PVDF 15 membranes, and lack of 125 I-VLDL binding to hLPL 378'448 containing fusion proteins.
  • Example 1 Production of recombinant fusion proteins containing C-terminal fragments of human lipoprotein lipase (hLPL)
  • a BamHI restriction site and a sequence coding for the factor X substrate site was introduced at the 5'-end, and at the 3'-end a stop codon as well as a Hind III restriction site was introduced.
  • the reactions were carried out in 50 ⁇ l Taq polymerase buffer (Perkin Elmer) containing:
  • the reaction mixture was overlaid with PCR oil and amplified on a Hybaid thermo cycler according to the following scheme: Cycle 1-3: Denaturation at 93°C for 1 minute Annealing at 50 ⁇ C for 30 seconds Extension at 72 ⁇ C for 30 seconds Cycle 3-24: Denaturation at 93°C for 1 minute Annealing at 60°C for 30 seconds
  • PCR products were extracted with phenol/chloroform, digested with 5 units BamHI (Amersham) and 5 units Hindlll (Amersham) for one hour and purified on a 0.8% low melting agarose (Seaplaque GTG) .
  • the fragments were ligated (18) into the E. coli T7 expression vectors (19,20) pT7H6FX, pT7CIIH6FX and pT7CIIMLCH6FX.
  • H6FX refers to the hexahistidine-Factor X substrate sequence MGSH6SIEGR.
  • CII refers to the N-terminal 30 amino acids of the lambda CII phage protein
  • MLC refers to the N-terminal 116 amino acids of chicken myosin light chain.
  • the ligated products were transformed into XL1 Blue competent cells (Stratagene) and grown on an ampicillin (4 ⁇ g/ml) agar plate (18) .
  • the plasmid constructs were amplified in 50 ml LB medium. An aliquot (1 ml) was stored at -80°C in 15% glycerol, and the rest was used for DNA purification using Quiagene's midi-prep kit. Expression of recombinant peptides.
  • LB medium supplemented with 4 ⁇ g a picilin pr ml medium
  • the expression in E. coli was then initiated with T7 lambda phage (21) . After four hours of expression, the cells were harvested in a Sorvall GS-3 rotor after centrifugation for 10 min at 4000 rpm.
  • the cell pellet was resuspended in 30 ml 0.5 M NaCl, 50 mM TRIS-base and 10 mM EDTA, pH 8, and mixed with 50 ml of phenolsaturated with TRIS buffer. After strong sonication for 3 min, the phenol phase was separated by centrifugation in a Sorvall GS-3 rotor for 20 minutes at 8000 rpm. Protein was precipitated from the phenol phase by the addition of 2.5 volumes absolute ethanol and collected by centrifugation in a Sorvall GS-3 rotor for 10 minutes at 6000 rpm.
  • the protein was resuspended in 25 ml 6 M guanidium hydrochloride, 50 mM TRIS- base, pH 8, and 0.1 M dithiotreitol.
  • the buffer was changed to 8 M urea, 50 mM TRIS-base, pH 8, 0.5 M NaCl and 10 mM ⁇ - mecaptoethanol by use of a G25 gel filtration column (Pharmacia) .
  • the protein was loaded onto a Ni-NTA column (19,20) and washed with the above buffer until OD was constant.
  • Example 2 Assay for binding of LPL fragments to ⁇ R/LRP.
  • ⁇ -MR/LRP is a two chain receptor with a 85 kDa membrane spanning .-chain and a 500 kDa non-covalently attached extracellular and ligand binding ⁇ -chain.
  • purified 125 I-labeled ⁇ 2 MR/LRP either immobilized or in solution, is capable of binding ligands with high affinity (7) . This property provides the basis for procedures to measure binding of LPL fragments to the purified receptor.
  • 2 MR/LRP was purified from human placenta and 125 I-labeled as described in detail (3,4,7) .
  • the membranes were blocked by incubation in buffer containing 5% bovine serum albumin, 150 mM NaCl, 2 mM CaCl 2 , 50 mM Tris, pH 7.8, for 2 hours at 20°C, washed, and incubated with 50 pM 125 I- ⁇ 2 MR/LRP for 16 hours at 4°C in 140 mM NaCl, 10 mM Hepes, 2 mM CaCl 2 , 1 mM MgCl 2 , 1% bovine serum albumin, pH 7.8 (buffer a). After washing, autoradiography (1-4 days) was performed using Hyperfilm (Amersham) . The reaction is taken as a semiquantitative measure of cinding activity of the immobilized fragment.
  • Half-maximal inhibition taken as a measure of affinity, was about 200 nM for C ⁇ MLC-LpL 378 ' 448 and C jj MLC-LpL 378"423 . This was not different from the apparent affinity of onomeric bovine LPL prepared by treatment with guanidinium hydrochloride (7) .
  • the cells were centrifuged after washing steps, solubilized in 20 mM ethylmorpholin and 5% SDS followed by SDS-PAGE and autoradiography.
  • 125 I-C ⁇ -LPL 378'448 bound to a protein corresponding to the location of ⁇ 2 MR/LRP on the SDS gel, and the binding was abolished in the presence of excess fusion protein.
  • LPL concentrated on the cell surface via binding to heparan sulfate proteoglycans (1,7,9), can mediate cellular binding and uptake of triglyceride-rich lipoproteins since it is capable of binding to lipoprotein on one hand and to ⁇ -MR/LRP on the other (7,11) . Since the C-terminal LPL fragment bound to purified and cellular receptor, but not to lipoprotein, it might function as an inhibitor of LPL-mediated lipoprotein uptake. Rabbit /3-VLDL, a chylomicron remnant surrogate, was used as model lipoprotein (11-13) .
  • the FH fibroblasts were first incubated for 6 h at 37°C with 20 ⁇ g unlabeled /3-VLDL protein/ml, 2 nM bovine LpL and with or without 1 ⁇ M C ⁇ -LPL 378"448 or C ⁇ MLC-LPL 378"448 . 14 C-oleate (0.4 ⁇ Ci/ml, NEN) was then added for an additional 2 hour incubation at 37°C. Parallel incubations were performed in the absence of 2 nM bovine LPL.
  • the fusion proteins containing LPL 378" 448 were found to inhibit the LPL induced incorporation of 14 C- oleate in the presence of /3-VLDL by 60-80% (cf. Fig. 3, bottom panel) .

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Abstract

La présente invention se rapporte à des compositions pharmaceutiques contenant des peptides pouvant se lier à la protéine associée au récepteur de macroglobuline-α2/récepteur de lipoprotéine de faible densité (MR-α2/LRP), ainsi qu'à un procédé de prévention ou de traitement d'états impliquant l'interaction entre MR-α2/LRP et une lipoprotéine ou une lipoprotéine-lipase ou un complexe lipoprotéine/lipoprotéine-lipase.
PCT/DK1995/000161 1994-04-22 1995-04-18 PEPTIDES SE LIANT A LA PROTEINE ASSOCIEE AU RECEPTEUR DE MACROGLOBULINE-α2/RECEPTEUR DE LIPOPROTEINE DE FAIBLE DENSITE WO1995028955A1 (fr)

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WO1998051788A2 (fr) * 1997-05-12 1998-11-19 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Procede et construction pouvant inhiber une migration cellulaire
WO2001015729A1 (fr) * 1999-08-27 2001-03-08 Fibrogen, Inc. Recepteur du facteur de croissance du tissu conjonctif, agonistes et antagonistes dudit recepteur, et leurs utilisations therapeutiques et diagnostiques

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO1998051788A2 (fr) * 1997-05-12 1998-11-19 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Procede et construction pouvant inhiber une migration cellulaire
WO1998051788A3 (fr) * 1997-05-12 1999-05-20 Tno Procede et construction pouvant inhiber une migration cellulaire
WO2001015729A1 (fr) * 1999-08-27 2001-03-08 Fibrogen, Inc. Recepteur du facteur de croissance du tissu conjonctif, agonistes et antagonistes dudit recepteur, et leurs utilisations therapeutiques et diagnostiques
US6555322B1 (en) 1999-08-27 2003-04-29 Fibrogen, Inc. α2-macroglobulin receptor as a receptor for CTGF

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