WO1994002589A1 - Mammalian cationic proteins having lipopolysaccharide binding and anti-coagulant activity - Google Patents
Mammalian cationic proteins having lipopolysaccharide binding and anti-coagulant activity Download PDFInfo
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- WO1994002589A1 WO1994002589A1 PCT/US1993/006731 US9306731W WO9402589A1 WO 1994002589 A1 WO1994002589 A1 WO 1994002589A1 US 9306731 W US9306731 W US 9306731W WO 9402589 A1 WO9402589 A1 WO 9402589A1
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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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates generally to compositions and methods for the treatment and diagnosis of lipopolysaccharide (LPS)-associated diseases, such as Gram-negative sepsis. More particularly, the present invention relates to the preparation and use of certain mammalian cationic proteins for such treatment and diagnosis.
- LPS lipopolysaccharide
- a rational approach for the control of Gram- negative sepsis is to neutralize the toxic effects of lipopolysaccharides which are released during the treatment of the underlying bacterial infection.
- Cationic antibiotics such as polymyxin B
- bind to and neutralize some types of lipopolysaccharide but their use is limited by toxicity.
- Certain LPS-neutralizing monoclonal antibodies recognize common types of LPS, but are not effective against all species of Gram-negative bacteria.
- Certain mammalian polypeptides have been identified that bind to LPS.
- LBP LPS-binding protein
- BPI bactericidal permeability increasing protein
- CAP18 a cationic protein having a molecular weight of approximately 16.6 kD has been identified in rabbit granulocytes.
- the protein known as CAP18, has been found to bind to and attenuate the activity of LPS in certain in vitro assays.
- proteins and polypeptides useful for therapy and diagnosis may be truncated or shortened forms of the natural human or other mammalian protein.
- the therapeutic forms of the proteins and polypeptides may also be modified at certain residues in order to enhance activity of the protein in some desirable manner.
- lipopolysaccharide-associated diseases such as Gram-negative sepsis.
- compositions and methods capable of inhibiting or neutralizing lipopolysaccharide-associated damage which occurs in such diseases.
- mammalian cationic protein capable of binding to lipopolysaccharide and inhibiting or neutralizing its activity.
- mammalian CAP18 a synthetically or recombinantly produced mammalian cationic protein, such as mammalian CAP18.
- isolated and purified human cationic proteins capable of binding to lipopolysaccharide and inhibiting or neutralizing its activity.
- modified forms of human and other mammalian cationic proteins which possess lipopolysaccharide binding activity in combination with other desirable activities, such as anti-coagulation activity.
- compositions and methods useful for the treatment and diagnosis of certain lipopolysaccharide-associated conditions such as Gram- negative sepsis and/or treatment of coagulation-related disorders, such as disseminated intravascular coagulation (DIC) .
- the compositions include ' certain isolated and purified mammalian cationic proteins and polypeptides having a molecular weight from 2 kD to 80 kD, which cationic proteins are capable of binding to lipopolysaccharide to inhibit lipopolysaccharide activity in vitro and in vivo .
- compositions further include isolated polynucleotides and genes which encode the mammalian cationic proteins as well as DNA constructs suitable for expressing the genes in cell culture.
- isolated and purified polypeptides may be recombinantly produced in cell culture.
- the isolated and purified polypeptides are then used in therapy by administering to a host at risk of or suffering from a lipopolysaccharide-associated condition and/or coagulation-related disorder.
- the polypeptide composition may be used for diagnosing patients suffering from such lipopolysaccharide- associated conditions.
- diagnostic methods will comprise exposing a patient sample, typically blood, to the cationic proteins and detecting binding between said proteins and lipopolysaccharide which may be present in the sample.
- Fig. 1 is a reverse-phase HPLC produced from neutrophil-derived, heparin-bound cationic proteins as described in the Experimental section.
- Fig. 2 is the hydropathic profile of CAP18 based on the amino acid sequence derived from DNA SEQ:1.
- the 29 amino acid amino-terminal signal sequence and 37 amino acid carboxy-terminal reactive nitrogen inhibitory protein (RNIP) are shown.
- Fig. 3 is a graph illustrating the dose response of lipopolysaccharide (LPS)-induced reactive nitrogen intermediate (RNI) production, as described in the Experimental section.
- Fig. 4 is a graph illustrating the inhibition of LPS-induced RNI production by poly yxin B, as described in the Experimental section.
- LPS lipopolysaccharide
- RNI reactive nitrogen intermediate
- Fig. 5 is a graph illustrating the activity of RNIP relative to certain other mammalian cationic proteins in inhibition of LPS-induced RNI production, as described in the Experimental section.
- Fig. 6A is a graph illustrating RNI production in RAW 264.7 cells as a function of LPS concentration.
- Fig. 6B is a graph illustrating the inhibitory effects of various concentrations of peptides #197 (RNIP) and #187 on RNI production in RAW 264.7 cells induced with 2.5 ng/ l LPS.
- Figs. 6C - 6E show the antibacterial activity of four synthetic RNIP peptides against various bacterial strains.
- Fig. 6F shows the antibacterial activity of synthetic RNIP peptides which do not exhibit LPS binding activity.
- Fig. 6G shows that peptide 32-1, which does not bind LPS, also does not inhibit the activity of RNIP peptide 197.
- Fig. 6H shows the antibacterial activity of
- Fig. 61 shows the antibacterial dose response of RNIP peptide 197 against both Gram-negative and Gram- positive bacteria.
- Fig. 7 is a graph illustrating the binding of biotin-labeled RNIP to RAW 264.7 murine macrophage cells.
- Fig. 8 is a graph illustrating the effect of RNIP on LPS-induced inhibition of RAW 264.7 cell proliferation.
- Fig. 9 is a comparison of CAP18 cDNA sequence homology in human and rabbit. SEQ ID NO:l sets forth the human cDNA sequence, and SEQ ID NO:3 sets forth the rabbit cDNA sequence.
- Fig. 10 is a comparison of the amino acid sequence homology of the proteins encoded by the cDNA sequences of Fig. 1 as well as pig cathelin.
- the human amino acid sequence is set forth in SEQ ID NO:2
- the rabbit amino acid sequence is set forth in SEQ ID NO:4
- the pig cathelin sequence is set forth in SEQ ID NO:5.
- Fig. 11 is a comparison of the inhibitory effects of human RNIP and rabbit RNIP on RAW cell RNI production, as described in detail in the Experimental section hereinafter.
- Fig. 12 is a chart comparing the effects of two active and two inactive RNIP peptides in inhibiting the LPS-induced generation of tissue factor, where Salmonella minnesota smooth LPS was incubated with peptides 197 (RNIP) , 36-1, 32-1, and 50-2 (1 ⁇ g/ml) for five minutes, with the mixture then being added to thioglycolate- stimulated murine macrophages and cultured for six hours prior to assay of tissue factor by clotting assay, as described in more detail in the Experimental section hereinafter.
- RNIP peptides 197
- Fig. 13 compares LPS-induced tissue factor generation by RNIP (peptide 197) at different concentrations at 0, 1, and 3 hours after LPS.
- Figs. 14A - 14C illustrate anti-coagulant activity of the four peptides of Fig. 12 as a function of dosage, for prothrombin time (PT) , partial thromboplastin time (PTT) , and activated partial thromboplastin time (aPTT) , respectively.
- PT prothrombin time
- PTT partial thromboplastin time
- aPTT activated partial thromboplastin time
- Figs. 15A and 15B illustrate the effect of active synthetic peptides 197 and 36-1 on factor Xa- induced clotting and factor X activating enzyme, respectively.
- Figs. 16A and 16B compare peptides 197 (RNIP) and 36-1 with respect to effect on factor X activating enzyme, and inhibition of factor Xa generation, respectively.
- Figs. 17A and 17B compare the effect of four test peptides on prothrombin activation by Echis carinadus venom and prothrombin activation by factor Xa, respectively.
- the present invention provides compositions and methods for treating and diagnosing certain lipopolysaccharide (LPS)-associated conditions, such as Gram-negative sepsis and/or coagulation-related disorders, such as disseminated intravascular coagulation (DIC) .
- LPS lipopolysaccharide
- DIC disseminated intravascular coagulation
- Gram-negative sepsis occurs as a result of infection of a host with Gram-negative bacteria, where treatment with antibiotics often exacerbates the condition by the massive release of LPS which can accelerate the inflainmatory process and cause significant tissue damage.
- Disseminated intravascular coagulation is a hemorrhagic syndrome which occurs following an uncontrolled activation of patient's clotting factors and/or fibrinolytic enzymes throughout the small blood vessels.
- LPS is known to bind to at least one LPS binding protein (LBP) which is present in plasma and which is known to be induced 10-fold from hepatocytes.
- LBP LPS binding protein
- LPS bound to this protein binds to CD14, a leukocyte differentiation antigen that transmits a second messenger signal to monocytes to augment synthesis of many important enzymes and mediators, such as interleukin 1 (IL1) , tumor necrosis factor (TNF) , tissue factor, and nitric oxide synthetase. These molecules contribute to the capillary leak syndrome and tissue injury associated with endotoxinemia.
- IL1 interleukin 1
- TNF tumor necrosis factor
- tissue factor tissue factor
- nitric oxide synthetase binds to CD14, a leukocyte differentiation antigen that transmits a second messenger signal to monocytes to augment synthesis of many important enzymes and mediators, such as interleukin 1 (IL1) , tumor necrosis factor (TNF) , tissue factor, and nitric oxide synthetase.
- IL1 interleukin 1
- TNF tumor necrosis factor
- tissue factor nitric oxide synthe
- Certain of the therapeutic and diagnostic aspects of the present invention rely on binding of LPS with a particular natural cationic protein (and derivatives thereof) produced by mammalian leukocytes, particularly granulocytes, such as rabbit granulocytes as described in the Experimental section hereinafter.
- a particular natural cationic protein (and derivatives thereof) produced by mammalian leukocytes, particularly granulocytes, such as rabbit granulocytes as described in the Experimental section hereinafter.
- These cationic proteins will generally have a molecular weight in the range from 2 kilodaltons (kD) to 80 kD, usually from 10 kD to 20 kD, and a pi in the range from 8 to 11, usually from 9 to 10.
- the cationic protein obtained from rabbit granulocytes has a calculated molecular weight (based on its amino acid sequence) of 16.6 kD and a PI of 10.
- This rabbit granulocyte cationic peptide has previously been designated CAP18, based on its previously estimated molecular weight.
- Homologous mammalian cationic proteins from other species, including human, will generally be referred to as CAP18 hereinafter and in the claims.
- CAP18 may be identified by conventional protein isolation and purification techniques in combination with in vitro assays which identify its capacity to bind and hemagglutinate LPS-sensitized sheep red blood cells (SRBC) .
- SRBC sheep red blood cells
- CAP18 was first cloned in rabbit, as reported in the Experimental section hereinafter.
- the human form which has been cloned and sequenced herein displays an amino acid sequence homology of only about 70% with the rabbit form, with amino acid sequence homology in the RNIP region (described below) being less than 38%.
- Such low sequence homology rendered cloning of the human analog difficult, as described in more detail hereinafter in the Experimental section.
- the human form of CAP18 and CAP18 RNIP have also been found to inhibit bacterial proliferation of Gram-negative bacteria and, to a lesser extent, Gram- positive bacteria.
- Rabbit RNIP in contrast, is able to inhibit the growth of both Gram-negative and Gram- .
- CAP18 appears to play a role in the vertebrate inflammatory response and is able* to bind most forms of LPS and neutralize LPS-mediated activation of monocytes.
- CAP18 includes a short peptide, usually present at its carboxy-terminus, referred to as the reactive nitrogen inhibitory peptide (RNIP) which by itself is able to inhibit activation of macrophages.
- RNIP reactive nitrogen inhibitory peptide
- LPS released by Gram-negative bacteria combines with LPS binding protein (LBP) to provide an activation signal by binding to CD14 on macrophages.
- LBP LPS binding protein
- Such activation of the macrophages can potentiate a variety of LPS-associated conditions, such as sepsis.
- CAP18 and active fragments thereof are able to inhibit or attenuate the activation of macrophages and can thus be useful in the treatment of such LPS- associated conditions.
- binding of CAP18 to LPS provides an inhibitory signal to the macrophages via the RNIP fragment. The correctness of this mechanism, however, is not central to the present invention which does not depend on the particular mechanism of activity.
- the RNIP fragment of CAP18 from certain species, particularly rabbit also displays anti ⁇ coagulant activity in standard in vitro clotting assays, including prothrombin time (PT) assays, partial thromboplastin time (PTT) assays, and activated partial thromboplastin time (aPTT) assays.
- the RNIP fragment also has a high binding affinity for heparin.
- the anti- coagulation activity of the RNIP peptide appears to occur at the activation of prothrombin (factor II to factor Ila) .
- RNIP is shown to inhibit both Echis carinatus venom activation of purified factor II to factor Ila as well as factor II activation of by factor Xa formed by incubation of tissue factor, factor VII and factor X, as demonstrated in the Experimental section hereinafter.
- the anti-coagulant activity appears to derive from inhibition of at least two sites in the clotting cascade, i.e., the activation of factor X and the activation of prothrombin. While the precise mechanism of inhibition is not clear, as both these coagulation steps require the binding of phospholipids, it is possible that the RNIP peptides interfere with such binding.
- isolated and purified CAP18 polypeptides are provided for use in diagnostic and therapeutic procedures.
- isolated and purified it is meant that the polypeptides have been isolated from their cellular source and purified to a desired degree of purity, as described in detail hereinafter.
- Such isolated and purified polypeptides will comprise at least about 9 amino acids, usually being from 25 to 175 amino acids, more usually being from 25 to 150 amino acids.
- the polypeptides will be identical or homologous to a natural sequence within a mammalian CAP18 molecule, such as the sequence of human CAP18 set forth in SEQ ID NO:2 and the sequence of rabbit CAP18 set forth in SEQ ID NO:4.
- polypeptides which comprise at least the active portion of the RNIP fragment of the CAP18 molecule and those which comprise substantially the entire CAP18 molecule.
- polypeptides of the present invention will generally have at least about 60% sequence homology with a natural mammalian CAP18 sequence, such as that provided in SEQ ID NO:2 and/or SEQ ID NO:4, usually having at least about 80% sequence homology, more usually having at least about 90% sequence homology, and frequently having 95% sequence homology or greater.
- Mere sequence homology will not necessarily be sufficient to provide a polypeptide according to the present invention. It will also be necessary that the polypeptide have the ability to bind with most or all forms of LPS and usually be able to inhibit the activation of macrophages, as described above.
- polypeptide will possess anti-coagulant activity, as measured by conventional assays, e.g., the PT assay, the PTT assay, and the aPTT assay, as described in more detail in the Experimental section hereinafter.
- conventional assays e.g., the PT assay, the PTT assay, and the aPTT assay, as described in more detail in the Experimental section hereinafter.
- polypeptides of the present invention be substantially identical to a natural mammalian CAP18 or RNIP polypeptide.
- substantially identical it is meant that amino acid sequence will either have the identical sequence as that set forth in SEQ ID NO:2 on a residue- by-residue basis, or will have a limited number of amino acid insertions, deletions, or substitutions, where such changes in the amino acid sequence do not significantly alter the activity of the protein.
- polypeptides will also retain their "human" nature, i.e. they will be generally recognized as human when administered to human hosts.
- the human RNIP is generally less active than the rabbit RNIP, particularly in anti ⁇ bacterial activity against gram-positive bacteria. It is believed that substitution of basic amino acids from rabbit RNIP, such as LYS and ARG for non-basic amino acids in human RNIP, such as GLY,. ASP, PHE, and LYS, would enhance the activity of the human peptide without loss of the human nature of the peptide.
- preferred RNIP peptides according to the present invention will be based on the human RNIP sequence (including amino acids 134-170 of SEQ ID NO:2) having at least one of the ARG-LYS-ARG sequence of rabbit RNIP (amino acids 137-139 of SEQ ID NO:4) substituted for at least one of the GLY-ASP-PHE sequence at positions 136-138 of human RNIP, where such substitutions would be expected to enhance LPS-binding activity and/or anti-coagulant activity of the human RNIP.
- Preferably two, and more preferably all three substitutions will be made.
- polypeptides will include at least 25 amino-terminal amino acids of the sequence set forth in SEQ ID NO: 6 which represents human RNIP with the preferred ARG-LYS-ARG substitution.
- polypeptides will include at least the 30 amino-terminal amino acids, more preferably including at least 32 amino-terminal amino acids, and may include the entire 37 amino acid sequence.
- the present invention provides isolated polynucleotides corresponding to all or part of a mammalian CAP18 gene in a variety of useful forms.
- Such polynucleotides include both DNA and RNA sequences corresponding to at least a portion of the CAP18 gene (coding or non-coding sequences) , usually including a sequence of at least about 10 contiguous bases within the gene and frequently including up to the entire length of the gene.
- the polynucleotide sequence may encode (or be complementary to a strand which encodes) the amino acids of the RNIP fragment of a CAP18 molecule, or of the entire CAP18 molecule, or any fragment of the CAP18 molecule which retains a useful activity of the intact molecule.
- the polynucleotides of the present invention may also include bases which do not correspond to the structural region of the CAP18 gene, for example, including control regions, linkers, or the like, when the polynucleotide is going to be used to produce recombinant gene product, and other regions which may facilitate the manipulation and/or expression of the polynucleotide.
- the polynucleotide may also include the structural region(s) of other unrelated genes, particularly when it is desired to produce a fused gene product.
- Correspondence between the polynucleotide and the CAP18 gene generally means that the polynucleotide will have a high degree of sequence homology with the naturally-occurring mammalian gene, usually having at least about 40% homology, more usually having at least about 65% sequence homology, and preferably having at least about 90% sequence homology. It will be appreciated, however, that such a high degree of sequence homology will not always be necessary, particularly when the polynucleotide is being used as a portion of a recombinant DNA construct to produce polypeptides corresponding to the natural CAP18 gene product.
- nucleotide substitutions can be made without significantly changing the amino acid constitution of the polypeptide being produced. It is only essential that the polynucleotide encode the desired polypeptide, i.e. one having an amino acid sequence having a particular homology with the product of the naturally occurring gene, as discussed above. In many cases, it may even be preferable to provide substitutions within the polynucleotide, e.g., when the recombinant DNA construct is to be expressed in a prokaryotic system, it will frequently be desirable to utilize codons preferentially recognized by the expression host.
- Homologous CAP18 genes from other species may be identified and cloned using probes based on the rabbit and/or CAP18 genes.
- Probes may be derived directly from the rabbit or human CAP18 cDNA, or degenerate probes may be prepared synthetically based on the nucleotide sequence and/or amino acid sequence provided in SEQ ID NOS:1, 2, 3, and 4. The probes may then be used to screen suitable gene libraries, particularly libraries derived from mammalian bone marrow cells.
- other mammalian CAP18 genes may be obtained by first isolating the homologous polypeptide from a suitable cellular source, at least partially sequencing the polypeptide, and preparing degenerate probes based on the deduced amino acid sequence.
- Such a screening technique is analogous to that used for identifying rabbit granulocyte CAP18, as described in the Experimental section hereinafter.
- CAP18 polypeptides may be utilized for synthesizing the
- CAP18 polypeptides of the present invention by expression in cultured cells of recombinant DNA molecules encoding a desired portion of the mammalian CAP18 gene.
- the mammalian CAP18 gene may itself be natural or synthetic, with the natural gene obtainable from cDNA or genomic libraries using degenerate probes as described above. Specific rabbit cDNA and human cDNA clones are described in detail in the Experimental section hereinafter.
- polynucleotides may be synthesized by well-known techniques. For example, single-stranded DNA fragments may be prepared by the phosphoramadite method first described by Beaucagen Carruthers (1981) Tett . Letters 2_2:1859-1862.
- a double-stranded fragment may then be obtained, either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
- the preparation of synthetic DNA sequences is conveniently accomplished using automated equipment available from the suppliers, such as Applied Biosystems, Inc., Foster City, California.
- DNA constructs coding for the desired CAP18 fragment will then be incorporated into a DNA construct capable of introduction to an expression in in vitro cell culture.
- the DNA constructs will be suitable for replication in a unicellular host, such as yeast or bacteria.
- DNA constructs prepared for introduction into bacteria or yeast will include a replication system recognized by the host, the CAP18 DNA fragment encoding the desired polypeptide product, transcriptional and translational initiation regulatory sequences joined to the 5 prime-end of the CAP18 DNA sequence, and transcriptional and translational termination regulatory sequences joined to the 3 prime-end of the CAP18 sequence.
- the transcriptional regulatory sequences will include a heterologous promoter which is recognized by the host.
- available expression vectors which include the replication system and transcriptional and translational regulatory sequences together with an insertion site for the CAP18 DNA sequences may be employed.
- co-transfection of cell lines in the presence of a suitable marker, such as the DHFR gene may be employed. Transfection may be accomplished using chemical, ballistic, or electroporation techniques.
- the CAP18 polypeptides are generally obtained in substantially pure form, that is, typically at least about 50% weight/weight (w/w) purity, being substantially free from interfering proteins and contaminants.
- the CAP18 polypeptides * are isolated or synthesized in a purity of at least about 80% w/w and, more preferably in at least about 95% w/w purity. Using conventional protein purification techniques, homogeneous polypeptide compositions of at least 99% w/w purity can be obtained.
- the CAP18 proteins may be purified by affinity chromatography using antibodies raised against the CAP18 protein.
- the isolated and purified polypeptides of the present invention can be incorporated as components of pharmaceutical compositions useful to attenuate, inhibit, or prevent LPS-associated conditions, such as Gram- negative sepsis, autoimmune disorders, inflammation, and the like.
- the composition should contain a therapeutic or prophylactic amount of at least one polypeptide according to the present invention present in a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier can be any compatible, non-toxic substance suitable to deliver the polypeptide to the patient. Sterile water, alcohol, fats, waxes, and inert solids may be used as the carrier.
- Pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like may also be incorporated into the pharmaceutical compositions.
- compositions can contain a single polypeptide or may contain two or more polypeptides according to the present invention to form a "cocktail".
- the pharmaceutical compositions just described are useful for oral or parenteral administration.
- the compositions will be administered parenterally, i.e. subcutaneously, intravascularly, or intravenously.
- Alternative modes of administration may also be employed, such as nasal delivery, respiratory delivery, transdermal delivery, and the like.
- the concentration of the CAP18 polypeptide in the form of suitable composition can vary widely, i.e., from less than about 0.1% by weight, usually being at least about 1% by weight, to as much as 20% by weight, or more.
- Specific methods for preparing pharmaceutical compositions are well known in the art and described in more detail in various publications, such as Remington's Pharmaceutical Science, 15th Edition, Mack Publishing Company, Easton, Pennsylvania (1980) , which is incorporated herein by reference.
- the pharmaceutical CAP18 polypeptide compositions of the present invention can be administered for prophylactic and/or therapeutic treatment of Gram- negative sepsis, autoimmune disorders, and other LPS- related conditions.
- the pharmaceutical compositions will be administered to a patient already showing signs of the condition, such as septic shock.
- the polypeptides will be administered to a patient prior to showing signs of septic shock, typically in conjunction with the primary antibiotic treatment for Gram-negative bacterial infection.
- the CAP18 polypeptide compositions of the present invention are also useful for detecting the presence of LPS in biological samples, such as patient samples in the diagnosis of Gram-negative sepsis.
- the isolated and purified CAP18 polypeptides may be utilized as a receptor for LPS in a variety of conventional assay formats, including competitive and non-competitive (sandwich) assay formats; radiometric, enzymatic, colormetric, luminescent, and phosphorescent formats; homogeneous and heterogenous formats, and the like.
- Protein sequence of CAP18 - Rabbit CAP18 was purified from rabbit peritoneal exudate cells elicited by IP injection of 500 ml of 0.25% sodium caseinate. Cells were washed and extracted with 0.1M citric acid. The acid soluble fraction was precipitated with 80% ethanol and applied to heparin-sepharose CL-6B column. The tightly bound cationic proteins eluting with 2M NaCl were used for further study. C8 reverse phase HPLC of the heparin bound material yielded two major peaks. The first peak (@26.1 min.) actively inhibited LPS induced tissue factor generation from murine macrophages (Hirata M, et al., unpublished data).
- the active peak was sequenced using an Applied Biosystems Model 477A protein/peptide sequencer with an on-line Applied Biosystems 120A PTH-amino acid analyzer.
- the sequence of the first 28 amino acids of the putative N-terminus of CAP18 was: GLY-LEU-ARG-LYS-ARG-LEU-ARG-LYS-PHE-ARG-ASN- LYS-ILE-LYS-GLU-LYS-LEU-LYS-LYS-ILE-GLY-GLN-(ASP or LYS)- ILE-GLN-(GLN or ILE)-(GLY or GLN)-LEU-LEU (SEQ ID NO:7) . Searches of the GenBank and the National Protein databases revealed that this sequence is unique. Note that this sequence was later found to correspond to the N-terminus of the
- Construction cDNA library Rabbit bone marrow cells were harvested and lysed with guanidinium thiocyanate, and spun on a cesium chloride gradient as per Maniatis, T. , et al., "Molecular Cloning: A Laboratory Manual . " (1982) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Poly(A)+mRNA was selected on an oligi dT- cellulose column (Pharmacia) . 5 ⁇ g mRNA was used to construct a cDNA library, using the cDNA Synthesis System Plus kit (Amersham) . EcoRl restriction sites were methylated, and EcoRl linkers were ligated overnight at room temperature using T4 DNA Ligase (New England Biolabs) .
- Excess linker was digested with EcoRl.
- the cDNA was size fractionated over a Sepharose S-400 column, ligated into lambda gtlO vector (Lambda Vector Kit, Stratagene) .
- C600Hf1 bacteria were infected with the packaged lambda and grown on NZYDT plates. Random plaques were amplified via polymerase chain reaction (PCR) (Perkin Elmer Cetus) using primers made to the Lambda arms flanking the insert site.
- PCR polymerase chain reaction
- a 200 bp PCR fragment was found to encode the known CAP18 amino acid sequence, some additional 3' information, and a poly A tail. This fragment was used to generate random primed probes as per manufacturer's instructions (Boehringer- Mannheim) , and used to screen the library for the full- length cDNA.
- oligonucleotide probes Two pairs of oligonucleotide probes were designed corresponding to amino acids #11-17 and #15-21 respectively. Initial attempts using conventional screening failed to identify a positive clone. An alternative approach using PCR was employed. CAP18 oligonucleotide #11-17 was matched with primers designed from the lambda phage arms. A 200 bp fragment corresponding to CAP18 was amplified, purified and used to screen the cDNA library. In the library of 400,000 plaques, greater than 100 clones hybridized to the probe. Twenty of these primary positives were replated and 4 were subcloned into plasmids for sequence analysis.
- the cDNA and predicted amino acid sequences of rabbit CAP18 are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.
- the rabbit CAP18 cDNA encodes a 29 amino acid signal peptide followed by a mature protein of 142 amino acids.
- the protein is predicted to be 16.6 kDa and to have a pi of 10. No N-linked glycosylation sites are predicted.
- cDNA library screening - Confluent plates were transferred to nitrocellulose filters. The filters were UV-cross-linked (Stratagene, UV Stratalinker) and incubated for 2 hours in 50% formamide, 50% PAM.
- Filters were hybridized at 42°C overnight with fresh solution containing the random primed probe. Filters were washed once at room temperature for 15 minutes in 2 X SSC, 0.1% SDS; then in 1 X SSC, 0.1% SDS; and a third time in 0.5xSSC, 0.1% SDS. Positives were subcloned into ml3mpl8 and/or pcDNA (Invitrogen) and sequenced.
- Fraction 25.8 is macrophage related protein 14 (MRP14) (Odink et al. (1987) Nature 330:80-82). The fraction at 20.3 minutes corresponds to a fragment of CAP18 of approximately 4-5 kD. This fragment was shown to inhibit LPS-induced reactive nitrogen intermediate (RNI) production and was named reactive nitrogen inhibitory protein (RNIP) (Fig. 2) .
- Fig. 3 demonstrates a dose response of LPS induced RNI production by cultured murine macrophage cell line (RAW 264.7).
- the murine macrophage cell line RAW 264.7 obtained from ATCC
- thioglycollate-elicited murine peritoneal exudate cells are used to produce RNI.
- Cells are cultured at lxl0 6 /ml. in RPMI-1640 +2.5% FCS in 24 well plates in the presence or absence of different concentrations of LPS or murine rlFN ⁇ . After 24 hr. incubation at 37°C, the cell free supernatant is collected and tested for the presence of RNI.
- Fig. 4 demonstrates inhibition of LPS [5ng/ml]- induced RNI production in RAW 264.7 cells by polymyxin B, a stoichiometric inhibitor of LPS. Such inhibition serves as a positive control for the induction of RNI activity hereinafter.
- RNIP inhibits RNI release from thioglycollate-elicited murine PECs stimulated by IFN ⁇ alone or the synergistic combination of IFN ⁇ plus LPS (Table 1) . Subsequently we showed that the IC50 for inhibition of gamma interferon stimulated nitrogen radical release by RNIP was less than 50 nM (Table 2) .
- RNIP inhibits both LPS and IFN ⁇ induced TNF release (Table 3) from human macrophages.
- RNIP is a novel peptide derived from an LPS binding protein that acts directly on macrophages to attenuate their activation by diverse stimuli.
- Table 1 RNIP blocks LPS and IFN ⁇ induced generation of nitrogen radicals from Raw 264.7 cells.
- RNIP blocks synergistic action of LPS and IFN ⁇ induced generation of nitrogen radicals from day 3 thioglycolate (4%/lml) elicted murine peritoneal exudate cells.
- Table 3 RNIP blocks TNF release from LPS and IFN ⁇ stimulated human monocytes.
- CAP18 a rabbit anti-LPS protein designated CAP18. From this sequence we have cloned a cDNA corresponding to CAP18. Apparently during the purification process a piece of CAP18 is proteolytically cleaved. This fragment designated RNIP can inhibit release of nitrogen radicals and cytokines by LPS and IFN ⁇ activated macrophages. 3. Synthetic Rabbit RNIP Peptides All of the data presented above were obtained with RNIP purified from rabbit granulocytes. It was therefore of interest to determine if synthetic RNIP retained similar or identical activities. Furthermore it was of interest to determine if a small fragment of RNIP might bind to LPS and inhibit LPS activation of macrophages.
- Synthetic rabbit RNIP and a series of rabbit RNIP-derived fragments were generated based on the sequence of SEQ ID NO:4. These fragments were made using automated solid phase synthesis (Merrifield synthesis) according to standard procedures. Each fragment was purified by HPLC. Table 4 lists the peptide fragments and their activity in LPS hemagglutination assays and inhibition of LPS-induced release of reactive nitrogen intermediate (RNI) radicals.
- RNI reactive nitrogen intermediate
- erythrocytes were sensitized with LPS as follows. One ml of 1% erythrocyte suspension (human 0 type, C3H/HeN mouse or sheep) was mixed with 0.2 ml of LPS solution and incubated at 37°C for 30 min, followed by washing with phosphate buffered saline (PBS) , and then the concentration of suspension was adjusted to 1.0%. In the case of S-LPS, the solution was heated at 100°C for 1 hour before sensitization of erythrocytes.
- PBS phosphate buffered saline
- peptide fragment #36-1 and the native RNIP fragment (#197) are active. Other fragments have much less activity. While there is some day-to-day variation in the bioassay, these peptides are active at concentrations of 10-50 nM.
- Figs. 6A and 6B compare the dose response of active synthetic RNIP (#197) with an inactive fragment (#187) in inhibition of LPS-induced reactive nitrogen intermediate (RNI) production in RAW 264.7 cells.
- Fig. 6A shows RNI production in the absence of inhibition
- Fig. 6B compares the inhibitory effects of peptides #197 (RNIP) and #187.
- the 50% inhibitory concentration (IC50) of RNIP in this assay is seen to be about 0.2 ⁇ g/ml (approximately 30 nM) .
- Table 4 RNIP peptide sequences.
- MAC minimum agglutinating concentration of Salmonella minnesota Re-LPS sensitized sheep erythrocytes.
- MIC minimal inhibitory concentration of LPS.
- Peptide #36-1 (16 ⁇ g/ml) and RNIP (#197) (25 ⁇ g/ml) were preincubated with each LPS preparation at 37° for 30 minutes, and each reaction mixture was added to sheep erythrocytes sensitized with Re-LPS.
- Bacterial cultures were collected at logarithmic phase and washed twice with phosphate-buffered saline pH 7.2, and adjusted to a final concentration of 5xl ⁇ 3 -lxl0 4 cells/ml.
- 50 ⁇ l of peptide was added and incubated at 37°C for 1 hour, and 100 ⁇ l of the reaction mixture was plated on the agar plate. After 24 hr-incubation at 37°C, colony-forming units (CFU) were counted.
- CFU colony-forming units
- PBS was added to bacterial suspension* and incubated for 1 hour, plated on agar, and cultured. For some experiments the percent of control CFU was determined.
- Bacteria were grown in trypticase broth overnight. The following day, bacteria were suspended at 10 5 /ml in RPMI with 10% fetal calf serum. The assay was set up in a 96 well plate containing 50 ⁇ l bacteria plus 50 ⁇ l peptides and incubated at 37°C for 1 hr, then 1 ⁇ Ci 3 H-thymidine (Amersham) is added to each well and incubated with the bacteria overnight. The assay is terminated by addition of 10% TCA and harvested and counted on a Matrix 96 Packard beta counter.
- Antibacterial activity of peptides 197 (rabbit RNIP) , 36-1, 32-1, and 50-2 were measured on the rough mutant strain, Salmonella minnesota Re .
- Figure 6C presents a dose-response of the four peptides on Salmonella minnesota Re CFU.
- Non-LPS binding peptides 32-1 and 50-2 are not active whereas LPS-binding peptides, 197 and 36-1 have significant anti-bacterial activity (IC50 ⁇ 100 ng/ml; 40 nM) .
- Figure 6E demonstrates that peptide 197 has significant activity versus two other clinical strains of bacteria, Pseudomonas aeruginosa (IC50 ⁇ 250 ⁇ g/ml; 40 nM) and Kiebsieiia pneumoniae (IC50 ⁇ 440 ng/ml; 70 nM) .
- Figure 6H compares the activity of peptide 197 versus encapsulated and non- encapsulated E . coli 09:K39.
- Table 5A demonstrates that peptide, 36-1 inhibits proliferation of several Gram positive bacterial strains including Streptococcus pneumoniae , Streptococcus pyogene ⁇ , and coagulase-positive Staphylococcus aureus .
- the highly active anti-bacterial peptides 197 and 36-1 were inactive over the range 0-20 ⁇ g/ml versus Candida albicans (data not shown) and versus multiple drug-resistant Mycobacterium tuberculosis and two strains of Mycobacterium avium (data not shown) .
- CAP18 The activity of CAP18 is to be contrasted with that of BPI which was originally shown to have anti ⁇ bacterial activity versus a variety of Gram-negative bacteria, with no activity versus' Gram-positive or fungal organisms.
- Both the amino-terminal fragment (rBPI 23 ) and the holoprotein (BPI 55 ) exhibited growth inhibitory activity (about 100-fold reduction over control) against an encapsulated E . coli . While both BPI 55 and rBPI 23 inhibited the growth of a rough mutant strain of Proteus mirabilis , only the rBPI 3 inhibited growth of the wild- type smooth organism.
- rBPI 23 nor holoprotein BPI 55 demonstrated inhibition of Gram-positive Staphylococcus aureus (Weiss et al. (1992) J. Clin. invest. 90:1122-1130).
- RNIP was prepared with a biotin group attached to a lysine residue. This biotin-RNIP was shown to be bioactive in an RNI assay (i.e., biotinylation did not alter activity of the molecule).
- RAW 264.7 cells were washed free of culture medium, and binding of the biotinylated RNIP was carried out at 4°C in Iscove's DMEM supplemented with 1% fetal calf serum. Cells were then washed free of excess biotin-RNIP, and streptavidin- phycoerythrin (PE) was added. Binding of biotin-RNIP to the cells was assessed using flow cytometry. The results are shown in Fig. 7.
- Fig. 8 shows that when RNIP is added to the cultures containing 5 ng/ml of LPS it reverses the inhibition of proliferation and activation of the RAW 264.7 macrophage cell line.
- Tissue factor assays were performed as follows. Mouse peritoneal cells were elicted with an intraperitoneal injection of thioglycolate medium and harvested 5-days later by irrigation with pyrogen-free saline.
- the cells were washed with RPMI 1640 medium, counted and resuspended at a concentration of 1.5 - 2.0 x 10 6 /ml in serum-free RPMI medium containing glutamine (0.29 mg/ml), penicillin (50 units/ml) and streptomycin (50 /ml) .
- Adherent cells were prepared by incubating 10 ml of a cell suspension (in RPMI 1640 media in sterile culture bottle (Nunclon, 50 ml, Inter Med) for 60 min. at 37°C in a 5% C0 2 atmosphere. The plastic adherent cells were recovered by scraping of the bottle with a sterile rubber policeman.
- the adherent cells were then washed twice with RPMI media, resuspended in serum-free medium (lxl ⁇ 6 /ml) in culture tube (Nunc cryotube, Inter Med) , and stimulated with graded doses of LPS for 6 hr. at 37°C in a 5% C0 2 .
- the cell suspension was centrifuged and cell pellet was frozen at -80°C until clotting assays were run.
- Human mononuclear cells were separated from citrated peripheral blood by centrifugation over Sodium- metrizoate-Ficoll (lymphocyte Separation Medium, Japan Antibody Institute, Gumma) .
- the resulting mixed mononuclear cell suspension was washed 3 times with RPMI 1640 media. Cell suspension (lxl0 6 /ml) was stimulated with LPS for 16 to 18 hr.
- Veronal buffered saline VBS
- Veronal buffered saline VBS
- a hand-held sonicator microbe Hexane-activated thromboplastin time (7) .
- Tissue factor activity was tested in modified unactivated partial thromboplastin time (7) .
- One tenth ml of cell lysate (lxl0 6 /ml) was preincubated with mouse plasma at 37°C for 3 min.
- tissue factor activity into arbitrary tissue factor units (1 ⁇ g/ml) suspension of rabbit brain was assigned a value of 1,000 units) .
- minnesota Smooth-LPS (2 ⁇ g/ml) was preincubated with equal volume of each LPS-binding protein (2-8HA) at 37° C for 45 minutes, and the mixture was added to the cells and cultured for 16 hours.
- Table 7 shows that another cationic peptide CAP37 (Morgan et al. (1991) J. Immunol. 147:3210-3214) is unable to inhibit tissue factor generation.
- Table 7 Effect of CAP-37 on tissue factor generation.
- t LPS was incubated with CAP-37 at 37° C for 2 hours, and the mixture was added to cells, then cultured for 6 hours.
- Galactosamine sensitizes mice to the lethal effects of LPS by a factor 1000 or more.
- CAP18 and LPS were mixed and injected IP into sensitized mice.
- CAP18 can neutralize the lethal effects of LPS in this model of endotoxemia (see Table 8) .
- Table 8 CAP18 attenuates LPS toxicity in galactosamine- sensitized mice.
- the cDNA revealed a protein with two domains: an amino terminal cystein protease inhibitor domain and a carboxy terminal endotoxin binding domain.
- Fig. 9 compares the cDNAs of human and rabbit CAP18.
- Fig. 10 compares the protein sequences of rabbit CAP 18, human CAP18 protein, and cathelin, a pig cysteine protease inhibitor. Table 9, below, compares the nucleic acid and amino acid composition homologies of the rabbit and human CAP18 proteins in both the N-terminal cysteine protease domain and the RNIP domain. There is a much higher level of amino acid conservation in the N-terminal cysteine protease inhibitor domain compared to the carboxy terminal RNIP domain.
- a human RNIP polypeptide comprising amino acids 134 to 170 of SEQ ID NO:2 was synthesized by the Merrifield solid phase synthesis method. LPS inhibition activity of human RNIP was compared with that of rabbit RNIP comprising amino acids 135 to 171 of the rabbit sequence in SEQ ID NO:4. Equimolar concentrations of both human and rabbit RNIP (1 ⁇ M, 0.25 ⁇ M, and 0.03 ⁇ M were introduced to stimulated cultures of RAW 264.7 which produce reactive nitrogen intermediates (nitric oxide) in the presence of LPS (2.5 ng/ml). The results are shown in Fig. 11.
- Blood coagulation and hemostasis are important components of the host defense system against traumatic injury and invasion by microorganisms. Overwhelming infections, particularly those associated with release of endotoxin can cause excessive activation of the coagulation cascade, a condition termed disseminated intravascular coagulation (DIC) .
- DIC disseminated intravascular coagulation
- the intrinsic pathway is activated by conversion of factor XII to factor Xlla secondary to exposure of collagen from damaged vascular endothelial cells.
- the extrinsic pathway is initiated by tissue factor (procoagulant, tissue thromboplastin) expressed by activated monocytes and macrophages.
- Tissue factor converts factor VII to factor Vila and the tissue factor-Vila complex converts factor X to Xa and factor IX to factor IXa. Both pathways converge by at the generation of a prothrombinase comprised of a complex of factor Xa, factor V, phospholipid and calcium ions.
- Prothrombinase converts prothrombin (factor II) to thrombin (Ila) .
- Thrombin generates fibrin monomers from fibrinogen. Because both monocytes and macrophages as well as endothelial cells are induced to synthesize tissue factor by LPS, the extrinsic pathway is thought to play an important role in LPS-induced DIC.
- LPS binding RNIP peptides inhibit LPS induced generation of tissue factor from murine macrophages. Furthermore, we demonstrate that RNIP peptides inhibit the coagulation cascade at two sites: activation of factor X and conversion of prothrombin to thrombin (factor II to factor Ila) .
- the putative heparin binding domain of RNIP is implicated in the inhibition of coagulation because of the high correlation of three observations: a) LPS-binding but not non-binding peptides inhibit coagulation; b) LPS- binding but not non-binding peptides contain a consensus heparin binding domain at the N-terminus of RNIP; c) peptides containing the heparin binding domain inhibit coagulation whereas those that do not contain the domain do not inhibit coagulation.
- Coagulation assays were run as follows: Clotting method: Human plasma (Ortho plasma control, Ortho Diagnostic Co.). ATPP reagent(Organo-technika, Tokyo, Japan).
- Standard tissue factor rabbit brain thromboplastin (Simplastin, ONO Pharmaceutical, Tokyo, Japan) .
- Purified human factor VII, factor X, factor Xa (Sigma Chemical Co., St. Louis, MO) . .Russell Viper venom (RW) and Echis Carinatus venom (ECV, Sigma Co.) .
- Synthetic chromogenic substrate method Substrate for factor Xa, S2222 (Kabi, Bz-Ile-Glu-Arg-pNA) was used.
- Substrate for factor II prothrombin
- Boc-Val-Pro-Arg-pNA (Sigma Chemical Co.) was used.
- Tissue factor assay Salmonella minnesota smooth LPS was incubated with each peptide (1 ⁇ g/ml) for 5 min., then the mixture was added to peritoneal murine macrophages obtained 4 days after thioglycollate stimulation. Cells were cultured for 6 hours prior to assay of tissue factor by clotting assay.
- Prothrombin time Human plasma (100 ⁇ l) was incubated with 100 ⁇ l of each peptide at 37°C for 3 min., then 100 ⁇ l of tissue factor (250 ⁇ g/ml) CaCl 2 was added and clotting time was measured.
- tissue factor 250 ⁇ g/ml
- Partial thromboplastin time Human plasma (100 ⁇ l) was incubated with each peptide (100 ⁇ l) at 37°C for 3 min., then 100 ⁇ l of CaCl 2 containing phospholipid was added and clotting time was measured.
- APTT Activated partial thromboplastin time
- Factor Xa activity using synthetic substrate A reaction mixture of factor VII (1 unit/ml, 200 ⁇ l) , factor X (1 unit/ml, 200 ⁇ l) , tissue factor (2.5 mg/ml, 50 ⁇ l) , 25mM-CaCl 2 (50 ⁇ l) and 50 ⁇ l of several concentration of peptide was incubated at 37°C for 10 min. To 200 ⁇ l of the reaction mixture, 60 ⁇ l of 4mM S-2222, a synthetic substrate for factor Xa, and 340 ⁇ l of Tris-HCl buffer, pH 8.2, was added and further incubated for 15 min. The reaction was stopped by adding 60 ⁇ l of 50 % acetic acid, and OD for p-nitroaniline was read at 405 nm.
- Echis Carinatus venom Echis Carinatus venom
- Prothrombin activation by factor Xa (divided into three steps) .
- b) Prothrombin (factor II) activation To 250 ⁇ l of the reaction mixture, factor II (1 unit/ml, 50 ⁇ l) , several concentration of peptide (25 ⁇ l) and phospholipid (50 ⁇ g/ml, 25 ⁇ l) was added and incubated at 37°C for 10 min.
- step b) Hydrolysis of thrombin substrate.
- thrombin 60 ⁇ l of substrate and 240 ⁇ l of Tris buffer was added and further incubated for 15 min. Interaction of heparin and synthetic peptide in a modified PT system.
- Each reaction mixture contained : 100 ⁇ l human plasma; 50 ⁇ l of buffer or heparin (2 units/ml) ; 50 ⁇ l buffer or peptide; 100 ⁇ l tissue factor (250 ⁇ g/ml) .
- Tissue factor lethality model Reference tissue factor (Simplastin, 1 mg/mouse) was IV injected into ddY mice (male, 20-week-old, 38-45g) incubated with or without peptide. 10. RNIP peptides inhibit LPS-induced tissue factor generation by macrophages.
- RNIP derived peptides having LPS binding activity inhibit LPS induction of tissue factor, whereas related cationic peptides without LPS binding activity do not inhibit LPS induction of tissue factor. 11. RNIP peptides inhibit in vitro clotting assays.
- tissue factor activity was associated with the surface membranes of granulocytes.
- ho ogenization or sonic disruption of these cells was observed to reduce tissue factor activity and this inhibition was ascribed to the release of cationic proteins.
- tissue factor utilizes the clotting cascade, it was reasoned that granulocyte-derived cationic proteins might directly inhibited other sites of the clotting cascade. Therefore, the RNIP peptides were tested in a series of in vitro clotting assays.
- RNIP peptide 197 reduced mortality by 57% and prolonged survival time of tissue factor treated mice, as shown in Table 12.
- Tissue factor (Simplastin, 10 mg/ml was mixed with equal volume of synthetic peptide 100 ⁇ g/ml or 50 ⁇ g/ml and 0.2 ml of the mixture injected IV. ddY mice (20 weeks, 38-45 gm) .
- the N-terminal domain is highly homologous to porcine cathelin originally purified as a cysteine protease inhibitor.
- Purified human CAP18 also demonstrates significant cysteine protease activity.
- the C-terminal 37 amino acid domain (RNIP) of CAP18 was identified as the LPS-binding domain when HPLC fractions were tested for their capacity to inhibit LPS-induced nitrogen radical production.
- Rabbit RNIP peptides demonstrated inhibition activity in a series of standard in vitro clotting assays, including the prothrombin time, the partial thromboplastin time and the activated partial thromboplastin time, with no evidence for a direct action on thrombin. No evidence was found for a direct effect on the activity of factor Xa. Both factor Xa induced clotting ( Figure 15A) and Russell Viper Venom (RW) activated clotting, however, were inhibited by the rabbit RNIP peptides. These results were confirmed using chromogenic assays. Thus, one activity of the active rabbit RNIP peptides is to inhibit the activation of factor Xa.
- rabbit CAP18 has a high affinity binding site for heparin because it was eluted from heparin-sepharose at high NaCl concentration (2.0 M) . Consistent with this inference is the finding of a high content of basic amino acids particularly in the C- terminal RNIP domain.
- Table 13 presents a sequence comparison of RNIP with a series of known heparin binding proteins. Many of these proteins contain a consensus peptide sequence, X-B-B-X-X-B-B-B-X-X-B-B-X-X (SEQ ID NO:9), where B is a basic amino acid and X is any amino acid (Sobel et al, (1992) J. Biol. Chem.
- Lipoprotein lipase K S S R K A R V K N I E (SEQ ID NO: 13)
- Heparin and heparinoids have been intensely studied in recent years as anti-coagulants. Heparin is thought to mediate its activity by binding to antithrombin III to increase its affinity for thrombin. The activity of protease inhibitor tissue factor pathway inhibitor (TFPI) an inhibitor of factor Vila and factor Xa is also enhanced by heparin.
- TFPI tissue factor pathway inhibitor
- RNIP may interact with anionic domains on target proteins, cell surface heparans or other negatively charged carbohydrates because of its high basic charge. Although the target molecules are unknown, limited structural studies indicate a high degree of specificity in the the activity of RNIP, a finding shared with the oppositely charged heparinoids.
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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AU46825/93A AU4682593A (en) | 1992-07-17 | 1993-07-15 | Mammalian cationic proteins having lipopolysaccharide binding and anti-coagulant activity |
JP6504605A JPH08504085A (en) | 1992-07-17 | 1993-07-15 | Mammalian cationic protein with lipopolysaccharide binding and anti-pseudoblood activity |
EP93917249A EP0746605A4 (en) | 1992-07-17 | 1993-07-15 | Mammalian cationic proteins having lipopolysaccharide binding and anti-coagulant activity |
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US91676592A | 1992-07-17 | 1992-07-17 | |
US91676192A | 1992-07-17 | 1992-07-17 | |
US07/916,765 | 1992-07-17 | ||
US07/916,761 | 1992-07-17 |
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WO1994002589A1 true WO1994002589A1 (en) | 1994-02-03 |
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ID=27129703
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PCT/US1993/006731 WO1994002589A1 (en) | 1992-07-17 | 1993-07-15 | Mammalian cationic proteins having lipopolysaccharide binding and anti-coagulant activity |
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EP (1) | EP0746605A4 (en) |
JP (1) | JPH08504085A (en) |
AU (1) | AU4682593A (en) |
WO (1) | WO1994002589A1 (en) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU684170B2 (en) * | 1994-07-08 | 1997-12-04 | A & H Brister Holdings Pty Ltd | Tank roof structure |
US5994306A (en) * | 1995-11-22 | 1999-11-30 | Intrabiotics Pharmaceuticals, Inc. | Fine-tuned protegrins |
US6025326A (en) * | 1995-07-07 | 2000-02-15 | Intrabiotics Pharmaceuticals, Inc. | Compositions and methods for the prevention and treatment of oral mucositis |
US6040291A (en) * | 1998-03-25 | 2000-03-21 | Seikagaku Corporation | Antimicrobial peptide |
US6103888A (en) * | 1992-07-17 | 2000-08-15 | Panorama Research, Inc. | Mammalian cationic proteins having lipopolysaccharide binding and anti-coagulant activity |
US6159936A (en) * | 1993-07-20 | 2000-12-12 | The Regents Of The University Of California | Compositions and methods for treating and preventing microbial and viral infections |
US6440690B1 (en) | 1995-06-29 | 2002-08-27 | Amram Mor | Peptides for the activation of the immune system in humans and animals |
WO2002095076A2 (en) * | 2001-05-23 | 2002-11-28 | Kabushiki Kaisha Toyota Chuo Kenkyusho | Modified polypeptides having protease-resistance and/or protease-sensitivity |
US7071293B1 (en) * | 1999-08-18 | 2006-07-04 | The University Of Iowa Research Foundation | Alpha helical peptides with broad spectrum antimicrobial activity that are insensitive to salt |
-
1993
- 1993-07-15 EP EP93917249A patent/EP0746605A4/en not_active Withdrawn
- 1993-07-15 JP JP6504605A patent/JPH08504085A/en active Pending
- 1993-07-15 WO PCT/US1993/006731 patent/WO1994002589A1/en not_active Application Discontinuation
- 1993-07-15 AU AU46825/93A patent/AU4682593A/en not_active Abandoned
Non-Patent Citations (5)
Title |
---|
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 179, No. 1, issued 30 August 1991, J.W. LARRICK et al., "Complementary DNA Sequence of Rabbit CAP18--A Unique Lipopolysaccharide Binding Protein", pages 170-175. * |
ENDOTOXIN, Volume 256, issued 1990, M. HIRATA et al., "Investigation of Endotoxin Binding Cationic Proteins from Granulocytes: Agglutination of Erythrocytes Sensitized with RE-LPS", pages 287-299. * |
INTERNATIONAL ENDOTOXIN SOCIETY, issued 10 May 1990, M. HIRATE et al., "Modification of LPS Activity by LPS-Binding Protein (CAP-18)", page II-P-87. * |
JOURNAL OF CELLULAR BIOCHEMISTRY, Volume 16, Part C, issued 01 March 1992, J.W. LARRICK et al., "Molecular Characterization of Rabbit CAP18--A Unique Lipopolysaccharide Binding Protein", page 172. * |
See also references of EP0746605A4 * |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6103888A (en) * | 1992-07-17 | 2000-08-15 | Panorama Research, Inc. | Mammalian cationic proteins having lipopolysaccharide binding and anti-coagulant activity |
US6159936A (en) * | 1993-07-20 | 2000-12-12 | The Regents Of The University Of California | Compositions and methods for treating and preventing microbial and viral infections |
AU684170B2 (en) * | 1994-07-08 | 1997-12-04 | A & H Brister Holdings Pty Ltd | Tank roof structure |
US6440690B1 (en) | 1995-06-29 | 2002-08-27 | Amram Mor | Peptides for the activation of the immune system in humans and animals |
US6025326A (en) * | 1995-07-07 | 2000-02-15 | Intrabiotics Pharmaceuticals, Inc. | Compositions and methods for the prevention and treatment of oral mucositis |
US5994306A (en) * | 1995-11-22 | 1999-11-30 | Intrabiotics Pharmaceuticals, Inc. | Fine-tuned protegrins |
US6040291A (en) * | 1998-03-25 | 2000-03-21 | Seikagaku Corporation | Antimicrobial peptide |
US7071293B1 (en) * | 1999-08-18 | 2006-07-04 | The University Of Iowa Research Foundation | Alpha helical peptides with broad spectrum antimicrobial activity that are insensitive to salt |
WO2002095076A2 (en) * | 2001-05-23 | 2002-11-28 | Kabushiki Kaisha Toyota Chuo Kenkyusho | Modified polypeptides having protease-resistance and/or protease-sensitivity |
WO2002095076A3 (en) * | 2001-05-23 | 2003-09-25 | Toyoda Chuo Kenkyusho Kk | Modified polypeptides having protease-resistance and/or protease-sensitivity |
Also Published As
Publication number | Publication date |
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EP0746605A1 (en) | 1996-12-11 |
AU4682593A (en) | 1994-02-14 |
EP0746605A4 (en) | 1997-04-16 |
JPH08504085A (en) | 1996-05-07 |
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