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Description  |
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FIELD OF THE INVENTION
The present invention relates to methods of making and using novel peptide fragments derived from CAP37, which is an approximately 37,000 dalton cationic granule protein normally synthesized by human polymorphonuclear leukocytes. In particular,
this invention relates to novel peptides having monocyte chemotactic activity, such as peptides having the amino acid sequences shown in the Sequence Listing as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 and novel
peptides having antibiotic activity or lipopolysaccharide-binding activity, such as peptides having the amino acid sequences shown in the Sequence Listing as SEQ ID NO:7 and SEQ ID NO:8.
Throughout this application, various publications are referenced. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are
hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
In the process of inflammation an initial wave of inflammatory cells, comprised primarily of polymorphonuclear leukocytes (PMN) is soon followed by a second wave of cells, which are predominantly monocytes (Hurley et al.; Wilkinson et al.).
Although it is widely held that monocytes arrive at an area of inflammation as a result of chemotaxis (Hayashi et al.), the specific mediator(s) responsible for the recruitment of monocytes has remained unresolved.
Monocytes are derived from pro-monocytes, found in bone marrow. The pro-monocytes differentiate into monocytes which are released into the blood. The monocytes circulate in the blood until they are attracted to the site of injury by the
inflammation process. Once monocytes enter into tissue they mature into macrophages, also referred to as mononuclear phagocytes. Macrophages are able to engulf and destroy foreign antigens; accordingly, macrophages play an important role in the body's
immunological defense system. The term "monocyte" as used herein refers collectively to both circulating monocytes and to macrophages present in tissue.
The preferential migration of monocytes during the latter phase of inflammation indicates the requirement for highly cell-specific chemoattractant, which has little or no effect on the migration of PMNs. Experiments indicate that a
granule-associated cationic protein (mol. wt. 37,000 daltons) from human PMN acts as a monocle-specific chemoattractant. This protein has been previously referred to in the literature as CAP37, cationic antimicrobial protein of mol. wt. 37,000 daltons.
CAP37 protein has been previously shown to (i) bind bacterial lipopolysaccharides with a high degree of specificity and affinity, and (ii) possess antimicrobial activity against a number of Gram negative bacteria, such as Salmonella typhimurium and
Escherichia coli (Shafer et al., 1986). Thus, CAP37 may play three important functional roles in host defense.
While use of purified CAP37 has far reaching and important functions involving the cellular progression of inflammation, antimicrobial and antineoplastic defenses of the host, the activity of CAP37 as isolated from human PMNs may be limited
because of (1) the very small quantities that can be purified and (2) the potential hazards of using human blood products. Use of recombinant CAP37 may overcome some of these problems, but CAP37 is still a large molecule. However, synthetic peptide
fragments derived from CAP37 that possess chemotactic, antimicrobial or LPS binding activity and are considerably smaller (e.g., about 25 amino acids in length; approx. 2500-3000 daltons) would overcome these problems. That is, these fragments would be
conveniently sized, capable of being produced in unlimited quantities and possess a non-infectious nature.
The instant specification discloses a conventional method for purifying CAP37 to homogeneity and a method for making CAP37 using recombinant DNA techniques. The characterization of the cDNA encoding human CAP37 protein is disclosed. The instant
specification has also advanced the art into unchartered areas by disclosing proteins and peptides related and/or derived from CAP37 that have monocyte chemotactic activity, antibiotic activity or lipopolysaccharide (LPS) binding activity. These
proteins and peptides can either be synthesized or produced using recombinant DNA techniques. Finally, the instant specification discloses methods for treating diseases and wounds using CAP37 and its related proteins and peptides as well as antibodies
to CAP37 and proteins and peptides derived from CAP37 and methods of using these antibodies.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a recombinant DNA molecule encoding a cationic granule protein having the amino acid sequence shown in the Sequence Listing as SEQ ID NO:9. This protein is a potent chemoattractant for monocytes,
is bactericidal and is capable of binding bacterial lipopolysaccharide.
The invention further includes DNA molecules encoding amino terminal extensions of the cationic granule protein such as, (a) the amino acid sequence shown in the Sequence Listing as SEQ ID NO:10 (Met Thr Arg Leu Thr Val Leu Ala Leu Leu Ala Gly
Leu Leu Ala Ser Ser Arg Ala Gly Ser Ser Pro Leu Leu Asp), and (b) Met. The invention also includes DNA molecules encoding carboxy terminal extensions such as Gly Pro Ala.
Another important object of the present invention is to provide a recombinant method of producing the cationic granule proteins encoded by the above described sequences. These proteins include the above described amino terminal extensions as
well as other modifications, such as the attachment, to the amino or the carboxy terminus of the sequence, of a second protein coding sequence. One example of such a second protein coding sequence is the ricin A chain. Other examples include abrin A
chain and trichosanthin. One method of recombinant production is the expression of the cationic granule protein coding sequence fused to the glutathione-S-transferase protein (Sj26) with an in-frame thrombin cleavage site separating the two coding
sequences. The fusion protein is then isolated by affinity chromatography for the Sj26 protein portion of the fusion protein, and the cationic granule protein is liberated from the fusion protein by digestion of the fusion protein with the thrombin
proteinase.
It is an object of this invention to identify the domains responsible for the antibacterial, chemotactic and LPS-binding activities of the 222 amino acid mature CAP37 protein (SEQ ID NO:9) as extracted from human neutrophils. Thus, it is an
object of the present invention to provide bioactive peptides having at least 5 continuous amino acids derived from the above described sequence. These bioactive peptides are either chemotactic for monocytes, able to bind bacterial lipopolysaccharide
and/or possess antibacterial qualities. The present invention further provides a recombinant process for the production of the bioactive peptides.
Additionally, it is an object of this invention to provide bioactive peptides (such as chemotactic, antibacterial and/or LPS-binding) derived from CAP37 that are capable of being produced in unlimited quantities and possess a non-infectious
nature. Particularly advantageous are those bioactive peptides comprising the peptides shown in the Sequence Listing as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7 and those peptides comprising the peptides shown in the Sequence Listing as
SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8.
Another object of this invention is to provide bioactive peptides derived from CAP37 that possess enhanced bactericidal activity with respect to the mature CAP37 protein against both gram negative and gram positive bacteria.
Another object of the present invention is to provide expression vectors containing DNA sequences encoding any of the above protein or peptide sequences. These vectors are useful for introducing the coding sequences into suitable hosts. The
expression vectors are effective to express the recombinant proteins in a variety of hosts, for example, bacteria, yeast, and mammalian cells. One vector useful for expression of the above described cationic granule protein contains a cytomegalo virus
promoter and a polyadenylation signal from SV40, in addition to the dihydrofolate reductase gene to facilitate selection of the vector in the transformed host.
The invention further includes a method of treating a wound by the application of a topical medication containing a cationic granule protein (CAP37) having the amino acid sequence shown in the Sequence Listing as SEQ ID NO:9, or proteins and/or
peptides derived from CAP37 that are chemotactic for monocytes or possess antibacterial activity, in the medication in a pharmacologically effective amount to promote wound healing and/or treat infection. Other additions to the medication may be
desirable such as the inclusion of epidermal growth factor also present in a pharmacologically effective amount to promote wound healing. The topical medication may take any number of standard forms such as pastes, gels, creams, and ointments.
Additionally, the invention includes methods of treating tumors and other diseases using CAP37 and its related proteins and peptides.
It is an object of this invention to treat infection, especially infection caused by gram negative bacteria, by administration of an effective amount of a peptide derived from CAP37 having bactericidal activity superior to that of the mature
CAP37 protein, most preferably by administration of the peptide defined in the Sequence Listing by SEQ ID NO:8.
Further, this invention provides for a method of treating endotoxemia or a prophylactic treatment, such as before surgery, for endotoxemia consisting of administration of an effective amount of the CAP37 mature protein or a peptide derived from
CAP37 having LPS-binding activity.
The invention also includes hybrid proteins containing the cationic granule protein having the amino acid sequence shown in the Sequence Listing as SEQ ID NO:9 covalently attached to a second protein molecule, such as an antibody, ricin A chain,
abrin A chain or trichosanthin.
These and other features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying examples.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the sequences of peptide fragments derived from the mature CAP37 protein as relevant to the Examples.
FIG. 2 shows a plot of the data which demonstrates the chemotactic properties of the CAP37 protein.
FIG. 3 shows the results of an ELISA showing binding of CAP37 to LPS and Lipid A.
FIG. 4 shows a comparison of the N-terminal amino acid sequence of the CAP37 protein with several inflammatory cell proteases.
FIG. 5 illustrates the pcDNAI cloning vector used for subcloning inserts from original lambda clones.
FIG. 6 shows the nucleic acid and protein sequences of the first isolated cDNA insert having homology to CAP37 protein.
FIG. 7 shows the nucleic acid and protein sequences of the 6a.1 clone insert.
FIG. 8 shows a hydropathy index computation for the entire coding sequence of the CAP37 protein including: the amino-terminal extension and the sequence encoding the mature protein.
FIG. 9 illustrates the expression vectors used for expression of CAP37 protein in mammalian cells.
FIG. 10 depicts the elution profile of the eluates from the hydrophobic HPLC of the impure product obtained after separation on a SEPHADEX G-75 chromatographic column.
FIG. 11 shows a plot of the data which demonstrates the chemotactic properties of peptides derived from the 113th to 122nd amino acid region of the mature CAP37 protein.
FIG. 12 shows the bactericidal effect of peptides derived from the mature CAP37 protein on Salmonella typhimurium SH9178.
FIG. 13 shows the bactericidal effect of peptides derived from the 1st to 44th amino acid region of the mature CAP37 protein on Salmonella typhimurium SH9178.
FIG. 14 shows the bactericidal effect of the peptide 20-44aa (SEQ ID NO:8) derived from the mature CAP37 protein on various gram negative and gram positive bacteria.
FIG. 15 shows a comparison of bactericidal activity of the peptide 20-44aa (SEQ ID NO:8) derived from the mature CAP37 protein against Salmonella typhimurium SH9178 with peptides derived from similar regions from cathepsin G and elastase peptide.
FIG. 16 shows the inhibition of the bactericidal effect of the peptide 20-44aa (SEQ ID NO:8) derived from the mature CAP37 protein on Salmonella typhimurium SH9178 by LPS and Lipid A.
FIG. 17(A) shows the neutralization of wild type LPS activity and FIG. 17(B) shows the neutralization of ReLPS by the peptide 20-44aa (SEQ ID NO:8) derived from the mature CAP37 protein as assayed by the limulus amebocyte lysate assay.
DETAILED DESCRIPTION OF THE INVENTION
I. PURIFICATION AND CHARACTERIZATION OF THE HUMAN CAP37 PROTEIN
A. Purification and Sequencing of CAP37 Protein.
CAP37 protein was purified from polymorphonuclear leukocytes (PMN) obtained from healthy adult donors (Example 1-A). The crude preparation generated from PMNs was dialyzed and applied to an ion exchange column. The eluent containing the CAP37
protein came off of the column at NaCl concentrations in the range of 0.6 to 0.7M, as judged by SDS-PAGE of the fractions. This eluent was dialyzed and concentrated. The concentrated preparation was size fractionated and the fractions analyzed by
SDS-PAGE to determine the fractions containing the CAP37 protein (Example 1).
Ascites fluid containing antibodies against CAP37 protein was prepared by injecting mice with alum-adsorbed CAP37 protein emulsified in Freund's complete and Freund's incomplete adjuvant (Example 1-C). The specificity of the ascites fluid
reactivity against the CAP37 protein was demonstrated in ELISA assays using CAP37, CAP57 (a cationic antimicrobial protein of mol. wt. 57,000 Da; Spitznagel et al.), lactoferrin, myeloperoxidase, cathepsin G and lysozyme as the antigens.
Immunocytochemical analysis of normal human PMN using the monospecific polyvalent mouse antiscrum to CAP37 established that CAP37 was a component of the cytoplasmic granules of human PMNs. All other peripheral blood cells which included
eosinophils, monocytcs, lymphocytes and red blood cells, did not stain positive for CAP37. Immunocytochemical analysis also demonstrated the presence of CAP37 in normal bone marrow, in cells belonging to the myeloid lineage, and in peripheral blood PMN
from patients with chronic myelogenous leukemia (CML).
For chemotactic assays, the CAP37 protein was further purified by fractionation of the preparation using hydrophobic high performance liquid chromatography (HPLC). For amino acid sequence determination, a final desalting step was added utilizing
reverse-phase HPLC (Example 1).
The N-terminal amino acid sequence of the CAP37 protein was determined by standard microsequencing procedures (Example 1-D). FIG. 1 (Example 1) shows the peptide fragment sequences which were determined for part of the mature CAP37 protein. The
first 42 amino acids at the amino terminus of CAP37 have received accession number AB3070 from the Biomedical Research Foundation, Georgetown University Medical Center, Washington, D.C. 20007.
B. Chemotactic and LPS-binding Properties of CAP37 Protein
Chemotaxis assays were performed using the modified Boyden chamber technique (Example 2). The results (FIG. 2) demonstrated that CAP37 was obviously chemotactic for human monocytes in the range of 10 to 10,000 pM. CAP37 appeared to be as
effective as FMLP in attracting monocytes. These experiments suggest CAP37 did not have any effect on PMN and lymphocyte chemotaxis.
In addition to the observed chemotactic effect on human monocytes, CAP37 at a higher concentration of 1000 ng/ml (2.7.times.10.sup.-8 M) was chemotactic for rabbit monocytes as well. This may reflect either (i) a reduction in the numbers of
receptors for CAP37 on rabbit monocytes as compared to the number of receptors on human monocytes, or, (ii) a difference in the K.sub.a values for the two species. The effect was selective for monocytes since, rabbit PMN did not show a chemotactic
response towards CAP37.
One of the important aspects of identification of a chemotaxin is to distinguish directed cell movement (chemotaxis) through the filters as opposed to merely accelerated random motion (chemokinesis). Using the checkerboard assay (Example 2) it
was demonstrated that, in addition to its chemotactic properties, CAP37 protein also has some chemokinetic effect on monocytes.
LPS-binding activity of the mature CAP37 protein was determined by measuring the LPS neutralization by CAP37 as determined by the Limulus Amebocyte Lysate (LAL) assay. CAP37 significantly neutralized LPS activity in the LAL assay. Further,
ELISA shows binding of CAP37 to LPS; CAP37 is capable of binding LPS and, in particular, Lipid A (FIG. 3).
C. Protein Sequence Comparisons with the Amino Terminus of the CAP37 Protein.
Using the amino terminal sequence of the CAP37 protein, a homology search was conducted of known protein sequences. This search revealed substantial homologies with the amino termini of a subset of serine proteases which mediate a number of
functions involved in the inflammatory response (FIG. 4).
Most of the serine proteases with which CAP37 protein shares its greatest homology (elastase, cathepsin G, rat mast cell proteases I and II, H Factor and cytotoxic T cell specific protein I) are derived from the granules of peripheral blood
cells. Further, these homologous proteins are known to play important roles in inflammation, such as cytolysis or degradation of extracellular matrices.
Two other serine proteases, bovine thrombin (Bing et al.) and a trypsin-like protease in guinea pig plasma (Kawaguchi et al.), have been described which exhibit chemotactic activity. Further, Wright et al. have described the presence of a
factor(s) in PMN specific granules which acts on serum to produce chemotactically active C5a and the opsonin C3b.
The experiments performed in support of the present invention show the isolation, characterization and purification to homogeneity of a PMN-granule associated protein which has monocyte specific chemotactic activity. Further, the CAP37 protein
possesses LPS-binding activity as well as bactericidal activity and thus may perform three very important functional roles in vivo.
EXAMPLE 1
Purification and sequencing of human CAP37 protein
A. Source and Preparation of Peripheral Blood PMN and Monocytes
Blood was collected into sterile sodium-EDTA tubes by venepuncture from healthy adult donors. The polymorphonuclear leukocytes (PMN) were separated from mononuclear cells essentially by the ficoll hypaque density gradient technique of Boyum,
followed by dextran sedimentation (T500, Pharmacia 3% in saline) and hypotonic lysis of contaminating red blood cells (RBC).
The mononuclear cell band was further purified to separate the monocytes from the lymphocytes. The mononuclear cells were washed once in phosphate buffered saline (PBS: 0.01M Na.sub.2 HPO.sub.4, containing 0.15M NaCl, pH 7.4) and resuspended in
PBS to a total volume of 5.1 ml. This cell suspension was then added to 6.7 ml of SEPRACELL-MN reagent (a colloidal, silica-based medium having a density of 1.099 g/ml, and available from Sepratech Corporation, Oklahoma City, Okla.), and centrifuged
(1500 g for 20 min at 22.degree. C.). The cells were washed twice in PBS (150 X g, 15 min) and resuspended in Geys buffered saline (Gibco) containing 2% bovine serum albumin (BSA-Fraction V, endotoxin free, Boehringer Mannheim Biochemicals) at a final
concentration of 2.times.10.sup.6 cells/ml. (Geys buffer has the following composition: CaCl.sub.2 (anhyd.) 0.17 g/L; KCl 0.37 g/L; KH.sub.2 PO.sub.4 0.03 g/L; MgCl.sub.2.6H.sub.2 0.21 g/L; MgSO.sub.4.7H.sub.2 O 0.07 g/L; NaCl 7 g/L; NaHCO.sub.3 2.27
g/L; Na.sub.2 HPO.sub.4.7H.sub.2 0.226 g/L; D-Glucose 1.00 g/L.) The monocytes were greater than 95% pure as determined by Wright's and non-specific esterase staining.
Peripheral blood mononuclear cells from adult female rabbits, were separated on a 61% PERCOLL (Pharmacia) gradient (Chambers et al.). This technique separated the mononuclear cells from the RBC and PMNs. No further separation of monocytes from
lymphocytes was undertaken with the rabbit blood.
B. Purification of CAP37
A granulocyte concentrate (>95% PMN) was obtained by leukophoresis from a normal human donor. The PMN were disrupted by homogenization in a Potter-Elvehjem tissue grinder (Kontes) for 60 sec at 4.degree. C. Mixed (specific and azurophil)
granules were harvested by differential centrifugation. The supernatant obtained by centrifuging at 126 x g for 15 minutes was further centrifuged at 20,000 x g for 20 minutes to yield a pellet of mixed granules which was extracted at 4.degree. C. with
0.2M sodium acetate (pH 4.0). Granule debris was collected by high-speed centrifugation at 20,000 x g for 30 min. The protein concentration was determined by the method of Bradford with chick egg white lysozyme as the standard (Bradford et al., 1976).
The crude granule extract was dialyzed against 50 mM sodium acetate (pH=5) and 0.15M NaCl at 4.degree. C. overnight. The dialysate was then applied to a carboxymethyl SEPHADEX ion exchange column (a weakly acidic cation exchanger having sodium
as a counter-ion and carboxymethyl as a functional group, and available from Pharmacia, Piscataway N.J.) which had been equilibrated with 50 mM sodium acetate (pH=5) and 0.15M NaCl. The column was extensively washed with 6M urea in 0.05M sodium acetate
(pH5) containing 1.5M sodium chloride. Protein bound to the column was eluted using a two-step linear salt gradient consisting of 0.15 to 0.4M and 0.4 to 1.0M NaCl in 50 mM sodium acetate pH=5. Protein elution was monitored by measuring the absorbance
of the eluent at 280 nm. Salt concentrations of the fractions were determined by conductivity measurements. Fractions from the CMS column were tested by ELISA using antiserum to CAP37 to determine those fractions which contained CAP37.
The positive fractions were pooled and dialyzed overnight against 0.2M sodium acetate (pH=4) at 4.degree. C., concentrated by membrane ultrafiltration at 4.degree. C. using a YM-5 membrane filter (an ultrafiltration membrane having very low
non-specific protein binding properties, a nominal molecular weight cut-off of 5000 daltons, a clean water flow rate of 0.07-0.1ml/min/cm.sup.2 and available from Amicon Corp., Lexington Ky.). The concentrated preparation was applied to a SEPHADEX G-75
SF (Pharmacia) column (0.5 by 50 cm) which had been equilibrated with 0.2M sodium acetate (pH=4). (SEPHADEX G-75 is a chromatographic medium comprised of a bead-formed gel prepared by crosslinking dextran with epichlorohydrin; said medium having a dry
bead size of 10-40 .mu., bed volume of 12-15 ml/g of dry SEPHADEX, and fractionation range for globular proteins and peptides of 3,000-70,000 daltons.) Fractions were eluted using the equilibration buffer and the A.sub.210 of each fraction determined.
The fractions were analyzed by ELISA and by SDS-PAGE to determine the fraction containing the CAP37 protein.
The CAP37-enriched fraction obtained from the molecular sieve column was further processed using hydrophobic high performance liquid chromatography (HPLC) (BIO-GEL TSK phenyl 5PW column, 7.5 mm.times.0.75 mm, packed with a 10 micron macroporous
support having 1,000 angstrom pores and containing a low density of phenyl groups which promote strong but nondenaturing hydrophobic interactions with proteins, said column available from Biorad Laboratories). The proteins were eluted using a 60 min
linear gradient from 1.7M to 0M (NH.sub.4).sub.2 SO.sub.4, which contained 0.1M sodium phosphate pH 7.0. The recovery was determined by the optical density of the proteins at 210 nm. The fractions obtained from the hydrophobic HPLC column were pooled
independently and the (NH4).sub.2 SO.sub.4 removed by dialysis employing a stirred cell concentrator (YM-5 membrane, Amicon) with 0.2M sodium acetate buffer, pH=4.0.
The purity of the fractions was determined by SDS-PAGE, western blot analysis and ELISA. All these results confirm that CAP37 prepared in this manner is devoid of other contaminating granule proteins which include the defensins, cathepsin G,
myeloperoxidase, lactoferrin, and CAP57, and thus indicates that the outlined method yields a highly purified preparation of CAP37.
An important aspect of the production of CAP37 was to keep it free from endotoxin contamination. All reagents and buffers were prepared using pyrogen-free water and tested for endotoxin contamination using the Limulus amoebocyte assay (Whittaker
Products). All glassware used was pyrogen-free. The starting material for each column was always checked for endotoxin contamination before it was applied to the column and, most importantly, the final product was always checked for the presence of
endotoxin before use.
As seen in FIG. 10, the elution profile from the hydrophobic HPLC column indicated the presence of two proteins. CAP37 was confined to Peak 2, the more hydrophobic peak. Peak 1 was found to contain cathepsin G, as judged by SDS-PAGE.
SDS-PAGE was performed to depict the stages of purification of CAP37 from normal human crude granule extract (CGE). The analysis was performed according to the method described by Laemmli (1970). The samples were solubilized in 0.625M Tris (pH
6.8), 4% (w/v) SDS, and 1% (v/v) beta mercapto ethanol, at 100.degree. C. for 5 min and analyzed on a 12.5% gel essentially according to the method of Laemmli. The analysis was performed under reducing conditions with a 12.5% separating gel and a 4%
stacking gel as described by Laemmli. The ratio of the acrylamide to the bisacrylamide was 37.5. Electrophoresis was carried out until the bromophenol blue dye reached the bottom of the separating gel. The gel was removed and fixed in a 25%
isopropanol, 7% acetic acid mixture. The gel was oxidized and stained with silver to visualize the protein bands.
The gel was silver stained according to well established protocols. Lane 1 contained 2 .mu.g "rainbow" molecular weight markers (Amersham). Lane 2 contained 5 .mu.g of crude granule extract (CGE). Lane 3 contained 400 ng of peak C material
obtained from the G-75 column which served as the starting material for the HPLC column. Lane 4 contained 350 ng of peak 2 (CAP37) from the HPLC column. Lane 5 contained 350 ng of peak 1 from the HPLC column and lane 6 contained purified cathepsin G.
The molecular weight standards used included myosin (M, 200,000), phophorylase b (M.sub.r 92,500), bovine serum albumin (M.sub.r 69,000), ovalbumin (M.sub.r 46,000), carbonic anhydrase (M.sub.r 30,000), trypsin inhibitor (M.sub.r 21,500) and lysozyme
(M.sub.r 14,300).
The CAP37 material from peak 2 (lane 4) migrated in SDS-PAGE as three extremely closely spaced bands with a molecular weight near 37,000 daltons. This electrophoretic behavior probably indicates glycosylation of the protein, rather than any
heterogeneity of the protein preparation. Furthermore, the fact that this preparation yielded an unambiguous amino acid sequence would also tend to indicate the absence of heterogeneity.
Judging from the nearly identical migration patterns of peak 1 from the HPLC Column (lane 6) and cathepsin G (lane 5), it may be concluded that this peak 1 material is cathepsin G. This was confirmed by the western blots.
The fractions obtained from the HPLC column were also analyzed by western blots. Western blot analysis was carried out according to the method of Towbin et al. (1979) with some modifications. Following electrophoresis on a 12.5% SDS-PAGE gel,
the proteins were transferred onto a nitrocellulose membrane (Biorad, Richmond, Calif.) in a TE series, Transphor Electrophoresis Unit (Hoefer Scientific Instruments, Calif.) under constant current (200 mA) at 11.degree. C. for 1.5 hr. The
concentration of the methanol in the buffer was reduced from 20% to 5% (v/v) and the pH raised to 9. The protein was detected on the nitrocellulose membrane using a mouse antibody to CAP37 (1:100 dilution) and an alkaline phosphatase immunoblotting
system with the addition system of 10 units per ml heparin sulfate buffer (Eastman, Kodak) (Spitznagel et al., (1987)) in the wash buffer (0.15M NaCl, 0.01M Tris HCl (pH 7.5), 0.01% v/v TWEEN 20). Color development was obtained with the nitroblue
tetrazolium/BCIP system.
The results of the western blot confirmed the SDS-PAGE results. Lane 1 contained molecular weight markers as described for the SDS-PAGE. Lanes 2 and 6 contained crude granule extract. Lanes 3 and 7 contained peak C material from the G-75
column. Lanes 4 and 8 contained peak 2 from the HPLC column and lanes 5 and 9 contained peak 1 from the HPLC column. The protein concentrations of all the samples loaded onto the gel were the same as described above.
Lanes 1 to 5 were probed with goat anti-human cathepsin G (1:2000 dilution) and lanes 6 to 9 were probed with mouse anti-human CAP37 (1:100 dilution). The second antibody was conjugated to alkaline phosphatase (1:7500 dilution, Promega). Color
development was obtained using the nitroblue tetrazolium/BCIP system as outlined in the manufacturers handbook (Promega). The goat anti-human cathepsin G antiserum reacted with crude granule extract, peak C material and peak 1 from the HPLC column. It
did not react with CAP37 (peak 2 material from the HPLC column). On the other hand, the antiserum to CAP37 reacted with peak 2 material but not with the cathepsin G. This indicates that CAP37 was totally separated from the contaminating cathepsin G in
the final step of the purification which is performed on the hydrophobic HPLC column.
The proteins obtained from the HPLC column were further analyzed by ELISA which confirmed the purity of the CAP37 preparation. The ELISA method used is described in Pereira, et al., (1989), and has proven to be a reproducible and sensitive
method for the detection of cationic proteins. Briefly, the protein was attached to a 96 well microtitre plate (NUNC Immunoplate I, VWR Scientific) which was pre-treated with poly-L-lysine. Following an overnight incubation at 4.degree. C., the plate
containing the antigen was washed four times in phosphate buffered saline (pH 7.4). Non-specific sites on the plate were then blocked with a heparin containing phosphate buffer, at room temperature for 1 hr (Pereira et al., 1989). The plates were
washed and the primary antibody was then added to the plate and incubated for 1 hr at 37.degree. C. Following this incubation the plate was washed and the second antibody was added and the plate incubated for 1 hr at 37.degree. C. The second antibody
was a goat anti-mouse immunoglobulin which is conjugated to horse-radish peroxidase. The color development step involves incubating the substrate (3.7 mM 0-phenylenediamine) in citrate phosphate buffer, pH 5.0, containing 0.24 .mu.l/ml 30% H.sub.2
O.sub.2 at room temperature for 30 min. The reaction is stopped with 2.5M H.sub.2 SO.sub.4 and the absorbance read immediately using a TITERTEK multiscan plate reader (Flow Laboratories) at 492 nm.
The results of the ELISA confirmed the results of the other analyses. The same samples as used for the SDS-PAGE were also used in the ELISA. Primary antibodies were prepared against each of these samples. The material from Peak 2 reacted only
with the antibodies prepared against a pure CAP37, while the material from Peak 1 reacted with the antibody prepared against Cathepsin G. The crude granule extract reacted with all the antibodies and the Peak C material reacted with antibodies for CAP37
and for Cathepsin G.
For amino acid sequencing of the CAP37 protein a final desalting step using reverse phase HPLC was employed. A DYNAMAX 300A C8 column was equilibrated with 0.1% Trifluoroacetic acid in water the above-purified material was then applied to the
column. Elution of the purified protein was effected by a 30 minute elution with a 0-80% gradient of 0.1% Trifluoroacetic acid in acetonitrile at a flow rate of 1 ml per minute. The CAP37 protein containing sample corresponded to the 16.14 minute peak,
as determined by SDS-PAGE: this fraction was concentrated in a Savant Instruments SPEED VAC.
C. Antibodies to Purified CAP37 Protein
Ascites fluid containing antibodies against CAP37 protein was prepared by injecting BALB/c mice (Jackson Laboratories) with 250 ng of alum-adsorbed (Herbert) CAP37 protein emulsified in an equal volume of Freund's complete and Freund's incomplete
adjuvant (Difco Laboratories). Mice were subcutaneously injected with a total volume of 200 .mu.l of the above suspension. The mice were boosted three weeks later with 250 ng of alum-adsorbed CAP37 injected intraperitoneally. At the same time the mice
were injected with 1 ml of pristane (2,6,10,14-tetramethyl pentadecane, Sigma). One week later 10.sup.7 SP2/0 mouse myeloma cells were injected intraperitoneally (Lacy et al.). Ascites fluid was collected. The specificity of the ascites fluid
reactivity against the CAP37 protein was demonstrated in ELISA assays (Pereira et al.) using CAP37, CAP57 (a cationic antimicrobial protein of mol. wt. 57,000 Da; Spitznagel et al.), lactoferrin, myeloperoxidase, cathepsin G and lysozyme as the antigens. Further, no cross reactivity between Cathepsin G and CAP37 protein was observed by western blot analysis (Ausubel et al.).
For the immunocytochemical studies, the final cell concentration was adjusted to 1.times.10.sup.6 nucleated cells per ml in 10% heat inactivated fetal bovine serum (Hyclone Laboratories Inc., Utah) in PBS. One hundred microlitres of the cell
suspension was cytocentrifuged onto glass slides. The cells were fixed in buffered formol acetone, pH 7.2 at 4.degree. C. for 60 seconds. The staining was performed using the VECTASTAIN Avidin Biotin Complex--Glucose oxidase (ABC-G0) technique
(Vectastain Laboratories, Burlingame, Calif.) as described previously (Spitznagel et al.). The above described monospecific polyvalent mouse anti CAP37 ascites fluid (1:100) was used to stain the cells. Color development was obtained using the
nitroblue tetrazolium salt (VECTASTAIN GO substrate kit I) at room temperature for 30 minutes. Normal mouse serum and phosphate buffered saline (PBS) served as the negative controls. Ascites fluid made against myeloperoxidase, a known marker of the
primary granule of PMNs, served as the positive control.
D. Microsequencing of CAP37
The protein sequence analysis of CAP37 was performed using an Applied Biosystems Model 477A Protein/Peptide Sequencer with an on-line Applied Biosystems 120A PTH-Amino Acid Analyzer. Reagents and solvents were from Applied Biosystems, Foster
City, Calif. Phenylthiohydantoin (PTH)-derivatized amino acids formed sequentially by Edman degradation were separated using an Applied Biosystems PTH C-18 HPLC reverse phase microbore column (2.1 mm ID.times.220 mm) by gradient elution. The sample was
applied to an acid-etched glass-fiber filter which had been treated with 3 mg Biobrene (polybrene) and precycled. Peak identification and yield quantitation was based on a standard PTH-amino acid profile. The N-terminal end of the CAP37 protein was
identified by standard microsequencing procedures (Applied Biosystems). Trypsin and chymotrypsin generated digestion fragments of CAP37, were separated and purified using hydrophobic and reverse-phase HPLC (Example 1-B). These fragments were then
sequenced, as described above. When the cloning of the cDNA was undertaken only the sequences shown in FIG. 1 had been determined. The sequences of the amino and carboxy termini of the mature CAP37 protein are designated in FIG. 1.
EXAMPLE 2
Chemotactic and LPS-binding Properties of the Purified Human CAP37
A. In Vitro Chemotaxis Assays
Chemotaxis was measured using the modified Boyden chamber technique (Snyderman et al.). The leading front method (Zigmond et al.) was used to assess migration of monocytes and lymphocytes through a 8 .mu.m filter (Millipore Corporation, Bedford,
Mass.). PMN chemotaxis was measured using a 3 .mu.m filter (Millipore). The purified CAP37 protein (Example 1) used in chemotaxis assays were endotoxin-free as determined by the Limulus Amoebocyte Lysate Assay (Whittaker Bioproducts, Inc.,
Walkersville, Md.). The dilutions of CAP37 and N-formyl-methionyl-leucyl-phenylalanine (FMLP) were made in Geys buffer (Gibco) containing 2% endotoxin-free bovine serum albumin (BSA). Geys buffer containing 2% BSA served as the negative control, and a
10.sup.-8 M solution of FMLP as the positive control.
The chambers were incubated in a humidified atmosphere (6.2% CO.sub.2) for 2 hours when testing monocytes and lymphocytes, and for 30 minutes when testing PMNs. The filters were then removed and processed as previously described (Snyderman et
al.). The cells were viewed using oil immersion and the distance the cells had migrated into the filter was determined over five different fields on the same slide (Zigmond et al.). Triplicate assays were performed for each experimental point.
The results are presented in FIG. 2. The data clearly show the strong chemotactic properties of the CAP37 protein.
CAP37 did not have any effect on PMN and lymphocyte chemotaxis in the range employed in these experiments. In addition to the observed chemotactic effect on human monocytes, CAP37 at a higher concentration of 1000 ng/ml (2.7.times.10.sup.-8 M)
was chemotactic for rabbit monocytes as well. The effect was selective for monocytes since rabbit PMN did not show a chemotactic response towards CAP37.
To distinguish directed cell movement (chemotaxis) through the filters of a Boyden chamber as opposed to accelerated random cell motion (chemokinesis), the chemokinetic activity of CAP37 was determined by the checkerboard assay of Zigmond et al.
The checkerboard assay demonstrated that in addition to its chemotactic properties, CAP37 has some chemokinetic effect on monocytes.
FIG. 3 shows the results of an ELISA procedure used for determining the binding of CAP37 to LPS from S. minnesota wild type (diamonds); LPS from S. minnesota Re mutant (triangles); Lipid A from S. minnesota R595 (circles); and Lipid A from S.
typhimurium Re mutant (squares). The LPS from S. minnesota wild type, S. minnesota Re mutant, Lipid A from S. minnesota R595, and Lipid A from S. typhimurium Re mutant were applied to microtitre wells at concentrations ranging from 0 to 50 .mu.g/ml and
incubated overnight. The trays were then incubated with CAP37 at 120 ng/well for 1 hour at 37.degree. C. The next incubation was with a rabbit antiserum against CAP37 (1:400 dilution) and the color development was as previously described (Pereira et
al., 1989) and the absorbance was read at 492 nm. The results shown in FIG. 3 indicate that CAP37 is capable of binding Re LPS and, in particular, is able to bind to Lipid A better than wild type LPS.
Further, the neutralization of LPS activity, as assayed by the limulus amebocyte lysate (LAL) assay, by CAP37 was measured. CAP37 was diluted to the desired concentrations (from 6.2 ng/ml to 500 ng/ml) in geys buffered saline pH 7 (GIBCO). To
this wild type LPS (RIBI IMMUNOCHEM) or Re LPS from Salmonella (David Morrison, Kansas Medical Center) was added so that the final concentrations were 0.1, 0.5, 1.0 and 2.0 ng/ml LPS. The tubes were incubated at 37.degree. C. for between 30 to 60
minutes and then assayed in the chromogenic LAL assay, using polymyxin E as a control. CAP37 significantly neutralized LPS activity in the LAL assay.
B. In Vivo Chemotactic Assay
The purpose of these experiments is to document that CAP37 when injected in vivo into mice will result in the emigration of monocytes/macrophages.
Female BALB/c mice (6-8 weeks) (Jackson Laboratory, Bar Harbor, Mich.) were injected intraperitoneally (i.p.) with 100 ng CAP37 (purified according to the method of the invention) per mouse in 2 ml RPMI-1640 serum-free medium (Mediatech). A
control group of mice were injected i.p. with 2 ml brewers thioglycollate (4% w/v) (Difco Laboratories), a known stimulator of inflammatory cells whose action is well documented. A second control group of mice were injected i.p. with 2 ml of RPMI-1640
alone.
(RPMI-1640 serum free medium from Mediatech has the following composition: CaNO.sub.3.4H.sub.2 O:100.00 mg/L; KCl:400.00 mg/L; MgSO.sub.4.7H.sub.2 O:100.00 mg/L; NaCl:6000.00 mg/L; NaHCO.sub.3 :2000.00 mg/L; Na.sub.2 HPO.sub.4. 7H.sub.2
O:1512.00 mg/L; D-Glucose:2000.00 mg/L; Glutathione (reduced):1.00 mg/L; Phenol red:5.00 mg/L; L-Arginine(free base):200.00 mg/L; L-Asparagine:50.00 mg/L; L-Aspartic acid:20.00 mg/L; L-Cystine:50.00 mg/L; L-Glutamic acid:20.00 mg/L; L-Glutamine:300.00
mg/L; Glycine:10.00 mg/L; L-Histidine (free base):15.00 mg/L; L-Hydroxyproline:20.00 mg/L; L-Isoleucine (Allo free):50.00 mg/L; L-Leucine (Methionine free):50.00 mg/L; L-Lysine HCl:40.00 mg/L; L-Methionine:15.00 mg/L; L-Phenylalanine:15.00 mg/L;
L-Proline (Hydroxy L-Proline free):20.00 mg/L; L-Serine:30.00 mg/L; L-Threonine (Allo free):20.00 mg/L; L-Tryptophan:5.00 mg/L; L-Tyrosine:20.00 mg/L; L-Valine:20.00 mg/L; Biotin:0.20 mg/L; D-Ca pantothenate:0.25 mg/L; Choline chloride:3.00 mg/L; Folic
acid:1.00 mg/L; i-Inositol:35.00 mg/L; Nicotinamide:1.00 mg/L; Para-aminobenzoic acid:1.00 mg/L; Pyridoxine HCl:1.00 mg/L; Riboflavin:0.20 mg/L; Thiamine HCl:1.00 mg/L; and Vitamin B.sub.12 :0.005 mg/L.)
At 6, 24, 48 and 72 hours following these i.p. injections, four mice in each group were sacrificed by CO.sub.2 anesthesia. Ten ml sterile medium was injected into the peritoneal cavity of each mouse and the abdominal area gently massaged to
dislodge the exudate cells. The medium containing the exudate cells was then aspirated from the peritoneal cavity using a 19 gauge needle attached to a syringe. The total number of cells in the peritoneal exudate was determined by counting the cells on
a COULTER COUNTER (Coulter Electronics, Hialeah, Fla.: The operation of the COULTER COUNTER is based on electrical conductivity difference between particles and common diluent. Particles act as insulators, diluents as good conductors. The particles,
suspended in an electrolyte, are forced through a small aperture through which an electrical current path has been established. As each particle displaces electrolyte in the aperture, a pulse essentially proportional to the particle volume is produced.
Thus a 3-dimensional particle volume response is the basis for all sizing, regardless of position or orientation of the particle in solution.) One hundred .mu.l of the peritoneal exudate was cytocentrifuged onto a glass slide and the cells stained with
Wright's stain. A differential count of the cells was made by counting the number of monocytes, lymphocytes, neutrophils, basophils and eosinophils per hundred consecutive cells. These experiments were performed to determine the time course of
migration of neutrophils, monocytes, lymphocytes and other cells into the peritoneal cavity, in response to an i.p. injection of CAP37.
Traditionally neutrophils are the first cells to migrate into the peritoneal cavity, and they do so about 6 hours after the i.p. injection. Monocytes emigrate much later, generally 72 hours to 6 days after the i.p. injection. The results,
shown in Table 1 below, indicated that an injection of 100 ng of CAP37 into the peritoneal cavity, moderately increased the overall numbers of cells emigrating into the peritoneal cavity. Nevertheless, monocytes appeared in the peritoneal cavity much
earlier (24 hours) than when thioglycollate was used. Furthermore, the percentage of monocytes elicited with CAP37 (55%) was much greater than when sodium thioglycollate (33%) was used even at the 72 hour time point. A further interesting observation
was the dramatic reduction in neutrophils by 24 hours with the CAP37 injection whereas, with thioglycollate, the neutrophils persist at a very high percentage up to 48 hours (36%), and are still present at 72 hours (21%).
TABLE 1 ______________________________________ Migration of cells following intraperitoneal injection with the inflammatory stimulus indicated. Inflammatory Hours Post Total % mono- Stimulus.sup.a Injection Cells.sup.b .times. 10.sup.6
cytes % PMN ______________________________________ None 0 3.1 18 30 RPMI-1640 6 1.5 15 42 24 2.2 12 11 48 3.1 14 28 72 2.8 43 5 Thioglycollate 6 9.5 4 65 24 9.1 5 77 48 8.4 20 36 72 11.1 33 21 CAP 37 6 3.6 7 65 24 3.8 27 26 48 3.5 48 4 72
3.3 55 3 ______________________________________ .sup.a Four mice per group .sup.b Mean value for each group of four mice
These responses with CAP37 contrasted markedly with responses to endotoxin in which far greater cell numbers are found to endotoxin in which far greater cell numbers are found and PMN persist as the predominant cell type for at least 48 hours
(Snyderman et al, (1971)). The migration of monocytes into the peritoneal cavity following CAP37 injection into the peritoneal cavity of mice demonstrates the potent monocyte chemoattractant capacity of CAP37 in this in vivo animal model.
EXAMPLE 3
Comparison of the CAP37 protein amino terminal sequence
Using the first 45 amino acids of the amino terminal end of the CAP37 protein, a search of a protein sequence data base was made using the FASTAMAIL program through the BIONET network. This search revealed substantial homologies with the amino
termini of a subset of serine proteases which mediate a number of functions involved in the inflammation response (see FIG. 4: elastase, ELAST; complement factor D, FACTD; bovine plasminogen, PLASM; cathepsin G, CATG; rat mast cell protease I and II,
RMCPI and RMCPII; cytotoxic T cell I, CCPI; cytotoxic T cell protease--H Factor, HF).
The closest over-all homologies were obtained with two PMN-derived granular proteins: a homology of 57.5% with human elastase (Sinha et al.) also known as medullasin (Okano et al.) and a homology of 45% with human cathepsin G (Salvesen et al.).
Other specific homologous sequences were seen with bovine plasminogen (45%) and human complement factor D (45.5%) (Johnson et al.; Nieman et al. et al.), which is the first enzyme involved in the activation of the alternative complement pathway.
Two other groups of proteins which demonstrated strong homology with the CAP37 protein were: (a) the group of serine protease-like molecules derived from granules of atypical rat mast cells--rat mast cell protease I (Woodbury et al.), RMCPI,
38.6%, and rat mast cell protease II, RMCPII, 40% (Benfey et al.); and (b) two proteins from cytotoxic T cells-cytotoxic T cell protease I (Lobe et al.), CCPI, 40%, and cytotoxic T cell protease factor H (Gershenfeld et al.), HF, 38.6%.
II. CLONING OF THE CAP37 PROTEIN CODING SEQUENCE: ISOLATION OF A FIRST CDNA HAVING HOMOLOGY TO CAP37 PROTEIN.
For the isolation of the CAP37 cDNA coding sequence, poly-A mRNA was isolated from tumor cell line HL60 (Example 4-A,B). Using the CAP37 protein specific antibodies generated in Example 1, the HL60 cell line was shown to contain antigens which
reacted with this antibody. Double-stranded (ds) cDNA was generated from the isolated RNA molecules (Example 4-C,D). These ds molecules were cloned int | | |
