JP2002505252A - Use of a heparin-binding protein to modulate or prevent apoptosis in mammalian cells - Google Patents

Abstract

  (57) [Summary] A heparin-binding protein for modulating or reducing apoptosis in mammalian cells of a mammal, comprising administering to the mammal an effective amount of a heparin-binding protein or a pharmaceutically acceptable active fragment thereof of the present invention. Regarding the use of

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to mammalian cells, comprising administering to a mammal an effective amount of heparin-binding protein (HBP) or a pharmaceutically acceptable fragment thereof. In particular, it relates to the use of heparin-binding proteins to modulate or reduce apoptosis in neurons, islets of Langerhans and endothelial cells.

BACKGROUND OF THE INVENTION Cell apoptosis: Cell apoptosis, also known as programmed cell death, is of great importance for metazoan animal evolution and homeostasis. Apoptosis is a catabolic enzyme, particularly within the limits of the plasma membrane, which is almost intact.
(catabolic enzyme) is the process by which essential macromolecules are degraded, leading to the characteristic biochemical and ultrastructural death phenotype of cells (Kroemer et al., 199).
7, Immunol. Today 18: 44-51, Zamzami et al., 1997, J. Bioenergetics and Bi.
omembranes, 29: 185-193).

[0003] In particular, cells are responsible for a variety of death-inducing stimuli, such as deficiency of unavoidable survival factors, lack of metabolic supply, death-signaling receptors (eg Fas / APO-1 / CD95) and tumor necrosis factor receptors. Suffers from subnecrotic damayl due to association with the body, combination of opposing signals, or toxin, heat or radiation. The stimuli are mitochondrial permeability transitions involved in pore formation (mitchondrial sermeabil
Initiate ity transition) (PT). The pores appear to consist of several proteins located on the inner and outer mitochondrial membranes that cooperate with each other at the site of contact where the two membranes are in close juxtaposition.

As a result, mitochondrial dysfunction, particularly disruption of mitochondrial transmembrane potential, separation of respiratory chains, release of soluble transmembrane proteins, efflux of matrix calcium and glutathione, disruption of mitochondrial biogenesis, and superoxide There is an overproduction of anions. This mitochondrial dysfunction results in secondary endonuclease activation and consequently oligonucleotide
Apoptogenic by DNA fragmentation Can cause activation and action of proteases. It is believed that cytochrome c plays a role in activating apoptosis-producing proteases (Read, 1997, Cell 91: 559-5).
62).

[0005] Apoptosis is a prominent force in forming embryonic organisms, as a major mechanism for precise regulation of cell membranes, and as a defense mechanism for removing unwanted and substantially dangerous cells. Can work. However, in addition to the beneficial effects of programmed cell death, inappropriate activation of apoptosis can cause or contribute to various disorders (Thompson, 1995, Science 267: 1456-).
146).

[0006] They include virus-induced lymphocyte depletion (AIDS); death in cells in neurodegenerative disorders characterized by the gradual loss of specific sets of neurons (eg, Alzheimer's disease, Parkinson's disease, ALS, Retinitis pigmentosa, spinal muscular atopy and various forms of cerebellar degeneration); death of cells in blood cell damage due to growth factor deprivation (
Anemias associated with chronic diseases, aplastic anemia, chronic depletion and myelodysplastic syndrome); and disorders resulting from acute loss of blood flow (eg, myocardial infarction and stroke).

[0007] Anti-apoptotic agents: Treatments that can increase the apoptotic threshold of specific cells may be beneficial in treating diseases associated with cell loss (Thompson, 1995, Science 267: 1).
456-1461). Examples of such treatments can be physiological inhibitors of apoptosis, such as growth factors, extracellular matrix, CD40 ligand, neutral amino acids, zinc, estrogens, androgens. On the other hand, pharmacological agents such as calpain inhibitors, cysteine protease inhibitors, tumor promoters such as PMA, phenobarbital, α-hexachlorocyclohexane are believed to act as apoptosis inhibitors.

Diabetes: Diabetes is a systemic disease characterized by the effects of insulin and other regulatory hormones on carbohydrate, fat and protein metabolism, and impairments in the structure and function of blood vessels. The main symptoms of diabetes are diabetes, which often involves the presence of large amounts of glucose in the urine, and hyperglycemia, which is accompanied by polyuria with high excretion of urine. Additional symptoms occur in chronic or long term diabetes. Those symptoms are
Includes vascular wall degeneration. Although many different organs are affected by their vascular changes, nerves, eyes and kidneys appear to be the most sensitive. Long-lasting diabetes is a major cause of blindness, even when treated with insulin.

[0009] There are two recognized types of diabetes. Type I diabetes begins in childhood, involves ketosis, progresses in the early stages of life with more severe symptoms, and is predicted for later vascular involvement. Control of this type of diabetes is difficult and requires exogenous insulin administration. Type II diabetes begins in adults, is ketosis-resistant, progresses in the middle stages of life, starts mildly and more slowly.

[0010] One of the most significant advances in the history of medicine came in 1992, when Banting and Best showed a therapeutic effect of insulin in diabetic dogs. However, even today, no clear picture of the underlying biochemical deficiencies of the disease is known, and diabetes remains a serious health problem. It appears that 2% of the United States population is afflicted by some forms of diabetes. The introduction of orally effective hypoglycemic agents has been an important advance in the treatment of hyperglycemia. Oral hyperglycemic agents are commonly used to treat diabetes beginning in adults.

Heparin-binding protein: The covalent structure of two closely related proteins isolated from peripheral neutrophil leukocytes of human and porcine origin has recently been determined (eg, H. Flodgaard et al., 1
991, FEBS Lett. 272: 200ff). Both proteins show high similarity to neutrophil elastase, but with active serine 195 and histidine 57 (chymotrypsin numbering (BS Hartley, “Homologies in Serine Proteinases”, Phil. Tran).
Due to the selective mutation of s. Roy. Sci. Series 257, 1970, P. 77ff.))), the protein lacks protease activity.

[0012] The proteins have been named as human heparin-binding protein (hHBP) and porcine heparin-binding protein (pHBP), respectively, due to their high affinity for heparin; Schafer et al. (WM Schafer et al.) ., Infec
t. Immun. 53. 1986, p. 651ff.) has been named as a protein cationic antimicrobial protein (CAP37) because of its antimicrobial activity.

HBP was first studied for its antibiotic and LPS binding properties (Gaba
y, et al., 1989, Proc. Natl. Acad. Sci. USS 90: 4733-7). However, the accumulation of evidence now suggests that, besides its bactericidal role, HBP may be responsible for monocyte recruitment and activation (Pe
reira et al., 1990, J. Clin. Invest. 85: 1468-1476 and Rasmussen et al., 1
996, FEBS Lett. 309: 109-112), T cell recruitment (Chertov et al., 1996, J. Bi.
ol. Chem. 271: 2935-2940) and its effects on induced contractions in endothelial cells and fibroblasts (Ostergaad and Flodgaard, 1992, J. Leuk. Biol. 51: 316-323). Support the concepts involved in the progress of Ostergaard and
See Flodgaard, 1992, supra.

It also discloses the enhanced viability of monocytes treated with heparin-binding proteins, but does not describe the underlying mechanism of this enhanced viability. Furthermore, in animal models of fecal peritonitis, HBP treatment has been shown to help mice from lethal trauma (Mercer-Jones et al., 1996, In Surgical F.
Orum, etc. pp.105-108 and ^{Wickel., 1997, In 4 th} International Congress on
the Immune Consequences of Trauma, Chock and Sepsis, Munich, Germany, p
p. 413-416).

[0015] From azurophil granules, hHBP and azurocidine (azuroc
idin), the first 20 identical to the first 20 N-terminal amino acid residues of CAP37
Proteins with N-terminal amino acid residues have also been isolated (JE Gab
ay., Proc. Natl. Acad. Sci. USA 86, 1989, P.5610ff .; CG Wilde, etc.
, J. Biol. Chem. 265, 1990, p. 2038 ff.) And its antibacterial properties have been reported (D. Campanelli et al., J. Clin. Invest. 85, 1990, p. 904ff.). .

The structure of HBP is clear from WO 89/08666 and H. Flodgaard et al., Supra. On the other hand, HBP is CAP37 (eg, WO 91/00907, US Pat. No. 5,458,
No. 874 and U.S. Pat. No. 5,484,885) and azulocidine (see, for example, CG Wilde et al., J. Biol. Chem. 265, 1990, p. 2038).

[0017] Diseases caused by apoptosis can be treated, but diseases associated with inhibition of apoptosis, such as cancer, autoimmune diseases such as systemic lupus erythematosus, immune-mediated glomerulonephritis, and viral infections such as herpes There is a need for effective inhibition of apoptosis without causing virus, poxvirus, or adenovirus infection. Accordingly, it is an object of the present invention to have effective inhibitors of apoptosis for use in treating diseases associated with enhanced apoptosis.

Summary of the Invention: It has surprisingly been found that heparin-binding protein (HBP) can reduce apoptosis in mammalian cells. As shown in the examples herein,
HBP is internalized in mitochondria and affects mitochondrial function and especially PT pore formation.

The present invention is directed to a method for modulating or reducing apoptosis in mammalian mammalian cells, particularly β-cells, endothelial cells and neural cells of the islets of Langerhans, wherein the method comprises glycosylated. In form, (i) having a molecular weight of about 28 kD as determined by SDS PAGE under reducing conditions; (ii) produced in azurophil granules of polymorphonuclear leukocytes; and (iii) A mammalian heparin-binding protein or a pharmaceutically acceptable active fragment thereof, which is a chemoattractant for a sphere, or a composition comprising said protein and a pharmaceutically acceptable carrier, is used to inhibit apoptosis in said cells. Administering to the mammal in need thereof an amount effective to adjust or lower it. Further, the present invention relates to the use of said heparin-binding protein (HBP) or a fragment thereof for the manufacture of a medicament for the treatment or prevention of a condition caused by apoptosis, and the use of said heparin-binding protein (HBP) or a fragment thereof for the treatment or prevention of said condition. Aimed at its use.

The present invention is further directed to a composition comprising said heparin-binding protein and a mitochondrial matrix targeting protein. As defined herein, a "mitochondrial matrix targeting protein" is a protein having an N-terminal extension that functions as a mitochondrial targeting signal. The targeting signal is a highly degenerate sequence with 20-30 residues that can fold into a positively charged amphipathic helix. The present invention is further directed to the use of those compositions for the manufacture of a medicament for treating or preventing a disease caused by cell apoptosis. Further, the present invention is directed to a method for modulating and preventing mammalian cell apoptosis and, therefore, diseases caused by cell apoptosis, comprising administering to a mammal an effective amount of the composition.

Specific Description of the Invention: The HBP may suitably be of mammalian, especially human or porcine origin. In particular, HB
P is the amino acid sequence set forth in SEQ ID NO: 1 that qualitatively retains the activity of the heparin-binding protein or fragment thereof, which inhibits entry of the pathogen into the patient's mononucleus (eg, monocytes or macrophages). Mature human HBP having at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 97% identity (hereinafter referred to as "homologous polypeptides") It is.

Alternatively, the HBP comprises the amino acid sequence set forth in SEQ ID NO: 2, which qualitatively retains the activity of said heparin-binding protein or fragment thereof, which inhibits, reduces or modulates apoptosis of mammalian cells in the mammal. Mature porcine HBP having at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 97% identity. In a preferred embodiment, the mammal is a human patient.

In a preferred embodiment, the homologous peptide comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2 and 5 amino acids, preferably 4 amino acids, more preferably 3 amino acids, even more preferably 2 amino acids And most preferably by one amino acid. The degree of identity between amino acid sequences can be determined by computer programs known in the art, for example,
GCG Program Package (Needleman and Wunsch, 1970, Journal of Molecul
ar Biology 48: 443-453). To determine the degree of identity between two amino acid sequences for the present invention, GAP is used with the following settings: a GAP creation penalty of 3.0 and a GAP extension penalty of 0.1.

The amino acid sequence of the homologous polypeptide may differ from the amino acid sequence shown in SEQ ID NO: 1 or 2 by insertion or deletion of one or more amino acid residues and / or one or more amino acid residues by different amino acid residues. Depends on amino acid residue substitution. Preferably, the amino acid changes are of a minor nature, ie, conservative amino acid substitutions that do not significantly affect the folding and / or activity of the protein; a few deletions, typically 1 to about 30 Deletion of one amino acid; few amino- or carboxyl-
Terminal extensions, eg, extension of amino-terminal methionine residues; small linker peptides of up to about 20 to 25 residues; or altering the net charge or other function, eg, poly-histidine system, antigenic epitope or binding domain Is a small extension that facilitates purification.

Examples of conservative substitutions include basic amino acids (eg, arginine, lysine and histidine), acidic amino acids (eg, glutamic acid and aspartic acid), polar amino acids (eg, glutamine and asparagine), hydrophobic amino acids (eg, leucine, isoleucine and histidine). (Valine), aromatic amino acids (eg phenylalanine, tryptophan and tyrosine) and small amino acids (eg glycine, alanine, serine, threonine and methionine).

In general, amino acid substitutions that do not alter a particular activity are known in the art and are described, for example, in H. Neurath and RL Hill, 1979, The Proteins, Aca
Written by demic Press, New York. The most routine exchanges are: Ala / Ser, Val / Ile, Asp / Glu, The / Ser, Ala / Gly, Ala / Thr, Ser / A
sn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / V
al, Ala / Glu, Asp / Gly, and vice versa.

The heparin binding protein is SEQ ID NO: 3 (mature human HBP shown in SEQ ID NO: 1).
SEQ ID NO: 5 (encoding human HBP including the mature protein sequence and prosequence shown in SEQ ID NO: 6), SEQ ID NO: 7 (signal sequence, prosequence and mature sequence shown in SEQ ID NO: 8) SEQ ID NO: 4 (encoding human HBP containing the protein sequence), SEQ ID NO: 4 (encoding porcine HBP shown in SEQ ID NO: 2), SEQ ID NO: 9
(Encoding porcine HBP comprising the sequence of the prosequence and mature protein shown in SEQ ID NO: 10), SEQ ID NO: 11 (encoding porcine HBP comprising the sequence of the signal sequence, prosequence and mature protein shown in SEQ ID NO: 12 Encoding), and
It may be encoded by a nucleic acid sequence having at least about 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 97% identity, as determined by agarose gel electrophoresis. . The nucleic acid sequence can be of genomic, cDNA, RNA, semi-synthetic, synthetic origin, or any combination thereof.

[0028]

[Table 1]

[0029]

[Table 2]

[0030]

[Table 3]

[0031]

[Table 4]

[0032]

[Table 5]

[0033]

[Table 6]

[0034]

[Table 7]

[0035]

[Table 8]

[0036]

[Table 9]

[0037]

[Table 10]

[0038]

[Table 11]

The degree of identity between two nucleic acid sequences can be determined by computer programs known in the art, such as GAP (provided in the GCG program package).
Needleman ando Wunsch, 1970, Journal of Molecular Biology 48: 443-453)
Can be determined by To determine the degree of identity between two nucleic acid sequences of the invention, GAP is used with the following settings: a GAP creation penalty of 5.0 and 0.3.
GAP extension penalty.

Modification of the nucleic acid sequence encoding HBP is necessary for the synthesis of polypeptide sequences that are substantially similar to HBP. The term "substantially similar" to HBP refers to a non-naturally occurring form of HBP. Their polypeptide sequences differ in some constructed manner from HBP isolated from its natural source. For example, it is of interest to synthesize variants of HBP whose variants differ in specific activity, thermostability, pH optima, or the like, for example, using site-directed mutagenesis.

A similar sequence may be based on a nucleic acid sequence provided as an HBP coding site of SEQ ID NO: 1, 2, 6, 8, 10 or 12, eg, a subsequence thereof, and / or It does not generate other amino acid sequences of the encoded HBP, but can generate different amino acid sequences, or by introducing nucleotide substitutions corresponding to the codon usage of the intended host organism for production of the enzyme. It can be constituted by the introduction of nucleotide substitutions. For a general description of nucleotide substitutions, see, for example, Ford ET, 1991, Protein Expression and Purification 2
: See 95-107.

It will be apparent to one skilled in the art that such substitutions can be made outside of the regions essential for the function of the molecule, and still result in an active polypeptide sequence. Amino acid residues that are essential for the activity of the polypeptide encoded by the isolated nucleic acid sequence of the present invention, and thus preferably are unsubstituted, can be obtained by methods known in the art, such as Specific mutagenesis or alanine-scanning mutagenesis (eg, Cuningham and Wells, 1989, Science 244: 1081-1085).
). In the latter technique, mutations are introduced at each residue in the molecule, and the resulting mutated molecule is tested for HBP activity to identify amino acid residues that are essential for the activity of the molecule.

[0043] Heparin-binding proteins also exhibit, under low to high stringency conditions, SEQ.
It can be encoded by a nucleic acid sequence that hybridizes to the nucleic acid sequences shown in 6, 8, 10 and 12. Under low to high stringency conditions, 5 × SSPE, 0.3% SDS, 200 μg / ml
Pre-hybridization and hybridization at 45 ° C. in sheared and denatured salmon sperm DNA, and 25, 35 or 50% formamide (for low, medium and high stringency, respectively). You.

The carrier material is preferably at least 50 ° C. (very low stringency), more preferably at least 55 ° C. (low stringency), more preferably at least 60 ° C. (medium stringency), more preferably at least 65 ° C (medium to high stringency), even more preferably at least 70 ° C (high stringency), and most preferably at least 75 ° C (
(Very high stringency) using 2 × SSC, 0.2% SDS for 30 minutes each
Washed once.

Preparation of HBP: The nucleic acid sequence encoding HBP can be obtained by standard established methods, such as SL Beauca
ge and MH Caruthers, Phosphoamidite method described by Tetrahedron Letters 22, 1981, pp. 185-189, or Matthes et al., EMBO journal 8, 1984, p.
It can be prepared synthetically by the method described on pages 801-805. According to the phosphoramidite method, oligonucleotides are synthesized, for example in an automatic DNA synthesizer, purified, annealed, ligated and cloned into a suitable vector.

The techniques used to isolate or clone the nucleic acid sequence encoding the heparin binding protein used in the methods of the present invention are known to those of skill in the art and can be isolated from genomic DNA. , Preparation from cDNA or combinations thereof. The cloning of the nucleic acid sequences of the present invention from such genomic DNA can be performed, for example, using the well-known polymerase chain reaction (PCR), or an expression library to detect cloned DNA fragments with sheared structural features. It can be effected by using rally antibody screening. For example, Innis et al., 1990, A Guide to Methods and Application, Academic Press, New York
checking ... Other nucleic acid amplification methods can be used, such as ligase chain reaction (LCR), ligated activated transcription (LAT), and nucleic acid sequence-based amplification (NASBA).

Next, the nucleic acid sequence is inserted into a recombinant expression vector, which may conveniently be any vector that can be subjected to recombinant DNA methods. Vector selection is often
It will depend on the host cell into which it is to be introduced. Thus, the vector can be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity, eg, a plasmid, whose replication is independent of chromosomal replication. On the other hand, the vector can be a vector which, when introduced into a host cell, is integrated into the host genome and replicated along with the chromosome (s) into which it has been integrated.

[0048] In the vector, the nucleic acid sequence encoding HBP should be operably linked to a suitable promoter sequence. A promoter can be any nucleic acid sequence that exhibits transcriptional activity in the host cell of choice, and can be derived from a gene encoding a protein that is homologous or heterologous to the host cell. Examples of suitable promoters for directing transcription of a nucleic acid sequence encoding HBP in mammalian cells include the SV40 promoter (Subramani et al., Mol. Cell Biol. 1,
1981, pp. 854-814) or adenovirus 2 major late promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter (Bo
Shart et al., 1981, Cell 41: 521-530), and the bovine papillomavirus promoter (BPV). A suitable promoter for use in insect cells is the polyhedrin promoter (Vasuvedan et al., FEBS Lett. 311, 1992, pp. 7-11).

Examples of suitable promoters for directing the transcription of a nucleic acid sequence encoding HBP, particularly in bacterial host cells, include the E. coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus・ Subtilis (Bacillus subtilis) levansululase gene (sacB), Bacillus
Licheniformis (Bacillus licheniformis) α-amylase gene (amyL), Bacillus stearothermophilus amylase gene (amyM), Bacillus amyloliquefaciens (Bacillus amyloliquefacien)
iens) α-amylase gene (amyQ),

The Bacillus licheformispenicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic β-lactamase gene (Villa-Kamaroff)
Et al., 1978, Proceedings of the National Academy of Sciences USA 75: 372
7-3731) and a tac promoter (DeBoer et al., 198)
3, Procedings of National Academy of Sciences USA 80: 21-25). Further promoters include “Useful Proteins from recombinant bacteria” in S
cientific American, 1980, 242: 74-94; and Sambrook et al., 1989, supra.

Examples of suitable promoters for directing transcription of a nucleic acid sequence encoding HBP in a filamentous fungal host cell include Aspergillus oryzae TA
KA amylase, Rhizomucor miehei aspartate proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid stable α-amylase, Aspergillus niger or Aspergillus awamori (
glaA),

[0052] Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (cited here by reference). And promoters derived from genes encoding their hybrids, as described in U.S. Patent No. 4,288,627, incorporated herein. Particularly preferred promoters for use in filamentous fungal host cells are TAKA amylase, NA
2-tpi (a hybrid of promoters from the genes encoding Aspergillus niger neutral amylase and Aspergillus oryzaetriose phosphate isomerase), and the glaA promoter.

In yeast hosts, useful promoters include the Saccharomyces cerevisiae enolase (ENO-1) gene, Saccharomyces cerevisiae
Cerevisiae galactokinase gene (GAL 1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase gene (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

The nucleic acid sequences encoding SEQ ID NOs: 1 and 2, eg, SEQ ID NOs: 3 and 9, can be operably linked to a nucleic acid encoding a heterologous prosequence. SEQ ID NOs: 6, 8, 10 and 12
For example, nucleic acids encoding SEQ ID NOs: 5, 7, 9, and 11 have a heterologous signal sequence and
And / or operably linked to a nucleic acid sequence encoding a prosequence.

[0055] The nucleic acid sequence encoding HBP can also be operably linked to a suitable terminator, such as a human growth factor hormone terminator (Palmiter et al., Supra). Preferred terminators for filamentous fungal host cells encode Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase and Fusarium oxysporum trypsin-like protease. Obtained from the gene.

Preferred terminators for yeast host cells are genes encoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome c (CYC 1), or Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Obtained from Other useful terminators for yeast host cells are described by Romanos et al., 1992 supra.

The vector may further comprise, for example, a polyadenylation signal (eg, from the SV40 or adenovirus 5Elb region), a transcription enhancer (eg, the SV40 enhancer).
, And translation enhancer sequences (eg, sequences encoding adenovirus VA RNA). In addition, preferred polyadenylation sequences for filamentous fungal host cells are obtained from genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glycoamylase, Aspergillus nidulans anthranilate synthase, and Aspergillus niger α-glucosidase. .

Useful polyadenylation sequences for yeast host cells are described in Cuo and Sherman, 1995,
Molecular Cellular Biology 15: 5983-5990. The recombinant expression vector can further include a DNA sequence that allows the vector to replicate in the host cell of interest. Examples of such sequences (where the host cell is a mammalian cell) are SV40 or a polyoma origin of replication. Examples of cellular origins of replication include the plasmids pBR322, pUC19, pACYC177, pACYC184, pUB110, pE194, pTA1060
And the replication origin of pAMβ1.

Examples of origins of replication for use in yeast host cells are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARS1. The origin of replication can have mutations to render its function thermosensitive in the host cell (
For example, Ehrlich, 1978, Proceeding of National Academy of Sciences USA7
5: 1433).

The vector may also confer resistance to a selectable marker, eg, a gene for a product that complements the defect in the host cell, eg, a gene encoding dihydrofolate reductase (DHFR), or a drug, eg, neomycin, geneticin, ampicillin, or hygromycin. The gene can be comprised of Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.

Selectable markers for use in filamentous fungal host cells can be selected from, but not limited to, the following: amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phos Phynotricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD
(Nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase,
sC (adenyl sulfate transferase), trpC (anthranilate synthase) and glufosinate resistance markers, and equivalents from other species.

For use on Aspergillus cells, the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the Streptomyces hygroscopicus bar marker are preferred. Further, selection can be accomplished by co-transformation, as described in WO 91/17243, wherein the selectable marker is on a separate vector.

The methods used to ligate the nucleic acid sequences encoding HBP, promoter and terminator, respectively, and insert them into a suitable vector containing the necessary information for replication are well known to those skilled in the art. It is known (eg, Sambrook et al., Supra).

The host cell into which the expression vector is introduced is capable of producing HBP, and preferably is a eukaryotic cell, such as an invertebrate (insect) cell or a vertebral cell, such as a Xenopus laevis oocyte. Any cell that is a cell or a mammalian cell, especially insect and mammalian cells. Examples of suitable mammalian cell lines include COS (eg, ATCC CRL1650), BHK (eg, ATCC CRL1632, ATCC CCL10) or CHO (
For example, ATCC CCL61) cell line.

The host cell can be a mammalian basophil or a mammalian hybrid cell. Mammalian basophil cells can be human, guinea pig, rabbit or rat basophil cells. In certain embodiments, the mammalian basophil cells are rat basophil cells. In the most particular embodiment, the rat basophil cells may be RBL-1 cells having the same characteristics as ATCC CRL-1378 or RBL-2H3 cells, or RBL-2H3 cells having the same characteristics as ATCC CRL2256. .

Methods for transfecting mammalian cells and expressing the introduced DNA sequence in the cells are described, for example, in Kaufman and Shaarp,
1982, J. Mol. Biol. 159: 601-621; Southern and Berg, 1982, J. Mol. Appl.
Genet. 1: 327-341; Loyter et al., 1982, Proc. Natl. Acad. Sci. USA 79: 422-42.
6; Wigler et al., 1978, Cell 14: 725; Corsaro and Pearson, 1981, Somatic C
ell Genetics 7: 603; Graham and van der Eb, 1973, Virology 52: 456; Fraley
1980, JBC 225: 10431; Capecchi, 1980, Cell 22: 479; Wiberg et al., 19
83, NAR 11: 7287; and Neumann et al., 1982, EMBO J. 1: 841-845. In certain embodiments, the mammalian basophil cells are treated with HBP using an electroporation device.
Transfected with DNA encoding

The medium used to culture the cells may be any conventional medium suitable for growing mammalian cells, such as serum-containing or serum-free medium with appropriate supplements, or mammalian cells. May be a suitable medium for growing S. cerevisiae. Suitable media are commercially available or publicly available Recipes (eg, American Type Cultur
e Collection catalog). Next, the cells are screened for antibiotic resistance. Subsequently, the selected clones are assayed for HBP activity and assayed for cytokine release from monocytes using assays known in the art, such as a chemotaxis assay (e.g., Rasmussen et al. , 1996, FEBS Lett. 390: 109-112)
.

On the other hand, the host cell can be a hybrid mammalian cell. Myeloma lines (eg, mouse, rat, human) are transfected with DNA encoding HBP using the methods described above. Subsequently, it can be fused with a mammalian cell expressing acidic proteoglycan, such as a mammalian basophil or mast cell, using the following method. In one embodiment, the parent cell is a culture medium, such as RPMI-16.
At 40 and exposed to a chemical fusing agent such as polyethylene glycol (see, eg, Gefter et al., 1997, Somat, Cell Genet. 3: 231-236). Subsequently, the fusion agent is diluted and the cells are incubated in medium and HAT. The selected clones are subsequently assayed for HBP activity as described above.

Alternatively, two parent cells can be fused by electrofusion. Membrane contact between cells
Achieved by non-uniform alternating electric fields that lead to dielectrophoresis and cell chain formation. Fusion is then triggered by the injection of a field pulse strong enough to trigger reversible degradation at the membrane contact area (eg, Okada et al., 1984, Biomed. Res, 5:51).
See 1-566). On the other hand, cell fusion can be induced by the Sendai virus (eg, Wainberg et al., 1973, J. Cell Biol. 57: 388-3.
96).

A host cell can be a unicellular pathogen, such as a prokaryote, or a non-unicellular pathogen, such as a eukaryote. Useful unicellular cells are bacterial cells such as:
However, they are not limited thereto: Gram-positive bacteria such as Bacillus cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis (
Bacillus brevis), Bacillus circulans, Bacillus coagulanns,

Bacillus lautus, Bacillus lentus
entus), Bacillus licheniformis (Bacillus licheniformis),
Megaterium (Bacillus megaterium), Bacillus stearothermophilus (
Bacillus stearothermophilus, Bacillus subtilis
Or Bacillus thuringiensis; or Streptomyces cells, such as Streptomyces livid.
ans) or Streptomyces murinus; or Gram-negative bacteria such as E. coli and Pseudomonas sp.
sp.).

In a preferred embodiment, the bacterial host cell is Bacillus lentus, Bacillus
Licheniformis, Bacillus stearothermophilus or Bacillus subtilis cells. Transformation of bacterial host cells can be achieved, for example, by protoplast transformation (see, eg, Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), using competent cells (eg, Young and Spizizin).
, 1961, Journal of Bacteriology 81: 823-829, or Dubnar and Davidoff Abel
son, 1971, Journal of Molecular biology 56: 209-221), by electroporation (eg, Shigekawa and Dower, 1988, Biotechni.
ques 6: 742-751) or by joining (eg, Koehler and Th
ome, 1987, Journal of Bacteriology 169: 5771-5278).

[0073] The host cell can be a fungal cell. "Fungi", as used herein, refers to phylum Ascomycota, Basidiomycota,
Chitridiomycota and Zygomycota (Howks
such worth., Ainsworth and Bisby's Dictionary of the Fungi, 8 th edition
, 1995, CAB International, University Press, Cambridge, UK), and Oomycota (Hawksworth et al., 1995, supra, p. 17).
1, as well as all vegetative spore fungi (Howksworth et al., 1).
995, supra).

A representative group of ascomokitas is, for example, neurospora
, Eupenicillium (= penicillium), Emerycela (Emer
icella) (= Aspergillus), Eurotum (= Aspergillus)
) And the genuine yeasts listed above. Examples of Basidiomycota include mushrooms, rusts and smut. Representative groups of chitridiomycota include, for example, Allomyces, Blastocladiella
), Coelomomyces and aqueous fungi.

A representative group of ohmicotas is, for example, Saprolegniomysetus
olegniomycetous) Includes aqueous fungi (water mold), such as cotton mold. Examples of vegetative spore fungi include Aspergillus, Penicillium, Candida and Alternaria. A representative group of Zuigomikota is, for example, Rhizopus
) And Mucor.

In a preferred embodiment. Fungal host cells are yeast cells. "Yeast"
As used herein, ascosporogenic yeasts (Endomycetales), basidiosporogenous yeasts, and yeasts belonging to the fungus Blastmycetes (Blastomycetes) are included. Ascosporogenic yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter include four subfamilies: Schizosaccharomycoideae (eg, genus Schizosaccharomyces), Nadsonioideae,
Lipomycoideae and Saccharomy
coideae) (for example, genus Pichia, Kluyveromyces
) And Saccharomyces).

[0077] Basidiosporogenic yeasts include the genus Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium and Filobasidiella. Yeasts belonging to the imperfect fungi are of two families, namely Sporobolomycetaceae (eg the genus Sorobolomyces and Bullera), and Cryptococcaceae (eg the genus Candida) Candida)).

As the classification of yeast will change in the future, for the purposes of the present invention, yeast will
Activities of Yeast (Skinner, FA, Passmore, SM, and Davenport, RR,
eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980). The manipulation of yeast biology and yeast genetics is well known in the art (eg, Biochemistry and Genetics of Yeast, Bacil, M., H.
orecker, BJ, and Stopani, AOM , editors, 2 nd edition, 1987; The Y
easts, Rose, AH, and Harrison , JS, editors, 2 nd edition, 1987; and
The Molecular Biology of the Yeast Saccharomyces, Strathern, etc., edito
rs, 1981).

In a more preferred embodiment, the yeast host cell is a cell of the species Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia or Yarrowia. In a most preferred embodiment, the yeast host cell is Saccharomyces calsbergensis (Saccharo
myces carlsbergensis), Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglassi
Saccharomyces douglasii),

Saccharomyces kluyveri, Saccharomyces kluyveri
Or Saccharomyces norvisis or Saccharomyces oviformis cells. In another most preferred embodiment, the yeast host cell is Kluyveromyces lactis.
Cells. In another most preferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

The medium used to culture the cells may be any conventional medium suitable for growing mammalian cells, such as serum-containing or serum-free medium with appropriate supplements, or insect, It may be a suitable medium for growing yeast or fungal cells.
Suitable media are commercially available or published Recipes (eg, American
(In the catalog of the Type Culture Collection).

The HBP produced by the cells is then separated from the medium by, for example, centrifugation or filtration, and the protein component of the supernatant or filtrate is precipitated with a salt, such as ammonium sulfate, and subjected to various chromatographic methods. It can be recovered from the culture medium by conventional methods, including purification by methods such as ion exchange chromatography, affinity chromatography or the like. Recombinant hosts can also produce acidic proteoglycans, such as heparin sulfate.

To obtain active HBP, acidic proteoglycans will need to be removed.
This can be achieved using a series of separation methods, namely precipitation or column chromatography, such as reverse phase HPLC, HIC, SEC, IEC and affinity based techniques. The separation method can be combined with other treatments such as increasing the salt concentration by changing the pH and by other means of reducing the interaction between acidic proteoglycans and HBP. Recombinant host cells can also produce acidic proteoglycans, such as heparin sulfate. To obtain active HBP, acidic proteoglycans will need to be removed.

Composition: In the pharmaceutical composition used in the method of the present invention, HBP is selected from Remington's Ph.
It can be formulated by any of the established methods of formulating pharmaceutical compositions, as described in armaceutical Sciences, 1985. The composition will typically be in a form adapted for local or systemic injection or infusion, and may be formulated with sterile water or an isotonic saline solution, or a glucose solution. The composition may be sterilized by conventional sterilization techniques well known in the art.

The resulting aqueous solution may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The composition may be any pharmaceutically acceptable auxiliary material, such as buffers, tonicity adjusting agents and the like, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, if needed to approach physiological conditions. , Calcium chloride,
Etc. can be included. The concentration of HBP may vary widely, i.e. from about 0.5% by weight or less, for example from 1% by weight to 15-20% by weight. A unit dose of the composition can typically contain from about 10 mg to about 1 g of HBP.

[0086] HBP or a pharmaceutically acceptable active fragment thereof is administered locally or by intravenous injection. The dosage will be prescribed by a physician according to the particular conditions and the particular individual being treated. Dosage amount and frequency will be adjusted carefully and adjusted according to the parameters determined by the attending physician. A preferred route of administration may be, for example, intraperitoneal injection. Intravenous intraperitoneal injections of HBP can be given every 24 hours in the range of 0.1-100 mg, in particular 0.1-20 mg, especially 0.1-10 mg per kg body weight. The dose is given 1 to 4 times per 24 hours or is administered continuously through a catheter.

The compositions used in the present invention can further include a mitochondrial matrix targeting protein. In certain embodiments, the mitochondrial matrix targeting protein has a molecular weight of about 33 kD and binds H-kininogen, but does not bind L-kininogen. In a more particular embodiment, the mitochondrial matrix targeting protein has an N-terminal amino acid sequence as shown in SEQ ID NO: 15, ie, p33 (Ghebrehiwet et al., 1994, J. Exp. Med. 179: 1
809) has the 32 N-terminal amino acid sequence: MLPLLRCVPRVLGSSVAGLRAAAPASPFRQLL (SEQ ID NO: 15).

[0088] In a most particular embodiment, the mitochondrial matrix targeting protein is a zinc-dependent p33 protein shown in SEQ ID NO: 16 below:

[Table 12]

The compositions used in the methods of the present invention are directed to use in treating or preventing a disease caused by apoptosis. They include, but are not limited to, HIV, neurodegenerative or neuromuscular disease, ischemic stroke, anoxia, ischemia / reperfusion injury and septic toxic shock. In certain embodiments, the composition can be used to prevent beta cell destruction in islets of Langerhans in the pancreas. Such β-cell damage causes diabetes.

[0090]

【Example】

Example 1 . Treatment of β-cells with HBP Generation of HBP: HBP is obtained from RBL-1 cells using the method described in application number PCT / DK98 / 00275.

Materials: The vectors pBlueBac III and pcDNA3 are obtained from Invitrogen. All primers and oligos are synthesized on an Applied Biosystems Model 394 DNA synthesizer. Restriction enzymes are obtained from New England Biolabs. Pfu polymerase used in the PCR reaction is obtained from Stratagene. RBL-1 cells (ATCC CRL-1378) and RBL
-2H3 cells (ATCC CRL-2256) were obtained from American Type Culture Coll. Of Manassas, VA.
section (ATCC).

Cells were either gamma-irradiated FCS (origin: NZ, Gibco, Life Technologies) or 10% heat-inactivated as recommended by the supplier or fetal calf serum (
Growth in RPMI 1640 culture medium (Gibco, Life Technologies) supplemented with FCS) (HyClone or North American origin from Bio Whittaker). Cells are grown at 37 ° C. in an 80% humidified atmosphere with 5% CO ₂ . Exponentially growing cells are used for all experiments.

Construction of Expression Vector: The 770 bp BamHI-HindIII fragment was replaced with a human HBP amino acid sequence (Flodgaar
d et al., 1991, Eur. J. Biochem. 197: 535-547) and CAP37 / azurocidin DNA
Sequences (Morgan et al., 1991, J. Immunol. 147: 3210-3214, and Almeida et al., 1
991, Biochem, Biophys. Res. Commun. 177: 688-695).
Construct using PCR technique from library A (Clontech).

This fragment contains the complete coding region for HBP, including a 19-residue signal peptide, a 7 amino acid pro-peptide, a mature portion of 22 amino acids, and a 3 amino acid C-terminal extension. The fragment is inserted into pBlue-BacIII, resulting in plasmid pSX556. For deletion of the pro-region, the 99 bp oligonucleotide linker (BamHI-EagI) encompassing the signal peptide and the first four amino acids of mature HBP was replaced by the original BamHI-EagI fragment in pSX556 and pSX559. Is given.

For expression of these two cDNA sequences in RBL-1 cells, the above pSX556 and pSX559 were templated in a PCR reaction with the following primers with Pfu polymerase according to the manufacturer's instructions (Stratagene). Used as: PBRa246 (5'-CCGGGGATCCAACTAGGCTGGCCCCGGTCCCGG-3 ') (SEQ ID NO: 13) PBRa247 (5'-CCGGGGATCCGATGACCCGGCTGACAGTCCTGG-3') (SEQ ID NO: 14). After BamHI excision of the PCR reaction product, the fragment is ligated in the correct orientation into the mammalian expression vector pcDNA3 (Invitrogen) linearized with BamHI, resulting in plasmids, pcDNA3-HBP and pcDNA3-HBP pro.

Transfection method: Transfection is performed according to the following method. 25 μg pcDNA3-HBP or pc
DNA3-HBP pro is transfected into RBL-1 or RBL-2H3 cells (8 × 10 ⁶ cells can be transfected into Gullberg et al., 1994, J. Biol. Chem. 269: 25219-252).
BioRad Electroporation Apparat with 960 μF and 300 V electrical settings as described in J. 25 and Garwicz et al., 1995, J. Biol. Chem. 270: 28413-28418.
transfected using us or as recommended by the supplier
transfect using fecAmine (Gibco, Life Technologies) or Superfect (Qiagen) transfection reagent).

Cells were purified from RPMI 1640 supplemented with 10% heat-inactivated gamma-irradiated FCS.
Grow in medium at 37 ° C. under 5% CO ₂ in an 80% grown atmosphere. Geneticin (2 mg / ml) is added 48 hours after transfection to select for recombinant clones.

[0098] Individual clones growing in the presence of geneticin were isolated and analyzed by ELISA.
Test for HBP expression. The HBP ELISA is a sandwich immunoassay using a monoclonal antibody as a catcher and a rabbit polyclonal antibody conjugated to horseradish peroxidase as a detector. Antibodies are prepared according to standard methods by immunizing mice and rabbits with HBP purified from human buffy coat cells (Flodgaard et al., 1991, Eur, J. Biochem. 19
7: 535-547).

In particular, individual cells are coated overnight with 0.5 μg of monoclonal anti-hHBP dissolved in 100 μl of PBS. 5% lactose, 0.5% coated wells
After washing three times with a solution of Byco A, 0.05% Tween20 and 0.024% Thiomersal, the plate is inverted on a piece of cloth and dried at room temperature. The coated plates can be taken with staniol and stored for up to 3 months.

[0100] Purified hHBP is used as a control preparation. 100 ng of hHBP / ml diluted solution is prepared with BSA-EDTA buffer and stored at -80 ° C for up to 2 weeks. BS
A series of dilutions containing 0, 0.3, 1, 4 and 12 ng of hHBP / ml, diluted in A-EDTA, are freshened and 100 μl are added to individual wells. The hHBP sample is also diluted with BSA-EDTA buffer and all samples are incubated and agitated for 1 hour at room temperature. The wells are emptied and washed three times with phosphate buffered saline, followed by addition of 100 μl / well of diluted (1: 1000) Fab-peroxidase conjugated rabbit anti-hHBP and stirring at room temperature. Incubate for 1 hour.

The peroxidase activity was reduced to 100 μl /
Measure using TMB perborate substrate solution in well. The control curve is a straight line when the log versus absorbance is plotted against the log versus volume. Clones with the most obvious expression are selected for further experiments, recloned, and retested for expression levels. The best HBP product is selected and propagated in large cultures or adapted to serum-free or protein-free media.

Isolation of HBP: Isolation of HBP from RBL-1 cells was performed as described by Rasmussen et al., 1996, FEBS Lett. 340:
Perform substantially as described by 109-112. The transfected and selected RBL-1 cells are first filtered to remove residual cells and cell debris. Subsequently, the culture medium is applied to a CM-Sepharose cation-exchange column (Pharmacia and Upjohn) previously equilibrated with 50 mM sodium phosphate, pH 7.3.

Unbound and loosely bound material is eluted with equilibration buffer until a baseline is reached, as measured by on-line UV detection at 280 nm. The column is then developed with a linear gradient of 0-1M sodium chloride in equilibration buffer. HBP is eluted with about 0.7 M sodium chloride and the fractions are combined based on UV absorbance. The pooled fractions are diluted with 2 volumes of distilled water and applied to a new CM-Sepharose column.

Following equilibration, HBP is eluted stepwise with 1 M sodium chloride in equilibration buffer and fractions are combined based on absorbance at 280 nm. Highly concentrated and pure HBP is obtained by this method. The final purification is performed on Sephadex G equipped with UV-flow cells and equilibrated and eluted with 0.02% trifluoroacetic acid.
Performed on a -25 gel-filtration column (Pharmacia & Upjohn). HBP is collected based on absorbance at 280 nm. Gel filtration acts primarily as a buffer exchange step to produce a stable preparation of HBP that is maintained at 4 ° C. until use.

HBP rescues β-cells from cytokine-induced apoptosis: The following protocol is used: Day 0: Rat insulinoma cells (RIN cells) are inoculated into 200 μl of medium at 10,000 cells per well . Day 1: After 24 hours, discard the medium and add 1 ml
Add fresh medium containing 0.20 or 50 μg HBP per medium to a total volume of 100 μl.

Day 2: Discard the media in wells without HBP and 0 or 1500 units of IL-1
New medium containing β and 20 μg / ml HBP is added in a total volume of 200 μl. HBP
0 or 1500 units of IL in wells (unbound cells) pre-treated with
Add -1β and 20 μg / ml HBP in a total volume of 100 μl to a total volume of 200 μl. Day 5: Measure NO content and accumulation of insulin in the medium. An MTT assay (measurement of succinate dehydrogenase activity, apoptosis) is performed on the cells.

The results are shown in FIGS. 1 to 6 indicate: 1: control (after preincubation, all medium is replaced by HBP and 200 μl of new medium with HBP and cytokines is added); same as 2: 1 Yes, but 20
containing HBP at μg / ml; same as 3: 1 except containing HBP at 50 μg / ml;
Control (100 ml of medium (containing HBP and 2 × cytokine) is added onto 100 μl of HBP pre-incubation medium); same as 5: 4, except that 20 μg /
Includes HBP of 6 ml; same as 6: 4, but with 50 μg / ml of HBP. It is clear from the results that treatment of β-cells with HBP results in reduced apoptosis, but no effect on NO production.

Effect of HBP on Streptozotocin-treated Cells: β cells are pre-incubated with HBP or control for 24 hours before treatment with various concentrations of NO donor, ie streptozotocin. Standard conditions (37 ° C,
After incubation of 5% CO ₂₎ 1~2 days, the cells are assayed for apoptosis. Cells pre-incubated with HBP show little or no apoptosis compared to control incubations. Therefore, HBP prevents beta cell destruction.

The β-cells are incubated with HBP and streptozotocin, or control vehicle and streptozotocin for 24 hours. Cells incubated with HBP show little or no apoptosis compared to control incubations.

Animal studies: Mature Wistar rats were sustained-release HB from a subcutaneously implanted mini-osmotic pump on the back
Pre-treat with P or control vehicle. After 24 or 48 hours, rats are rendered diabetic by intraperitoneal injection of streptozotocin into the tail. Blood glucose and urinary excretion of glucose and albumin are monitored for 2-3 weeks. HBP treated rats show substantially lower diabetes frequency than controls. Finally, the animals are sacrificed and a histological examination of the pancreas is performed. HBP treated rats show little or no beta cell destruction.

Spontaneous diabetic NOD (non-obese diabetic) mice are treated with HBP or control vehicle for 1-2 months. HBP-treated mice show a significantly lower frequency of diabetic symptoms than controls. Finally, the animals are sacrificed and a histological examination of the pancreas is performed. HBP-treated mice show little or no β-cell destruction. 3 month old obese Zucker rats and slender rats equivalent to age
Treat with HBP for one month by subcutaneous infusion using a mini-osmotic pump delivering 0.05 mg / kg body weight. One month later, the animals were sacrificed and the pancreas was evaluated for damage to the islets of Langerton by histological techniques known to those skilled in the art.

Example 2 HBP is internalized and targeted to the mitochondrial compartment : Experimental method: Cell culture: Human umbilical cord endothelial cells (HUVEC) were isolated by digestion with collagenase (Wort
hington Biochemical, Freehold, NJ. USA), and M199 containing Earle's salt (GI
BCO) (Jaffe et al., 1973, J. Clin. Invest. 52: 2745-2756, and Thornton et al., 1983, Science 222: 623-625), fetal bovine serum (ICN Biochemica).
ls Inc., Costa Mesa, Ca. USA), bovine serum (ICN Biochemicals Inc.), endothelial cell growth factor (Sigma), heparin (Sigma) and antibiotics (Gibco BRL., Pais).
gelled in the presence of ley, Scotland) (Sigma Chemical Co., St. Louis.
MO. USA) on the surface. The primary culture is passaged only once using trypsin-EDTA. Cells are used when expressed in a cobblestone morphology, except when semi-confluent cells are used, where microscopic studies are performed.

Wild-type CHO cells (CHO-K1) and heparin sulfate deletion mutant (pgsD-677) (
Murphy Ulrich et al., 1988, J. Biol. Chem. 272: 24363-243670) are grown in a F12K nutrient mixture containing Kaighn's denaturation (GIBCO) supplemented with fetal calf serum and antibiotics. White 96-well tissue culture plate, CulturePlates ™, Microscint-PS
Scintillation fluid and microplate Scintillation counter Topc
ount ™ is obtained from Packard, Meriden, USA. Standard 96-well tissue culture plates are obtained from Nunc, Denmark. Fetal calf serum (FCS) is obtained from Gibco BRL. Hydrogen peroxide is obtained from Merck. Formaldehyde and Triton X-100
Obtained from Sigma.

Antibodies and proteins: Recombinant human HBP was produced in Sf9 insect cells (BRL) using the baculovirus expression system and was described as described (Rasmussen et al., 1996, TEB).
S, Lett. 390: 109-112) and refine. Mouse monoclonal antibody (mab) 2F23
Rabbit antiserum to C3 and recombinant HBP (anti-HBP) was affinity-purified. An antiserum against the mitochondrial protein p33 / gClqR (anti-p33) was raised in rabbits and affinity-purified (Dedio et al., 1998, J. Immunol. 160
: 3534-3542).

The mouse mab 3G10, which recognizes desaturated glucuronic acid reactive with heparitinase-digested heparin sulfate proteoglycan core protein, has been previously characterized (David et al., 1992, J. Cell Biol. 119: 961-975). Texas
Red-conjugated goat anti-rabbit immunoglobulin IgG and goat anti-mouse IgG were from Jackson Immuno Research Laboratories Inc. (West Grove, PA, USA); a peroxidase-conjugated antibody to rabbit IgG was Bio-Red (Richmon
d, CA, USA); and alkaline phosphatase-conjugated rabbit anti-mouse antibodies are from Promega Corporation (Madison, WI, USA). Refined
Performs biotinylation of HBP.

[0116] Endothelial cell metabolism Labeling: Endothelial cells were diluted in endothelial cell culture medium, 100 mCi / ml of Na ₂ ³⁵ SO ₄ (specific activity
) (Amersham International, cultured in the presence of Buckinghamshire, UK) or 50 mCi / ml of ³ H-leucine (Amersham). After 24 hours, cells were washed with cold phosphate buffered saline (PB
(S, GIBCO) and dissolve for 1 hour at 4 ° C. on a shaker.
The lysis buffer was 1% Triton-X-100, 20 mM Tris-HCl, 1 mM CaCl ₂ , 1 mM
Of MgCl _2, 2 mM of PMSF, 5 mM 1,10-phenanthroline, 4 mg / ml leupeptin,
Consists of 4 mg / ml pepstatin A and 100 mg / ml aprotonin (pH 8.0). The lysate is centrifuged at 10,000g for 30 minutes at 4 ° C and the pellet discarded.

Affinity chromatography on HBP Sepharose: Endothelial cell lysate containing 0.2 M NaCl was added to 0.2 M NaCl, 20 mM Tris-HCl, 0.1%
Pre-equilibrated with 1 mM Triton-X-100 and 1 mM PMSF solution (pH 8.0)
Incubate with Sepharose Fast Flow (Pharmacia Biotech AB, Uppsala, Sweden). After 1 hour at 4 ° C, DEAE-Sepharose was added to 10 gel volumes of 0.2 M Na
Cl, 50 mM sodium acetate, 0.1% Triton-X-100, 1 mM PMSF solution (pH 4.0
) And then buffer A (20 mM Tris-HCl, 0.1% Triton-X-
Elute with 1M NaCl solution in 100 and / mM PMSF, pH 7.4).

The biotinylated HBP (5 mg) is conjugated to 2.5 ml of streptavidin-agarose (Sigma). Unbound agarose is used as a control column. Confirm the binding of HBP to the column by SDS-PAGE (data not shown). Equal gel volumes of HBP- and control agarose were mixed with 150 mM NaCl, 1 mM Ca
Equilibrate at 4 ° C. with buffer A containing Cl ₂ and 1 mM MgCl ₂ . The material eluted from DEAE-Sepharose is dialyzed against the equilibration buffer and incubated with the control column at 4 ° C for 2 hours, followed by the HBP-column at 4 ° C for 2 hours.
The HBP- and control columns are washed with 10 gel volumes of equilibration buffer and then eluted with 5 individual gel volumes of a discontinuous NaCl gradient of 250-1000 mM NaCl in buffer A.

[0119] Samples from the eluted material were subjected to 4-16% SDS-PA under reducing or non-reducing conditions.
Separate by GE. Analysis of radioactive materials was performed using X-ray film or Fuji Imaging plate (BioImaging Analyzer Bas200, Fuji Photo Film Co., LTD, Tokyo, Japan)
Perform on gels exposed to If the sample contains ³ H-leucine-labeled material, the gel is treated with a 1.3 M sodium salicylate solution (pH 7) in 5 mM NaHPO ₄ before exposure to the X-ray film (Chamberlain, 1979). , Anal. Bio
chem. 98: 132-135).

Enzymatic and chemical digestion of proteoglycans: radiolabeled HB eluted from HBP-streptavidin agarose
P-binding material was prepared using 60 mU / ml CABC (Sigma) and 4 U / ml Heparinase III (Sigm
a) or a combination of both in 50 mM Tris-HCl, 0.1 M NaCl solution (pH 7.3) at room temperature overnight. To decompose heparin sulfate, the sample is treated with HNO _{3 at} pH 1.5 for 10 minutes at room temperature (Shively an
d Conrad, 1976, Biochemistry 15: 3932-3942).

[0121] Proteoglycans are separated on the basis of their size in agarose gels where they run as separate units rather than as large smears (Bj
omsson, 1993, Anal. Chem. 210: 290-298). Some processed samples were separated on a 1.2% agarose gel, while others were 4-16% SDS
-Separated on PAGE. The dried gel is exposed to a Fuji Imaging plate.

Proteoglycan Western Blot: HBP-affinity purified material was applied to a 100 ml DEAE-Trisacryl column (BioSepra
SA, Villeneuve la Garenne Cedex, France) and 1 M NaCl
Elution with buffer A (5 × 50 ml) containing The concentrated sample and the sample from the original endothelial cell lysate were subjected to 100 mM NaCl, 1 mM CaCl ₂ , 0.1% Triton-X.
0.5 U / ml CA in 100, 50 mM 6-amino-hexanoic acid, 20 mg / ml leupeptin, 2.5 mg / ml pepstatin A, 1 mM PMSF and 50 mM HEPES solution (pH 7.0)
BC (Seikagaku Kogyo, Tokyo, Japan) and 10 mU / ml heparitinase (Seikag
Double digest with aku) at 37 ° C for 3 hours.

After separation under non-reducing conditions by 20% SDS-PAGE, the material was run at 70 V, 0.5 mA,
On a Zeta-Probe membrane (Bio-Rad Laboratories, Hercules, CA, USA) at 4 ° C.
Transfer for 4 hours. The membrane was blocked with 0.5% casein in PBS containing 0.6M NaCl (buffer B) at 4 ° C. overnight, followed by room temperature with mouse mAb 3G10 diluted in buffer B containing 0.15 mM NaCl. And incubate for 1 hour. After two washes with buffer B containing 0.6 M NaCl, the membrane was washed once with buffer B containing 0.15 M NaCl and then washed once with buffer B containing 0.15 M NaCl. : Rabbit anti-mouse IgG conjugated with alkaline phosphatase diluted at a ratio of: 5000. After two washes, bound antibody is detected using a CSPD detection system (Tropix, Bedford, MA, USA) according to the manufacturer.

Western blot of p33: Protein samples eluted from the HBP-bound column were analyzed using 12.5% SDS
-Separation by PAGE and transfer on nitrocellulose membrane at 100 mA for 30 minutes. The membrane was made up of 50% containing 5% (w / v) dry milk powder and 0.05% (w / v) Tween20.
Block with a solution of mM KH ₂ PO ₄ , 0.2 M NaCl (pH 7.4; buffer C). The transferred protein was diluted with a primary antibody (anti-rp33, anti-rp33, diluted to 1 mg / ml in buffer C).
(Anti-CDR31, anti-MBP, anti-HBP). The bound primary antibody was purified by a peroxidase-conjugated secondary antibody to rabbit IgG, followed by EC
L (Amersham) Detection method.

Indirect ELISA: The microtiter plates were incubated with HBP, H-kininogen or the control peptide KLH (1 mM each) in a solution of 100 mM sodium acetate and 100 mM NaCl, pH 5.5, pH 5.5.
g / ml) in a greenhouse overnight (Herwald et al., supra). Binding of the recombinant fusion protein rp33 or the fusion partner MBP (2x dilution starting at a concentration of 2 mg / ml) to the immobilized protein is detected by a-MBP (1: 2500, v / v),
Incubate with a peroxidase-conjugated secondary antibody directed against rabbit IgG (1: 3000, v / v) (Herwald et al., Supra).

The incubation step was performed in a buffer containing 50 mM KH ₂ PO ₄ , 0.2 M NaCl, 2% (w / v) bovine serum albumin (BSA) and 0.05% (w / v) Tween20.
Performed at pH 7.4. For visualization, 0.15% in 100 mM citric acid (pH 4.5)
w / v) of diammonium 2, 2-azino - bis - (3-ethyl-2, 3-dihydro-benz-thia-6-sulfonate, (ABTS), 0.012% of substrate solution H ₂ O _2, 30 minutes , Applied, and the change in absorbance is determined at 405 nm.

Measurement of Affinity between HBP and p33: The specific interaction between HBP and p33 was determined by surface plasmon resonance spectroscopy (BIAlite,
Pharmacia, Freiburg, Germony). HBP is coupled to a CM5 sensor chip according to the manufacturer's instructions. MBP and rp33 were purified from HE in the presence or absence of 50 μM Zn ²⁺ at two-fold serial dilutions with a starting concentration of 100 μg / ml.
Dissolve in PES-Tyrodes buffer.

To follow the binding of rp33 and MBP to the bound HBP, 30 μl of the protein sample here is applied using a flow rate of 10 ml / min. Three minutes later, the tip
Wash with PBS and follow dissociation for 3 minutes. The chip is regenerated by washing with 30 mM HCl. Use the BIA Evaluation 2.0 program (Pharmacia, Freiburg, Germany) to calculate the dissociation constant.

FACS-Analysis: Endothelial cells grown to confluence in 12-well plates were treated with Hanks' solution (
GIBCO), washed once in M199 and then incubated with unconjugated recombinant HBP (50 μg / ml) diluted in the same medium at 37 ° C. for various times. Cells were purified from 0.5% human serum albumin in PBS (Calbiochem, La Jolla
, CA, USA) and once with Ca2 ⁺ and Mg2 ⁺ free PBS. Cell dissociation solution (500 μl; Sigma) is added, the cells are left at 37 ° C. for 15 minutes and then harvested using a cell scrapper.

After fixing in 1% formaldehyde at room temperature for a minimum of 1 hour, cells were
PBS containing heat-inactivated human serum / 0.02% acid solution or 1% saponin (Si
gma) and 0.0125% digitonin (Sigma) in the same solution.
g / ml). The cells are then incubated with the FITC-conjugated secondary goat-anti-mouse antibody diluted 1: 100.

The cells (5,000 cells / experiment) are analyzed on a FACSort (Becton Dickinson, Palo Alto, CA, USA) using the FACStation with Cellquest software. Control cells are incubated with either primary antibody or both primary and secondary antibodies (without HBP). The mean fluorescence intensity (MFI) is calculated based on the channel values. The results are given as average SD.

In some experiments, endothelial cells were treated with NH ₄ C prior to the addition of HBP (50 μg / ml).
Pre-treat with l (50 mM), cytochalasin D (1 μM) or cycloheximide (1 nM). Various substances are present in the 30 minute incubation with HBP. In another experiment, HBP is incubated with heparin (100 μg / ml) before addition of the HBP-heparin mixture to the cells. Binding and internalization of HBP to endothelial cells is also examined at 4 ° C. Wild type (CHO-K1)
And CHO cells lacking heparin sulfate proteoglycan (pgsD-677) (Murphy-Ulri
ch, et al., supra) are treated as for endothelial cells and examined for HBP internalization.

Confocal laser microscopy: Endothelial cells grown overnight on microscope slides were treated with MFC containing Hanks' solution (GIBCO) for various times with 50 μg / ml FITC-labeled or unconjugated HBP. Incubate. After a brief wash with PBS, the endothelial cells were
Fix in 1% formaldehyde for 1 hour. The cells are washed with 100 mM glycine for 1 hour and infiltrated with cold methanol for 10 minutes. Cells
Incubate with 1% BSA in PBS before incubating with antibodies. A staining step is performed to avoid cross-reaction from secondary reagents.

If FITC-conjugated HBP is used, the cells are incubated for up to 24 hours, fixed in formaldehyde and stained for p33. Anti-rp33 (10 μg /
cells for 30 minutes after washing with Texas Red −
Fix with conjugated goat-anti-rabbit IgG. Equilibrate slides, and
Fix with SlowFade Antifade (Molecular Probes, Leiden, The Netherlands) according to the manufacturer's instructions.

The cells are analyzed using a Zeiss LSM310 (Laser Scan Microscope, Oberkochen, Germany). A 590 nm filter is used to prevent interference of the emitted light of the green-red signals. A horizontal portion of the double-stained cells is taken and examined for co-localization. Co-localization resulted in a transition from green and red to yellow / orange. In some experiments, the signal from the green channel is subtracted from the red channel, resulting in a black region representing co-localization.

If unconjugated HBP is used, fixed cells are incubated with biotinylated rabbit-anti-human-HBP antibody (50 mg / ml) for 30 minutes followed by FITC-conjugated streptavidin (10 mg). / ml). Next, the cells
A mouse mAb specific for human mitochondria (mAb127, diluted 1:50)
3) Incubate with Texas Red-conjugated goat-anti-mouse IgG followed by Cells are fixed and examined for co-localization as described above.

Cell sorting: Confluent endothelial cells grown in 5 12-wells were transferred to Hanks' solution (GIBC
Wash once with M199 with M199. Individual plates were incubated with unconjugated recombinant wild-type HBP (25 mg / ml) in 5 ml of the same buffer at 37 ° C. for 24 hours. Cells are washed once with Ca ²⁺ and Mg ²⁺ free PBS and scraped from the plate. After centrifugation (800 × g, 10 minutes), cells were harvested with 50 mM phosphate (pH 7.4), 0.28 M sucrose, 100 μg / ml phenylmethanesulfonyl fluoride, 1 μg / ml aprotinin, 0.5 μg / ml ml leupeptin, 1 μg / pepstatin A, 3.6 μg / ml trans-epoxysuccinyl-L-leucylamide- (
Resuspend in 2.5 ml of a solution of 4-guanidino) butane.

The washed cells are pressurized with N _{2 at} 350 psi and 4 ° C. for 5 minutes and the cavities are collected. The homogenate is centrifuged at 800 × g for 10 minutes and the nuclear pellet P1 is discarded. The supernatant (S1) was further analyzed as described above and the supernatant S1 was
Centrifuge at 00xg for 20 minutes and collect the resulting supernatant S2, the membrane pellet P2,
Wash with 5 ml of 50 mM phosphate buffer containing proteinase inhibitor and centrifuge again. The washed pellet P2 is resuspended in 1 ml of phosphate buffer containing 12% (w / v) sucrose and a 33% (w / v) sucrose cushion (
10 ml), followed by centrifugation at 100,000 xg for 3 hours.

The pellet P3 (vesicular fraction) is resuspended in phosphate buffer. The membrane at the gradient interface is collected, diluted with 30 ml of phosphate buffer and centrifuged (30,000
xg, 45 minutes). The pellet P4 (membrane fraction) is resuspended in phosphate buffer. Centrifugation (100,000xg, 3 hours) of supernatant S2 is performed by pellet P3 (microsome fraction) and supernatant
S3 (cytosole fraction) was applied. All processing is performed at 4 ° C.

Induction of apoptosis in HUVEC cells: HUVEC cells are harvested and subcultured. The cells in passage # 1 are trypsinized and seeded into 96-well culture plates pre-coated with gelatin (10,000 cells per well containing 100 μl complete growth medium). The cells are cultured overnight to obtain confluency. Cells are co-inoculated into a standard 96-well culture plate (Nunc) as a visible control.

After overnight cultivation, the medium was changed to M199 + 10% FCS, hHBP was added, and the final concentration was changed to 0.
Make up to 10 or 50 μg / ml and incubate the cells for 24 hours. After a pre-incubation period, apoptosis was simultaneously washed with hHBP, and the medium was washed with
Induced by changing to either M199 + 10% FCS or M199 without additives. Subsequently, the cells are incubated for 24 hours. DNA fragmentation was performed with TUNEL as described in the kit marketed by PharMingen, with some denaturation.
Measured by

At the end of the assay, the culture medium was carefully removed and the cells were removed by adding 200 μl of 10% formaldehyde buffered in PBS (20 mM phosphate, 150 mM NaCl, pH 7.4). Fix for 30 minutes at room temperature. Wash cells once with PBS,
100 μl of a mixture of 0.1% sodium citrate and 0.1% Tritonx-100
For 5 minutes at room temperature, followed by washing with PBS. TUNEL reaction 50
μl of TUNEL reaction mixture (200 mM sodium cacodylate, 25 mM Tris-HCl
5 U Tdt enzyme and 0.3 μl in 1 mM CoCl ₂ , 0.25 g / l BSA solution (pH 6.6)
Of [ ³² P] dCTP (per well)) and plate
Incubate at <RTIgt; 1 C </ RTI> for 1 hour, determine background levels, and simultaneously incubate wells with reaction mixture without Tdt enzyme.

The reaction is stopped by carefully removing the TUNEL reaction mixture and washing the cells twice with PBS. Subsequently, the plate was dried completely under vacuum, 200 μl of Microscint-PS scintillation fluid was added, the plate was sealed and a microplate scintillation counter (Packard TopCount)
Count by. The degree of labeling is determined by subtracting the background level from the value obtained in the sample containing the Tdt enzyme.

Effect of HBP on hydrogen peroxide-treated HUVEC cells: HUVEC cells are treated with medium (control) and medium containing hydrogen peroxide as indicated for 18 hours. Apoptosis is determined as described above.

Results: Release of HBP from activated human neutrophils: HBP is an almost exclusively synthesized and stored protein in polymorphonuclear (PMN) leukocytes. To show that HBP can be released from those cells, PMN leukocytes were cultured in the presence or absence of human umbilical vein endothelial cells (HUVEC).
Elevated concentrations of phorbol myristate acetate (PMA) or f-Met-Leu
Stimulate from human plasma by -Phe (fMLP).

Agonist-induced HBP release was followed by sandwich ELISA and illustrated for PMA (FIG. 3A) and fMLP (FIG. 3B). PMA and fMLP
Induces HBP secretion from PMN in a dose-dependent manner. The presence of endothelial cells further enhances HBP secretion. Thus, human PMNs secrete HBP effectively upon stimulation with PMA or fMLP in close proximity to HUVEC.

Affinity Purification of HBP Binding Site: Endothelial cells are metabolically labeled with Na ₂ [ ³⁵ S] SO ₄ to allow incorporation of proteoglycans into glycosaminoglycan chains. The cells are lysed, the total cell lysate is applied to DEAE-Sepharose, and the eluted material is affinity purified based on HBP covalently attached to agarose. Radioactively labeled material, 250-50
The HBP-column was eluted with a step gradient of 0 mM NaCl and the resulting fractions were analyzed by SDS-PAGE (FIG. 4A, left panel).

[0148] The eluted material is about 40, as often seen for proteoglycans.
Appeared as broad band smears covering the molecular weight range from kDa to 400 kDa, and a small amount of non-specifically bound material eluted from the unbound matrix (FIG. 4A, right panel). Thus, ^[35 S] - heterogeneous population of labeled molecules, attached to HBP -Sepharose representing at least a portion of the proteoglycans; they are referred to as HBP binding sites.

Glucosidase treatment of HBP-bound proteoglycans: The radiolabeled material from the HBP column is treated with chondroitinase _ABC (C _ABC ) to remove chondroitin sulfate side chains or treated with HNO ₂ ,
Selectively destroy heparin sulfate side chains. The resulting mixture is separated electrophoretically on an agarose gel (FIG. 4B).

The ³⁵ SO ₄ -labeled HBP-binding site was identified as C _ABC (left panel) or HNO ₂ (
(Right panel), which forms part of the HBP binding site from which proteoglycans containing chondroitin sulfate, dermatan sulfate and / or heparin sulfate side chains have been isolated. Treatment with heparinase III, which degrades the heparin sulfate side chain, partially digests the HBP binding site, whereas heparinase
The combined action of III and C _ABC results in complete digestion. These data further indicate that proteoglycans are involved in HBP binding.

Identification of HBP-Binding Proteoglycans: In particular, endothelial cells contain six important types of proteoglycans, including glycosaminoglycans of the heparin sulfate type: perlecan, glypican and syndecan-1,
-2, -3 and -4 are expressed. The affinity-purified HBP binding sites were treated by double digestion with heparinase III and C _ABC to completely remove their heparin side chains (see above). The resulting degradation products are separated by SDS-PAGE and using a mouse monoclonal mAb3G10 which recognizes a new epitope of heparin sulfate proteoglycan exposed on the basis of immobilized glucuronate, a heparinase treatment. (Figure 4C)
).

For comparison, the proteoglycans of all HUVEC lysates are run simultaneously and identified by the relative molecular mass of their core proteins: syndecan-4 (35
K), syndecan-2 (48K), glypican (64K), syndecan-1 (90K)
, Syndecan-3 (125K), and perlecan (> 200K), FIG. 4 (right panel,
Low to top). All heparin sulfate-containing proteoglycans, present in all HUVEC lysates, are also present in the HBP-binding fraction (left panel), indicating that they represent a binding site for HBP.

Inhibition of HBP internalization: To further characterize the HBP internalization process, FACS analysis was performed
Performed on HUVECs preincubated with μg / ml HBP for 0.5 h, followed by intensive washing to remove free ligand (FIG. 5A). HU infiltrated
VEC showed a significantly higher mean fluorescence index than intact cells, suggesting that a significant fraction of exogenously applied HBP had entered the cells.
Incubation in the absence of HBP resulted in an MFI similar to that of intact cells, indicating that the ligand was effectively removed.

Pre-incubation of cells with 100 μg / ml heparin effectively prevented HBP uptake, probably due to competition with HBP for binding sites on heparin sulfate-containing proteoglycans. Incubation at 4 ° C was performed with HU infiltrated
HBP internalization for VEC was significantly reduced, but not for uninfiltrated cells, indicating that HBP internalization is an energy-dependent process. It has been shown to prevent ligand release from the internalized receptor and thus lead to destruction rather than recirculation of the internalized receptor (Gekle et al., 1995, Am.
J. Physiol. 268: F899-906 and Rao et al., 1983, FEBS Lett. 160: 213-216)
The addition of NH ₄ Cl dramatically reduces HBP internalization.

Cytochalasin D, an inhibitor of actin filament polymerization (Coop
er, 1987, J. Cell Biol. 105: 1473-1478) describes HBP internalization.
40.6% lower and cycloheximide, an inhibitor of protein synthesis (Ennis, 1964, FEBS Lett. 399: 255-258), lowers HBP internalization. Colchicine, an inhibitor of microtubule assembly (Olmstead and
Borisy, 1973, Ann. Rev. Biochem. 42: 507-540) reduces HBP internalization by 21%. Together, their findings point to the fact that HBP internalization is an active step that requires an intact and functional cytoskeleton.

Role of Proteoglycans in HBP Internalization: To more precisely point out the possible role of proteoglycans in HBP internalization, heparin sulfate proteoglycan-deficient Chinese hamster ovary (CHO) cells, pgsD-677 ( Murphy-Ulrich et al., 1988, J. Bi
ol. Chem. 272: 24363-243670) and its corresponding wild-type CHO cells.
Study HBP internalization by FACS analysis. Transfer cells to 50 μg / ml HBP
And analyzed for HBP content in fixed and infiltrated cells (FIG. 5B).

[0157] Progressive internalization of exogenously added HBP was seen over 3 hours in wild-type CHO cells. Heparin sulfate-deficient cells also internalize HBP, albeit at a much lower efficiency; internalization is pgsD compared to wild-type CHO-K1 (100% at each time point). In -677 cells, they were reduced by 33% (30 minutes), 38% (1 hour) and 57% (3 hours), respectively. This finding suggests that chondroitin sulfate-containing proteoglycans, which are known to be overexposed by heparin sulfate-type proteoglycans, such as pgsD-677 cells (Murphy-Ulrich, 1988, supra), are other sites where HBP can be internalized. Should be present, but suggests that it is related to HBP internalization.

[0158] Therefore, the present inventors have proposed that pgsD-677 cells be incubated with CA prior to incubation with HBP.
Treated with BC for 30 minutes. CABC treatment also reduced HBP uptake by 13% (30 minutes), although it had no significant effect on wild-type CHO-K1 cells. These findings indicate that both heparin sulfate- and chondroitin sulfate-type proteoglycans are critical to HBP internalization, although other sites may be involved in HBP internalization by CHO cells. Help support your thoughts. The inventor has also: FIG. 4 shows that cells other than HUVEC specifically bind and internalize HBP.

Subcellular fractionation of HBP-treated HUVECs: Subcellular localization of HBP was performed by homogenous fractionation of HUVEC cells preincubated with unlabeled HBP at 37 ° C. for 24 hours. To be tested. Equal amounts of proteins from different cell fractions are subjected to Western blots using anti-HBP (
(Figure 7, upper panel). A major 35 kDa band and a minor 29 kDa band were present in the vesicles (lane 2) and microsomal fraction (lane 4), but not in cytosole (lane 4).
It was not present in lane 1) or the membrane fraction (lane 3).

Most of the internalized HBP retains the molecular mass of the native protein (35K), suggesting that it is still present in an intact form. Control HUVEC maintained with buffer only showed no specific immunoreactive bands. The endogenous protein, p33 / gClqR (Dedio et al., 1996, FEBS Lett. 399: 255-258), shown in the vesicle fraction of HUVEC, is used to validate the fractionation method (FIG. 7). , Lower panel). HBP, which is absent from native HUVEC, appears to be taken up by internal cells and routed to their vesicles and / or microsomal compartments in their intact form.

Co-localization of HBP and p33 / gClqR: As shown in FIG. 7, HBP binds to p33 / gClqR. Since the internalized HBP co-localizes with p33 / gClqR in the vesicle fraction of HUVEC, double staining for those two proteins is performed. Endothelial cells, FETC
-Incubate with labeled HBP for up to 24 hours, fix and
3 Double stained for p33 (from rabbit) followed by an antibody against Texas Red-conjugated anti-rabbit immunoglobulin (from goat). Both FITC-conjugated HBP (green) and p33 / gClqR (red) are prominent in accessory nuclear spots (FIG. 8A).

The color shift to yellow / orange in the confocal overlay and to the black spot in the subtraction overlay was at least HBP and p33 fractions were HUVE
It appears to indicate co-localization within C (FIG. 7B). Since p33 / gClqR has been shown to be a mitochondrial protein, their expression seems to indicate that the internalized HBP is juxtaposed to mitochondria or targeted to a compartment associated with mitochondria. .

Effect of hHBP on apoptosis induced by removal of FCS: HUVEC are incubated with hHBP for 24 hours at the indicated concentrations. The medium is changed and, following the preincubation, incubated in serum-free medium for 18 hours to induce apoptosis. Control cells are incubated with M199 supplemented with 10% FCS. DNA fragmentation is measured by the TUNEL method.
The result is shown in FIG. They show that there is a reduction in apoptosis in hHBP-treated cells.

Effect of hHBP on cells treated with hydrogen peroxide: HUVEC cells are treated with medium (control) and medium containing hydrogen peroxide for 18 hours in the presence or absence of hHBP. Apoptosis is determined as described above. The invention described and claimed herein is not limited by the specific embodiments disclosed herein, as those embodiments are illustrative of some aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to be within the scope of the present invention.

Various documents are cited herein, the disclosures of which are incorporated by reference in their entirety.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the effect of human HBP on MTT values in control and cytokine exposed RIN cells.

FIG. 2 shows the effect of human HBP on NO (nitrite) accumulated in control and cytokine exposed RIN cells.

FIG. 3A shows the release of HBP from PMN in the presence of PMA (FIG. 3A) in the presence or absence of HUVEC.

FIG. 3B shows the release of HBP from PMN in the presence of fMLP (FIG. 3B) in the presence or absence of HUVEC.

FIG. 4A shows the characterization of HBP binding to proteoglycans. FIG. 4A shows that ³⁵ S
-Shows that HBP-bound proteoglycans from labeled endothelial cells are enriched by ion exchange chromatography and affinity purified on HBP-agarose. The fractions eluted by a stepwise ionic strength gradient (250-1000 mM NaCl) are separated on SDS-PAGE and visualized by Phospho-Imaging. Unbound agarose is used as a control column. The molecular marker is given to its left. Molecular weight markers are given on the left.

FIG. 4B shows the characterization of HBP binding to proteoglycans. FIG. 4B shows 300,
Shows that ³⁵ S-labeled HBP-affinity purified proteoglycans, eluted with 400 and 500 mM NaCl, were treated with CABC or HNO ₂ (+). Degradation products were separated on an agarose gel and visualized by Phospho-Imaging. As a control, untreated material (-) is analyzed under the same conditions.

FIG. 4C shows the characterization of the binding of HBP to proteoglycans. Fig. 4C shows 300m
Unlabeled endothelial cell proteoglycans or endothelial cell lysates eluted from the HBP-column with M NaCl were digested (+) or remained undigested (-) by CABC and heparitinase. Show. Decomposition products are converted to SDS-
Separated by PAGE and transferred to Zeta-Probe membrane. Membranes were incubated with mAb 3G10 directed against heparinase-digested heparin sulfate proteoglycan core protein, the immobilized glucuronate. The positions of the different proteoglycans are indicated by (★). Molecular weight markers are given on the left.

FIG. 5A shows the characterization of the internalization process. The endothelial cells are pre-treated with various substances capable of inhibiting receptor-ligand interaction, protein synthesis or actin polymerization before the addition of HBP. On the other hand, cells were treated with NH ₄ Cl (50 mM), cycloheximide (1 nM) or cytochalasin D (1
nM) for 60 minutes. Bound and / or internalized HBP is quantified by FACS analysis in intact or infiltrated cells. Endothelial cells are incubated with HBP or buffer alone (control) as positive and negative controls, respectively, followed by primary and secondary antibodies as described. Data shows MFI ± SD (n = 3).

FIG. 5B shows CHO wild type (CHO-K1) and heparin sulfate proteoglycan deletion.
pgsD677 cells are treated with HBP for the indicated times. Cells are permeated and analyzed as described above.

FIG. 6 shows HBP-treated HUVEC subcellular fractionation. Equal amounts of proteins from different cell fractions are subjected to Western blots using anti-HBP (top panel) and anti-p33 (bottom panel).

FIG. 7A shows binding of HBP to rp33. In FIG. 7A, microtiter plates were coated with H kininogen, HBP or control protein KLH (1 mg / ml) followed by a series of dilutions of rp33 (fusion protein) or fusion partner MBP (2 mg / ml). Starting with the concentration (2 parts dilution). Bound proteins are detected by a rabbit antiserum raised against the fusion partner MBP (1: 2500 v / v) and a peroxidase-conjugated secondary antibody against rabbit IgG (1: 3000 v / v). . Absorbance at 405 nm is expressed in arbitrary units.

FIG. 7B shows binding of HBP to rp33. Using a plasmon resonance spectrometer, an overlay plot of rp33 binding to immobilized HBP is shown in the presence of Zn ²⁺ (FIG. 7B). Elevated concentrations of rp33 (6.25, 12.5, 25, 50 or 100 mg / ml) or MBP (
100 mg / ml) is applied for 3 minutes during the association phase, followed by injection of buffer only,
The dissociation of the complex formed is monitored.

FIG. 7C shows binding of HBP to rp33. Using a plasmon resonance spectrometer, an overlay plot of binding of rp33 to immobilized HBP in the absence of Zn ²⁺ (FIG. 7C) is shown. Elevated concentrations of rp33 (6.25, 12.5, 25, 50 or 100 mg / ml) or MBP
(100 mg / ml) is applied during the association phase for 3 minutes, followed by injection of buffer only and monitoring the dissociation of the complex formed.

FIG. 8A shows the co-localization of p33 with HBP and mitochondria with HBP.
In FIG. 8A, endothelial cells were incubated with FITC-labeled HBP (green) for 6 hours, fixed in 4% formaldehyde, and an antibody to rp33, followed by a Texas Red conjugated secondary antibody to rabbit IgG (red). ). Panel DOUBLE shows overlay of FITC and Texas Red staining. panel
In SUBTRACT, the FITC signal is subtracted from the Texas Red signal. Magnification = 310X.

FIG. 8B shows the co-localization of p33 with HBP and mitochondria with HBP.
In FIG. 8B, endothelial cells were incubated with FITC-labeled HBP (green) for 6 hours, fixed in 4% formaldehyde, and an antibody against rp33 followed by a secondary antibody conjugated to Texas Red to rabbit IgG (red). ). Panel DOUBLE shows overlay of FITC and Texas Red staining. panel
In SUBTRACT, the FITC signal is subtracted from the Texas Red signal. Magnification = 310X.

FIG. 8C shows co-localization of p33 with HBP and mitochondria with HBP.
In FIG. 8C, endothelial cells were incubated with unconjugated HBP for 30 minutes, fixed in 4% formaldehyde, and first incubated with a biotinylated rabbit-anti-HBP antibody followed by FITC-streptavidin. ,
And secondly, it is incubated with mAb1273 against human mitochondria, followed by a Texas Red conjugated secondary antibody against mouse IgG. Co-localization is a panel
DOUBLE is shown, and the deduction in panel SUBTRACT is shown. Magnification = 310X.

FIG. 9 shows the effect of human heparin-binding protein on apoptosis induced by removal of FCS.

FIG. 10 shows the effect of HBP on cells treated with hydrogen peroxide.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] December 24, 1999 (December 24, 1999)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

[Procedure amendment 2]

[Document name to be amended] Drawing

[Correction target item name] FIG. 7A

[Correction method] Change

[Correction contents]

FIG. 7A

[Procedure amendment]

[Submission date] August 30, 2000 (2000.8.30)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0030

[Correction method] Change

[Correction contents]

[0030]

[Table 3]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0035

[Correction method] Change

[Correction contents]

[0035]

[Table 8]

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0037

[Correction method] Change

[Correction contents]

[0037]

[Table 10]

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name]

[Correction method] Change

[Correction contents]

Subcellular fractionation of HBP-treated HUVECs: Subcellular localization of HBP was performed by homogenous fractionation of HUVEC cells preincubated with unlabeled HBP at 37 ° C. for 24 hours. To be tested. Equal amounts of proteins from different cell fractions are subjected to Western blots using anti-HBP (
(Figure 6, upper panel). A major 35 kDa band and a minor 29 kDa band were present in the vesicles (lane 2) and microsomal fraction (lane 4), but not in cytosole (lane 4).
It was not present in lane 1) or the membrane fraction (lane 3).

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0160

[Correction method] Change

[Correction contents]

Most of the internalized HBP retains the molecular mass of the native protein (35K), suggesting that it is still present in an intact form. Control HUVEC maintained with buffer only showed no specific immunoreactive bands. An endogenous protein, p33 / gClqR, shown in the vesicle fraction of HUVEC (Dedio et al., 1996, FEBS Lett. 399: 255-258), is used to validate the fractionation method (FIG. 6). , Lower panel). HBP, which is absent from native HUVEC, appears to be taken up by internal cells and routed to their vesicles and / or microsomal compartments in their intact form.

[Procedure amendment 6]

[Document name to be amended] Statement

[Correction target item name] Brief description of drawings

[Correction method] Change

[Correction contents]

[Brief description of the drawings]

FIG. 1 shows the effect of human HBP on MTT values in control and cytokine exposed RIN cells.

FIG. 2 shows the effect of human HBP on NO (nitrite) accumulated in control and cytokine exposed RIN cells.

FIG. 3A shows the release of HBP from PMN in the presence of PMA (FIG. 3A) in the presence or absence of HUVEC.

FIG. 3B shows the release of HBP from PMN in the presence of fMLP (FIG. 3B) in the presence or absence of HUVEC.

FIG. 4A shows the characterization of HBP binding to proteoglycans. FIG. 4A shows that ³⁵ S
-Shows that HBP-bound proteoglycans from labeled endothelial cells are enriched by ion exchange chromatography and affinity purified on HBP-agarose. The fractions eluted by a stepwise ionic strength gradient (250-1000 mM NaCl) are separated on SDS-PAGE and visualized by Phospho-Imaging. Unbound agarose is used as a control column. The molecular marker is given to its left. Molecular weight markers are given on the left.

FIG. 4B shows the characterization of HBP binding to proteoglycans. FIG. 4B shows 300,
Shows that ³⁵ S-labeled HBP-affinity purified proteoglycans, eluted with 400 and 500 mM NaCl, were treated with CABC or HNO ₂ (+). Degradation products were separated on an agarose gel and visualized by Phospho-Imaging. As a control, untreated material (-) is analyzed under the same conditions.

FIG. 4C shows the characterization of the binding of HBP to proteoglycans. Fig. 4C shows 300m
Unlabeled endothelial cell proteoglycans or endothelial cell lysates eluted from the HBP-column with M NaCl were digested (+) or remained undigested (-) by CABC and heparitinase. Show. Decomposition products are converted to SDS-
Separated by PAGE and transferred to Zeta-Probe membrane. Membranes were incubated with mAb 3G10 directed against heparinase-digested heparin sulfate proteoglycan core protein, the immobilized glucuronate. The positions of the different proteoglycans are indicated by (★). Molecular weight markers are given on the left.

FIG. 5A shows the characterization of the internalization process. The endothelial cells are pre-treated with various substances capable of inhibiting receptor-ligand interaction, protein synthesis or actin polymerization before the addition of HBP. On the other hand, cells were treated with NH ₄ Cl (50 mM), cycloheximide (1 nM) or cytochalasin D (1
nM) for 60 minutes. Bound and / or internalized HBP is quantified by FACS analysis in intact or infiltrated cells. Endothelial cells are incubated with HBP or buffer alone (control) as positive and negative controls, respectively, followed by primary and secondary antibodies as described. Data shows MFI ± SD (n = 3).

FIG. 5B shows CHO wild type (CHO-K1) and heparin sulfate proteoglycan deletion.
pgsD677 cells are treated with HBP for the indicated times. Cells are permeated and analyzed as described above.

FIG. 6 shows HBP-treated HUVEC subcellular fractionation. Equal amounts of proteins from different cell fractions are subjected to Western blots using anti-HBP (top panel) and anti-p33 (bottom panel).

FIG. 7A shows binding of HBP to rp33. In FIG. 7A, microtiter plates were coated with H kininogen, HBP or control protein KLH (1 mg / ml) followed by a series of dilutions of rp33 (fusion protein) or fusion partner MBP (2 mg / ml). Starting with the concentration (2 parts dilution). Bound proteins are detected by a rabbit antiserum raised against the fusion partner MBP (1: 2500 v / v) and a peroxidase-conjugated secondary antibody against rabbit IgG (1: 3000 v / v). . Absorbance at 405 nm is expressed in arbitrary units.

FIG. 7B shows binding of HBP to rp33. Using a plasmon resonance spectrometer, an overlay plot of rp33 binding to immobilized HBP is shown in the presence of Zn ²⁺ (FIG. 7B). Elevated concentrations of rp33 (6.25, 12.5, 25, 50 or 100 mg / ml) or MBP (
100 mg / ml) is applied for 3 minutes during the association phase, followed by injection of buffer only,
The dissociation of the complex formed is monitored.

FIG. 7C shows binding of HBP to rp33. Using a plasmon resonance spectrometer, an overlay plot of binding of rp33 to immobilized HBP in the absence of Zn ²⁺ (FIG. 7C) is shown. Elevated concentrations of rp33 (6.25, 12.5, 25, 50 or 100 mg / ml) or MBP
(100 mg / ml) is applied during the association phase for 3 minutes, followed by injection of buffer only and monitoring the dissociation of the complex formed.

FIG. 8A shows the co-localization of p33 with HBP and mitochondria with HBP.
In FIG. 8A, endothelial cells were incubated with FITC-labeled HBP (green) for 6 hours, fixed in 4% formaldehyde, and an antibody to rp33, followed by a Texas Red conjugated secondary antibody to rabbit IgG (red). ). Panel DOUBLE shows overlay of FITC and Texas Red staining. panel
In SUBTRACT, the FITC signal is subtracted from the Texas Red signal. Magnification = 310X.

FIG. 8B shows the co-localization of p33 with HBP and mitochondria with HBP.
In FIG. 8B, endothelial cells were incubated with FITC-labeled HBP (green) for 6 hours, fixed in 4% formaldehyde, and an antibody against rp33 followed by a secondary antibody conjugated to Texas Red to rabbit IgG (red). ). Panel DOUBLE shows overlay of FITC and Texas Red staining. panel
In SUBTRACT, the FITC signal is subtracted from the Texas Red signal. Magnification = 310X.

FIG. 8C shows co-localization of p33 with HBP and mitochondria with HBP.
In FIG. 8C, endothelial cells were incubated with unconjugated HBP for 30 minutes, fixed in 4% formaldehyde, and first incubated with a biotinylated rabbit-anti-HBP antibody followed by FITC-streptavidin. ,
And secondly, it is incubated with mAb1273 against human mitochondria, followed by a Texas Red conjugated secondary antibody against mouse IgG. Co-localization is a panel
DOUBLE is shown, and the deduction in panel SUBTRACT is shown. Magnification = 310X.

FIG. 8D shows co-localization of p33 with HBP and mitochondria with HBP.
In FIG. 8D, endothelial cells were incubated with unconjugated HBP for 30 minutes, fixed in 4% formaldehyde, and first incubated with a biotinylated rabbit-anti-HBP antibody, followed by FITC-streptavidin. ,
And secondly, it is incubated with mAb1273 against human mitochondria, followed by a Texas Red conjugated secondary antibody against mouse IgG. Co-localization is a panel
DOUBLE is shown, and the deduction in panel SUBTRACT is shown. Magnification = 310X.

FIG. 9 shows the effect of human heparin-binding protein on apoptosis induced by removal of FCS.

FIG. 10 shows the effect of HBP on cells treated with hydrogen peroxide.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/00 A61P 37/06 37/06 43/00 43/00 111 111 C07K 14/47 C07K 14/47 A61K 37/02 C12N 15/09 ZNA C12N 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE) , LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, Z, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL , IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZWF term (reference) 4B024 AA01 BA80 CA04 DA03 EA04 GA11 4C084 AA02 BA01 BA22 BA44 DC50 ZA012 ZA022 ZA162 ZA332 ZA512 ZA542 ZA552 ZB212 ZB332 ZC032 ZC352 ZC552 4H045 AA30 BA10 BA53 CA40 EA20 FA72 FA73 FA74 HA05

Claims (20)

[Claims] 1. A method for modulating or reducing apoptosis in mammalian cells of a mammal selected from the group consisting of β-cells, endothelial cells and neurons of the islets of Langerhans, comprising: i) SDS PAGE under reducing conditions
Has a molecular weight of about 28 kD as determined by: (ii) produced in the azulous granules of polymorphonuclear leukocytes; and (iii) a mammalian heparin-binding protein or drug that is a chemoattractant for monocytes An active fragment that is chemically acceptable
Administering to the mammal in need thereof in an amount effective to modulate or reduce apoptosis in said cells. 2. The method of claim 1, wherein said mammalian heparin-binding protein (HBP) is human or porcine HBP. 3. The amino acid sequence having at least about 80% identity to the amino acid sequence shown in SEQ ID NO: 1, 2, 5, 7, 9 or 11, or an allelic or natural variant thereof. The method of claim 1 having a body. 4. The nucleic acid sequence wherein the HBP hybridizes to the nucleic acid sequence shown in SEQ ID NO: 3, 4, 6, 8, 10, or 12; (ii) its complementary strand; or (iii)
2. The method of claim 1, wherein the method is encoded by the subsequence of (i) or (ii). 5. The method according to claim 2, wherein the HBP has an amino acid sequence represented by SEQ ID NO: 1, 2, 5, 7, 9 or 11. 6. The method according to claim 1, wherein the HBP is encoded by a nucleic acid sequence that hybridizes to the nucleic acid sequence shown in SEQ ID NO: 3, 4, 6, 8, 10 or 12. 7. The method of claim 1, wherein the heparin-binding protein comprises about 10 mg per unit dosage form.
The method of claim 1, wherein the method is present in an amount of from about 1 g. 8. The method of claim 1 wherein said heparin-binding protein is present in an amount of about 0.1 to 10 per kg body weight.
The method of claim 1 which is present in an amount of 0 mg. 9. The method of claim 1 wherein said heparin-binding protein is present in an amount of about 0.5 to 50 per kg body weight.
2. The method of claim 1, wherein the method is present in an amount of mg. 10. The method of claim 1 wherein said heparin-binding protein is present in an amount of about 1 to 25 per kg body weight.
2. The method of claim 1, wherein the method is present in an amount of mg. 11. A method for modulating or reducing apoptosis in mammalian cells of a mammal selected from the group consisting of β-cells, endothelial cells and neural cells of the islet of Langerhans, comprising: (a) a glycosylated form. And (i) under reducing conditions
A mammalian heparin-binding protein having a molecular weight of about 28 kD as determined by SDS PAGE; (ii) produced in azure granules of polymorphonuclear leukocytes; and (iii) a chemoattractant for monocytes Or a composition comprising a pharmaceutically acceptable active fragment thereof, and (b) a pharmaceutically acceptable carrier, in an amount effective to modulate or reduce apoptosis in said cells. Administering to the mammal. 12. A method for preventing or treating a disease caused by apoptosis of mammalian cells in a mammal, wherein the method is selected from the group consisting of β cells, endothelial cells and neural cells of the islets of Langerhans, wherein the method comprises the steps of: In form, (i) has a molecular weight of about 28 kD as determined by SDS PAGE under reducing conditions; (ii) is produced in azuric granules of polymorphonuclear leukocytes; and (iii) Administering to the mammal in need thereof a chemoattractant mammalian heparin-binding protein or a pharmaceutically acceptable active fragment thereof in an amount effective to modulate or reduce apoptosis in the cell. A method comprising: 13. The method of claim 12, wherein said disease is selected from the group consisting of a state of insufficient function of insulin production or insulin action, a neurodegenerative disease, a neuromuscular disease, a human immunodeficiency virus and ischemic stroke. Method. 14. The method of claim 1, wherein said mammal is a human patient. 15. A method for preventing or treating a disease caused by apoptosis of a mammalian cell of a mammal selected from the group consisting of β-cells, endothelial cells and neurons of the islets of Langerhans, comprising: (a) In glycosylated form, (i) has a molecular weight of about 28 kD as determined by SDS PAGE under reducing conditions; (ii) is produced in azuric granules of polymorphonuclear leukocytes; and A composition comprising a mammalian heparin-binding protein or a pharmaceutically acceptable active fragment thereof, which is a chemoattractant for a sphere, and (b) a pharmaceutically acceptable carrier.
Administering to the mammal in need thereof an amount effective to modulate or reduce apoptosis in the cell. 16. SDS in glycosylated form (i) under reducing conditions
Has a molecular weight of about 28 kD as determined by PAGE; (ii) produced in the azuloid granules of polymorphonuclear leukocytes; and (iii) a mammalian heparin-binding protein which is a chemoattractant for monocytes or A composition comprising a pharmaceutically acceptable active fragment thereof, and (b) a proteoglycan that binds to said heparin-binding protein. 17. SDS in glycosylated form (i) under reducing conditions
Has a molecular weight of about 28 kD as determined by PAGE; (ii) produced in the azuloid granules of polymorphonuclear leukocytes; and (iii) a mammalian heparin-binding protein which is a chemoattractant for monocytes or A composition comprising a pharmaceutically acceptable active fragment thereof, and (b) a protein that is a mammalian mitochondrial matrix targeting protein and binds to said heparin-binding protein. 18. The composition of claim 17, wherein said mitochondrial matrix binding protein comprises a mitochondrial targeting sequence set forth in SEQ ID NO: 13. 19. A method for modulating or reducing apoptosis in mammalian cells of a mammal selected from the group consisting of β-cells, endothelial cells and neurons of the islets of Langerhans, wherein the composition according to claim 16 is used. Administering to the mammal in need thereof in an amount effective to modulate or reduce apoptosis in the cell. 20. A method for modulating or reducing apoptosis in a mammalian cell of a mammal selected from the group consisting of β-cells, endothelial cells and neurons of the islets of Langerhans, wherein the composition according to claim 17 is used. Administering to the mammal in need thereof in an amount effective to modulate or reduce apoptosis in the cell.