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WO1993015195A1 - Granulins from leukocytes - Google Patents

Granulins from leukocytes Download PDF

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Publication number
WO1993015195A1
WO1993015195A1 PCT/CA1992/000089 CA9200089W WO9315195A1 WO 1993015195 A1 WO1993015195 A1 WO 1993015195A1 CA 9200089 W CA9200089 W CA 9200089W WO 9315195 A1 WO9315195 A1 WO 9315195A1
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WIPO (PCT)
Prior art keywords
cys
granulin
gly
asp
ala
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Application number
PCT/CA1992/000089
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French (fr)
Inventor
Samuel Solomon
Original Assignee
Samuel Solomon
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Publication of WO1993015195A1 publication Critical patent/WO1993015195A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to novel peptides and pharmaceutical formulations containing them; the pep- tides are useful as inhibitors of keratinocytes.
  • leukocytes are pedtidergic cells.
  • Neutrophil granules contain large amounts of basic, cystine-rich peptides of 29 to 34 amino acids, that have been variously called de- fensins (1), coricostatins (2), myeloid-related sequences (3), and cryptidins (4).
  • de- fensins (1) basic, cystine-rich peptides of 29 to 34 amino acids
  • coricostatins (2) coricostatins (2)
  • myeloid-related sequences (3) myeloid-related sequences
  • cryptidins cryptidins
  • corti- costatins have potential regulatory functions, includ- " ing the ability to inhibit the action of the hormone adrenocorticotropin on glucocorticoid secretion (2,6,7) and to stimulate nifedipine-sensitive L-type Ca ⁇ + channels in villus enterocytes (8). It has also been reported that a human defensin is a monocyte chemotactic agent (9). Other granulocyte-associated peptides have also been shown to have regulatory activities. For example, hemoregulatory peptide 1 is a granulocyte-associated thiol containing pentapep- tide, with potent inhibitory actions on myelopoieses (10).
  • Granulocyte enriched extracts contain several cystine-rich components at levels approximately three orders of magnitude lower than the defensin/corticostatins. From their compositional analysis and chromatographic behaviour these peptides appear unrelated to any known hormone, including the definsin/corticostatins.
  • Gran- ulocyte-derived peptides have potential both as immunoregulatory molecules, and in host resistance.
  • Granulins are novel candidate growth factors recently discovered in human and rat inflammatory leukocytes (22). Two rat granulin homologs, epithelin 1 and 2, occur in the kidney (23). Epithelin 1, which is probably identical to rat leukocyte granulin (22,23) exhibits activities similar to epidermal growth factor on epithelian cells .in vitro (23) .
  • the invention relates to a family of novel leukocyte-associated peptides that are cystine-rich.
  • the peptides are approximately 6 Kda and may be cytokines.
  • Granulin A DVKCDMEVSCPDGYTCCRLQSGAWGC- CPFTQAVCCEDHIHCCPAGFTCDTQKGTCE, SEQ ID NO: 1.
  • Rat Granulin EVKCDLEVSCPDGYTCCRLNTGA G(CCPFSB)AVCCEDHIHCCPAGFTCXTQ, SEQ ID NO: 5.
  • Granulin C VPCDXVSSCPSSDTCCOLTSGEHGCCPIPEAVC, SEQ ID NO: 3.
  • Granulin D IGCDQXDTSSCCPDG, SEQ ID NO: 4.
  • carp Granulin VIHCDAATICPDGTICCLSPYGMBGQCCRDGIHCCRHGYHCDSRTTHC , SEQ ID NO: 6.
  • Granulin B VMCPDARSRCPDGHTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSK Cl, SEQ ID NO: 2.
  • Granulin E Asp Val Glu Cys Gly Clu Gly His Phe Cys His Asp Asp Gin Thr Cys Cys Arg Asp Asn Arg Glu Gly Trp Ala Cys Cys Pro Try Ala Gin Gly Cla Cys Cys Ala Asp Arg Arg His Cys Cys Pro Ala Gly Phe Arg Cys Ala Arg Arg Gly Thr Lys Cys Leu, SEQ ID NO: 7.
  • Granulin F Ala lie Gin Cys Pro Asp Ser Gin Phe Glu Cys Pro Asp Phe Ser Thr Cys Cys Val Met Val Asp Gly Ser Trp Gly Cys Cys Pro Met Pro Gin Ala Ser Cys Cys Glu Asp Arg Val His Cys Cys Pro His Gly Ala Phe Cys Asp Leu Val His Thr Arg Cys Lie, SEQ ID NO: 8.
  • Granulin G Gly Gly Pro Cys Gin Val Asp Ala His Cys Ser Ala Gly His Ser Cys lie Phe Thr Val Ser Gly Thr Ser Ser Cys Cys Pro Phe Pro Glu Ala Cys Gly Asp Gly His His Cys Cys Pro Arg Gly Phe His Cys Ser Ala Asp Gly Arg Ser Cys Phe, SEQ ID NO: 9.
  • a topical formulation comprising an effective amount of a Granulin of the invention in association with a pharmaceutically acceptable carrier for topical formulation.
  • a method of healing wounds comprising applying to a wound site a Granulin of the invention.
  • FIG. 1 Figure IA shows the HPLC chromatogram. of a crude granule extract from inflammatory exudate cells, and B shows the chromatogram of a whole cell extract. The position of the granulins are marked by arrows. Note the absence in A of thymosin-/3-4, a cyto- plasmic marker peptide.
  • the granule peptide markers HP-1 and HP-4 were identified as previously described (6), lysozyme was identified by a ino terminal sequence analysis (unpublished). Thymosin- ⁇ -4 and its oxidation product were identified by Fast Atom Bombardment mass spectrometry;
  • FIG.2 Size-exclusion purification of granulin A, (panel A), granulin B, (panel B), and granulins C and D, (panel C). Size markers were substance P, CLIP and ACTH1.-39. Apparent m.wts for native granulin A, 2700; granulin B, 3200; granulin C, 1700; and granulin D 7. 3900; FIG. 3 Purification of rat granulin from bone marrow; panel A shows the first HPLC chromatogram in ace onitrile/TFA, and B shows the second step of purification in
  • FIG. 4A Shows the sequence of the probe used to screen a human bone marrow cDNA library in gt 11
  • FIG. 4B Shows a sequencing strategy
  • FIG. 4C Shows the complete nucleotide sequence and deduced polypeptide sequence of granulin
  • FIG. 5 Shows a comparison of the granulin-like domains
  • FIG. 6 Is a Southern blot analysis of digested human DNA
  • FIG. 7A Is a Northern blot analysis of granulin precursor mRNA
  • FIG. 7B Is a Northern blot analysis of RNA from
  • FIG. 7C Shows distribution of granulin mRNA. MODES FOR CARRYING OUT THE INVENTION MATERIALS AND METHODS Tissue Sources
  • S-pyridylethylated peptides were digested using trypsin (TPCK-treated, Sigma) chymotrypsin (Sigma), and S. aureus V8 protease (Sigma), at enzyme to substrate ratios of approximately 1 to 50 by weight. Digestions were performed at 37°C in 100 ⁇ l
  • proteolytic fragments were then fractionated by rp-HPLC on a C-ig ⁇ Bondapak column using a gradient of
  • HPLC profile of a typical extract of human inflammatory cells is shown in Figure 1.
  • HP-1 and HP-4 several low abundance components are present.
  • three low abundance components that had unusually high levels of cystine were identified... These are labelled A, B and C/D, and were present in both whole cell extracts (FIG. IB) and crude granule preparations (FIG. IA) .
  • Each of these extracts was then further purified using size-exclusion HPLC, revealing that the component C/D
  • SUBSTITUTE SHEET contained two peptides, one of which, D, eluted as a larger molecule than the other three. Each peptide was further purified on rp-HPLC. Their amino acid compositions are given in Table 1. The purified peptides were S-pyridylethylated, and amino terminal sequence analyses were performed, revealing that the four peptides were distinct but related molecules with no homology to any known protein. Because these peptides were associated with the granule fraction, we call them granulins A, B, C and D. Granulin D was run on reducing and non-reducing SDS-PAGE, and ran as a smaller molecule after reduction, indicating that it is probably a dimer.
  • Granulin A is the most abundant of the human granulins, and was subject to a more detailed analysis. S-pyridylethylated peptide was digested with trypsin, chy otrypsin or S. aureus V8 protease. The fragments were isolated by HPLC, one fifth aliquots analysed by amino acid analysis, and appropriate fragments were then submitted to gas phase Edman microsequencing. Final recovery of the digestion products was between 150 and 300 picomoles. The overlap of the granulin A fragments is described in the legend to Table 2 together, with similar data for the rat granulin. The two sequences are highly
  • SUBSTITUTE SHEET conserved, as would be expected for regulatory molecules. Inflammatory exudates and bone marrow preparations are mixtures of cells, the exudates containing typically 70 to 95% granulocytes. When leukocytes from the blood of healthy donors was fractionated by density gradient centrifugation, granulins could be detected in the granulocyte pellet, but not in the interface where the mononuclear cells partition (data not shown). The isolation and characterization of a novel family of leukocyte associated cystine-rich peptides, which are called granulins has been described.
  • SUBSTITUTESHEET present it is not certain if both peptides are the product of the same, or different genes. It is also too early to determine whether the epithelins are intrinsic to renal cells, or if they are derived from blood borne cells trapped in the kidney. It is clear, however, that the two peptides from the kidney are members of a larger family, the granulins, and that a major source of the granulins, and probably also the epithelins, is from circulating leukocytes. Rat granulin was isolated from bone marrow, indicating that granulins are of myeloid origin. Whether granulins are also synthesized in circulating leukocytes remains to be determined.
  • Granulins were extracted from granulocyte rich preparations, and are recoverable from the granulocyte pellet after Ficoll- Hypaque density gradient centrifugation. This suggests that their cellular origin may be the neutrophil, however, it is possible that other granulocytes such as eosinophils, or contaminating monocytes, contribute to the granulin content of these extracts. Extracts of human platelets contained no detectable granulins (data not shown). It is possible that each granulin belongs to a distinct cell type, or that sub-classes of the same cell-type contain different granulins. These are issues best answered using immunolocalization procedures. The granulins co-purify in a crude granule extract.
  • Granulocytes have several different granule subclasses, including a true secretory compartment (21) that can be activated independently of phagocytosis.
  • a true secretory compartment (21) that can be activated independently of phagocytosis.
  • suitable immunoassay techniques will permit the unambiguous location .of the granulins to a cell type, and a subcellular compartment.
  • the granulins are a novel family of cystine- rich immunoinflammatory peptides. Their presence in circulating leukocytes and inflammatory exudates, and
  • granulins a novel class of leukocyte peptides with possible cytokine-like activities which are called granulins. They are cystine-rich with molecular weights of approximately 6Kda, except for granulin D, which appears to be a dimer.
  • the sequence of one member of this family, a 56 residue peptide, granulin A, and amino-terminal sequences for three other granulins from human peripheral leukocytes are described.
  • a fifth related peptide was isolated and partially sequenced from rat bone marrow, suggesting that at least some of the granulin in peripheral leukocytes is preformed in the marrow.
  • Rat granulin, and human granulin A are closely related, showing that the granulin structures are highly conserved between species. It has now been found that the precursor for the human granulins is a 593 residue glycoprotein, containing seven repeats of the 12-cys mecanic granulin domain. Gene expression is seen in the kidney, leukemic cell lines and fibroblasts, and very strongly in transformed epithelial cell lines, which both express the gene, and respond to the mature polypeptide.
  • Human leukocytes contain four granulin homologs, designated A, B, C and D. Their cellular distribution, and whether they are products of one or several genes under co-ordinate or independent control was unknown. To address these, and related issues, two oligonucleotide primers for PCR were synthesized corresponding to the amino terminal and mid-portion regions of granulin A (grnA). GrnA was chosen because it is the most abundant granulin in human leukocyte
  • the sequence (Fig. 4C) predicts a protein of 593 residues, with a probable signal peptide (24) extending to residue 17. It contains the 56 residue grnA sequence, as expected, and also six other 12- cysteine granulin-like domains, including B, C and D, hitherto known only from N-terminal sequences.
  • the precursor contains two novel Cys-12 domains, E and F, and a degenerate granulin domain, G, with 10 cysteines.
  • the positions of the cysteines are highly conserved (Fig. 5) as are certain other residues such as ASP39, His42 and Pr ⁇ 4g.
  • An eighth domain at the N- terminus, (paragranulin, Fig. 5) contains only 6 cysteines corresponding to the amino-terminal half of a granulin domain.
  • the inter-domain sequences show little homology, except for a pro-ala dipeptide, found midway between each domain except F and G.
  • Mono- or dibasic sequences which are frequent sites of proteolysis in peptide processing (25), flank granulin domains C, D and E, but not A or B, both of which have been isolated as excised peptides. Thus nothing can be
  • EGF and granulin/epithelins have similar actions on epithelial cells (23), but probably different receptors (23). Whether the similarities are significant, or merely coincidental, is unclear. Unlike the EGF-precursor, pro-granulin has no transmembrane segment or cytoplasmic domain (28,29) implying different post-translational pathways for the two proteins. Multiple sequential repeats of cysteine-rich domains is a recurrent structural motif among regulatory proteins (28,29,30,31), wherein typically, one exon delineates one cysteine-rich domain (30,32,33). In contrast, the PCR amplified fragment of grnA is bisected by an intron (Fig. 4A), and preliminary results show that introns bisect domains G, F, B, C and D.
  • cysteines align with an approximately mirror-image symmetry around the bisecting intron. Genomically, therefore, pro- granulin bears little resemblance to the EGF- precursor.
  • Proerythroid leukemic cells express granulin mRNA (Fig. 7A) , but seem unlikely to be involved in regulating epithelial proliferation. Similarly, the
  • SUBSTITUTE SHEET strong expression of granulin in RNA in kidney, but not other tissues, may imply functions unrelated to epithelial cell mitogenesis.
  • granulins may have multiple biological activities; indeed distinct activities may be associated with different domains, as already proposed for epithelins 1 and 2 (23) .
  • the availability of cloned granulins permit these questions to be addressed.
  • FIG. 4. shows the sequence of the probe used to screen a human bone marrow cDNA library in gtll (Clontech, Palo Alto, CA. ) .
  • the probe was generated by PCR of human DNA, with forward and reverse primers (underlined) based on the amino acid sequence of granulin A.
  • the nucleotides in lower case represent an intron in the coding region of granulin A.
  • Fig. 4B shows the sequencing strategy.
  • the nucleotide sequence is a composite of three sequences HBM12, HBM3 and HNM4.
  • Fig. 4C shows the complete nucleotide sequence and deduced polypeptide sequence of granulin. The nucleotides are numbered from the initiator codon (ATG), and the amino acids from the probable signal peptide cleavage site. Underlined sequences correspond to sequences previously determined by gas-phase microsequencing of purified granulins.
  • N-glycosylation sites are indicated by an asterisk (*) , and the stop codon is shown by a #.
  • the boxed nucleotides corresponds to a polyadenylation signal.
  • Clone HBM12 starts at nucleotide 6.
  • the sequences of clones HBM3 and HBM4 differ in the 5'-untranslated region as indicated by the bifurcation in the nucleotide sequence.
  • oligonucleotide primers forward primer, 5'- CGATGTGAAGTG(T/C)GA(T/C)ATGGA-3 ' ; reverse primer, 5'- CTGGCATGTGGTT(T/C)TC(A/G)CA(G/A)CA-3 ' ) were synthesized (Sheldon Biotechnology Centre, McGill University) and used in the polymerase chain reaction (PCR) with 2 ⁇ g of genomic DNA as template (34).
  • the amplified product was subcloned into the plasmid vector Bluescript KSII+ and its identity confirmed by double stranded dideoxynucleotide sequencing with Sequenase (USB). This fragment was labeled with 32 P by nick-translation (Boehringer Mannheim) and used to probe a human bone marrow cDNA library in ⁇ gtll (Cl ⁇ ntech, Palo Alto, CA. ) consisting of 1.51 X10 6 independent clones. Duplicate nitrocellulose filters (Schleicher and Schuell) were prehybridized in 5X SSC, 5X Denhardt's reagent, 0.2% SDS at 37°C for 5 hours.
  • Hybridization was in 5X SSC, 2.5X Denhardt's reagent, 0.2% SDS, 50% formamide, 10% polyethylene glycol with 1x10? cpm probe at 37°C for 12 hours. Filters were washed twice for 45 minutes in 2X SSC, 0.1% SDS at 58°C and exposed at -70°C with Kodak X-Omat film with an intensifying screen. 16 positives were obtain from 3x10 ⁇ clones screened. The cDNA insert from clone HMB12 was digested with Kpnl and Sad, and the resulting cDNA fragments subcloned into Bluescript KSII+.
  • Nucleotide sequence was determined by double stranded dideoxy sequencing using Sequenase (USB).
  • the sequence of HBM12 lacked an initiator methionine.
  • the remaining positive clones were analysed using PCR with ⁇ gtll sequence specific primers in combination with a primer, cll2rp, corresponding to nucleotides 45 to 61 of clone HBM12.
  • Fig.5. Comparison of the granulin-like domains. Residues occuring three or more times are boxed, and dashes have been introduced to align the cysteines.
  • the domain boundaries were determined by gas-phase microsequencing and by comparison with the sequence of grnA. The domains are located at amin ⁇ acids, 264-319, grnA; 169-244, grnB; 347-400, 425-479, grnD; 501-506, grnE; 106-162, grnF; 4i grnG and 1-27, paragranulin.
  • the order of domains is: paragranulin-G-F-B-A-C-D-E. They are not alphabetically aligned because several domains were isolated and named as discrete proteins before their common origin was known.
  • Fig. 7 Northern blot analysis of: A. granulin precursor mRNA in the following cell lines,
  • HL-60 promycelocytic leukemia
  • U937 histocytic leukemia
  • K562 histocytic leukemia
  • KMOE proerythroid leuker ⁇ ias
  • A431 epidermoid carcinoma
  • A54 lung epithelial
  • RNA from A431 (Chinese hamster ovaries), and murine Balb/C 3T3 fibroblasts, exposed for 10 days. (B). RNA from A431,
  • RNA was denatured with glyoxal, electrophoresed on a 1.1% agarose gel in 10 mM NaH2P ⁇ 4, pH 6.8, transferred to nylon membranes (ZetaProbe, BioRad) by capillary blotting with lOmM NaOH and fixed by baking at 80°C for 2 hours.
  • the membranes were hybridized at 65°C for RNA extracted from cell lines or 60°C for rabbit tissue RNA in 0.5M NaH 2 P ⁇ 4, pH 6.8, 7% SDS, lmM EDTA for 24 hours with the same probe described in Figure 6.
  • the membranes were washed at hybridization temperature for 30 mins. in 40 mM NaH2P ⁇ , pH 6.8, 5%
  • VIHCDAATICPDGTICCLSPYGMBGQCCRDGIHCCRHGYHCDSRTTHCL was isolated and characterized. It was found to be homologous with rat and human granulins but not identical.
  • the carp Granulin causes an increase in ⁇ E- thymidine incorporation in a keratinocyte cell line which indicates increased cell growth in keratinocytes and thus wound healing.
  • Granulin A causes a fall in 3n-t,hymidine incorporation in A-431 cells, a human epidermal carcinoma cell line.
  • the human granulins of the invention retard growth of A-431 cells.
  • the granulins of the invention are suitably employed as the active ingredient in pharmaceutical compositions for topical application.
  • the granulins may be incorporated in a topical formulation in an amount of 5 to 50 ⁇ g/ml of
  • SUBSTITUTE SHEET ointment and are applied to a wound site in an amount of 5 to 50 ⁇ g/cm 2 of wound site.
  • A. preferred treatment amount of the granulins is about lO ⁇ g/cm 2 of wound site suitably applied three times a day.
  • the topical pharmaceutical composition may be in the form of a cream, ointment, gel, lotion or other formulation suitable for application to the skin, and thus comprise a granulin of the invention admixed with an acceptable carrier for topical application.
  • SUBSTITUTESHEET TABLE 1 The amino acid compositions of purified granulins. The recovery of cysteine in this system is variable and is between 65 to 80%. Values have not been corrected for background contamination or oxidation. Tryptophan was not determine. Predicted values for granulin A from the gas-phase sequence determinations are given in brackets.
  • SUBSTITUTE SHEET TABLE 2 Structural analyses of five members of the granulin family.
  • the proposed structure for granulin A was determined by overlapping two amino terminal sequences, (1-11), and (1-23); tryptic fragments (4- 18), (19-52), and V8 protease fragment (37-56), and chymotryptic fragment (47-56).
  • the proposed structure of rat granulin is based partly upon the direct sequencing of the peptide itself (i.e., the sequence 1 through 21) and its proteolytic fragments (i.e., fragments corresponding to 4 to 18, 19 to 31, 32 to 46, and 32 to 51 sequences).
  • Rat Granulin EVKCDLEVSCPDGYTCCRLNTGAWG(CCPFSB)AVCCE-
  • Granulin B VMCPDARSRCPDGHTCCELPSGKYGCCPMPNATCCSDH- LHCCPQDTVCDLIQSKCI.
  • Granulin C VPCDXVSSCPSSDTCCOLTSGEHGCCPIPEAVC.
  • Granulin D IGCDQXDTSSCCPDG.
  • TFA trifluoroacetic acid: HFBA, heptafluorobutyric acid; rp-HPLC reverses phase high performance liquid chromatography; PTH, phenylthiohydantoin.
  • Cys Cys Pro lie Pro Glu Ala Val Cys Cys Ser 25 30 35 Asp His Gin His Cys Cys Pro Gin Arg Tyr Thr Cys
  • position 31 is not yet determined; it is either Asp or Asn.
  • identity of position 49 is not known.
  • position 23 is not yet determine; it is either Asp or Asn.

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Abstract

Novel leukocyte peptides are useful in healing wounds; the peptides are cystine rich and approximately 6 Kda.

Description

GRANULINS PROM LEUKOCYTES TECHNICAL FIELD
This invention relates to novel peptides and pharmaceutical formulations containing them; the pep- tides are useful as inhibitors of keratinocytes. BACKGROUND ART
It has recently become clear that leukocytes are pedtidergic cells. Neutrophil granules contain large amounts of basic, cystine-rich peptides of 29 to 34 amino acids, that have been variously called de- fensins (1), coricostatins (2), myeloid-related sequences (3), and cryptidins (4). Some of these pep¬ tides are antimicrobial agents at micromolar concen¬ trations (5), and it was initially thought that their only biological activity was in non-oxidative, non- enzymatic, destruction of phagocytosed microorganisms. More recently, however, it has been shown that corti- costatins have potential regulatory functions, includ- " ing the ability to inhibit the action of the hormone adrenocorticotropin on glucocorticoid secretion (2,6,7) and to stimulate nifedipine-sensitive L-type Ca^+ channels in villus enterocytes (8). It has also been reported that a human defensin is a monocyte chemotactic agent (9). Other granulocyte-associated peptides have also been shown to have regulatory activities. For example, hemoregulatory peptide 1 is a granulocyte-associated thiol containing pentapep- tide, with potent inhibitory actions on myelopoieses (10). Several groups have reported the existence of immunomodulatory or cytokine-like activities associated with neutrophil extracts or supernatants (11,12,13,14). These activities include mast cell de- granulation, chemotaxis, and the inhibition of myelopoetic-colony formation. Despite these reports, and the evidence for regulatory actions associated with known granulocyte peptides, few systematic
SUBSTITUTESHEET attempts to characterize the regulatory molecules of the granulocyte seem to have been made. Granulocyte enriched extracts contain several cystine-rich components at levels approximately three orders of magnitude lower than the defensin/corticostatins. From their compositional analysis and chromatographic behaviour these peptides appear unrelated to any known hormone, including the definsin/corticostatins. Gran- ulocyte-derived peptides have potential both as immunoregulatory molecules, and in host resistance.
Granulins are novel candidate growth factors recently discovered in human and rat inflammatory leukocytes (22). Two rat granulin homologs, epithelin 1 and 2, occur in the kidney (23). Epithelin 1, which is probably identical to rat leukocyte granulin (22,23) exhibits activities similar to epidermal growth factor on epithelian cells .in vitro (23) . DISCLOSURE OF THE INVENTION
It is an object of this invention to provide novel granulins useful in wound healing.
In particular the invention relates to a family of novel leukocyte-associated peptides that are cystine-rich.
The peptides are approximately 6 Kda and may be cytokines.
In accordance with this invention there is provided Granulin A: DVKCDMEVSCPDGYTCCRLQSGAWGC- CPFTQAVCCEDHIHCCPAGFTCDTQKGTCE, SEQ ID NO: 1.
In accordance with another aspect of the invention there is provided Rat Granulin: EVKCDLEVSCPDGYTCCRLNTGA G(CCPFSB)AVCCEDHIHCCPAGFTCXTQ, SEQ ID NO: 5.
In accordance with still another aspect of the invention there is provided Granulin C: VPCDXVSSCPSSDTCCOLTSGEHGCCPIPEAVC, SEQ ID NO: 3.
SUBSTITUTESHEET In accordance with yet another aspect of the invention there is provided Granulin D: IGCDQXDTSSCCPDG, SEQ ID NO: 4.
In accordance with a still further aspect of the invention there is provided carp Granulin: VIHCDAATICPDGTICCLSPYGMBGQCCRDGIHCCRHGYHCDSRTTHC , SEQ ID NO: 6.
In accordance with another aspect of the invention there is provided Granulin B: VMCPDARSRCPDGHTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSK Cl, SEQ ID NO: 2.
In accordance with still another aspect of the invention there is provided Granulin E: Asp Val Glu Cys Gly Clu Gly His Phe Cys His Asp Asp Gin Thr Cys Cys Arg Asp Asn Arg Glu Gly Trp Ala Cys Cys Pro Try Ala Gin Gly Cla Cys Cys Ala Asp Arg Arg His Cys Cys Pro Ala Gly Phe Arg Cys Ala Arg Arg Gly Thr Lys Cys Leu, SEQ ID NO: 7.
In accordance with yet another aspect of the invention there is provided Granulin F: Ala lie Gin Cys Pro Asp Ser Gin Phe Glu Cys Pro Asp Phe Ser Thr Cys Cys Val Met Val Asp Gly Ser Trp Gly Cys Cys Pro Met Pro Gin Ala Ser Cys Cys Glu Asp Arg Val His Cys Cys Pro His Gly Ala Phe Cys Asp Leu Val His Thr Arg Cys Lie, SEQ ID NO: 8.
In accordance with still another aspect of the invention there is provided Granulin G: Gly Gly Pro Cys Gin Val Asp Ala His Cys Ser Ala Gly His Ser Cys lie Phe Thr Val Ser Gly Thr Ser Ser Cys Cys Pro Phe Pro Glu Ala Cys Gly Asp Gly His His Cys Cys Pro Arg Gly Phe His Cys Ser Ala Asp Gly Arg Ser Cys Phe, SEQ ID NO: 9.
In accordance with yet another aspect of the invention there is provided Paragranulin: Thr Arg Cys Pro Asp Gly Gin Phe Cys Pro Val Ala Cys Cys Leu Asp
SUBSTITUTE SHEET Pro Gly Gly Ala Ser Tyr Ser Cys Cys Arg Pro Leu Leu Asp, SEQ ID NO: 10.
In accordance with an embodiment of the invention there is provided a topical formulation comprising an effective amount of a Granulin of the invention in association with a pharmaceutically acceptable carrier for topical formulation.
In accordance with another aspect of the invention there is provided a method of healing wounds comprising applying to a wound site a Granulin of the invention.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 Figure IA shows the HPLC chromatogram. of a crude granule extract from inflammatory exudate cells, and B shows the chromatogram of a whole cell extract. The position of the granulins are marked by arrows. Note the absence in A of thymosin-/3-4, a cyto- plasmic marker peptide. The granule peptide markers HP-1 and HP-4 were identified as previously described (6), lysozyme was identified by a ino terminal sequence analysis (unpublished). Thymosin-β-4 and its oxidation product were identified by Fast Atom Bombardment mass spectrometry;
FIG.2 Size-exclusion purification of granulin A, (panel A), granulin B, (panel B), and granulins C and D, (panel C). Size markers were substance P, CLIP and ACTH1.-39. Apparent m.wts for native granulin A, 2700; granulin B, 3200; granulin C, 1700; and granulin D7.3900; FIG. 3 Purification of rat granulin from bone marrow; panel A shows the first HPLC chromatogram in ace onitrile/TFA, and B shows the second step of purification in
SUBSTITUTESHEET acetonitrile/HFBA. The bar in A corres¬ ponds to the region where the rat granulin elutes, and its elution position in B is marked with an arrow; FIG. 4A Shows the sequence of the probe used to screen a human bone marrow cDNA library in gt 11; FIG. 4B Shows a sequencing strategy; FIG. 4C Shows the complete nucleotide sequence and deduced polypeptide sequence of granulin;
FIG. 5 Shows a comparison of the granulin-like domains; FIG. 6 Is a Southern blot analysis of digested human DNA; FIG. 7A Is a Northern blot analysis of granulin precursor mRNA; FIG. 7B Is a Northern blot analysis of RNA from
A431, A549 and SKMES-1; and FIG. 7C Shows distribution of granulin mRNA. MODES FOR CARRYING OUT THE INVENTION MATERIALS AND METHODS Tissue Sources
Blood was taken from healthy volunteers, prepared and fractionated using Ficoll-Hypaque (Pharmacia, Upsalla, Sweden) as previously described
(6) . Differential counts were obtained from the'
Hematology Department, Royal Victoria Hospital,
Montreal, Canada. For structural studies, the first wash peritoneal exudate from patients with peritonitis was used as a source of leukocytes. Typically, this comprises from 75 to 95% neutrophils. Crude granule preparations were made by lysing the cells in Hank's buffered saline solution using a Cole Palmer
Ultrasonic Homogeniser 4710 Series. Preparations were inspected visually under a microscope to ensure complete cell lysis. The cellular debris was pelleted
SUBSTITUTE SHEET by centrifugation at 500 x g for 10 mins. , and the supernatant inspected to ensure the complete removal of broken cells. The supernatant was then pelleted by centrifugation for 20 minutes at 5000 x g, and the crude granule pellet washed twice in HBSS. The two human granule peptides, HP-1 and HP-4, and the granule enzyme lysozyme, were used as granule markers, and the cytoplasmic peptide thymosin-/3-4 was used as a marker for cytoplasmic contamination. Extraction and Purification
Whole cell preparations or crude granule fractions were extracted by sonication using an acidic high-salt extraction medium as described elsewhere
(15). The extract was then centrifuged at 2000 x g for 15 mins., and the pellets . re-extracted. Pooled supernatants were then adsorbed on SepPak Cig cartridges (Waters Associates, Milford, Mass.) and eluted in 5 to 10 ml 80% acetonitrile in 0.1% TFA and the eluate lyophilized. The SepPak eluate was fractionated by reversed phase HPLC using a Waters CigLiBondapak column (7.8mm x 30cm) eluted over a three hour period using a gradient of 0 to 80% acetonitrile in 0.1% TFA throughout at an elution rate of 1.5ml min"1. Aliquots of the eluted fractions were then screened by amino acid analysis. The fractions of interest were further purified by size-exclusion HPLC using two 1-125 ProteinPak columns (Waters) connected in series, eluted isocratically in 40% acetonitrile in 0.1% TFA at 1 ml min"1 (16). Partially purified peptides were then purified to homogeneity using a second C-18μBondapak HPLC column (3.9mm x 30cm), with a gradient of 10 to 40% acetonitrile in 0.1% TFA throughout at 1.5 ml min-1 for 90 mins. The rat peptide was purified from the aspirated femural bone marrow of 50 Sprague-Dawley rats (Charles Rivers, St.. Constant, Quebec), and extracted directly as outlined
SUBSTITUTE SHEET above. SepPak eluates were fractionated on a CigμBondapak column using a gradient of 4 to 48% acetonitrile in 0.1% TFA throughout over 1 hour at 1.5 ml min-1. Fractions were screened by amino acid 5 analysis, and granulin-like material further purified using the same column with a gradient of 20 to 40% acetonitrile in 0.13% HFBA throughout over one hour (15). Final purification was by size-exclusion HPLC as described above. 0 Amino Acid Analysis and Microsequencinq
For amino acid analysis aliquots of the peptide were lyophilized in borosilicate glass tubes and hydrolyzed in an evacuated reacti-vial for 16 hours at 105°C with 6N HCl. Amino acid analysis was 5 performed using a model 6300A Analyser (Beckman Instruments, Palo Alto, CA. ) . For microsequence analysis purified peptides were reduced with 10 mM dithiothreitol, or 2- mercaptoethanol, in 8M guanidine-HCl, 1 mM EDTA, 0.25M Tris, pH 8.5 for 1.5 0 hours at 37°C and then pyridylethylated with 2 μl 4- vinylpyridine (Aldrich Chemicals) under the same conditions.
The S-pyridylethylated peptides were then purified using a gradient of 5% to 60% acetonitrile
25 containing 0.1% TFA throughout over 60 minutes with an initial 40 minute isocratic stage at 5% acetonitrile to elute polymeric vinylpyridine side products. The derivatized peptides were then submitted directly to sequence analysis or further processed by enzymatic
30 digestion. The amino acid sequence determinations were carried out with an Applied Biosystem gas-phase sequenator (model 470A) as described in (17) but using a sequence program adapted from Speicher (18). The resulting phenylthiohydantoin (PTH)-amino acids were
35. analysed by reverse-phase HPLC on the on line PTff— analyser (Applied Biosystem model 120A) and/or a stand
SUBSTITUTE SHEET alone Varian HPLC unit as described previously (17). The PTH-yields for each standard were normalized according to a PTH-NorLeucine internal standard while the initial and repetitive yields were obtained by linear regression from the yields of selected stable PTH-derivatives. Sequence analysis of rat granulin and its fragments was undertaken at the McGill Peptide and Protein Sequencing facility located in the laboratory of Dr. Michael van der Rest at the Shriner's Hospital for Crippled Children in Montreal. Enzymatic Digestion
S-pyridylethylated peptides wer digested using trypsin (TPCK-treated, Sigma) chymotrypsin (Sigma), and S. aureus V8 protease (Sigma), at enzyme to substrate ratios of approximately 1 to 50 by weight. Digestions were performed at 37°C in 100 μl
50mM ammonium bicarbonate buffer, pH 8.3 for 3 hours, and terminated by the addition of 1 ml of 0.1% TFA.
The proteolytic fragments were then fractionated by rp-HPLC on a C-igμBondapak column using a gradient of
0 to 40% acetonitrile in 0.1% TFA throughout over 60 mins. at 1.5ml min-1. Fractions were collected, aliquots removed for amino acid analysis, and then stored frozen at -80°C. RESULTS
The HPLC profile of a typical extract of human inflammatory cells is shown in Figure 1. In addition to HP-1 and HP-4, several low abundance components are present. On the basis of amino acid composition analysis of these components three low abundance components that had unusually high levels of cystine were identified... These are labelled A, B and C/D, and were present in both whole cell extracts (FIG. IB) and crude granule preparations (FIG. IA) . Each of these extracts was then further purified using size-exclusion HPLC, revealing that the component C/D
SUBSTITUTE SHEET contained two peptides, one of which, D, eluted as a larger molecule than the other three. Each peptide was further purified on rp-HPLC. Their amino acid compositions are given in Table 1. The purified peptides were S-pyridylethylated, and amino terminal sequence analyses were performed, revealing that the four peptides were distinct but related molecules with no homology to any known protein. Because these peptides were associated with the granule fraction, we call them granulins A, B, C and D. Granulin D was run on reducing and non-reducing SDS-PAGE, and ran as a smaller molecule after reduction, indicating that it is probably a dimer. Only one peptide was recovered after S-pyridylethylation of granulin D . suggesting that it is a homodimer. However, until a full sequence is determined the possibility that it is a heterodimer of closely related subunits can not be excluded. The rat defensin/corticostatins elute in the same region of the chromatogram as the granulins (19). Rat granulin (marked with a bar in Fig. 3a) was purified to apparent homogeneity by a further rp-HPLC step using HFBA as the counterion (Fig. 3b) and subjected to structural analysis essentially as described for the human peptides. Granulin A is the most abundant of the human granulins, and was subject to a more detailed analysis. S-pyridylethylated peptide was digested with trypsin, chy otrypsin or S. aureus V8 protease. The fragments were isolated by HPLC, one fifth aliquots analysed by amino acid analysis, and appropriate fragments were then submitted to gas phase Edman microsequencing. Final recovery of the digestion products was between 150 and 300 picomoles. The overlap of the granulin A fragments is described in the legend to Table 2 together, with similar data for the rat granulin. The two sequences are highly
SUBSTITUTE SHEET conserved, as would be expected for regulatory molecules. Inflammatory exudates and bone marrow preparations are mixtures of cells, the exudates containing typically 70 to 95% granulocytes. When leukocytes from the blood of healthy donors was fractionated by density gradient centrifugation, granulins could be detected in the granulocyte pellet, but not in the interface where the mononuclear cells partition (data not shown). The isolation and characterization of a novel family of leukocyte associated cystine-rich peptides, which are called granulins has been described. The sequence of one human granulin, A, and identi ication of three other human granulins, B, C and D has been set out and a fifth granulin isolated from rat bone marrow is partially sequenced. The most striking feature of their primary sequence is the high content of oxidised cysteine, over 20%, suggesting that the secondary structure of the granulins are an essential determinant of their biological activity. This is supported by evidence of rigid evolutionary constraint on their structures; the rat partial sequence is almost identical with human granulin A.
When the sequences were entered into the National Biomedical Research Foundation PIR data bank no homologies were found with other proteins, indicating that the granulins are a novel polypeptide family. Subsequently, however, two amino terminal sequences were published, epithelin 1 and 2, isolated from the rat kidney, which are homologous with the granulins. The kidney peptides are putative cytokines that have growth inhibitory and stimulatory properties on some epithelial cells in. vitro (20). Rat granulin and the reported amino terminal sequence of epithelin 1 differ at only one residue; epithelin lacks the amino terminal glutamyl residue of rat granulin A. At
SUBSTITUTESHEET present it is not certain if both peptides are the product of the same, or different genes. It is also too early to determine whether the epithelins are intrinsic to renal cells, or if they are derived from blood borne cells trapped in the kidney. It is clear, however, that the two peptides from the kidney are members of a larger family, the granulins, and that a major source of the granulins, and probably also the epithelins, is from circulating leukocytes. Rat granulin was isolated from bone marrow, indicating that granulins are of myeloid origin. Whether granulins are also synthesized in circulating leukocytes remains to be determined. Granulins were extracted from granulocyte rich preparations, and are recoverable from the granulocyte pellet after Ficoll- Hypaque density gradient centrifugation. This suggests that their cellular origin may be the neutrophil, however, it is possible that other granulocytes such as eosinophils, or contaminating monocytes, contribute to the granulin content of these extracts. Extracts of human platelets contained no detectable granulins (data not shown). It is possible that each granulin belongs to a distinct cell type, or that sub-classes of the same cell-type contain different granulins. These are issues best answered using immunolocalization procedures. The granulins co-purify in a crude granule extract. Granulocytes have several different granule subclasses, including a true secretory compartment (21) that can be activated independently of phagocytosis. The availability of suitable immunoassay techniques will permit the unambiguous location .of the granulins to a cell type, and a subcellular compartment.
The granulins are a novel family of cystine- rich immunoinflammatory peptides. Their presence in circulating leukocytes and inflammatory exudates, and
SUBSTITUTE SHEET their structural similarity with the rat epithelins (20), indicates their use in inflammation, wound repair and tissue remodeling.
Thus the isolation and characterization of a novel class of leukocyte peptides with possible cytokine-like activities which are called granulins is described. They are cystine-rich with molecular weights of approximately 6Kda, except for granulin D, which appears to be a dimer. The sequence of one member of this family, a 56 residue peptide, granulin A, and amino-terminal sequences for three other granulins from human peripheral leukocytes are described. A fifth related peptide was isolated and partially sequenced from rat bone marrow, suggesting that at least some of the granulin in peripheral leukocytes is preformed in the marrow. Rat granulin, and human granulin A, are closely related, showing that the granulin structures are highly conserved between species. It has now been found that the precursor for the human granulins is a 593 residue glycoprotein, containing seven repeats of the 12-cys eine granulin domain. Gene expression is seen in the kidney, leukemic cell lines and fibroblasts, and very strongly in transformed epithelial cell lines, which both express the gene, and respond to the mature polypeptide.
Human leukocytes contain four granulin homologs, designated A, B, C and D. Their cellular distribution, and whether they are products of one or several genes under co-ordinate or independent control was unknown. To address these, and related issues, two oligonucleotide primers for PCR were synthesized corresponding to the amino terminal and mid-portion regions of granulin A (grnA). GrnA was chosen because it is the most abundant granulin in human leukocyte
SUBSTITUTE SHEET extractε, and the complete sequence of A, but only partial sequences of the other granulins, was available (22,23). Two PCR amplification products were obtained from human genomic DNA. One was unrelated to the granulins. The other fragment which represented part of the GrnA gene split by a 79 nucleotide intron (Fig. 4A) was 32p labelled and used to probe a human bone marrow cDNA library (Clontech). Positive clones were analyzed and the nucleotide sequence of the grnA precursor was then obtained as outlined in Fig. 4B.
The sequence (Fig. 4C) predicts a protein of 593 residues, with a probable signal peptide (24) extending to residue 17. It contains the 56 residue grnA sequence, as expected, and also six other 12- cysteine granulin-like domains, including B, C and D, hitherto known only from N-terminal sequences. The precursor contains two novel Cys-12 domains, E and F, and a degenerate granulin domain, G, with 10 cysteines. The positions of the cysteines are highly conserved (Fig. 5) as are certain other residues such as ASP39, His42 and Prθ4g. An eighth domain at the N- terminus, (paragranulin, Fig. 5) contains only 6 cysteines corresponding to the amino-terminal half of a granulin domain.
There are 5 potential N-glycosylation sites (Fig. 4C), including one at Asns of grnC. GnrC hydrolysates contain amino sugars and microsequencing gave a blank at residue 5 (N=2), consistent with glycosylation. The inter-domain sequences show little homology, except for a pro-ala dipeptide, found midway between each domain except F and G. Mono- or dibasic sequences, which are frequent sites of proteolysis in peptide processing (25), flank granulin domains C, D and E, but not A or B, both of which have been isolated as excised peptides. Thus nothing can be
SUBSTITUTE SHEET reliably inferred about post-translational cleavage mechanisms of the granulin precursor. There are no obvious trans-membrane sequences beyond the signal peptide (26). Southern blot analysis of human genomic DNA suggests that only one gene hybridizes with the granulin probe (Fig. 6). Significant interspecies sequence conservation exists, since DNA blots (Clontech) from human, monkey, rat, mouse, dog, bovine and rabbit, but not chicken, or yeast, hybridized with the granulin probe (data not shown). The blots probably underestimate species distribution since granulin-like peptides have been isolated from teleost hematopoietic tissue (H.P.J. Bennett, personal communication).
Four myelogenous leukemic cell lines of diverse linage, HL-60, U937, K562, KMOE, each expressed a granulin mRNA of approximately 2.3 Kb, as did murine 3T3 fibroblasts, but by far the strongest hybridization was detected in epithelial cell lines (Fig. 7A) . These cells produce two granulin mRNA species, at 2.3 and 2.5 Kb (Fig. 7B), the origin and significance of which is under investigation. The abundant expression of granulin mRNA in epithelial cell lines is particularly interesting, since epithelial cells, including A431 (23), respond to, and presumably have receptors for, epithelin 1 (and grnA). The majority of solid tumors are epithelial in origin, thus the propensity of transformed epithelial cells to express granulin mRNA, and respond to the mature peptide, may have important pathophysiological consequences. In tissues, very strong hybridization was obtained with rabbit kidney RNA (Fig. 7C) . This resembles EGF gene expression, which is a hundred times higher in the kidney than any other organ, except the submaxillary gland in rodents (27).
SUBSTITUTE SHEET The widespread expression of the granulin gene in cells of diverse lineage; the effects of some of the granulins/epithelins on cell proliferation (23), and the ' high degree of sequence conservation noted earlier (22), and corroborated here by interspecies DNA hybridization, cumulatively support a fundamental regulatory role for granulins/epithelins. The presence of all four known granulins, plus three novel granulin-like sequences in a common precursor, was highly unexpected. The iterative nature of pro- granulin draws unavoidable comparisons with the EGF precursor (28,29). Both the pro-granulin and EFT- precursor (27) mRNAs are very abundant in the kidney. Biologically, EGF and granulin/epithelins have similar actions on epithelial cells (23), but probably different receptors (23). Whether the similarities are significant, or merely coincidental, is unclear. Unlike the EGF-precursor, pro-granulin has no transmembrane segment or cytoplasmic domain (28,29) implying different post-translational pathways for the two proteins. Multiple sequential repeats of cysteine-rich domains is a recurrent structural motif among regulatory proteins (28,29,30,31), wherein typically, one exon delineates one cysteine-rich domain (30,32,33). In contrast, the PCR amplified fragment of grnA is bisected by an intron (Fig. 4A), and preliminary results show that introns bisect domains G, F, B, C and D.
In each case the cysteines align with an approximately mirror-image symmetry around the bisecting intron. Genomically, therefore, pro- granulin bears little resemblance to the EGF- precursor.
Proerythroid leukemic cells express granulin mRNA (Fig. 7A) , but seem unlikely to be involved in regulating epithelial proliferation. Similarly, the
SUBSTITUTE SHEET strong expression of granulin in RNA in kidney, but not other tissues, may imply functions unrelated to epithelial cell mitogenesis. Thus granulins may have multiple biological activities; indeed distinct activities may be associated with different domains, as already proposed for epithelins 1 and 2 (23) . The availability of cloned granulins permit these questions to be addressed.
Fig. 4. (A) shows the sequence of the probe used to screen a human bone marrow cDNA library in gtll (Clontech, Palo Alto, CA. ) . The probe was generated by PCR of human DNA, with forward and reverse primers (underlined) based on the amino acid sequence of granulin A. The nucleotides in lower case represent an intron in the coding region of granulin A. Fig. 4B shows the sequencing strategy. The nucleotide sequence is a composite of three sequences HBM12, HBM3 and HNM4. Restriction sites for EcoRI, Kpnl and SacI are shown; sequence was obtained using M13 or T3 primers (f,g,l,n,o) or custom-synthesized 17-mer sequence specific primers (a,b,c,d,e,h,i,j,k,m,p) . Arrows indicate the direction of sequencing and overlap. Fig. 4C shows the complete nucleotide sequence and deduced polypeptide sequence of granulin. The nucleotides are numbered from the initiator codon (ATG), and the amino acids from the probable signal peptide cleavage site. Underlined sequences correspond to sequences previously determined by gas-phase microsequencing of purified granulins. Possibly N-glycosylation sites are indicated by an asterisk (*) , and the stop codon is shown by a #. The boxed nucleotides corresponds to a polyadenylation signal. Clone HBM12 starts at nucleotide 6. The sequences of clones HBM3 and HBM4 differ in the 5'-untranslated region as indicated by the bifurcation in the nucleotide sequence.
SUBSTITUTESHEET METHODS. To generate the grnA probe, degenerate oligonucleotide primers (forward primer, 5'- CGATGTGAAGTG(T/C)GA(T/C)ATGGA-3 ' ; reverse primer, 5'- CTGGCATGTGGTT(T/C)TC(A/G)CA(G/A)CA-3 ' ) were synthesized (Sheldon Biotechnology Centre, McGill University) and used in the polymerase chain reaction (PCR) with 2μg of genomic DNA as template (34). The amplified product was subcloned into the plasmid vector Bluescript KSII+ and its identity confirmed by double stranded dideoxynucleotide sequencing with Sequenase (USB). This fragment was labeled with 32P by nick-translation (Boehringer Mannheim) and used to probe a human bone marrow cDNA library in λgtll (Clσntech, Palo Alto, CA. ) consisting of 1.51 X106 independent clones. Duplicate nitrocellulose filters (Schleicher and Schuell) were prehybridized in 5X SSC, 5X Denhardt's reagent, 0.2% SDS at 37°C for 5 hours. Hybridization was in 5X SSC, 2.5X Denhardt's reagent, 0.2% SDS, 50% formamide, 10% polyethylene glycol with 1x10? cpm probe at 37°C for 12 hours. Filters were washed twice for 45 minutes in 2X SSC, 0.1% SDS at 58°C and exposed at -70°C with Kodak X-Omat film with an intensifying screen. 16 positives were obtain from 3x10^ clones screened. The cDNA insert from clone HMB12 was digested with Kpnl and Sad, and the resulting cDNA fragments subcloned into Bluescript KSII+. Nucleotide sequence was determined by double stranded dideoxy sequencing using Sequenase (USB). The sequence of HBM12 lacked an initiator methionine. The remaining positive clones were analysed using PCR with λgtll sequence specific primers in combination with a primer, cll2rp, corresponding to nucleotides 45 to 61 of clone HBM12. Only two clones, HBM3 and HBM4 contained inserts longer than HBM12. Their 5' sequence was obtained by dideoxy sequencing of single stranded templates generated by asymmetric PCR (35)
SUBSTITUTESHEET using a forward primer specific for λgtll and primer cll2rp as the reverse primer. Only the 5'-ends of HBM3 and HBM4 were sequenced. The probable identity with HBM12 was established by PCR mapping using primer pairs b,d; e,k; i,k; h,grnAr; grnBf,grnAr; b,grnFr. grnAr, grnBf and grnFr are primers specific for granulins A, B and F and were not used for sequencing.
Fig.5. Comparison of the granulin-like domains. Residues occuring three or more times are boxed, and dashes have been introduced to align the cysteines. The domain boundaries were determined by gas-phase microsequencing and by comparison with the sequence of grnA. The domains are located at amin^ acids, 264-319, grnA; 169-244, grnB; 347-400, 425-479, grnD; 501-506, grnE; 106-162, grnF; 4i grnG and 1-27, paragranulin. The order of domains is: paragranulin-G-F-B-A-C-D-E. They are not alphabetically aligned because several domains were isolated and named as discrete proteins before their common origin was known.
Fig. 6. Southern blot analysis of human
DNA, digester with Bσlll. Pstl. BamHI, Hindlll, and
EcoRI. 5ug per lane (Clontech, Palo Alto, CA. ) , probed with a 32P nick-translated 1890 bp EcoRi/SacI fragment from clone HBM12. Hybridization was in 0.5M NaH2P0 , pH 7.2, 7% SDS, lmM EDTA at 65°C for 20 hours. The blot was washed twice at 65°C, 30 minutes each, in 40 mM NaH2Pθ4, pH 7.2, 5% SDS, lmM EDTA and 40mM NaH2Pθ4, pH 7.2, 1% SDS, lmM EDTA. The autoradiogram was obtained by exposing the blot to Kodak X-Omat film with intensifying screen at -70°C for 5 hours.
Fig. 7. Northern blot analysis of: A. granulin precursor mRNA in the following cell lines,
HL-60 (promycelocytic leukemia), U937 (histocytic leukemia), K562 and KMOE (proerythroid leukerαias) ,
A431 (epidermoid carcinoma), A54 (lung epithelial
SUBSTITUTESHEET carcinoma), SKMES-1 (lung squamous carinoma), CHO-KI
(Chinese hamster ovaries), and murine Balb/C 3T3 fibroblasts, exposed for 10 days. (B). RNA from A431,
A549 and SKMES-1, exposed for 20 hours. (C). Distribution of granulin mRNA in rabbit tissues; heart, liver, spleen, kidney, bone marrow, exposed for
4 days.
METHODS. Total cellular RNA was isolated from cell lines and rabbit tissues by the acid guanidinium thiocyanate-phenol-chloroform extraction method (15),
30μg (cell lines) or 50μg (rabbit tissues) RNA was denatured with glyoxal, electrophoresed on a 1.1% agarose gel in 10 mM NaH2Pθ4, pH 6.8, transferred to nylon membranes (ZetaProbe, BioRad) by capillary blotting with lOmM NaOH and fixed by baking at 80°C for 2 hours. The membranes were hybridized at 65°C for RNA extracted from cell lines or 60°C for rabbit tissue RNA in 0.5M NaH2Pθ4, pH 6.8, 7% SDS, lmM EDTA for 24 hours with the same probe described in Figure 6. The membranes were washed at hybridization temperature for 30 mins. in 40 mM NaH2Pθ , pH 6.8, 5%
SDS, lmM EDTA and 30 minutes in 40mM NaH2P04, pH 6.8,
1% SDS, lmM EDTA. Autoradiograms were obtained by exposing the blots to Kodak X-Omat film with intensifying screens at -70°C. REFERENCES
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Following the procedures described herein before carp Granulin:
VIHCDAATICPDGTICCLSPYGMBGQCCRDGIHCCRHGYHCDSRTTHCL was isolated and characterized. It was found to be homologous with rat and human granulins but not identical.
The carp Granulin causes an increase in ^E- thymidine incorporation in a keratinocyte cell line which indicates increased cell growth in keratinocytes and thus wound healing.
The above described Granulin A causes a fall in 3n-t,hymidine incorporation in A-431 cells, a human epidermal carcinoma cell line. The human granulins of the invention retard growth of A-431 cells. Pharmaceutical Compositions
The granulins of the invention are suitably employed as the active ingredient in pharmaceutical compositions for topical application. Suitably the granulins may be incorporated in a topical formulation in an amount of 5 to 50μg/ml of
SUBSTITUTE SHEET ointment and are applied to a wound site in an amount of 5 to 50 μg/cm2 of wound site. A. preferred treatment amount of the granulins is about lOμg/cm2 of wound site suitably applied three times a day.
The topical pharmaceutical composition may be in the form of a cream, ointment, gel, lotion or other formulation suitable for application to the skin, and thus comprise a granulin of the invention admixed with an acceptable carrier for topical application.
SUBSTITUTESHEET TABLE 1: The amino acid compositions of purified granulins. The recovery of cysteine in this system is variable and is between 65 to 80%. Values have not been corrected for background contamination or oxidation. Tryptophan was not determine. Predicted values for granulin A from the gas-phase sequence determinations are given in brackets.
Amino Granulin Granulin Granulin Granulin Rat Acid A B C D Granulin
Figure imgf000026_0001
SUBSTITUTE SHEET TABLE 2: Structural analyses of five members of the granulin family. The proposed structure for granulin A was determined by overlapping two amino terminal sequences, (1-11), and (1-23); tryptic fragments (4- 18), (19-52), and V8 protease fragment (37-56), and chymotryptic fragment (47-56). The proposed structure of rat granulin is based partly upon the direct sequencing of the peptide itself (i.e., the sequence 1 through 21) and its proteolytic fragments (i.e., fragments corresponding to 4 to 18, 19 to 31, 32 to 46, and 32 to 51 sequences). Alignment of the fragments is based upon the clear homology of rat granulin with human granulin A. The sequence of residues 26 to 31 (in parenthesis) could not be determined and the sequence shown is based upon the amino acid composition of fragment 19 to 31. This fragment is a major tryptic clevage product. No evidence of arginine was found, however, it is possible that the fragment contains a lysine residue which co-elutes with pyridylethyl-cysteine in the amino acid analysis system. B signifies either aspartic acid or asparagine, and X signifies an unassigned residue. Granulin A: DVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDH-
IHCCPAGFTCDTQKGTCE. Rat Granulin: EVKCDLEVSCPDGYTCCRLNTGAWG(CCPFSB)AVCCE-
DHIHCCPAGFTCXTQ. Granulin B: VMCPDARSRCPDGHTCCELPSGKYGCCPMPNATCCSDH- LHCCPQDTVCDLIQSKCI.
Granulin C: VPCDXVSSCPSSDTCCOLTSGEHGCCPIPEAVC. Granulin D: IGCDQXDTSSCCPDG.
Abbreviations. TFA, trifluoroacetic acid: HFBA, heptafluorobutyric acid; rp-HPLC reverses phase high performance liquid chromatography; PTH, phenylthiohydantoin.
SUBSTITUTE SHEET GENERAL INFORMATION
(i)APPLICANT: SOLOMON, SAMUEL (ii)TITLE OF INVENTION: GRANULINS FROM LEUKOCYTES (iii) UMBER OF SEQUENCES: Six (6) (iv)CORRESPONDENCE ADDRESS:
(A) Dr. Samuel Solomon, Ph.D., FRIC Royal Victorial Hospital
(B) STREET: 687 Pine Avenue West
L2.05 (C) CITY: Montreal
(D) STATE: Quebec
(E) COUNTRY: Canada
(F) ZIP: H3A 1A1
(v)COMPUTER READABLE FORM: (A) Medium Type: Diskette 3.50 Inch 1.44 Mb
(B) COMPUTER: Olivetti M300-386 SX
(C) OPERATING SYSTEM: MS DOS
(D) SOFTWARE: ASCII Format (Word for Windows) (vi)CURRENT APPLICATION DATA: (A) APPLICATION NUMBER - U.S. Patent
Application, Ser. No. Unknown
(B) FILING DATE: 3 Feb. 1992
(C) CLASSIFICATION: Unknown (vii)PRIOR APPLICATION DATA: (A) APPLICATION NUMBER - U.S. Patent
Application Ser. No. 07/627,490
(B) FILING DATE: 14 Dec. 1990
(C) CLASSIFICATION: 154
SUBSTITUTE SHEET SEQ ID NO:l
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 AMINO ACIDS
(B) TYPE: AMINO ACID (C) STRANDEDNESS: NOT APPLICABLE
(D) TOPOLOGY: LINEAR (ii)MOLECULAR TYPE: PEPTIDE (vi)ORIGINAL SOURCE: HUMAN NEUTROPHILS
Asp Val Lys Cys Asp Met Glu Val Ser Cys Pro Asp Gly
5 10
Tyr Thr Cys Cys Arg Leu Gin Ser Gly Ala Trp Cys Cys 15 20 25
Pro Phe Thr Gin Ala Val Cys Cys Glu Asp His lie His 30 35
Cys Cys Pro Ala Gly Phe Thr Cys Asp Thr Gin Lys Gly 40 45 so
Thr Cys Glu 55
SUBSTITUTE SHEET SEQ ID NO: 2
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 AMINO ACIDS
(B) TYPE: AMINO ACID (C) STRANDEDNESS: NOT APPLICABLE
(D) TOPOLOGY: LINEAR (ii) OLECULE TYPE: PEPTIDE (vi)ORIGINAL SOURCE: HUMAN NEUTROPHILS .
Val Met Cys Pro Asp Ala Arg Ser Arg Cys Pro Asp
5 10
Gly Ser Thr Cys Cys Glu Leu Pro Ser Gly Lys Tyr Gly 15 20 25
Cys Cys Pro Met Pro Asn Ala Thr Cys Cys Ser Asp His
30 35
Leu His Cys Cys Pro Gin. Asp Thr Val Cys Asp Leu lie 40 45 50
Gin Ser Lys Cys Leu 55
SUBSTITUTE SHEET SEQ ID NO : 3
(i)SEQUENCE CHARACTERISTICS:
(A) SEQUENCE LENGTH: 54 AMINO ACIDS
(B) TYPE: AMINO ACID (C) STRANDEDNESS: NOT APPLICABLE
(D) TOPOLOGY: LINEAR (ii) OLECULAR TYPE: PEPTIDE (iv)ORIGINAL SOURCE: HUMAN NEUTROPHILS
Val Pro Cys Asp Asn Val Ser Ser Cys Pro Ser
5 10
Ser Asp Thr Cys Cys Gin Leu Thr Ser Gly Glu Trp 15 20
Gly Cys Cys Pro lie Pro Glu Ala Val Cys Cys Ser 25 30 35 Asp His Gin His Cys Cys Pro Gin Arg Tyr Thr Cys
40 45
Val Ala Glu Gly Gin Cys Gin 50
SUBSTITUTESHEET SEQ ID NO: 4
(i) SEQUENCE CHARACTERISTICS:
(A) SEQUENCE LENGTH: 55 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NOT APPLICABLE
(D) TOPOLOGY: LINEAR (ii)MOLECULE TYPE: PEPTIDE
(vi) ORIGINAL SOURCE: HUMAN NEUTROPHILS
lie Gly Cys Asp Gin His Thr Ser Cys Pro Val
5 10
Gly Gly Thr Cys Cys Pro Ser Gin Gly Gly Ser Trp 15 20
Ala Cys Cys Gin Leu Pro His Ala Val Cys Cys Glu 25 30 35 Asp Arg Gin His Cys Cys Pro Ala Gly Tyr Thr Cys
40 45
Asn Val Lys Ala Arg Ser Cys Glu 50 55
SUBSTITUTESHEET SEQ ID NO: 5
(i) SEQUENCE CHARACTERISTICS
(A) SEQUENCE LENGTH: 51 AMINO ACIDS
(B) TYPE: AMINO ACID (C) STRANDEDNESS: NOT APPLICABLE
(D) TOPOLOGY: LINEAR (ii) OLECULAR TYPE: PEPTIDE (vi)ORIGINAL SOURCE: RAT BONE MARROW
Glu Val Lys Cys Asp Leu Glu Val Ser Cys Pro Asp
5 10
Gly Tyr Thr Cys Cys Arg Leu Asn Thr Gly Ala Trp 15 20
Gly Cys Cys Pro Phe Ser Asp Ala Val Cys Cys Glu 25 30 35
Asp His lie His Cys Cys Pro Ala Gly Phe Thr Cys 40 45 Thr Gin
50
The exact identity of position 31 is not yet determined; it is either Asp or Asn. The identity of position 49 is not known.
SUBSTITUTE SHEET SEQ ID NO: 6
(i) SEQUENCE CHARACTERISTICS:
(A) SEQUENCE LENGTH: 49 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NOT APPLICABLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULAR TYPE: PEPTIDE
(vi) ORIGINAL SOURCE: CARP SPLEEN
Val Iso His Cys Asp Ala Ala Thr lie Cys Pro Asp
5 10
Gly Thr lie Cys Cys Leu Ser Pro Tyr Gly Asp Met 15 20
Gly Gin Cys Cys Arg Asp Gly lie His Cys Cys Arg 25 30 35 His Gly Tyr His Cys Asp Ser Arg Thr Thr His Cys
40 45
Leu
The exact identity of position 23 is not yet determine; it is either Asp or Asn.
SUBSTITUTE SHEET SEQ ID NO: 7
(i)SEQUENCE CHARACTERISTICS:
(A) SEQUENCE LENGTH: 56 AMINO ACIDS
(B) TYPE: AMINO ACID (C) STRANDEDNESS: NOT APPLICABLE
(D) TOPOLOGY: LINEAR (ii)MOLECULAR TYPE: PEPTIDE (v)ORIGINAL SOURCE: HUMAN NEUTROPHILS
Asp Val Glu Vys Gly Glu Gly His Phe Cys His. Asp Asp
5 10
Gin Thr Cys Cys Arg Asp Asn Arg Glu Gly Trp Ala Cys 15 20 25
Cys Pro Tyr Ala Gin Gly Val Cys Cys Ala Asp Arg Arg 30 35 His Cys Cys Pro Ala Gly Phe Arg Cys Ala Arg Arg Gly 40 45 50
Thr Lys Cys Leu 55
SUBSTITUTE SHEET SEQ ID NO: 8
(i)SEQUENCE CHARACTERISTICS:
(A) SEQUENCE LENGTH: 57 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NOT APPLICABLE
(D) TOPOLOGY: LINEAR
(ii)MOLECULAR TYPE: PEPTIDE
(vi)ORIGINAL SOURCE: HUMAN NEUTROPHILS
Ala lie Gin Cys Pro Asp Ser Gin Phe Glu Cys Pro Asp
5 10
Phe Ser Thr Cys Cys Val Met Val Asp Gly Ser Trp Gly 15 20 25
Cys Cys Pro Met Pro Gin Ala Ser Cys Cys Glu Asp Arg 30 35 Val His Cys Cys Pro' His Gly Ala Phe Cys Asp Leu Val 40 45 50
His Thr Arg Cys lie 55
SUBSTITUTE SHEET SEQ ID NO: 9
(i) SEQUENCE CHARACTERISTICS:
(A) SEQUENCE LENGTH: 57 AMINO ACIDS
(B) TYPE: AMINP ACID (C) STRANDEDNESS: NOT APPLICABLE
(D) TOPOLOGY: LINEAR (ii)MOLECULE TYPE: PEPTIDE (vi) ORIGINAL SOURCE: HUMAN NEUTROPHILS
Gly Gly Pro Cys Gin Val Asp Ala His Cys Ser Ala Gly
5 10
His Ser Cys lie Phe Thr Val Ser Gly Thr Ser Ser Cys 15 20 . 25
Cys Pro Phe Pro Glu Ala Cys Gly Asp Gly His His Cys 30 35 Cys Pro Arg Gly Phe His Cys Ser Ala Asp Gly Arg Ser 40 45 50
Cys Phe
SUBSTITUTE SHEET SEQ ID NO: 10
(i) SEQUENCE CHARACTERISTICS:
(A) SEQUENCE LENGTH: 30 AMINO ACIDS
(B) TYPE: AMINO ACID (C) STRANDEDNESS: NOT APPLICABLE
(D) TOPOLOGY: LINEAR (ii)MOLECULAR TYPE: PEPTIDE (vi)ORIGINAL SOURCE: HUMAN NEUTROPHILS
Thr Arg Cys Pro Asp Gly Gin Phe Cys Pro Val Ala Cys
5 10
Cys Leu Asp Pro Gly Gly Ala Ser Tyr Ser Cys Cys Arg 15 20 25
Pro Leu Leu Asp 30
SUBSTITUTE SHEET

Claims

1. Novel cystine-rich leukocyte granulins.
2. A granulin of claim 1, which is Granulin A: DVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGT CE, SEQ ID NO: 1.
3. A granulin of claim 1, which is Granulin B: VMCPDARSRCPDGGTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSK CL, SEQ ID NO: 2.
4. A granulin of claim 1, which is Granulin C: VPCDXVSSCPSSDTCCQLTSGEHGCCPIPEAVC, SEQ ID NO: 3.
5. A granulin of claim 1, which is Granulin D: IGCDQXDTSSCCPDG, SEQ ID NO: 4.
6. A granulin of claim 1, which is Rat Granulin: EVKCDLEVSCPDGYTCCRLNTGAWG(CCPFSB)AVCCEDHIHCCPAGFTCXTQ, SEQ ID NO: 5.
7. A granulin of claim 1, which is Carp Granulin: VIHCDAATICPDGTICCLSPYGBMGQCCRDGIHCCRHGYHCDSRTTHCL, SEQ ID NO: 6.
8. A granulin of claim 1, which is Granulin E: Asp Val Glu Cys Gly Glu Gly His Phe Cys His Asp Asp Gin Thr Cys Cys Arg Asp Asn Arg Glu Gly Trp Ala Cys Cys Pro Tyr Ala Gin Gly Val Cys Cys Ala Asp Arg Arg His Cys Cys Pro Ala Gly Phe Arg Cys Ala Arg Arg Gly Thr Lys Cys Leu, SEQ ID NO: 7.
9. A granulin of claim 1, which is Granulin F: Ala lie Gin Cys Pro Asp Ser Gin Phe Glu Cys Pro Asp Phe Ser Thr Cys Cys Val Met Val Asp Gly Ser Trp Gly Cys Cys Pro Met Pro Gin Ala Ser Cys Cys Glu Asp. Arg
SUBSTITUTE SHEET Val His Cys Cys Pro His Gly Ala Phe Cys Asp Leu Val His Thr Arg Cys lie, SEQ ID NO: 8.
10. A granulin of claim 1, which is Granulin G: Gly Gly Pro Cys Gin Val Asp Ala His Cys Ser Ala Gly His Ser Cys lie Phe Thr Val Ser Gly Thr Ser Ser Cys Cys Pro Phe Pro Glu Ala Cys Gly Asp Gly His His Cys Cys Pro Arg Gly Phe His Cys Ser Ala Asp Gly Arg Ser Cys Phe, SEQ ID NO: 9.
11. A granulin of claim 1, which is Paragranulin: Thr Arg Cys Pro Asp Gly Gin Phe Cys Pro Val Ala Cys Cys Leu Asp Pro Gly Gly Ala Ser Tyr Ser Cys Cys Arg Pro Leu Leu Asp, SEQ ID NO: 10.
12. A topical formulation containing a granulin of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, in association with a pharmaceutically acceptable carrier for topical application.
13. A topical formulation comprising an effective keratinocytic amount of the granulin of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, in association with a pharmaceutically acceptable carrier for topical application.
14. A granulin of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, for use in healing a wound site.
SUBSTITUTESHEET
PCT/CA1992/000089 1992-02-03 1992-02-28 Granulins from leukocytes WO1993015195A1 (en)

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US6720159B1 (en) * 1997-12-16 2004-04-13 A&G Pharmaceutical, Inc. 88KDA tumorigenic growth factor and antagonists
US6872703B2 (en) 2000-06-07 2005-03-29 Ajinomoto Co., Inc. Insulin receptor-related receptor binding protein
US7091047B2 (en) 1997-05-23 2006-08-15 A&G Pharmaceutical, Inc. Methods and kits for diagnosing tumorigenicity
WO2009010045A1 (en) * 2007-07-16 2009-01-22 Johann Wolfgang Goethe-Universität Frankfurt am Main Use of a granulin or a granulin-like compound in the therapy or prophylaxis of chronic pains
US7651854B2 (en) 2003-02-26 2010-01-26 A & G Pharmaceutical, Inc. Methods for increasing the proliferation of B cells
US20100111928A1 (en) * 1997-05-23 2010-05-06 A & G Pharmaceutical, Inc. Methods and kits for diagnosis tumorgenicity
US7815906B2 (en) 2003-08-01 2010-10-19 A & G Pharmaceutical, Inc. Compositions and methods for restoring sensitivity to treatment with HER2 antagonists
US8088373B2 (en) 2002-11-19 2012-01-03 A&G Pharmaceutical, Inc. Autocrine growth factor receptor antibodies and methods
US8911950B2 (en) 1997-05-23 2014-12-16 A&G Pharmaceutical, Inc. Methods and compositions for inhibiting the growth of hematopoietic malignant cells
JP2014238343A (en) * 2013-06-07 2014-12-18 株式会社島津製作所 Quantitative analysis method of progranulin and granulin peptide using mass spectroscope, and analysis program
US8999365B2 (en) 2005-02-01 2015-04-07 Sinclair Pharmaceuticals Limited Prevention of bacterial contamination
WO2018039748A1 (en) 2016-09-02 2018-03-08 James Cook University Wound healing peptide
CN110903377A (en) * 2019-11-08 2020-03-24 上海交通大学 A kind of biologically active polypeptide IGCDQHTSCPVGQTCPS and its preparation method and application

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US7091047B2 (en) 1997-05-23 2006-08-15 A&G Pharmaceutical, Inc. Methods and kits for diagnosing tumorigenicity
US20100111928A1 (en) * 1997-05-23 2010-05-06 A & G Pharmaceutical, Inc. Methods and kits for diagnosis tumorgenicity
US8911950B2 (en) 1997-05-23 2014-12-16 A&G Pharmaceutical, Inc. Methods and compositions for inhibiting the growth of hematopoietic malignant cells
US6720159B1 (en) * 1997-12-16 2004-04-13 A&G Pharmaceutical, Inc. 88KDA tumorigenic growth factor and antagonists
US6872703B2 (en) 2000-06-07 2005-03-29 Ajinomoto Co., Inc. Insulin receptor-related receptor binding protein
US8088373B2 (en) 2002-11-19 2012-01-03 A&G Pharmaceutical, Inc. Autocrine growth factor receptor antibodies and methods
US7651854B2 (en) 2003-02-26 2010-01-26 A & G Pharmaceutical, Inc. Methods for increasing the proliferation of B cells
US7815906B2 (en) 2003-08-01 2010-10-19 A & G Pharmaceutical, Inc. Compositions and methods for restoring sensitivity to treatment with HER2 antagonists
US8999365B2 (en) 2005-02-01 2015-04-07 Sinclair Pharmaceuticals Limited Prevention of bacterial contamination
DE102007033359A1 (en) * 2007-07-16 2009-01-29 Johann Wolfgang Goethe-Universität Frankfurt am Main Use of a granule or a granulin-like compound for the therapy or prophylaxis of chronic pain
DE102007033359B4 (en) * 2007-07-16 2010-12-02 Johann Wolfgang Goethe-Universität Frankfurt am Main Use of a granule or a granulin-like compound for the therapy or prophylaxis of chronic pain
US8367616B2 (en) 2007-07-16 2013-02-05 Johann Wolfgang Goethe-Universitat Frankfurt Am Main Use of a granulin or a granulin-like compound in the therapy or prophylaxis of chronic pains
JP2010533660A (en) * 2007-07-16 2010-10-28 ヨハン ウォルフガング ゲーテ−ウニベルジテート フランクフルト アム マイン Use of granulin or granulin-like compounds for the treatment or prevention of chronic pain
WO2009010045A1 (en) * 2007-07-16 2009-01-22 Johann Wolfgang Goethe-Universität Frankfurt am Main Use of a granulin or a granulin-like compound in the therapy or prophylaxis of chronic pains
US9655947B2 (en) 2007-07-16 2017-05-23 Johann Wolfgang Goethe-Universitat Frankfurt Am Main Use of a granulin or a granulin-like compound for the therapy or prophylaxis of chronic pain
JP2014238343A (en) * 2013-06-07 2014-12-18 株式会社島津製作所 Quantitative analysis method of progranulin and granulin peptide using mass spectroscope, and analysis program
WO2018039748A1 (en) 2016-09-02 2018-03-08 James Cook University Wound healing peptide
EP3507300A4 (en) * 2016-09-02 2020-04-08 James Cook University Wounds healing peptide
CN110903377A (en) * 2019-11-08 2020-03-24 上海交通大学 A kind of biologically active polypeptide IGCDQHTSCPVGQTCPS and its preparation method and application

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