[go: up one dir, main page]

WO2007142381A1 - Peptide-1 hélicoïdal court humain qui présente une activité antimicrobienne, antitumorale et immune stimulante et ses utilisations - Google Patents

Peptide-1 hélicoïdal court humain qui présente une activité antimicrobienne, antitumorale et immune stimulante et ses utilisations Download PDF

Info

Publication number
WO2007142381A1
WO2007142381A1 PCT/KR2006/002936 KR2006002936W WO2007142381A1 WO 2007142381 A1 WO2007142381 A1 WO 2007142381A1 KR 2006002936 W KR2006002936 W KR 2006002936W WO 2007142381 A1 WO2007142381 A1 WO 2007142381A1
Authority
WO
WIPO (PCT)
Prior art keywords
hshp
peptide
antimicrobial
cells
antitumor
Prior art date
Application number
PCT/KR2006/002936
Other languages
English (en)
Inventor
Yeon Sook Kim
Suk Keun Lee
Sang Chol Park
Je Geun Chi
Soo Il Chung
Original Assignee
Seoul National University Industry Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Seoul National University Industry Foundation filed Critical Seoul National University Industry Foundation
Publication of WO2007142381A1 publication Critical patent/WO2007142381A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a human short helical peptide-1 (referred as 'hSHP-1' hereinafter) which has antimicrobial, antitumor and immune stimulating activity.
  • 'hSHP-1' human short helical peptide-1
  • defensin As a human antimicrobial protein, defensin, cathelicidin (LL-37), dermicidin, histatin and lysozyme have been identified but their antimicrobial activities were proved to be very low, compared with magainin separated from a toad.
  • antimicrobial proteins In variety of antimicrobial proteins have been found in various animals including a frog, a pig, a snake, a crocodile, a scorpion and a fly, etc, which cannot be applied to human, though, since they cause immune response right after being introduced. Thus, it is required to establish techniques to prepare an antimicrobial peptide having a strong antimicrobial activity with less immunogenicity based on genetic recombinant technology.
  • the present inventors have searched a human specific antimicrobial peptide and separated hSHP-1 as a result. And the present inventors finally completed this invention by confirming that the hSHP-1 or its recombinant peptides have antimicrobial, antitumor and immune stimulating activity.
  • the present invention provides a peptide having antimicrobial, antitumor and immune stimulating activity which is selected from the following group consisting of:
  • N-terminal is selected from between the 12 th - the 31 st amino acid residue of the sequence represented by SEQ. ID. NO: 2
  • C-terminal of the peptide is either the 45 th or the 46 th amino acid residue of the sequence represented by SEQ. ID. NO: 2
  • the peptide is selected from peptides with more than 16 mer
  • the peptide is preferably one of peptides having sequences represented by SEQ. ID. NO: 3 - NO: 8 or their tandem repeats and the peptide can be glycosylated.
  • the preferable peptide in the present invention includes
  • peptide of the invention can further include those peptides having similar sequences but the same functions as the above sequences.
  • the present invention provides a polynucleotide encoding the peptide.
  • the present invention provides a pharmaceutical composition for the treatment of infectious diseases developed in oral cavity, eyeball, hair, skin, nasal cavity, gastrointestinal track, peritoneum, pleura, vagina, prostate, urinary bladder, urinary track, joint capsule, pituitary membrane, outer ear, inner ear, bone marrow or other internal organs, comprising the peptide or the full length hSHP-1 peptide or a vector harboring a nucleic acid sequence encoding the peptide as an effective ingredient.
  • the pathogen of the infectious disease is Gram negative or Gram positive bacteria, fungi, or encapsulated virus. It is more preferred that the pathogen of the infectious disease is methicillin resistant bacteria.
  • the present invention provides a pharmaceutical composition for the prevention and treatment of precancerous lesion or cancer comprising the peptide as an effective ingredient.
  • the cancer is selected from the group consisting of skin cancer, osteosarcoma, oral cancer, stomach cancer, colon cancer, brain cancer and hepatoma.
  • the present invention provides a composition for stimulating immune system comprising the peptide as an effective ingredient.
  • the present invention provides antimicrobial household goods comprising the peptide as an effective ingredient .
  • the household goods are preferably selected from the group consisting of cosmetics, toothpaste, skin cleansing, intima detergent, artificial tears for treating scheroma, artificial saliva for treating xerostomia, artificial rhinorrhea for treating nasal dryness, food additives or feed additives.
  • a living thing has an antimicrobial activity to get rid of a pathogen, which is a survival system of its own. And ex vivo sterilization comes first and then the antimicrobial protein starts working to intercept the invasion of a pathogen. Every living thing produces antimicrobial proteins of its own and human is not an exception.
  • antimicrobial proteins in human which are histatin, lysozyme, LL-37, defensin and dermicidin, but their involvement in in vivo bactericidal action has not been explained, yet.
  • LL- 37 comprising 37 amino acids cut out of the protein called ⁇ cathelicidin' exhibits a very strong bactericidal action.
  • hSHP-1 can be easily attached on the biofilm or virus capsule by glycosylation, suggesting that it can be applied to the treatment of infectious or viral diseases.
  • the present inventors identified hSHP-1, a novel secretory antimicrobial protein produced in human, and have studied on its characteristics, functions, structure, components and usages, and thus it is an object of the invention to provide a usage of hSHP-1 to treat infectious disease or to prevent hSHP-1 deficiency-related diseases.
  • the human antimicrobial protein hSHP-1 has a function of inhibiting vicarious hypertrophy of mucous membrane-cutaneous epithelium caused by infection or physical-chemical stimuli. So, hSHP-1 can inhibit the formations of keloid, polyp and precancerous lesion.
  • hSHP-1 gene and nomenclature hSHP-1 gene has been recorded at GeneBank in the name of Salvic by the accession number AY177672.
  • the open reading frame (ORF) of hSHP-1 is 141 bp and 138 bp of it designates the sequence of 46 amino acids (SEQ. ID. NO: 2) (Fig. 5) .
  • the present inventors presumed that 11 amino acids out of
  • hSHP-1 cationic short helical peptides
  • SHP cationic short helical peptides
  • hSHP-1 is a short protein having an ⁇ -helical structure and comprises 8 serines. It includes two serine-serine structures, which might be the major targets of fast sialidization leading to glycosylation.
  • glycosylated hSHP-ls are linked by disulfide bond, forming a macromolecule.
  • the macromolecule of the glycosylated hSHP-1 suppresses the denaturation resulted from the strong activity of proteases included in mucus such as saliva or tears, can be easily adhered on the cell membrane of a bacterium, is active in acidic or basic solution and maintains the functions of hSHP-1.
  • the presumed pi value of hSHP-1 by amino acid sequence was 9.45 and the presumed molecular weight of the early hSHP-1 was 5.25 kDa.
  • the actual pi (sioelectrophoresis) value of hSHP- 1 obtained from saliva, tears or prostate was approximately 6 - 6.3 and the molecular weight determined by Western blot using immuno-precipitation was approximately 6, 35, 45, 52 and 77 kDa and among those scattered bands, the strongest one was found at 45 kDa (Fig. 11) .
  • hSHP-1 is generated in mucus-secreting cells and is growing in molecular weight by continuous sialidization and glycosylation in serine-serine region which is the helical structured hydrophilic amino acid region. Then, upon completion of forming complicated helical structure by disulfide bond based on cysteine, hSHP-1 is secreted and glutamine-lysine (Q-K) cross-linked right way with proteins in saliva or other proteins existing on the surface of mucosal epithelium by transglutaminase-3 to form a complex.
  • Q-K glutamine-lysine
  • hSHP-1 antibody hSHP-1 is a short protein comprising 46 amino acids and its 11 amino acids at amino terminus are presumed to be digested as a signal peptide. So, an antibody can be prepared based on 35 amino acids in fact. 23 amino acids at carboxyl terminus are believed to have antimicrobial activity but antigenicity of those amino acids is very low, making antibody production very difficult. Therefore, a peptide corresponding to 13 - 23 amino acids of hSHP-1, which seem to have high antigenicity, is preferably used for the production of an antibody.
  • the hSHP-1 antibody can be prepared in both forms of polyclonal antibody and monoclonal antibody.
  • a polyclonal antibody New Zealand white rabbit, goat, bovine, mouse, rat, etc. can be used.
  • a monoclonal antibody 35 amino acids at carboxy terminus are used.
  • the hSHP-1 antibody can have high specificity.
  • a monoclonal antibody can be continuously prepared by using myeloma hybrid.
  • Expression level and pattern of hSHP-1 in human The expression pattern of hSHP-1 can be investigated by immunohistochemical observation and RNA in situ hybridization.
  • the expression levels and patterns of hSHP-1 in each mucous tissue and organ are as follows (Figs. 2, 9 and 12) .
  • hSHP-1 Oral mucosa and salivary gland hSHP-1 is largely expressed in duct cells of salivary gland and rarely in serous gland cells. The strongest expression is observed in parotid gland and the significantly high expression level is observed also in maxillary gland, among major salivary glands. Low level of hSHP-1 expression is observed in sublingual gland producing mucus or minor salivary glands. hSHP-1 exists in duct cells as particles and localizes around the nucleus, and then secreted. As a result, a huge amount of hSHP-1 is included in secreted saliva.
  • hSHP-1 is not observed in normal mucosal membrane cells of oral cavity, but a thin film of hSHP-1 is formed on the surface of mucosal membrane contacting saliva, suggesting that hSHP-1 in saliva can be strongly attached onto the surface of mucosal membrane.
  • the exfoliated keratinocytes of oral mucosa were collected, which were put in a mixing solution of 6 M urea and 1 mM EDTA, followed by double-boiling for one week. Immunohistochemical staining with the exfoliated keratinocytes of oral mucosa using hSHP-1 was performed. As a result, hSHP-1 expression was focused on the outside of the epithelia rather than on the inside of the epithelia. The above result suggests that hSHP-1 is so strongly attached onto the epithelia of the exfoliated keratinocytes by cross-linking that it is not separated in the mixing solution of 6 M urea and 1 mM EDTA even by the one-week boiling. And the outer surfaces of the keratinocytes of oral mucosa were highly hSHP-1 positive, suggesting that hSHP-1 included in saliva is adhered onto the epithelia of the keratinocytes .
  • the dysplastic variation of the epithelium of oral mucosa causes vitiligo, and at this time, the cornified epithelium cannot be easily exfoliated from the oral mucosa, making the epithelium of mucosal membrane thick and over- proliferated. Then, hSHP-1 generation increases, resulting in intracellular accumulation of the same.
  • the well-differentiated tumor cells in the early stage of metastasis exhibit the high level of hSHP-1, that is hSHP-1 is abundant in cytoplasm and some of hSHP-1 move around the nucleus and are accumulated in nucleoplasm.
  • the tumor cells with hSHP-1 accumulation in their nuclei shrink gradually or are dead as vacuolation is progressed in the nuclei.
  • hSHP-1 is not expressed in malignant tumor cells where cell differentiation is intercepted and the amount of nucleoplasm increases abnormally (Fig. 14) .
  • M ⁇ bomian gland which is a lacrimal gland located in the back of palpebra exhibits the highest hSHP-1 expression.
  • the lacrimal gland generates very important functional tears which clean the surface of eye balls to get rid of bacteria and to protect thereof, whenever eyes are blinked.
  • hSHP-1 is also expressed highly in the lacrimal gland, a major secretory gland, and particularly high expression of hSHP-1 is observed in duct cells.
  • hSHP-1 expression in lacrimal gland increases significantly, suggesting that hSHP-1 expression is closely related to inflammatory disease of eyes.
  • Nasal mucosa and mucus secretory gland hSHP-1 diffuses in mucus secretory cells of nasal mucosa, in particular localizes in those mucus secretory cells of mucosal membrane of maxillary sinus.
  • Nasal mucosa comprises long pillar-shaped cells whose outer face exhibits a film showing positive reaction to hSHP-1. From the observation, it was confirmed that hSHP-1 is actively secreted in nasal mucosa and adhered tightly on the surface of the nasal mucosa to protect it.
  • Gastric mucosa hSHP-1 is highly expressed in gastric mucosa, and especially high level of hSHP-1 is observed in parietal cells of the gastric mucosa. hSHP-1 expression is also observed in some mucus secretory cells. hSHP-1 is tightly adhered on the surface of the gastric mucosa, forming a hSHP-1 positive film. Although hSHP-1 is still observed in the papillary structure of the mucosa, the expression level is very low in premature cells of crypt but is increased on the surface of terminal mature enterocytes.
  • hSHP-1 is continuously expressed in parietal cells secreting gastric acid, making gastric juice strong acidic condition with pH 2.5 - 3, which means that the activity of hSHP-1 is not reduced by acidic condition, suggesting that the antimicrobial and antitumor activity of hSHP-1 also might remain unchanged.
  • hSHP-1 diffuses in mucus secretory epithelia to be able to be confirmed as positive to hSHP-1 and is adhered on the surface of the mucosa to form a film on the mucosal epithelia but the level of hSHP-1 thereon seems to be reduced and instead, most hSHP-ls are found in cytoplasm of macrophages permeated in connective tissue under the mucosal epithelia.
  • hSHP-1 therein is tightly adhered onto or surrounds an antigen causing inflammation, leading to opsonization to make macrophages engulf the antigen.
  • hSHP-1 is believed to act as a catalyst to promote immune response against a non-specific antigen causing inflammation.
  • hSHP-1 expression is very low in intestinal cells of fundus but is strong on the surface of those intestinal cells on top of the papillary epithelia. This expression pattern is clearly observed particularly in large intestine. That is, high hSHP-1 expression localizes on the surfaces of intestinal cells on the top of papillary epithelia where stools are gathered.
  • hSHP-1 positive reaction focused on the top of papillary epithelia is reduced and diffused hSHP-1 reaction is generally observed on mucosal epithelia.
  • hSHP-1 film formed on the top of papillary epithelia of intestinal membrane to protect from stools is disappeared and then scattered hSHP-ls start acting as an accelerator for chronic inflammatory reaction on the intestinal membrane.
  • hSHP-1 High level of hSHP-1 expression is observed in Bartholin' s gland of vaginal mucosa and also hSHP-1 positive reaction forming a film on the surface of cornified epithelia of vaginal mucosa is observed.
  • Vaginal fluid which is a weak acidic (pH 4-5), provides a good condition for hSHP-1 to be activated well to exhibit antimicrobial, antitumor and immune stimulating activity.
  • hSHP-1 diffuses on the well-developed endometrium mucosal epithelia of uterine mucosa. The expression of hSHP-1 on the mucosal epithelia decreases rapidly during menstruation, but increases again in the regeneration phase of uterine mucosa-.
  • hSHP-1 expression is also observed in such skin organs as sweat gland and sebaceous gland.
  • hSHP-1 expression is mainly observed in duct cells of sweat gland.
  • the secreted sweat was tested by the method of the present invention, resulting in that glycosylated hSHP-1 was abundant in the sweat.
  • hSHP-1 also localizes in sebaceous gland where hairs grow.
  • the hSHP-ls concentrated in cytoplasms of fatty cells are discharged in sebaceous gland.
  • the discharged hSHP-1 is spread on the surface of the skin with being mixed with fat.
  • hSHP-1 is tightly adhered onto the outside of cornified epithelia of skin, forming a film positive to hSHP-1. This hSHP-1 film is presumed to be functioning as a skin protecting film and to maintain its antimicrobial activity.
  • the highest hSHP-1 expression is observed in the duct cells of prostate gland among human secretory glands. Numbers of hSHP-1 positive particles exist in the duct cells and are slowly discharged. The hSHP-1 expression is increased in patients with prostatic hypertrophy. From the molecular biological investigation on the ejaculated semen, it was confirmed that glycosylated hSHP-1 is abundant in the semen. This result indicates that hSHP-1 secreted from prostate gland has an antimicrobial activity in semen.
  • Tonsil and mucosal immune organ hSHP-1 forms a thin hSHP-1 positive film on the surface of normal skin or mucosal epithelia, while the whole cytoplasm of tonsil, an immune perception organ, or mucosal epithelial cells of intestinal mucosa are highly positive to hSHP-1 owing to its concentration.
  • the whole cytoplasm is strongly hSHP-1 positive and immune cells like lymphocytes are permeated and proliferated in the interior of epithelium, suggesting that hSHP-1 copes with an antigen invading from outside of mucosa to adhere onto the antigen and carry the antigen to connective tissues to cause immune cells to be functioning.
  • hSHP-1 The cationic short helical peptide of hSHP-1 is reacted nonspecifically with an invading antigen and thus mucosal epithelial cells are functioning as superficial antigen presenting cells. This is another characteristic function of hSHP-1 as an immune stimulator.
  • Liver cells also produce abundant hSHP-1 even though they are not exocrine cells.
  • small amount of hSHP-1 is expressed diffusely in cytoplasm but once liver cells are stimulated by cholestasis, chronic inflammation or liver cirrhosis, etc, the hSHP-1 expression increases.
  • the produced hSHP-1 is discharged in liver vascular system through liver vessel wall and spread to all over a body. Therefore, an abundant amount of hSHP-1 is detected in normal human blood.
  • hSHP-1 As mentioned above, once the liver cells are stimulated by cholestasis, chronic inflammation or liver cirrhosis, hSHP-1 is over-accumulated in cytoplasms of the liver cells. If cholestasis, chronic inflammation or liver cirrhosis continues to progress, some of hSHP-ls are gathered around nuclei. However, the invasion of hSHP-1 into nuclei has not been observed, yet. Only when the liver cells are affected by tumor growth and thus are fast proliferated, hSHP-1 permeates into nuclei and binds nonspecifically to nucleoplasms.
  • hSHP-1 is believed to play a role as a protective shield having antimicrobial and anti-proliferating and immune stimulating activity.
  • hSHP-1 Cerebrospine and cerebrospinal fluid hSHP-1 is also abundant in cerebrospinal fluid.
  • the origin of hSHP-1 was investigated and as a result it was confirmed that ependymal cells producing spinal fluid secrete hSHP-1 abundantly and meningeal membrane covering the brain also expresses hSHP-1.
  • hSHP-1 is distributed widely in cerebrospinal fluid and exhibits antimicrobial, anti-proliferating and also immune stimulating activity.
  • hSHP-1 peptide of C-23 (SEQ. ID. NO: 6) was co- cultured with HUVECs (human umbilical vascular endothelial cells), followed by ex vivo tests. HUVEC migration was accelerated by VEGF (vascular endothelial growth factor, 5 ng/mL) .
  • VEGF vascular endothelial growth factor, 5 ng/mL
  • C-23 hSHP-1 peptide inhibited HUVEC migration in proportion to the C-23 peptide concentration, precisely C-23 hSHP-1 peptide inhibited cell migration 35% with the C-23 peptide concentration of 1 ⁇ M and 55% with the concentration of 2.5 ⁇ M. From the results, it was confirmed that the C-23 peptide could effectively inhibit HUVEC migration and thereby inhibits angiogenesis effectively.
  • Antimicrobial activity of hSHP-1 Antimicrobial activity against bacteria and fungi hSHP-1 presumably contains 16 helical structures and among them 11 helical structures are responsible for its antimicrobial activity. Hydrophobic helix at amino terminal adheres very well on bacteria and a motif at carboxy terminal is involved in the cross-linking of glutamine-lysine via transglutamine, which allows antimicrobial activity to be continued by adhesion on the surface of a cell or mucosa.
  • hSHP-1 helical structure of approximately 12 th - 45 th amino acids is responsible for the major antimicrobial activity of hSHP-1, which is the helical structure of 12 th - 45 th amino acids adheres strongly onto the cell membrane and then permeates through cell membrane to make a hole on the cell wall, resulting in the destruction of the cell.
  • a minimum size peptide (16 amino acid peptide; SRVLNRSLQVKWKIT) showing the equal antimicrobial activity with hSHP-1 has +4 value of positive charge.
  • the antimicrobial activity of hSHP-1 was investigated by using a peptide comprising 23 amino acids of carboxy terminal (SEQ. ID. NO: 6) .
  • hSHP-1 strong antimicrobial activity against such pathogens as Staphylococcus aureus, Candida albicans, Aspergillus niger and Pseudomonas aeruginosa
  • hSHP-1 C-23 mer, MIC: about 10 ⁇ M/10 7 cells
  • MIC medium level of bactericidal activity against Escherichia coli
  • Fig. 7 methicillin-resistant Staphylococcus aureus
  • hSHP- 1 also exhibited a medium level of bactericidal activity against Helicobacter pylori (MIC: about 150 ⁇ M/10 7 cells), which has been known as a responsible pathogen of human gastritis.
  • Antimicrobial activity against virus hSHP-1 contains two serine-serine (SS) motifs which are adhered on sialic acid.
  • SS serine-serine
  • glycoproteins are generated by multiple sialidization.
  • the glycosylated hSHP-1 is now functioning as an antivirus, just like glycosylated basic proline rich protein. Therefore, the glycosylated hSHP-1 is able to be adhered tightly on enveloped virus and cationic SHP damages the envelope and thereby brings an effective antiviral activity.
  • glycosylated hSHP-1 is able to be adhered on the biofilm generated by bacteria or fungi. Thus, it can act on bacteria in the biofilm and further intercept the generation of the biofilm and loosen the structure of the biofilm, resulting in the inhibition of the biofilm generation and elimination of the old biofilm.
  • hSHP-1 secretion causes multiple infectious diseases by various pathogens. Therefore, it is required to supplement hSHP-1.
  • hSHP-1 concentration in mucous secretion is measured by ELISA, dot blotting or Western blotting. The antimicrobial activity can be tested with the secreted mucus and determined by MIC
  • hSHP-1 protein is produced in ribosome, gathered in Golgi complex and glycosylated by glycosylation. At this time, if sialic acid is adhered on hSHP-1, it (original pi: 9.4) becomes acidic by the sialic acid and thus pi of the protein is lowered to approximately 6.2. The resulting hSHP-1 can be easily discharged and be functioning as an antimicrobial factor.
  • hSHP-ls are not fully glycosylated by sialic acid and thus accumulated in cells and as a result pH of the accumulated hSHP-1 is raised to 8 - 9 and some of hSHP-ls are migrated into acidic nuclei. Then, the helical structure of hSHP-1 and the repeated hydrophobic and hydrophilic substrates become capable of binding non-specifically to DNA helical structure.
  • hSHP-1 accumulation is clearly observed in primary tumor cells or precancerous lesion with fast proliferation. It was also detected from the immunohistochemical observation that hSHP-1 migrates into the nucleus and diffuses in nucleoplasm in the center of nucleus. Particularly, hSHP-1 permeates into nucleus and then adheres to nucleoplasm, resulting in the inhibition of cell proliferation and further apoptosis.
  • the above action of hSHP-1 in precancerous lesion or tumor cells can be understood as an antitumor activity.
  • hSHP-1 secretion in malignant tumor cells is significantly reduced and almost stopped in the late stage tumor cells, indicating that it is very difficult to expect that hSHP-1 can maintain its antitumor activity in cancer cells.
  • hSHP-1 inhibits the proliferation of atypical cells which are proliferated fast in the early stage of tumor, leading to the antitumor effect. Sialidization and glycosylation of hSHP-1 are not easily induced since lactic acid is increased by hypoxia and ATP is excessively consumed in precancerous lesion proliferating abnormally or in the early stage of tumor development. Thus, hSHP-1 cannot be discharged and is accumulated in cytoplasm instead.
  • hSHP-1 migrates into nucleus slowly and then adheres nonspecifically on nucleoplasm, DNA, resulting in the decrease of the activity or the apoptosis of the cell.
  • Those cells with damaged nucleoplasms by hSHP-1 are early exfoliated from the mucosal epithelium before apoptosis, suggesting that abnormally proliferating precancerous lesion cells or primary tumor cells can be eliminated by being fallen off early from the mucosa.
  • C-35 SEQ. ID. NO: 8
  • cell proliferation was at least 30% inhibited within a day in immortalized cell lines obtained from gingival fibroblasts, periodontal ligament fibroblasts and human epithelial keratinocytes .
  • cell proliferation was also inhibited by the C-35 peptide in various cancer cell lines such as human osteogenic sarcoma cell line (HOS) , stomach cancer cell line (SNU-I), etc.
  • C-23 peptide of hSHP-1 was also examined by the same experiment as the above. AS a result, it was observed that the C-23 peptide migrated into cell nucleus quickly but cell proliferation inhibiting effect was rather weak (Fig. 17) .
  • C-35 peptide of hSHP-1 was functioning to cope with tumor cell proliferation, genetic materials expressed in SNU-I, HOS and RHEK were identified in the meantime.
  • C-35 peptide reduced the expressions of cell growth factors such as PCNA, CDK4, c-erB2 and EGFR, etc, but increased the expressions of apoptosis-involved proteins such as caspase-9, caspase-8, caspase-3, FAS, p53, Bax, BAD, FADD, etc, and cytopathic- related proteins such as MMP-3, MMP-9 and MMP-IO.
  • C-35 peptide of hSHP-1 effectively inhibits proliferation of immortalized cells or tumor cells so as to induce apoptosis of tumor cells.
  • hSHP-1 accumulation is increased particularly in cytoplasms of tumor cells or other fast proliferating cells, suggesting that hSHP-1 is nonspecifically but tightly adhered onto nucleoplasms of those cells to inhibit cell proliferation and induce apoptosis (Fig. 18).
  • Cationic short helical peptide hSHP-1 has unique characteristics of adhering tightly on an antigenic substance and cross-linking on the surface of mucosal epithelium, suggesting that hSHP-1 can catch a passing-by antigen around mucous cells to adhere it tightly on the mucous cells.
  • Some of mucosal epithelium can engulf the adhered antigenic substance by endocytosis and then carry it into immune cells near, for example into fissured mucous tissue cells of tonsil or intestinal membrane.
  • hSHP-1 Classification of hSHP-1 peptides hSHP-1 is determined whether be useful or not according to whether it harbors unit amino acid involved in antimicrobial or antitumor activity.
  • N-Il peptide of amino terminal is strongly hydrophobic, indicating that it strongly adheres on a hydrophobic lipid component under the hydrophobic condition.
  • the amino acid sequence of N-Il has a signal peptide structure, so that it can play a certain role in keeping the stability of hSHP-1 from the extracellular discharge to the arrival at the destination.
  • the minimum size peptide 3O SRVL1NRSLQVKWKIT 45 having antimicrobial activity includes two arginines (R) and two lysines (K) is an important amino acid sequence.
  • C- 23 peptide (SEQ. ID. NO: 6) comprises the amino acid sequence of SSVSS, and thereby glycosylation can be induced by sialic acid and ⁇ -helical structure can be persistently produced, which is appropriate for an antimicrobial peptide.
  • C-23 peptide was tested for antigenicity. An antibody was not produced by C- 23 in a rabbit and a rat, suggesting that C-23 peptide has a low antigenicity. From the above results, it is suggested that the possibility of immunological side effects is very rare even though C-23 is continuously and with huge amount administered into human.
  • C-35 peptide (SEQ. ID. 6) comprises the amino acid sequence of SSVSS, and thereby glycosylation can be induced by sialic acid and ⁇ -helical structure can be persistently produced, which is appropriate for an antimicrobial peptide.
  • C-23 peptide was tested for antigenicity. An antibody was not produced by C- 23 in a rabbit and a rat, suggesting that
  • c-35 peptide exhibits antitumor activity against fast proliferating cells or tumor cells, making it a multifunctional peptide .
  • hSHP-1 needs at least 8 amino acids, indicating that the minimum size peptide to show an antimicrobial effect is the length of 8 amino acids theoretically and its modification might bring stronger antimicrobial effect.
  • hSHP-1 Production of a recombinant protein from bacteria using a plasmid vector hSHP-1 is a short peptide having a short nucleotide sequence, so a vector can be prepared by tandem repeated code.
  • DNA code optimization is required. It is possible for hSHP-1 itself to inhibit the proliferation of bacteria, a host. So, it is required to inhibit the function of hSHP-1 by linking a fusion protein such as KSI, His-tag, GFP and GST with hSHP-1 code and to employee a method to help the absorption on chromatography during the separation and purification of hSHP-1.
  • a commercial vector, particularly pET32b or pET series can be used.
  • a recombinant protein produced in bacteria is not glycosylated or disulfurated. Thus, the separated and purified hSHP-1 sometimes needs to be glycosylated and sulfurated.
  • Pichia pastoris a kind of yeast, favors the production of pure functional hSHP-1 because the level of endotoxin is comparatively low and intracellular glycosylation is induced more easily.
  • Any commercial vector for yeast which enables fast proliferation of yeast can be used herein for the mass- production of a recombinant protein.
  • Insect cells were transfected with baculovirus, a virus vector, to produce a hSHP-1 recombinant peptide.
  • glycosylation and sulfuration are better induced in a peptide, so that a highly functional hSHP-1 recombinant peptide can be prepared thereby.
  • Insect cells are characterized by high proliferation and low endotoxin, which are advantages for culture. But, the insect sells have a problem at the same time that the host cells might recognize hSHP as another functional protein so the glycosylation or sulfuration process therein might be changed, resulting in the decrease of antimicrobial, antitumor and immune stimulating activity of hSHP-1. Therefore, the culture conditions, harvesting time and purifying method have to be carefully determined and conducted.
  • CMV promoter containing vector can be directly inserted into immortalized cells or tumor cells of human, rat or bovine, which are commonly used for cell culture, to produce a hSHP-1 recombinant protein. Glycosylation and sulfuration processes are well induced in hSHP-1 by this method but hSHP-1 is not glycosylated and rather accumulated in highly proliferative host cells, resulting in the inhibition of the proliferation and further apoptosis of the host cells, suggesting that hSHP-1 is functioning as an antitumor agent. Therefore, the mass- production of hSHP-1 might be impossible.
  • Another problem of using animal cells is the slower cell differentiation speed and protein production speed than those of bacteria or yeast, making the mass-production of hSHP-1 difficult.
  • hSHP-1 Separation method of hSHP-1 from human secretion hSHP-1 is converted into a multifunctional protein by glycosylation and disulfide bond in secretory cells and then discharged out of the cells.
  • a recombinant protein produced from bacteria, prokaryotes is deficient in glycosylation and disulfide bond
  • a recombinant protein produced from yeast or animal cells, eukaryotes includes incomplete glycosylation and disulfide bond.
  • hSHP-1 produced in human secretory gland has an authentic molecular structure to have antimicrobial and antitumor activity in the inside of human body. Therefore, it is preferred to separate hSHP-1 directly from the secreted human body fluid.
  • the representative secretions expressing significantly high level of hSHP-1 are secreted from salivary glands, lacrimal glands, vagina, prostate, mammary glands, sebaceous glands and gastrointestines.
  • salivary glands and lacrimal glands are comparatively easy to obtain secretions.
  • an adult secrets saliva approximately 1.5 L per day, which is comparatively large amount.
  • parotid gland largely secreting serous fluid produces a large amount of hSHP-1 and maxillary gland, sublingual gland and accessory salivary gland follows.
  • the component of hSHP-1 included in saliva was examined by Western blotting.
  • parotid saliva directly collected from the duct of parotid gland was confirmed to produce more hSHP-1 than the early mixed saliva collected from the whole saliva secreted in the mouth, but the components of the two were not different.
  • the mixed saliva stayed more than 1 minute in the mouth, protein decomposition and denaturation were observed by SDS-gel electrophoresis.
  • Enough amount of hSHP-1 can be separated and purified by gathering the primary mixed saliva from numbers of people. hSHP-1 purification can be performed by various methods.
  • hSHP-1 is highly resistant against acid. So it can endure under pH 4.
  • hSHP-1 can also be separated from the supernatant obtained from 50% ethanol solution.
  • cation exchange column (SP; Pharmacia, USA) chromatography can be applied to extract a cationic substance which is abundant in saliva by taking advantage of the fact that hSHP-1 is a cationic peptide.
  • gel filtration chromatography can be performed to separate hSHP-1 from the individual cationic substance.
  • HPLC is performed using C-8 column to separate hSHP-1. The supernatant resulted from the extraction using 50% ethanol solution still contains abundant other proteins.
  • hSHP-1 can be separated from secretions using hSHP-1 antibody. According to this method, the amount of hSHP-1 separated is limited.
  • glycosylated component in the heavy chain of the antibody was substituted with amide of the bead.
  • the prepared hSHP-1 antibody column was used to adhere hSHP-1 included in the secretion to the antibody, followed by separation and purification of hSHP-1 using sialic acid (citric acid, pH 2.5) .
  • hSHP-1 peaks are confirmed by ELISA, dot blotting or Western blotting and then glycosylated hSHP-1 is separated and purified.
  • hSHP-1 is not denaturated under pH 2.5, so it can be stored as it is. Or, sialic acid, salt and ethanol are eliminated therefrom by dialysis according to the purpose of use and lyophilization can be carried out.
  • the glycosylated hSHP-1 in human secretions contains glycosylated early protein (6 kDa) , heavy glycosylated hSHP-1
  • hSHP-1 (30-50 kDa) and hSHP-1 cross-linked to other proteins (53-70 kDa) , which are all positive to hSHP-1 antibody.
  • the glycosylated hSHP-1 band of 55 kDa was observed most clearly. Once this protein is separated and the glucose therein is eliminated by neuraminidase (sialidase) , hSHP-1 protein is easily decomposed and rapidly denaturated.
  • C-16 peptide the minimum size antimicrobial peptide of hSHP-1
  • longer hSHP-1 peptides which are C-19 (SEQ. ID. NO: 4), C-21 (SEQ. ID. NO: 5), C-23 (SEQ. ID. NO: 6), C-30 (SEQ. ID. NO: 7) and C-35 (SEQ. ID. NO: 8) can be used.
  • hSHP-1 has a strong antimicrobial activity against both Gram-positive and Gram-negative bacteria. In the meantime, it shows a low antimicrobial activity against E.
  • hSHP-1 has equal antimicrobial activity against ⁇ methicillin-resistant bacteria, and maintains its antimicrobial activity even in neutral solutions such as saliva and tears and in acidic solutions such as gastric juice and serous fluid.
  • hSHP-1 keeps its antimicrobial activity under the hydrophobic condition such as in sebaceous glands. Therefore, hSHP-1 is determined to be a human antimicrobial protein widely applicable to a human body.
  • hSHP-1 glycosylated by sialic acid it is preferred to use to inhibit the decomposition of hSHP-1 and strengthen the antimicrobial activity thereof. It is also preferable for hSHP- 1 to form a dimer with another hSHP-1 to build a complicated- SHP structure, which favors the antimicrobial activity. In conclusion, dimer formation and glycosylation are two important factors for hSHP-1 to maintain its antimicrobial activity in a solution. hSHP-1 exists in native form in human secretions, and this native form of hSHP-1 exhibits antimicrobial activity against not only bacteria but also virus, precisely it can inhibit the formation of biofilm or eliminate already formed biofilm, making it a highly functional peptide.
  • the synthetic or recombinant hSHP-1 protein can be used as an alternative fluid for tears, saliva, rhinorrhea, gastrointestinal fluid, vaginal fluid, prostatic fluid and sweat or a therapeutic agent.
  • hSHP-1 combined treatment is required.
  • hSHP-1 The antitumor effect of hSHP-1 is resulted from the anti- proliferating activity of hSHP-1 inhibiting dysplasia in early stage of tumor development during which proliferation is accelerated.
  • the level of lactic acid is increased by hypoxia and ATP is consumed excessively, so sialidization and glycosylation of hSHP-1 are not induced properly, causing hSHP-1 not to be discharged out of cells and to be accumulated in cytoplasm instead.
  • the accumulated hSHP-1 slowly migrates into nucleus and adheres nonspecifically on nucleoplasm DNA, resulting in the decrease of cellular activity and further apoptosis.
  • hSHP-1 The severely damaged cells by hSHP-1 are exfoliated from the mucosal epithelium before apoptosis, suggesting that hSHP-1 might be used to eliminate abnormally proliferating precancerous lesion cells or early stage tumor cells from the mucosa.
  • the synthetic or recombinant hSHP-1 peptide obtained from cell culture using hSHP-1 without glycosylation can migrate into nucleus more easily, which is an advantage as a therapeutic agent for the treatment of precancerous lesion or cancer .
  • antitumor effect of hSHP-1 is expected by applying an ointment containing hSHP-1. But, when treating inaccessible areas such as gastrointestinal tracks, regular oral-administration is encouraged to bring the antitumor effect of hSHP-1.
  • hSHP-1 The antitumor activity of hSHP-1 is more effective in precancerous lesion or early stage tumor cells into which hSHP- 1 can easily invades.
  • hSHP-1 cannot permeate into malignant tumor cells in which cell wall is unclear and thickened by unknown glycoproteins and hypoxia is increased in cytoplasm.
  • hSHP-1 By using a virus vector constructed from the mutant virus that is available for the primary infection but not for the secondary infection, hSHP-1 can be introduced into malignant tumor cells to be functioning as an antitumor agent.
  • the cationic short helical peptide ⁇ hSHP-l' is characterized by adhering tightly on the antigenic substance and cross-linking on the surface of mucosal epithelium, suggesting that hSHP-1 catches a passing-by antigen around mucous cells and attach the antigen on the mucous cells.
  • the mucosal epithelium engulfs the attached antigenic substance by endocytosis and then carries it into nearby immune cells, which is observed frequently in fissured mucous tissue cells of tonsil or intestinal membrane.
  • a medicine or an ointment containing hSHP-1 with the attachment of a specific antigen can be administered or applied to skin or mucosa for a while, by which immune system can recover its functions .
  • hSHP-1 peptide, glycDsylation and disulfide dimer formation can be varied with the characteristics of a specific antigen.
  • partially glycosylated short hSHP-1 is used to catch a small antigen like influenza virus envelope in order to stimulate immune response on mucous cells.
  • a pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is selected from the general carriers such as carbohydrate compounds (ex: lactose, amylose, dextrose, sucrose, sorbitol, manitol, starch, cellulose, etc) , acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, gum arabic, vegetable oil (ex: corn oil, cotton seed oil, soybean oil, olive oil, coconut oil, etc) , polyethylene glycol, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil, but not always limited thereto.
  • carbohydrate compounds ex: lactose, amylose, dextrose, sucrose, sorbitol, manitol, starch, cellulose, etc
  • acacia rubber calcium phosphate, alginate, gelatin, calcium si
  • the pharmaceutical composition of the present invention can additionally include, in addition to the above carrier, lubricants, wetting agents, sweetening agents, flavors, emulsifying agents, suspension agents, preserving agents, etc, but not always limited thereto.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally.
  • the parenteral administration is exemplified by intravenous injection, hypodermic injection, intramuscular injection, etc.
  • the effective dosage of the pharmaceutical composition of the present invention varies from the formulation, administration pathway, age, weight, gender, severity of a disease, diet, administration frequency and pathway, excretion and sensitivity.
  • the dosage can be determined by an experienced doctor with consideration of the goal of the treatment or preventive effect.
  • a proper dosage for an adult is 50 mg - 10 g once a day.
  • the pharmaceutical composition of the present invention can be formulated as a unit dose medicine or as a medicine in multidose vehicle by mixing with pharmaceutically acceptable carriers and/or excipients by the method well known to those in the art.
  • the pharmaceutical formulation can be selected from the group consisting of ointments, solutions, suspensions or emulsions, extracts, powders, granules, tablets or capsules and additionally includes a dispersing agent or a stabilizing agent.
  • hSHP-1 of the present invention is identified as an antimicrobial and antitumor protein secreted in human. So, there is no worry about immunological allergic reaction to this protein. And hSHP-1 has stronger antimicrobial activity than any other human antimicrobial protein.
  • hSHP-1 shows the strongest antimicrobial activity among found human antimicrobial proteins and is expressed in most secretory glands and mucous tissues. The deficiency of this protein can cause infectious mucosal disease. Thus, hSHP-1 can be effectively used for the prevention and treatment of infectious mucosal diseases.
  • hSHP-1 of the present invention can be applied to highly proliferating precancerous lesion cells or tumor cells to inhibit the proliferation of those cells and induce early detachment of tumor cells or early apoptosis, resulting in the inhibitory effect on tumor development and reducing infiltrative growth of tumor cells. Therefore, by such antitumor functions, hSHP-1 is expected to be developed as an effective antitumor agent.
  • hSHP-1 of the present invention has a characteristic helical peptide structure and at the same time, it is a hydrophobic peptide showing cationic charge characteristics. This protein can be attached to another protein by cross- linking with glutamine-lysine and disulfide bond.
  • hSHP-1 can be adhered on the mucosal epithelium and intrude between heterologous proteins by cross-linking, forming salivary complex.
  • hSHP-1 reacts with a foreign protein that is unable to be recognized to attach this protein onto mucosal epithelium by forming a complex with it, inducing the primary immune response.
  • mucosa associated immunity can be stimulated by using hSHP-1, precisely via its mechanism to form a complex with a small antigen that is difficult to be recognized to adhere the antigen on mucosal epithelium.
  • Fig. 1 is a table showing the result of the investigation on sequence homology of hSHP-1 amino acid sequence with other antimicrobial peptide sequences in the antimicrobial peptide database (APD; University of Kansas Medical Center, USA, http: //aps. unmc.edu/AP/main. html) ,
  • Fig. 2 is a table showing the immunohistochemical expression patterns of hSHP-1 in different human organs
  • Fig. 3 is a table showing the MIC (minimum inhibitory concentration) of hSHP-1 for different bacterial strains
  • Fig. 4 is a table showing the MBC (minimum bactericidal concentration) according to the length of hSHP-1 peptide
  • Fig. 5 is a diagram showing the nucleotide sequence and amino acid sequence of hSHP-1 gene
  • Fig. 6 is a set of photographs showing the cross-linking of hSHP with monodansylcadaverine by TGase
  • Fig. 7 is a set of graphs showing the result of colony killing assay of hSHP-1 against Staphylococcus aureus and E. coli.
  • hSHP-1 killed Staphylococcus aureus completely from the concentration of 2 ⁇ M, but showed low bactericidal activity to E. coli. It only killed 50% E. coli at the concentration of 30 ⁇ M,
  • Fig. 8 is a set of photographs showing that oral exfoliated squamous cells were double-boiled in 6 M urea, 1 inM EDTA solution for one week to eliminate all the non- specifically adhered components. Then, immunohistochemical staining was performed using hSHP-1 antibody, confirming that the outsides of the exfoliated squamous cells were positive to hSHP-1 (indicating that hSHP-1 in saliva was cross-linked on the exfoliated squamous cells) ,
  • Fig. 9 is a set of photographs showing the hSHP expressions in different human organs. From the immunohistochemical staining results, it was confirmed that hSHP-1 was highly expressed in mucosa and exocrine glands. Particular in secretory glands, small granule-like hSHP-1 positive materials were observed in cytoplasms of secretory cells, and hSHP-1 localized mainly on the surface of oral mucosa and the top of epithelium of gastrointestinal mucosa,
  • Fig. 10 is a photograph showing that proteins separated from saliva directly obtained from the human parotid gland, tears and semen were tested by Western blotting and as a result was confirmed that hSHP-1 was equally expressed in those secretions,
  • Fig. 11 is a photograph showing the result of immunoprecipitation with mixed saliva by using hSHP-1 antibody column. Various sizes hSHP-1 positive bands were observed, suggesting that hSHP-1 caused protein modification fast,
  • Fig. 12 is a set of photographs showing the result of mRNA hybridization of hSHP-1 in different human organs. hSHP-1 expression was observed in most secretory glands and particularly the expression level was comparatively high in gastrointestinal track tissue and prostatic gland,
  • Fig. 13 is a set of SEM photographs showing the antimicrobial activity of hSHP-1 against Staphyococcus aureus and Candida albicans (their cellular membranes were completely destroyed within 30 minutes by hSHP-1) ,
  • Fig. 14 is a set of photographs showing the hSHP-1 expression in the progression of oral squamous cell carcinoma
  • hSHP-1 expression was weak in normal oral mucosal epithelium but the expression was increased in leukoplakia, the precancerous lesion, and significantly increased in carcinoma in situ. Also strong hSHP-1 expression was observed in well differentiated primary tumor cells but the expression was no more observed in malignant tumor cells where differentiation was poor) ,
  • Fig. 15 is a set of photographs showing the hSHP-1 expressions in hepatoma cells and chondrosarcoma cells. Some of hSHP-1 migrated into nucleus and adhered on nucleoplasm,
  • Fig. 16 is a set of EM photographs showing the hSHP-1 expression in hepatoma cells. hSHP-1 is expressed as being infiltrated in cytoplasm and nucleoplasm (hSHP-1 localizes in
  • Fig. 17 is a set of graphs showing the anti-proliferative effect of hSHP-1 in the culture of human gingival fibroblasts and periodontal ligament fibroblasts. hSHP-1 inhibited the proliferation of those cells significantly,
  • Fig. 18 is a set of a diagram and a graph showing the antitumor effect of hSHP-1.
  • Various oncogenes, apoptosis- related genes and tissue-modification related genes were screened using human stomach cancer cell line (SNU-I cells) to investigate the effect of hSHP-1 on the gene expression.
  • hSHP-1 inhibited the activity of SNU-I cells as a whole.
  • Example 1 Chemical synthesis of hSHP-1 and preparation of a recombinant hSHP-1
  • a recombinant protein from bacteria using a plasmid vector hSHP-1 is a short peptide having a short nucleotide sequence. So, a vector for the peptide can be constructed by tandem repeated code. DNA code had to be optimized to produce human protein code properly in bacteria.
  • hSHP-1 itself can inhibit the proliferation of bacteria, host cells, so a fusion protein such as KSI, His-tag, GFP and GST had to be fused to hSHP-1 code to suppress the functions of hSHP-1 and to make the separation using chromatography easy. Any commercial vector such as pET32b and pET series can be used for this method.
  • a recombinant protein produced from bacteria is not through glycosylation or di-sulfuration. Thus, the recombinant hSHP-1 prepared thereby might need to be glycosylated or sulfurated.
  • Pichia pastoris a kind of yeast, favors the production of pure functional hSHP-1 because the level of endotoxin is comparatively low and intracellular glycosylation is induced more easily therein.
  • Any commercial vector for yeast which enables fast proliferation of yeast can be used herein for the mass-production of a recombinant protein.
  • Insect cells were transfected with baculovirus, a virus vector, to produce a hSHP-1 recombinant peptide.
  • glycosylation and sulfuration are better induced in a peptide so that a highly functional hSHP-1 recombinant peptide can be prepared thereby.
  • Insect cells are characterized by high proliferation and low endotoxin, which are advantages for culture. But, the insect sells have a problem at the same time that the host cells might recognize hSHP as another functional protein so the glycosylation or sulfuration processes therein might be changed, resulting in the decrease of antimicrobial, antitumor and immune stimulating activity of hSHP-1. Therefore, the culture conditions, harvesting time and purifying method have to be carefully determined and conducted.
  • CMV promoter containing vector can be directly inserted into immortalized cells or tumor cells of human, rat or bovine, which are commonly used for cell culture, " to produce a hSHP-1 recombinant protein. Glycosylation and sulfuration processes are well induced in hSHP-1 by this method but hSHP-1 is not glycosylated and rather accumulated in highly proliferative host cells, resulting in the inhibition of the proliferation and further apoptosis of the host cells, suggesting that hSHP-1 is functioning as an antitumor agent. Therefore, the mass- production of hSHP-1 might be impossible.
  • Another problem of using animal cells is the slower cell differentiation speed and protein production speed than those of bacteria or yeast, making the mass-production of hSHP-1 difficult.
  • Example 2 Separation of hSHP-1 complex from human mucus hSHP-1 plays an important role as a complex by being combined with other proteins in human mucus . It is very difficult to separate and purify hSHP-1 alone from human saliva. Thus, hSHP-1 combined saliva protein complex was extracted instead. The extraction method using hSHP-1 antibody column facilitates direct extraction but is not good for the mass- production. So, to mass-produce the protein, hSHP-1 characteristics were examined and utilized. hSHP-1 has a helical structure where hydrophobic region and hydrophilic region are repeated, providing strong adherence. This property is utilized herein.
  • hSHP-1 For example, to separate hSHP-1 from saliva of parotid gland or mixed saliva, amylase, mucin and proline rich proteins, which were abundant in saliva, were eliminated by ethanol precipitation using 70% ethanol. Hydrophobic protein was precipitated by 80% and 90% ethanol. And strong hydrophobic protein was collected from the remaining supernatant by drying. The protein precipitated by using 90% ethanol exhibited the highest hSHP-1 content and the protein precipitated by using 80% ethanol and the protein precipitated by using 70% ethanol followed in that order. The obtained saliva proteins were applied to cationic ion exchange column (SP-column) to separate cationic proteins.
  • SP-column cationic ion exchange column
  • the separated proteins still include histidine or basic proline rich proteins, so those proteins were purified by HPLC using C-8 column considering non-specific binding property of hSHP-1 helical structure.
  • Distilled water was used as a hydrophilic solution and methanol was used as a hydrophobic solution. 0.01% trifluoroacetate was added to each solution.
  • Most of hSHP-1 binding saliva complexes were attached on C-8 column and some of them were dissolved by methanol moving therein, resulting in separation of them with exhibiting various complicated bands . This result indicates that hSHP-1 binding saliva complex includes various kinds of heterologous proteins and different structures thereof.
  • hSHP-1 binding saliva complexes were absorbed on C-8 column, they were dissolved in acetic acid solution (pH 2.5), followed by freeze- drying. 0.1 M DTT solution was added thereto, followed by culture at 70 ° C for 20 minutes to break off disulfide bond. Hydrophobic silicated gel filtration column with less adsorption capacity was used to separate hSHP-1 binding saliva complexes. The reduced hSHP-1 binding saliva complexes became multifunctional proteins with high activity, which could be freeze-dried or stored at 4 ° C until use and can be used for antimicrobial and antitumor activity tests at any time.
  • hSHP-1 Antimicrobial activity of hSHP-1 against bacteria and fungi hSHP-1 is believed to have 16 helical structures in total, and 11 out of those 16 helical structures have been confirmed to have characteristic antimicrobial activity.
  • the hydrophobic helix at amino terminal characteristically adheres to bacteria and the motif at carboxy terminal is responsible for the glutamine-lysine cross-linking by transglutamine.
  • hSHP-1 might have antimicrobial activity by adhering tightly on mucosa or other relevant cells.
  • the antimicrobial activity of hSHP-1 seems to be attributed to the mechanism in which helical structure comprising 12 th - 45 th amino acids strongly adheres on the cell membrane of bacteria and then invades through the cell membrane to make a hole thereon, leading to the destruction of cells.
  • the minimum size peptide (16 amino acid peptide; SRVLNRSLQVKWKIT) showing the equal antimicrobial activity with hSHP-1 has +4 value of positive charge.
  • the antimicrobial activity of hSHP-1 was investigated by using a peptide comprising 23 amino acids at carboxy terminal.
  • hSHP-1 exhibited same antimicrobial activity to both methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus.
  • hSHP-1 also exhibited a medium level of bactericidal activity against Helicobacter pylori (MIC: about 150 ⁇ M/10 7 cells), which has been known as a responsible pathogen of human gastritis.
  • MIC Helicobacter pylori
  • Antimicrobial activity of hSHPl against virus hSHP-1 has two sialic acid binding serine-serine motifs and two hSHP-ls can be linked together by disulfide bond to form a dimer. During the process of forming a dimer, glycoproteins were produced by sialidation.
  • the glycosylated hSHP-1 has an antiviral activity like the glycosylated basic proline rich protein.
  • the hSHP-1 can strongly adhere on enveloped virus and the cationic SHP might damage the envelope, suggesting the antiviral activity.
  • glycosylated hSHP-1 is able to adhere on biofilm formed by bacteria or fungi, suggesting that the glycosylated hSHP-1 not only exhibits antimicrobial activity against bacteria existing on biofilm but also interrupts biofilm formation and loosens the biofilm structure, resulting in the inhibition of biofilm formation and elimination of the already formed biofilm.
  • hSHP-1 glycosylated hSHP-1
  • secretions on various mucosa or skin are significantly reduced in patients with senile or degenerative disease or auto-immune disease. In these patients, the decrease of hSHP-1 secretion causes multiple infectious diseases by various pathogens. Therefore, it is required to supplement hSHP-1.
  • concentration of hSHP-1 in mucous secretion can be measured by ELISA, dot blotting, western blotting, etc.
  • the antimicrobial activity can be tested with the secreted mucus and determined by MIC (minimum inhibitory concentration) value via bacteria killing assay using a medium.
  • the minimum antimicrobial peptide of hSHP-1 which is C- 16 peptide, can be synthesized biochemically or produced as a recombinant protein, which can be directly applied to the infected areas.
  • a longer hSHP-1 peptide can be used, which is exemplified by C-19 (SEQ. ID. NO: 4), C- 21 (SEQ. ID. NO: 5), C-23 (SEQ. ID. NO: 6), C-30 (SEQ. ID. NO: 7) and C-35 (SEQ. ID. NO: 8) .
  • hSHP-1 exhibits strong antimicrobial activity for both Gram-positive and Gram-negative bacteria.
  • hSHP-1 exhibits comparatively low antimicrobial activity against the human resident microorganism, E. coli, suggesting that it can be developed as a medicine safe for human resident microorganisms.
  • hSHP-1 exhibits the same antimicrobial activity against methicillin-resistant bacteria.
  • the peptide also exhibits antimicrobial activity in neutral solution such . as saliva and tears and further maintains strong antimicrobial activity even in acidic solution like gastric juice and serous fluid.
  • hSHP-1 still maintains its antimicrobial activity under hydrophobic environment such as in sebaceous gland. Therefore, hSHP-1 is a far-reaching human antimicrobial protein.
  • hSHP-1 is observed as a native form in human secretions .
  • the native form of hSHP-1 exhibits antimicrobial activity against bacteria and virus, inhibits the formation of biofilm of bacteria and eliminates the old biofilm, indicating that the native form of hSHP-1 is highly functional peptide.
  • the synthetic or recombinant hSHP-1 protein can be used as- an alternative fluid for tears, saliva, rhinorrhea, gastrointestinal fluid, vaginal fluid, prostatic fluid and sweat or a therapeutic agent.
  • hSHP-1 combined treatment is required.
  • Example 4 Antitumor activity of hSHP-1 hSHP-1 expression patterns in normal cells and tumor cells were investigated.
  • the expression of hSHP-1 in dysplasia increased and stronger hSHP-1 expression was observed in early stage of tumor cells.
  • hSHP-1 expression was scarcely observed. So, antitumor activity of hSHP-1 is attributed to the anti-proliferative function of hSHP-1 inhibiting early dysplasia.
  • the antitumor effect of hSHP-1 is characterized by that endogenous hSHP-1 precursor peptide is functioning directly in cytoplasm and nucleus to inhibit tumor cell proliferation, unlike other antitumor agents mediated by extracellular signal transduction pathway.
  • hSHP-1 Most secretory proteins are modified by phosphorylation, sulfuration and glycosylation in ribosome and Golgi complex of cytoplasm to be discharged.
  • hSHP-1 has a strong acidity and a high hydrophobic adsorption capacity, so it is modified by glycosylation, accumulated as inactivated, granulated and then discharged out of cells .
  • the level of lactic acid is increased by hypoxia and ATP is consumed excessively, making sialidization and glycosylation of hSHP-1 difficult.
  • hSHP-1 cannot be discharged and is still accumulated in cytoplasm.
  • hSHP-1 migrates into nucleus and adheres nonspecifically on nucleoplasm DNA, resulting in the rapid decrease of cell activity but increase of apoptosis (Fig. 15) .
  • Cells with damaged nucleoplasm by hSHP-1 lead to apoptosis very fast, during which epithelial cell cornification is progressed and thus cell membrane is hardened and the level of keratin in cytoplasm increases.
  • apoptosis occurs because of the break-down of intracellular bridges between epithelial cells (Fig. 14) .
  • the synthetic (16-46 mer) or recombinant hSHP-1 (16-46 mer) exhibits no protein modification.
  • the hSHP- 1 migrates easily into the inside of cells and accumulated in cytoplasm because of its hydrophobicity and adsorption capacity.
  • the basic hSHP-1 is highly reactive to acidic nucleoplasm, so that it can permeate into nucleus. Then, hSHP-1 is reacted with nucleoplasm non-specifically to increase apoptosis, suggesting that hSHP-1 can be used as a therapeutic agent applicable to precancerous lesion or cancer (Figs. 14, 15 and 16).
  • an ointment or periodic galgling liquid containing hSHP-1 can be applied with expecting an antitumor effect.
  • a pharmaceutical formula containing hSHP-1 can be orally administered periodically.
  • hSHP-1 treatment is more effective in precancerous lesion or early stage tumor cells because hSHP-1 can easily permeate into these cells.
  • hSHP-1 cannot permeate into malignant tumor cells where cell wall is thickened by glycoprotein and hypoxia is increased.
  • hSHP-1 can be introduced into malignant tumor cells to be functioning as an antitumor agent.
  • Example 5 Immune stimulating activity of hSHP-1
  • the cationic short helical peptide hSHP-1 is characterized by strong adhesion onto an antigenic substance.
  • hSHP-1 is cross-linked on the surface of mucosal epithelium where it catches an antigen passing-by around mucous cells and adheres it on the mucous cells.
  • Some mucous cells engulf the attached antigenic substance by endocytosis and then carries into nearby immune cells. This phenomenon is frequently observed in fissured mucous tissue cells of tonsil or intestinal membrane.
  • a specific antigen binding hSHP-1 can be included in a pharmaceutical formula or an ointment which is applied to skin or mucosa for a while, leading to the immune stimulating effect.
  • the size of hSHP-1 peptide, glycosylation and disulfide dimer formation can be varied with the characteristic of a specific antigen. For example, for a small antigen like influenza virus envelope, partially glycosylated short hSHP-1 is used to enhance immune response on mucous cells.
  • the hSHP-1 peptide of the present invention has antimicrobial, antitumor and immune stimulating activity.
  • an effective pharmaceutical composition or antimicrobial household goods can be prepared by including the peptide as an effective ingredient.
  • SEQ. ID. NO: 1 is the nucleotide sequence of hSHP-1 gene
  • SEQ. ID. NO: 2 is the amino acid sequence of the full- length hSHP-1 peptide
  • SEQ. ID. NO: 3 - NO: 8 are the amino acid sequences of shortened hSHP-1 peptides.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne une composition pharmaceutique qui possède une activité antimicrobienne, antitumorale et immune stimulante et contient un peptide-1 hélicoïdal court humain (hSHP-1) doté d'une activité antimicrobienne, antitumorale et immune stimulante. L'invention concerne également un procédé de traitement du hSHP-1, destiné à augmenter son activité antimicrobienne, antitumorale et immune stimulante, ledit procédé incluant l'étape qui consiste à administrer le peptide hSHP-1.
PCT/KR2006/002936 2006-06-05 2006-07-26 Peptide-1 hélicoïdal court humain qui présente une activité antimicrobienne, antitumorale et immune stimulante et ses utilisations WO2007142381A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2006-0050487 2006-06-05
KR1020060050487A KR100847287B1 (ko) 2006-06-05 2006-06-05 항균, 항암, 면역자극 활성을 가지는, 사람의 짧은 나선형펩티드-1 및 이의 용도

Publications (1)

Publication Number Publication Date
WO2007142381A1 true WO2007142381A1 (fr) 2007-12-13

Family

ID=38801612

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2006/002936 WO2007142381A1 (fr) 2006-06-05 2006-07-26 Peptide-1 hélicoïdal court humain qui présente une activité antimicrobienne, antitumorale et immune stimulante et ses utilisations

Country Status (2)

Country Link
KR (1) KR100847287B1 (fr)
WO (1) WO2007142381A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8835602B2 (en) 2009-03-06 2014-09-16 Indian Institute Of Science Templates for nucleation and propagation of peptide secondary structure
CN108904813A (zh) * 2018-09-30 2018-11-30 派生特（福州）生物科技有限公司 一种禽用疫苗稀释液的制备方法
US10800822B2 (en) 2015-11-30 2020-10-13 The Board Of Trustees Of The University Of Illinois Histatins and method of use thereof
WO2021108482A1 (fr) 2019-11-27 2021-06-03 The Board Of Trustees Of The University Of Illinois Pentapeptide et ses méthodes d'utilisation
WO2021236879A1 (fr) 2020-05-20 2021-11-25 The Board Of Trustees Of The University Of Illinois Méthode de traitement de maladies lysosomales à l'aide de peptides d'histatine

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE NCBI [online] XP008092829, Database accession no. (AAO18648) *
GURURAJA T.L. ET AL.: "Structural features of the human salivary mucin, MUC7", GLYCOCONJUGATE JOURNAL, vol. 15, no. 5, April 1998 (1998-04-01), pages 457 - 467, XP008090520 *
SCOTT M.G. ET AL.: "An alpha-helical cationic Antimicrobial peptide selectively modulates macrophage responses to lipopolysaccharide and directly alters macrophage gene expression", THE JOURNAL OF IMMUNOLOGY, vol. 165, 2000, pages 3358 - 3365, XP002263247 *
SITU H. ET AL.: "Human salivary MUC7 mucin peptides: effect of size, charge and cysteine residues on antifungal activity", BIOCHEM. J., vol. 375, no. 1, October 2003 (2003-10-01), pages 175 - 182, XP008090521 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8835602B2 (en) 2009-03-06 2014-09-16 Indian Institute Of Science Templates for nucleation and propagation of peptide secondary structure
US10800822B2 (en) 2015-11-30 2020-10-13 The Board Of Trustees Of The University Of Illinois Histatins and method of use thereof
US11370816B2 (en) 2015-11-30 2022-06-28 The Board Of Trustees Of The University Of Illinois Histatins and method of use thereof
CN108904813A (zh) * 2018-09-30 2018-11-30 派生特（福州）生物科技有限公司 一种禽用疫苗稀释液的制备方法
WO2021108482A1 (fr) 2019-11-27 2021-06-03 The Board Of Trustees Of The University Of Illinois Pentapeptide et ses méthodes d'utilisation
WO2021236879A1 (fr) 2020-05-20 2021-11-25 The Board Of Trustees Of The University Of Illinois Méthode de traitement de maladies lysosomales à l'aide de peptides d'histatine

Also Published As

Publication number Publication date
KR20070116442A (ko) 2007-12-10
KR100847287B1 (ko) 2008-07-21

Similar Documents

Publication Publication Date Title
US20230279048A1 (en) Compositions containing hc-ha/ptx3 complexes and methods of use thereof
CA2032059C (fr) Traitement des blessures faisant appel a des peptides biologiquement actifs
CN105705160B (zh) Il-22二聚体在制备用于治疗胰腺炎的药物中的用途
CN103764669A (zh) 抗炎药物产品
WO2007142381A1 (fr) Peptide-1 hélicoïdal court humain qui présente une activité antimicrobienne, antitumorale et immune stimulante et ses utilisations
US8815812B2 (en) Synthetic arginine substituted peptides and their use
JP5778692B2 (ja) 疾病抑制剤
EP1080111B1 (fr) INDUCTION DE PROTEINES ET DE PEPTIDES ANTIBIOTIQUES AU MOYEN DE LA PROTEINE sCD14/LAIT (PROTEINE IMMUNOTROPHIQUE ASSOCIEE A LA LACTATION)
US5527775A (en) Reduction of mammalian neoplasms with phospholipase A2 activating substances
KR100879628B1 (ko) 사람의 짧은 나선형 펩티드-1 을 유효성분으로 함유하는항균활성 약학적 조성물
KR20200061573A (ko) 신규 펩타이드 및 이를 포함하는 약제학적 조성물
WO2006025646A1 (fr) Peptide, fragments et derives de ce peptide favorisant l'adhesion et l'etalement des cellules
JP4048565B2 (ja) 「抗ピロリ菌剤」
WO2022204331A1 (fr) Compositions comprenant des peptides dérivés de kgf-2 ramifiés et procédés d'utilisation dans un traitement oculaire
WO2008077938A1 (fr) Modulateurs d'association taline/integrine et leur utilisation
HK1223556B (zh) Il-22二聚體在製備用於治療胰腺炎的藥物中的用途
HK1149769A (en) New synthetic arginine substituted peptides and their use

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 06783413

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 06783413

Country of ref document: EP

Kind code of ref document: A1