JP2003265183A - Trypsin-like enzyme desc1 and method for using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an active-form DESC1 protein of true structure having activity, to provide a method for screening a medicine for inhibiting inflammation accentuation action, intracellular calcium inflow causing action, PAR activation action and plasminogen activator activation action caused by mucus production accentuation action, EGFR route activation action, cytokinin production accentuation, etc., by the active-form DESC1 protein and to construct an evaluation system for detecting the activity. <P>SOLUTION: The DESC1 has structure in which a propeptide part represented by a specific amino acid sequence is bonded through a disulfide bond to a trypsin-like protein part. The inhibitor has the mucus production accentuation action, PAR activation action, the intracellular calcium inflow causing action, IL-8 production enhancement action or cell growth promotion action of an enzyme having the structure as an index. The screening system of inhibitory polypeptide is provided. The antibody is bonded to the enzyme having the structure. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an active protein of type II membrane-bound trypsin-like enzyme protein DESC1. In addition, the present invention uses a detection or measurement system of mucus secretion promoting action, inflammation inducing action, intracellular calcium influencing inducing action, protease activating receptor activating action of the enzyme, and a compound or polypeptide (protein and antibody (Including)). Furthermore, it relates to a method for utilizing the inhibitory substance obtained by the method.

[0002]

2. Description of the Related Art Recently, squamous epithelial cells of tongue cancer and invasive neck cancer (metastatic neck), which are present in human oral mucosa, are present.
DE as a gene whose expression is not found in nodal tissue)
SC1 (differentially expressed in squamous cell
carcinoma) gene was registered in GeneBank (Acc
ession No. AF064819), its amino acid sequence and cd
The NA sequence has been revealed. As a tissue expressing the mRNA, normal tissues are expressed in the oral mucosa, tonsils, prostate, testis, pancreas, thymus, appendix, lymph node, gallbladder, ovary, spleen, colon, small intestine, neutrophil, heart,
It has been reported that no expression is found in the brain, placenta, lung, liver, skeletal muscle, and kidney (PCT WO00 / 5).
0061). Furthermore, the recombinant protein is Dye-Pr.
o-Leu-Ser-Arg-Thr-Leu-Ser
Since it cleaves a synthetic substrate such as -Val-Ala-Ala-Lys (PCT WO00 / 50061),
It is expected that this enzyme has protease activity. Furthermore, since the above-mentioned synthetic substrate after cleavage does not have amino acids after Arg, Pro, Leu, S
It is presumed that it was cut at any of the er.

On the other hand, a protease activated receptor (hereinafter referred to as "PA") is defined as a receptor whose signal transduction is triggered by protease activity.
Also referred to as "R". ) Is a receptor that mediates a signal by a protease, and a thrombin receptor (human PAR-1) present in human platelets was cloned in 1991 (Vu U KH et al., Cell, 64: 1057). -1068,
1991). After that, mouse PAR-1 (Coughlin, SR,
Unpublished, Accession No. L03529, 1992), rat PAR-1 (Runge MS et al., J. Biol. Chem., 26.
7: 16975-16979,1992), mouse PAR-2 (Nystedt
S. et al., Proc Natl Acad Sci USA, 91: 9208-9212, 1
994), human PAR-2 (Bohm SK, Biochem. J., 31.
4: 1009-1016, 1996), rat PAR-2 (Saifeddine
M. et al., Br. J. Pharmacol., 118 (3): 521-530, 199.
6), human and mouse PAR-3 (Ishihara H. et a
l., Nature, 386: 502-506, 1997), mouse PAR-4
(Kahn ML et al., Nature, 394: 690-694, 1998),
Human PAR-4 (Xu WF et al., Proc Natl Acad Sci
USA, 95: 6642-6646, 1998) have been cloned so far. Many reports have been made so far regarding PAR-1 and PAR-2 involved in various pathological conditions such as inflammatory reaction (Dery O. et al., Am. J.
Physiol., 274: C1429-C1452, 1998).

Regarding the activation of PAR-2, trypsin (Nystedt S. et al., Proc. Natl. Acad. Sci. USA,
91: 9208-9212, 1994) and tryptase (Schwart
z, LB et al., J. Immunol., 126: 1290-1294, 198.
1) has been reported to activate PAR2 (Mol
ino M. et al., J. Biol. Chem., 272: 4043-4049, 199
7). PAR2 regulates the secretion of pancreatic juice in the pancreas (Bohm SK, Biochem. J., 314: 1009-1016, 199).
6), Ion channel activation (Nguyen TD et al.,
J. Clin. Invest., 103: 261-269, 1999) and others, as well as the regulation of duodenal motility (Kawabata A. et al.,
Br. J. Pharmacol., 128 (4): 865-872, 1999).

On the other hand, tryptase is released from mast cells and shows enzymatic activity only as a tetramer (Schwartz, L.
B. et al., J. Biol. Chem., 256: 11939-11943, 198.
1), Skin inflammation (Steinhoff M. et al., Exp. Dermato
l., 8: 282-294, 1999) and lung inflammation (Am. J. Physiol.
Lung Cell. Mol. Physiol., 278: L193-L201, 2000)
There are reports suggesting that they are involved in.

[0006] In addition, matriptase, a type II membrane-bound serine protease (Chen-Yong Lin et a
l., J. Biol. Chem., 274: 18231-6, 1999) is a plasminogen activator (prourokinase) activating action (Dano, K. et al., Adv. Cancer Res. 44: 139-26).
6, 1985) with enzyme activity (Sheau-Ling
Lee et al., J. Biol. Chem., 275: 36720-5, 2000),
From this activity, it is considered that these enzymes may be involved in cancer metastasis. Furthermore, it has been reported that ecotin, which directly inhibits matriptase, suppressed the growth of prostate cancer cells (Takeuchi, T. et al.,
Proc. Natl. Acad. Sci. USA, 96: 11054-11061, 199
9).

On the other hand, regarding DESC1, it is disclosed in PCT WO00 / 50061 that the expression of mRNA is reduced in the tongue squamous cells with canceration, and this is utilized to diagnose squamous cell carcinoma. A patent with the contents such as has been applied.

[0008]

The object of the present invention is to obtain an active DESC1 protein having a true structure and having activity.

[0009] Further, the true structure of DESC1 having activity
(Hereinafter, simply referred to as “DESC1”) has an effect of enhancing mucus production, EGFR (Epidermal Growth Factor Recepto)
r) Pathway activating action, inflammatory action due to increased cytokine production, intracellular calcium influx action, PAR
Method for screening drug that inhibits activation activity (defined as protease activation receptor, receptor whose signal transduction is triggered by protease activity) and plasminogen activator (prourokinase) activation activity, and detection of the activity This is the construction of an evaluation system for doing so.

Furthermore, compounds or polypeptides (including proteins and antibodies) that inhibit the activity of DESC1 having the structure, or mucus production-promoting action, EGFR pathway-activating action, and inflammation-promoting action of DESC1 having the structure,
Induction of intracellular calcium influx, PAR activation, plasminogen activator (prourokinase)
It is an object of the present invention to provide an evaluation system for a compound or polypeptide that inhibits the activating effect.

Further, a means for diagnosing abnormalities of the secretory system, inflammatory diseases, abnormalities of the coagulation / fibrinolysis system, cancer, etc. by obtaining an antibody that binds to the enzyme of the structure and constructing a measuring system is provided. It is to be.

That is, a compound or polypeptide which inhibits the DESC1 activity obtained by utilizing the present invention, or a mucus production promoting action by DESC1, E
Compounds or polypeptides that inhibit GFR pathway activating action, inflammatory promoting action, intracellular calcium influx action, PAR activating action, and plasminogen activator (prourokinase) activating action are secretory promoting action of DESC1 on secretory cells. , Action on coagulation / fibrinolysis system, action on inflammation, fibroblast / epithelial cell (ciliary cell)
・ Action against proliferation and damage of smooth muscle cells / goblet cells,
It can be expected to suppress or modify physiological effects such as effects on cancer growth and metastasis, and effects on mucociliary movement, and to improve and treat the pathological condition.

Further, since DESC1 activates PAR, the secretory promoting action of PAR on secretory cells (secretory promoting action of mucous gland cells, serous gland cells, goblet cells, secretory promoting action on nerve cells / neuroendocrine cells) , Relaxation of blood vessels, respiratory tract, esophagus, intestinal tract, etc. through epithelial cells or endothelial cells, contraction of smooth muscle, induction and enhancement of inflammatory cytokine production of fibroblasts and epithelial cells, fibroblast / epithelium Suppress or modify physiological effects such as cell proliferation and disorders of cells (ciliocytes, goblet cells, basal cells), smooth muscle cells, secretory gland cells, and enhance hypersensitivity to nerve cells, and improve and treat pathological conditions Can be expected to be used for.

From the above direct and indirect actions of DESC1, it can be used as an evaluation system and a diagnostic agent for a new therapeutic drug screening for inflammatory diseases, cancer, and diseases caused by abnormalities in coagulation / fibrinolytic system.

[0015]

[Means for Solving the Problems] In view of the above circumstances, the present inventors have earnestly studied the expression method and the purification method of a DESC1 recombinant, and as a result, have the activity of DESC1.
Was successfully expressed in recombinant insect cells, and a method for efficient purification from recombinant insect cells was established. In addition, this recombinant DESC1 is Phe-Ser-A
rg-pNA (synthetic substrate for trypsin), Ser-Ly
It was found for the first time that it cleaves synthetic substrates such as s-Gly-Arg-pNA (PAR2 activation site). As a result of determining the amino acid sequence using the obtained purified protein, the structure of the purified active form of DESC1 was a structure in which, in addition to the amino acid structure similar to serine protease, a propeptide and a serine protease moiety were linked by a disulfide bond. It was revealed for the first time that it is a trypsin-like enzyme with a protein structure containing Furthermore, in the ovary and large intestine, where expression in normal tissues was scarcely found, it was found that DESC1 expression was enhanced in adenocarcinoma cells derived from those tissues, and the relationship between the increased expression of DESC1 and canceration was found for the first time. It was It was also found that in normal tissues, it is expressed in the respiratory tract and esophagus. The present inventors have reached the present invention as a result of further research based on these findings.

The present inventors have found that in insect cells c of DESC1
A recombinant baculovirus vector was prepared based on the DNA sequence (PCT WO00 / 50061), and the recombinant protein was expressed. A protein having activity was purified by the purification method disclosed in the present invention, and the primary structure of the active protein was determined. As a result, it was reported previously (PCT WO00 / 5.
0061, PCT WO01 / 36604) in addition to the structural protein similar to serine protease (protein portion starting from isoleucine (Ile) at position 191),
A trypsin-like structure having a structure in which a partial partial sequence of the propeptide consisting of the amino acid sequence between the 1st methionine (Met) and the 190th arginine (Arg) is linked to the serine protease protein part via a disulfide bond It was revealed to be a protein, and it was discovered that this structural protein is the main component of active DESC1.

The present inventors conducted a BLAST search based on the gene sequence of human DESC1 and found that mouse ES
A partial sequence having homology was obtained from the T database. CDNA prepared from mouse respiratory tract based on this sequence
PCR was performed using the DNA as a template and the sequence of the obtained DNA fragment was determined.
I found that there is one counterpart. Based on this information, the full-length cDNA encoding the mouse DESC1 counterpart was converted to 5'-RACE (Rapid Amplif
ication of cDNA Ends) was obtained by performing 3'-RACE. Furthermore, when the mouse DESC1 gene thus obtained was expressed as a recombinant and the enzyme activity was evaluated, it was revealed that it retains the same trypsin-like enzyme activity as human. Further, the primary structure of the protein was determined, and as a result, mouse DESC1 has the same first methionine (Met) as human DESC1.
To the 191st arginine (Arg), the partial sequence of the propeptide consisting of the amino acid sequence is linked to the trypsin-like protein portion starting from the 192nd isoleucine (Ile) via a disulfide bond. It revealed that.

It was also found that the DESC1 counterpart is present in higher mammals such as rabbits using the same method as described above. The full-length cDNA encoding the rabbit DESC1 counterpart was 5'-RA
It was obtained by performing CE, 3'-RACE. Furthermore, when the rabbit DESC1 gene thus obtained was transiently expressed in human cultured cells, it was revealed that the same activity as human DESC1 could be expressed.

The above-mentioned discovery of the presence of DESC1 in animals makes it possible to evaluate the gene level or protein level expression of animal DESC1, and to evaluate the expression and activity of animal DESC1 in animal disease models. Is very useful for clarifying the involvement of DESC1 in the pathological condition. In addition, by suppressing the expression of DESC1 using an antisense oligonucleotide or by increasing the expression of DESC1 by introducing an expression vector, D
The involvement of ESC1 can be investigated. Furthermore, DES
It also provides important information for studying and evaluating species differences in animals that are important in evaluating the drug efficacy of a DESC1 inhibitor or an inhibitory polypeptide (including protein and antibody) in an animal model involving C1.

A synthetic substrate of recombinant DESC1 having the above structure (Boc-Ser-Lys-Gly-Arg-pN)
A) From the results of the cleavage experiment, it was expected that DESC1 activates PAR2. From this, DESC1 is PAR
2 activates calcium influx, and DESC1 of the structure causes I through a PAR-mediated signal transduction system.
It is expected that L-8 production is enhanced and cell proliferation is caused, and that in disease, it is expected that DESC1 modifies its pathological state by a signal transduction system mediated by PAR. Therefore, based on this, it is possible to construct a screening system based on the enzymatic activity or PAR activating effect of the recombinant protein having the above structure.

Furthermore, in human mucus-secreting cell lines, DESC1 having the above structure enhances mucus production, induces intracellular calcium influx by a PAR-mediated signal transduction system, or enhances IL-8 production and cell proliferation. In the chronic disease in which mucus production is enhanced, DESC1 enhances mucus production, induces intracellular calcium influx by a PAR-mediated signal transduction system, and enhances IL-8 production and cell proliferation It is expected that the disease state is modified by.
Then, based on this, a screening system based on the enzymatic activity of the recombinant protein having the above-mentioned structure or mucus production enhancing action, PAR activating action, intracellular calcium influencing action, IL-8 production enhancing action or cell growth promoting action is provided. They have found that they can be constructed and have completed the present invention.

That is, the present invention is DESC1 having a structure in which a propeptide portion is bound to a trypsin-like protein portion via a disulfide bond.

Further, screening of an inhibitor or an inhibitory polypeptide using the enzyme having the structure as described above for promoting mucus production, PAR activation, intracellular calcium influx, IL-8 production enhancing activity or cell growth promoting activity. It is a system.

The present invention further provides a DESC having such a structure.
Animal cells such as insect cells that produce 1 are also included.

Further, an antibody that binds to an enzyme having the above structure and has a binding property capable of detecting the enzyme in vivo by an enzyme immunoassay, or in tissue by immunohistochemical staining or A monoclonal antibody or a polyclonal antibody having a binding property capable of detecting the enzyme in cells, and a monoclonal antibody or a polyclonal antibody capable of inhibiting the enzyme activity.

Furthermore, the present invention also includes animal cells such as hybridoma cells which produce such monoclonal antibodies.

Further, the DESC1 inhibitor obtained by utilizing the present invention can be a new therapeutic drug for inflammatory diseases, cancer and diseases caused by abnormalities in coagulation / fibrinolytic system.

The present invention will be described in more detail below. (1) A protein consisting of all or part of the amino acid sequence shown in SEQ ID NO: 1, which is 1 from the first Met
The propeptide part consisting of all or part of the amino acid sequence between the 90th Arg and the trypsin-like protein part consisting of the 232 amino acid sequence from the 191th Ile to the 422nd Ile are formed by a single disulfide bond. DESC1 with linked structure. (2) A protein having a homology of 75% or more with the amino acid sequence represented by SEQ ID NO: 1, wherein a portion corresponding to the propeptide portion and a portion corresponding to the trypsin-like protein portion are one disulfide bond. DESC1 with linked structure. (3) A protein consisting of all or part of the amino acid sequence shown in SEQ ID NO: 2, which is 1 to the first Met
A polypeptide part consisting of all or part of the amino acid sequence between the 90th Arg and a polypeptide part consisting of a 232 amino acid sequence from the 191th Ile to the 422nd Ile are linked by a single disulfide bond. DESC1 having the structure shown. (Hereinafter, the DESC1 having the structure of any one of (1) to (3) above
Is simply referred to as "DESC1". (4) An antibody that specifically binds to DESC1. (5) A monoclonal antibody that inhibits human DESC1 activity or inhibits its activation. (6) A method of detecting or measuring DESC1 using the above antibody. (7) An enzyme substrate and a compound to be measured or a polypeptide are mixed, and these and DESC1 or the enzyme-expressing cell or the enzyme-expressing tissue are incubated under appropriate conditions to react with the enzyme substrate, and the reaction product is obtained. D of the target compound or polypeptide by measuring
A method for detecting ESC1 inhibitory activity. (8) DESC1 or the enzyme-expressing cell or the enzyme-expressing tissue, the compound to be measured or the polypeptide, and the PAR-expressing cell are mixed, and DE of the compound to be measured or the polypeptide is measured using PAR activation as an index.
A method for detecting an inhibitory activity of PAR activation by SC1. (9) DESC1 or the enzyme-expressing cell or the tissue expressing the enzyme, the compound or polypeptide to be measured, and a cell having an intracellular calcium influx ability are mixed, and the compound to be measured or intracellular polycalcium is used as an index. A method for detecting the inhibitory activity of a peptide for intracellular calcium influx by DESC1. (10) DES1 of a compound or polypeptide to be measured using DESC1 or cells expressing the enzyme or tissue expressing the enzyme, a compound to be measured or a polypeptide and a cell having a secretory ability as a mixture, and using the secretion product as an index
A method for detecting the inhibitory activity of C1 secretagogue action. (11) mixing DESC1 or the enzyme-expressing cell or the enzyme-expressing tissue, the compound to be measured or the polypeptide, and the cell having EGFR-L, and EG
A method of detecting the inhibitory activity of the compound to be measured or the polypeptide's ability to release EGFR-L by DESC1 using the amount of FR-L released as an index. (12) A step of determining a three-dimensional structure of DESC1, analyzing a three-dimensional structure suitable for binding to a substrate, synthesizing a compound that binds to a predicted reaction site, and determining the enzyme inhibitory activity of the compound And a method for evaluating the enzyme inhibitor. (13) Coding region of natural mammalian DESC1 gene. (14) Mammalian DESC1 gene-derived mRNA or cDNA (for example, those represented by SEQ ID NO: 3 or SEQ ID NO: 4). (15) Mammalian DESC1 protein. (16) An expression vector prepared by using the nucleic acid of (13) or (14). A cell into which it is transfected or transformed. DESC from the cell
1. A method for purifying 1 or purified DESC1. (17) By using information on all or part of the nucleic acid,
A method for detecting the expression level of the DESC1 gene corresponding to those nucleic acids. (18) DESC1 over-activation or up-regulation (the definition of "DESC1 over-activation or up-regulation" is "increase in active DESC1 protein or up-regulation of DESC1 gene") A method of screening a substance that inhibits the activity of DESC1 or the activation of the enzyme, or a method of determining the therapeutic effect thereof, which comprises administering a compound or polypeptide to be measured to a mammal having a disease. (19) Mammals with diseases characterized by increased mucus production, increased inflammation, intracellular calcium influx, EGFR pathway activation, plasminogen activator (prourokinase) activation, PAR overactivation or upregulation by DESC1 Against mucous membrane, administration of a compound or polypeptide to be measured, DESC1 enhances mucus production, inflammation, intracellular calcium influx, EGFR pathway activation, plasminogen activator (prourokinase) activity A method for screening a substance that inhibits the activating effect and PAR activating effect, or a method for determining the therapeutic effect thereof. (20) For mammals having a disease characterized by over-activation or up-regulation of DESC1,
A method for determining a therapeutic effect due to the inhibition of DESC1 activity or the inhibition of the activation thereof by administering a drug containing the antisense oligonucleotide. (21) A method for diagnosing cancer using hyperactivation or upregulation of DESC1 as an index. (22) The above (7) to (12) or (18)
A method for treating cancer using an active DESC1 inhibitor (compound or polypeptide) obtained by using the screening method described in any one of (1) to (19).

[0029]

BEST MODE FOR CARRYING OUT THE INVENTION In DESC1 of the present invention consisting of all or part of the amino acid sequence shown in SEQ ID NO: 1, it is preferable that the propeptide portion and the trypsin-like protein portion are linked by a disulfide bond.
Regarding DESC1 of the present invention consisting of all or part of the amino acid sequence shown in SEQ ID NO: 2, it is preferable that the portion corresponding to the propeptide portion and the portion corresponding to the trypsin-like protein portion are linked by a disulfide bond. Regarding DESC1 of the present invention having a homology of 75% or more with the amino acid sequence shown in SEQ ID NO: 1,
It is preferable that the portion corresponding to the propeptide portion and the portion corresponding to the trypsin-like protein portion are linked by a disulfide bond.

The antibody which specifically binds to DESC1 of the present invention is preferably a monoclonal antibody, and particularly preferably an antibody which inhibits human DESC1 activity or inhibits its activation.

As a method for detecting or measuring DESC1 using the antibody of the present invention, an enzyme immunoassay method is preferable.

The DESC1 or DESC1 expressing cells used in the method for screening the DESC1 inhibitory activity of the compound to be measured or the polypeptide of the present invention are the DESC1 having the structure of the present invention or the DES.
Any cells producing C1 may be used, and DESC1-expressing cells or cells or cell lines derived from DESC1-expressing tissues are preferable. The cells or cell lines derived from the tissue expressing DESC1 include cancer cells and cancer cell lines.
The purified DESC1 of the present invention having the above structure is most preferable for evaluation.

In the method of the present invention for detecting the inhibitory activity of the compound to be measured or the polypeptide against the mucus production promoting action by DESC1, the mucus glycoprotein is stained by the AB-PAS staining method as an index of the mucus production promoting action. Amount, release amount of high molecular weight sulfate labeled product, secretion amount of mucin, mRNA expression amount of mucin, transcriptional activity of mucin promoter, production amount or release amount of ligand of EGF receptor, and protein involved in EGF receptor signal transduction pathway Amount of tyrosine phosphorylated form, MAPK (MAP
The amount of phosphorylated form of Kinase) is preferably mentioned.

In the screening method of the inhibitory activity against the EGFR pathway activating action of DESC1 of the compound to be measured or the polypeptide of the present invention, EGFR-L (EGFR-L) is used as an index of EGFR pathway activation.
igand) cleavage and release, or the previously reported EGFR signaling system (Wells A., Int. J. of
Biochem. & Cell Biology, 31: 637-643, 1999) can be used to measure the activation of the EGFR signal transduction system by DESC1 using a measurement target or a phenomenon resulting from signal transduction thereof.

For example, cell proliferation assay, NFkB,
Genes containing transcriptional elements such as AP-1 (IL-
(8, IL-6, GM-CSF, etc.), measurement of protein and produced protein, measurement of intracellular inositol phosphate degradation activity, measurement of prostaglandin E ₂ production, measurement of thromboxane A ₂ , cAMP ( cyclic AM
P), mucus production, mucin production, mucin mRNA expression, mucin promoter transcriptional activity, tyrosine phosphorylation of proteins involved in EGF receptor signaling pathway (EGFR and MAPK ( M
Amount of phosphorylated form such as AP Kinase).

In the screening method of the inhibitory activity against the PAR activating action of DESC1 of the compound to be measured or the polypeptide of the present invention, as the index of the signal transduction of PAR activation, the object to be measured already reported is used as DESC1. It is possible to measure the activation of the PAR signal transduction system.

For example, intracellular calcium influx, cell proliferation assay, transcription activity of genes (IL-8, IL-6, GM-CSF, etc.) containing transcription elements such as NFkB and AP-1, and measurement of produced protein, Measurement of intracellular degrading activity of inositol phosphate, measurement of prostaglandin E ₂ production, measurement of thromboxane A ₂ , cAMP
(Cyclic AMP) measurement, NO (Nitric
Oxide) is directly or indirectly measured, calcium release and current are measured using Xenopus Oocyte, chloride ion transport is measured via chloride channels, and mucus secretion is measured.

The DESC1 expressing cells are preferably recombinant cells expressing the enzyme. Furthermore, DESC1
The expression cells, the tissue expressing the enzyme, or cell lines derived from them include cancer cells and cancer cell lines.

Examples of the mRNA or cDNA derived from the rodent DESC1 gene of the present invention include those of mouse. An mRNA or cDNA encoding DESC1 represented by SEQ ID NO: 4. Similarly, rabbit is exemplified as the mammalian DESC1 of the present invention, and specifically, mRNA encoding DESC1 represented by SEQ ID NO: 20.
Alternatively, cDNA may be used.

The present invention provides an expression vector for directing the expression of DESC1 having the structure of the present invention prepared by using the information of all or part of these nucleic acid sequences, cells transfected or transformed therewith, A method for producing active DESC1, which comprises culturing the cells and recovering the active DESC1 having the structure of the present invention from the culture solution or the cultured cells, and further, the active DESC1 obtained by the production method. The present invention also includes native mammalian DESC1 purified from mammalian body fluids, cells, or cell lines in which the native mammalian DESC1 is present.

Furthermore, the present invention uses information on all or part of these nucleic acids to determine the DE corresponding to those nucleic acids.
A method for detecting the expression level of the SC1 gene is exemplified by Northern hybridization method and gene amplification method. The present invention also includes a sense primer or an antisense primer prepared based on the information of the natural mammalian DESC1 gene sequence used for carrying out such gene amplification method, for example, the information of its cDNA sequence.

The present invention further inhibits the activity of DESC1, which comprises administering a compound or polypeptide to be measured to a mammal having a disease characterized by the above-mentioned hyperactivation or upregulation of DESC1. Or a method for screening a substance that inhibits activation of the enzyme or a method for determining a therapeutic effect thereof.

The diseases characterized by the over-activation or up-regulation of DESC1 include
Examples include inflammatory diseases, cancer, and diseases caused by abnormal coagulation / fibrinolysis system. In particular, inflammatory diseases or secretory abnormalities, extracellular matrix synthesis abnormalities, cell proliferation abnormalities, cell differentiation abnormalities, nervous system abnormalities, immune system abnormalities, or diseases caused by pathological conditions due to coagulation / fibrinolytic abnormalities, adenocarcinoma in the large intestine, Examples include adenocarcinoma in the ovary.

The diseases characterized by the increased mucus production, increased inflammation, intracellular calcium influx, EGFR pathway activation, and PAR activation by DESC1 are as follows:
Examples include inflammatory diseases, cancer, and diseases caused by abnormalities in the coagulation / fibrinolytic system. In particular, inflammatory diseases or secretory abnormalities, extracellular matrix synthesis abnormalities, cell proliferation abnormalities, cell differentiation abnormalities, nervous system abnormalities, immune system abnormalities, or diseases caused by pathological conditions due to coagulation / fibrinolytic abnormalities, adenocarcinoma in the large intestine, Examples include adenocarcinoma in the ovary.

The present invention is also an antisense oligonucleotide prepared by using a part of the above-mentioned nucleic acid of the present invention. The present invention is characterized by the above-mentioned over-activation or up-regulation of DESC1. By administering a drug containing such an antisense oligonucleotide to a diseased mammal, DESC1
It also includes a method for determining the activity inhibition of or the therapeutic effect by inhibiting the activation thereof, and a therapeutic agent for the disease containing such an antisense oligonucleotide as an active ingredient. Specific examples of such diseases characterized by over-activation or up-regulation of DESC1 include the same diseases as those mentioned above.

As an embodiment of the measurement index in the DESC1 inhibitor screening system based on the mucus production promoting action, (1) at the glycoprotein level, monoclonal antibody 17Q2 (human airway mucin is used as an antigen) (antibody against mucin antigen) ( Thomas, E. et al., Am. J. Respir. C
ell Mol. Biol., 14: 104-112, 1996) and the like, enzyme immunoassay, mucus staining (AB-PAS staining), and measurement of release amount of polymer sulfate label are preferable. (2) At the gene level, as a method for measuring the amount of mucin mRNA, a real-time PCR method and a method for evaluating the transcriptional activity of a gene include a reporter gene assay system using a mucin promoter region.

In addition, the signal transduction of the EGF-R pathway is D
The main pathway of increased mucus production by ESC1 (Takeyama,
K. et al., Proc. Natl. Acad. Sci. USA, 96: 3081-30
86,1999), it is estimated that
Reactions involved in this EGF-R signal transduction pathway (Lo
uis, ML et al., Current Opinion in Cell Biolog
y, 11: 177-183, 1999).
It is possible to measure the effect of SC1 inhibitors. If it can be quantified in the routes related to the above,
All can be used as measurement indicators. For example, phosphorylation of tyrosine residues of EGF-R and phosphorylation of MAPK (MAP Kinase) in target cells can be used as an index using an immunoassay.

As an embodiment of the DESC1 inhibitor screening system based on the action of promoting mucus production, DESC1 can be applied to tissues, organs or cultured cells having a mucus-secreting ability.
The amount of secretion, mR
A method of measuring the mucus production inhibitory effect by measuring the NA amount or the transcriptional activity of the gene. EGFR-L in the above-mentioned organ or cell culture supernatant
The inhibitory effect can also be determined by a method of directly measuring the content of, or a method of indirectly adding it to another cell line and measuring it. The EGFR-L to be measured is EGF (Epidermal Groth Factor, Carpenter,
G. et al., J. Biol. Chem., 265: 7709-7712, 199
0), HB-EGF (Heparin-binding EGF-like Groth
Factor, Abraham, JA et al., Biochem. Biophys. R
es. Commun., 190: 125-133, 1993) TGF- (Transfo
rming Groth Factor-, Lee, DC et al., Pharm.
Rev., 47: 51-85, 1995), Amphiregulin
(Plowman, GD et al., Mol. Cell. Biol., 10: 1969
-1981, 1990), Betacellulin (Shing, Y.
et al., Science, 259: 1604-1607, 1993) and the like are preferred (Gerhard, R. et al., Biochim. Bioph.
ys. Acta, 1333: F179-F199, 1997). Especially EGF, H
B-EGF is preferred.

[0049]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

[Example 1] Collection of human DESC1 cDNA
Obtaining and Recombinant Expression (1) Obtaining Human Airway cDNA PolyA ⁺ RNA derived from human airway was used for Clontec.
Purchased from company h. 2.5 g of this PolyA ⁺ RNA
And oligo (dT) _12-18 using SuperScr
ipt ^™ Preamplification Sys
tem forFirst Strand cDNA
Using the Synthesis Kit (manufactured by GIBCO-BRL), cDNA was synthesized according to the attached protocol. (2) Full-length DESC1 cDNA from human respiratory tract cDNA
Human disclosed in the acquisition WO00 / 50061 Desc1
Based on the cDNA sequence of SEQ ID NO: 5, the Forward Primer and the Reverse shown in SEQ ID NO: 6
Amplified DNA of full-length DESC1 cDNA of desired length by synthesizing se Primer and performing Pyrobest ^™ PCR (manufactured by Takara Shuzo) using human airway cDNA as a template.
Got the fragment. (3) Recombinant baculo for expressing human DESC1 in insect cells
Virus production Bac-to-Bac System (GIBCO-B
(Manufactured by RL) and transferred to the transfer vector pFAST-BAC1 according to the attached protocol.
The SC1 cDNA was inserted. Further, according to the attached protocol, recombinant Bacmid for expressing insect cells was prepared and transfected into Sf9 cell line to obtain recombinant baculovirus. (4) Expression of human DESC1 by insect cells and enzyme
Activity measurement Recombinant human DESC1 was expressed by mass-culturing the insect cell line Tn5, infecting it with the above recombinant baculovirus, and culturing for 3 days. The expressed cell pellet is MP
Solubilized with ER (PIERCE) and recovered, DES
Buffer for C1 activity measurement (50 mM TrisHC
Cell-lysate was prepared by adding an equal amount of 1-0.01% BSA (pH = 8.6)). This CellL
Ysate and cell culture supernatant (= Sup) were used for activity measurement.

When the cleavage activity was evaluated using three trypsin-like synthetic substrates, the synthetic substrate: Boc-Ser-L
ys-Gly-Arg-pNA, Boc-Phe-Se
r-Arg-pNA and <Glu-Gly-Arg-
It was revealed that the recombinant human DESC1 has a pNA (S-2444 (Daiichi Pure Chemicals)) cleaving activity and retains a trypsin-like activity.

[0052]

[Table 1]

[Example 2] Purification of recombinant DESC1 Regarding the purification of recombinant DESC1, a recombinant baculovirus vector was prepared based on Example 1, and recombinant DESC1 (rDESC) was prepared by infecting insect cells.
1) was produced, and recombinant DESC1 was purified from the cell fraction. That is, insect cells (Tn-5 cells) were grown in a monolayer to a density of 5 × 10 ⁶ cells / mL, the medium was removed, and then MOI (Multiplicity of Infectio) per cell.
A serum-free medium containing recombinant DESC1 expressing baculovirus was added to the cells so that n) = 0.2-0.5, infection was performed, and the cells were cultured for 3 days to express DESC1. Confirmation of the expressed protein was confirmed by SDS-PAGE and anti-DESC1.
Western blotting using peptide antibody (Anal. Bi
ochem., 112, 195-203, 1981).

The supernatant and cells were separated by centrifugation (about 500 g). Cells equivalent to 450 mL were transferred to M-PER (Mamm
alian Protein Extraction Reagent, manufactured by PIERCE) was suspended in 100 mL and incubated at room temperature for 30 minutes. In addition, sonicate for 15 minutes on ice,
Solubilized. The obtained solubilized product was added at 10,000 rpm, 4
Centrifugation was performed at 30 ° C. for 30 minutes, and the supernatant was collected. One liter of 50 mM sodium acetate (pH 4) / 0.
After dialysis with 01% PEG6000 at room temperature for 2 hours with stirring, centrifugation was carried out at 10,000 rpm for 30 minutes at 4 ° C., and the supernatant was recovered.

Next, BSA (final concentration 100 g /
mL) and 2 liters of 50 mM TrisHC
1 (pH 8) /0.5M NaCl / 0.01% PEG
After dialysis twice at 6000, 10,000 rpm at 4 ° C
The supernatant was recovered by centrifugation for 30 minutes. The collected supernatant was placed on benzamidine sepharose 6B (10 mL bed volume) and 100 mL of 50 mM TrisHCl was added.
(PH 8) /0.5M NaCl / 0.01% PEG6
The column was washed at 000. Further 80mL 10m
After washing the column with M sodium phosphate buffer (pH 7.5), the bound protein was eluted with 10 mM HCl (pH 2). After elution, the absorbance (280 nm) of each fraction was measured, and the main peak was collected. This was cryopreserved as a purified recombinant DESC1 preparation.

[Example 3] Specific activity of purified recombinant DESC1
Sex measurement Synthetic substrate for trypsin Boc-Phe-Ser-Arg
-100 MCA (MCA: Methylcoumarin amide)
50 mM Tris- containing 0.01% of M and BSA
50 L of the DESC1-containing solution was added to 1.0 mL of HCl buffer (pH 8.6), and the mixture was incubated at 37 ° C. for 1 hour. Then, 1 mL of 30% acetic acid was added to produce 7-
The amount of amino-4-methylcoumarin (AMC) was quantified by fluorescence measurement (fluorescence 460 nm, excitation light 380 nm), and the activity of the enzyme was calculated. The activity of producing 1 pM AMC in 1 minute is defined as 1 unit.

[Example 4] Normal cells of human DESC1 and
RT-PC in beauty gland cancer (Adenocarcinoma)
Comparison of mRNA expression by R (1) Preparation of human DESC1 PCR primer Based on the nucleotide sequences disclosed in WO00 / 50061, Forward Primer, Reverse P
A trimer was created. The sequences are SEQ ID NO: 5 and SEQ ID NO: 6.
It was shown to. (2) Examination of mRNA expression by PCR of human DESC1 Human Tumor MTC P from Clontech
Anel was purchased and Pyrobest ^™ DNA Polymer was prepared using the primer prepared in (1).
PCR was performed using ase. As a control internal standard, the GAPDH gene was used as a comparative control. As a result, it was observed that the expression of DESC1 mRNA was elevated in adenocarcinomas of the large intestine and ovary, which are not normally expressed. The results are shown in Fig. 1.

[Example 5] D using recombinant DESC1
Evaluation of ESC1 Inhibitory Substance A substance having an activity of inhibiting DESC1 was evaluated by adding an evaluation substance to the DESC1 activity measurement system shown in Example 1. Of the three synthetic substrates used, B
oc-Phe-Ser-Arg-pNA.

[0059]

[Table 2]

[Example 6] Mouse DESC1 counter
Acquisition of Part cDNA (1) Acquisition of Mouse Airway cDNA The airway of a mouse (C57Black) was collected and homogenized by using 1 mL of ISOGEN per airway. 0.2 mL of chloroform was added to this homogenate, mixed by vortexing, and mixed at room temperature for 2 to 2 times.
Let stand for 3 minutes. After centrifugation at 12000 rpm for 10 minutes at 4 ° C, the upper layer was transferred to a new tube. 0.5m to this
Add L isopropyl alcohol, mix, and mix at room temperature for 1
It was left for 0 minutes. This is 15000 rpm, 4 ℃ 15
Total RNA was precipitated by centrifugation for minutes. 75% for pellets
After adding 1 mL of ethanol and mixing well, 4 ° C, 1
It was centrifuged at 0000 rpm for 5 minutes. Air dry the pellets,
Dissolve in DNase / RNase free water, -80 ° C
Saved in.

Using 2.5 g of total RNA from mouse respiratory tract and oligo (dT) _12-18 , SuperScript
t ^TM Preamplification System
m for First Strand cDNA S
synthesis Kit (manufactured by GIBCO-BRL)
Was used according to the attached protocol to synthesize cDNA.

(2) DESC from mouse respiratory tract cDNA
1 Acquisition of PCR fragment A BLAST search was performed on the mouse EST database based on the sequence of human DESC1, and a gene sequence homologous to human DESC1 shown in SEQ ID NO: 7 was obtained (Accession No. BB627871). ).

(3) Full length mouse D by 3'-RACE
3'-R based on the partial base sequence obtained by obtaining ESC1 cDNA (2)
Primers for ACE (Rapid Amplification of cDNA Ends) were synthesized. The primer sequence for 1stRACE is shown in SEQ ID NO: 8, and the primer sequence for 2ndRACE is shown in SEQ ID NO: 9. On the other hand, Total collected from the respiratory tract
Using 1 g of RNA, SMART-RACE Kit
(Manufactured by Clontech) was used to synthesize 3'-RACE cDNA, and 3'-RACE was performed using the above-mentioned RACE primer. The PCR reaction was carried out by Advantage 2 Polymerase Mi.
x (manufactured by Clontech) was used according to the attached protocol. P obtained by 3'-RACE
The CR product was separated by agarose gel electrophoresis, the main amplified DNA fragment was cut out from the agarose gel, and the TA cloning vector (Invitroge
(manufactured by N. Co.) and transformed into E. coli to obtain several clones. A plasmid was prepared from these transformants according to a standard method, and the nucleotide sequence of the inserted DNA fragment was determined. As a result, it was confirmed that the base sequence homologous to human DESC1 was extended in the 3 ′ direction starting from the sequence that matched the partial base sequence obtained in (2). Determined full-length mouse DESC
1 cDNA sequence and amino acid sequence are shown in SEQ ID NO: 4 and SEQ ID NO: 2.

(4) Ratio of homology with human DESC1
Comparison Results of comparison of amino acid homology between human DESC1 and mouse DESC1 are shown in FIG. Gene analysis software GENETYX-MAC (SOFTWARE)
DEVELOPMENT CO. LTD. ) Was used. As a result, the amino acid sequences were 76% in agreement as a whole.

[Example 7] Mammal DESC1 counter
Acquisition of Part cDNA (1) Acquisition of Mammalian Airway cDNA An airway of a mammal (rabbit) was collected and homogenized by using 5 mL of ISOGEN per about 0.5 g of the airway. The homogenate was centrifuged at 3000 rpm for 10 minutes, and 1 mL of the supernatant was collected. 0.2 mL of chloroform was added to this supernatant, mixed by vortexing, and left at room temperature for 2-3 minutes. 1 at 12000 rpm
After centrifuging for 0 minutes at 4 ° C., the upper layer was transferred to a new tube.
To this, 0.5 mL of isopropyl alcohol was added, mixed, and allowed to stand at room temperature for 10 minutes. This is 15,000 rp
Total RNA was precipitated by centrifugation at 4 ° C. for 15 minutes. After adding 1 mL of 75% ethanol to the pellet and mixing well, centrifugation was performed at 10,000 rpm for 5 minutes at 4 ° C. The pellet was air-dried, dissolved in water free of DNase and RNase, and stored at -80 ° C.

Airway-derived total R obtained above
SuperScript ^™ Preamplifi using 2.5 g NA and Oligo (dT) _12-18.
Cation System for First S
tran cDNA Synthesis Kit
(Manufactured by GIBCO-BRL) was used to synthesize cDNA according to the attached protocol.

(2) DESC1 P from airway cDNA
Acquisition of CR Fragment The homology of the nucleotide sequences of human DESC1 and mouse DESC1 was compared, and the Forward Primer shown in SEQ ID NO: 12 or SEQ ID NO: 13 and SEQ ID NO: 1
4 or Reverse P shown in SEQ ID NO: 15
Created the rimer. Using these primers in combination, using TaKaRa Taq DNA polymerase (Takara Shuzo) or Pyrobest DNA polymerase (Takara Shuzo), the mammalian airway-derived cD
PCR reaction was performed using NA as a template according to the attached protocol. As a result of subjecting the PCR product to agarose gel electrophoresis, an amplified band was observed near the desired 0.5 Kbp. This amplified DNA fragment was cut out from an agarose gel, inserted into a TA cloning vector, and transformed into E. coli to obtain a clone. A plasmid was prepared according to a standard method, and the nucleotide sequence of the inserted DNA fragment was determined. As a result, it was revealed that a gene fragment showing homology with human DESC1 was cloned.

(3) By 5'-RACE and 3'-RACE
Acquisition of full-length mammalian DESC1 cDNA for rabbit DESC1 based on the partial base sequence obtained in (2), 3'-RACE (Rapid A
mplicification of cDNAEnds)
And primers for 5'-RACE were synthesized. 1s
As the primer sequence for tRACE, 3'-RACE: SEQ ID NO: 16 and 5'-RACE: SEQ ID NO: 17 were used,
The primer sequence for 2nd RACE is 3'-RACE
For: SEQ ID NO: 18, for 5′-RACE: SEQ ID NO: 19 was used. On the other hand, using 1 g of total RNA collected from the respiratory tract, SMART-RACE Kit (Clontech
5'-RACE, 3'-RACE c
5'-RACE and 3'-R for synthesizing DNA
ACE was performed. PCR reaction is Advantage2
Polymerase Mix (manufactured by Clontech) was used according to the attached protocol. 5 '
-RACE, PCR products obtained by 3'-RACE were subjected to agarose gel electrophoresis, and the main amplified DN
The A fragment was cut out from an agarose gel. This was inserted into a TA cloning vector (manufactured by Invitrogen) and transformed into E. coli to obtain several clones. A plasmid was prepared from these transformants according to a standard method, and the nucleotide sequence of the inserted DNA fragment was determined. As a result, starting from the sequence that matched the partial base sequence obtained in (2), 5'-direction, 3'-
It was confirmed that a base sequence homologous to human DESC1 was extended in the direction. The determined full length rabbit DESC1 cDNA and amino acid sequences are shown (SEQ ID NOs: 20, 21).

[Example 8] Enzymatic activity of various mammalian DESC1s
Sex measurement (1) Various mammalian DESC1 animal cell expression plasmids
Preparation of Forward primer (SEQ ID NO: 10 in mouse) and Reverse primer (SEQ ID NO: 11 in mouse) based on the gene sequences encoding N-terminal and C-terminal of various mammalian DESC1, and respiratory tract cDNA
CDNA encoding full-length DESC1 using as a template Pyrobest DNA polymerase (Takara Shuzo)
PCR amplified by.

On the other hand, pcDNA3 (Invitroge
(manufactured by company n) was cleaved with restriction enzymes Nru-I and Kpn-I. From the human genome Elongation Fact
the promoter region of or-1 (EF-1)
rd Primer; GACTTCCGCGACGTGA
GGCTCCGGTGCCCCGTC, ReverseP
rimer; GACTGGTACCAAGCTTTTC
PC using ACGACACCTGAAATGGAAG
Amplified by R, and the amplified fragment is restricted with Nru-
It was cleaved with I and Kpn-I and inserted into the above plasmid vector to construct a pEF9 expression vector having a human EF-1 promoter. DE amplified by PCR
The SC1 fragment was inserted into the multi-cloning site downstream of the human EF-1 promoter of the pEF9 expression vector to prepare various animal DESC1 expression plasmids. The nucleotide sequence was confirmed by sequencing using an automatic sequencer.

(2) For transient expression in animal cultured cells
Evaluation of enzyme activity by human fetal kidney-derived 293EBNA cells (Invitro
gen) is cultured in a 6-well plate and about 10 ⁶ ce
5 μg of the various expression plasmids prepared in (1) for lls were added to LipofectAMINE2000 (G
10 μL (manufactured by ibcoBRL) was used for transfection according to the attached manual. Two days after transfection, 1 mL of M-PER (PIERC
B) for DESC1 activity measurement
buffer (50 mM TrisHCl-0.01% B
1 mL of SA (pH = 8.6) was added to Cel.
I Lysate. The Cell Lysate and cell culture supernatant (= Sup) were subjected to activity measurement. Synthetic substrate solution [10 mM Boc-Phe-Ser-Arg-M
Buffer of CA in DMSO for DESC1 activity measurement
20 μL diluted with 50 times with r] and 40 μL of the above measurement sample were mixed, and an increase in fluorescence intensity per minute was measured (fluorescence wavelength 460 nm, excitation wavelength 380 n
m) Enzyme activity was measured. The results are shown in Table 3.

[0072]

[Table 3]

(3) For expressing insect cells of mammalian DESC1
Production of recombinant baculovirus Mouse DESC1, rat DESC1, monkey DESC1
With respect to the Bac-to-Bac System (GI
Recombinant Bacmid for insect cell expression was prepared using BCO-BRL) according to the attached protocol, and Sf
Recombinant baculovirus was obtained by transfecting 9 cell lines.

(4) For expression of insect cells of mammalian DESC1
Large-scale expression and enzyme activity measurement by insect cell line Tn5 was mass-cultured, infected with the above recombinant baculovirus, and cultured for 3 days to express recombinant mammalian DESC1. The same procedure as in Example 1 was carried out from the expressed cell pellet to obtain purified recombinant mammalian DESC1. When the cleavage activity was evaluated using three trypsin-like synthetic substrates, the synthetic substrate: Boc-Phe-Ser-Arg-pNA was found as in human DESC1.
And Boc-Ser-Glu-Gln-Arg-pN
A, Boc-Ser-Lys-Gly-Arg-pNA
It was found that it has a trypsin-like activity similar to human DESC1.

[0075]

INDUSTRIAL APPLICABILITY According to the present invention, there is provided DESC1 having a structure in which all or part of a propeptide moiety is bound to a trypsin-like protein moiety via a disulfide bond. Using this to construct a screening system for inhibitors that activates PAR of the enzyme having the structure or enhances mucus production, activates EGFR signal transduction system, and induces intracellular calcium influx, and screens the inhibitors Thus, a DESC1 inhibitor or an inhibitory polypeptide (including an inhibitory antibody) can be obtained. DESC1 inhibitor or PA of DESC1 thus obtained
R activation inhibitor or mucus production acceleration inhibitor, EGFR
By using a signal transduction system activation inhibitor, an intracellular calcium influx induction inhibitor, or a plasminogen activator (prourokinase) activation inhibitor as a therapeutic agent, the direct or PAR activation action of DESC1 and EGFR signal transduction can be obtained. Suppresses physiological actions such as system activation action, action on inflammation by mediating intracellular calcium influx action, mucus secretion enhancement action, action in coagulation / fibrinolysis system, cell proliferation action or action relating to cancer growth or metastasis It can be expected to be modified and used for prevention of onset or improvement or treatment in disease state.

Further, by measuring or controlling the protein expression or gene expression of animal DESC1 based on the sequence of animal DESC1 such as mouse DESC1 of the present invention, the disease state of DESC1 in any animal model can be determined. Can be determined to be involved in. Further, by utilizing the present invention, it is possible to judge the therapeutic effect of the selected DESC1 inhibitor or the inhibitory polypeptide (including the inhibitory antibody) and estimate the clinical dose.

Further, by immunohistochemical staining using the monoclonal antibody or the polyclonal antibody of the present invention,
The expression site in the tissue can be detected. Further, according to the enzyme immunoassay system, the diagnostic agent, or the diagnostic kit using the monoclonal antibody or the polyclonal antibody of the present invention, for example, the DESC1 concentration in serum can be measured, which is useful for grasping and diagnosing the pathological condition related to DESC1. Is.

[0078]

[Sequence list] <110> Teijin Limited <120> Trypsin-like enzyme DESC1 and its use <130> P35640 <160> 21 <210> 1 <211> 422 <212> PRT <213> Homo sapiens <222> 1..190 propeptide portion 191..422 trypsin-like protein portion <400> 1 Met Tyr Arg Pro Asp Val Val Arg Ala Arg Lys Arg Val Cys Trp Glu   1 5 10 15 Pro Trp Val Ile Gly Leu Val Ile Phe Ile Ser Leu Ile Val Leu Ala              20 25 30 Val Cys Ile Gly Leu Thr Val His Tyr Val Arg Tyr Asn Gln Lys Lys          35 40 45 Thr Tyr Asn Tyr Tyr Ser Thr Leu Ser Phe Thr Thr Asp Lys Leu Tyr      50 55 60 Ala Glu Phe Gly Arg Glu Ala Ser Asn Asn Phe Thr Glu Met Ser Gln  65 70 75 80 Arg Leu Glu Ser Met Val Lys Asn Ala Phe Tyr Lys Ser Pro Leu Arg                  85 90 95 Glu Glu Phe Val Lys Ser Gln Val Ile Lys Phe Ser Gln Gln Lys His             100 105 110 Gly Val Leu Ala His Met Leu Leu Ile Cys Arg Phe His Ser Thr Glu         115 120 125 Asp Pro Glu Thr Val Asp Lys Ile Val Gln Leu Val Leu His Glu Lys     130 135 140 Leu Gln Asp Ala Val Gly Pro Pro Lys Val Asp Pro His Ser Val Lys 145 150 155 160 Ile Lys Lys Ile Asn Lys Thr Glu Thr Asp Ser Tyr Leu Asn His Cys                 165 170 175 Cys Gly Thr Arg Arg Ser Lys Thr Leu Gly Gln Ser Leu Arg Ile Val             180 185 190 Gly Gly Thr Glu Val Glu Glu Gly Glu Trp Pro Trp Gln Ala Ser Leu         195 200 205 Gln Trp Asp Gly Ser His Arg Cys Gly Ala Thr Leu Ile Asn Ala Thr     210 215 220 Trp Leu Val Ser Ala Ala His Cys Phe Thr Thr Tyr Lys Asn Pro Ala 225 230 235 240 Arg Trp Thr Ala Ser Phe Gly Val Thr Ile Lys Pro Ser Lys Met Lys                 245 250 255 Arg Gly Leu Arg Arg Ile Ile Val His Glu Lys Tyr Lys His Pro Ser             260 265 270 His Asp Tyr Asp Ile Ser Leu Ala Glu Leu Ser Ser Pro Val Pro Tyr         275 280 285 Thr Asn Ala Val His Arg Val Cys Leu Pro Asp Ala Ser Tyr Glu Phe     290 295 300 Gln Pro Gly Asp Val Met Phe Val Thr Gly Phe Gly Ala Leu Lys Asn 305 310 315 320 Asp Gly Tyr Ser Gln Asn His Leu Arg Gln Ala Gln Val Thr Leu Ile                 325 330 335 Asp Ala Thr Thr Cys Asn Glu Pro Gln Ala Tyr Asn Asp Ala Ile Thr             340 345 350 Pro Arg Met Leu Cys Ala Gly Ser Leu Glu Gly Lys Thr Asp Ala Cys         355 360 365 Gln Gly Asp Ser Gly Gly Pro Leu Val Ser Ser Asp Ala Arg Asp Ile     370 375 380 Trp Tyr Leu Ala Gly Ile Val Ser Trp Gly Asp Glu Cys Ala Lys Pro 385 390 395 400 Asn Lys Pro Gly Val Tyr Thr Arg Val Thr Ala Leu Arg Asp Trp Ile                 405 410 415 Thr Ser Lys Thr Gly Ile             420 422 <210> 2 <211> 423 <212> PRT <213> Mus musculus <222> 1..191 propeptide portion 192..423 trypsin-like protein portion <400> 2 Met Tyr Arg Ser Cys Val Val Arg Ala Arg Lys Arg Thr Cys Val Glu   1 5 10 15 Pro Trp Val Ile Gly Ile Ile Ser Phe Leu Ser Leu Ile Val Leu Ala              20 25 30 Val Cys Ile Gly Leu Thr Val His Tyr Val Arg Tyr Asn His Arg Arg          35 40 45 Thr Tyr Asn Tyr Tyr Ser Thr Leu Ser Phe Thr Ser Asp Lys Leu Tyr      50 55 60 Ser Glu Phe Gly Arg Glu Ala Ser Lys Asn Phe Thr Glu Met Ser Gln  65 70 75 80 Arg Ile Glu Thr Met Val Lys His Ala Phe His Lys Ser Pro Leu Arg                  85 90 95 Gly Gln Leu Val Lys Ala His Ile Ile Lys Phe Ser Lys Glu Asp Asp             100 105 110 Gly Val Leu Ala His Met Leu Leu Ile Phe Arg Ile Arg Ser Thr Glu         115 120 125 Asp Pro Glu Thr Val His Lys Ile Ile Glu Tyr Val Leu His Glu Lys     130 135 140 Leu Lys Tyr Ala Thr Gly Pro Pro Asn Val Asp Pro Glu Ser Val Lys 145 150 155 160 Ile Lys Lys Ile Asn Lys Thr Glu Ser Asp Asn Tyr Phe Asn His Cys                 165 170 175 Cys Gly Thr Arg Arg Asn Lys Ser Thr Val Gln Thr Ser Val Arg Ile             180 185 190 Val Gly Gly Thr Pro Val Glu Glu Glu Glu Trp Pro Trp Gln Ser Ser         195 200 205 Leu Arg Trp Asp Gly Ser His Arg Cys Gly Ala Thr Leu Ile Asn Asn     210 215 220 Thr Trp Leu Val Thr Ala Ala His Cys Phe Arg Thr His Lys Asp Pro 225 230 235 240 Ser Arg Trp Ser Ala Thr Phe Gly Ala Thr Leu Gln Pro Arg Lys Leu                 245 250 255 Thr Thr Gly Ile Arg Arg Ile Ile Val His Glu Lys Tyr Lys Tyr Pro             260 265 270 Ser His Asp Tyr Asp Ile Ala Leu Ala Glu Leu Ser Lys Pro Val Pro         275 280 285 Cys Thr Asn Ala Val His Lys Val Cys Leu Pro Asp Ala Asn His Glu     290 295 300 Phe Gln Pro Gly Gln Arg Met Phe Val Thr Gly Phe Gly Ala Leu Lys 305 310 315 320 Asn Asp Gly Phe Thr Gln Asn Asn Leu Arg Gln Val Gln Val Asp Tyr                 325 330 335 Ile Asp Thr Gln Thr Cys Asn Gln Pro Gln Ser Tyr Asn Gly Ala Ile             340 345 350 Thr Pro Arg Met Leu Cys Ala Gly Phe Leu Lys Gly Glu Lys Asp Ala         355 360 365 Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Thr Ala Asp Val Arg Asp     370 375 380 Ile Trp Tyr Leu Ala Gly Val Val Ser Trp Gly Asp Glu Cys Gly Gln 385 390 395 400 Pro Asn Lys Pro Gly Val Tyr Thr Arg Val Thr Ala Phe Arg His Trp                 405 410 415 Ile Ala Ser Asn Thr Gly Ile             420 423 <210> 3 <211> 1471 <212> DNA <213> Homo sapiens <222> 53 .. 1324 CDS 53 ..625 propeptide portion 626..1324 trypsin-like protein portion <400> 3 TGACTTGGAT GTAGACCTCG ACCTTCACAG GACTCTTCAT TGCTGGTTGG CA ATG ATG 58                                                           Met Met                                                             1 TAT CGG CCA GAT GTG GTG AGG GCT AGG AAA AGA GTT TGT TGG GAA CCC 106 Tyr Arg Pro Asp Val Val Arg Ala Arg Lys Arg Val Cys Trp Glu Pro           5 10 15 TGG GTT ATC GGC CTC GTC ATC TTC ATA TCC CTG ATT GTC CTG GCA GTG 154 Trp Val Ile Gly Leu Val Ile Phe Ile Ser Leu Ile Val Leu Ala Val      20 25 30 TGC ATT GGA CTC ACT GTT CAT TAT GTG AGA TAT AAT CAA AAG AAG ACC 202 Cys Ile Gly Leu Thr Val His Tyr Val Arg Tyr Asn Gln Lys Lys Thr  35 40 45 50 TAC AAT TAC TAT AGC ACA TTG TCA TTT ACA ACT GAC AAA CTA TAT GCT 250 Tyr Asn Tyr Tyr Ser Thr Leu Ser Phe Thr Thr Asp Lys Leu Tyr Ala                  55 60 65 GAG TTT GGC AGA GAG GCT TCT AAC AAT TTT ACA GAA ATG AGC CAG AGA 298 Glu Phe Gly Arg Glu Ala Ser Asn Asn Phe Thr Glu Met Ser Gln Arg              70 75 80 CTT GAA TCA ATG GTG AAA AAT GCA TTT TAT AAA TCT CCA TTA AGG GAA 346 Leu Glu Ser Met Val Lys Asn Ala Phe Tyr Lys Ser Pro Leu Arg Glu          85 90 95 GAA TTT GTC AAG TCT CAG GTT ATC AAG TTC AGT CAA CAG AAG CAT GGA 394 Glu Phe Val Lys Ser Gln Val Ile Lys Phe Ser Gln Gln Lys His Gly     100 105 110 GTG TTG GCT CAT ATG CTG TTG ATT TGT AGA TTT CAC TCT ACT GAG GAT 442 Val Leu Ala His Met Leu Leu Ile Cys Arg Phe His Ser Thr Glu Asp 115 120 125 130 CCT GAA ACT GTA GAT AAA ATT GTT CAA CTT GTT TTA CAT GAA AAG CTG 490 Pro Glu Thr Val Asp Lys Ile Val Gln Leu Val Leu His Glu Lys Leu                 135 140 145 CAA GAT GCT GTA GGA CCC CCT AAA GTA GAT CCT CAC TCA GTT AAA ATT 538 Gln Asp Ala Val Gly Pro Pro Lys Val Asp Pro His Ser Val Lys Ile             150 155 160 AAA AAA ATC AAC AAG ACA GAA ACA GAC AGC TAT CTA AAC CAT TGC TGC 586 Lys Lys Ile Asn Lys Thr Glu Thr Asp Ser Tyr Leu Asn His Cys Cys         165 170 175 GGA ACA CGA AGA AGT AAA ACT CTA GGT CAG AGT CTC AGG ATC GTT GGT 634 Gly Thr Arg Arg Ser Lys Thr Leu Gly Gln Ser Leu Arg Ile Val Gly     180 185 190 GGG ACA GAA GTA GAA GAG GGT GAA TGG CCC TGG CAG GCT AGC CTG CAG 682 Gly Thr Glu Val Glu Glu Gly Glu Trp Pro Trp Gln Ala Ser Leu Gln 195 200 205 210 TGG GAT GGG AGT CAT CGC TGT GGA GCA ACC TTA ATT AAT GCC ACA TGG 730 Trp Asp Gly Ser His Arg Cys Gly Ala Thr Leu Ile Asn Ala Thr Trp                 215 220 225 CTT GTG AGT GCT GCT CAC TGT TTT ACA ACA TAT AAG AAC CCT GCC AGA 778 Leu Val Ser Ala Ala His Cys Phe Thr Thr Tyr Lys Asn Pro Ala Arg             230 235 240 TGG ACT GCT TCC TTT GGA GTA ACA ATA AAA CCT TCG AAA ATG AAA CGG 826 Trp Thr Ala Ser Phe Gly Val Thr Ile Lys Pro Ser Lys Met Lys Arg         245 250 255 GGT CTC CGG AGA ATA ATT GTC CAT GAA AAA TAC AAA CAC CCA TCA CAT 874 Gly Leu Arg Arg Ile Ile Val His Glu Lys Tyr Lys His Pro Ser His     260 265 270 GAC TAT GAT ATT TCT CTT GCA GAG CTT TCT AGC CCT GTT CCC TAC ACA 922 Asp Tyr Asp Ile Ser Leu Ala Glu Leu Ser Ser Pro Val Pro Tyr Thr 275 280 285 290 AAT GCA GTA CAT AGA GTT TGT CTC CCT GAT GCA TCC TAT GAG TTT CAA 970 Asn Ala Val His Arg Val Cys Leu Pro Asp Ala Ser Tyr Glu Phe Gln                295 300 305 CCA GGT GAT GTG ATG TTT GTG ACA GGA TTT GGA GCA CTG AAA AAT GAT 1018 Pro Gly Asp Val Met Phe Val Thr Gly Phe Gly Ala Leu Lys Asn Asp             310 315 320 GGT TAC AGT CAA AAT CAT CTT CGA CAA GCA CAG GTG ACT CTC ATA GAC 1066 Gly Tyr Ser Gln Asn His Leu Arg Gln Ala Gln Val Thr Leu Ile Asp         325 330 335 GCT ACA ACT TGC AAT GAA CCT CAA GCT TAC AAT GAC GCC ATA ACT CCT 1114 Ala Thr Thr Cys Asn Glu Pro Gln Ala Tyr Asn Asp Ala Ile Thr Pro     340 345 350 AGA ATG TTA TGT GCT GGC TCC TTA GAA GGA AAA ACA GAT GCA TGC CAG 1162 Arg Met Leu Cys Ala Gly Ser Leu Glu Gly Lys Thr Asp Ala Cys Gln 355 360 365 370 GGT GAC TCT GGA GGA CCA CTG GTT AGT TCA GAT GCT AGA GAT ATC TGG 1210 Gly Asp Ser Gly Gly Pro Leu Val Ser Ser Asp Ala Arg Asp Ile Trp                 375 380 385 TAC CTT GCT GGA ATA GTG AGC TGG GGA GAT GAA TGT GCG AAA CCC AAC 1258 Tyr Leu Ala Gly Ile Val Ser Trp Gly Asp Glu Cys Ala Lys Pro Asn             390 395 400 AAG CCT GGT GTT TAT ACT AGA GTT ACG GCC TTG CGG GAC TGG ATT ACT 1306 Lys Pro Gly Val Tyr Thr Arg Val Thr Ala Leu Arg Asp Trp Ile Thr         405 410 415 TCA AAA ACT GGT ATC TAA GAGAGAAAAG CCTCATGGAA CAGATAACAT TTTTTTTT 1362 Ser Lys Thr Gly Ile     420 423 GTTTTTTGGG TGTGGAGGCC ATTTTTAGAG ATACAGAATT GGAGAAGACT TGCAAAACAG 1422 CTAGATTTGA CTGATCTCAA TAAACTGTTT GCTTGATGCA AAAAAAAAA 1471 <210> 4 <211> 1430 <212> DNA <213> Mus musculus <222> 25 .. 1296 CDS 25 .. 597 propeptide portion 598..1296 trypsin-like protein portion <400> 4 GGACTCGTAA CTTGCTGGTC AGCA ATG TAT CGG TCA TGT GTG GTG AGG GCC 51                            Met Tyr Arg Ser Cys Val Val Arg Ala                              1 5 AGG AAG AGA ACG TGT GTG GAG CCC TGG GTT ATC GGC ATC ATT TCT TTT 99 Arg Lys Arg Thr Cys Val Glu Pro Trp Val Ile Gly Ile Ile Ser Phe  10 15 20 25 TTA TCC CTG ATT GTC TTG GCA GTG TGC ATT GGG CTC ACT GTT CAC TAT 147 Leu Ser Leu Ile Val Leu Ala Val Cys Ile Gly Leu Thr Val His Tyr                  30 35 40 GTC AGA TAC AAT CAC AGG AGA ACC TAC AAT TAC TAC AGC ACG TTG TCA 195 Val Arg Tyr Asn His Arg Arg Thr Tyr Asn Tyr Tyr Ser Thr Leu Ser              45 50 55 TTC ACA AGT GAC AAG CTA TAT TCT GAG TTT GGA AGA GAG GCT TCT AAA 243 Phe Thr Ser Asp Lys Leu Tyr Ser Glu Phe Gly Arg Glu Ala Ser Lys          60 65 70 AAT TTC ACA GAA ATG AGC CAG AGA ATT GAA ACT ATG GTA AAA CAT GCA 291 Asn Phe Thr Glu Met Ser Gln Arg Ile Glu Thr Met Val Lys His Ala      75 80 85 TTT CAT AAA TCT CCA CTA CGG GGA CAA CTT GTC AAG GCT CAC ATT ATC 339 Phe His Lys Ser Pro Leu Arg Gly Gln Leu Val Lys Ala His Ile Ile  90 95 100 105 AAG TTC AGT AAA GAA GAT GAT GGA GTG TTG GCT CAC ATG CTG CTG ATT 387 Lys Phe Ser Lys Glu Asp Asp Gly Val Leu Ala His Met Leu Leu Ile                  110 115 120 TTT CGA ATT CGC TCT ACC GAG GAT CCT GAA ACT GTG CAT AAA ATT ATT 435 Phe Arg Ile Arg Ser Thr Glu Asp Pro Glu Thr Val His Lys Ile Ile             125 130 135 GAG TAT GTT CTG CAT GAA AAG CTG AAG TAT GCT ACG GGA CCC CCC AAT 483 Glu Tyr Val Leu His Glu Lys Leu Lys Tyr Ala Thr Gly Pro Pro Asn         140 145 150 GTT GAT CCT GAG TCA GTT AAA ATT AAA AAA ATC AAC AAG ACA GAA TCA 531 Val Asp Pro Glu Ser Val Lys Ile Lys Lys Ile Asn Lys Thr Glu Ser     155 160 165 GAC AAC TAT TTC AAC CAT TGT TGT GGG ACA CGA CGG AAC AAA TCT ACA 579 Asp Asn Tyr Phe Asn His Cys Cys Gly Thr Arg Arg Asn Lys Ser Thr 170 175 180 185 GTA CAG ACT AGT GTG AGG ATT GTT GGA GGG ACA CCA GTA GAA GAG GAA 627 Val Gln Thr Ser Val Arg Ile Val Gly Gly Thr Pro Val Glu Glu Glu                 190 195 200 GAG TGG CCA TGG CAA AGC AGC CTG CGG TGG GAT GGG TCC CAC CGC TGT 675 Glu Trp Pro Trp Gln Ser Ser Leu Arg Trp Asp Gly Ser His Arg Cys             205 210 215 GGA GCA ACT TTG ATC AAC AAC ACA TGG CTT GTG ACT GCT GCT CAC TGC 723 Gly Ala Thr Leu Ile Asn Asn Thr Trp Leu Val Thr Ala Ala His Cys         220 225 230 TTT CGA ACA CAC AAG GAC CCA TCC AGA TGG AGT GCT ACC TTT GGG GCA 771 Phe Arg Thr His Lys Asp Pro Ser Arg Trp Ser Ala Thr Phe Gly Ala     235 240 245 ACA TTA CAA CCT CGA AAA CTG ACG ACA GGC ATC CGG AGG ATA ATT GTT 819 Thr Leu Gln Pro Arg Lys Leu Thr Thr Gly Ile Arg Arg Ile Ile Val 250 255 260 265 CAT GAA AAA TAC AAA TAC CCG TCC CAT GAC TAT GAC ATT GCA CTG GCA 867 His Glu Lys Tyr Lys Tyr Pro Ser His Asp Tyr Asp Ile Ala Leu Ala                 270 275 280 GAA CTG TCT AAG CCG GTC CCC TGC ACA AAT GCA GTG CAT AAG GTT TGT 915 Glu Leu Ser Lys Pro Val Pro Cys Thr Asn Ala Val His Lys Val Cys             285 290 295 CTC CCA GAT GCA AAC CAT GAG TTT CAG CCG GGG CAG AGG ATG TTT GTG 963 Leu Pro Asp Ala Asn His Glu Phe Gln Pro Gly Gln Arg Met Phe Val         300 305 310 ACA GGA TTT GGA GCA TTG AAA AAT GAT GGT TTC ACC CAG AAT AAC CTT 1011 Thr Gly Phe Gly Ala Leu Lys Asn Asp Gly Phe Thr Gln Asn Asn Leu     315 320 325 CGG CAA GTA CAG GTG GAC TAC ATA GAT ACC CAG ACC TGC AAT CAG CCT 1059 Arg Gln Val Gln Val Asp Tyr Ile Asp Thr Gln Thr Cys Asn Gln Pro 330 335 340 345 CAA TCT TAC AAC GGA GCC ATC ACT CCT AGG ATG TTG TGC GCT GGC TTC 1107 Gln Ser Tyr Asn Gly Ala Ile Thr Pro Arg Met Leu Cys Ala Gly Phe                 350 355 360 TTA AAA GGA GAA AAA GAT GCG TGC CAG GGT GAC TCG GGA GGT CCA CTT 1155 Leu Lys Gly Glu Lys Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu             365 370 375 GTT ACT GCA GAT GTT AGA GAT ATA TGG TAC CTT GCT GGA GTA GTG AGC 1203 Val Thr Ala Asp Val Arg Asp Ile Trp Tyr Leu Ala Gly Val Val Ser         380 385 390 TGG GGA GAT GAA TGT GGC CAA CCC AAT AAG CCT GGT GTT TAC ACT AGA 1251 Trp Gly Asp Glu Cys Gly Gln Pro Asn Lys Pro Gly Val Tyr Thr Arg     395 400 405 GTG ACC GCC TTC CGA CAC TGG ATT GCT TCC AAC ACT GGT ATC TAA ACCAG1301 Val Thr Ala Phe Arg His Trp Ile Ala Ser Asn Thr Gly Ile 410 415 420 423 AAAGACCCAG AGGGATGAAA TGGATGGTTT TATCTGTTGA CAGTTCTTGG AGACACACAA 1361 ATGGAAAAGA AGCCTTGCAA ACCAGGAGGG CCCGAAACAC CACACTGAAC AATTCTGAAG 1421 TGTACATTT 1430 <210> 5 <211> 37 <212> DNA <213> Homo sapiens <400> 5 GGTTGAATTC AAAATGTATC GGCCAGATGT GGTGAGG 37 <210> 6 <211> 40 <212> DNA <213> Homo sapiens <400> 6 GGTTGCGGCC GCTAGATACC AGTTTTTGAA GTAATCCAGT 40 <210> 7 <211> 676 <212> DNA <213> Mus musculus <400> 7 TGGACTCGTA ACTTGCTGGT CAGCA ATG TAT CGG TCA TGT GTG GTG AGG GCC 52 AGG AAG AGA ACG TGT GTG GAG CCC TGG GTT ATC GGC ATC ATT TCT TTT 100 TTA TCC CTG ATT GTC TTG GCA GTG TGC ATT GGG CTC ACT GTT CAC TAT 148 GTC AGA TAC AAT CAC AGG AGA ACC TAC AAT TAC TAC AGC ACG TTG TCA 196 TTC ACA AGT GAC AAG CTA TAT TCT GAG TTT GGA AGA GAG GCT TCT AAA 244 AAT TTC ACA GAA ATG AGC CAG AGA ATT GAA ACT ATG GTA AAA CAT GCA 292 TTT CAT AAA TCT CCA CTA CGG GGA CAA CTT GTC AAG GCT CAC ATT ATC 340 AAG TTC AGT AAA GAA GAT GAT GGA GTG TTG GCT CAC ATG CTG CTG ATT 388 TTT CGA ATT CGC TCT ACC GAG GAT CCT GAA ACT GTG CAT AAA ATT ATT 436 GAG TAT GTT CTG CAT GAA AAG CTG AAG TAT GCT ACG GGA CCC CCC AAT 484 GTT GAT CCT GAG TCA GTT AAA ATT AAA AAA ATC AAC AAG ACA GAA TCA 532 GAC AAC TAT TTC AAC CAT TGT TGT GGG ACA CGA CGG AAC ATA TCT ACA 580 GTA CAG ACT AGT GTG AGG ATT GTT GGA GGG ACA CCA GTA GAA GAG GAA 628 GAG TGG CCA TGG CAA AGC AGC CTG CGG TGG GAT GGG TCC CAC CGC TGT 676 <210> 8 <211> 24 <212> DNA <213> Mus musculus <400> 8 CTTGCTGGTC AGCAATGTAT CGGT 24 <210> 9 <211> 24 <212> DNA <213> Mus musculus <400> 9 TGGTCAGCAA TGTATCGGTC ATGT 24 <210> 10 <211> 35 <212> DNA <213> Mus musculus <400> 10 GGTTAGATCT CAGCAATGTA TCGGTCATGT GTGGT 35 <210> 11 <211> 41 <212> DNA <213> Mus musculus <400> 11 GGTTGCGGCC GCTTAGATAC CAGTGTTGGA AGCAATCCAG T 41 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> PCR Primer <400> 12 CCTGATTGTC YTGGCAGTGT GCATTGG 27 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> PCR Primer <400> 13 CTAAMAATTT YACAGAAATG AGCCAGAGA 29 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> PCR Primer <400> 14 GAAATAGYTG TCTGWTTCTG TCTTGTTG 28 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> PCR Primer <400> 15 TGCCAKGGCC AYTCWYCCTC TTCTACT 27 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> PCR Primer <400> 16 CCTGATTGTC YTGGCAGTGT GCATTGG 27 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> PCR Primer <400> 17 GAAATAGYTG TCTGWTTCTG TCTTGTTG 28 <210> 18 <211> 29 <212> DNA <213> Artificial Sequence <220> PCR Primer <400> 18 CTAAMAATTT YACAGAAATG AGCCAGAGA 29 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> PCR Primer <400> 19 CCAACACTCC ATCTTCCTCC TGACT 25 <210> 20 <211> 1874 <212> DNA <213> Oryctolagus cuniculus <222> 88 ..1356 CDS 88 ..657 propeptide portion 658..1356 trypsin-like protein portion <400> 20 CAAGAAGCTG GATGCACTGA CAGAGCTGGA GGCGCACACT TGACTGCCAA CCTCTGTGTC 60 CACAGGACTC CTCCTTGCTG GTCAGCG ATG TAT CGG TCA GCT GTG GTG AAA GAC 114                               Met Tyr Arg Ser Ala Val Val Lys Asp                                 1 5 AGG AAA AGT GCC TGT TTG GAG CCC TGG GTT ATC GGC CTC ATC ACC TTC 162 Arg Lys Ser Ala Cys Leu Glu Pro Trp Val Ile Gly Leu Ile Thr Phe  10 15 20 25 CTA TCT CTG ATT GTC CTG GCA GTG TGT ATT GGA GTC ACT GTT CAT TAC 210 Leu Ser Leu Ile Val Leu Ala Val Cys Ile Gly Val Thr Val His Tyr                  30 35 40 GTG AGA CAC AAT CAC AGA GAG ACC CAC CAT TAC TAT AGC ACA TTG TTA 258 Val Arg His Asn His Arg Glu Thr His His Tyr Tyr Ser Thr Leu Leu              45 50 55 TTC ACA AGT GAC AAA GTA TAT GGT GAC TTT GGA AAG GAG GCT TCT AAA 306 Phe Thr Ser Asp Lys Val Tyr Gly Asp Phe Gly Lys Glu Ala Ser Lys          60 65 70 AAT TTC ACA GAA ATG AGC CAG AGA ATT GAA TCA ATG GTG AAA AAT GCA 354 Asn Phe Thr Glu Met Ser Gln Arg Ile Glu Ser Met Val Lys Asn Ala      75 80 85 TTC CAT AAA TCT CCA TTA AGG GGA GAA TTT GTC AAG TCT CAC ATT ATC 402 Phe His Lys Ser Pro Leu Arg Gly Glu Phe Val Lys Ser His Ile Ile  90 95 100 105 AAG TAC AGT CAG GAG GAA GAT GGA GTG TTG GCC CAT ATG CTG CTG ATT 450 Lys Tyr Ser Gln Glu Glu Asp Gly Val Leu Ala His Met Leu Leu Ile                 110 115 120 TTT CGA TTC CGC TCT ACT GAA GAT ACT GAA AAC ATC AAT AAA ATT ATT 498 Phe Arg Phe Arg Ser Thr Glu Asp Thr Glu Asn Ile Asn Lys Ile Ile             125 130 135 CAG CGT ATT CTA AAT GAA AAG CTA CAA GAT GCT ACG GGA CCT CCT AAG 546 Gln Arg Ile Leu Asn Glu Lys Leu Gln Asp Ala Thr Gly Pro Pro Lys         140 145 150 CTA GAT CCT GAA TCA GTT GAA ATT AAA AAA ATC AAC AAG ACA GAA ACA 594 Leu Asp Pro Glu Ser Val Glu Ile Lys Lys Ile Asn Lys Thr Glu Thr     155 160 165 GAC AAA TTT CTG AAC AAC TGT TGT GGG ACA CGA AGG AGT AAA TCC GCA 642 Asp Lys Phe Leu Asn Asn Cys Cys Gly Thr Arg Arg Ser Lys Ser Ala 170 175 180 185 GGT CAG AGT GTC AGG ATA GTT GGT GGT ACA GAG GTC GAA GAA GGA GAA 690 Gly Gln Ser Val Arg Ile Val Gly Gly Thr Glu Val Glu Glu Gly Glu                 190 195 200 TGG CCG TGG CAA GCC AGC CTG CAA TGG GAT GGG TTC CAT CGC TGT GGA 738 Trp Pro Trp Gln Ala Ser Leu Gln Trp Asp Gly Phe His Arg Cys Gly             205 210 215 GCC ACT CTA ATT AAC AGC ACA TGG CTT CTG AGT GCT GCT CAC TGC TTT 786 Ala Thr Leu Ile Asn Ser Thr Trp Leu Leu Ser Ala Ala His Cys Phe         220 225 230 ATC TTG TAT AAG GAC CCA ACC AGA TGG ACG GCA TCG TTT GGA GTA ACA 834 Ile Leu Tyr Lys Asp Pro Thr Arg Trp Thr Ala Ser Phe Gly Val Thr     235 240 245 CTA AAT CCT CTG AAG ATG AAA CAA GGC CTC CGG AGG ATA ATT GTC CAT 882 Leu Asn Pro Leu Lys Met Lys Gln Gly Leu Arg Arg Ile Ile Val His 250 255 260 265 GAG AAA TAC AAT CAC TCA ACG CAT GAC TAT GAT ATT TCA CTG GCA GAG 930 Glu Lys Tyr Asn His Ser Thr His Asp Tyr Asp Ile Ser Leu Ala Glu                 270 275 280 CTT TCT AGG CCT GTT CCC TAC ACA AAC GCC GTA CAT AGA ATC TGT CTC 978 Leu Ser Arg Pro Val Pro Tyr Thr Asn Ala Val His Arg Ile Cys Leu             285 290 295 CCT GAG GCA AGT CAT GAG TTC CGC CCA GGT GAG GAG ATG TTT GTG ACA 1026 Pro Glu Ala Ser His Glu Phe Arg Pro Gly Glu Glu Met Phe Val Thr         300 305 310 GGA TTT GGR GCA CGA CAG AAT GAT GGT TTC AGT CAA AAT CAC CTT CGA 074 Gly Phe Xaa Ala Arg Gln Asn Asp Gly Phe Ser Gln Asn His Leu Arg     315 320 325 GAA GTA CAG GTG GAT CTT ATA GAC ACT GAT ACC TGC AAT GAT CCA CAA 1122 Glu Val Gln Val Asp Leu Ile Asp Thr Asp Thr Cys Asn Asp Pro Gln 330 335 340 345 GTT TAT AAT GGT GCC ATA ACT CCT AGA ATG CTA TGT GCT GGC CCC TTA 1170 Val Tyr Asn Gly Ala Ile Thr Pro Arg Met Leu Cys Ala Gly Pro Leu                 350 355 360 CAA GGC AAA AGA GAT GCA TGT CAG GGT GAC TCT GGT GGA CCG CTG GTT 1218 Gln Gly Lys Arg Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Val             365 370 375 ACT TCA GAT CCT AGG AAC ATC TGG TAC ATT GCT GGG ATT GTG AGC TGG 1266 Thr Ser Asp Pro Arg Asn Ile Trp Tyr Ile Ala Gly Ile Val Ser Trp         380 385 390 GGA GAT GAA TGC GGG CAA CCC AAT AAG CCT GGT GTC TAT ACT AGA GTG 1314 Gly Asp Glu Cys Gly Gln Pro Asn Lys Pro Gly Val Tyr Thr Arg Val     395 400 405 ACT GCC CTC CGT GAC TGG ATC ACT TCC AAA ACT GGC CTT TAA GAGAGAAAA1365 Thr Ala Leu Arg Asp Trp Ile Thr Ser Lys Thr Gly Leu 410 415 420 422 ACCCAGTGAA GTGGAATATG CCTTTGTGTG TGGAGGCCGT TTTTAGACAT AGAGAATCGA 1425 AGAGGAAACC TTGTGATTCA CGGGAATCTG ATCCATCTCT CCAAAGTGTG TGCTTGATGC 1485 ATACATATCT CTTCCAGCTC TGCTCCATGC CAGAGCTTCC TGCTTCTACC AGATGGRCTC 1545 TGTCCCAACC TGCATTTATT TGTTTTCTGG AAGTTTYCAT TCTTCAAACT TTTGGCTTTT 1605 TGGCATAAAT TTTTCATGCA TATATTCATC TTCTTAGTTT CTCTCCTTTC AGTAAATTTC 1665 TACTTTCAAG GTGTGGCATR AAGAATTAAA GAAAATATAT AAAGGAAAAA ACTACATTTT 1725 TTATGTACAC AGAAGTATTA TGTGTTTTCT TTTTCTCCCT GTGGAATGTG GAAGATGTTC 1785 ATAGTCATTA TGAAAGGTCA AACAAAGAGA CATCAGAATA CCAAATACTT CATGATGGAG 1845 AATGGGGACT AAAGAAAGGA AATCAGAAA 1874 <210> 21 <211> 422 <212> PRT <213> Oryctolagus cuniculus <222> 1..190 propeptide portion 191..423 trypsin-like protein portion <400> 21 Met Tyr Arg Ser Ala Val Val Lys Asp Arg Lys Ser Ala Cys Leu Glu   1 5 10 15 Pro Trp Val Ile Gly Leu Ile Thr Phe Leu Ser Leu Ile Val Leu Ala              20 25 30 Val Cys Ile Gly Val Thr Val His Tyr Val Arg His Asn His Arg Glu          35 40 45 Thr His His Tyr Tyr Ser Thr Leu Leu Phe Thr Ser Asp Lys Val Tyr      50 55 60 Gly Asp Phe Gly Lys Glu Ala Ser Lys Asn Phe Thr Glu Met Ser Gln  65 70 75 80 Arg Ile Glu Ser Met Val Lys Asn Ala Phe His Lys Ser Pro Leu Arg                  85 90 95 Gly Glu Phe Val Lys Ser His Ile Ile Lys Tyr Ser Gln Glu Glu Asp             100 105 110 Gly Val Leu Ala His Met Leu Leu Ile Phe Arg Phe Arg Ser Thr Glu         115 120 125 Asp Thr Glu Asn Ile Asn Lys Ile Ile Gln Arg Ile Leu Asn Glu Lys     130 135 140 Leu Gln Asp Ala Thr Gly Pro Pro Lys Leu Asp Pro Glu Ser Val Glu 145 150 155 160 Ile Lys Lys Ile Asn Lys Thr Glu Thr Asp Lys Phe Leu Asn Asn Cys                 165 170 175 Cys Gly Thr Arg Arg Ser Lys Ser Ala Gly Gln Ser Val Arg Ile Val             180 185 190 Gly Gly Thr Glu Val Glu Glu Gly Glu Trp Pro Trp Gln Ala Ser Leu         195 200 205 Gln Trp Asp Gly Phe His Arg Cys Gly Ala Thr Leu Ile Asn Ser Thr     210 215 220 Trp Leu Leu Ser Ala Ala His Cys Phe Ile Leu Tyr Lys Asp Pro Thr 225 230 235 240 Arg Trp Thr Ala Ser Phe Gly Val Thr Leu Asn Pro Leu Lys Met Lys                 245 250 255 Gln Gly Leu Arg Arg Ile Ile Val His Glu Lys Tyr Asn His Ser Thr             260 265 270 His Asp Tyr Asp Ile Ser Leu Ala Glu Leu Ser Arg Pro Val Pro Tyr         275 280 285 Thr Asn Ala Val His Arg Ile Cys Leu Pro Glu Ala Ser His Glu Phe     290 295 300 Arg Pro Gly Glu Glu Met Phe Val Thr Gly Phe Gly Ala Arg Gln Asn 305 310 315 320 Asp Gly Phe Ser Gln Asn His Leu Arg Glu Val Gln Val Asp Leu Ile                 325 330 335 Asp Thr Asp Thr Cys Asn Asp Pro Gln Val Tyr Asn Gly Ala Ile Thr             340 345 350 Pro Arg Met Leu Cys Ala Gly Pro Leu Gln Gly Lys Arg Asp Ala Cys         355 360 365 Gln Gly Asp Ser Gly Gly Pro Leu Val Thr Ser Asp Pro Arg Asn Ile     370 375 380 Trp Tyr Ile Ala Gly Ile Val Ser Trp Gly Asp Glu Cys Gly Gln Pro 385 390 395 400 Asn Lys Pro Gly Val Tyr Thr Arg Val Thr Ala Leu Arg Asp Trp Ile                 405 410 415 Thr Ser Lys Thr Gly Leu             420 422

[Brief description of drawings]

FIG. 1 is a diagram comparing the expression of the DESC1 gene in normal tissues and cancer tissues.

FIG. 2 is a diagram comparing the amino acid sequence homology between human DESC1 and mouse DESC1.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07K 16/40 C12N 1/15 4B065 C12N 1/15 1/19 4C084 1/19 1/21 4H045 1/21 9/48 5/10 C12Q 1/02 9/48 1/37 C12Q 1/02 1/68 A 1/37 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 33/53 S 33/50 33/577 B 33/53 C12P 21/08 33/577 G01N 33/574 A // C12P 21/08 C12N 15/00 ZNAA G01N 33/574 5/00 AF Term (reference) 2G045 AA34 AA35 BB10 BB20 BB46 BB51 CB01 DA13 DA36 FA29 FB01 FB02 FB03 FB05 FB12 GC10 GC15 4B024 AA01 AA11 AA12 BA14 CA04 EA03 GA11 HA12 HA15 4B050 CC01 CC03 DD11 LL01 LL03 4B063 QA18 QQ36 AQAQAQAQAQAQAQBQQQAQQQAQQQAQQQAQQQAQQQAQQQAQQQAQQAQQQAQQAQA AB01 AC14 B A02 CA33 CA44 CA46 4C084 AA17 NA14 ZA662 ZA812 ZB262 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA76 DA86 DA89 EA24 EA28 EA50 EA51 FA72 FA74

Claims (34)

[Claims] 1. A protein consisting of all or part of the amino acid sequence shown in SEQ ID NO: 1, which is the first Me
One disulfide is composed of a propeptide part consisting of all or part of the amino acid sequence between t and 190th Arg and a trypsin-like protein part consisting of 232 amino acid sequence from 191st Ile to 423nd Ile. Active DES having a structure connected by a bond
C1. 2. The active DESC1 shown in SEQ ID NO: 1.
Mammalian active DESC1 having a structure in which a portion corresponding to the propeptide portion and a portion corresponding to the trypsin-like protein portion are linked by one disulfide bond. 3. An enzyme substrate and a compound to be measured or a polypeptide are mixed, and the active DESC1 or the enzyme-expressing cell or the enzyme-expressing tissue according to claim 1 or 2 is incubated under appropriate conditions. The active DES of the compound to be measured or the polypeptide by reacting with the enzyme substrate and measuring the reaction product
A method for detecting the inhibitory activity of C1. 4. The activated DE according to claim 1 or 2.
SC1 or the enzyme-expressing cell or the enzyme-expressing tissue, the compound or polypeptide to be measured, and the protease-activated receptor-expressing cell are mixed, and the activation of the protease-activated receptor signal transduction pathway is used as an index to be measured. Active DES of compound or polypeptide
A method for detecting an inhibitory activity against activation of a protease-activated receptor signal transduction pathway by C1. 5. The detection method according to claim 4, wherein the protease-activated receptor is PAR2. 6. A method for evaluating the inhibitory activity of a compound to be measured or a polypeptide against the induction of intracellular calcium influx by the activated DESC1 according to claim 1 or 2, using the intracellular calcium concentration as an index. 7. Inhibition of a compound or a polypeptide to be measured against the activity of enhancing IL-8 release by the active DESC1 according to claim 1 or 2, using the release amount or expression level of IL-8 or the transcription activity of a promoter as an index. How to assess activity. 8. The active DE according to claim 1 or 2.
SC1 or the enzyme-expressing cell or the enzyme-expressing tissue, the compound or polypeptide to be measured, and the cell having mucus-producing ability are mixed, and the active form DE of the compound or polypeptide to-be-measured using mucus-producing ability as an index
A method for detecting the inhibitory activity against the enhancement of mucus production by SC1. 9. A method for measuring mucus producing ability is AB-PA.
The detection method according to claim 8, which is an S staining method. 10. The detection method according to claim 8, wherein the indicator of mucus-producing ability is mucin expression level. 11. The detection method according to claim 8, wherein the index of mucus-producing ability is the amount of EGFR-L released or produced. 12. The detection method according to claim 8, wherein the indicator of mucus-producing ability is activation of EGF-R signal transduction pathway. 13. The active form D according to claim 1 or 2.
ESC1 or cells expressing the enzyme, tissues expressing the enzyme, compound to be measured or polypeptide, and EGF
A method of mixing with a cell having an R signal transduction system, and detecting an inhibitory activity against activation of EGFR pathway by an active DESC1 of a compound to be measured or a polypeptide using EGFR signal transduction system activation as an index. 14. The active form D according to claim 1 or 2.
A natural mammalian DESC1 gene encoding ESC1, mRNA derived from the gene, or cDNA. 15. The gene according to claim 14, mRN.
The expression vector of active DESC1 according to claim 1 or 2, which is prepared by using A or all or part of cDNA. 16. A cell transfected or transformed with the expression vector according to claim 15. 17. The cell according to claim 16 is cultured, and the active DESC1 according to claim 1 or 2 is cultured.
How to purify. 18. The activated DESC1 according to claim 1 or 2, which is obtained by using the method according to claim 17. 19. A DESC corresponding to a nucleic acid according to claim 14, which uses information on all or part of the nucleic acid.
A method for detecting the expression level of one gene. 20. The DESC according to claim 1 or 2.
1. Inhibiting the activity of activated DESC1 or activating the enzyme, which comprises administering a compound or polypeptide to be measured to a mammal having a disease characterized by hyperactivation or upregulation of 1. A method for screening an inhibitory substance, or a method for determining a therapeutic effect thereof. 21. The active form D according to claim 1 or 2.
Protease-activated receptor by activated DESC1 comprising administering a compound or polypeptide to be measured to a mammal having a disease characterized by hyperactivation or upregulation of protease-activated receptor by ESC1 A method for screening a substance that inhibits activation, or a method for determining a therapeutic effect thereof. 22. The active form D according to claim 1 or 2.
A method for screening a substance that inhibits the enhanced mucus production by active DESC1, which comprises administering a compound or polypeptide to be measured to a mammal having a disease characterized by the enhanced mucus production or upregulation by ESC1 or How to determine the therapeutic effect. 23. The activated form D according to claim 1 or 2.
A substance for inhibiting EGFR pathway activation by active DESC1, which comprises administering a compound or polypeptide to be measured to a mammal having a disease characterized by ESC1 activation or upregulation of EGFR pathway Or a method for determining the therapeutic effect thereof. 24. The activated form D according to claim 1 or 2.
An antibody that specifically binds to ESC1. 25. The active form D according to claim 1 or 2.
A monoclonal antibody that specifically binds to ESC1. 26. The active form D according to claim 1 or 2.
A monoclonal antibody that inhibits ESC1 activity or inhibits its activation. 27. The active DESC according to claim 1 or 2, which comprises using the antibody according to claim 24 or 26.
A method for detecting or measuring 1. 28. The DESC according to claim 1 or 2.
A method for diagnosing cancer using the over-activation or up-regulation of 1 as an index. 29. The DESC according to claim 1 or 2.
1. A method for diagnosing cancer in the large intestine using the hyperactivation or upregulation of 1 as an index. 30. The DESC according to claim 1 or 2.
A method for diagnosing cancer in the ovary using the hyperactivation or upregulation of 1 as an index. 31. The DESC according to claim 1 or 2.
A method for diagnosing adenocarcinoma in the large intestine using the hyperactivation or upregulation of 1 as an index. 32. A method for treating cancer in the large intestine using an active DESC1 inhibitor (compound or polypeptide) obtained by using the screening method according to any one of claims 3 to 13 or 20 to 23. . 33. A method for treating cancer in the ovary using an active DESC1 inhibitor (compound or polypeptide) obtained by using the screening method according to any one of claims 3 to 13 or 20 to 23. . 34. A method for treating adenocarcinoma in the large intestine using an active DESC1 inhibitor (compound or) obtained by using the screening method according to any one of claims 3 to 13 or 20 to 23.