U.S. patent application number 10/860844 was filed with the patent office on 2005-09-15 for novel endonuclease of immune cell, process for producing the same and immune adjuvant using the same.
This patent application is currently assigned to Cheil Jedang Corporation. Invention is credited to Jeon, Yeong Joong, Jung, Sang Bo, Kim, Doo Sik, Kwon, Hyung Joo, Lee, Na Gyong, Park, Wan Je.
Application Number | 20050203039 10/860844 |
Document ID | / |
Family ID | 34525544 |
Filed Date | 2005-09-15 |
United States Patent
Application |
20050203039 |
Kind Code |
A1 |
Jeon, Yeong Joong ; et
al. |
September 15, 2005 |
Novel endonuclease of immune cell, process for producing the same
and immune adjuvant using the same
Abstract
The present invention relates to a novel endonuclease enzyme
which is secreted from immune cell and recognizes bacterial DNA as
foreign agent and processes it to produce about 10 bp
single-stranded oligonucleotide including CpG motif which is
involved in immune response. Also, the present invention relates to
a process for producing the endonuclease which comprises culturing
human B-lymphoblastic IM9 cell line or TPA-treated myelogenous U937
cell line on an appropriate medium to produce the said endonuclease
and isolating the said endonuclease from the cell lysate or the
culture medium. In addition, the present invention relates to an
immune adjuvant comprising about 10 bp single-stranded
oligonucleotide having CpG motif produced by treatment of bacterial
DNA by the endonuclease.
Inventors: |
Jeon, Yeong Joong; (Seoul,
KR) ; Park, Wan Je; (Kyungkwido, KR) ; Lee, Na
Gyong; (Kyungkwido, KR) ; Jung, Sang Bo;
(Seoul, KR) ; Kim, Doo Sik; (Seoul, KR) ;
Kwon, Hyung Joo; (Seoul, KR) |
Correspondence
Address: |
AKERMAN SENTERFITT
P.O. BOX 3188
WEST PALM BEACH
FL
33402-3188
US
|
Assignee: |
Cheil Jedang Corporation
500, 5-ga, Namdaemun-ro, Chung-ku
Seoul
KR
100-095
|
Family ID: |
34525544 |
Appl. No.: |
10/860844 |
Filed: |
June 4, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10860844 |
Jun 4, 2004 |
|
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09722776 |
Nov 27, 2000 |
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6881561 |
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Current U.S.
Class: |
514/44R ;
435/6.16 |
Current CPC
Class: |
C12N 9/22 20130101 |
Class at
Publication: |
514/044 ;
435/006 |
International
Class: |
A61K 048/00; C12Q
001/68 |
Foreign Application Data
Date |
Code |
Application Number |
May 27, 1998 |
KR |
1998/19176 |
Claims
What is claimed is:
1. An immune adjuvant comprising about 10 bp single-stranded
oligonucleotide having CpG motif produced by a purified
endonuclease enzyme which is secreted from human B lymphoblatic IM9
cell line or 12-tetradecanoylphorbol 13-acetate-treated U937 cell
line, wherein the oligonucleotide is produced by recognition and
processing of bacterial DNA as foreign agent by the endonuclease
enzyme.
2. The immune adjuvant of claim 1, wherein the oligonucleotide
activates the immune cell.
3. The immune adjuvant of claim 1, wherein the oligonucleotide
promotes the secretion of cytokine and IgM.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a novel endonuclease enzyme
which is secreted from immune cells and recognizes bacterial DNA as
foreign substance and processes it to generate approximately 10
bases single-stranded oligonucleotide including CpG motif known to
involve immune response. In addition, the present invention relates
to an immune adjuvant comprising the said single-stranded
oligonucleotides of approximately 10 bases generated by the said
endonuclease enzyme.
[0003] 2. Description of the Prior Art
[0004] Mammalian animals develop immune systems to defend against
foreign agents. The immune systems is classified into natural
(nonspecific) immunity or acquired (antigen-specific) immunity. The
innate or nonspecific immunity is a primary resistance against
diseases caused by one species, and creates defence barrier as four
types such as structural, physiological, endocytic and phagocytic,
and inflammatory response. Representative examples of the
structural defence barrier include skin and mucus. The
physiological defence barrier include, for example, temperature,
pH, oxygen pressure, and various aqueous soluble factors. The
endocytic and phagocytic defence barrier refers to endocytosis and
phagocytosis degradation systems in which foreign macromolecules
are incorporated into and subsequently degraded by certain cells.
The inflammation defence barrier is an inflammatory response which
is evolved by various vasoactive and chemotactic agents generated
by penetration of bacteria and followed by skin damage. Then,
enzyme systems such as clotting, kinin, fibrinolytic or complement
are activated. The acquired immunity is different from the innate
immunity in that the former possesses specificity, diversity,
memory and self and/or non-self recognition. The properties of the
acquired immunity are derived from the humoral and cellular
immunities which respond by B lymphocytes, T lymphocytes, antibody,
cytokine, etc.
[0005] The immune response by penetration of microorganisms is
generated by the innate mechanism which rapidly recognizes certain
molecules of the microorganisms at the initiation stage of the
penetration. The proteins and lipids present in microorganisms are
well known as agents inducing immune systems which specifically
respond to antigen. LPS, formyl methionine, lipoarabinomannan,
peptidoglycan, etc. are well known as agents which directly
activate the complement system (Marrack, P., and Kapple, J. W.
(1994) Cell 76, 323-332). Recently, it has been uncovered by many
researchers that in mammalian animals, humoral and cellular
immunities are activated by distinguishing their intrinsic DNA from
bacterial DNA and recognizing the bacterial DNA as foreign agent
and that also such bacterial DNA involves innate immunity.
[0006] From the fact that a great quantity of anti-DNA antibody is
generated during systemic lupus erythematosus (SLE), autoimmune
disease, DNA has been investigated in view of antigen or
autoantigen. The anti-DNA antibody is serologically considered to
be most important in connection with SLE, and functions as a major
mediator involving kidney damage, skin eruption, arthritis, etc.
(Tan, E. M. (1989) A Texbook in Rheumatology, 11 th Ed. D. J.
McCarty, ed. Led and Febiger, Philadelphia, Pa., 1049; Isenberg, D.
A, et al (1997) The role of antibodies to DNA in systemic lupus
erythematosus-A review and introduction to an international
workshop on DNA antibodies held in London, May 1996 Lupus 6,
290-304; Swaak, A. J. G., et al. (1979) Arthritis Rheum. 22,
226-235; and Isenberg, D. A., et al (1994) Arthritis Rheum. 37,
169-180). These antibodies were shown to bind to
structure-determining factor present in ssDNA and dsDNA (Isenberg,
D. A., et al (1994) Arthritis Rheum. 37, 169-180; Pisetskyi, D. S.
(1992) Rheum. Dis. Clin. North Am. 18, 437-454; and Shoenfeld, Y.,
and Isenberg, D. A.(1989) Immunol. Today 10, 123-126). Although the
cause of SLE has not yet exactly revealed, recent studies strongly
demonstrate that DNA antigen is significantly implicated in the
diseases (Shlomchik, M. J., et al (1987) Proc. Natl. Acad. Sci. USA
84, 9150-9154; Shlomchik, M. J., et al (1990) J. Exp. Med 171,
265-292; and Tillman, D. M., et al (1992) J. Exp. Med. 176,
761-779). Researchers used normal mouse and autoimmune disease
mouse to examine immune response to bacterial DNA (Gilkeson, G. S.,
et al.(1989) Clin. Immunol. Immunopathol. 51, 1482-1486; Gilkeson,
G. S., et al (1993) J. immunol. 151, .1353-1364; and Gilkeson, G.
S., et al (1995) J. Clin. Invest. 95, 1398-1402). Unlike mammalian
DNA, bacterial DNA possesses potent immunological properties which
activate polyclonal B cell and produce antibodies having
specificity in mouse (Gidkeson, G. S., et al (1995) J. Clin.
Invest. 95, 1398-1402; and Gilkeson, G. S., et al (1991) Clin.
Immunol. Immunopathol. 59, 288-300). The activity degree is due to
the fact that the base sequence motif present in bacterial DNA is
different from the base sequence motif of mammalian DNA and may be
recognized as foreign agent, i.e., non-self (Messina, J. P. et al
(1993) Cell. Immunol. 147, 148-157; Krieg, A. M. et al (1995)
Nature 374, 546-549; and Halpern, M. D. et al (1996) Cell. Immunol.
167, 72-78). When normal mouse is challenged by bacterial DNA, it
produces antibody capable of binding to not only bacterial dsDNA
but also mammalian and bacterial ssDNA (Gilkeson, G. S. et al
(1991) Clin. Immunol. Immunopathol. 59, 288-300). However, any
autoantibody which is cross-reactive to mammalian dsDNA was not
produced. Unlike normal mouse, preautoimmune (NZB X NZW)F1(NZB/W)
mouse challenged by dsDNA produced cross-reactive antibody which is
bound to mammalian dsDNA (Gilkeson, G. S., et al (1995) J. Clin.
Invest. 95, 1398-1402). As such, when autoimmune disease mouse is
immunized with bacterial DNA, the animal has the ability to produce
anti-dsDNA antibody which is cross-reactive to mammalian DNA. That
is because mistaken tolerence which auto-responding anti-dsDNA B
cells produced by bacterial DNA in lack of immune-regulator in
NZB/W mouse respond to their intrinsic DNA was taken place and thus
pathogenic auto-antibodies responding to not only bacterial DNA but
also their own DNA were increased (Whock, M. K. et al (1997) J.
Immunol. 158, 4500-4506).
[0007] A production of antibody by stimulating and activating B
cell with protein antigen is well known as the process in which
protein antigen is processed by antigen presentation cell.(APC) and
is bound to major histocompatibility complex (MHC) to induce a
presentation of antigen so that MHC-restricted T cell is activated
and the activated T cell secrets cytokine to activate B cell
(Parker, D. C. (1993) ) Annu. Rev. Immunol. 11, 331-60; and Clark,
E. A., and Ledbetter, J. A. (1994) Nature 367, 425-428). It is also
well known that lipoarabinomannan lipoglycans (LAMs), mycolic acid
lipids, processed form of constitutive element of mycobacterial
cell wall which are distinct from protein antigen can be presented
by hCD1b (Beckman, E. M. et al (1994) Nature 372, 691-694;
Bendelac, A. (1995) Science 269, 185-186; Sieling, P. A., et al
(1995) Science 269, 227-230; and Prigozy, T. I., et al (1997)
Immunity 6, 187) and hCD1c (Beckman E. M., et al (1996) J. Immunol.
157, 2795-2803). CD1 family is nonpolymorphic cell surface
glycoprotein which is encoded at the different sites from MHC
molecule. Although CD1-T cell binding has not yet clearly defined,
it was apparently suggested that mCD1d1 is recognized by CD8.sup.+
and CD4.sup.+ T cells (Castano A. R., et al (1995) Science 269,
223-226; Cardell, S., et al (1995) J. Exp. Med. 182, 993-1004) and
hCD1b is recognized by CD4.sup.- and CD8.sup.- T cells (Bendelac,
A. (1995) Science 269, 185-186). It is thus assumed that CD1 is
involved in presentation of various antigens other than proteins
found in pathogenic microorganisms. Many studies reported that DNA
is involved in anti-DNA-specific B cell stimulation. Krishnan and
Marion showed that immunization of mouse with the combination of
DNA and peptide could induce anti-DNA antibody (Krishnaa, M. R.,
and Marion, T. N. (1993) J. Immunol. 150, 4948-4957). Accordingly,
in light of the fact that anti-DNA antibody is generated during
various autoimmune disease, it is important to confirm as to
whether activation of B cell for production of anti-DNA antibody
depends on MHC-restricted T cell stimulation. Waisman suggested
that specific activation of T cell by DNA is involved in DNA
presentation by MHC class II molecule (Waisman, A., et al (1996)
Cell, Immunol. 173, 7-14). That is, he showed the fact that DNA is
bound to MHC class II molecule on APC surface and, as results, T
cell can be proliferated specifically by DNA and, on the basis of
the fact, proposed that DNA takes a critical role in autoimmune
disease. However, there are no further studies and knowledge
regarding processing and presentation mechanism of DNA antigen. For
instance, the matters as to whether bacterial DNA is processed in
APC and presented by MHC molecule as in protein antigen or whether
other molecules are involved in such a presentation have not yet
been explained.
[0008] Many researchers showed that vertebrate animals distinguish
their intrinsic DNA from bacterial DNA and thereby the immune cell,
is activated by the bacterial DNA. The bacterial DNA recognized as
non-self by vertebrate animals is characterized by generating
nonmethylated CpG dinucleotide at high level. The extraordinary
difference between bacterial DNA and vertebral DNA may be
summarized as follows. First, bacterial DNA generates CpG
dinucleotide of 16 dinucleotides at most level, but vertebral DNA
generates 1/4 of bacterial DNA. This means that CpG suppression
exists in vertebral DNA. Second, methylation frequency of CpG
dinucleotide present in bacterial DNA is low. While vertebral DNA
shows 80% methylation, methylation of microbial cytosine is hardly
found (Bird, A. P. (1995) Trends Genet. 11, 94-100). Third,
bacterial DNA is higher than vertebral DNA in the frequency of
flanking two 5'-purines and two 3'-pyrimidines at both ends of CpG
dinucleotide (Razin, A, and Friedman, J. (1981) Prog. Nucleic Acid
Res. Mol. Biol. 25, 33-52). The specific structure of the bacterial
DNA called "CpG motif" was reported to activate immune response.
That is, the activation of immune cell when two 5'-purines and two
3'-pyrimidines were flanked at both ends of CpG dinucleotide
(mitogenic CpGs) is much higher compared to when other bases are
flanked at both ends of CpG dinucleotide (non-stimulatory
CpGs).
[0009] Many researchers used chemically synthesized
oligodeoxyribonucleotide (ODN) in order to elucidate activation and
action of immune cell by specific base sequence of bacterial DNA.
Yamamoto and other researchers showed that bacterial DNA increased
lyric activation of NK cell and induced the production of
interferon-.gamma. (IFN-.gamma.) (Yamamoto, S., et al (1992) J.
Immunol. 148, 4072-4076; Cowdery, J. S., et al (1996) J. Immunol.
156, 4570-4575; and Ballas, Z. K., et al (I 996) J. Immunol. 157,
1840-1845). Kuramoto reported that such effects are associated with
palindromic base sequence of CpG motif included in bacterial DNA
(Kuramoto, E., et al (1992) J. Cancer Res. 83, 1123-1131; and
Kimura, Y., et al (1994) J. Biochem. 116, 991-994). In addition, it
was reported that bacterial DNA is bound to DNA-binding protein and
induces activation of B cell (Gilkeson, G. S., et al (1989) J.
Immunol. 142, 1398-1402; Yamamoto, S., et al (1992) J. Immunol.
148, 4072-4076; Gilkeson, G. S., et al (1989) J. Immunol. 142,
1482-1486; Messina, J. P., et al (1991) J. Immunol. 147, 1759-1764;
Field, A. K., et al (1967) Proc. Natl. Acad. Sci USA 58, 1004-1010;
and Oehler; J. R., and Herverman, R. B. (1978) Int. J. Cancer 21,
221-220). That is, it is understood that the activation of B cell
is promoted by CpG motif which consists of six(6) bacterial bases.
Immune response by bacterial infection including B cell activation
is characterized by producing immune-regulator cytokine(Van Damme,
J., et al (1989) Eur. J. Immunol. 19, 163-168; and Paul, W. E., et
al Adv. Immunol. 53, 1-29). It was also reported that CpG motif
takes part in secretion of IL-12 involving cellular immunity and
IL-6 involving humoral immunity (Halpern, M. D. et al (1996) Cell.
Immunol. 167, 72-78; Yi, A. K., et al (1996) J. Immunol. 157,
5394-5402; and Klinman, D. M., et al (1996) Proc. Natl. Acad. Sci.
USA 93, 2879-2883). Cytokines generated therefrom include IL-6
which plays a role in activating T cell and B cell (Uyttenhove, C.,
et al (1988) J. Exp. Med 167, 1417-1427: Muraguchi, A., et al
(1988) J. Exp. Med. 167, 332-344; Le, J. M., and Vilcek, J. (1989)
Lab. Invest. 61, 588-602; and Hirano, T., et al (1990) Immunol.
Today 11, 443-449) IFN-.gamma. which promotes the function of
macrophage to eliminate intra- and extra-cellular pathogenic
bacteria (Murray, H. W. (1990) Diagn, Microbial. Infect. Dis. 13,
411-421) and IL-12 which regulates production of IFN-.gamma. and
activates NK cell (Trinchieri, G. (1994) Blood 84, 4008-4027; Zhan,
Y, and Cheers C. (1995) Infect. Immune. 63, 1387-1390; and Bohn,
E., et al (1994) Infect. Immun. 62, 3027-3032). IL-12 and
IFN-.gamma. take an important role to eliminate human pathogenic
bacteria by increasing Type 1 cytokine (Klinman, D. M., et al
(1996) Proc. Natl. Acad. Sci. USA 93, 2879-2883; Zhan, Y, and
Cheers, C. (1995) Infect. Immun. 63, 1387-1390; Bohn, E., et al
(1994) Infect. Immun. 62, 3027-3032; and Heinzel, F. P., et al
(1991) Proc. Natl. Acad. Sci. USA 88, 7011-7015). IL-6 stimulates
the production of antibody by promoting growth and differentiation
of T cell and B cell by type 2 cytokine (Uyttenhove, C., et al
(1988) J. Exp. Med 167, 1417-1427; Muraguchi, A., et al (1988) J.
Exp. Med. 167,332-344; Le. J. M., and Vilcek, J.(1989) Lab, Invest.
61, 588-602; and Hirano, T., et al (1990) Immunol. Today 11,
443-449). Indeed, it was observed that mouse with knockout IL-6
gene was easily infected (Yi, A. K. et al (1996) J. Immunol. 157,
5394-5402; and Libert, C. et al (1994) Eur. J. Immunol.
24,2237-2242). Thus, bacterial DNA is understood to induce the
production of cytokine which is involved in cellular and humoral
immunity. Recently, it has been further reported that the
proliferation and generation of B cell is led by bacterial DNA
(Krieg, A. M. et al (1995) Nature 374, 546-549; Liang, H., (1996)
J. Clin. Invest. 98, 1119-1129; and Yi, A. K., et al (1996) J.
Immunol. 156, 558-564). Study of Krieg showed that CpG motif
present in ODN is essential to induce secretion of IgM while
activating and proliferating B cell and that the expression of
class II MHC molecule, typical phenomenon occurred when B cell was
activated, is increased and cell cycle starts from G.sub.0 to
G.sub.1. According to report of Sato (Sato, Y. et al (1996) Science
273, 352-354), it can be seen that when plasmid DNA including
immunostimulatory DNA sequence (ISS) with short CpG motif is
transfected into monocyte, amounts of IFN-.alpha. IFN-.beta. and
IL-12 are increased. This result indicates that if plasmid
including ISS is transfected into bone marrow stem cell, then
surrounding macrophage and T cell are activated and in vivo
rearrangement of the stem cell may be mistakenly occurred. Thus,
vector for somatic or stem cell-replacing therapy should be
designed not to include ISS. As contrast, one approach to improve
the efficacy of vaccine is to design plasmid DNA so as to include
many repetitive ISS.
[0010] In order to activate cell, bacterial DNA should be
incorporated into the cell. It was found that ODN adsorbed on cell
culture vessel fails to activate B cell (Krieg, A. M. et al (1995)
Nature 374, 546-549) and that when oligonucleotide was lipofected,
the incorporation thereof is increased while the activation of NK
cell is greatly increased (Yamamoto, T., et al (1994) Microbiol.
Immunol. 38, 831-836). It was also found that there was no
significant difference between abilities of oligonucleotides to be
bound to cell surface whether or not they include CpG motif (Krieg,
A. M. et al (1995) Nature 374, 546-549; and Yamamoto, T., et al
(1994) Microbiol. Immunol. 38, 831-836). Bennet showed that DNA
incorporated into mononuclear cell was degraded in endosomal
compartment (Bennett, R. M., et al (1985) J. Clin. Invest. 76,
2182-2190). Stacey showed that bacterial DNA was bound to
transcription factor nuclear factor-kb in macrophage and the
expression of TNF-.alpha., IL1-.beta. and plasminogen activator
inhibitor-2 mRNA was greatly augmented (Stacey, K. J., et al (1996)
J. Immuno. 157, 2116-2122). It was expected that incorporation of
oligonucleotides into cell faith mediation of receptor on cell
surface would be taken place by endocytosis (Bennett, R. M. et al
(1985) J. Clin. Invest. 76, 2182-2190). Also, a study was performed
by using fluorescent-labelled phosphorothioate
oligodeoxynucleotides in peripheral blood, bone mellow cell and
leukemia cell line (Loke, S. L., et al (1989) Proc. Natl. Acad.
Sci. USA 86, 3474-3478; Yakubov, L. A., et al (1989) Proc. Natl.
Acad. Sci. USA 86, 6454-6458; Zhao, Q., et al (1996) Blood 88,
1788-1795; Ribeiro J. M., and Carson D. A.(1993) Biochemistry 32,
9129-9136), but any property and mechanism thereof have not yet
been defined.
[0011] Bacterial DNA has been so far understood to take a critical
role in the immune system. It has been known that autoimmune
disease SLE is occurred by the generation of anti-DNA antibody by
bacterial DNA and that CpG motif of bacterial DNA is incorporated
into immune cell to activate the cell and thereby promote secretion
of cytokine and IgM. However, there is no report as to what
mechanism enables such critical bacterial DNA to produce antibody
and how oligonucleotide having CpG motif is made in cell.
[0012] A novel endonuclease was identified by the inventors from
human B-lymphoblastic IM9 cell and 12-O-tetradecanoylphorbol
13-acetate-treated differentiated myelogeneous U937 and culture
medium thereof using DNA-native-polyacrylamide gel electrophoresis
(DNA-native-PAGE).
SUMMARY OF THE INVENTION
[0013] In one aspect, the present invention provides novel
endonuclease enzyme which is secreted from immune cell and which
recognizes bacterial DNA as foreign agent and processes it to
produce about 10 bp single-stranded oligonucleotide having CpG
motif which is involved in immune response.
[0014] In another aspect, the present invention provides a process
for producing the endonuclease of the present invention which
comprises culturing human B-lymphoblastic IM9 or TPA-treated
myelogeneous U937 cell line on an appropriate medium to produce the
said endonuclease and isolating the said endonuclease from cell
lysates or culture medium.
[0015] In further aspect, the present invention provides an immune
adjuvant comprising about 10 bp single-stranded oligonucleotide
having CpG motif which is produced by treating bacterial DNA with
the endonuclease of the present invention.
BRIEF DESCRIPTION OF THE DRAWING
[0016] FIG. 1 shows the endonuclease activity of IM9 cells
according to the present invention analyzed by DNA-native-PAGE. The
endonuclease activity in IM9 cell lysates (A), medium (B) and
medium cultured in serum-free (C) was detected by in-gel system.
Bovine DNase 1 (lane) and culture medium endonuclease activity
(lane2) were compared (D).
[0017] FIG. 2 shows the endonuclease activity of DNase 1 (A) and
IM9 culture medium (B) analyzed by DNA-native-PAGE.
[0018] FIG. 3 shows the immunoprecipitation of endonuclease
activity by anti-bovine DNase 1 antibody. The endonuclease
activities in supernatant after immunoprecipitation (IP) was
estimated indicated anti-serum treatment by the DNA-native-PAGE as
described under "Materials and Methods". A, 10% FBS medium and
human serum; B, IM9 cell culture medium.
[0019] FIG. 4 shows the pretreatment of IM9 cells with actinomycin
D and secretion of the endonuclease. The endonuclease activity in
cell lysates (A) and culture medium (B) according to the present
invention was analyzed at indicated culture periods after
pretreatment.
[0020] FIG. 5 shows the comparative analysis of endonuclease
activity in immune cell lines according to the present invention.
The endonuclease activity in lysates (A) and culture medium (B) of
each cell line was analyzed by the DNA-native-PAGE. Lane 1, 10% FBS
containing medium; lane 2, IM9; lane 3, RPMI1788; lane 4, Molt-4;
lane 5, Jurkat; lane 6, U937.
[0021] FIG. 6 shows the endonuclease activity of IFN-.gamma. or
IL-1.beta. treated IM9 cell culture medium and cell lysates
according to the present invention analyzed by DNA-native-PAGE. IM9
cells were treated with 10 units/ml of IFN-.gamma. or IL-1.beta.
for the indicated culture times. Control, IM9 culture medium in RPM
16-40 medium containing 10% FBS for 12 hours.
[0022] FIG. 7 shows the optimal pH for the endonuclease activity
according to the present invention.
[0023] FIG. 8 shows the requirement of divalent cation for the
endonuclease activity according to the present invention. A, the
enzyme was reacted with 100 ng of plasmid DNA for 10 min at 37 in
the presence or absence of the indicated concentration of Ca.sup.2+
and/or Mg.sup.2- in 20 mM Tris-HCl, pH 7.0. B, the enzyme reaction
was allowed for 180 minutes in the presence of 10 mM Mg.sup.2-;
[0024] FIG. 9 shows the growth curve of TPA, LPS, and CHX treated
U937 cells. U937 cells were treated with TPA (10 ng/ml), LPS (1
ng/ml), and CHX (10/ml) as the indicated times. Cell number and
viability were esteminated by trypan blue exclusion in a
hemocytometer during culture time.
[0025] FIG. 10 shows the TPA-concentration dependent synthesis and
secretion of endonuclease in U937 cells were analyzed by
DNA-native-PAGE. Cell lysates (A) and culture medium (B) was
prepared by incubation for 24 hours at the indicated TPA
concentration.
[0026] FIG. 11 shows the endonuclease activity of TPA treated U937
cell lysates and culture medium analyzed by in-gel system. U937
cells were treated with 10 ng/ml TPA for the indicated culture
times.
[0027] FIG. 12 shows the endonuclease activity of LPS treated U937
cell lysates and culture medium. The endonuclease activity in cell
lysates (A) and medium (B) was analyzed at the indicated culture
periods after 1 ng/ml LPS treatment by DNA-native-PAGE.
[0028] FIG. 13 shows the endonuclease activity of U937 cells
treated with stimulatory factors (lane 1, U937 cell culture in
RPML1640 medium containing 10% FBS for 48 hr; lane 2, TPA(10 ng/ml)
treatment for 48 hr; lane 3, LPS(1 ng/ml) treatment for 24 hr; and
lane 4, CHX(10/ml) treatment for 12 hr).
[0029] FIG. 14 shows the endonuclease activity in nuclei isolated
from IM9 cells by autodigestion method (A, Nuclei were incubated
for 2 hr at 37.degree. C. either alone or on the presence of the
indicated concentration of Ca.sup.2+ and/or Mg.sup.2+; B,
Inhibition of internucleosomal DNA fragmentation; C and D, Nuclei
were incubated at 37.degree. C. in the presence of 10 mM Mg.sup.2+
(C) or 10 mM Ca.sup.2+ (D) for 0-240 min as indicated; and Marker,
1 kb ladder)
[0030] FIG. 15 shows the apoptotic cell death of IM9 cells by CHX
treatment (A, 1.8% agarose gel electrophoreis of DNA from IM9 cells
treated with CHX (10/ml); Untreated (B) and treated (C) with CHX of
IM9 cells were cultured for 24 hr and cytocentrifuge preparations
stained with Wright-Giemsa).
[0031] FIG. 16 shows the endonuclease activity of IM9 cell lysates
and nuclei analyzed by DNA-native-PAGE according to the present
invention. The endonuclease activity was detected in IM9 cell
lysates during culture time (A), cell lysates (B) and nuclei (C)
treated with 10 ug/ml CHX for the indicated times. Control: FBS
DNase 1.
[0032] FIG. 17 shows the requirement of divalent cation for the
endonuclease activity according the present invention. The
endonuclease was isolated from nuclei and the enzyme activity was
determined by resolving the reaction products on 1% agarose
gel.
[0033] FIG. 18 shows the time-course degradation of plasmid DNA by
the endonuclease of the present invention. The endonuclease
activity of RPMI medium containing 10% FBS (A), IM9 cell culture
medium in serum free (B), and IM9 cell culture containing 10% FBS
(C) was estimated by resolving the reaction products on 1% agarose
gel.
[0034] FIG. 19 shows the products of endonuclease reaction of the
present invention on E. coli DNA, IM9 cells DNA, and salmon sperm
DNA.
[0035] FIG. 20 shows the southern blot analysis of foreign DNA
incorpotated into IM9 cells. 100-200 bp fragments of endonuclease
reaction products were used as a probe. Blot were hybridized for 6
hr at 55.degree. C. with probe prepared by the random-priming
method.
[0036] FIG. 21 shows the construction scheme for the cloning and
sequencing of the DNA fragments obtained by endonuclease reaction
according to the present invention.
[0037] FIG. 22 shows the DNA fragments produced by processing of
bacterial DNA in IM9 cells according to the present invention.
[0038] FIG. 23 shows the DNA fragments produced by processing of
bacterial DNA in U937 cells according to the present invention (A,
labelled DNA incorporation after U937 cells culture in RPMI1640
medium containing 10% FBS for 48 hr; and B, labelled DNA
incorporation after TPA (10 ng/ml) treatment for 12 hr).
[0039] FIG. 24 shows the detection of endonuclease reaction
products sequence of the present invention. The endonuclease
reaction products were hybridized with synthetic oligonucleotides
as described under "Materials and Methods".
[0040] FIG. 25 shows the induction of IgM secretion by CpG motifs
in bacterial DNA or oligonucleotides. IM9 cells were stimulated
with oligonucleotides (25/ml), or E. coli DNA (25/ml) for 24
hr.
[0041] FIG. 26 shows the products of endonuclease reaction using
EC1 PCR product as a substrate according to the present
invention.
[0042] FIG. 27 shows the products of endonuclease reaction using
EC2 PCR product as a substrate according to the present
invention.
[0043] FIG. 28 shows the product of endonuclease reaction using HC1
PCR product as a substrate according to the present invention.
[0044] FIG. 29 shows the inhibition of endonuclease activity by
Zn.sup.2- and EDTA.
[0045] FIG. 30 shows the product of endonuclease reaction from EC1
PCR product reacted with indicated IM9 cell culture medium amounts
according to the present invention.
[0046] FIG. 31 shows the product from endonuclease reacted with EC1
PCR product for the indicated times according to the present
invention.
[0047] FIG. 32 shows the endonuclease activity comparing EC2 PCR
product (157 bp) with short DNA fragment cleaved by Alu I (lane 1,
.sup.32p-labelled PCR product; lane 2, endonuclease digested
product of lane 1; lane 3, .sup.32p-labelled short DNA fragment
cleaved by Alu I; lane 4, 30 min reaction of lane 3; lane 5, 1 hr
reaction of lane 3; lane 6, 2 hr reaction of lane 3).
[0048] FIG. 33 shows the identification of 3'-exonuclease activity
in IM9 cell secreted endonuclease (lane 1, labelled PCR product;
lane 2, products of endonuclease reaction on labelled double
stranded DNA; lane 3, products of endonulease reaction on labelled
single stranded DNA).
[0049] FIG. 34 shows the comparison between endonuclease reaction
product and processed product by IM9 cells with DNase I reaction
product (lane 1, labelled PCR product; lane 2, reaction product of
IM9 cell culture medium; lane 3, processed product in IM9, cells;
lane 4, DNase I reaction product; Panel A, 20% native-PAGE in TBE
buffer; Panel B, denatured-urea (8.3 M)-PAGE in TBE buffer)
[0050] FIG. 35 shows the identification of single stranded
fragments derived from endonuclease reaction by S1 nuclease
reaction (A, 20% native-PAGE in TBE buffer; B, 20% denatured-Urea
(8.3 M)-PAGE in TBE buffer).
[0051] FIG. 36 shows the chromatographic fractionation of IM9 cell
culture medium by Mono S HR5/5 ion exchange chromatography (A, ion
exchange profile of IM9 culture medium; B, the enzyme activity was
determined at the indicated retention times by resolving the
reaction products on a 1% agarose gel).
[0052] FIG. 37 shows the purification of endonuclease by RESOURCE
PHE hydrophobic interaction chromatography (A, hydrophobic
interaction profile of activity fraction obtained from ion exchange
chromatography; B, the enzyme activity was determined at the peak
fractions by resolving the reacction products on 1% agarose
gel.
[0053] FIG. 38 shows the SDS-PAGE of purified endonuclease by ion
exchange chromatography and hydrophobic interaction
chromatography.
[0054] FIG. 39 shows the molecular weight determination of purified
endonuclease by SDS-PAGE (marker proteins are myosin (200 kD),
.beta.-galactosidase (116.3 kD), phosphorylase B (97.4 kD), bovine
serum albumin (66.2 kD) and ovalbumin (45 kD)).
[0055] FIG. 40 shows the native-pore gradient gel electrophoreis
(4-15%) of Mono S chromatography fraction containing activity (B)
and endonuclease activity of eluted protein from gel band of panels
B on agarose gel electrophoresis (A).
[0056] FIG. 41 shows the SDS polyacrylamide gel electrophoresis of
purified endonuclease by ion exchange chromatography and
native-gradient PAGE gel elution of activity band.
[0057] FIG. 42 shows the effects of cation on purified endonuclease
activity in isolated U937 cell nuclei.
DETAILED DESCRIPTION OF THE INVENTION
[0058] The novel endonuclease was identified from IM9 cell lysates
and culture medium using DNA-native-PAGE nuclease assay system. The
molecular weight of the endonuclease was determined as 72.4 kD by
SDS-PAGE. The endonuclease activity of the present invention was
detected in IM9 cell nuclei during culture time and accumulation of
the enzyme activity was shown in the IM9 cell nuclei protein
extracts of the apoptotic cells. The signals for proliferation and
differentiation of myelogeneous U937 cells are provided by
extracellular stimuli such as lipopolysaccharide (LPS) and
12-O-tetradecanoylphorbil 13-acetate (TPA). Experimental results
indicated that TPA has significant effect on the degree of
endonuclease secretion. The enzyme activity was induced in U937
cells by LPS treatment, while the secretion of the enzyme was not
detected in the culture medium. Using supercoiled plasmid DNA as a
substrate, the endonuclease activity determined with the enzyme
isolated from the cell culture medium. The endonuclease, with
Mg.sup.2- alone, was able to catalyze the conversion of the plasmid
DNA into linear form followed by further degradation. The pH
optimum required for the catalytic activity was determined to be in
the range of pH 6.6-7.4. Experimental results clearly demonstrated
that the endonuclease activity of the immune cell lines is distinct
from that of Dnase I in the DNA-native-PAGE assay system.
Immunoprecipitation analysis using anti-DNase I antibody showed
that the secreted endonuclease is not recognized by the antibody.
The Mg.sup.2+ dependent endonuclease characterized by the present
invention appears to be distinct from the nuclease reported so far
in several aspects including cation dependence for enzyme activity,
electrophoretic mobility in native-PAGE, and optimum pH required
for catalysis. The DNA fragments processed by the endonuclease
activity were detected by Southern blot analysis in immune cell
lines. The foreign DNA antigen partially processed in cell culture
medium appears to be bound to the cell surface followed by
incorporation into the cell. Using radiolabelled DNA fragments as a
foreign antigen, the further processing of DNA antigen in the
immune cell lines was demonstrated by autoradiography. Experimental
results showed that the single-stranded DNA fragments of
approximately 10 bases that are generated by the endonuclease we
redegraded by S1 nuclease reaction. The short single-stranded DNA
sequence was successful to be hybridized with complementary
synthetic oligonucleotide containing a CpG motif with unmethylated
CpG dinucleotide flanked by two 5'-purines and two 3'-pyrimidines.
The present invention demonstrates the presence and characteristics
of a novel endonuclease that exists both in human immune cell lines
and in their culture media. Also, the present invention shows that
the endonuclease from immune cell recognizes bacterial DNA as a
foreign substance and carries out immunological process by
generating DNA fragments containing a CpG motif.
[0059] The present invention will be illustrated through the
examples below.
EXAMPLE 1
Biosynthesis and Secretion of the Endonuclease from Immune Cell
[0060] From the fact that the enzymatic activity of DNase I is
widely distributed over human tissue and body fluid, it was assumed
that the enzyme possesses certain physiological in vivo functions
in addition to the digestive function (Nadano D., et al (1993)
Clin. Chem. 39, 448-452; and Yasuda T., et al (1993) Clin. Chim.
Acta. 218, 5-16). DNase I is known to cleave internucleosomal DNA
during apoptosis (Peitsch M. C., et al (1993) EMBO J. 12, 371-377).
The presence of DNase I in human scrum and the biochemical
properties thereof were reported (Love J. D., and Hewitt R.
R.(1979) J. Biol. Chem. 254, 12588-12594; and Kishi K., et al
(1990) Am. J Hum. Genet. 47, 121-126). It was taught that serum
DNase I is secreted from pancreas (Love J. D., and Hewitt R.
R.(1979) J. Biol. Chem. 254, 12588-12594; and Ito K. et al (1984)
J. Biochem. 95, 1399-1406) but studies on the secretion from other
tissues are still required. A study of Messina showed that DNase I
fails to produce CpG motif which activates B cell and macrophage
since it completely degrades the bacterial DNA (Messina, J. P. et
al (1991) J. Immunol. 147, 1759-1764). In addition, it was revealed
from the study of Messina that bacterial DNA not treated by DNase I
activates immune cell and promotes secretion of cytokine and IgM.
These are consistent with the results obtained when cell was
treated with ODN having CpG motif. Therefore, under the assumption
that a new type of endonuclease which can recognize foreign DNA and
produce CpG motif by processing is present in immune cell, the
inventors conducted experiments using various immune cell lines to
confirm the presence of the activity of such an endonuclease.
[0061] 1-1 Cell Culture and Pretreatment
[0062] Human B-lymphoblastic (IM9 and RPMI1783) cell line,
T-lymphoblastic (Molt-4 and Jurkat) cell line and myelogeneous
(U937) cell line were purchased from American Type Culture
Collection. Cells were cultured on RPMI1640 containing heated fetal
bovine serum (FBS, Gibco BRL) 10% while maintaining
4-5.times.10.sup.5 cells/ml. Cell culture was carried out in
incubator (Forma) including 5% CO.sub.2 at 37.degree. C. The number
of cells and the viability of cells during culture were
periodically measured by trypan blue exclusion method using
hemocytometer. The viability of cells was kept at 95% or more over
whole experiment. IM9 cell line was pretreated with actinomycin D
(ACD, Sigma) at 0.33 ug/ml to confirm the biosynthesis of the
endonuclease in cells (Cooper H. L., and Braverman R. (1977) Nature
269, 527-529). IM9 cell line was treated with ACD, cultured for 30
minutes and washed. Then, the enzymatic activity of the
endonuclease was measured at regular intervals while the cells were
cultured for 48 hours on RPMI1640 containing 10% FBS.
[0063] DNA-native-PAGE nuclease activity assay was performed to
detect endonuclease enzyme activity in cell culture solution and
cell lysate. The activity of the secreted endonuclease in tested
cell cultures was observed only in IM9 cell line (FIG. 5B, lane 2).
However, the endonuclease activity was always detected in cell
lysate of human T-lymphoblastic Molt4 cell line (FIG. 5A, lane 4)
but not in cell culture solution. As for myelogeneous U937 cell
line, B-lymphoblastic RPMI1788 cell line and T-lymphoblastic Jurkat
cell line, the endonuclease activity was detected in neither cell
lysate nor cell culture solution. As shown in FIG. 4, the
biosynthesis of endonuclease in cell (FIG. 4A) and the secretion of
endonuclease into cell culture solution (FIG. 4B) were remarkably
decreased at the earlier period following the ACD pretreatment.
These results indicate that there is close co-relationship between
biosynthesis and secretion of endonuclease in IM9 cell line.
[0064] Cytokine involving immune response was treated with
interferon-.gamma. (IFN-.gamma., 10 units/ml, Genetech Inc.) and
interleukine-1.beta. (IL-1.beta., 10 units/ml, Genetech Inc.) to
confirm the effects of cytikine on the biosynthesis and secretion
of endonuclease. DNA-native-PAGE assay was conducted while
culturing cells for 24 hours on medium containing interleukine. As
shown in FIG. 6, such cytokines did not significantly influence on
the secretion of endonuclease. Also, it was observed that neither
lipopolysaccharide (LPS) nor tetradecanoylphorbol 13-acetate (TPA,
Sigma) influenced on the secretion of endonuclease.
[0065] The biosynthesis and secretion were observed while U937
cells were treated with TPA at different concentrations over
indicated hours. U937 cell treated by cycloheximide (CHX, 10 ug/ml,
Sigma) and LPS (1 ng/ml, Sigma) was compared with U937 cell treated
by TPA. Cells were washed by phosphate-buffered saline (PBS, 137 mM
NaCl, 2.7 mM Kcl, 10 mM Na.sub.2HPO.sub.4, 1.8 mM KH.sub.2PO.sub.4,
pH 7.4) and stained by Wright-Giemsa (Sigma) over cytocentrifuge
slides to observe cell shape following the treatment of cells by
various agents. As results, the cell shape of differentiated human
myelogeneous leukemia cell line with TPA stimuli was takes to be
manure and growth was stopped, but the proliferation of cell
following stimulation of LPS was taken place. The growth curve of
cell stimulated by such mitogens is shown in FIG. 9. It can be seen
from FIG. 9 that U937 cell lines were differentiated by TPA to
change the cell shape and ultimately growth was stopped and that
the cells were proliferated by LPS. In addition, it was observed
that the cells were killed by treatment of CHX, apoptosis-occurring
agent. Additional experiments were carried out to confirm as to
whether endonuclease is produced and secreted under such culture
conditions. FIG. 10A shows that the biosynthesis of endonuclease in
cell was increased in line with increased TPA concentration. FIG.
10B shows that endonuclease enzyme was secreted from cell at the
same time that the biosynthesis of endonuclease in cell was
increased Also FIG. 10A shows that the biosynthesis of endonuclease
was rapidly increased at 10 ng/ml of TPA or more. FIG. 11 shows the
results obtained when endonuclease activity was observed at regular
intervals following treatment of U937 cell culture solution by 10
ng/ml of TPA. It can be seen that the biosynthesis and secretion of
endonuclease enzyme was initiated at 6 hours after treatment of TPA
and the peak thereof was achieved at 24 hours after treatment of
TPA. FIG. 12 shows the results obtained when endonuclease activity
was determined at regular intervals after treatment of cell by 1
ng/ml of LPS. The results indicate that endonuclease activity was
detected in cell lysate at 12 hours after treatment of LPS and any
endonuclease activity was not detected in cell culture solution
over 24 hours. It was microscopically observed that U937 cell line
was killed upon treatment by CHX, apoptosis-inducing agent, and
such a treatment did not effect on the biosynthesis of endonuclease
(FIG. 13, lane 4). The endonuclease activity after treatment of
U937 cell line by TPA, LPS and CHX was shown in FIG. 13.
[0066] 1-2 Preparation of Cell Lysate and Determination of
Endonuclease Activity on DNA-Native-PAGE
[0067] The cell culture was centrifuged at 1,500 rpm for five(5)
minutes and supernatant was removed. The centrifuged cells were
washed twice with cold PBS and resuspended in 0.5% Nonidet
P-40(NP-40) buffer solution (lysis buffer solution) containing 150
mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA and 1 mM PMSF to
1.times.10.sup.7 cells/ml. After standing the solution at 4.degree.
C. for 15 minutes, it was centrifuged at 12,000 rpm, 4.degree. C.
for 15 minutes and supernatant was used as cell lysate.
[0068] A modified native polyacrylamide gel assay system was used
to determine the endonuclease activity and identify its
characteristics. By using Hoefer Tall Mighty Small(0.75 mm.times.8
cm.times.11 cm) vertical electrophoresis device, 7% polyacrylamide
gel was ploymerized with supercoiled plasmid DNA(PGEM-T vector, 3.0
Kb, Promega) to the final concentration of 150/ml. 10 ug of protein
sample of the cell culture solution or the cell lysate per well was
loaded and then an electrophoresis was carried out at 4.degree. C.
After electrophoresis, the gel was washed three times with
distilled water and reacted in a reaction buffer solution
containing 20 mM Tris-HCl, pH 7.0, 1 mM CaCl.sub.2 and 10 mM
MgCl.sub.2 (TCM buffer) at 37.degree. C. for 4 hours while
stirring. An enzyme activity of the endonuclease enzyme was
observed while changing the reaction time to identify the reaction
specificity of the enzyme on DNA-native-PGAE. The reacted gel was
stained with TCM buffer solution comprising 1/ml ethidium bromide
at 37.degree. C. for 30 minutes and photographed with 30 nm
transilluminator. The sites exhibiting nuclease activity on the gel
were observed as black band on orange background. The standard for
endonuclease activity was bovine pancreatic DNase 1 (RNase-free
10-50.times.10.sup.3 units/ml, Boehringer Mannheim).
[0069] Enzyme activity in cell culture and cell lysate, along with
sensitivity, can be determined by DNA-native-PAGE nuclease assay
system designed for the present invention (FIG. 1). Intensive
reaction bands were observed at the site exhibiting endonuclease
activity in cell culture solution and cell lysate containing 10 ug
of protein. The analyzed endonuclease biosynthesis and secretion of
IM9 cells for indicated times are shown in FIG. 1. The endonuclease
activity in cell lysate was constantly detected during 48 hour
culture (FIG. 1A), but under the same reaction conditions, the
endonuclease activity in cell culture solution was considerably
accumulated (FIG. 1B). When IM9 cell culture was washed several
times with PBS and then transferred to serum-free medium, the major
band for endonuclease activity shown in FIG. 1B was detected as it
was, but any DNase I enzyme activity was not detected (FIG. C).
This result indicates that the endonuclease detected in IM9 cell
culture and cell lysate was not derived from FBS which is an
ingredient of medium composition on which the cells were cultured,
but was synthesized in the cell and secreted into the medium. The
weak band of nuclease activity rapidly migrated on the
electrophoresis in the cell culture of FIG. 1B was identified as
DNase I by comparing with bovine DNase I obtained from Boehringer
Mannheim.
[0070] FIG. 2 shows the enzyme activity for 1 hour and 4 hours
detected by DNA-native-PAGE under the conditions of different
reaction solutions. When a reaction was performed in 20 mM
Tris-HCl, pH 7.0, buffer solution containing 10 mM Mg.sup.2- for 1
hour, the activity was detected only in the endonuclease secreted
by IM9 cell as shown in FIG. 2A. However, when an reaction was
carried out for an extended time of 4 hours under the same reaction
condition, DNase I present in FBS as well as purchased DNase I
showed the enzyme activity on the same site. This result shows that
the migration distance of the endonuclease synthesized and secreted
by IM9 cell is different from the migration distance of DNase I on
native-PAGE and the enzyme reactivity is also different from each
other under a given reaction condition.
[0071] 1-3 Production of Antibodies against DNase I and
Immunoprecipitation
[0072] Blood was taken from tail of Sprague Dawley (SD, 150-250 g)
to obtain preimmune serum. Bovine pancreatic DNase I (Sigma) was
digested into small fragments in sterile PBS for immunization. SD
rats were immunized with 100 ug of the protein according to the
standard method (Harlow, E., and Lane D.(1988) Antibodies, A
laboratory manual, Cold Spring Harbor, New York). 10 days after the
rats were four times challenged, blood was taken and the antibody
titer and specificity were assayed. Anti-DNase I antibodies in the
serum containing DNase I were purified by using Immunopure plus
protein A/G IgG purification kit (Pierce) and Separose-CL 4B bound
by bovine DNase I. 50 ml of cell culture or 50 ml of human serum 10
times diluted to PBS was immunoprecipitated by beads to which the
purified anti-DNase I antibodies and Protein A-Sepharose CL 4B were
bound. The immunoprecipitation was performed at 4.degree. C. for 6
hours while stirring. Immunoprecipitated supernatant was collected
and the enzyme activity of endonuclease was detected on
DNA-native-PAGE.
[0073] FIG. 3A shows that the prepared antibodies recognize the
DNase I derived from FBS and are cross-reactive with DNase I
present in human serum. The preimmune serum did not recognize FBS
and human serum DNase I. By using the DNase I prepared therefrom,
the cross reactivity of the secreted endonuclease to each DNase I
was detected. The supernatant immunoprecipitated from IM9 cell
culture solution by anti-DNase I antibodies showed rapidly mobile
DNase I enzyme activity, but the enzyme activity of the secreted
endonuclease was not immunoprecipitated an recovered as it was
(FIG. 3B). This result demonstrates that the endonuclease secreted
from IM9 cell line is immunologically distinct from DNase I.
[0074] 1-4 Partial Purification of Endonuclease Activity and
Characterization
[0075] The protein band exhibiting enzyme activity by
electrophoresis according to the above Example 1-2 was eluted to
partially purify the endonuclease from the culture solution of IM9
cell. The protein band exhibiting nuclease activity was cut,
fragmented into small pieces and -transferred to eppendorf
microtubes. Then, protein elution was carried out by using 20 mM
Tris-HCl, pH 7.0, buffer solution at 4.degree. C. for 10 hours
while stirring. The sample was centrifuged at 4.degree. C., 14,000
rpm for 10 minutes and the supernatant was divided by 20 ul. 20 ng
of eluted protein sample was reacted with supercoiled plasmid DNA
in 20 mM Tris-HCl buffer solution (pH 7.0) at 37.degree. C. for 10
minutes. To determine the effect of pH on the enzyme activity, the
enzyme activity was determined in 20 mM MOPS buffer solutions
containing 1 mM CaCl.sub.2 and 10 mM MgCl.sub.2 having each
different pH. To analyze the enzyme activity of endonuclease for
indicated reaction time, enzyme reaction was observed in 20 mM
Tris-HCl buffer solution (pH 7.0) containing 10 mM MgCl.sub.2 at
37.degree. C. for 180 minutes at regular intervals. After adding TE
(10 mM Tris-HCl, pH 7.6, 1 mM EDTA) buffer solution containing DNA
sample buffer solution (30% glycerol, 0.5% Bromophenol Blue and
0.5% xylene cynol), the reaction was stopped by adding ice. The
reaction product was identified by conducting an electrophoresis on
1% agarose gel containing ethidium bromide (0.5 ng/ml). The
isolated enzyme was determined as having the optimal activity at pH
7. However, the catalytic activity was detected at relatively broad
range of pH (FIG. 7). The enzyme activity of endonuclease in 20 mM
Tris-HCl (pH 7.0) buffer solution was dependent on Mg.sup.2+ and
was not affected by Ca.sup.2- (FIG. 8A). The enzyme was not
activated in the range of 1 to 10 mM Ca.sup.2+, but linear DNA was
formed from plasmid by the enzyme activity depending on the
concentration of Mg.sup.2+ a formation of linear DNA by
endonuclease for indicated reaction time was observed.
[0076] The conversion reaction of supercoiled plasmid DNA into
linear DNA was started after 10 minutes reaction and was gradually
increased until 60 minutes reaction. The nuclease activity was
lasted for 180 minutes under the same experimental condition (FIG.
8B). Thus, it was confirmed that this enzyme converts supercoiled
plasmid DNA into linear DNA and subsequently digest the DNA. This
result demonstrates that the endonuclease activity secreted by IM9
immune cell is Mg.sup.2+-dependent.
[0077] The present invention showed that the Mg.sup.2+-depending
endonuclease activity is produced in a constant amount and
consistently secreted into the cell culture. Also, the fact that
the endonuclease is different from the DNase I present in FBS and
human serum was confirmed by the difference of migrating distance
on native-PAGE and the immunoprecipitation result obtained by using
anti-DNase I antibody. These indicate that the endonuclease of the
present invention is distinct from the endonuclease reported so far
to digest DNA in the process of apoptosis, in aspects of various
biochemical properties such as cation-dependence for enzyme
activity, mobility distance in native-PAGE, optimal pH required for
activity, etc. Also, the fact that the endonuclease was produced
and secreted at the time that U937 cell line was differentiated
confirms that the endonuclease has an very important biological
function which can recognize foreign DNA in immune reaction and
digest it into a suitable size. The function of the endonuclease
according to the present invention supports the report of Stacey
that macrophage is activated when bacterial DNA is incorporated
into the cell and the report of Higashi that cell toxicity mediated
by mononuclear cell/macrophage may be occurred by nuclease (Stacey,
K. J. et al.(1986) J. Immunol. 157, 2116-2122; and Higashi, N. et
al.(1993) Cell. Immunol. 150, 333-342).
EXAMPLE 2
Identification of Mg.sup.2+-dependent Endonuclease Inducing
Internucleosomal DNA Fragmentation
[0078] Apoptosis is defined as specific type of "cell death" such
as chromatin condensation, membrane blebbing or chromatin
fragmentation as various nucleosome sizes by endonuclease activity
(Wyllie, A. H., et al (1984) J. Pathol. 142, 67-77; Wyllie, A. H.
(1980) Nature 284, 555-556; and Kerr, J. F. R., et al (I972)
Cancer. 26, 239-257).
[0079] Endonuclease activation is significantly responsible for
apoptosis process (Arends, M. J., and Wyllie, A, H. (1990) J.
Pathol. 136, 593-608). Many researchers suggested that there are
various enzymes which involve nucleosome fragmentation. Examples of
enzymes involving internucleosomal DNA fragmentation include DNase
I (Peitsch M. C., et al (1993) EMBO J. 12, 71-377), DNase II
(Torriglia A., (1995) J. Biol. Chem. 270, 28579-28585; and Barry M.
A., and Eastman A. (1993) Arch. Biochem. Biophys. 300.440450), and
NUC-18 (Wawabata, H., et al (1997) Biochem. Biophys. Res. Commun.
233, 133-138). Also, there are many reports proposing that
internucleosomal DNA in various types of tissue and cell can be
fragmented by Ca.sup.2+/Mg.sup.2+-dependent endonuclease (Stratling
W. H., et al (1984) J. Biol. Chem. 259, 5893-5898; Pandey S., et al
(1997) Biochemistry 36, 71 1-720; and Ribeira J. M. and Carson D.
A.(1993) Biochemistry 32,9129-9136) or Mg.sup.2+-dependent
endonuclease (Anzai N., et al (1995) Blood 86, 917-923; Kawabata
H., et al (1993) Biochem. Biophys. Res. Commun. 191, 247-254; Sun
X. M., and Cohen G. M. (1994) J. Biol. Chem. 269, 14857-14860; and
Wawabata, H., Anzai, N., et al (1997) Biochem. Biophys. Res.
Commun. 233, 133-138). However, it has not yet demonstrated that
each of many endonucleases is implicated with different multiple
cell systems or that when cell death is actively taken place in all
types of cell, there exists certain unknown enzyme which works for
chromtin fragmentation. Therefore, it has been required to
characterize endonuclease involving apoptosis and define mechanism
thereof.
[0080] The biosynthesis of endonuclease enzyme by human
B-lymphoblastic IM9 cell line and the isolation of the endonuclease
by DNA-native-PAGE were illustrated in the above Example 1. It was
now found that the isolated endonuclease enzyme is distinguished
from any endonuclease known so far in aspects of electrophoretic
mobility in native-PAGE, optimum pH required for catalysis and
divalent cation dependence for enzyme activity.
[0081] It was also found by the inventors that the activity of the
enzyme which appears to be identical with the endonuclease present
in the cell culture solution and cell lysate prepared by the above
Example is detected in cell nucleus and the enzyme is deposited in
the nucleus during apoptosis process. The fact that such
endonuclease induces nucleosomal fragmentation in nucleus may be
interpreted as depense action in that the cell tarketed to be
killed is eliminated by such action. It was demonstrated by agarose
gell eletrophoresis that when IM9 cell line was treated by CHX,
oligonucleosomal fragments of DNA were generated. The generation of
the fragments is known as an apex of biochemical phenomenon on
apoptosis. In addition, typical DNA fragmentation was found when
the nucleus isolated from IM9 cell line was reacted in the presence
of Mg.sup.2+. DNA fragmentation by autodigestion was identified to
be Mg.sup.2+-dependent and Ca.sup.2--independent. The activity of
endonuclease enzyme present in nucleus was also observed by
DNA-native-PAGE assay system. The optimum pH for the activity of
the enzyme was between 6.5 and 7.5. These results demonstrate that
the endonuclease synthesized and secreted by IM9 cell line as
described in the above Example 1 is identical with the enzyme
present in nucleus of IM9 cell line. It is assumed that the
endonuclease is closely related to Mg.sup.2+-dependent endonuclease
reported by many reasearchers to be present in various tissues and
cells (Anzai N., et al (1995) Blood 86, 917-923; Kawabata H., et al
(1993) Biochem. Biophys. Res. Commun. 191, 247-254; Sun X. M., and
Cohen G. M. (1994) J. Biol. Chem. 269, 14857-14860; and Wawabata,
H., Antzai, N., et al (1997) Biochem. Biophys. Res. Commun. 233,
133-138). However, any reports on the endonuclease involving
apoptosis published so far have not yet taught that
Mg.sup.2--dependent endonuclease was identified as protein band or
enzyme activity band.
[0082] In aspects of calcium dependence, mobility distance in
native-PAGE and optimum pH, the endonuclease identified by the
inventors is distinct from enzymes involving internucleosomal
fragmentation of DNA reported so far, for example,
Ca.sup.2+/Mg.sup.2--dependent endonuclease (Stratling W. H. et al
(1984) J. Biol. Chem. 259, 5893-5898; Pandey S. et al (1997)
Biochemistry 36, 711-720; and Ribeiro J. M. and Carson D. A.(1993)
Biochemistry 32, 9129-9136), DNase I (Peitsch M. C., et al (1993)
EMBO J. 12, 371-377), DNase II (Totriglia A. et al (1995) J. Biol.
Chem. 270,28579-28585; and Barry M. A., and Eastman A. (1993) Arch.
Biochem. Biophys. 300;440-450) and NUC-18 (Kawabata, H. et al
(1997) Biochem. Biophys. Res. Commun. 233,133-138). Since the
endonuclease of the present invention is observed in cellular
nucleus during apoptosis process, it is believed that the
endonuclease takes an important role in the apoptosis process.
[0083] 2.1 Induction of Apoptosis
[0084] Human B-lymphoblastic (IM9) cells were cultured and then
apoptosis was induced by treating the cells with 10 ug/ml
CHX(Sigma) and then culturing them for 24 hours (Chow, S. C. et at.
(1995) Exp. Cell. Res. 216, 149-159). A changed cell shape of the
apoptosis cells induced by CHX was identified by staining the cells
with Wright-Giernsa (Sigma) on centrifuge slide.
[0085] FIG. 15A shows that the treatment of IM9 cells with
CHX(10/ml) for 24 hours provides the DNA fragments as a
characteristic form of internucleosomal DNA digestion. After 24
hours, the effect of CHX was exhibited and after further 24 hours
large amount of DNA fragments were formed. The cell was stained
with Wright-Giemsa dye to observe the cell shape during the cell
death caused by apoptosis. The result is shown in FIG. 15C. Many
apoptosis cells were generated by CHX treatment and the cell shape
characterized by distortion, chromosomes aggregation and nuclei
fragmentation was distinct from that of normally growing cells.
[0086] 2-2 Identification of DNA Fragmentation by Endonuclease of
IM9 Cells Treated with CHX
[0087] IM9 cell lines were cultured to 5.times.10.sup.5 cells/ml
and treated with 10 ug/ml CHX (Sigma). While culture was performed
for 24 hours, 1.times.10.sup.6 cells were collected at regular
intervals and then DNA was extracted according to modified method
of Blin and Stafford (Blin, N., and Stafford, D. W. (1976) Nucleic
Acids Res. 3, 2303-2308. 1.times.10.sup.6 cells were washed two
times with PBS and resuspended in TE buffer solution. After adding
1 ml of DNA extraction buffer solution (10 mM Tris-HCl, pH 7.6, 10
mM EDTA. 100 mM NaCl, 0.2% SDS and 100 ug/ml proteinase K), the
reaction mixture was reacted at 50.degree. for 8 hours and treated
twice with phenol/chloroform to remove protein. After adding 0.3M
sodium acetate(pH 5.2), the reaction mixture was precipitated with
cold ethanol. The precipitate was dissolved in TE(10 mM Tris-HCl,
pH 7.6, 1 mM EDTA) buffer solution and treated with RNase A. Then
an electrophoresis was, carried out on 1.8% agarose gel. The
electrophoresis gel was placed in the solution containing 0.5 ug/ml
ethidium bromide for 30 minutes and photographed under UV.
[0088] IM9 cells treated with CHX were collected at regular
intervals and cell lysate and nuclei were prepared. After the cell
lysate and nuclei were dissolved, a gel assay of DNA-native-PAGE
nuclease activity was performed to identify the enzyme activity of
the endonuclease present in nucleus. FIG. 16A shows that when IM9
cells were cultured for 24 hours, the enzyme activity of the
endonuclease was constantly detected in cell lysate over the
period. However, the enzyme activity in the cell lysate treated by
CHX was reduced for 6 to 12 hours (FIG. 16B), whereas the enzyme
activity in the nucleus was accumulated (FIG. 16C).
[0089] 2-3 Isolation of Cell Nucleus and Autolysis
[0090] 1.times.10.sup.7 cells was washed three times with cold PBS
and dissolved in 0.5 ml cold buffer solution comprising 50 ml
Tris-HCl, pH8.0, 5 mM MgCl.sub.2 and 0.9 M sucrose at 40.degree. C.
for 20 minutes to isolate nucleus of cells. The prepared cell
lysate was placed in 0.5 ml of 1.2 M sucrose solution and
centrifuged at 800 g for 40 minutes. The resulting precipitate was
suspended in 20 mM Tris-HCl, pH 7.0, buffer solution to obtain
nuclei.
[0091] Autolysis of the isolated nucleus was carried out by
changing the concentration of Mg.sup.2+ and Ca.sup.2- at 37.degree.
C. at regular intervals. The reaction was stopped by adding 0.5 ml
DNA extraction buffer solution comprising 10 mM Tris-HCl, pH 7.5,
100 ml NaCl, 1 mM EDTA, 1% SDS proteinase K The mixture was reacted
at 50.degree. C. for 30 minutes, treated twice with
phenol/chloroform to remove proteins. After adding 0.3M sodium
acetate, pH 5.2, the mixture was precipitated with cold ethanol
After dissolving the precipitate in TE buffer solution, the
solution was treated with Rnase A. Then, electrophorosis was
carried out on 1.8% agarose gel. The electrophoresis gel was placed
in a solution containing 0.5/ml ethidium bromide and photographed
under UV.
[0092] By using autolysis method of the isolated cell nuclei, the
enzyme activity of the endonuclease present in the nucleus of IM9
cell was determined to observe the phenomenon in which DNA
fragments are produced depending on the Mg.sup.2+ concentration.
However, when under the same experiment condition Mg.sup.2+ was
replaced with Ca.sup.2+ such DNA fragmentation was not observed. In
the presence of both of Ca.sup.2+ and Mg.sup.2+, DNA fragments were
produced as the same type as in the presence of Mg.sup.2- only. The
result indicates that DNA is digested by the endonuclease depending
on the concentration of Mg.sup.2+. In the presence of 1 mM
Zn.sup.2+ and 5 mM EDTA, DNA fragments were not produced in nucleus
(FIG. 14B). DNA fragmentation was carried out at the constant
concentration of Mg.sup.2+.(10 mM) and Ca.sup.2+ (10 mM) for 0-4
hours. As results, 30 minutes after the reaction was initiated in
the presence of Mg.sup.2+ alone, the DNA fragments was produced,
and at 60 to 240 minutes, the DNA fragments were accumulated (FIG.
14C). But, in the presence of Ca.sup.2+, the formation of DNA
fragments was not observed for 4 hours (FIG. 14D). These results
indicate that Mg.sup.2+ is required for the endonuclease activity
present on the nucleus of IM9 cell to form DNA fragments.
[0093] 2-4 Partial Purification of Endonuclease Present on the
Nucleus of Cells and Characterization
[0094] The endonuclease present in the nucleus of cells was
partially purified by dissolving the nucleus and eluting the
protein band exhibiting the endonuclease activity as described in
the above Examples 1-4. The enzyme activity was measured by using
the supercoiled plasmid DNA as a substrate and the divalent ion
dependence was observed according to the above Examples 1-4. Also,
the change of enzyme activity by treatment of ZnCl.sub.2,
apoptosis-inhibiting substance, and EDTA, chelating agent was
observed.
[0095] The enzyme was isolated and eluted by native-PAGE to
characterize the enzyme activity of endonuclease present in the
nucleus of IM9 cell which was identified by DNA-native-PAGE. The
observation revealed. that the endonuclease is Mg.sup.2--dependent
(FIG. 17). The endonuclease activity was completely inhibited by
Zn.sup.2-, apoptosis-inhibiting substance, and EDTA, chelating
agent. The results indicate that Mg.sup.2+ is required to convert
the supercoiled plasmid DNA into the linear DNA and is consistent
with the result shown in FIG. 8.
EXAMPLE 3
Action of Endonuclease to Foreign DNA in Immune Cell and
Characterization of the Reaction Product
[0096] It was known that bacterial DNAs so far recognized as
foreign agent include various structure-determining factors which
are not present in mammalian DNA and that such factors are involved
in the activation of immune cell (Gilkeson, G. S. et al (1995) J.
Clin. Invest. 95, 1398-1402; Gilkeson, G. S. et al (1991) Clin.
Immunol, Immunopathol. 59, 288-300; Messina, J. P. et al (1993)
Cell. Immunol. 147, 148-157; Krieg, A. M. et al (1995) Nature
374,546-549; and Halpern, M. D. et al (1996) Cell. Immunol. 167,
72-79 ). One of the differences of mammalian DNA from bacterial DNA
is that the mammalian DNA was subject to considerable CpG
restriction and was selectively methylated on cytosine of CpG
dinucleotide (Bird, A. P. (1995) Trends Genet. 11, 94-100; Razin,
A., and Friedman, J. (1981) Prog. Nucleic Acid Res. Mol. Biol. 25,
33-52; and Han, J. et al (1994) Antisense Res. Dev. 4, 53-65).
Recent reports show that CpG motif present in bacterial DNA
activates polyclonal B cell to promote secretion of IgM (Krieg, A.
M. et al (1995) Nature 374, 546-549; Liang, H. et al (1996)j. Clin.
Invest. 98,1119-1129; and Yi, A. K et al (1996) J. Immunol. 156,
558-564) and suggest that cell cycle is stopped by anti-IgM
antibody and bacterial CpG motif inhibits the expression of c-myc
of B ceU while increasing the expression of myn, blc.sub.2 and
bcl-X.sub.L mRNA to protect the cell from apoptosis (Y, A. K. et al
(1996) J. Immunol. 157, 4918-4925). Halpern reported that CpG motif
directly activates B cell to promote secretion of IL-6 and IL-12
for short period (Halpern, M. D. et al (1996) Cell. Immunol.
167,72-78; Yi. A. K. et al (1996) J. Immunol. 157,5394-5402; and
Klinman, D. M. et al (1996) Proc. Natl. Acad. Sci. USA 93,
2879-2883). Bird showed that CpG motif weakly act for NK cell to
induce IFN-.gamma. from CD4.sup.4 (Bird, A. P. (1995) Trends Genet.
11, 94-100; aid Yamamoto; S. et al (1992) Microbiol. Immunol. 36,
983-997). Accordingly, the activation of immune cell by CpG DNA
increases humoral immunity by IL6 and increases cellular immunity
by IFN-.gamma. secretion. It was known that bacterial DNA is
digested by macrophage and then the macrophage is activated to
produce TNF-.alpha., IL-1.beta. and plasminogen activator
inhibitor-2 mRNA (Stacey, K. J. et al (1996) J. Immunol. 157,
2116-2122)
[0097] 3-1 Bacteria DNA Processing by Endonuclease
[0098] The culture solution of IM9 cell was used as enzyme source
to analyse the specificity of the enzyme activity of the
endonuclease and the property of the final reaction product. IM9
cells were cultured in RPMI 1640 medium containing 10% FBS in 5%
CO.sub.2 incubator at 37.degree. C. for 48 hours and then the
culture solution was used as enzyme source. Also, the culture
solution obtained by culturing the cells in FBS-free medium for 36
hours was used as enzyme source including only endonucleases
secreted by IN9 cell without DNase I.
[0099] The plasmids (PGEM-T vector, 3.0 Kb) used as the substrate
of enzyme were obtained by disrupting the E. coli by the alkaline
lysis method (Birboim, H. C., and Doly, J.(1979) Nucleic Acids Res.
7, 1513-1523), followed by extracting twice with phenol/chloroform
and precipitating with ethanol. Genomic DNA of E. coli and DNA of
IM9 cell were extracted by the modified method of Blin and
Stafford. Cells were twice washed with PBS and was floated by TE
buffer solution. DNA extraction buffer solution (10 mM Tris-HCl, pH
7.6, 10 mM EDTA, 50 mM NaCl, 0.2% SDS, 20 ug/ml. Rnase A) was added
and the solution was stood at 37.degree. C. for 10 minutes.
Proteinase K was added to 100 ug/ml and the mixture solution was
reacted at 50.degree. C. for 8 hours. The reaction solution was
extracted three times with phenol/chloroform to remove protein and
ethanol precipitation reaction was carried out to genomic DNA.
Salmon sperm DNA was also extracted and used as described above.
The amount of LPS present in DNA was measured by Limulus amebocyte
lysate assay (Sulliyan, J. D. et al (1976) in Mechanisms in
Bacterial Toxicity, A. W. Beheimner, ed. Wiley, New York, p. 217)
and was 2.5 ng/ml or less.
[0100] To compare the enzyme activity in IM9 cells cultured on the
medium containing 10% FBS and the FBS-free medium; 100 ng of
plasmid DNA was mixed with 20 ul of each culture and reacted at
37.degree. C. at regular intervals. In the assay of enzyme activity
of endonuclease by using the cell culture, the cell culture on the
FBS-free medium was used in order to to eliminate the effects of
DNase I present in FBS. Also, 100 ng of each of genomic DNAs of E.
coli, IM9 cell and salmon sperm was reacted under the same
condition as described above and thus the enzyme activity was
determined on 1% agarose gel electrophoresis.
[0101] RPMI1640 medium containing 10% FBS and IM9 cell culture was
directly reacted with bacterial DNA to observe the digestion degree
of bacteria DNA in vitro. FIG. 18A show the digestion degree of
plasmid DNA in RPMI1640 medium containing 10% FBS. Unlike the
results shown in FIG. 1D, the result showed that despite DNase I
was present in the medium, the weak enzyme activity was detected.
This indicates that the medium includes ions or inhibitors which
can effect on DNase I activity. However, plasmid DNA was degraded
by FBS-free cell culture solution or 10% FBS-containing cell
culture solution (FIGS. 18B and 18C). This indicates that plasmid
DNA was degraded by the endonuclease secreted by IM9 cell line. The
comparison of FIG. 18B and FIG. 18C shows that the enzyme activity
in 10% FBS-containing cell culture solution is higher. In the case
where cell culture was initiated at 2.times.10.sup.5 cells/ml, cell
growth in the presence of FBS was actively taken placed and a great
quantity of endonuclease was secreted. FIG. 19 shows that all of E.
coli DNA, IM9 cell DNA and salmon sperm DNA was degraded by
endonuclease.
[0102] 3.2 Incorporation of Bacterial DNA into Cells and Analysis
of the DNA Base Sequence
[0103] IM9 cell line was cultured on a 1:1 mixed medium of RPMI1640
medium containing heated 10% FBS and IM9 cell culture solution
which was cultured for 48 hours The bacterial DNA (25 ug/ml) was
added to the culture solution. While the IM9 cell line was cultured
in cell density of 1.times.10.sup.6 cells/ml, the cells were
collected at regular intervals and were used to extract the DNAs
incorporated into the cells.
[0104] After IM9 cell line was treated with the bacterial DNA, the
cells were collected at regular intervals and washed three times
with PBS. The cells were resuspended in a cold lysis buffer (10 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1 mM PMSF, and 0.5%
NP-40), and the cell lysates were obtained by the method described
in the above Example 1-2. The same volume of DNA extraction buffer
was added to the cell lysates and placed at 42.degree. C. for 3
hours and then treated twice with phenol/chloroform to remove
protein. Then the DNA present in the cell lysates was extracted by
ethanol precipitation. The precipitate was dissolved in TE buffer
and treated with RNase A and used for a sample of southern
blotting.
[0105] The DNA extracted from the cell lysates was separated by
electrophoresis on a 1.8% agarose gel and southern transfer was
carried out. After electrophoresis, the agarose gel was shaken for
20 minutes in about 200 ml of alkaline solution (1.5 M NaCl and 0.5
M NaOH), washed with distilled water, and placed for 20 minutes in
about 200 ml of the neutralization solution (1.5 M NaCl, 0.5 M
Tris-HCl, pH 7.5). Subsequently, the agarose gel was shaken in a
fresh neutralization solution. 175.3 g of NaCl, 88.2 g of sodium
citrate and 2 ml of 0.5 M EDTA were added to distilled water to
make 1 l solution and the resulting solution was autoclaved at
121.degree. C. to obtain SSC solution. One rectangular vessel A was
reversely placed on another vessel B containing 20.times.SSC so
that the bottom of the vessel A could be positioned on the surface
of the solution contained in the vessel B. Whatman No. 3 paper with
a width of 2 mm, saturated with 20.times.SSC, was placed on the
bottom of the vessel A without any air bubbles so that both
20.times.SSC could be connected. The agarose gel was reversely
placed on the paper, and Hybond nylon membrane was placed on the
agarose gel to prevent air bubbles from being generated. Three
Whatman No. 3 papers, smaller than the agarose gel in four facets
by 2 mm each, were placed on the Hybond nylon membrane with no air
bubbles. Then, paper towels were piled to reach the height of 10 to
20 cm and were weighed down with about 500 g. The bottom around the
gel was surrounded with parafilm and the capillary transfer was run
for 8 hours. The membrane carrying the. DNA was exposed to UV
radiation of 120,000 uJ/cm for 2 minutes to be crosslinked by
UV.
[0106] The bacterial DNA was reacted with the endonuclease at
37.degree. C. for 30 minutes and 100 to 200 bp of the resulting
product was electrophoresed on 1% agarose gel. Using the Gene Clean
Kit (Promega Inc.), the DNA was recovered from the gel and was used
as a DNA probe. 50 to 100 ng of the DNA was heated to 100.degree.
C. in water for 3 minutes, and cooled rapidly on ice to separate
the DNA fragments and then labeled with .sup.32P. DNA labeling
reaction was conducted according to the random priming method in
which 20 ul of a mixture consisting of 10 of random primer, 4 ul of
buffer solution (50 mM Tris-HCl, pH 80, 5 mM MgCl.sub.2, 2 mM DTT,
0.5 mM HEPES, pH 6.6), 25 uM dATP, 25 uM dGTP, 25 uM dTTP, 60 uCi
[.alpha.-.sup.32P]dCTP, and 5 ul of Klenow enzyme was reacted at
37.degree. C. for 2 hours. 2 ul of 0.5 M EDTA was added to the
reaction mixture to terminate the reaction, and then the same
volume of 3 M sodium acetate, pH 5.2, and 20 ug of salmon sperm DNA
were added. A double volume of ethanol was added to the reaction
mixture to precipitate the desired DNA. One precipitate was
dissolved in TE buffer, heated at 100.degree. C., and cooled
rapidly on ice.
[0107] UV-crosslinked Hybond.sup.+ nylon membrane was added to a
prehybridization solution (6.times.SSC, 5.times. Denhardt's
solution, 0.05% sodium pyrophosphate, 0.5% SDS, and 100 ug/ml of
salmon sperm DNA) prebathed at 68.degree. C., and shaken at
68.degree. C. for at least 1 hour. The labeled probe, prepared by
the random priming method and cooled on ice, was added to be
hybridized for about 12 hours. The filter was transferred to the
washing solution I (2.times.SSC, 0.1% SDS);and washed for about 10
minutes at ambient temperature The filter was then transferred to
the washing solution 1 prebathed at 68.degree. C., and shaken and
washed about 25 minutes. Subsequently, the filter was transferred
to the washing solution 11 (0.2.times.SSC, 0.1% SDS) prebathed at
68.degree. C. and washed while the radioactivity of the filter was
measured by Geiger counter. The filter was inserted to a cassette
fit with intensifying screen and X-ray film. After 12 to 24 hours
at -70.degree. C. the filter was developed.
[0108] The bacterial DNA or the plasmid DNA were added during cell
culture, and incubated for 1 hour at 37.degree. C. The DNA
fragments present in the cell lysates were electrophoresed ona 1.8%
agarose gel. Then, the gel of 50-200 bp sites by southern transfer
was recovered using Gene Clean Kit (Promega Inc.). The DNA
fragments were introduced into the pGEM-T vector (Promega Inc.) as
shown in FIG. 21 to identify base sequences. The resulting vector
was cloned in E. coli and analyzed by a SEQUENASE.TM. version 2.0
DNA sequencing kit (USB), according to the Sanger
dideoxyribonucleotide chain termination method (Sanger, F. et al.
(1977) Proc. Natl. Acad. Sci. USA 74,5463-5467). MB forward/reverse
primer described in Table 1 below was used as the primer for
sequencing. Cloned DNA sequence was confirmed as bacterial or
plasmid DNA fragments by a BLAST (Basic Local Alignment Search
Tools) program.
1 TABLE I oligonucleotides using A TTAAAACGTTCAC CpG motif B
AAGTGAACGTTTT CpG motif C AGCAGCGCTAA CpG motif D AATTAGCGCTG CpG
motif E CTCCCGGCCGCCATG PCR primer F TTGGGAGCTCTCCC PCR primer G
GTTTTCCCAGTCACGAC sequencing (puC/M13 forward) H CAGGAAACAGCTATGAC
sequencing (pUC/M13 reverse)
[0109] The test results show that when 25 ug/ml of bacterial DNA
was incubated with IM9 cell, the DNA was properly processed in the
cell culture and incorporated into the cells. This was confirmed by
southern hybridization (FIG. 20). The presence of 100-200 bp DNA
was found 30 minutes after the cell culture. The amount of 100-200
bp DNA in the cells was decreased with the lapse of the culture
time. The results suggest that properly processed DNA was
incorporated into IM9 cell line and the processing was continued in
cells.
[0110] To identify the property of the product obtained by the
enzyme activity of the endonuclease, the bacterial DNA incorporated
into cells was isolated and its DNA sequence was analyzed as shown
in FIG. 21. As shown in Table 2, the reaction product of the
endonuclease had a characteristic base sequence, i.e., CpG motif
carrying two Purina bases at 5' end and two pyridine bases at 3'
end. It is known that CpG motif activates B cell or macrophage in
an immune system and promotes the secretion of cytosine and IgM and
is present in bacterial DNA at high frequency. Accordingly, it was
now found that the endonuclease present in IM9 cell culture
solution and the DNA processing by the enzyme activity in the cell
generate CpG motif which functions to activate immune cells.
2TABLE 2 50-200 bp DNA fragments produced by endonuclease reaction
in cell Sequence Precursor EC1 . . . AGAGCAGCGCTAATGTCTATCGATGATTT
. . . GTCAAAACGTT E. coli from bases 2874223 to 2885223 CACCA . . .
of the complete genome (5416-5614) EC2 . . .
TTAACAACGTTGGGGCGATTGAGAGCGATGGCGTT- GATTTCATGTAAACGAAGCTA E. coli
from bases 4067762-4083201 ACGTGGTGAAAACGATGATGGCGCACGCGAGAAAT . .
. of the complete genome (4741-4852) EC3 . . . CCCATGACGCACCGCA . .
. ATTCCATCGCCATCTCAAACTTCGGTAA E. coli from bases 2244905 to
2255428 of the complete genome (2027-2108) EC4
TGCCTCGGAGTTACCTAATTCCATCGCCATCTCAAACTTCGGTAAA E. coli from bases
2244905 to 2255428 of the complete genome (2064-2109) EC5 . . .
CCTTTGACGTTGAGTCCACGTTCTTTA . . . CCTATCTCGGTCTATT . . . E. coli
plasmid synthetic cloning vector pET31F (268 376) EC6 TTTACGGTTCCTG
. . . TTTCCTGCGTTATCCC . . . Plasmid pKF3 from E. coli (2004-2066)
EC7 GTCGACCATATGGGAGAGCTCC . . ACGCGTTGGATG . . . AGCTTGGCGTAAT
Cloning vector pGEM-5Z1(-) (75-170) CAT . . . EC8 . . .
TTTCCTGCGTTATCCC . . . GCTGATACGCTCGCCGCAGCCGAACGACCGAG Sequence
from patent U.S. Pat. CGCAGCGAGTCAGTGAGCGAGGAAGCG No. 4921698 PD1
CTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGACGGTATCAG . . .
pGEM-T vector (401-519) PD2 . . . GGTCTGACGCTCAGTGGAACGAAAACTCAC-
GTTAAGGG . . . pGEM-T vector (1130-1306) PD3 . . .
TAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGG pGEM-T
vector (2513-2610) CCC . . . PD4 . . . TTCCCAACGATCAAGGCGAGTTACA .
. . CTCCGATCGTTGTCAG . . . pGEM-T vector (1685-1795) EC9 . . .
TGCTGTTCGGCACCAACAATCACGCCGACTTTAA . . . E. coli from bases 220025
to 231029 of the complete genome (829-872) HC1 . . . TTCGATTCGAT .
. . TCGAT . . . TTCGAG . . . Homo sapiens satellite 2 repetitive
element DNA
[0111] 3-3 Confirmation on the Processing of DNA Incorporated into
Cells
[0112] To confirm as to how far the cloned DNA fragments of 50-200
bp incorporated into cell by processing are further processed
within cells and how the base sequences of the DNA fragments
influence on the cells, a PCR amplification was performed. The PCR
reactive solution contained 0.2 mM dNTP, 10 pmole of primer, 10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl.sub.2, and 2.5 units of
Taq polymerase. 5'-CTCCCGGCCGCCATG-3' and 5'-TTGGGAGCTCTCC-3'
(Table I) were synthesized and used as a PCR primer. The PCR
reaction was repeated 35 times under the following condition:
duration.(at 94.degree. C. for 30 sec.);primer annealing (at
42.degree. C. for 30 sec.); primer extension (at 72.degree. C. for
50 sec.). The PCR product was run on 8% native-PAGE in a TBE buffer
solution and was stained with ethidium bromide. Subsequently, DNA
was separated from the gel of the PCR reaction product. The
separated DNA was precipitated by ethanol, and was dissolved in PBS
for cell culture or was dissolved in TE buffer solution for the
detection of the activity of the endonuclease.
[0113] To confirm as to which form of the end product exists after
the product obtained by the PCR amplification in a vector cloning
100-200 bp of bacteria DNA, which was cut by the activity of the
endonucleotide, is introduced into cells, the EC1, EC2, and HC1 DNA
fragments were labeled with .sup.32P by a random-priming method
using random primer and Klenow enzyme. 100 ng/ml of the DNA
fragments labeled with .sup.32P were incorporated into IG9 cell
line, U937 cell line, and U937 cell line treated with TPA. Whether
DNA was incorporated into cells was confirmed at regular intervals.
The endonuclease was treated with 0.2 mM ZnSO.sub.4(Sigma) to
terminate its enzyme activity. The DNA incorporated into cells was
recovered in a cell lysate and run on 20% native-PAGE in the TBE
buffer solution. The gel was dried and autoradiogaphed to confirm
the processing of DNA within cells and the end product obtained by
the endonuclease reaction.
[0114] The EC1 DNA fragment labeled with .sup.32P was incorporated
into IM9 cell line. The DNA incorporated into cells were extracted
from cell lysates and 20% native-PAGE was carried out. The
processed DNA labeled with .sup.32P was extracted from the gel and
annealed with a various synthesized oligonucleotides to compare
their base sequences with each another. A predetermined amount of
the extracted DNA labeled with .sup.32P and 10 ng of each of
oligonucleotides were mixed with 6.times.SSC. The mixture was
boiled at a temperature of 100.degree. C. for 5 minutes, and
annealed at temperatures lowered by 1.degree. C. per 30 seconds.
The annealed reactant was run on 20% natural-PAGE at 4.degree. C.,
and an autoradiography for the reactant was carried out. Thus,
oligonucleotides complimentarily conjugated to the DNA incorporated
into cells were confirmed. As standards, a mixture of DNA and
oligonucleotides which were not annealed, and a DNA denaturated and
annealed with only the DNA incorporated into cells were used. The
base sequences of synthesized oligonucleotides used oiligo A and B
which exist in EC1 DNA fragments and oligo C, D, and E which do not
exist in EC1 DNA (Table 1 above).
[0115] It was confirmed that exterior DNA was incorporated into
human B-lymphoblastic IM9 cell line. U937 cell line, and U937 cell
line differentiated by the treatment of TPA and was processed in
the cell lines. As a result of a test, the processed product was
identified as PCR amplified .sup.32P-labeled bacterial DNA
fragments. The incorporation and processing of exterior DNA in IM9
cell line were confirmed as shown in FIG. 22. 157 bp(EC1) of DNA
fragments were not incorporated into cells at 4.degree. C. but was
conjugated to the cell membranes. At a temperature of 37.degree.
C., the DNA fragments were processed in the cells with the lapse of
culture time. In 24 hours, about 10 bp DNA fragments were produced
as the end product in 20% native-PAGE. In a subsequent test, the
DNA fragments of 10 bp were identified as a single-stranded DNA
structure. The DNA fragment also was identified as a
single-stranded DNA structure on 20% denaturated-PAGE (8.3 M urea).
FIG. 23 shows the incorporation of exterior DNA observed in U937
cell line and U937 cell line differentiated by TPA treatment and
the processing of the DNA in the cells. However, FIG. 23B shows the
incorporation of exterior DNA and the processing in cells by U937
cell line differentiated by the treatment of TPA. As shown in FIG.
23B, the DNA processing in the cells was inhibited by 0.2mM of
Zn.sup.2+.
[0116] The test results demonstrate that the immune cells produce
the end product of 10 bases by processing the exterior DNA. The
properties of the base sequence of the DNA fragments were also
confirmed. After EC1 DNA products amplified by PCR were labeled
with .sup.32P and were incorporated into cells, the DNA fragments
of 10 bases processed by cellular endonucleases were annealed with
several types of synthetic oligonucleotides consistent with partial
sequences of EC1 to identify the base, sequences bound
complementarily with the oligonucleotides (FIG. 24). As a result of
annealing, the oligonucleotides, the most bound complementarily
with the base sequences, were oligo A sequences of Table 1, i.e.,
the base sequences which have AACGTT motif and are present in EC1
DNA. From the above results, it was clearly confirmed that CpG
motif known as the base sequences of bacterial DNA which activates
immune cells by enzymatic activity of endonuclease in cells was
produced.
[0117] 3-4 Confirmation on IgM Secretion of IM9 Cell Line by
Bacterial DNA
[0118] By activating B cells, the oligonucleotides having CpG motif
which is known to secrete cytokines and IgM were synthesized. Among
the DNA fragments produced by the action of endonuclease, the oligo
A present in EC1 and oligo B present in EC2, and oligo E which does
not have CpG motif were synthesized (Table 1). EC1 or EC2 PCR
products containing several CpG motifs, and several types of DNA
were also used in this experiment. To methylate CpG motif,
bacterial DNA was digested with endonuclease therey obtaiing the
fragments having 100-200 bp. The methylation was carried out by
mixing 30 .mu.g of DNA with the buffer solution (10 mM Tris-HCl, pH
7.9, 10 mM MgCl.sub.2, 50 mM NaCl, and 1 mM DTT) containing CpG
methylase and 160 uM of S-adenosylmethionine, and reacting for 3
hours at 37.degree. C. CpG methylation was confirmed by digesting
with Hpa II.
[0119] IM9 cell line, human B-lymphoblastic cell line, was cultured
in RPMI1640 medium containing 10% FBS with the cell counts being
2.times.10.sup.5/ml at the beginning of culture. After the culture
was treated with several types of preprepared oligonucleotides and
DNA in 25 ug/ml and was cultured for 24 hours, the cell culture was
obtained.
[0120] Anti-human-.mu.-chain-specific IgM (5 ug/ml, Sigma) in 0.1M
carbonate buffer solution (pH 9.6) was incorporated into flat
bottom plate in 100 ul/well and then the plate was placed for 16
hours at 4.degree. C. The plate was washed three times with PBS and
placed for 2 hours at room temperature. Subsequently, the plate was
washed thre times with TPBS (0.05% Tween-20 in PBS). Approximately
diluted cell culture or purified human IgM (Sigma) in 100 ul/well
was introduced. After being placed for 2 hours at room temperature,
it was washed three times with TPBS. Horseradish peroxidase-linked
anti-human Ig diluted in 1/4,000 in PBS containing % BSA was
introduced, and placed for 1 hour at room temperature. and then
washed three times with TPBS. The plate was subsequently treated
with O-phenylenediamine dihidrochioride in 0.05M phosphate buffer
solution (pH 5.0) for 30 minutes. The plate was treated with 0.67 N
H.sub.2SO.sub.4 to terminate the reaction and IgM was quantifed
using microplate reader.
[0121] While measuring the amount of lgM secreted 48 hours after
treating IM9 cell line with synthetic oligonucleotides, it was
confirmed that in oligo A and oligo C having CpG motif, 2-3 ng/ml
of IgM was secreted but in oligo E having no CpG motif, 0.5 ng/ml
of IgM was synthesized (FIG. 25). Further, it was observed that 2-3
ng/ml of IgM was also secreted in bacterial DNA and, PCR products
(Table 1, EC1, EC2 DNA) of bacterial DNA having several CpG motif.
However, if CpG motif of bacterial DNA IgM was methylated and
treated in cell line culture, it was not secreted (FIG. 25). In
comparison with bacterial DNA, the secretion of IgM was not
increased by salmon sperm DNA. From the above results, it was
confirmed that CpG motif of bacterial DNA increased the secretion
of IgM by activating IM9 cell line and it was consistent with the
results of study manifested by using CpG motif synthesized by
previous several researchers.
[0122] 3-5. Properties of the Enzyme Activity of Endonuclease
Involving the Processing of Foreign DNA
[0123] EC1, EC2, and HCl DNA amplified by PCR were labeled with
.sup.32P random-priming method employing random primer and Kienow
enzyme. To obtain shorter DNA. EC2 DNA was digested with Alu I
(Promega Inc.) and the fragments whose 5'-end was labeled with
.sup.32P were used as substrate. To identify the properties of
enzyme activity of the endonuclease, EC1 DNA was 5'-end or 3'-end
labeled and utilized 5'-end labeling was carried out by mixing 10
ng, EC1 DNA, [.gamma.-.sup.32P]ATP 50 uCi, kinase buffer solution
and 5 units of polynucleotide kinase (Promega Inc.) and reacting
form 1 hour at 37.degree. C. 3'-end labeling was carried out by
mixing 10 ng EC1 DNA, [.alpha.-.sup.32P]CTP 50 uCi, TdT buffer
solution and 5 units of terminal deoxytransferase (Boehringer
Mannheim) and reacting for 1 hour at 37.degree. C. and
utilized.
[0124] 5ng of EC1, EC2 and HCl DNA, prepared above, labeled by
random priming method, and EC2 DNA digested with Alu I and 5'-end
labeled was mixed IM9 cell culture which does not have FBS and is
reacted with constant time interval. Enzyme activities were also
measured by increasing the amount of the cell cultures. The
reaction mixture was treated with phenoltchloroform to remove
protein, precipitated with cold ethanol, and electrophoresed with
20% natural-PAGE and 20% denatured-PAGE (8.3 M urea) in TBE buffer
solution. The gel was dried, and autoradiographed. The reaction
products were analyzed for enzymatic activities. To compare with
the enzymatic activity of DNase I in FBS, 10 ml of RPMI1640 medium
containing 10% FBS was also reacted with several substrates.
Further, what effect do Zn.sup.2+ (1 mM), the inhibitor of
endonuclease and EDTA (10 mM), chelating agent was confirmed to
have enzmatic activities. At this time, Bromophenol Blue (BPB) and
xylene cynol (XC) were employed as standards of molecular weights.
In denatured-PAGE; BPB was positioned at 8 bases and XC was at 28
bases. In native-PAGE, BPB and XC were at 12 bps and 45 bps,
respectively.
[0125] To identify the properties of the enzyme activity of the
endonuclease and the properties of the resulting products, EC1,
EC2, and HCl DNA listed in Table 2 were labeled with .sup.32P by
random-priming method and utilized as substrates. It was confirmed
that the reaction of these substrates with IM9 cell line cultured
in FBS-free medium resulted in DNA reaction products having about
10 bases as shown in FIG. 26, 27 and 28. The results show that the
endonuclease does not recognize specifically the base sequence of
DNA. The endonuclease acted on foreign DNA regardless of base
sequences, thereby resulting in DNA fragments having about 10
bases. It was also confirmed that the enzytae activities are
inhibited by Zn.sup.2- the inhibitor of endonuclease, and EDTA, the
chelating agent (FIGS. 26, 27, 28 and 29). By in vitro experiment,
it was confirmed that the resulting products are consistent with
the reaction products produced by processing the DNA incorporated
into cells by the action of endonuclease. It was confirmed that the
reaction resultant product of enzymatic activities of endonuclease
consists of the DNA fragments having about 10 bases. Even in case
the amount of endonuclease was increased (FIG. 30) and the reaction
time was kept up to 24 hours, the DNA fragments having about 10
bases, i.e., the reaction resultant product formed by enzymatic
reaction were not degradcd further. Namely, the endonuclease
manifested by this invention does not exert any activity on the DNA
fragments having less than about 10 bases.
[0126] FIG. 32 shows several DNA fragments, i.e., endonuclease
reaction products formed before EC2 DNA product amplified by PCR
was digested with Alu I and the resulting DNA fragments were
reacted with endonuclease to produce the end product. The
substrates employed in this experiment were labeled with .sup.32P
in 5'-end of DNA reacted with Alu I. The labeled enzymatic reaction
resultant product was DNA fragment having about 10 bases. From this
fact, it is confirmed that the enzymatic activity of 5'-exonuclease
for producing single stranded DNA is not present.
[0127] To confirm whether endonuclease of IM9 cell line has
3'-exonuclease or 5'-exonuclease activity, EC2 DNA labeled with
.sup.32P at 5'- or 3'-end was employed as substrate and reacted
with endonuclease for 1 hour at 37.degree. C. The reaction
resultant products were compared by autoradiography by the same
method as stated above. Also, to confirm how enzymatic activity of
endonuclease appears in single stranded DNA, labeled DNA was boiled
for 5 minutes at 100.degree. C., cooled on ice and reacted with
endonuclease as stated above.
[0128] FIG. 33 shows that the DNA fragments having about 10 bases
are formed by the action of endonuclease on the substrate labeled
in 5'-end (5'-end label, lane 2). The results are the same as shown
in FIG. 32. That is, 5'-exonuclease activity was not present in
endonuclease. However, the DNA fragments having about 10 bases,
i.e., the enzymatic reaction resultant products, were not formed in
the substrates labeled with .sup.32P in 3'-end. After the
substrates labeled with .sup.32P were boiled for 10 minutes at
100.degree. C., cooled on ice to convert to single stranded DNA,
and then reacted with endonuclease, labeled DNA fragments having
about 10 bases were formed as enzymatic reaction products in DNA
substrates labeled in both 5'- and 3'-ends. Based on the test
results, 3'-exonuclease activity does not act on single stranded
chains.
[0129] To compare the reaction resultant products produced by
enzymatic activity of endonuclease present in cell cultures, with
the products produced by processing in cell and the reaction end
product produced by the action of DNase I, 5 units of bovine
pancreatic DNase I (Boehringer Mannheim) and EC1 DNA labeled with
.sup.32P by random-priming method were reacted in 10 mM Tris-HCl,
pH7.6, 10 mM MgCl.sub.2 buffer solution for 1 hour at 37.degree. C.
The reaction resultant was run on 20% of natural-PAGE and 20% of
denatured-PAGE (8.3M urea) autoradiographed, and compared with the
enzymatic reaction resultant product of endonuclease. Also, to
confirm tat the resultant product of endonuclease activity is
present in single stranded chains, it was treated with S1 nuclease.
Enzymatic activity of S1 nuclease was confirmed by mixing
endonuclease reaction resultant product by the action on the
substrates labeled with .sup.32P by random-priming method with 3
units of S1 nuclease contained in S1 nuclease reaction buffer
solution (7.times. buffer solution, 0.3M Potassium acetate, pH4.6,
2.5M NaCl, 10 mM ZnSO.sub.4, 50% glycerol), and reacting the
mixtures for 1 hour at 37.degree. C. The reaction resultants were
confirmed by autoradiography by the same method as stated
above.
[0130] By the comparison of the result product produced by the
enzymatic activity of endonuclease and the reaction product
produced by the action of DNase I, as shown in FIG. 34, the DNA
reaction products processed by the endonuclease of cell cultures
(lane 2) and cellular endonuclease (lane 3) are all the DNA
fragments having about 10 bases. However, by the action of DNase I
(lane 4), the DNA fragments were degraded into less than 10 bases.
The fragments were subsequently degraded into mononucleotides.
Also, to confirm whether the products obtained by endonuclease
reaction are present in single stranded form, the products were
treated with S1 nuclease and the enzymatic reaction products were
observed. When the reaction products having about 10 bases, i.e.,
the products of endonuclease, were reacted again with S1 nuclease,
as shown in FIG. 35, they were completely degraded into
mononucleotides. This is a clear evidence proving that the DINA
fragments having about 10 bases, produced by the enzymatic activity
of endonuclease are present in single stranded form.
EXAMPLE 4
Purification and Identification or Endonuclease Secreted from IM9
Cell Line
[0131] It was now found that IM9 cell line synthesized and secreted
Mg.sup.2+-dependent endonuclease which is distinct from nuclease
known so far. It was also found that the endonuclease is present in
the nucleus of IM9 cell line and participates in the apoptosis
process. The endonucleases involved in the apoptosis include
Mg.sup.2--dependent endonuclease (Anzai N. et al (1995) Blood 86,
917-923; Kawabata H. et al (1993) Biochem. Biophys. Res. Commun.
191, 247-254; Sun X. M., and Cohen G. M. (1994) J. Biol. Chem. 269,
14857-14860; and Kawabata, H. et al (1997) Biochem. Biophys. Res.
Commun. 233, 133-138),Ca.sup.2+/Mg.sup.2+-d- ependent endonuclease
(Stratling V. H. et al (1984) J. Biol. Chem. 259,5893-5898; Pandey
S. et al (1993) Biochemistry 32, 9129-9136). DNase I (Peitech M. C.
et al (1993) EMBO J. 12,371-377), NUC18 (Kawabata, H. et al (1997)
Biochem. Biophys. Res. Commun. 233, 133-138) and so on. However,
the purification, biochemical characteristics, and physiological
function or the above known endonucleases have not been clearly
defined. Furthermore, there was no report on any endonucleases
which may recognize bacterial DNAs as foreign agents and process
them. By the present invention, the endonuclease synthesized and
secreted by IG9 cell line was purified and the property thereof was
now identified.
[0132] 4-1 Protein Source and Assay for the Enzyme Activity of
Endonuclease
[0133] IM9 cell line secreting the endonuclease was massively
cultured in RPMI1640 medium containing heated 10% FBS and the
resulting cell culture was used as enzyme source. After the cell
culture was centrifuiged at 1,500.times. g for 5 minutes, IM9 cell
line was discarded and the cell culture solution was recovered. 101
of cell culture solution was centrifuged at 14,000.times. g for 30
minutes at 4.degree. C., and the resulting supernatant was used as
enzyme source.
[0134] During the purification of the endonuclease, the enzyme
activity was measured as the degree of the formation and digestion
of linear DNA from supercoiled plasmid DNA used as a substrate. 100
ng of plasmid DNA was added to 20 mM Tris-HCl pH 7.0, buffer
solution containing 10 mM MgCl.sub.2, and the mixture was reacted
with 20 ul of a sample obtained during the protein purification at
37.degree. C. for 10 minutes. The enzyme reaction was terminated
with the DNA sample buffer and the enzyme activity was assayed by
electrophoresis on a 1% agarose gel.
[0135] 4.2 Purification of Endonuclease
[0136] The cell culture solution was saturated to a concentration
of 80% by slowly adding NH.sub.1SO.sub.4, and then centrifuged at
14,000.times. g. The resulting precipitate was dialyzed overnight
in 20 mM sodium acetate buffer solution, pH 5.2. A 5 ml aliquot of
the enzyme solution was loaded on Mono-S column (0.5.times.5.0 cm,
Pharmacia LKB) preequilibrated with 5 ml of the same buffer
solution. The protein was first eluted by a linear concentration
gradient of 0-0.08 M NaCl (15 ml) in the same buffer solution and
then was passed over 15 ml of the same buffer solution containing
0.08 M NaCl. Subsequently, the protein was again eluted with a
linear concentration gradient of 0.08-0.2 M NaCl (20 ml). The
volume of each fraction was 1 ml and the flow rate was 0.5 ml/min.
The above procedure was repeated and the fraction containing the
endonuclease was pooled and concentrated using Centricon, and then
equilibrated with 50 mM of sodium phosphate buffer solution, pH.
7.0, containing 1.5 M (NH.sub.4).sub.2SO.sub.4. This enzyme source
was loaded on a RESOURCE PHE column (0.64.times.30mm, 1 ml.
Pharmacia LKB) and the hydrophobic interaction chromatography was
carried out. The column was washed with 15 ul of the same buffer
solution and the protein was eluted with a linear concentration
gradient of 1.5-0 M (NH.sub.4).sub.2SO.sub.4 (25 ml). The fraction
with the enzyme activity was concentrated in Centricon and
equilibrated with 20 mM Tris-HCl buffer solution, pH 7.0.
[0137] IM9cell culture solution was concentrated over ammonium
sulfate and used for purification. The differential precipitation
over ammonium sulfate was conducted depending on the concentration
difference, but did not significantly effect on the isolation of
the enzyme. Thus, culture solution was concentrated to 80% and the
protein was precipitated for the purification. The enzyme activity
in the sample passed over Mono S column was recovered by a linear
concentration gradient of 0.08-0.2 M NaCl (FIG. 36). The fraction
exhibiting the activity on cation-exchange resin was pooled and
passed over RESOURCE PHE column in a hydrophobic interaction
chromatography. When the protein was eluted with 1.5-0 M
(NH.sub.4).sub.2SO.sub.4 linear concentration gradient, the
activity of the endonuclease was detected in the protein fraction
obtained from 0.7M (NH.sub.4).sub.2SO.sub.4 gradient among the
above gradients (FIG. 37). 10 ug of the endonuclease was purified
from 10 l of IM9 cell culture solution containing about 10 g of
protein.
[0138] 4-3 Determination of Molecular Weight by the Nature Porous
Gradient PAGE and SDS-PAGE
[0139] The fraction exhibiting the enzyme activity on the
cation-exchange resin chromatography was pooled and concentrated in
Centricon. The concentrated solution was loaded on 4-15% linear
gradient of acrylamide gradient gel and electrophoresis was
performed with 4-5 mA for 18 hours at 4.degree. C. Then, the gel
was stained with coomnassie brilliant blue R-250 to detect the
protein band. The gel portion of the protein band was cut to make a
small piece prior to the staining and was eluted with 20 mM
Tris-HCl buffer solution, pH 7.0, for 8 hours at 4.degree. C. The
enzyme activity among the eluted protein fraction was detected
using the supercoiled plasmid DNA as a substrate. The enzyme
activity fraction was concentrated and separated by
SDS-polyacrylamide gel electrophoresis. The concentrations of the
staking gel and the running gel were 4% and 7%, respectively. The
standard protein in the native porous gradient PAGE was a mixture
of ferritin (440 kD), catalase (232 kD), lactate dehydrogenase (140
kD) and bovine serum albumin (87 kD). The standard protein in the
SDS-PAGE was a mixture of myosin (200 kD), .beta.-galactosidase
(116.3 kD), phosphorylase B (97.4 kD), bovine serum albumin (66.2
kD) and ovalbumin (45 kD).
[0140] The enzyme activity fraction eluted on Mono S column was
concentrated and 4-15% native porous gradient gel electrophoresis
was carried out to detect the protein band showing the enzyme
activity (FIG. 40). The protein band exhibiting the enzyme,
activity was not shown as clear single band and was, spread around
140 kD as compared with the standard protein. The protein band
exhibiting the enzyme activity was eluted. After concentration;
electrophoresis was conducted on SDS-PAGE (FIG. 41). The purified
protein band was shown at the same site as the endonuclease
purified by chromatography and the molecular weight of the protein
was determined as about 72.4 kD (FIG. 39). A comparison of the
results of the native porous gradient gel electrophoresis and
SDS-PAGE revealed that the endonuclease exhibits the enzyme
activity in the form of homodimer.
[0141] 4-4 Characterization of the Purified Enzyme
[0142] Nuclei were isolated from U937 cell from which any
endonuclease enzyme activity was not detected for 4 hours, and were
used as substrate. The specificity of the enzyme activity was
confirmed by adding 1 mM Ca.sup.2+, 1 mM Mg.sup.2+, 1 mM Zn.sup.2-,
and EDTA to 20 mM Tris-HCl buffer solution, pH 7.0 containing the
isolated nuclei and the reaction was allowed for 10 minutes at
37.degree. C.
[0143] When the nuclei isolated from U937 cell line were used as
substrate, the activity of the purified enzyme was exhibited in the
presence of Mg.sup.2+ and was completely inhibited by Zn.sup.2+ ,
apoptosis inhibitor, and EDTA, chelating agent (FIG. 42). The
characteristic enzyme activity was consistent with that of the
above enzymatic activity.
[0144] Industrial Availability of the Invention
[0145] The endonuclease of the present invention is able to degrade
foreign bacterial DNA and incorporate the DNA fragments into cells.
The DNA incorporated into cells is processed by the intracellular
endonuclease to produce oligonucleotide including CpG motif and
then the immune cell is activated by the CpG motif to promote the
secretion of antibody. Accordingly, the endonuclease of the present
invention is industrially valuable as an pharmaceutical immune
adjuvant.
Sequence CWU 1
1
30 1 13 DNA Escherichia coli 1 ttaaaacgtt cac 13 2 13 DNA
Escherichia coli 2 aagtgaacgt ttt 13 3 11 DNA Escherichia coli 3
agcagcgcta a 11 4 11 DNA Escherichia coli 4 aattagcgct g 11 5 15
DNA Escherichia coli 5 ctcccggccg ccatg 15 6 14 DNA Escherichia
coli 6 ttgggagctc tccc 14 7 17 DNA pUC/M13 forward 7 gttttcccag
tcacgac 17 8 17 DNA pUC/M13 forward 8 caggaaacag ctatgac 17 9 29
DNA Escherichia coli 9 agagcagcgc taatgtctat cgatgattt 29 10 16 DNA
Escherichia coli 10 gtcaaaacgt tcacca 16 11 91 DNA Escherichia coli
11 ttaacaacgt tggggcgatt gagagcgatg gcgttgattt catgtaaacg
aagctaacgt 60 ggtgaaaacg atgatggcgc acgcgagaaa t 91 12 16 DNA
Escherichia coli 12 cccatgacgc accgca 16 13 28 DNA Escherichia coli
13 attccatcgc catctcaaac ttcggtaa 28 14 46 DNA Escherichia coli 14
tgcctcggag ttacctaatt ccatcgccat ctcaaacttc ggtaaa 46 15 27 DNA
plasmid pET31F 15 cctttgacgt tgagtccacg ttcttta 27 16 15 DNA
plasmid pET31F 16 cctatctcgg tctat 15 17 13 DNA plasmid pKF3 from
E. coli 17 tttacggttc ctg 13 18 16 DNA plasmid pKF3 from E. coli 18
tttcctgcgt tatccc 16 19 22 DNA plasmid pGEM-5Zf(-) 19 gtcgaccata
tgggagagct cc 22 20 12 DNA plasmid pGEM-5Zf(-) 20 acgcgttgga tg 12
21 11 DNA plasmid pGEM-5Zf(-) 21 agcttggcgt a 11 22 16 DNA Sequence
from US patent 4921698 22 tttcctgcgt tatccc 16 23 59 DNA Sequence
from US patent 4921698 23 gctgatacgc tcgccgcagc cgaacgaccg
agcgcagcga gtcagtgagc gaggaagcg 59 24 56 DNA plasmid pGEM-T 24
cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgacgg tatcag 56 25
38 DNA plasmid pGEM-T 25 ggtctgacgc tcagtggaac gaaaactcac gttaaggg
38 26 58 DNA plasmid pGEM-T 26 taaagaacgt ggactccaac gtcaaagggc
gaaaaaccgt ctatcagggc gatggccc 58 27 25 DNA plasmid pGEM-T 27
ttcccaacga tcaaggcgag ttaca 25 28 16 DNA plasmid pGEM-T 28
ctccgatcgt tgtcag 16 29 34 DNA Escherichia coli 29 tgctgttcgg
caccaacaat cacgccgact ttaa 34 30 11 DNA Homo sapiens 30 ttcgattcga
t 11
* * * * *