U.S. patent application number 09/952663 was filed with the patent office on 2002-08-29 for arginase ii.
Invention is credited to Dillon, Patrick J., Vockley, Joseph G..
Application Number | 20020119554 09/952663 |
Document ID | / |
Family ID | 24812513 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119554 |
Kind Code |
A1 |
Vockley, Joseph G. ; et
al. |
August 29, 2002 |
Arginase II
Abstract
The invention relates to Arginase II polypeptides,
polynucleotides encoding the polypeptides, methods for producing
the polypeptides, in particular by expressing the polynucleotides,
and agonists and antagonists of the polypeptides. The invention
further relates to methods for utilizing such polynucleotides,
polypeptides, agonists and antagonists for applications, which
relate, in part, to research, diagnostic and clinical arts.
Inventors: |
Vockley, Joseph G.;
(Downingtown, PA) ; Dillon, Patrick J.;
(Gaithersburg, MD) |
Correspondence
Address: |
LICATA & TYRRELL P.C.
66 E. Main Street
Marlton
NJ
08053
US
|
Family ID: |
24812513 |
Appl. No.: |
09/952663 |
Filed: |
September 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09952663 |
Sep 14, 2001 |
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09541762 |
Apr 3, 2000 |
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09541762 |
Apr 3, 2000 |
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09116115 |
Jul 15, 1998 |
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09116115 |
Jul 15, 1998 |
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08914981 |
Aug 20, 1997 |
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08914981 |
Aug 20, 1997 |
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08700186 |
Aug 20, 1996 |
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60013395 |
Mar 14, 1996 |
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Current U.S.
Class: |
435/226 ;
435/320.1; 435/325; 435/6.11; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61P 13/02 20180101;
A61K 38/00 20130101; A61K 48/00 20130101; A61P 9/12 20180101; A61P
43/00 20180101; A61P 15/00 20180101; C12N 9/78 20130101 |
Class at
Publication: |
435/226 ;
435/325; 435/320.1; 435/69.1; 536/23.2; 435/6 |
International
Class: |
C12N 009/64; C12Q
001/68; C07H 021/04; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated polynucleotide comprising a nucleotide sequence that
has at least 80% identity over its entire length to a nucleotide
sequence encoding the HUMAN ARGINASE II polypeptide of SEQ ID NO:2;
or a nucleotide sequence complementary to said isolated
polynucleotide.
2. The polynucleotide of claim 1 wherein said polynucleotide
comprises the nucleotide sequence contained in SEQ ID NO:1 encoding
the HUMAN ARGINASE II polypeptide of SEQ ID NO:2.
3. The polynucleotide of claim 1 wherein said polynucleotide
comprises a nucleotide sequence that is at least 80% identical to
that of SEQ ID NO:1 over its entire length.
4. The polynucleotide of claim 3 which is polynucleotide of SEQ ID
NO:1.
5. The polynucleotide of claim 1 which is DNA or RNA.
6. A DNA or RNA molecule comprising an expression system, wherein
said expression system is capable of producing a HUMAN ARGINASE II
polypeptide comprising an amino acid sequence, which has at least
80% identity with the polypeptide of SEQ ID NO:2 when said
expression system is present in a compatible host cell.
7. A host cell comprising the expression system of claim 6.
8. A process for producing a HUMAN ARGINASE II polypeptide
comprising culturing a host of claim 7 under conditions sufficient
for the production of said polypeptide and recovering the
polypeptide from the culture.
9. A process for producing a cell which produces a HUMAN ARGINASE
II polypeptide thereof comprising transforming or transfecting a
host cell with the expression system of claim 6 such that the host
cell, under appropriate culture conditions, produces a HUMAN
ARGINASE II polypeptide.
10. A HUMAN ARGINASE II polypeptide comprising an amino acid
sequence which is at least 80% identical to the amino acid sequence
of SEQ ID NO:2 over its entire length.
11. The polypeptide of claim 10 which comprises the amino acid
sequence of SEQ ID NO:2.
12. An antibody immunospecific for the HUMAN ARGINASE II
polypeptide of claim 10.
13. A method for the treatment of a subject in need of enhanced
activity or expression of HUMAN ARGINASE II polypeptide of claim 10
comprising: (a) administering to the subject a therapeutically
effective amount or an agonist to said polypeptide; and/or (b)
providing to the subject an isolated polynucleotide comprising a
nucleotide sequence that has at least 80% identity to a nucleotide
sequence encoding the HUMAN ARGINASE II polypeptide of SEQ ID NO:2
over its entire length; or a nucleotide sequence complementary to
said nucleotide sequence in a form so as to effect production of
said polypeptide activity in vivo.
14. A method for the treatment of a subject having need to inhibit
activity or expression of HUMAN ARGINASE II polypeptide of claim 10
comprising: (a) administering to the subject a therapeutically
effective amount of an antagonist to said polypeptide; and/or (b)
administering to the subject a nucleic acid molecule that inhibits
the expression of the nucleotide sequence encoding said
polypeptide; and/or (c) administering to the subject a
therapeutically effective amount of a polypeptide that competes
with said polypeptide for its ligand, substrate, or receptor.
15. A process for diagnosing a disease or a susceptibility to a
disease in a subject related to expression or activity of HUMAN
ARGINASE II polypeptide of claim 10 in a subject comprising: (a)
determining the presence or absence of a mutation in the nucleotide
sequence encoding said HUMAN ARGINASE II polypeptide in the genome
of said subject; and/or (b) analyzing for the presence or amount of
the HUMAN ARGINASE II polypeptide expression in a sample derived
from said subject.
16. A method for identifying compounds which inhibit (antagonize)
or agonize the HUMAN ARGINASE II polypeptide of claim 10 which
comprises: (a) contacting a candidate compound with cells which
express the HUMAN ARGINASE II polypeptide (or cell membrane
expressing HUMAN ARGINASE II polypeptide) or respond to HUMAN
ARGINASE II polypeptide; and (b) observing the binding, or
stimulation or inhibition of a functional response; or comparing
the ability of the cells (or cell membrane) which were contacted
with the candidate compounds with the same cells which were not
contacted for HUMAN ARGINASE II polypeptide activity.
17. An agonist identified by the method of claim 16.
18. An antagonist identified by the method of claim 16.
19. A recombinant host cell produced by a method of claim 9 or a
membrane thereof expressing a HUMAN ARGINASE II polypeptide.
Description
[0001] This invention relates, in part, to newly identified
polynucleotides and polypeptides; variants and derivatives of the
polynucleotides and polypeptides; processes for making the
polynucleotides and the polypeptides, and their variants and
derivatives; agonists and antagonists of the polypeptides; and uses
of the polynucleotides, polypeptides, variants, derivatives,
agonists and antagonists. In particular, in these and in other
regards, the invention relates to polynucleotides and polypeptides
of human Arginase II.
BACKGROUND OF THE INVENTION
[0002] Clinically significant hyperagininemia results from
mutations in the Type I arginase gene (Arginase I) (Cederbaum S D,
et al., 1979, Pediat Res 13:827-833; Cederbaum S., et al., 1977, J.
Pediatr 90:569-573; Michel V V, et al., 1978, Clin Genet 13:61-67)
which is predominantly expressed in the liver and red blood cells
(Gasiorowska I et al., 1970, Biochim Biophys Acta 17:19-30; Hermann
B G and Frischauf A- M., 1987, Meth Enzymol 152: 180-183; Spector E
B, et al., 1982, Biochem Med 28: 165-175.) Arginase I deficient
patients present with spasticity, growth retardation, progressive
mental impairment and episodic hyperammonemia (Cederbaum S D, et
al., 1979, Pediat Res 13:827-833: Cederbaum S D, et al., 1977, J.
Pediatr 90:569-573; Thomas K R and Capecchi M R, 1987, Cell
51:503-512.) Although significantly devastating, Arginase I
deficiency has a milder clinical phenotype than other urea cycle
disorders. It has been proposed that the presence of a second
isoform of arginase might be responsible for this milder
presentation (Grody W W., et al., 1989, J Clin Invest 83:602-609;
Grody W W, et al., 1993, Hum Genet 91: 1-5) The existence of an
extra-urea cycle form of arginase (Arginase II), which localizes to
the mirochondria (Wissmann P B, et al., 1994, Am J Hum Genet
55:A139), has been demonstrated utilizing various non-cross
reacting antibodies to Arginase I and Arginase II (Spector E B. et
al, 1983, Pediatr Res 17:941-944). In patients deficient in type I
arginase activity, a compensatory up regulation of Arginase II has
been observed (Gasiorowska I, et al., 1970, Biochim Biophys Acta
17:19-30; Hermann B G, et al., 1987. Meth Enzymol 152:180-183).
Using these antibodies, it has been established that Arginase II is
expressed predominantly in the kidney, but is also found in the
brain, activated macrophage, the gastrointestinal tract and in the
lactating mammary gland (Spector E B, et al., 1983, Pediatr Res
17:941-944.) Arginase II is not expressed at a significant level in
the liver or red blood cells. In addition to a hypothetical role in
the production of proline and glutamate it has been postulated that
Arginase II may play an important role in nitric oxide biosynthesis
through the production of ornithine as a precursor of glutamate
(Mezl V A, et al., 1977, Biochem J 164:105-113; Wang W W et al.,
1995, Biochem Biophys Res Comm 210:1009-1016.) It is because of the
many potential extra-urea cycle, metabolic roles of Arginase II and
its up regulation in the hyperargininemic patient, with its
implications for gene therapy, that cloning of the Arginase II gene
is important. Many different techniques have been utilized to
isolate the gene for Arginase II. The presence of six highly
conserved regions in the protein, present in the arginases of most
species examined, has been critical to the discovery process
(Johnson J L, et al., 1984, J Neurochem 43:1123-1126; Ikeda Y, et
al., 1987, Arch Biochem Biophys 252:662-674.)
SUMMARY OF THE INVENTION
[0003] Toward these ends, and others, it is an object of the
present invention to provide polypeptides, inter alia, that have
been identified as novel Arginase II by homology between the amino
acid sequence set out in FIG. 2 and known amino acid sequences of
other proteins such as Type I arginase and agmatinase.
[0004] It is a further object of the invention, moreover, to
provide polynucleotides that encode Arginase II, particularly
polynucleotides that encode the polypeptide herein designated
Arginase II.
[0005] In a particularly preferred embodiment of this aspect of the
invention the polynucleotide comprises the region encoding human
Arginase II in the sequence set out in FIG. 2 or in the cDNA in
ATCC deposit No. 75,656 deposited Jan. 26, 1994 (referred to herein
as the deposited clone).
[0006] In accordance with this aspect of the invention there are
provided isolated nucleic acid molecules encoding human Arginase
II, including mRNAs, cDNAs, genomic DNAs and, in further
embodiments of this aspect of the invention, biologically,
diagnostically, clinically or therapeutically useful variants,
analogs or derivatives thereof, or fragments thereof including
fragments of he variants, analogs and derivatives.
[0007] Among the particularly preferred embodiments of this aspect
of the invention are naturally occurring allelic variants of human
Arginase II.
[0008] It also is an object of the invention to provide Arginase II
polypeptides, particularly human Arginase II polypeptides, to treat
diseases associated with or caused by a defect in the Arginase II
gene or Arginase II gene expression, such as, for example, urea
cycle diseases, hypertension, hypotension, episodic hyperammonemia,
defects in biosynthesis of proline, glutamate, nitric oxide and
ornithine, as well as hyperagininemia and its related spasticity,
growth retardation, and progressive mental impairment, and prostate
disease, particularly prostate cancer, prostatitis and benign
prostatic hyperplasia or hypertrophy, and also prostate damage,
kidney disease, and kidney damage.
[0009] In accordance with another object of the invention is a
method of using Arginase II Lo control nitric oxide formation in an
individual having a need to control such formation.
[0010] In accordance with yet another object of the invention is a
method of treating systemic hypotension caused by sepsis or
cytokines using Arginase II alone or in combination with an alpha
sub-1 adrenergic agonist.
[0011] Another object of the invention is a method to deplete
systemic arginine levels in an individual having a need for a
depletion of such levels.
[0012] In accordance with this aspect of the invention there are
provided novel polypeptides of human origin referred to herein as
Arginase II as well as biologically, diagnostically or
therapeutically useful fragments, variants and derivatives thereof,
variants and derivatives of the fragments, and analogs of the
foregoing.
[0013] Among the particularly preferred embodiments of this aspect
of the invention are variants of human Arginase II encoded by
naturally occurring alleles of the human Arginase II gene.
[0014] It is another object of the invention to provide a process
for producing the aforementioned polypeptides, polypeptide
fragments, variants and derivatives, fragments of the variants and
derivatives and analogs of the foregoing.
[0015] In a preferred embodiment of this aspect of the invention
there are provided methods for producing the aforementioned
Arginase II polypeptides comprising culturing host cells having
expressibly incorporated therein an exogenously-derived human
Arginase II-encoding polynucleotide under conditions for expression
of human Arginase II in the host and then recovering the expressed
polypeptide.
[0016] In accordance with another object the invention there are
provided products, compositions, processes and methods that utilize
the aforementioned polypeptides and polynucleotides for research,
biological, clinical and therapeutic purposes, inter alia.
[0017] In accordance with certain preferred embodiments of this
aspect of the invention, there are provided products, compositions
and methods, inter alia, for, among other things: assessing
Arginase II expression in cells by determining Arginase II
polypeptides of Arginase II-encoding mRNA; in a sample from a host
organism having or suspected of having a disease associated with or
caused by a defect in the Arginase II gene or Arginase II gene
expression, such as, for example, urea cycle diseases,
hypertension, hypotension, episodic hyperammonemia, defects in
biosynthesis of proline, glutamate, nitric oxide and ornithine, as
well as hyperagininemia and its related spasticity, growth
retardation, and progressive mental impairment, and prostate
disease, particularly prostate cancer, prostatitis and benign
prostatic hyperplasia or hypertrophy, and also prostate damage,
kidney disease, and kidney damage, in vitro, ex vivo or in vivo by
exposing cells to Arginase II polypeptides or polynucleotides as
disclosed herein; assaying genetic variation and aberrations, such
as defects, in Arginase II genes; and administering a Arginase II
polypeptide or polynucleotide to an organism to augment Arginase II
function or remediate Arginase II dysfunction.
[0018] Another object of the invention is a method for the
preparation of L-ornithine salts.
[0019] Yet another object of the invention is a method for the
diagnosis of a disorder arising from Arginase II deficiency in an
individual having or suspected of having a defect in the nitric
oxide pathway and the urea cycle.
[0020] Also provided is a method to monitor progression or therapy
of disorders with hypotension as a clinical sign.
[0021] Further provided is a method to monitor progression of or
therapy of disorders of the immune or nervous systems associated
with or caused by nitric oxide regulation.
[0022] Another object of the invention is a method to monitor
activation state of macrophage.
[0023] A further object of the invention is a method to monitor
mitochondrial activity as a function of the transportation of
arginase II into the mirochondria.
[0024] In accordance with certain preferred embodiments of this and
other aspects of the invention there are provided probes that
hybridize specifically to human Arginase II sequences.
[0025] In certain additional preferred embodiments of this aspect
of the invention there are provided antibodies against Arginase II
polypeptides. In certain particularly preferred embodiments in this
regard, the antibodies are highly selective for human Arginase
II.
[0026] In accordance with another aspect of the present invention,
there are provided Arginase II agonists. Among preferred agonists
are molecules that mimic Arginase II, that bind to Arginase
II-binding molecules or receptor molecules, and that elicit or
augment Arginase II-induced responses. Also among preferred
agonists are molecules that interact with Arginase II or Arginase
II polypeptides, or with other modulators of Arginase II
activities, and thereby potentiate or augment an effect of Arginase
II or more than one effect of Arginase II.
[0027] In accordance with yet another aspect of the present
invention, there are provided Arginase II antagonists. Among
preferred antagonists are those which mimic Arginase II so as to
bind to Arginase II receptor or binding molecules but not elicit a
Arginase II-induced response or more than one Arginase II-induced
response. Also among preferred antagonists are molecules that bind
to or interact with Arginase II so as to inhibit an effect of
Arginase II or more than one effect of Arginase II.
[0028] The agonists and antagonists may be used to mimic, augment
or inhibit the action of Arginase II polypeptides. They may be
used, for instance, to treat diseases associated with or caused by
a defect in the Arginase II gene or Arginase II gene expression,
such as, for example, urea cycle diseases, hypertension
hypotension, episodic hyperammonemia, defects in biosynthesis of
proline, glutamate, nitric oxide and ornithine, as well as
hyperagininemia and its related spasticity, growth retardation, and
progressive mental impairment, and prostate disease, particularly
prostate cancer, prostatitis and benign prostatic hyperplasia or
hypertrophy, and also prostate damage, kidney disease, and kidney
damage.
[0029] In a further aspect of the invention there are provided
compositions comprising a Arginase II polynucleotide or a Arginase
II polypeptide for administration to cells in vitro, to cells ex
vivo and to cells in vivo, or to a multicellular organism. In
certain particularly preferred embodiments of this aspect of the
invention, the compositions comprise a Arginase II polynucleotide
for expression of a Arginase II polypeptide in a host organism for
treatment of disease. Particularly preferred in this regard is
expression in a human patient for treatment of a dysfunction
associated with aberrant endogenous activity of Arginase II or to
provide therapeutic. Such compositions may be used to treat, for
example, urea cycle diseases, hypertension, hypotension, episodic
hyperammonemia, defects in biosynthesis of proline, glutamate,
nitric oxide and ornithine, as well as hyperargininemia and its
related spasticity growth retardation, and progressive mental
impairment, and prostate disease, particularly prostate cancer,
prostatitis and benign prostatic hyperplasia or hypertrophy, and
also prostate damage, kidney disease, and kidney damage.
[0030] Other objects, features, advantages and aspects of the
present invention will become apparent to those of skill from the
following description. It should be understood, however, that the
following description and the specific examples, while indicating
preferred embodiments of the invention, are given by way of
illustration only. Various changes and modifications within the
spirit and scope of the disclosed invention will become readily
apparent to those skilled in the art from reading the following
description and from reading the other parts of the present
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The following drawings depict certain embodiments of the
invention. They are illustrative only and do not limit the
invention otherwise disclosed herein
[0032] FIG. 1(A) shows the contig of the Arginase II cDNA with the
EST numbers from FIG. 1(B) aligned with the EST, and (B) shows the
ESTs that form the Arginase II contig.
[0033] FIG. 2 shows the nucleotide (SEQ ID NO:1) and deduced amino
acid (SEQ ID No:2) sequence of human Arginase II.
[0034] FIG. 3 shows the primers used in cloning the Arginase II
cDNA.
[0035] FIG. 4 shows the regions of similarity between
polynucleotide sequences of Arginase II (top) and Arginase I
(bottom) polynucleotides.
[0036] FIG. 5 shows the regions of similarity between polypeptide
sequences of Arginase I (top) and Arginase II (bottom).
[0037] Glossary
[0038] The following illustrative explanations are provided to
facilitate understanding of certain terms used frequently herein,
particularly in the examples. The explanations are provided as a
convenience and are not limitative of the invention
[0039] DIGESTION of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes referred to herein are
commercially available and their reaction conditions, cofactors and
other requirements for use are known and routine to the skilled
artisan.
[0040] For analytical purposes, typically, 1 mg of plasmid or DNA
fragment is digested with about 2 units of enzyme in about 20 ml of
reaction buffer. For the purpose of isolating DNA fragments for
plasmid construction, typically 5 to 50 mg of DNA are digested with
20 to 250 units of enzyme in proportionately larger volumes.
[0041] Appropriate buffers and substrate amounts for particular
restriction enzymes are described in standard laboratory manuals,
such as those referenced below, and they are specified by
commercial suppliers.
[0042] Incubation times of about 1 hour at 37.degree. C. are
ordinarily used, but conditions may vary in accordance with
standard procedures, the supplier's instructions and the
particulars of the reaction. After digestion, reactions may be
analyzed, and fragments may be purified by electrophoresis through
an agarose or polyacrylamide gel, using well known methods that are
routine or those skilled in the art.
[0043] GENETIC ELEMENT generally means a polynucleotide comprising
a region that encodes a polypeptide or a region that regulates
transcription or translation or other processes important to
expression of the polypeptide in a host cell, or a polynucleotide
comprising both a region that encodes a polypeptide and a region
operably linked thereto that regulates expression.
[0044] Genetic elements may be comprised within a vector that
replicates as an episomal element; that is, as a molecule
physically independent of the host cell genome. They may be
comprised within mini-chromosomes, such as those that arise during
amplification of transfected DNA by methotrexate selection in
eukaryotic cells. Genetic elements also may be comprised within a
host cell genome; not in their natural state but, rather, following
manipulation such as isolation, cloning and introduction into a
host cell in the form of purified DNA or in a vector, among
others.
[0045] SIMILARITY between two polpeptides is determined by
comparing the amino acid sequence and its conserved amino acid
substitutes of one polypeptide to the sequence of a second
polypeptide. Moreover, also known in the art is "IDENTITY" which
means the degree of sequence relatedness between two polypeptide or
two polynucleotide sequences as determined by the identity of the
match between two strings of such sequences Both identity and
similarity can be readily calculated (Computational Molecular
Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds, M Stockton Press, New York, 1991). While
there exist a number of methods to measure identity and similarity
between two polynucleotide or polypeptide sequences, the terms
"identity" and "similarity" are well known to skilled artisans
(Sequence Analysis in Molecular Biology, von Heinje, G., Academic
Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux,
J., eds., M Stockton Press, New York. 1991; and Carillo, H., and
Lipman, D., SIAM J. Applied Math, 48:1073 (1988). Methods commonly
employed to determine identity or similarity between two sequences
include, but are not limited to disclosed in Guide to Huge
Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994,
and Carillo, H., and Lipman, D, SIAM J. Applied Math., 48: 1073
(1988). Preferred methods to determine identity are designed to
give the largest match between the two sequences tested. Methods to
determine identity and similarity are codified in computer
programs. Preferred computer program methods to determine identity
and similarity between two sequences include, but are not limited
to, GCG program package (Devereux, J., et al., Nucleic Acids
Research 12(1): 387 (1984)), BLASTP, BLASTN, FASTA (Atschui, S. F.
et al., J. Molec. Biol. 215: 403 (1990)).
[0046] ISOLATED means altered "by the hand of man" from its natural
state; i.e., that if it occurs in nature, it has been changed or
removed from its original environment, or both.
[0047] For example, a naturally occurring polynucleotide or a
polypeptide naturally present in a living animal in its natural
state is not "isolated," but the same polynucleotide or polypeptide
separated from some or all of the coexisting materials of its
natural is "isolated", as the term is employed herein.
[0048] As part of or following isolation, such polynucleotides can
be joined to other polynucleotides, such as DNAs, for mutagenesis,
to form fusion proteins, and for propagation or expression in a
host, for instance. The isolated polynucleotides, alone or joined
to other polynucleotides such as vectors, can be introduced into
host cells, in culture or in whole organisms. Introduced into host
cells in culture or in whole organisms, such DNAs still would be
isolated, as the term is used herein, because they would not be in
their naturally occurring form or environment. Similarly, the
polynucleotides and polypeptides may occur in a composition, such
as a media formulations, solutions for introduction of
polynucleotides or polypeptides, for example, into cells,
compositions or solutions for chemical or enzymatic reactions, for
instance, which are not naturally occurring compositions, and,
therein remain isolated polynucleotides or polypeptides within the
meaning of that term as it is employed herein.
[0049] LIGATION refers to the process of forming phosphodiester
bonds between two or more polynucleotides, which most often are
double stranded DNAs. Techniques for ligation are well known to the
art and protocols for ligation are described in standard laboratory
manuals and references, such as, for instance, Sambrook et al.,
MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Maniatis et
al., pg. 146, as cited below.
[0050] OLIGONUCLEOTIDE(S) refers to relatively short
polynucleotides. Often the term refers to single-stranded
deoxyribonucleotides, but it can refer as well to single-or
double-stranded ribonucleotides, RNA:DNA hybrids and
double-stranded DNAs, among others.
[0051] Oligonucleotides, such as single-stranded DNA probe
oligonucleotides, often are synthesized by chemical methods, such
as those implemented on automated oligonucleotide synthesizers.
However, oligonucleotides can be made by a variety of other
methods, including in vitro recombinant DNA-mediated techniques and
by expression of DNAs in cells and organisms.
[0052] Initially, chemically synthesized DNAs typically are
obtained without a 5' phosphate. The 5' ends of such
oligonucleotides are not substrates for phosphodiester bond
formation by ligation reactions that employ DNA ligases typically
used to form recombinant DNA molecules. Where ligation of such
oligonucleotides is desired, a phosphate can be added by standard
techniques, such as those that employ a kinase and ATP.
[0053] The 3' end of a chemically synthesized oligonucleotide
generally has a free hydroxyl group and, in the presence of a
ligase, such as T4 DNA ligase, readily will form a phosphodiester
bond with a 5' phosphate of another polynucleotide, such as another
oligonucleotide. As is well known, this reaction can be prevented
selectively, where desired, by removing the 5' phosphates of the
other polynucleotide(s) prior to ligation.
[0054] PLASMIDS generally are designated herein by a lower case p
preceded and/or followed by capital letters and/or numbers, in
accordance with standard naming conventions that are familiar to
those of skill in the art. Starting plasmids disclosed herein are
either commercially available, publicly available on an
unrestricted basis, or can be constructed from available plasmids
by routine application of well known, published procedures. Many
plasmids and other cloning and expression vectors that can be used
in accordance with the present invention are well known and readily
available to those of skill in the art. Moreover, those of skill
readily may construct any number of other plasmids suitable for use
in the invention. The properties, construction and use of such
plasmids, as well as other vectors, in the present invention will
be readily apparent to those of skill from the present
disclosure.
[0055] POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide
or polydeoxribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. Thus, for instance, polynucleotides as used
herein refers to, among others, single- and double-stranded DNA,
DNA that is a mixture of single- and double-stranded regions,
single- and double-stranded RNA, and RNA that is mixture of single-
and double-stranded regions, hybrid molecules comprising DNA and
RNA that may be single-stranded or, more typically, double-stranded
or a mixture of single- and double-stranded regions.
[0056] In addition, polynucleotide as used herein refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The strands in such regions may be from the same molecule or from
different molecules. The regions may include all of one or more of
the molecules, but more typically involve only a region of some of
the molecules. One of the molecules of a triple-helical region
often is an oligonucleotide.
[0057] As used herein, the term polynucleotide includes DNAs or
RNAs as described above that contain one or more modified bases.
Thus, DNAs or RNAs with backbones modified for stability or for
other reasons are "polynucleotides" as that term is intended
herein. Moreover, DNAs or RNAs comprising unusual bases, such as
inosine, or modified bases, such as tritylated bases, to name just
two examples, are polynucleotides as the term is used herein.
[0058] It will be appreciated that a great variety of modifications
have been made to DNA and RNA that serve many useful purposes known
to those of skill in the art. The term polynucleotide as it is
employed herein embraces such chemically, enzymatically or
metabolically modified forms of polynucleotides, as well as the
chemical forms of DNA and RNA characteristic of viruses and cells,
including simple, and complex cells, inter alia.
[0059] POLYPEPTIDES, as used herein includes all polypeptides as
described below. The basic structure of polypeptides is well known
and has been described in innumerable textbooks and other
publications in the art. In this context, the term is used herein
to refer to any peptide or protein comprising two or more amino
acids joined to each other in a linear chain by peptide bonds. As
used herein, the term refers to both short chains, which also
commonly are referred to in the art as peptides, oligopeptides and
oligomers, for example, and to longer chains, which generally are
referred to in the art as proteins, of which there are many
types.
[0060] It will be appreciated that polypeptides often contain amino
acids other than the 20 amino acids commonly referred to as the 20
naturally occurring amino acids, and that many amino acids,
including the terminal amino acids, may be modified in a given
polypeptide, either by natural processes, such as processing and
other post-translational modifications, but also by chemical
modification techniques which are well known to the art. Even the
common modifications that occur naturally in polypeptides are too
numerous to list exhaustively here, but they are well described in
basic texts and in more detailed monographs, as well as in a
voluminous research literature, and they are well known to those of
skill in the art. Among the known modifications which may be
present in polypeptides of the present are, to name an illustrative
few, acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
cross-links, formation of cysteine, formation of pyroglutamate,
formylation, gamma-carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0061] Such modifications are well known to those of skill and have
been described in great detail in the scientific literature.
Several particularly common modifications, glycosylation, lipid
attachment, sulfation, gamma-carboxylation of glutamic acid
residues, hydroxylation and ADP-ribosylation, for instance, are
described in most basic texts, such as, for instance
PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York. (1993). Many
detailed reviews are available on this subject, such as, for
example, those provided by Wold, F., Posttranslational Protein
Modifications: Perspectives and Prospect, pgs. 1-12 in
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,
Ed., Academic Press, New York (1983); Seifter et al., Analysis for
protein modifications and nonprotein cofactors, Meth. Enzymol. 182:
626-646 (1990) and Rattan et al., Protein Synthesis:
Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci.
663: 48-62 (1992).
[0062] It will be appreciated, as is well known and as noted above,
that polypeptides are not always entirely linear. For instance
polypeptides may be branched as a result of ubiquitination, and
they may be circular, with or without branching generally as a
result of postranslation events, including natural processing event
and events brought about by human manipulation which do not occur
naturally. Circular, branched and branched circular polypeptides
may be synthesized by non-translation natural process and by
entirely synthetic methods, as well.
[0063] Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. In fact, blockage of the amino or carboxyl group
in a polypeptide, or both, by a covalent modification, is common in
naturally occurring and synthetic polypeptides and such
modifications may be present in polypeptides of the present
invention, as well. For instance, the amino terminal residue of
polypeptides made in E. coli, prior to proteolytic processing,
almost invariably will be N-formylmethionine.
[0064] The modifications that occur in a polypeptide often will be
a function of how it is made. For polypeptides made by expressing a
cloned gene in a host, for instance, the nature and extent of the
modifications in large part will be determined by the host cell
posttranslational modification capacity and the modification
signals present in the polypeptide amino acid sequence. For
instance, as is well known, glycosylation often does not occur in
bacterial hosts such as E. coli. Accordingly, when glycosylation is
desired, a polypeptide should be expressed in a glycosylating host,
generally a eukaryotic cell. Insect cell often carry out the same
posttranslational glycosylations as mammalian cells and, for this
reason, insect cell expression systems have been developed to
express efficiently mammalian proteins having native patterns of
glycosylation, inter alia. Similar considerations apply to other
modifications.
[0065] It will be appreciated that he same type of modification may
be present in the same or varying degree at several sites in a
given polypeptide. Also, a given polypeptide may contain many types
of modifications.
[0066] In general, as used herein, the term polypeptide encompasses
all such modifications, particularly those that are present in
polypeptides synthesized by expressing a polynucleotide in a host
cell.
[0067] VARIANT(S) of polynucleotides or polypeptides, as the term
is used herein, are polynucleotides or polypeptides that differ
from a reference polynucleotide or polypeptide, respectively.
Variants in this sense are described below and elsewhere in the
present disclosure in greater detail.
[0068] (1) A polynucleotide that differs in nucleotide sequence
from another, reference polynucleotide. Generally, differences are
limited so that the nucleotide sequences of the reference and the
variant are closely similar overall and, in many regions,
identical.
[0069] As noted below, changes in the nucleotide sequence of the
variant may be silent. That is, they may not alter the amino acids
encoded by the polynucleotide. Where alterations are limited to
silent changes of this type a variant will encode a polypeptide
with the same amino acid sequence as the reference. Also as noted
below, changes in the nucleotide sequence of the variant may alter
the amino acid sequence of a polypeptide encoded by the reference
polynucleotide. Such nucleotide changes may result in amino acid
substitutions, additions, deletions, fusions and truncations in the
polypeptide encoded by the reference sequence, as discussed
below.
[0070] (2) A polypeptide that differs in amino acid sequence from
another, reference polypeptide. Generally, differences are limited
so that the sequences of the reference and the variant are closely
similar overall and, in many region, identical.
[0071] A variant and reference polypeptide may differ in amino acid
sequence by one or more substitutions, additions, deletions,
fusions and truncations, which may be present in any
combination.
[0072] RECEPTOR MOLECULE, as used herein, refers to molecules which
bind or interact specifically with Arginase II polypeptides of the
present invention, including not only classic receptors, which are
preferred, but also other molecules that specifically bind to or
interact with polypeptides of the invention (which also may be
referred to as "binding molecules" and "interaction molecules,"
respectively and as "Arginase II binding molecules" and "Arginase
II interaction molecules." Binding between polypeptides of the
invention and such molecules, including receptor or binding or
interaction molecules may be exclusive to polypeptides of the
invention, which is very highly preferred, or it may be highly
specific for polypeptides of the invention, which is highly
preferred, or it may be highly specific to a group of proteins that
includes polpeptides of the invention, which is preferred, or it
may be specific to several groups of proteins at least one of which
includes polypeptides of the invention.
[0073] Receptors also may be non-naturally occurring, such as
antibodies and antibody-derived reagents that bind specifically to
polypeptides of the invention.
DESCRIPTION OF THE INVENTION
[0074] The present invention relates to novel Arginase II
polypeptides and polynucleotides, among other things, as described
in greater detail below. In particular, the invention relates to
polpeptides and polynucleotides of a novel human Arginase II, which
is related by amino acid sequence homology to Arginase I, non-type
I arginase from Xenopus and agmatinase. The invention relates
especially to Arginase II having the nucleotide and amino acid
sequences set out in FIG. 2, and to the Arginase II nucleotide and
amino acid sequences obtained from a cDNA library in ATCC Deposit
No. 75,656, which is herein referred to as "the deposited clone" or
as the "cDNA of the deposited clone." It will be appreciated that
the nucleotide and amino acid sequences set out in FIG. 2 were
obtained by sequencing the cDNA of the deposited clone. Hence, the
sequence of the deposited clone is controlling as to any
discrepancies between the two.
[0075] Polynucleotides
[0076] In accordance with one aspect of the present invention,
there are provided isolated polynucleotides which encode the
Arginase II polypeptide having the deduced amino acid sequence of
FIG. 2 or the Arginase II polypeptide encoded by the cDNA in the
deposited clone.
[0077] Using the information provided herein, such as the
polynucleotide sequence set out in FIG. 2, a polypeptide of the
present invention encoding human Arginase II polypeptide may be
obtained using standard cloning and screening procedures, such as
those for cloning cDNAs using mRNA from cells of a Jurkat cell line
library as starting material. Illustrative of the invention, the
polynucleotide set out in FIG. 2 was discovered in a cDNA library
derived from cells of a human Jurkat cell line library.
[0078] Human Arginase II of the invention is structurally related
to other proteins of the arginase family, as shown by the results
of sequencing the cDNA encoding human Arginase II in the deposited
clone. The cDNA sequence thus obtained is set out in FIG. 2. It
contains an open reading frame encoding a protein of about 355
amino acid residues with a deduced molecular weight of about 35
kDa. The protein exhibits greatest homology to Arginase I and
non-type I arginase from Xenopus, among known proteins. The 355
residues of the Arginase II of FIG. 2 have about 55% identity and
about 68% similarity with the amino acid sequence of human type I
arginase.
[0079] Arginase II is expressed in kidney and to a much greater
extent in prostate as determined by northern blot analysis using
the Arginase II gene as a probe. Other tissues showed no observable
message on northern blots. It is known that express nitric oxide is
expressed at extremely high levels in the prostate.
[0080] Polynucleotides of the present invention may be in the form
of RNA, such as mRNA, or in the form of DNA, including, for
instance, cDNA and genomic DNA obtained by cloning or produced by
chemical synthetic techniques or by a combination thereof. The DNA
may be triple-stranded, double-stranded or single-stranded.
Single-stranded DNA may be the coding strand, also known as the
sense strand, or it may be the non-coding strand, also referred to
as the anti-sense strand.
[0081] The coding sequence which encodes the polypeptide may be
identical to the coding sequence of the polynucleotide shown in
FIG. 2 or that of the deposited clone. It also may be a
polynucleotide with a different sequence, which, as a result of the
redundancy (degeneracy) or the generic code, encodes the
polypeptide of the DNA of FIG. 2 or of the deposited cDNA.
[0082] Polynucleotides of the present invention which encode the
polypeptide of FIG. 2 or the polypeptide encoded by the deposed
cDNA may include, but are not limited to the coding, sequence for
the mature polypeptide, by itself; the coding sequence for the
mature polypeptide and additional coding sequences, such as those
encoding a leader or secretory sequence, such as a pre-, or pro- or
prepro- protein sequence; the coding sequence of the mature
polypeptide, with or without the aforementioned additional coding
sequences, together with additional, non-coding sequences,
including for example, but not limited to introns and non-coding 5'
and 3' sequences, such as the transcribed, non-translated sequences
that play a role in transcription, mRNA processing--including
splicing and polyadenylation signals, for example--ribosome binding
and stability of mRNA, additional coding sequence which codes for
additional amino acids, such as those which provide additional
functionalities. Thus, for instance, the polypeptide may be fused
to a marker sequence, such as a peptide, which facilitates
purification of the fused polypeptide. In certain preferred
embodiments of this aspect of the invention, the marker sequence is
a hexa-histidine peptide, such as the tag provided in the vector
pQE-9, among others, many of which are commercially available. As
described in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824
(1989), for instance, hexa-histidine provides for convenient
purification of the fusion protein. The HA tag corresponds to an
epitope derived of influenza hemagglutinin protein which has been
described by Wilson et al., Cell 37: 767 (1984), for instance.
[0083] In accordance with the foregoing the term "polynucleotide
encoding a polypeptide" as used herein encompasses polynucleotides
which include a sequence encoding a polypeptide of the present
invention, particularly the human Arginase II having the amino acid
sequence set out in FIG. 2 or the amino acid sequence of the human
Arginase II encoded by the cDNA of the deposited clone. The term
encompasses polynucleotides that include a single continuous region
or discontinuous regions encoding the polypeptide (for example,
interrupted by introns) together with additional regions, that also
may contain coding and/or non-coding sequences.
[0084] The present invention further relates to variants of the
herein above described polynucleotides which encode for fragments,
analogs and derivatives of the polypeptide having the deduced amino
acid sequence of FIG. 2 or the polypeptide encoded by the cDNA of
the deposited clone. A variant of the polynucleotide may be a
naturally comprising variant such as a naturally occurring allelic
variant, or it may be a variant that is not known to occur
naturally. Such non-naturally occurring variants of the
polynucleotide may be made by mutagenesis techniques, including
those applied to polynucleotides, cells or organisms.
[0085] Among variants in this regard are variants that differ from
the aforementioned polynucleotides by nucleotide substitutions,
deletions or additions. The substitutions, deletions or additions
may involve one or more nucleotides. The variants may be altered in
coding or non-coding regions or both. Alterations in the coding
regions may produce conservative or non-conservative amino acid
substitutions, deletions or additions.
[0086] Among the particularly preferred embodiments of the
invention in this regard are polynucleotides encoding polypeptides
having the amino acid sequence of Arginase II set out in FIG. 2 or
the amino acid sequence of Arginase II of the cDNA of the deposited
clone; variants, analogs, derivatives and fragments thereof, and
fragments of the variants, analogs and derivatives.
[0087] Further particularly preferred in this regard are
polynucleotides encoding Arginase II variants, analogs, derivatives
and fragments, and variants, analogs and derivatives of the
fragments, which have the amino acid sequence of the Arginase II
polypeptide of FIG. 2 or of the deposit in which several, a few, 5
to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are
substituted, deleted or added, in any combination. Especially
preferred among these are silent substitutions, additions and
deletions, which do not alter the properties and activities of the
Arginase II. Also especially preferred in this regard are
conservative substitutions. Most highly preferred are polpeptides
having the amino acid sequence of FIG. 2 or of the deposit, without
substitutions.
[0088] Further preferred embodiments of the invention are
polynucleotides that are at least 70% identical to a polynucleotide
encoding the Arginase II polypeptide having the amino acid sequence
set out in FIG. 2, or variants, close homologs, derivatives and
analogs thereof, as described above, and polynucleotides which are
complementary to such polynucleotides. Alternatively, most highly
preferred are polynucleotides that comprise a region that is at
least 80% identical to a polynucleotide encoding the Arginase II
polypeptide of the cDNA of the deposited clone and polynucleotides
complementary thereto. In this regard, polynucleotides at least 90%
identical to the same are particularly preferred, and among these
particularly preferred polynucleotides, those with at least 95% are
especially preferred. Furthermore, those with at least 97% are
highly preferred among those with at least 95%, and among these
those with at least 98% and at least 99% are particularly highly
preferred, with at least 99% being the more preferred.
[0089] Particularly preferred embodiments in this respect,
moreover, are polynucleotides which encode polpeptides which retain
substantially the same biological function or activity as the
mature polypeptide encoded by the cDNA of FIG. 2 or the cDNA of the
deposited clone.
[0090] The present invention further relates to polynucleotides
that hybridize to the herein above-described sequences. In this
regard, the present invention especially relates to polynucleotides
which hybridize under stringent conditions to the herein
above-described polynucleotides. As herein used, the term
"stringent conditions" means hybridization will occur only if there
is at least 95% and preferably at least 97% identity between the
sequences.
[0091] As discussed additionally herein regarding polynucleotide
assays of the invention, for instance, polynucleotides of the
invention as discussed above, may be used as a hybridization probe
for cDNA and genomic DNA to isolate full-length cDNAs and genomic
clones encoding Arginase II and to isolate cDNA and genomic clones
or other genes that have a high sequence similarity to the human
Arginase II gene. Such probes generally will comprise at least 15
bases. Preferably, such probes will have at least 30 bases and may
have at least 50 bases. Particularly preferred probes will have at
least 30 bases and will have 50 bases or less.
[0092] For example, the coding region of the Arginase II gene may
be isolated by screening using the known DNA sequence to synthesize
an oligonucleotide probe. A labeled oligonucleotide having a
sequence complementary to that of a gene of the present invention
is then used to screen a library of human cDNA, genomic DNA or mRNA
to determine which members of the library the probe hybridizes
to.
[0093] The polynucleotides and polypeptides of the present
invention may be employed as research reagents and materials for
discovery of treatments and diagnostics to human disease, as
further discussed herein relating to polynucleotide assays, inter
alia.
[0094] The polynucleotides may encode a polypeptide which is the
mature protein plus additional amino or carboxyl-terminal amino
acids, or amino acids interior to the mature polypeptide (when the
mature form has more than one polypeptide chain, for instance).
Such sequences may play a role in processing of a protein from
precursor to a mature form, may facilitate protein trafficking, may
prolong or shorten protein half-life or may facilitate manipulation
of a protein for assay or production, among other things. As
generally is the case in situ, the additional amino acids may be
processed away from the mature protein by cellular enzymes.
[0095] A precursor protein, having the mature form of the
polypeptide fused to one or more prosequences may be an inactive
form of the polypeptide. When prosequences are removed such
inactive precursors generally are activated. Some or all of the
prosequences may be removed before activation. Generally, such
precursors are called proproteins.
[0096] In sum, a polynucleotide of the present invention may encode
a mature protein, a mature protein plus a leader sequence (which
may be referred to as a preprotein), a precursor of a mature
protein having one or more prosequences which are not the leader
sequences of a preprotein, or a preproprotein, which is a precursor
to a proprotein, having a leader sequence and one or more
prosequences, which generally are removed during processing steps
that produce active and mature forms of the polypeptide.
[0097] Deposited Materials
[0098] A deposit containing a human Arginase II cDNA has been
deposited with the American Type Culture Collection, as noted
above. Also as noted above, the cDNA deposit is referred to herein
as "the deposited clone" or as "the cDNA of the deposited
clone."
[0099] The deposited clone was deposited with the American Type
Culture Collection, 12301 Park Lawn Drive, Rockville, Md. 20852,
USA, on Jan. 26, 1994, and assigned ATCC Deposit No. 75,656.
[0100] The deposited material is a pBluescript SK (-) plasmid
library (Stratagene, La Jolla, Calif.) that contains cDNA clones,
including those of Arginase II.
[0101] The deposit has been made under the terms of the Budapest
Treaty on the international recognition of the deposit of
micro-organisms for purposes of patent procedure. The strain will
be irrevocably and without restriction or condition released to the
public upon the issuance of a patent. The deposit is provided
merely as convenience to those of skill in the art and is not an
admission that a deposit is required for enablement, such as that
required under 35 U.S.C. section 112.
[0102] The sequence of the polynucleotides contained in the
deposited material, as well as the amino acid sequence of the
polypeptide encoded thereby, are controlling in the event of any
conflict with any description of sequences herein.
[0103] A license may be required to make, use or sell the deposited
materials, and no such license is hereby granted.
[0104] Polypeptides
[0105] The present invention further relates to a human Arginase II
polypeptide which has the deduced amino acid sequence of FIG. 2 or
which has the amino acid sequence encoded by the deposited
clone.
[0106] The invention also relates to fragments, analogs and
derivatives of these polypeptides. The terms "fragment,"
"derivative" and "analog" when referring to the polypeptide of FIG.
2 or that encoded by the deposited cDNA, means a polypeptide which
retains essentially the same biological function or activity as
such polypeptide. Thus, an analog includes a proprotein which can
be activated by cleavage of the proprotein portion to produce an
active mature polypeptide.
[0107] The polypeptide of the present invention may be a
recombinant polypeptide, a natural polypeptide or a synthetic
polypeptide. In certain preferred embodiments it is a recombinant
polypeptide.
[0108] The fragment, derivative or analog of the polypeptide of
FIG. 2 or that encoded by the cDNA in the deposited clone may be
(i) one in which one or more of the amino acid residues are
substituted with a conserved or non-conserved amino acid residue
(preferably a conserved amino acid residue) and such substituted
amino acid residue may or may not be one encoded by the genetic
code, or (ii) one in which one or more or he amino acid residues
includes a substituent group, or (iii) one in which the mature
polypeptide is fused with another compound, such as a compound to
increase the half-life of the polypeptide (for example,
polyethylene glycol), or (iv) one in which the additional amino
acids are fused to the mature polypeptide, such as a leader or
secretory sequence or a sequence which is employed for purification
of the mature polypeptide or a proprotein sequence. Such fragments,
derivatives and analogs are deemed to be within the scope of those
skilled in the art from the teachings herein.
[0109] Among the particularly preferred embodiments of the
invention in this regard are polypeptides having the amino acid
sequence of Arginase II set out in FIG. 2, variants, analogs,
derivatives and fragments thereof, and variants, analogs and
derivatives of the fragments. Alternatively, particularly preferred
embodiments of the invention in this regard are polypeptides having
the amino acid sequence of the Arginase II of the cDNA in the
deposited clone, variants, analogs, derivatives and fragments
thereof, and variants, analogs and derivatives of the
fragments.
[0110] Among preferred variants are those that vary from a
reference by conservative amino acid substitutions. Such
substitutions are those that substitute a given amino acid in a
polypeptide by another amino acid of like characteristics.
Typically seen as conservative substitutions are the replacements,
one for another, among the aliphatic amino acids Ala, Val, Leu and
Ile; interchange of the hydroxyl residues Ser and Thr, exchange of
the acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr.
[0111] Further particularly preferred in this regard are variants,
analogs, derivatives and fragments, and variants, analogs and
derivatives of the fragments, having the amino acid sequence of the
Arginase II polypeptide of FIG. 2 or of the cDNA in the deposited
clone, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no
amino acid residues are substituted, deleted or added, in any
combination. Especially preferred among these are silent
substitutions, additions and deletions, which do not alter the
properties and activities of the Arginase II. Also especially
preferred in this regard are conservative substitutions. Most
highly preferred are polypeptides having the amino acid sequence of
FIG. 2 or the deposited clone without substitutions.
[0112] The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
[0113] The polypeptides of the present invention include the
polypeptide of SEQ ID NO: 2 (in particular the mature polypeptide)
as well as polypeptides which have at least 70% similarity
(preferably at least 70% identity) to the polypeptide of SEQ ID
NO:2 and more preferably at least 90% similarity (more preferably
at least 90% identity) to the polypeptide of SEQ ID NO:2 and still
more preferably at least 95% similarity (still more preferably at
least 95% identity) to the polypeptide of SEQ ID NO:2 and also
include portions of such polypeptides with such portion of the
polypeptide generally containing at least 30 amino acids and more
preferably at least 50 amino acids.
[0114] Fragments or portions of the polpeptides of the present
invention may be employed for producing the corresponding
full-length polypeptide by peptide synthesis; therefore, the
fragments may be employed as intermediates for producing the
full-length polypeptides. Fragments or portions of the
polynucleotides of the present invention may be used to synthesize
full-length polynucleotides of the present invention.
[0115] Fragments
[0116] Also among preferred embodiments of this aspect of the
present invention are polypeptides comprising fragments of Arginase
II, most particularly fragments of the Arginase II having the amino
acid set out in FIG. 2, or having the amino acid sequence of the
Arginase II of the deposited clone, and fragments of variants and
derivatives of the Arginase II of FIG. 2 or of the deposited
clone.
[0117] In this regard a fragment is a polypeptide having an amino
acid sequence that entirely is the same as part but not all of the
amino acid sequence of the aforementioned Arginase II polypeptides
and variants or derivatives thereof
[0118] Such fragments may be "free-standing," i.e., not part of or
fused to other amino acids or polypeptides, or they may be
comprised within a larger polypeptide of which they form a part or
region. When comprised within a larger polypeptide, the presently
discussed fragments most preferably form a single continuous
region. However, several fragments may be comprised within a single
larger polypeptide. For instance, certain preferred embodiments
relate to a fragment of a Arginase II polypeptide of the present
comprised within a precursor polypeptide designed for expression in
a host and having heterologous pre and pro-polypeptide regions
fused to the amino terminus of the Arginase II fragment and an
additional region fused to the carboxyl terminus of the fragment.
Therefore, fragments in one aspect of the meaning intended herein,
refers to the portion or portions of a fusion polypeptide or fusion
protein derived from Arginase II.
[0119] As representative examples of polypeptide fragments of the
invention, there may be mentioned those which have from about 8 to
about 16 amino acids which include the regions of high
cross-section homology and polypeptide fragments of the invention
which have from about 20 to 50 amino acids which include the
regions of lowest homology between type I arginase and the sequence
reported in FIG. 2.
[0120] In this context about includes the particularly recited
range and ranges larger or smaller by several, a few,5, 4, 3, 2 or
1 amino acid at either extreme or at both extremes. For instance,
about 65-90 amino acids in this context means a polypeptide
fragment of 65 plus or minus several, a few, 5, 4, 3, 2 or 1 amino
acids to 90 plus or minus several a few, 5, 4, 3, 2 or 1 amino acid
residues, i.e, ranges as broad as 65 minus several amino acids to
90 plus several amino acids to as narrow as 65 plus several amino
acids to 90 minus several amino acids.
[0121] Highly preferred in this regard are the recited ranges plus
or minus as many as 5 amino acids at either or at both extremes.
Particularly highly preferred are the recited ranges plus or minus
as many as 3 amino acids at either or at both the recited extremes.
Especially particularly highly preferred are ranges plus or minus 1
amino acid at either or at both extremes or the recited ranges with
no additions or deletions. Most highly preferred of all in this
regard are fragments from about 8 to about 50 amino acids to
include six areas of high cross-species homology which most likely
contribute to the active size of the enzyme and three areas of low
cross species homology which may be of greatest diagnostic
significance.
[0122] Among especially preferred fragments of the invention are
truncation mutants of Arginase II. Truncation mutants include
Arginase II polpeptides having the amino acid sequence of FIG. 2,
or of the deposited clone, or of variants or derivatives thereof,
except for deletion of a continuous series of residues (that is, a
continuous region, part or portion) that includes the amino
terminus, or a continuous series of residues that includes the
carboxyl terminus or, as in double truncation mutants, deletion of
two continuous series of residues, one including the amino terminus
and one including the carboxyl terminus. Fragments having the size
ranges set out about also are preferred embodiments of truncation
fragments, which are especially preferred among fragments
generally.
[0123] Also preferred in this aspect of the invention are fragments
characterized by structural or functional attributes of Arginase
II. Preferred embodiments of the invention in this regard include
fragments that comprise alpha-helix and alpha-helix forming regions
("alpha-regions"), beta-sheet and beta-sheet-forming regions
("beta-regions"), turn and turn-forming regions ("turn-regions"),
coil and coil-forming regions ("coil-regions"), hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions and
high antigenic index regions of Arginase II.
[0124] Certain preferred regions in these regards are set out in
FIG. 2 and include, but are not limited to, regions of the
aforementioned types identified by analysis of the amino acid
sequence set out in FIG. 2. As set out in FIG. 2, such preferred
regions include Garnier-Robson alpha-regions, beta-regions,
turn-regions and coil-regions, Chou-Fasman alpha-regions,
beta-regions and turn-regions, Kyte-Doolittle hydrophilic regions
and hydrophilic regions, Eisenberg alpha and beta amphipathic
regions, Karplus-Schulz flexible regions, Emini surface-forming
regions and Jameson-Wolf high antigenic index regions.
[0125] Among highly preferred fragments in this regard are those
that comprise regions of Arginase II that combine several
structural features, such as several of the features set out above.
In this regard, the regions defined by the residues about amino
acids 116-123, amino acids 141-153, amino acids 156-168, amino
acids 193-206, amino acids 249-261, amino acids 265-236, amino
acids 96-115, amino acids 213-248, and amino acids 306-356 of FIG.
2, which are regions of either high cross-species homology, thus
potentially involved in the active site of the enzyme, or regions
of low-cross species homology which may increase their usefulness
in generating arginase II specific antibodies or use arginase II
specific DNA based diagnostics. Such regions may be comprised
whithin a larger polypeptide or may be by themselves a preferred
fragment of the present invention, as discussed above. It will be
appreciated that the term "about" as used in this paragraph has the
meaning set out above regarding fragments in general.
[0126] Further preferred regions are those that mediate activities
of Arginase II. Most highly preferred in this regard are fragments
that have a chemical, biological or other activity of Arginase II,
including those with a similar activity or an improved activity, or
with a decreased undesirable activity. Highly preferred in this
regard are fragments that contain regions that are homologs in
sequence, or in position, or in both sequence and to active regions
of related polpeptides, such as, for example, the related
polypeptides set out in FIG. 4, which include arginases. Among
particularly preferred fragments in these regards are truncation
mutants, as discussed above.
[0127] It will be appreciated that the invention also relates to,
among others, polynucleotides encoding the aforementioned
fragments, polynucleotides that hybridize to polynucleotides
encoding the fragments, particularly those that hybridize under
stringent condition and polynucleotides, such as PCR primers, such
as, for example SEQ ED NO:3-15, for amplifying polynucleotides that
encode the fragments. In these regards, preferred polynucleotides
are those that correspondent to the preferred fragments, as
discussed above.
[0128] Vectors, Host Cells, Expression
[0129] The present invention also relates to vectors which include
polynucleotides of the present invention, most cells which are
genetically engineered with vectors of the invention and the
production of polypeptides of the invention by recombinant
techniques.
[0130] Host cells can be genetically engineered to incorporate
polynucleotides and express polypeptides of the present invention.
For instance, polynucleotides may be introduced into host cells
using well known techniques of infection, transduction,
transfection, transvection and transformation. The polynucleotides
may be introduced alone or with other polynucleotides. Such other
polynucleotides may be introduced independently, co-introduced or
introduced joined to the polynucleotides of the invention.
[0131] Thus, for instance, polynucleotides of the invention may be
transfected into host cells with another, separate, polynucleotide
encoding a selectable marker, using standard techniques for
co-transfection and selection in, for instance, mammalian cells. In
this case the polynucleotides generally will be stably incorporated
into the host cell genome.
[0132] Alternatively, the polynucleotides may be joined to a vector
containing a selectable marker for propagation in a host. The
vector construct may be introduced into host cells by the
aforementioned techniques. Generally, a plasmid vector is
introduced as DNA in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. Electroporation
also may be used to introduce polynucleotides into a host. If the
vector is a virus, it may be packaged in vitro or introduced into a
packaging cell and the packaged virus may be transduced into cells.
A wide variety of techniques suitable for making polynucleotides
and for introducing polynucleotides into cells in accordance with
this aspect of the invention are well known and routine to those of
skill in the art. Such techniques are reviewed at length in
Sambrook et al. cited above, which is illustrative of the many
laboratory manuals that detail these techniques.
[0133] In accordance with this aspect of the invention the vector
may be, for example, a plasmid vector, a single or double-stranded
phage vector, a single or double-stranded RNA or DNA viral vector.
Such vectors may be introduced into cells as polynucleotides,
preferably DNA, by well known techniques for introducing DNA and
RNA into cells. The vectors, in the case of phage and viral vectors
also may be and preferably are introduced into cells as packaged or
encapsidated virus by well known techniques for infection and
transduction. Viral vectors may be replication competent or
replication defective. In the latter case viral propagation
generally will occur only in complementing host cells.
[0134] Preferred among vectors, in certain respects, are those for
expression of polynucleotides and polypeptides of the present
invention. Generally, such vectors comprise cis-acting control
regions effective for expression in a host operatively linked to
the polynucleotide to be expressed. Appropriate trans-acting
factors either are supplied by the host, supplied by a
complementing vector or supplied by the vector itself upon
introduction into the host.
[0135] In certain preferred embodiments in this regard, the vectors
provide for specific expression. Such specific expression may be
inducible expression or expression only in certain types of cells
or both inducible and cell-specific. Particularly preferred among
inducible vectors are vectors that can be induced for expression by
environmental factors that are easy to manipulate, such as
temperature and nutrient additives. A variety of vectors suitable
to this aspect of the invention, including constitutive and
inducible expression vectors for use in prokaryotic and eukaryotic
hosts, are well known and employed routinely by those of skill in
the art.
[0136] The engineered host cells can be cultured in conventional
nutrient media, which may be modified as appropriate for, inter
alia, activating promoters, selecting transformants or amplifying
genes. Culture conditions, such as temperature, pH and the like,
previously used with the host cell selected for expression
generally will be suitable for expression of polypeptides of the
present invention as will be apparent to those of skill in the
art
[0137] A great variety of expression vectors can be used to express
a polypeptide of the invention. Such vectors include chromosomal,
episomal and virus-derived vectors e.g., vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, from viruses such as baculoviruses,
papova viruses, such as SV40, vaccinia viruses, aderoviruses, fowl
pox viruses, pseudorabies viruses and retroviruses and vectors
derived from combinations thereof, such as those derived from
plasmid and bacteriophage genetic elements, such as cosmids and
phagemids, all may be used for expression in accordance with this
aspect of the present invention. Generally, any vector suitable to
maintain, propagate or express polynucleotides to express a
polypeptide in a host may be used for expression in this
regard.
[0138] The appropriate DNA sequence may be inserted into the vector
by any of a variety of well-known and routine techniques. In
general, DNA sequence for expression is joined to an expression
vector by cleaving the DNA sequence and the expression vector with
one or more restriction endonucleases and then joining the
restriction fragments together using T4 DNA ligase. Procedures for
restriction and ligation that can be used to this end are well
known and routine to those of skill. Suitable procedures in this
regard, and or constructing expression vectors using alternative
techniques, which also are well known and routine to those skill,
are set forth in great detail in Sambrook et al. cited elsewhere
herein.
[0139] The DNA sequence in sequence in the expression vector is
operatively linked to appropriate expression control sequence(s),
including, for instance, a promoter to direct mRNA transcription.
Representatives of such promoters include the phage lambda PL
promoter, the E. coli lac, trp and tac promoters, the SV40 early
and late promoters and promoters of retroviral LTRs, to name just a
few of the well-known promoters. It will be understood that
numerous promoters not mentioned are suitable for use in this
aspect of the invention are well known and readily may be employed
by those of skill in the manner illustrated by the discussion and
the examples herein.
[0140] In general, expression constructs will contain sites for
transcription initiation and termination, and, in the transcribed
region, a ribosome binding site for translation. The coding portion
of the mature transcripts expressed by the constructs will include
a translation initiating AUG at the beginning and a termination
codon appropriately positioned at the end of the polypeptide to be
translated.
[0141] In addition, the constructs may contain control regions that
regulate as well as engender expression. Generally, in accordance
with many commonly practiced procedures, such regions will operate
by controlling transcription, such as repressor binding sites and
enhancers, among others.
[0142] Vectors for propagation and expression generally will
include selectable markers. Such markers also may be suitable for
amplification or the vectors may contain additional markers for
this purpose. In this regard, the expression vectors preferably
contain one or more selectable marker genes to provide a phenotypic
trait for selection of transformed host cells. Preferred markers
include dihydrofolate reductase or neomycin resistance for
eukaryotic cell culture, and tetracycline or ampicillin resistance
genes for culturing E. coli and other bacteria.
[0143] The vector containing the appropriate DNA sequence as
described elsewhere herein, as well as an appropriate promoter, and
other appropriate control sequences, may be introduced into an
appropriate host using a variety of well known techniques suitable
to expression there of a desired polypeptide. Representative
examples of appropriate hosts include bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal cells,
such as yeast cells; insect cells such as Drosophila S2 and
Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes
melanoma cells; and plant cells. Hosts for of a great variety of
expression constructs are well known, and those of skill will be
enabled by the present disclosure readily to select a host for
expressing a polypeptides in accordance with this aspect of the
present invention.
[0144] More particularly, the present invention also includes
recombinant constructs, such as expression constructs, comprising
one or more of the sequences described above. The constructs
comprise a vector, such as a plasmid or viral vector, into which
such a sequence of the invention has been inserted. The sequence
may be inserted in a forward or reverse orientation. In certain
preferred embodiments in this regard, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and there are
many commercially available vectors suitable for use in the present
invention.
[0145] The following vectors which are commercially available, are
provided by way of example. Among vectors preferred for use in
bacteria are pQE70, pQE60 and pQE-9, available from Qiagen; pBS
vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a,
pNH18A , pNH46A, available from Stratagene; and ptrc99a, pKK223-3,
pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred
eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pKT1 and pSG
available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available
from Pharmacia. These vectors are listed solely by way of
illustration of the many commercially available and well known
vectors that are available to those of skill in the art for use in
accordance with this aspect of the present invention. It will be
appreciated that any other plasmid or vector suitable for, for
example, introduction, maintenance, propagation or expression of a
polynucleotide of the invention in a host may be used in this
aspect of the invention.
[0146] Promoter regions can be selected from any desired gene using
vectors that contain a reporter transcription unit lacking a
promoter region, such as a chloramphenicol acetyl transferase
("CAT") transcription unit, downstream of restriction site or sites
for introducing a candidate promoter fragment; i.e., a fragment
that may contain a promoter. As is well known, introduction into
the vector of a promoter-containing fragment at the restriction
site upstream of the cat gene engenders production of CAT activity,
which can be detected by standard CAT assays. Vectors suitable to
this end are well known and readily available. Two such vectors are
pKK232-8 and pCM7. Thus, promoters for expression of
polynucleotides of the present invention include not only well
known and readily available promoters, but also promoters that
readily may be obtained by the foregoing technique, using a
reporter gene.
[0147] Among known bacterial promoters suitable for expression of
polynucleotides and polypeptides in accordance with the present
invention are the E. coli lacI and lacZ and promoters, the T3 and
T7 promoters, the gpt promoter, the lambda PR, PL promoters and the
trp promoter.
[0148] Among known eukaryotic promoters suitable in this regard are
the CMV immediate early promoter, the HSV thymidine kinase
promoter, the early and late SV40 promoters, the promoters of
retroviral LTRs, such as those of the Rous sarcoma virus ("RSV"),
and metallothionein promoters, such as the mouse metallothionein-I
promoter.
[0149] Selection of appropriate vectors and promoters for
expression in a host cell is a well known procedure and the
requisite techniques for expression vector construction,
introduction of the vector into the host and expression in the host
are routine skills in the art.
[0150] The present invention also relates to host cells containing
the above-described constructs discussed above. The host cell can
be a higher eukaryotic cell, such as a mammalian cell, or a lower
eukaryotic cell, such as a yeast cell, or the host cell can be a
prokaryotic cell, such as a bacteria cell.
[0151] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran medicated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al. BASIC METHODS IN MOLECULAR BIOLOGY, (1986).
[0152] Constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Alternatively, the polpeptides of the invention can be
synthetically produced by conventional peptide synthesizers.
[0153] Mature proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989).
[0154] Generally, recombinant expression vectors will include
origins of replication, a promoter derived from a highly-expressed
gene to direct transcription of a downstream structural sequence,
and a selectable marker to permit isolation of vector containing
cells after exposure to the vector. Among suitable promoters are
those derived from the genes that encode glycolytic enzymes such as
3-phosphoglycerate kinase ("PGK"), a-factor, acid phosphatase, and
heat shock proteins, among others. Selectable markers include the
ampicillin resistance gene of E. coli and the trp1 gene of S.
cerevisiae.
[0155] Transcription of the DNA encoding the polpeptides of tie
present invention by higher eukaryotes may be increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA usually about from 10 to 300 bp that act
to increase transcriptional activity of a promoter in a given host
cell-type. Examples of enhancers include the SV40 enhancer, which
is located on the late side of the replication origin at bp 100 to
270, the cytomegalovirus early promoter enhancer, the polyoma
enhancer on the late side of the replication origin, and adenovirus
enhancers.
[0156] Polynucleotides of the invention, encoding the heterologous
structural sequence of a polypeptide of the invention generally
will be inserted into the vector using standard techniques so that
it is operably linked to the promoter for expression. The
polynucleotide will be positioned so that the transcription start
site is located appropriately 5' to a ribosome binding site. The
ribosome binding site will be 5' to the AUG that initiates
translation of the polypeptide to be expressed. Generally, there
will be no other open reading frames that begin with an initiation
codon, usually AUG, and lie between the ribosome binding site and
the initiating AUG. Also, generally, there will be a translation
stop codon at the end of the polypeptide and there will be a
polyadenylation signal and a transcription termination signal
appropriately disposed at the 3' end of the transcribed region.
[0157] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the expressed polypeptide. The signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0158] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals but
also additional heterologous functional regions. Thus, for
instance, a region of additional amino acids, particularly charged
amino acids, may be added to the N-terminus of the polypeptide to
improve stability and persistence in the host cell, during
purification or during subsequent handling and storage. Also,
region also may be added to the polypeptide to facilitate
purification. Such regions may be removed prior to final
preparation of the polypeptide. The addition of peptide moieties to
polypeptides to engender secretion or excretion, to improve
stability and to facilitate purification, among others, are
familiar and routine techniques in the art.
[0159] Suitable prokaryotic hosts for propagation, maintenance or
expression of polynucleotides and polypeptides in accordance with
the invention include Escherischia coli, Bacillus subtilis and
Salmonella typhimurium. Various species of Pseudomonas,
Streptomyces, and Staphylococcus are suitable hosts in this regard.
Moreover, many other hosts also known to those of skill may be
employed in this regard.
[0160] As a representative but non-limiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed.
[0161] Following transformation or a suitable host strain and
growth of the host strain to an appropriate cell density, where the
selected promoter is inducible it is induced by appropriate means
(e.g., temperature shift or exposure to chemical inducer) and cells
are cultured for an additional period.
[0162] Cells typically then are harvested by centrifugation,
disrupted by physical or chemical means, and the resulting crude
extract retained for further purification.
[0163] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents,
such methods are well know to those skilled in the art.
[0164] Various mammalian cell culture systems can be employed for
expression, as well. Examples of mammalian expression systems
include the COS-7 lines of monkey kidney fibroblast, described in
Gluzman et al., Cell 23: 175 (1981). Other cell lines capable of
expressing a compatible vector include for example, the C127, 3T3,
CHO, HeLa, human kidney 293 and BHK cell lines.
[0165] Mammalian expression vectors will comprise an origin of
replication, a suitable promoter and enhancer, and also any
necessary ribosome binding sites, polyadenylation sites, splice
donor and acceptor sites, transcriptional termination sequences,
and 5' flanking non-transcribed sequences that are necessary for
expression. In certain preferred embodiments in this regard DNA
sequences derived from the SV40 splice sites, and the SV40
polyadenylation sites are used for required non-transcribed genetic
elements of these types.
[0166] The Arginase II polypeptide can be recovered and purified
from recombinant cell cultures by well-known methods including
ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification. Well known techniques for refolding
protein may be employed to regenerate active conformation when the
polypeptide is denatured during isolation and or purification.
[0167] Polypeptides of the present invention include naturally
purified products, products of chemical synthetic procedures, and
products produced by recombinant techniques from a prokaryotic or
eukaryotic host including for example, bacterial yeast, higher
plant, insect and mammalian cells. Depending upon the host employed
in a recombinant production procedure, the polypeptides of the
present invention may be glycosylated or may be non-glycosylated.
In addition, polypeptides of the invention may also include an
initial modified methionine residue, in some cases as a result of
host-mediated processes.
[0168] Further Illustrative Aspects and Preferred Embodiments of
the Invention
[0169] Arginase II polynucleotides and polypeptides may be used in
accordance with the present invention for a variety of
applications, particularly those that make use of the chemical and
biological properties Arginase II. Among these are applications in
diagnosis and treatment of urea cycle diseases, hypertension,
hypotension, episodic hyperammonemia, defects in biosynthesis or
proline, glutamate, nitric oxide and ornithine, as well as
hyperargininemia and its related spasticity, growth retardation,
and progressive mental impairment, and prostate disease,
particularly prostate cancer, prostatitis and benign prostatic
hyperplasia or hypertrophy, and also prostate damage, kidney
disease, and kidney damage. Additional applications relate to
diagnosis and to treatment of disorders of cells, tissues and
organisms.
[0170] Methods of using Arginase II are also provided to control
nitric oxide formation in an individual having a need to control
such formation, and to treat systemic hypotension caused by sepsis
or cytokines using Arginase II alone or in combination with an
alpha sub-1 adrenergic agonist.
[0171] A method for using Arginase II polypeptides to prepare or
synthesize of L-ornithine salts is provided. Such salts are useful
in chemical reactions, industrial and pharmaceutical applications.
Skilled artisans will readily be able to produce useful quantities
of such salts using large scale cell culture or fermentation with
cells comprising the Arginase II gene.
[0172] These aspects of the invention are illustrated further by
the following discussion.
[0173] Polynucleotide Assays
[0174] This invention is also related to the use of the Arginase II
polynucleotides to detect complementary polynucleotides such as,
for example, as a diagnostic reagent. Detection of a mutated form
of Arginase II associated with a dysfunction will provide a
diagnostic tool that can add or define a diagnosis of a disease or
susceptibility to a disease which results from under-expression
over-expression or altered expression of Arginase II, such as, for
example, urea cycle diseases, hypertension, hypotension, episodic
hyperammonemia, defects in biosynthesis of proline, glutamate,
nitric oxide and ornithine, as well as hyperargininemia and its
related spasticity, growth retardation, and progressive mental
impairment, and prostate disease, particularly prostate cancer,
prostatitis and benign prostatic hyperplasia or hypertrophy, and
also prostate damage, kidney disease, and kidney damage.
[0175] Host organisms carrying mutations in the human Arginase II
gene may be detected at the DNA level by a variety of techniques.
Nucleic acids for diagnosis may be obtained from a patient's cells,
such as from blood, urine, saliva, tissue biopsy and autopsy
material. The genomic DNA may be used directly for detection or may
be amplified enzymatically by using PCR prior to analysis. PCR
(Saiki et al., Nature, 324: 163-166 (1986)). RNA or cDNA may also
be used in the same ways. As an example, PCR primers complementary
to the nucleic acid encoding Arginase II can be used to identify
and analyze Arginase II expression and mutations. For example,
deletions and insertions can be detected by a change in size of the
amplified product in comparison to the normal genotype. Point
mutations can be identified by hybridizing amplified DNA to
radiolabeled Arginase II RNA or alternatively, radiolabeled
Arginase II antisense DNA sequences. Perfectly matched sequences
can be distinguished from mismatched duplexes by RNase A digestion
or by differences in melting temperatures.
[0176] Sequence differences between a reference gene and genes
having mutations also may be revealed by direct DNA sequencing. In
addition, cloned DNA segments may be employed as probes to detect
specific DNA segments. The sensitivity of such methods can be
greatly enhanced by appropriate use of PCR or another amplification
method. For example, a sequencing primer is used with
double-stranded PCR product or a single-stranded template molecule
generated by a modified PCR. The sequence determination is
performed by conventional procedures with radiolabeled nucleotide
or by automatic sequencing procedures with fluorescent-tags. In
situ nucleic acid amplification methods, such as, for example, PCR
reactions can also be carried out using biopsied tissues and cell
samples using well known methods.
[0177] One embodiment of the invention is the use of
polynucleotides of the invention to detect Arginase II gene
expression in prostate cells as a marker for a disease or
susceptibility to a disease which results from under-expression
over-expression or altered expression of Arginase II, such as, for
example, urea cycle diseases, hypertension, hypotension, episodic
hyperammonemia, defects in biosynthesis of proline, glutamate,
nitric oxide and ornithine, as well as hyperargininemia and its
related spasticity, growth retardation, and progressive mental
impairment, and prostate disease, particularly prostate cancer,
prostatitis and benign prostatic hyperplasia or hypertrophy, and
also prostate damage, kidney disease, and kidney damage prostate
disease or prostate damage. A preferred embodiment of the invention
is using polynucleotides of the invention to detect cells which are
expressing an aberrant or variant Arginase II gene or expressing
Arginase II gene in an inappropriate tissue to diagnose and
prognose disease. Another preferred embodiments of such methods is
to detect cells circulating in the blood which express an Arginase
II gene, particularly to prognose or stage prostate disease.
Circulating cells of the prostate or kidney expressing an Arginase
II gene are useful to diagnose or prognose late prostate or kidney
cancer respectively, particularly, with respect to prostate cancer,
stages C or D, as such stages are known in the art (see, for
example, Dugan J A, et al. JAMA 275: 288 (1996)). A more preferred
embodiment of the invention provides that polynucleotides of the
invention may be used to detect and quantitate levels of Arginase
II expression in cells. As compared with normal Arginase II
expression in a cell, detection of higher levels of expression is
useful for prognosis of the stage of a disease, particularly for
cancer, especially kidney and prostate cancer. Detection of
Arginase II expression in cells of the prostate or kidney with no
detectable concomitant expression in cells circulating in the blood
is useful to diagnose early stage prostate or kidney cancer
respectively, particularly, with respect to prostate cancer, stages
A or B, as such stages are known in the art.
[0178] Other preferred embodiments include a method for the
diagnosis of a disorder arising from Arginase II deficiency in a
host organism having or suspected of having a defect in the nitric
oxide pathway and the urea cycle using Arginase II polynucleotides,
polypeptides and antibodies, including but not limited to fragments
of either. Arginase II polynucleotides, polpeptides, and
antibodies, can be used in a method to monitor progression or
therapy of disorders with hypotension as a clinical sign, to
monitor progression of or therapy of disorders of the immune or
nervous systems associated with or caused by nitric oxide
regulation, to monitor activation state of macrophage, or to
monitor mitochondrial activity as a function of the transportation
of arginase II into the mirochondria.
[0179] RT-PCR is a well known method (Kawasaki E S, et al., Proc
Natl Acad Sci. USA 85: 5698 (1988)). Polynucleotides of the
invention are particularly useful for RT-PCR for detection of
Arginase II gene expression. Polynucleotides of the invention may
be used as primers to mediate the reverse transcription reaction of
RT-PCR, and may also be used as primers for the PCR reaction.
[0180] Genetic testing based on DNA sequence differences may be
achieved by detection of alteration in electrophoretic mobility of
DNA fragments in gels, with or without denaturing agents. Small
sequence deletions and insertions can be visualized by high
resolution gel electrophoresis. DNA fragments of different
sequences nay be distinguished on denaturing formamide gradient
gels in which the mobilities of different DNA fragments are
retarded in the gel at different positions according to their
specific melting or partial melting temperatures (see, e.g., Myers
et al., Science, 230: 1242 (1985)).
[0181] Sequence changes at specific locations also may be revealed
by nuclease protection assays, such as RNase and S1 protection or
the chemical cleavage method (e.g., Cotton et al., Proc. Natl.
Acad. Sci., USA, 4397-4401(1985)).
[0182] Thus, the detection of a specific DNA sequence may be
achieved by methods such as hybridization, RNase protection,
chemical cleavage, direct DNA sequencing or the use of restriction
enzymes, (e.g., restriction fragment length polymorphisms ("RFLP")
and Southern blotting of genomic DNA. In situ hybridization
reactions can also be carried out using biopsied tissues and cell
samples using well known methods.
[0183] In addition to more conventional gel-electrophoresis and DNA
sequencing, mutations also can be detected by in situ analysis.
[0184] Chromosome assays
[0185] The sequences of the present invention are also valuable for
chromosome identification. The sequence is specifically targeted to
and can hybridize with a particular location on an individual human
chromosome. Moreover, there is a current need for identifying
particular sites on the chromosome. Few chromosome marking reagents
based on actual sequence data (repeat polymorphisms) are presently
available for marking chromosomal location. The mapping of DNAs to
chromosomes according to the present invention is an important
first step in correlating those sequences with genes associated
with disease.
[0186] In certain preferred embodiments in this regard, the cDNA
herein disclosed is used to clone genomic DNA of a Arginase II
gene. This can be accomplished using a variety of well known
techniques and libraries, which generally are available
commercially. The genomic DNA the is used for in situ chromosome
mapping using well known techniques for this purpose. Typically, in
accordance with routine procedures for chromosome mapping some
trial and error may be necessary to identify a genomic probe that
gives a good in situ hybridization signal.
[0187] In some cases, in addition, sequences can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp) from the
cDNA. Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than one
exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic
cell hybrids containing individual human chromosomes. Only those
hybrids containing the human gene corresponding to the primer will
yield an amplified fragment.
[0188] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular DNA to a particular chromosome. Using the
present invention with the same oligonucleotide primers,
sublocalization can be achieved with panels of fragments from
specific chromosomes or pools of large genomic clones in an
analogous manner. Other mapping strategies that can similarly be
used to map to its chromosome include in situ hybridization,
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0189] Fluorescence in situ hybridization ("FISH") of a cDNA clone
to a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
cDNA as short as 50 or 60. For a review of this technique, see
Verma et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES,
Pergamon Press, New York (1988).
[0190] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V McKusick, MENDELIAN INHERITANCE IN MAN, available on
line through Johns Hopkins University, Welch Medical Library. The
relationship between genes and diseases that have been mapped to
the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0191] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0192] With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a chromosomal
region associated with the disease could be one of between 50 and
500 potential causative genes. (This assumes 1 megabase mapping
resolution and one gene per 20 kb).
[0193] Polypeptide assays
[0194] The present invention also relates to a diagnostic assays
such as quantitative and diagnostic assays for detecting levels of
Arginase II protein in cells and tissues, including determination
of normal and abnormal levels. Thus, for instance, a diagnostic
assay in accordance with the invention for detecting
over-expression of Arginase II protein compared to normal control
tissue samples may be used to detect the presence of urea cycle
diseases, hypertension, hypotension, episodic hyperammonemia,
defects in biosynthesis of proline, glutamate, nitric oxide and
ornithine, as well as hyperargininemia and its related spasticity,
growth retardation, and progressive mental impairment, and prostate
disease, particularly prostate cancer, prostatitis and benign
prostatic hyperplasia or hypertrophy, and also prostate damage,
kidney disease, and kidney damage, or example. Assay techniques
that can be used to determine levels of a protein, such as an
Arginase II protein of the present invention, in a sample derived
from a host are well-known to those of skill in the art. Such assay
methods include radioimmunoassays, competitive-binding assays,
Western Blot analysis and ELISA assays. Preferred samples to be
assayed using these assays, include, but are not limited to cells,
tissues and bodily fluids. Among these ELISAs frequently are
preferred. An ELISA assay initially comprises preparing an antibody
specific to Arginase II, preferably a monoclonal antibody. In
addition a reporter antibody generally is prepared which binds to
the monoclonal antibody. The reporter antibody is attached a
detectable reagent such as radioactive, fluorescent or enzymatic
reagent, in this example horseradish peroxidase enzyme.
[0195] To carry out an ELISA a sample, such as, for example, bodily
fluid, is removed from a host and incubated on a solid support,
e.g. a polystyrene dish, that binds the proteins in the sample. Any
free protein binding sites on the dish are then covered by
incubating with a non-specific protein such as bovine serum
albumin. Next, the monoclonal antibody is incubated in the dish
during which time the monoclonal antibodies attach to any Arginase
II proteins attached to the polystyrene dish. Unbound monoclonal
antibody is washed out with buffer. The reporter antibody linked to
horseradish peroxidase is placed in the dish resulting in binding
of the reporter antibody to any monoclonal antibody bound to
Arginase II. Unattached reporter antibody is then washed out.
Reagents for peroxidase activity, including a colorimetric
substrate are then added to the dish. Immobilized peroxidase,
linked to Arginase II through the primary and secondary antibodies,
produces a colored reaction product. The amount of color developed
in a given time period indicates the amount of Arginase II protein
present in the sample. Quantitative results typically are obtained
by reference to a standard curve.
[0196] A competition assay may be employed wherein antibodies
specific to Arginase II attached to a solid support and labeled
Arginase II and a sample derived from the host are passed over the
solid support and the amount of label detected attached to the
solid support can be correlated to a quantity of Arginase II in the
sample.
[0197] One embodiment of the invention is the use of polypeptide
assays of the invention to detect Arginase II gene expression in
prostate cells as a marker for a disease or susceptibility to a
disease which results from under-expression over-expression or
altered expression of Arginase II, such as, for example, urea cycle
diseases, hypertension, hypotension, episodic hyperammonemia,
defects in biosynthesis of proline, glutamate, nitric oxide and
ornithine, as well as hyperargininemia and its related spasticity,
growth retardation, and progressive mental impairment, and prostate
disease, particularly prostate cancer, prostatitis and benign
prostatic hyperplasia or hypertrophy, and also prostate damage,
kidney disease, and kidney damage, prostate disease or prostate
damage. A preferred assay embodiment of the invention is to detect
cells which are expressing an aberrant or variant Arginase II gene
or expressing Arginase II gene in an inappropriate tissue to
diagnose and prognose disease. Another preferred embodiment of such
methods is to detect cells circulating in the blood which express
an Arginase II gene, particularly to prognose or stage prostate
disease. Circulating cells of the prostate or kidney expressing an
Arginase II gene are useful to diagnose or prognose late prostate
or kidney cancer respectively, particularly, with respect to
prostate cancer, stages C or D, as such stages are known in she
art. A more preferred embodiment of the invention provides that
assays of the invention may be used to detect and quantitate levels
of Arginase II expression in cells. As compared with normal
Arginase II expression in a cell, detection of higher levels of
expression is useful for prognosis of the stage of a disease,
particularly for cancer, especially kidney and prostate cancer.
Detection of Arginase II expression in cells of the prostate or
kidney with no detectable concomitant expression in cells
circulating in the blood is useful to diagnose early stage prostate
or kidney cancer respectively, particularly, with respect to
prostate cancer, stages A or B, as such stages are known in the
art.
[0198] Antibodies
[0199] The polypeptides, their fragments or other derivatives, or
analogs thereof, or cells expressing them can be used as an
immunogen to produce antibodies thereto. These antibodies can be,
for example, polyclonal or monoclonal antibodies. The present
invention also includes chimeric, single chain, and humanized
antibodies, as well as Fab fragments, or the product of an Fab
expression library. Various procedures known in the art may be used
for the production of such antibodies and fragments.
[0200] Antibodies generated against the polypeptides corresponding
to a sequence of the present invention can be obtained by direct
injection of the polypeptides into an animal or by administering
the polypeptides to an animal, preferably a nonhuman. The antibody
so obtained will then bind the polypeptides itself. In this manner,
even a sequence encoding only a fragment of the polypeptides can be
used to generate antibodies binding the whole native polypeptides.
Such antibodies can then be used to isolate the polypeptide from
tissue expressing that polypeptide
[0201] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler, G.
and Milstein, C., Nature 256: 495-497 (1975), the trioma technique,
the human B-cell hybridoma technique (Kozbor et al., Immunology
Today 4:72 (1983) and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole et al., pg. 77-96 in MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
[0202] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products of this
invention. Also, transgenic mice, or other organisms such as other
mammals, may be used to express humanized antibodies to immunogenic
polypeptide products of this invention.
[0203] Thus, among others, such antibodies can be used to treat
such as, for example, urea cycle diseases, hypertension,
hypotension, episodic hyperammonemia, defects in biosynthesis of
proline, glutamate, nitric oxide and ornithine, as well as
hyperargininemia and its related spasticity, growth retardation,
and progressive mental impairment, and prostate disease,
particularly prostate cancer, prostatitis and benign prostatic
hyperplasia or hypertrophy, and also prostate damage, kidney
disease, and kidney damage.
[0204] The methods of the invention to detect and/or quantitate
Arginase II polynucleotide sequence, Arginase II expression levels
and gene expression products, particularly the immunological and
immunohistochemical methods and methods using oligonucleotides, can
be used with bodily tissues and fluids from individuals. Preferred
bodily tissues and fluids useful with the methods of the invention
include, but are not limited to, cells and tissues of the liver,
kidney, brain, gastrointestinal tract, mammary gland, particularly
while lactating, and blood, particularly red blood cells, and
activated macrophages. Preferred bodily samples useful with the
immunohistochemical methods of the invention include, but are not
limited to, tissues and cells.
[0205] Arginase II Binding Molecules and Assays
[0206] This invention also provides a method for identification of
molecules, such as receptor molecules, that bind Arginase II. Genes
encoding proteins that bind Arginase II, such as receptor proteins,
can be identified by numerous methods known to those of skill in
the art, for example, ligand panning and FACS sorting. Such methods
are described in many laboratory manuals such as, for instance,
Coligan et al., Current Protocols in Immunology 1(2): Chapter 5
(1991).
[0207] For instance, expression cloning may be employed for this
purpose. To this end polyadenylated RNA is prepared from a cell
responsive to Arginase II, a cDNA library is created from this RNA,
the library is divided into pools and the pools are transfected
individually into cells that are not responsive to Arginase II. The
transfected cells then are exposed to labeled Arginase II.
(Arginase II can be labeled by a variety of well-known techniques
including standard methods of radio-iodination or inclusion of a
recognition site for a site-specific protein kinase.) Following,
exposure, the cells are fixed and binding of Arginase II is
determined. These procedures conveniently are carried out on glass
slides.
[0208] Pools are identified of cDNA that produced Arginase
II-binding cells. Sub-pools are prepared from these positives,
transfected into host cells and screened as described above. Using
an iterative sub-pooling and re-screening process, one or more
single clones that encode the putative binding molecule, such as a
receptor molecule, can be isolated.
[0209] Alternatively a labeled ligand can be photoaffinity linked
to a cell extract, such as a membrane or a membrane extract,
prepared from cells that express a molecule that it binds, such as
a receptor molecule. Cross-linked material is resolved by
polyacrylamide gel electrophoresis ("PAGE") and exposed to X-ray
film. The labeled complex containing the ligand-receptor can be
excised, resolved into peptide fragments, and subjected to protein
microsequencing. The amino acid sequence obtained from
microsequencing can be used to design unique or degenerate
oligonucleotide probes to screen cDNA libraries to identify genes
encoding the putative receptor molecule.
[0210] Polypeptides of the invention also can be used to assess
Arginase II binding capacity of Arginase II binding molecules, such
as receptor molecules, in cells or in cell-free preparations.
[0211] Agonists and Antagonists--Assays and Molecules The invention
also provides a method of screening compounds to identity those
which enhance or block the action of Arginase II on cells, such as
its interaction with Arginase II-binding molecules such as receptor
molecules. An agonist is a compound which increases the natural
biological functions of Arginase II, while antagonists decrease or
eliminate such functions.
[0212] For example, a cellular compartment, such as a membrane or a
preparation thereof, such as a membrane-preparation, may be
prepared from a cell that expresses a molecule that binds Arginase
II, such as a molecule of a signaling or regulatory pathway
modulated by Arginase II. The preparation is incubated with labeled
Arginase II in the absence or the presence of a candidate molecule
which may be a Arginase II agonist or antagonist. The ability of
the candidate molecule to bind the binding molecule is reflected in
decreased binding of the labeled ligand. Molecules which bind
gratuitously, i.e., without including the effects of Arginase II on
binding the Arginase II binding molecule, are most likely to be
good antagonists. Molecules that bind well and elicit effects that
are the same as or closely related to Arginase I, for example, are
agonists.
[0213] Arginase II-like effects of potential agonists and
antagonists may by measured, for instance, by determining activity
of a second messenger system following interaction of the candidate
molecule with a cell or appropriate cell preparation and comparing
the effect with that of Arginase II or molecules that elicit the
same erects as Arginase II. Second messenger systems that may be
useful in this regard include but are not limited to AMP guanylate
cyclase, ion channel or phosphoinositide hydrolysis second
messenger systems.
[0214] Another example of an assay for Arginase II antagonists is a
competitive assay that combines Arginase HI and a potential
antagonist with membrane-bound Arginase II receptor molecules or
recombinant Arginase II receptor molecules under appropriate
conditions for a competitive inhibition assay. Arginase II can be
labeled, such as by radioactivity, such that the number of Arginase
II molecules bound to a receptor molecule can be determined
accurately to assess the effectiveness of the potential
antagonist.
[0215] Potential antagonists include small organic molecules,
peptides, polypeptides and antibodies that bind to a polypeptide of
the invention and thereby inhibit or extinguish its activity.
Potential antagonists also may be small organic molecules, a
peptide, a polypeptide such as a closely related protein or
antibody that binds the same sites on a binding molecule, such as a
receptor molecule, without inducing Arginase II-induced activities,
thereby preventing the action of Arginase II by excluding Arginase
II from binding.
[0216] Potential antagonists include a small molecule which binds
to and occupies the binding site of the polypeptide thereby
preventing binding to cellular binding molecules, such as receptor
molecules, such that normal biological activity is prevented.
Examples of small molecules include but are not limited to small
organic molecules, peptides or peptide-like molecules.
[0217] Other potential antagonists include antisense molecules.
Antisense technology can be used to control gene expression through
antisense DNA or RNA or through triple-helix formation. Antisense
techniques are discussed, for example, in Okano, J. Neurochem.
56:560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF
GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988). Triple helix
formation is discussed in, for instance Lee et al, Nucleic Acids
Research 6:3073 (1979); Cooney et al., Science 241:456 (1988); and
Dervan et al., Science 251:1360 (1991). The methods are based on
binding of a polynucleotide to a complementary DNA or RNA. For
example, the 5' coding portion of a polynucleotide that encodes the
mature polypeptide of the present invention may be used to design
an antisense RNA oligonucleotide of from about 10 to 40 base pairs
in length. A DNA oligonucleotide is designed to be complementary to
a region of the gene involved in transcription thereby preventing
transcription and the production of Arginase II. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into Arginase II polypeptide. The
oligonucleotides described above can also be delivered to cells
such that the antisense RNA or DNA may be expressed in vivo to
inhibit production of Arginase II.
[0218] The antagonists may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as the naive
described.
[0219] The antagonists nay be employed for instance to treat an
individual having or suspected of having a disease associated with
or caused by a defect in the Arginase II gene or Arginase II gene
expression, such as, for example, urea cycle diseases,
hypertension, hypotension episodic hyperammonemia, defects in
biosynthesis of proline, glutamate, nitric oxide and ornithine, as
well as hyperargininemia and its related spasticity, growth
retardation, and progressive mental impairment, and prostate
disease, particularly prostate cancer, prostatitis and benign
prostatic hyperplasia or hypertrophy, and also prostate damage,
kidney disease, and kidney damage.
[0220] Antagonist and agonists of the invention, including but not
limited to antibodies, can be used in method to deplete systemic
arginine levels in an individual having a need for a depletion of
such levels.
[0221] Arginase II is involved in the nitric oxide pathway. Nitric
oxide is a very important regulatory molecule and arginase
regulates nitric oxide synthesis through the production of
ornithine from arginine. This regulation is a key to the
involvement of arginase, and as provided herein Arginase II, in
hypotension, macrophage killing and immune function, and in the
nervous system.
[0222] Compositions
[0223] The invention also relates to compositions comprising the
polynucleotide or the polypeptides discussed above or the agonists
or antagonists. Thus, the polypeptides of the present invention may
be employed in combination with a non-sterile or sterile carrier or
carriers for use with cells, tissues or organisms, such as a
pharmaceutical carrier suitable for administration to a subject.
Such compositions comprise, for instance, a media additive or a
therapeutically effective amount of a polypeptide of the invention
and a pharmaceutically acceptable carrier or excipient. Such
carriers may include, but are not limited to, saline, buffered
saline, dextrose, water, glycerol, ethanol and combinations
thereof. The formulation should suit the mode of
administration.
[0224] Kits
[0225] The invention further relates to pharmaceutical packs and
kits comprising one or more containers filled with one or more of
the ingredients of the aforementioned compositions of the
invention. Associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
reflecting approval by the agency of the manufacture, use or sale
of the product for human administration.
[0226] Administration
[0227] Polypeptides and other compounds of the present invention
may be employed alone or in conjunction with other compounds, such
as therapeutic compounds.
[0228] The pharmaceutical compositions may be administered in any
effective, convenient manner including, for instance,
administration by topical, oral, anal, vaginal, intravenous,
intraperitoneal, intramuscular, subcutaneous, intranasal or
intradermal routes among others.
[0229] The pharmaceutical compositions generally are administered
in an amount effective for treatment or prophylaxis of a specific
indication or indications. In general, the compositions are
administered in an amount of at least about 10 mg/kg body weight.
In most cases they will be administered in an amount not in excess
of about 8 mg/kg body weight per day. Preferably, in most cases,
dose is from about 10 mg/kg to about 1 mg/kg body weight daily. It
will be appreciated that optimum dosage will be determined by
standard methods for each treatment modality and indication, taking
into account the indication, its severity, route of administration,
complicating conditions and the like.
[0230] Gene Therapy
[0231] The Arginase II polynucleotides, polypeptides, agonists and
antagonists that are polypeptides may be employed in accordance
with the present invention by expression of such polypeptides in
vivo, in treatment modalities often referred to as "gene
therapy".
[0232] A preferred embodiment of :he invention is treatment of
disease associated or caused by defects in nitric oxide
biosynthesis using Arginase II in gene therapy. Arginase II may
also be used in gene therapy to treat defects of type I arginase,
particularly those defects associated with of which cause nitric
oxide biosynthesis disorders. Also provided is a method of using
gene therapy to treat immunologic and/or neurologic disorders
associated with or caused by nitric oxide dysfunction, particularly
those which are a result of Arginase II deficiency.
[0233] A further preferred embodiment of the invention is a method
of using gene therapy to treat disorders resulting in
hyperargininemia, to include but not be limited to defects in
arginase Type I and Type II genes.
[0234] Thus, for example, cells from a patient may be engineered
with a polynucleotide, such as a DNA or RNA encoding a polypeptide
ex vivo, and the engineered cells then can be provided to a patient
to be treated with the polypeptide. For example, cells may be
engineered ex vivo by the use of a retroviral plasmid vector
containing RNA encoding a polypeptide of the present invention.
Such methods are well-known in the art and their use in the present
invention will be apparent from the teachings herein.
[0235] Similarly, cells may be engineered in vivo for expression of
a polypeptide in vivo by procedures known in the art. For example,
a polynucleotide of the invention may be engineered for expression
in a replication defective retroviral vector, as discussed above.
The retroviral expression construct then may be isolated and
introduced into a packaging cell is transduced with a retroviral
plasmid vector containing RNA encoding a polypeptide of the present
invention such that the packaging cell now produces infectious
viral particles containing the gene of interest. These producer
cells may be administered to a patient for engineering cells in
vivo and expression of the polypeptide in vivo. These and other
methods for administering a polypeptide of the present invention by
such method should be apparent to those skilled in the art from the
teachings of the present invention.
[0236] Retroviruses from which the retroviral plasmid vectors
herein above mentioned may be derived include, but are not limited
to, Money Murine Leukemia Virus, spleen necrosis virus,
retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus,
avian leukosis virus, gibbon ape leukemia virus, human
immunodeficiency virus, adenovirus, Myeloproliferitive Sarcoma
Virus, and mammary tumor virus. In one embodiment, the retroviral
plasmid vector is derived from Moloney Murine Leukemia Virus.
[0237] Such vectors well include one or more promoters for
expressing the polypeptide. Suitable promoters which may be
employed include, but are not limited to, the retroviral LTR; the
SV40 promoter, and the human cytomegalovirus (CMV) promoter
described in Miller et al., Biotechniques 7: 980-990 (1989), or any
other promoter (e.g., cellular promoters such as eukaryotic
cellular promoters including, but not limited to, the histone, RNA
polymerase II, and .infin.-actin promoters). Other viral promoters
which may be employed include, but are not limited to, adenovirus
promoters, thymidine kinase (TK) promoters, and B19 parvovirus
promoters. The selection of a suitable promoter will be apparent to
those skilled in the art from the teachings contained herein.
[0238] The nucleic acid sequence encoding the polypeptide of the
present invention will be placed under the control or a suitable
promoter. Suitable promoters which may be employed include, but are
not limited to, adenoviral promoters, such as the adenoviral major
late promoter, or heterologous promoters, such as the
cytomegalovirus (CMV) promoter, the respiratory syncytial virus
(RSV) promoter, inducible promoters, such as the MMT promoter, the
metallothionein promoter; heat shock promoters; the albumin
promoter; the ApoAI promoter; human globin promoters; viral
thymidine kinase promoters, such as the Herpes Simplex thymidine
kinase promoter; retroviral LTRs (including the modified retroviral
LTRs herein above described); the .infin.-actin promoter; and human
growth hormone promoters. The promoter also may be the native
promoter which controls the gene encoding the polypeptide.
[0239] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, Y-2, Y-AM, PA12, T19-14X,
VT-19-17-H2, YCRE, YCRIP, GPTE-86, GPTenvAm12, and DAN cell lines
as described in Miller, A., Human Gene Therapy 1:5-14 (1990). The
vector may be transduced into the packaging cells through any means
known in the art. Such means include, but are not limited to,
electroporation, the use of liposomes, and CaPO.sub.4
precipitation. In one alternative, the retroviral plasmid vector
may be encapsulated into a liposome, or coupled to a lipid, and
then administered to a host.
[0240] The producer cell line will generate infectious retroviral
vector particles, which include the nucleic acid sequence(s)
encoding the polypeptides. Such retroviral vector particles then
may be employed to transduce eukaryotic cells, either in vitro or
in vivo. The transduced eukaryotic cells will express the nucleic
acid sequencer(s) encoding the polypeptide. Eukaryotic cells which
may be transduced include, but are not limited to, embryonic stem
cells, embryonic carcinoma cells, as well as hematopoietic stem
cells, hepatocytes, fibroblasts, myoblasts, keratinocytes,
endothelial cells, and bronchial epithelial cells.
EXAMPLES
[0241] The present invention is further described by the following
examples. The examples are provided solely to illustrate the
invention by reference to specific embodiments. These
exemplification's, while illustrating certain specific aspects of
the invention, do not portray the limitations or circumscribe the
scope of the disclosed invention.
[0242] Certain terms used herein are explained in the foregoing
glossary.
[0243] All examples were carried out using standard techniques,
which are well known and routine to those of skill in the art,
except where otherwise described in detail. Routine molecular
biology techniques of the following examples can be carried out as
described in standard laboratory manuals, such as Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989), herein referred
to as "Sambrook."
[0244] All parts or amounts set out in the following examples are
by weight, unless otherwise specified.
[0245] Unless otherwise stated size separation of fragments in the
examples below was carried out using standard techniques of agarose
and polyacrylamide gel electrophoresis ("PACE") in Sambrook and
numerous other references such as, for instance, by Goeddel et al.,
Nucleic Acids Res. 8: 4057 (1980).
[0246] Unless described otherwise, ligations were accomplished
using standard buffers, incubation temperatures and times,
approximately equimolar amounts of the DNA fragments to be ligated
and approximately 10 units of T4 DNA ligase ("ligase") per 0.5 mg
of DNA.
Example 1
Cloning of the Human Arginase II cDNA
[0247] The cDNA sequence of Arginase I was used as a probe sequence
for a computer search (Kerlavage et al., In: Proceedings of the
26th Hawaii International Symposium on System Sciences 585-594
(1993)) of a cDNA database comprising EST sequences (the "research
database") made by random sequencing of human cDNA libraries with
an ABI 373 sequencer using well known methods (see, for example,
Adams, et al. Nature 368: 447 (1994)). The sequences were stored in
an electronically searchable database. This database comprises
sequences of the deposited clone. A number of ESTs were identified
that had 100% homology to Arginase I. Another subset of ESTs were
identified which had between 50% and 60% homology to Arginase I.
Upon closer examination most of these ESTs had regions which were
approximately 70% homologous at the nucleic acid level to Arginase
I but other regions were significantly different. Utilizing the
Univesity of Wisconsin GCG programs these ESTs (FIG. 1(B)) were
compiled into a contiguous DNA sequence (FIG. 1(A)). By utilizing
flanking non-homologous sequences as new probe sequences, the
contig was expanded both 5' and 3' until the research database was
exhausted of overlapping clones. The sequence of all overlapping
clones was used to generate a consensus sequence of 1075 bases. PCR
primers were designed to the extreme 3' and 5' ends of the
consensus sequence (FIG. 3 (SEQ D NO:3-15)). Skilled artisans can
readily obtain such amplification using the primers of the
invention, including, for example, those in FIG. 3 (SEQ ID
NO:3-15). A Jurkat cell line cDNA library was PCR amplified with
the flanking primers resulting in a single, clear band when the
amplicon was visualized on an agarose gel after ethidium bromide
staining. The resulting amplicon was cloned into the TA cloning
vector (Clontech Inc) and sequenced in both directions. The final
sequence was compared to the consensus derived from the research
database search and is presented in FIG. 2 along with a computer
generated translation of 355 amino acids (SEQ ID NO:2).
[0248] Comparison of the nucleic acid and protein translation of
the proposed Arginase II clone to that of Arginase I identified six
highly conserved regions between the two molecules (FIG. 5). These
are the same six regions of cross-species homology identified in
Arginase I from virtually all species studied. The nucleic acid
homology in the conserved regions is approximately 70% while in the
adjacent non-conserved regions, homology drops to below 40%.
Similarly, when the amino acid sequence of the putative Arginase II
protein was compared to the know sequence of Arginase I, the six
regions of cross-species homology were conserved between Arginase I
and Arginase II (FIG. 5). Only six amino acid residues out of the
79 residues included in the conserved region are different between
Arginase I and Arginase II giving a homology within the conserved
region of 92%. Outside of the conserved regions amino acid homology
averages around 42%.
[0249] The sequence of known arginase molecules was used to
generate an phylogenetic tree using GCG software well known in the
art. This analysis demonstrated that the human Arginase II sequence
is more closely related to "non-type I" arginase from Xenopus than
it is related to human Arginase I.
[0250] The full length Arginase II clone identified from a Jurkat
cDNA library may be subcloned into the expression vector pTRC99A
for arginase expression studies.
[0251] The DNA sequence encoding human Arginase II in the deposited
polynucleotide can be amplified using PCR oligonucleotide primers
specific to the amino acid carboxyl terminal sequence of the human
Arginase II protein and to the end of the contig (FIG. 3 (SEQ ID
NO:3-15)) Additional nucleotides containing restriction sites to
facilitate cloning were added to the 5' and 3' sequences
respectively. No additional nucleotides were added. It was cloned
into a TA cloning vector.
[0252] The 5' oligonucleotide primer had the sequence
5'-ATGTCCCTAAGGGGCAGCCTCTCGCGTC-3' (SEQ ID NO:3) which encodes a
start AUG, followed by 25 nucleotides of the human Arginase II
coding sequence set out in FIG. 2 beginning with the first base of
the AUG start codon.
[0253] The 3' primer had the sequence
5'-TAAATTCTCACACGTGCTTGATTTCTG-3' (SEQ ID NO:4) complimentary to
the last 28 nucleotides of the Arginase II coding sequence set out
in FIG. 2, including the stop codon.
[0254] The restrictions sites were generated using primers 5F and
5R in FIG. 3 and were convenient to restriction enzyme sites in the
bacterial expression vectors pTRC99A, which were used for bacterial
expression in these examples (Pharmacia). pTRC99A encodes
ampicillin antibiotic resistance ("Ampr") and contains a bacterial
origin of replication ("ori"), an IPTG inducible promoter, a
ribosome binding site ("RBS") and restriction enzyme sites.
[0255] The amplified human Arginase II DNA and the vector pTRC99A
both were digested with EcoRI and PstI and the digested DNAs then
were ligated together. Insertion of the Arginase II DNA into the
pTRC99A restricted vector placed the Arginase II coding region
downstream of and operably linked to the vector's IPTG-inducible
promoter and in-frame with an initiating AUG appropriately
positioned for translation of Arginase II.
[0256] The ligation mixture was transformed into competent E. coli
cells using standard procedures. Such procedures are described in
Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.;
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989).
[0257] E. coli strain M15/rep4, containing multiple copies of the
plasmid pREP4, which expresses lac repressor and confers kanamycin
resistance ("Kanr"), is used in carrying out the illustrative
example described here. This strain, which is only one of many that
are suitable for expressing Arginase II, is available commercially
from Qiagen.
[0258] Transformants are identified by their ability to grow on LB
plates in the presence of ampicillin. Plasmid DNA is isolated from
resistant colonies and the identity of the cloned DNA and is
confirmed by restriction analysis.
[0259] Clones containing the desired constructs are grown overnight
("O/N") in liquid culture in LB media supplemented with both
ampicillin (100 ug/ml) and kanamycin (25 ug/ml).
[0260] The O/N culture is used to inoculate a large culture, at a
dilution of approximately 1:100 to 1:250. The cells are grown to an
optical density at 600 nm ("OD") of between 0.4 and 0.6.
Isopropyl-B-D-thiogalact- opyranoside ("IPTG") is then added to a
final concentration of 1 mM to induce transcription from lac
repressor sensitive promoters, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
then are harvested by centrifugation and disrupted, by standard
methods. Inclusion bodies are purified from the disrupted cells
using routine collection techniques, and protein is solubilized
from the inclusion bodies into 8 M urea. The 8 M urea solution
containing the solubilized protein is passed over a PD-10 column in
2X phosphate buffered saline ("PBS"), thereby removing the urea,
exchanging the buffer and refolding the protein. The protein is
purified by a further step of chromatography to remove endotoxin.
Then, it is sterile filtered. The sterile filtered protein
preparation is stored in 2X PBS at a concentration of 95 micrograms
per ml.
[0261] Analysis of the preparation by standard methods of
polyacrylamide gel electrophoresis is expected to reveal that the
preparation contains monomeric Arginase II.
Example 2
Analysis of Human Arginase II cDNA
[0262] Utilizing the Arginase I cDNA sequence, 21 overlapping
clones were identified in the research database which have sequence
homology to, but were distinct from, Arginase I. These clones were
used to generate a full length cDNA of 1075 bases, which, upon
examination, proved to be a previously novel arginase gene,
Arginase II (FIG. 2 (SEQ ID NO: 1-2)). The pattern of expression of
Arginase II, as determined by antibody studies, has striking
similarity to the origin of the libraries that contained positive
clones. The clones listed in FIG. 1 are derived from white cell
lineage, the gastrointestinal tract and brain. However, clones from
the kidney and lactating mammary gland, two tissues known to
express Arginase II may be present. In addition, a major source of
clones from the research database is from prostate tissue library
sequence. These include normal and cancerous prostatic tissues and
tissue from patients with benign hyperplasia of the prostate.
[0263] The sequence of the clone recovered from a Jurkat cDNA
library was identical to the consensus deviated by the overlapping
clones from the research database verifying the correct sequence of
the gene. Comparison of this sequence to the known sequence of
arginase I identifies six conserved regions in Arginase II. The
homology within these conserved regions is approximately 70%.
Divergence between the sequences is limited to the wobble position
in almost every one of the codons in the conserved region,
resulting in 92% conservation at the amino acid level. Considering
the low level of homology between the two types of arginase outside
of the conserved region, maintenance of the bases essential to
conservation of the amino acid sequence in these regions, gives
some indication of how important these residues are to enzymatic
activity. In spite of evolutionary pressure for the two sequences
to diverge, selection, based on function has preserved the
conserved regions. While Arginase II contains all of the conserved
regions present in Arginase I, the phylogenetic tree demonstrated
that human Arginase II is more closely related to the non-hepatic
form of arginase from Xenopus than to human Arginase I.
Example 3
Cloning and Expression of Human Arginase II in a Baculovirus
Expression System
[0264] The cDNA sequence encoding the full length human Arginase II
protein, in the deposited clone can be amplified using PCR
oligonucleotide primers corresponding to the 5' and 3' sequences of
the gene using methods well known in the art. Inserted into an
expression vector, as described below, the 5' end of the amplified
fragment encoding human Arginase II may provide an efficient signal
peptide. An efficient signal for initiation of translation in
eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196,
947-950 (1987) is appropriately located in the vector portion of
the construct.
[0265] An amplified fragment may be isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101 Inc., La
Jolla, Calif.). The fragment then is digested with the appropriate
restriction endonucleases and again is purified on a 1% agarose
gel. This fragment is designated herein F2.
[0266] Expression vectors known in the art will be useful to
express the Arginase II protein in a baculovirus expression system,
using standard methods, such as those described in Summers et at, A
MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE
PROCEDURES, Texas Agricultural Experimental Station Bulletin No.
1555 (1987). A preferred expression vector contains the strong
polyhedron promoter of the Autographa clifornica nuclear
polyhedrosis virus (AcMNPV) followed by convenient restriction
sites. Preferably, the signal peptide of AcMNPV gp67, including the
N-terminal methionine, is located just upstream of a BamHl site.
Preferably the polyadenylation site of the simian virus 40 ("SV40")
is used for efficient polyadenylation. Preferably, for an easy
selection of recombinant virus the beta-galactosidase gene from E.
coli is inserted in the same orientation as the polyhedrin promoter
and is followed by the polyadenylation signal of the polyhedrin
gene. Preferably, the polyhedrin sequences are flanked at both
sides by viral sequences for cell-mediated homologous recombination
with wild-type viral DNA to generate viable virus that express the
cloned polynucleotide.
[0267] Many other baculovirus vectors could be used in place of
pA2-GP, such as pAc373, pVL941 and pAcIM1 provided, as those of
skill readily will appreciate, that construction provides
appropriately located signals for transcription, translation,
trafficking and the like, such as an in-frame AUG and a signal
peptide, as required. Such vectors are described in Luckow et al.,
Virology 170: 31-39, among others.
[0268] The plasmid is digested with appropriate restriction enzymes
and then is dephosphorylated using calf intestinal phosphatase,
using routine procedures known in the art. The DNA is then isolated
from a 1% agarose gel using a commercially available kit
("Geneclean" BIO 101 Inc., La Jolla Calif.). This vector DNA is
designated herein "V2".
[0269] Fragment F2 and the dephosphorylated plasmid V2 are ligated
together with T4 DNA ligase. E. coli HB101 cells are transformed
with ligation mix and spread on culture plates. Bacteria are
identified that contain the plasmid with the human Arginase II gene
by digesting DNA from individual colonies using restriction enzymes
appropriate to remove the inserted sequence using known methods and
then analyzing the digestion product by gel electrophoresis. The
sequence of the cloned fragment is confirmed by DNA sequencing.
This plasmid is designated herein pBacArginase II.
[0270] 5 mg of the plasmid pBacArginase II is co-transfected with
1.0 mg of a commercially available linearized Baculovirus DNA
(BaculoGold baculovirus DNA, Pharmingen, San Diego, Calf.), using
the lipofection method described by Felgner et al., Proc. Natl.
Acad. Sci. USA 84: 7413-7417 (1987) 1 mg of BaculoGold virus DNA
and 5 mg of the plasmid pBacArginase II are mixed in a sterile well
of a microtiter plate containing 50 ml of serum free Grace's medium
(Life Technologies Inc., Gaithersburg, Md.). Afterwards 10 ml
Lipofectin plus 90 ml Grace's medium are added, mixed and incubated
for 15 minutes at room temperature. Then the transfection mixture
is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a
35 mm tissue culture plate with 1 ml Grace's medium without serum.
The plate is rocked back and forth to mix the newly added solution.
The plate is then incubated for 5 hours at 27.degree. C. After 5
hours the transfection solution is removed from the plate and 1 ml
of Grace's insect medium supplemented with 10% fetal calf serum is
added. The plate is put back into an incubator and cultivation is
continued at 27.degree. C. for four days.
[0271] After four days the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, cited above.
An agarose gel with Blue Gal (Life Technologies Inc., Gaithersburg)
is used to allow easy identification and isolation of
gal-expressing clones, which produce blue-stained plaques. (A
detailed description of a "plaque assay" of this type can also be
found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
page 9-10).
[0272] Four days after serial dilution, the virus is added to the
cells. After appropriate incubation, blue stained plaques are
picked with the tip of an Eppendorf pipette. The agar containing
the recombinant viruses is then resuspended in an Eppendorf tube
containing 200 ml of Grace's medium. The agar is removed by a brief
centrifugation and the supernatant containing the recombinant
baculovirus is used to infect Sf9 cells seeded in 35 mm dishes.
Four days later the supernatants of these culture dishes are
harvested and then they are stored at 4.degree. C. A clone
containing properly inserted Arginase II is identified by DNA
analysis including restriction mapping and sequencing. This is
designated herein as V-Arginase II.
[0273] Sf9 cells are grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells are infected with the recombinant
baculovirus V-Arginase II at a multiplicity of infection ("MOI") of
about 2 (about 1 to about 3). Six hours later the medium is removed
and is replaced with SF900 II medium minus methionine and cysteine
(available from Life Technologies Inc., Gaithersburg). 42 hours
later, 5 mCi of .sup.35S-methionine and 5 mCi .sup.35S cysteine
(available from Amersham) are added. The cells are further
incubated for 16 hours and then they are harvested by
centrifugation, lysed and the labeled proteins are visualized by
SDS-PAGE and autoradiography.
Example 4
Expression of Arginase II in COS Cells
[0274] The expression plasmid, Arginase II HA, is made by cloning a
cDNA encoding Arginase II into the expression vector pcDNAI/Amp
(which can be obtained from Invitrogen, Inc.).
[0275] The expression vector pcDNAI/amp contains: (1) an E. coli
origin of replication effective for propagation in E. coli and
other prokaryotic cell; (2) an ampicillin resistance gene for
selection of plasmid-containing prokaryotic cells; (3) an SV40
origin of replication for propagation in eukaryotic cells; (4) a
CMV promoter, a polylinker, an SV40 intron, and a polyadenylation
signal arranged so that a cDNA conveniently can be placed under
expression control of the CMV promoter and operably linked to the
SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker.
[0276] A DNA fragment encoding the entire Arginase II precursor and
a HA tag fused in frame to its 3' end is cloned into the polylinker
region of the vector so that recombinant protein expression is
directed by the CMV promoter. The HA tag corresponds to an epitope
derived from the influenza hemagglutinin protein described by
Wilson et al., Cell 37: 767 (1984). The fusion of the HA tag to the
target protein allows easy detection of the recombinant protein
with an antibody that recognizes the HA epitope.
[0277] The plasmid construction strategy is as follows. The
Arginase II cDNA of the deposit clone is amplified using primers
that contained convenient restriction sites, much as described
above regarding the construction or expression vectors for
expression of Arginase II in E. coli and S. furgiperda.
[0278] To facilitate detection, purification and characterization
of the expressed Arginase II, one of the primers contains a
heamaglutinin tag ("HA tag") as described above.
[0279] PCR amplified DNA fragments and the vector, pcDNAI/Amp, are
digested with and then ligated. The ligation mixture is transformed
into E. coli strain SURE (available from Stratagene Cloning
Systems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037) the
transformed culture is plated on ampicillin media plates which then
are incubated to allow growth of ampicillin resistant colonies.
Plasmid DNA is isolated from resistant colonies and examined by
restriction analysis and gel sizing for the presence of the
Arginase II-encoding fragment.
[0280] For expression of recombinant Arginase II, COS cells are
transfected with an expression vector, as described above, using
DEAE-DEXTRAN, as described, for instance, in Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Laboratory
Press, Cold Spring Harbor, N.Y. (1989).
[0281] Cells are incubated under conditions for expression of
Arginase II by the vector.
[0282] Expression of the Arginase II HA fusion protein is detected
by radiolabelling and immunoprecipitation, using methods described
in, for example Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2nd
Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1988). To this end, two days after transfection, the cells are
labeled by incubation in media containing .sup.35S-cysteine for 8
hours. The cells and the media are collected, and the cells are
washed and the lysed with detergent-containing RIPA buffer: 150 mM
NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH 7.5, as
described by Wilson et al. cited above. Proteins are precipitated
from the cell lysate and from the culture media using an
HA-specific monoclonal antibody. The precipitated proteins then are
analyzed by SDS-PAGE gels and autobiography. An expression product
of the expected size is seen in the cell lysate, which is not seen
in negative controls.
Example 5
Tissue Distribution of Arginase II Expression
[0283] Northern blot analysis is carried out to examine the levels
of expression of Arginase II in human tissues, using methods
described by, among others, Sambrook et al, cited above. Premade
Clontech were used for northern blots. Total cellular RNA samples
are isolated with RNAzol B system (Biotecx Laboratories, Inc. 6023
South Loop East, Houston, Tex. 77033).
[0284] About 1 mg of Total RNA is isolated from tissue samples. The
RNA is size resolved by electrophoresis through a 1% agarose gel
under strongly denaturing conditions. RNA is blotted from the gel
onto a nylon filter, and the filter then is prepared for
hybridization to a detectably labeled polynucleotide probe.
[0285] As a probe to detect mRNA that encodes Arginase II, the
antisense strand of the coding region of the cDNA insert in the
deposited clone is labeled to a high specific activity. The cDNA is
labeled by primer extension, using the Prime-It kit, available from
Stratagene. The reaction is carried out using 50 ng of the cDNA,
following the standard reaction protocol as recommended by the
supplier. The labeled polynucleotide is purified away from other
labeled reaction components by column chromatography using a
Select-G-50 column, obtained from 5-Prime-3-Prime, Inc. of 5603
Arapahoe Road, Boulder, Colo. 80303.
[0286] The labeled probe is hybridized to the filter, at a
concentration of 1,000,000 cpm/ml, in a small volume of 7% SDS, 0.5
M NaPO4, pH 7.4 at 65.degree. C., overnight.
[0287] Thereafter the probe solution is drained and the filter is
washed twice at room temperature and twice at 60.degree. C. with
0.5X SSC, 0.1% SDS. The filter then is dried and exposed to film at
-70.degree. C. overnight with an intensifying screen.
[0288] Autoradiography shows that mRNA for Arginase II is abundant
in prostate and to a lesser extent (approximately 5-10-fold less)
in kidney.
Example 6
Gene Therapeutic Expression of Human Arginase II
[0289] Fibroblasts are obtained from a subject by skin biopsy. The
resulting tissue is placed in tissue-culture medium and separated
into small pieces. Small chunks of the tissue are placed on a wet
surface of a tissue culture flask, approximately ten pieces are
placed in each flask. The flask is turned upside down, closed tight
and left at room temperature overnight. After 24 hours at room
temperature, the flask is inverted--the chunks of tissue remain
fixed to the bottom of the flask--and fresh media is added (e.g.,
Ham's F12 media, with 10% FBS, penicillin and streptomycin). The
tissue is then incubated at 37.degree. C. for approximately one
week. At this time, fresh media is added and subsequently changed
every several days. After an additional two weeks in culture, a
monolayer of fibroblasts emerges. The monolayer is trypsinized and
scaled into larger flasks
[0290] A vector for gene therapy is digested with restriction
enzymes for cloning a fragment to expressed. The digested vector is
treated with calf intestinal phosphatase to prevent self-ligation.
The dephosphorylated, linear vector is fractionated on an agarose
gel and purified.
[0291] Arginase II cDNA capable of expressing active Arginase II,
is isolated. The ends of the fragment are modified, if necessary,
for cloning onto the vector. For instance, 5' overhanging may be
treated with DNA polymerase to create blunt ends. 3' overhanging
ends may be removed using S1 nuclease. Linkers may be ligated to
blunt ends with T4 DNA ligase.
[0292] Equal quantities of the Moloney murine leukemia virus linear
backbone and the Arginase II fragment are mixed together and joined
using T4 DNA ligase. The ligation mixture is used to transform E.
coli and the bacteria are then plated onto agar-containing
kanamycin. Kanamycin phenotype and restriction analysis confirm
that the vector has the properly inserted gene.
[0293] Packaging cells are grown in tissue culture to confluent
density in Dulbecco's Modified Eagles Medium. (DMEM) with 10% calf
serum (CS), penicillin and streptomycin. The vector containing the
Arginase II gene is introduced into the packaging cells by standard
techniques. Infectious viral particles containing the Arginase II
gene are collected from the packaging cells, which now are called
producer cells.
[0294] Fresh media is added to the producer cells, and after an
appropriate incubation period media is harvested from the plates of
confluent producer cells. The media, containing the infectious
viral particles, is filtered through a Millipore filter to remove
detached producer cells. The filtered media then is used to infect
fibroblast cells. Media is removed from a sub-confluent plate of
fibroblasts and quickly replaced with the filtered media. Polybrene
(Aldrich) may be included in the media to facilitate transduction.
After appropriate incubation, the media is removed and replaced
with fresh media. If the titer of virus is high, then virtually all
fibroblasts will be infected and no selection is required. If the
titer is low, then it is necessary to use a retroviral vector that
has a selectable marker, such as neo or his, to select out
transduced cells for expansion.
[0295] Engineered fibroblasts then may be injected into rats,
either alone or after having been grown to confluence on
microcarrier beads, such as cytodex 3 beads. The injected
fibroblasts produce Arginase II product, and the biological actions
of the protein are conveyed to the host.
Example 7
Northern Blots
[0296] Six northern blots were hybridized with an Arginase I and an
Arginase II specific cDNA probe. Arginase I appears to be expressed
in normal liver, as previously known, but is absent in the one
Liver tumor sample tested. Arginase II has a complex pattern of
expression. Arginase II mRNA levels increase in gastric, colon,
rectal, prostate, lymph node, and liver cancer (over normal
adjacent tissue) with the greatest difference being in the lymph
node. Arginase II mRNA levels decrease in brain, kidney, esophagus,
thymus, adrenal gland, parotid, and bladder cancer (over normal
adjacent tissue) with the greatest difference being in the kidney.
These data suggest that arginase mRNA levels change with the
formation of cancer. The implication is that a change in arginase
mRNA expression results in a change in the level of arginase
activity which would affect the availability of arginine within the
cell. Cellular arginine levels are important in polyamine, proline,
GABA, nitric oxide and aginatine biosynthesis.
Example 3
Solid Tumor Arginase Tumor Activity and Serum Arginase Activity in
Cancer Patients
[0297] Arginase activity was tested on solid tumor samples from
patients with a number of different types or tumors. The results
are summarized in the table below.
1TABLE 1 Arginase Activity (ng/mg protein) Cancer Type n Cancer
Normal Adjacent Tissue Breast 2 46.7 +/- 9.1 15.6 +/- 4.5 Ovarian 3
14.4 +/- 12.6 4.7 +/- 1.3 Lung 2 21.1 +/- 7.0 6.6 +/- 1.9 Colon (I,
II) 7 27.6 +/- 11.1 9.8 +/- 3.7 Colon (III, IV) 4 53.4 +/- 23.2 7.5
+/- 2.2 Testicular 4 24.7 +/- 11.1 5.4 +/- 0.8 Prostate 11 64.7 +/-
28.6 12.5 +/- 3.3
[0298] Serum arginase activity was also tested from a number of
different cancer patients. The results are summarized in the table
below and are compared to standard cancer markers.
2TABLE 2 Arginase Activity (ng/ml) Arginase Activity Cancer Type n
(ng/ml) Marker (ng/ul) Breast 12 4.0 +/- 0.4 CEA 1.5 +/- 0.2
Ovarian 10 4.8 +/- 0.4 CEA 1.3 +/- 0.1 Lung 17 6.5 +/- 0.1 CEA 10.0
+/- 1.3 Colon (I, II) 10 4.4 +/- 0.7 CEA 1.5 +/- 0.15 Testicular 3
2.2 +/- 0.4 AFP 4.6 +/- 0.7 Testicular (met) 8 12.8 +/- 0.6 AFP
1255 +/- 187 Prostate 15 2.3 +/- 0.3 PSA 6.8 +/- 2.3 Prostate (met)
21 29.8 +/- 1.7 PSA 187.8 +/- 31.5 Control 44 2.1 +/- 0.4
[0299] These data suggest that arginase enzymatic activity is
elevated in tumors over normal adjacent tissue and that it is
expressed at highest levels in the serum of metastatic cancer
patents.
[0300] All of these data suggest that arginase expression and
activity may be a key factor in the formation and metastasis of
cancer. As arginase depletes the arginine concentration within the
cell, it shuts down nitric oxide synthesis disabling
tumor-infiltrating macrophage and limiting the cytotoxic effect of
nitric oxide. At the same time, polyamine synthesis may be
stimulated by the production of large amounts of ornithine, the end
product of the arginase reaction, through ornithine decarboxylase,
while proline biosynthesis is enhanced through the action of
ornithine aminotransferase on the excess ornithine. The effect of
elevated arginase activity in the serum is to have a systemic
interference with the immune system to include inhibiting
lymphocytes and three different splenic derived killer cells.
Combined, there is a stimulation of cancer growth while at the same
time a local and systemic inhibition of the immune system.
[0301] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples.
[0302] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
Sequence CWU 1
1
17 1 1303 DNA HOMO SAPIENS UNSURE (6)(1271)(1278)(1293)(1294) 1
gcgganctct gccttggaga ttctcagtgc tgcggatcat gtccctaagg ggcagcctct
60 cgcgtctcct ccagacgcga gtgcattcca tcctgaagaa atccgtccac
tccgtggctg 120 tgataggagc cccgttctca caagggcaga aaagaaaagg
agtggagcat ggtcccgctg 180 ccataagaga agctggcttg atgaaaaggc
tctccagttt gggctgccac ctaaaagact 240 ttggagattt gagttttact
ccagtcccca aagatgatct ctacaacaac ctgatagtga 300 atccacgctc
agtgggtctt gccaaccagg aactggctga ggtggttagc agagctgtgt 360
cagatggcta cagctgtgtc acactgggag gagaccacag cctggcaatc ggtaccatta
420 gtggccatgc ccgacactgc ccagaccttt gtgttgtctg ggttgatgcc
catgctgaca 480 tcaacacacc ccttaccact tcatcaggaa atctccatgg
acagccagtt tcatttctcc 540 tcagagaact acaggataag gtaccacaac
tcccaggatt ttcctggatc aaaccttgta 600 tctcttctgc aagtattgtg
tatattggtc tgagagacgt ggaccctcct gaacatttta 660 ttttaaaagg
aactatggat atccagtatt ttttccatgg aggagatatt ggatcgaact 720
tggtatccag gaaggtcatg ggaacggaac atttgatctg gctgattggc aagagacaaa
780 gaccaatcca tttgagtttt gatattgatg catttgaccc tacactggct
ccagccacag 840 gaactcctgt tgtcggggga ctaacctatc gagaaggcat
gtatattgct gaggaaatac 900 acaatacagg gttgctatca gcactggatc
ttgttgaagt caatcctcag ttggccacct 960 cagaggaaga ggcgaagact
acagctaacc tggcagtaga tgtgattgct tcaagctttg 1020 gtcagacaag
agaaggaggg catattgtct atgaccaact tcctactccc agttcaccag 1080
atgaatcaga aaatcaagca cgtgtgagaa tttaggagac actgtgcact gacatgtttc
1140 acaacaggca ttccagaatt atgaggcatt gaggggatag atgaatactt
aaatggttgt 1200 tctgggtcaa tactgcctta atggggacat ttacacattc
tcacattgta aagttttccc 1260 ccctatttgg ngaccatnat tactgtaaat
ggnntttggg ttt 1303 2 358 PRT HOMO SAPIENS 2 Met Ser Leu Arg Gly
Ser Leu Ser Arg Leu Leu Gln Thr Arg Val His 1 5 10 15 Ser Ile Leu
Lys Lys Ser Val His Ser Val Ala Val Ile Gly Ala Pro 20 25 30 Phe
Ser Gln Gly Gln Lys Arg Lys Gly Val Glu His Gly Pro Ala Ala 35 40
45 Ile Arg Glu Ala Gly Leu Met Lys Arg Leu Ser Ser Leu Gly Cys His
50 55 60 Leu Lys Asp Phe Gly Asp Leu Ser Phe Thr Pro Val Pro Lys
Asp Asp 65 70 75 80 Leu Tyr Asn Asn Leu Ile Val Asn Pro Arg Ser Val
Gly Leu Ala Asn 85 90 95 Gln Glu Leu Ala Glu Val Val Ser Arg Ala
Val Ser Asp Gly Tyr Ser 100 105 110 Cys Val Thr Leu Gly Gly Asp His
Ser Leu Ala Ile Gly Thr Ile Ser 115 120 125 Gly His Ala Arg His Cys
Pro Asp Leu Cys Val Val Trp Val Asp Ala 130 135 140 His Ala Asp Ile
Asn Thr Pro Leu Thr Thr Ser Ser Gly Asn Leu His 145 150 155 160 Gly
Gln Pro Val Ser Phe Leu Leu Arg Glu Leu Gln Asp Lys Val Pro 165 170
175 Gln Leu Pro Gly Phe Ser Trp Ile Lys Pro Cys Ile Ser Ser Ala Ser
180 185 190 Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro Pro Glu His
Phe Ile 195 200 205 Leu Lys Gly Thr Met Asp Ile Gln Tyr Phe Phe His
Gly Gly Asp Ile 210 215 220 Gly Ser Asn Leu Val Ser Arg Lys Val Met
Gly Thr Glu His Leu Ile 225 230 235 240 Trp Leu Ile Gly Lys Arg Gln
Arg Pro Ile His Leu Ser Phe Asp Ile 245 250 255 Asp Ala Phe Asp Pro
Thr Leu Ala Pro Ala Thr Gly Thr Pro Val Val 260 265 270 Gly Gly Leu
Thr Tyr Arg Glu Gly Met Tyr Ile Ala Glu Glu Ile His 275 280 285 Asn
Thr Gly Leu Leu Ser Ala Leu Asp Leu Val Glu Val Asn Pro Gln 290 295
300 Leu Ala Thr Ser Glu Glu Glu Ala Lys Thr Thr Ala Asn Leu Ala Val
305 310 315 320 Asp Val Ile Ala Ser Ser Phe Gly Gln Thr Arg Glu Gly
Gly His Ile 325 330 335 Val Tyr Asp Gln Leu Pro Thr Pro Ser Ser Pro
Asp Glu Ser Glu Asn 340 345 350 Gln Ala Arg Val Arg Ile 355 3 28
DNA HOMO SAPIENS 3 atgtccctaa ggggcagcct ctcgcgtc 28 4 28 DNA HOMO
SAPIENS 4 taaattctca cacgtgcttg attttctg 28 5 30 DNA HOMO SAPIENS 5
ctctgccttg gagattctca gtgctgcgga 30 6 30 DNA HOMO SAPIENS 6
attgtctcct aaattctcac acgtgcttga 30 7 27 DNA HOMO SAPIENS 7
gaggtggtta gcagagctgt gtcagat 27 8 22 DNA HOMO SAPIENS 8 caacaggagt
tcctgtggct gg 22 9 30 DNA HOMO SAPIENS 9 gctgattggc aagagacaaa
gaccaatcca 30 10 29 DNA HOMO SAPIENS 10 gggcatggcc actaatggta
ccgattgcc 29 11 47 DNA HOMO SAPIENS 11 gattgcctta tggaattcat
gtcccctaag gggcagcctc tcgcgtc 47 12 43 DNA HOMO SAPIENS 12
gattgccttc tgcagtaaat tctcacacgt gcttgatttt ctg 43 13 15 DNA HOMO
SAPIENS 13 aggaactatg gatat 15 14 15 DNA HOMO SAPIENS 14 cttgccaatc
tgcct 15 15 21 DNA HOMO SAPIENS 15 atgtgattgc ttcaagcttt g 21 16
1075 DNA HOMO SAPIENS 16 atgtccctaa ggggcagcct ctcgcgtctc
ctccagacgc gagtgcattc catcctgaag 60 aaatccgtcc actccgtggc
tgtgatagga gccccgttct cacaagggca gaaaagaaaa 120 ggagtggagc
atggtcccgc tgccataaga gaagctggct tgatgaaaag gctctccagt 180
ttgggctgcc acctaaaaga ctttggagat ttgagtttta ctccagtccc caaagatgat
240 ctctacaaca acctgatagt gaatccacgc tcagtgggtc ttgccaacca
ggaactggct 300 gaggtggtta gcagagctgt gtcagatggc tacagctgtg
tcacactggg aggagaccac 360 agcctggcaa tcggtaccat tagtggccat
gcccgacact gcccagacct ttgtgttgtc 420 tgggttgatg cccatgctga
catcaacaca ccccttacca cttcatcagg aaatctccat 480 ggacagccag
tttcatttct cctcagagaa ctacaggata aggtaccaca actcccagga 540
ttttcctgga tcaaaccttg tatctcttct gcaagtattg tgtatattgg tctgagagac
600 gtggaccctc ctgaacattt tattttaaaa ggaactatgg atatccagta
ttttttccat 660 ggaggagata ttggatcgaa cttggtatcc aggaaggtca
tgggaacgga acatttgatc 720 tggctgattg gcaagagaca aagaccaatc
catttgagtt ttgatattga tgcatttgac 780 cctacactgg ctccagccac
aggaactcct gttgtcgggg gactaaccta tcgagaaggc 840 atgtatattg
ctgaggaaat acacaataca gggttgctat cagcactgga tcttgttgaa 900
gtcaatcctc agttggccac ctcagaggaa gaggcgaaga ctacagctaa cctggcagta
960 gatgtgattg cttcaagctt tggtcagaca agagaaggag ggcatattgt
ctatgaccaa 1020 cttcctactc ccattcacca gatgaatcag aaaatcaagc
acgtgtgaga attta 1075 17 357 PRT HOMO SAPIENS 17 Thr Glu Gly Leu
Thr Gly Glu Leu Lys Cys Ser Lys Glu Lys Cys Gln 1 5 10 15 Ser Met
Ser Ala Lys Ser Arg Thr Ile Gly Ile Ile Gly Ala Pro Phe 20 25 30
Ser Lys Gly Gln Pro Arg Gly Gly Val Glu Glu Gly Pro Thr Val Leu 35
40 45 Arg Lys Ala Gly Leu Leu Glu Lys Leu Lys Glu Gln Glu Cys Asp
Val 50 55 60 Lys Asp Tyr Gly Asp Leu Pro Phe Ala Asp Ile Pro Asn
Asp Ser Pro 65 70 75 80 Phe Gln Ile Val Lys Asn Pro Arg Ser Val Gly
Lys Ala Ser Glu Gln 85 90 95 Leu Ala Gly Lys Val Ala Gln Val Lys
Lys Asn Gly Arg Ile Ser Leu 100 105 110 Val Leu Gly Gly Asp His Ser
Leu Ala Ile Gly Ser Ile Ser Gly His 115 120 125 Ala Arg Val His Pro
Asp Leu Gly Val Ile Trp Val Asp Ala His Thr 130 135 140 Asp Ile Asn
Thr Pro Leu Thr Thr Thr Ser Gly Asn Leu His Gly Gln 145 150 155 160
Pro Val Ser Phe Leu Leu Lys Glu Leu Lys Gly Lys Ile Pro Asp Val 165
170 175 Pro Gly Phe Ser Trp Val Thr Pro Cys Ile Ser Ala Lys Asp Ile
Val 180 185 190 Tyr Ile Gly Leu Arg Asp Val Asp Pro Gly Glu His Tyr
Ile Leu Lys 195 200 205 Thr Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu
Val Asp Arg Leu Gly 210 215 220 Ile Gly Lys Val Met Glu Glu Thr Leu
Ser Tyr Leu Leu Gly Arg Lys 225 230 235 240 Lys Arg Pro Ile His Leu
Ser Phe Asp Val Asp Gly Leu Asp Pro Ser 245 250 255 Phe Thr Pro Ala
Thr Gly Thr Pro Val Val Gly Gly Leu Thr Tyr Arg 260 265 270 Glu Gly
Leu Tyr Ile Thr Glu Glu Ile Tyr Lys Thr Gly Leu Leu Ser 275 280 285
Gly Leu Asp Ile Met Glu Val Asn Pro Ser Leu Gly Lys Thr Pro Glu 290
295 300 Glu Val Thr Arg Thr Val Asn Thr Ala Val Ala Ile Thr Leu Ala
Cys 305 310 315 320 Phe Gly Leu Ala Arg Glu Gly Asn His Lys Pro Ile
Asp Tyr Leu Asn 325 330 335 Pro Pro Lys Met Trp Lys His Pro Ile Ile
Ser Leu Met Ala Leu Glu 340 345 350 Ser Ser Phe Ser Ala 355
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