Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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EUKARYOTIC PEPTIDE UPTAKE SYSTEM FOR
TRANSPORTATION OF ENKEPHALINS
RELATED CASES
This application is a conversion of provisional application Serial No.
60/122,312, filed on March l, 1999, entitled: Enkephalins are Transported by a
Novel
Eucaryotic Peptide Uptake System.
FIELD OF THE INVENTION
The invention relates to plant molecular genetics, and more specifically, to
an
oligopeptide transporter in the yeast Saccharomyces cerevisiae. The
transporter
mediates the uptake of tetra- and penta- peptides, including leucine
enkephalin and
methionine enkephalins.
BACKGROUND OF THE INVENTION
Peptide uptake is the process by which individual cells are able to transport
intact peptides across their plasma membranes. The process is a general
physiological
phenomenon of bacteria, fungi, plant cells and mammalian cells (Becker, J. M.
et al,
In: Microorganisms and Nitrogen Sources, Payne, J. W. (ed.), John Wiley and
Sons,
Inc., pp. 257-279 (1980); Matthews, D. M. et al., Curr. Top. Membr. Transp.
14:331-
425 ( 1980)). In every case studied so far, peptide transport is a specific
biochemical
process in which small peptides (_< 6 amino acids) are transported across a
membrane
by energy-dependent, saturable carriers.
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Three genetically distinct systems of peptide uptake have been identified in
gram-negative bacteria. An oligopeptide permease (Opp) system has been
identified in
bacteria such as E. coli, and S. typhimurium (Andrews, J. C. et al., J.
Bacteriol.
161:484-492 (1985); Hogarth, B. G. et al., J. Bacteriol. 153:1548-1551
(1983)). The
Opp system is capable of transporting peptides having up to 5 amino acid
residues,
regardless of their side chains (Payne, J. W. et al, J. Biol. Chem.
243:3395=3403
( 1968); Payne, J. W. et al., J. Biol. Chem. 243:6291-6299 ( 1968)). In
contrast,
tripeptide permease (Tpp) systems, such as that of S. typhimurium, exhibit an
apparent
affinity for peptides having hydrophobic amino acid residues (Gibson, M. M. et
al., J.
Bacteriol. 160:122-130 (1984)). The third system, a dipeptide permease (Dpp)
system,
has a preference for transporting dipeptides (Abouhamad, W. N., et al., Mol.
Microbiol. 5:1035-1047 (1991)). Functionally similar systems have been
described in
fungi and yeast (Naider, F. et al., In: Current Topics in Medial Mycology,
volume II,
McGinnis, M. M. (ed.) (1987)), but have not been well characterized.
The genes that encode the protein components of the oligopeptide transporters
ofE. coli (Kashiwagi, K. et al, J. Biol. Chem. 265:8387-8391 (1990)),
Salmonella
typhimurium (Hiles, I. D. et al., Eur. J. Biochem. 158:561-567 (1986); Hiles,
I. D. et
al, J. Molec. Biol 195:125-142 (1987)), Bacillus subtilis (Rudner, D. Z. et
al., J.
Bacteriol. 173:1388-1398 (1991); Perego, M. et al., Mol. Microbiol. 5:173-185
( 1991 )), Streptococcus pneumoniae (Alloing, G. et al., Mol. Microbiol. 4:633-
644
1990)), and Lactococcus lactis as well as two dipeptide permeases, one in E.
coli
(Abouhamad, W. N., et al., Mol. Microbiol. 5:1035-1047 (1991)), and the other
in
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Bacillus subtilis (Mathiopoulos, C. et al., Mol. Microbial. 5:1903-1913 ( 1991
)) have
been cloned and sequenced.
The ability of bacteria and plant cells to accumulate peptides has been found
to
be dependent upon peptide transport systems (Becker, J. M. et al., In:
Microorganisms
and Nitrogen Sources, Payne, J. W. (ed.), John Wiley and Sons, Inc., pp. 257-
279
(1980); Matthews, D. M. et al., Curr. Top. Membr. Transp. 14:331-425 (1980);
Higgins, C. F. et al., In: Microorganisms and Nitrogen Sources, Payne, J. W.
(ed.),
John Wiley and Sons, Inc., pp. 211-256 (1980); Naider, F. et al., In: Current
Topics in
Medial Mycology, volume II, McGinnis, M. M. (ed.) (1987)). These systems are
distinct from the mechanisms that mediate the uptake of amino acids.
The existence of peptide transport systems in plants was demonstrated by
showing that plants could accumulate non-hydrolyzable, non-physiological
peptide
substrates, intact and against a concentration gradient (Higgins, C. F. et
al., Planta
134:205-206 (1977); Higgins, C. F. et al., Planta 136:71-76 (1977); Higgins,
C. F. et
al, Planta 138:211-216 (1978); Higgins, C. F. et al., Planta 142:299-305
(1978);
Sopanen, T. et al., FEBS Lett. 79:4-7 (1977)). The transport system was found
to
exhibit saturation kinetics and to be inhibited by a range of metabolic
inhibitors
(Higgins, C. F. et al., Planta 136:71-76 (1977)). The plant peptide transport
system
can transport both di- and tripeptides (Sopanen, T. et al., FEBS Lett. 79:4-7
(1977);
Higgins, C. F. et al., Planta 142:299-305 (1978)). Plant peptide transport
systems are
capable of transporting a wide variety of peptides. These systems exhibit
broad
transport specificity with respect to amino acid side-chains. The presence of
D-amino
acids, however, reduces the transport rate, thus indicating that the
transporters have
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strong stereospecificity. Two proteins, approximately 66 D and 41 D, have been
suggested as components of the plant peptide transport system in barley grains
(Payne,
J. W. et al., Planta 170:263-271 (1987).
The primary function of peptide transport is to supply amino acids for
nitrogen
nutrition (Payne, J. W. et al., In: Microorganisms and Nitrogen Sources,
Payne, J. W.
(ed.), John Wiley and Sons, Inc., pp. 257-279 (1980); Matthews, D. M. et al.,
Curr.
Top. Membr. Transp. 14:331-425 (1980); Becker, J. M. et al., In:
Microorganisms and
Nitrogen Sources, Payne, J. W.:(ed.), John Wiley and Sons, Inc., pp. 257-279
(1980);
Adibi, S. A. et al., Metabolism 36:1001-1011 (1987); Higgins, C. F. et al.,
Planta
138:211-216 (1978); Sopanen, T. et al., FEBS Lett. 79:4-7 (1977); Higgins, C.
F. et
al., Planta 138:217-221 (1978)). In bacteria, peptide transport has, however,
also been
associated with sporulation (Perego, M. et al., Mol. Microbiol. 5:173-185
(1991);
Mathiopoulos, C. et al., Mol. Microbiol. 5:1903-1913 ( 1991 )); chemotaxis
(Manson,
M. D. et al., Nature 321:253-256 (1986), and the recycling of cell wall
peptides
(Goodell, E. W. et al., J. Bacteriol 169:3861-3865 (1987)).
Small peptides containing four to five amino acid residues are transported by
a
recently identified class of peptide transporters named the OPTl family
(Lubkowitz et
al. Mol. Microbiol. (1998) 28(4):729-741, incorporated herein by reference).
The
amino acid sequence of this family is distinct from that of the PTR family, a
ubiquitous group of proton-coupled transporters which selectively transports
di- and
tripeptides. Phylogenetic analysis suggests that the OPT family is also
distinct from
the major facilitator superfamily (MFS), a diverse collection of proteins
which
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catalyzes the transport of a wide variety of substrates, including sugars,
amino acids,
neurotransmitters, and drugs.
Members of the OPT family have been identified and characterized in the
yeasts Candida albicans, Schizosaccharomyces pombe, and Saccharomyces
cerevisiae. Additional members exist in plants, as indicated by searches of
publicly
accessible data bases. In mammalian tissues, reports in the literature suggest
that the
enkephalins, endogenous pentapeptides involved in analgesia in the central
nervous
system, are transported across the blood-brain barrier by a specific,
saturable transport
system. The existence of enkephalin transporters has been inferred from data
obtained
by measuring whole brain flux of the peptides in rodents. To date, no protein
has
been identified in eukaryotes as the discrete enkephalin carrier.
SUMMARY OF THE INVENTION
It has now been discovered that the endogenous opioids Met-enkephalin and
Leu-enkephalin, pentapeptides of amino acid sequence YGGFM and YGGFL,
respectively, can be transported by cells expressing the S. cerevisiae ORF
YJL212C.
When expressed under the control of a constitutive promoter in a high copy
number
vector, this OPT family member is necessary and sufficient to transport Leu-
enkephalin into yeast cells. This is the first example of a genetically
defined
eukaryotic transport protein which can transport enkephalins across the cell
membrane. This gene has been named OPTl.
The invention also comprises a method for obtaining mammalian enkephalin
transporters by functional complementation of OPT1 deficient yeast.
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The invention also comprises a fungicidal composition comprising a toxic
analogue of enkephalin as an active ingredient, and a method of killing fungi
comprising applying a toxic analogue of enkephalin to a substrate or organism
to be
treated.
The invention also comprises a method of preventing or reducing fungal
growth on substrates.
The invention also comprises OPT1 transformed plants, methods of such
transformation and methods of growing transformed plants.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 shows shows the growth of S. cerevisiae BY4730 expressing OPT family
members.
Fig. 2 shows uptake of [3H]Leu-enkephalin by S. cerevisiae BY4730
transformants.
Fig. 3 shows a chromatographic analysis of Leu-enkephalin.
Fig. 4 shows the effects of naloxone and naltrexone on the uptake of [3H]Leu-
enkephalin and [3H]leucyl-leucine.
Fig. 5 shows the amino acid sequence of the OPT family member (isp4-like
protein)
fromArabidopsis thaliana designated emb CAB43855.1
Fig. 6 shows the amino acid sequence of the OPT family member (isp4-like
protein)
from Arabidopsis thaliana designated emb CAB 10414.1
Fig. 7 shows the amino acid sequence of the OPT family member(isp4 like
protein)
fromArabidopsis thaliana designated Genbank GB:D14061.
Fig. 8 shows the amino acid sequence of the OPT family member (previously
unknown protein) from Arabidopsis thaliana designated emb CAB38285.
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Fig. 9 shows the amino acid sequence of the OPT family member (isp4 like
protein)
from Arabidopsis thaliana designated Genbank GB:D83992.
Fig. 10 shows the amino acid sequence of the OPT family member (Optlp) from
Candida albicans designated Genbank GB:AAB69628.1.
Fig. 11 shows the amino acid sequence of the OPT family member (Optl) from
Saccharomyces cerevisiae designated YJL212c.
Fig. 12 shows the amino acid sequence of the OPT family member (YPR194c) from
Saccharomyces cerevisiae designated YPR194c.
Fig. 13 shows the amino acid sequence of the OPT family member (previously
unknown protein) from Schizosaccharomyces pombe designated emb CAB 16254:'1.
Fig. 14 shows the amino acid sequence of the OPT family member from
Schizosaccharomyces pombe designated emb CAA19062.1.
Fig. 15 shows the amino acid sequence of the OPT family member from
Schizosaccharomyces pombe designated sp40900 or Gi 729859.
Fig. 16 shows a sequence comparison of Optl family members.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention arises, in part, from the exploitation of the S.
cerevisiae
OPTIp. S. cerevisiae strains that carry mutations in OPT1 may be created by
knocking out OPTl by any means known in the art, such as homologous
recombination with a defective OPTl , by homologous recombination replacing
OPTI
with another gene, by mutation and selection, and the like. These knock-out
yeasts
will be completely deficient for enkephalin transport, which can be determined
by
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their resistance to toxic derivatives of enkephalin and lack of uptake of
radiolabeled
Met- and Leu-enkephalin.
In addition to obtaining mammalian homologues to OPTl by functional
complementation, functional augmentation may also be used. In all the wild-
type
yeast strains tested, enkephalin transport was deficient in the absence of a
strong
promoter such as the ADH promoter. Therefore, yeast vectors carrying exogenous
mammalian DNA may be transformed into wild-type yeast and examined for
enkephalin transport. Preferably, these vectors comprise a strong promoter to
augment expression of a functional enkephalin transport protein.
Thus, one aspect of the present invention is the construction of a stable S.
cerevisiae OPTI mutant. Methods for isolating such mutants are described
below, and
by Perry, J. R. et al. (In: "Isolation and Characterization of a Saccharomyces
cerevisiae Peptide Transport Gene," Molecular and Cellular Biology, volume 14
(1994), herein incorporated by reference in its entirety). Polynucleotides
that encode
the peptide transport genes of higher plants have been identified and isolated
by their
capacity to complement the peptide transport deficiency of the stable S.
cerevisiae
ptr2 strain. In a like manner, knock-out S. cerevisiae may be used in
functional
complementation assays using polynucleotides that encode mammalian proteins.
Transformation of knock-out S. cerevisiae with mammalian sequences and
subsequent
growth on Leu-enkephalin as the source of leucine may reveal mammalian
homologues of OPTI. In addition, functional augmentation may be employed to
obtain mammalian homologues.
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The present invention relates in part to the isolation of a novel
polynucleotide
that is capable of hybridizing to, or recombining with, a plant gene that
encodes a
peptide transport protein. The polynucleotides of the present invention are
"substantially purified," in that they have been purified from undesired yeast
genes
with which they are associated in nature. The molecules maybe in either a
double-stranded or single-stranded form. Such polynucleotides are capable of
augmenting the transport capacity of a recipient plant, and thus may be used
to
facilitate the delivery of desired compounds to the plant. In an alternative
embodiment, the polynucleotides of the present invention can be used to
disrupt or
otherwise inactivate endogenous transport systems. Such disruption renders the
plant
incapable of transporting toxic peptides, and thus resistant to pathogens that
produce
such peptides.
The capacity of the polynucleotides of the present invention to hybridize to a
plant gene arises out of the extent of homology between the respective
sequences of
the polynucleotides. As used herein, a polynucleotide of the present invention
is said
to be able to "hybridize" to a plant gene if the two molecules are capable of
forming
an anti-parallel, double-stranded nucleic acid structure. The molecules are
said to be
"minimally complementary" if they can hybridize to one another with sufficient
stability to permit them to remain annealed to one another under at least
conventional
"low-stringency" conditions. Similarly, the molecules are said to be
"complementary"
if they can hybridize to one another with sufficient stability to permit them
to remain
annealed to one another under conventional "high-stringency" conditions. Such
conventional stringency conditions are described by Sambrook, J., et al., (In:
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Molecular Cloning, a Laboratory Manual, 2nd Edition, Cold Spring Harbor Press,
Cold Spring Harbor, N.Y. ( 1989)), and by Haymes, B. D., et al. (In: Nucleic
Acid
Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985)), both
herein incorporated by reference).
Complementary molecules thus need not exhibit "complete complementarity,"
but need only be sufficiently complementary in sequence to be able to form a
stable
double-stranded structure. Departures from complete complementarity are,
therefore,
permissible, so long as such departures do not completely preclude the
capacity of the
molecules to form a double-stranded structure. In contrast, where two nucleic
acid
molecules exhibit "complete complementarity," every nucleotide of one of the
molecules is complementary to a nucleotide of the other; such molecules need
not
have the same lengths.
The capacity of the polynucleotides of the present invention to recombine with
a plant gene is determined by the extent of sequence "homology" between the
polynucleotide and the plant gene. Homologous recombination is a well-studied
natural cellular process which involves the exchanges of a region of one
polynucleotide with a region of another (see, Sedivy, J. M.,Bio-Technol.
6:1192-1196
(1988)). Sufficient homology for recombination requires only minimal homology
in
regions of the polynucleotide that flank the portion of the polynucleotide
that
undergoes recombination. The region may be of any length from a single base to
a
substantial fragment of a chromosome. Generally, a region having a length of
about
ten nucleotide residues is sufficient. Recombination is catalyzed by enzymes
which
are naturally present in both prokaryotic and eukaryotic cells.
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The polynucleotides of the present invention comprise isolated nucleic acid
molecules that can complement or augment a tetra or pentapeptide transport
deficiency of S. cerevisiae. The term "polynucleotide" encompasses nucleic
acid
molecules that encode a complete protein, as well as nucleic acid molecules
that
encode fragments of a complete protein. The polynucleotides may comprise the
wild-type allele (or a portion of such allele) of a functional peptide
transport gene, or
they may comprise mutated or disrupted (as by the insertion of additional DNA
or
RNA) alleles of such genes. As used, herein a "fragment" of a polynucleotide
is an
oligonucleotide whose nucleotide sequence is identical to that of a region of
the
polynucleotide, and whose length is greater than about 15 nucleotide residues,
and
preferably greater than about 20 nucleotide residues.
Functional complementation or augmentation
The isolation and cloning of polynucleotides that encode S. cerevisiae
enkephalin transport proteins permits the isolation of analogous,
complementary
polynucleotides from mammalian cells. The functional role of such isolated
polynucleotides can be readily determined by transforming them into the
above-described stable enkephalin transport-deficient yeast strain, and
evaluating
whether transformants acquire the capacity to transport intact enkephalin.
Thus, the
methods of the present invention permit the isolation of polynucleotides from
mammalian cells. Such polynucleotides are the equivalents of the preferred
polynucleotides of the present invention.
In one embodiment of the invention, mammalian protein encoding sequences
are cloned into suitable yeast expression vectors. Such vectors comprise
regulatory
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sequences such as promoters, termination signals and restriction endonuclease
recognition sequences to permit the introduction of heterologous sequences,
such as
the mammalian protein encoding sequences. Any suitable vector for expression
of
proteins in yeast known in the art may be used.
Examples of suitable yeast vectors include the yeast 2-micron circle, the
expression plasmids YEP13, YCP and,YRP, etc., or their derivatives. Such
plasmids
are well known in the art (Botstein, D., et al., Miami Wntr. Syrup. 19:265-274
(1982);
Broach, J. R., In: The Molecular Biology of the Yeast Saccharomyces: Life
Cycle and
Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 445-
470
(1981); Broach, J. R., Cell 28:203-204 (1982); Sherman, F. et al., In: Methods
in
Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1986)).
Yeast transformation may be accomplished by any means known in the art.
Selection of transformed yeast with functional complementation or augmentation
of
the deleted OPT1 may be on enkephalin containing media as the source of
leucine, for
example.
Yeast transformants that display functional complementation or augmentation
may be further analyzed by isolating the heterologous DNA and determining the
nucleic acid sequence. The deduced amino acid sequence may be analyzed for
identity and/or similarity by any of the available sequence analysis programs.
A score
of high identity or similarity, and predicted structural features of the
deduced amino
acid sequence (particularly the presence and number of transmembrane domains
and
the presence of the putative consensus sequence for OPT1 family members)
indicates
homology of the mammalian sequence to S. cerevisiae OPTlp.
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For complementation, OPTI knock-out yeast strains may be generated and
used. Alternatively, wild-type yeast may be used in augmentation studies. In
either
case, yeast cells are transformed with yeast expression vectors carrying
heterologous
mammalian DNA. Preferably, in augmentation studies, the expression of the
heterologous sequence in the yeast expression vector is under the control of a
strong
promoter, such as the ADH promoter.
Antifungal Compositions
Another embodiment of the mention comprises an antifungal composition
comprising at least one toxic derivative of enkephalin as an active
ingredient.
Another embodiment is a method of using the antifungal compositions comprising
applying the antifungal composition to a substrate to reduce or prevent fungal
growth.
There is a plethora of toxic moieties that may be employed in the antifungal
composition and method using the antifungal composition. Virtually any toxic
moiety
may be used that satisfies the following criteria: the toxic moiety or
moieties must be
able to be associated with the enkephalin peptide, and the moiety or moieties
must not
interfere with the uptake of the peptide into the cells.
Substantial evidence suggests that the uptake of toxic peptides is mediated by
peptide transport systems (McCarthy, P. J. et al.,Antimicrob. Agents
Chemother.
28:494-499 (1985); McCarthy, P. J. et al., J. Gen. Micro. 131:775-780 (1985);
Moneton, P. et al., J. Gen. Micro. 132:2147-2153 (1986); Yadan, J. C. et al.,
J.
Bacteriol. 160:884-888 (1984)); Payne, J. W. et al., FEMS Microbiol. Letts.
28:55-60
(1985); Mehta, R. J. et al., Antimicrob. Agents Chemother. 25:373-374 (1984)).
Since the polynucleotides of the present invention define the genetic loci
responsible
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for enkephalin transport in yeast, fungi would be take up toxic enkephalin
derivatives
by transport, and fungal growth would be prevented or reduced.
In this regard, the present invention provides a method for conjugating an
antimicrobial or antifungal agent or a pesticide to a peptide in order to
provide a more
effective treatment against fungal growth. In a similar manner, toxic peptide
derivatives maybe used as herbicides to eliminate fungal growth around plants
(particularly crops).
Examples of toxic peptide or peptidyl molecules that may be used include, but
are not limited to:
(A) metabolic toxins (such as the antifungal agent FMDP >N<sup>3</sup>
-(4-methoxyfumaroyl)-L-2,3 diaminopropanoic acid), toxic nucleotides (such as
halogenated nucleotides (e.g., 5-fluoroorotic acid), dideoxynucleotides,
mutagenic
nucleotide or nucleoside analogs, etc. (Kingsbury, W. D. et al., J. Med. Chem.
27:1447-1451 (1984); Andruszkiewicz, R. et al., J. Med. Chem.30:1715-1719
(1987);
Andruszkiewicz, R. et al., J. Med. Chem. 33:132-135 (1990); Andruszkiewicz, R.
et
al., J. Med.Chem. 33:2755-2759 (1990); Milewski, S. et al., J. Drugs Expt.
Clin. Res.
14:461-465 (1988));
(B) peptides that contain toxic amino acids (such as oxalysine,
fluorophenylalanine, ethionine, unusual D amino acids, etc.)(McCarthy, P. J.
et al.,
Antimicrob. Agents Chemother. 28:494-499 (1985); Marder, R. et al., J.
Bacteriol.
36:1174-1177(1978); Moneton, P. et al., J. Gen. Micro. 132:2147-2153 (1986);
Mehta, R. J. et al., Antimicrob. Agents Chemother.25:373-374 (1984); Bosrai,
M. et
al., J. Gen. Microbiol. 138:2353-2362 (1992));
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(C) toxic peptides and peptidyl molecules such as bacilysin (Milewski, S. et
al., Arch. Microbiol. 135:130-136 (1983);Moneton, P. et al., J. Gen.
Microbiol.
132:2147-2153 (1986); Kenig, M. et al. J. Gen. Microbiol. 94:37-45 (1976)),
polyoxins (especially polyoxin D) (Becker, J. M. et al., Antimicrob. Agents
Chemother. 23:926-929 (1983)), nikkomycins(especially nikkomycin Z) (Dahn, U.
et
al., Arch. Microbiol. 107:143-160 (1976)), and their analogs (Smith, H. A. et
al.,Antimicrob. Agents Chemother. 29:33-39 (1986); Naider, F. et al.,
Antimicrob.
Agents Chemother. 24:787-796 (1983);Krainer, E. et al., J. Med. Chem. 34:174-
180
(1991); Shenbagamurthi. P. et al., J. Med. Chem. 26:1518-1522 (1983);
Shenbagamurthi. P. et al., J. Med. Chem. 29:802-809 (1986); Khare, R. K. et
al., J.
Med. Chem. 83:650-656 (1988); Emmer, G. et al., J. Med. Chem. 28:278-281
(1985);
Decker, H. et al., J. Gen Microbiol. 137:1805-1813 (1991); Delzer, J.et al.,
J.
Antibiot. 37:80-82 (1984); all herein incorporated by reference).
In a preferred embodiment, the peptides of such conjugates will be
N-.alpha.-acetylated, since such modification facilitates theuptake of peptide
molecules.
In the method of reducing or preventing fungal growth, a composition
containing at least one toxic analogue of enkephalin as an active ingredient
are applied
to a substrate or plant in an amount suitable to prevent or reduce fungal
growth.
OPT1 Vectors
Vectors to allow the expression of OPT1 in plants comprise nucleic acid
molecules comprising the coding sequence of OPT1, regulatory sequences
suitable for
use and functional in plants (such as promoters, enhancers, termination
sequences and
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the like), and may include selectable marker genes as is well known in the art
(such as
antibiotic resistance genes, and the like). Such vectors may be used to
transform plant
cells to provide expression of OPTIp in plants.
In one embodiment of the invention, the polynucleotides will be operably
linked to regulatory sequences sufficient to permit the polynucleotide's
transcription.
Such polynucleotides may be incorporated into nucleic acid vectors that are
sufficient
to permit either the propagation or maintenance of the polynucleotide within a
host
cell. The nature of the regulatory elements will depend upon the host cell,
and the
desired manner of expressing the polynucleotide. Examples of suitable
regulatory
elements include constitutive or inducible prokaryotic promoters, such as the
.lambda.
pL or pR promoters, or other well-characterized promoters (e.g., lac, gal,
trp, ara, hut,
etc.). Other promoters which may be employed are the nos, ocs and CaMv
promoters.
Efficient plant promoters that may be used are over-producing plant promoters
such as
the small subunit (ss) of the ribulose 1, 5 biphosphate carboxylase from
soybean
(Berry-Lowe, et al., J. Molec. App. Gen. 1:483-498 ( 1982)) and the promoter
of the
chlorophyll a/b binding protein. These two promoters are known to be light
induced in
eukaryotic plant cells (see Genetic Engineering of Plants, An Agricultural
Perspective," Cashmore, A. (ed), Plenum, N.Y., pp. 29-38 (1983); Coruzzi, G.
et al.,
J. Biol. Chem. 258:1399 (1983); and Dunsmeier, P. et al., J. Molec. App. Gen.
2:285
(1983)). The 35S promoter is particularly preferred.
Preferred prokaryotic vectors include plasmids such as those capable of
replication in E. coli such as, for example, pBR322,Co1El, pSC101, pACYC 184,
.pi.VX. Such plasmids are, for example, disclosed by Maniatis, T., et al. (In:
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Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring
Harbor, N.Y. (1982)). Bacillus plasmids include pC194, pC221, pT127, etc. Such
plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the
Bacilli,
Academic Press, N.Y. (1982), pp. 307-329). Suitable Streptomyces plasmids
include
p1J101 (Kendall, K. J., et al., J. Bacteriol.169:4177-4183 (1987)), and
Streptomyces
bacteriophages such as .o slashed.C31 (Chater, K. F., et al., In: Sixth
International
Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary
(1986), pp. 45-54). Pseudomonas plasmids are reviewed by John, J. F., et al.
(Rev.
Infect. Dis. 8:693-704 (1986)), and Izaki, K. (Jpn. J. Bacteriol. 33:729-742
(1978)).
As indicated, the invention particularly contemplates providing the
polynucleotides of the present invention to plants, especially tobacco,
coffee, wheat
and other cereals, apple and other non-citrus fruit producers, and citrus
fruit crops.
Suitable plants include, for example, species from the genera Fragaria, Lotus,
Medicago, Onobrychis, Trifolium, Trigonefla, Vigna, Citrus, Linum, Geranium,
Manicot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, A tropa, Capsicum,
Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis,
Majorana,
Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis,
Nemesia, Pelargonium, Panicurn, Pennisetum, Ranunculus, Senecio, Salpiglossis,
Cucumis, Browallia, Glycine, Lolium, Zea, Triticum, Sorghum, Ipomoea,
Passiflora,
Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, Santalure, Allium, Lilium,
Narcissus, Ananas, Arachis, Phaseolus, Pisum and Datura.
In one embodiment, OPT1 polynucleotide is provided without promoters or
other regulatory elements, but under conditions sufficient to permit the
polynucleotide
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to recombine with and replace a region of the endogenous plant peptide
transport
gene. In an alternative embodiment, the polynucleotides will be administered
to the
plant operably linked to regulatory elements and/or vector elements.
Any of a variety of methods may be used to introduce the OPT1
polynucleotide of the present invention into a plant cell. The genetic
material can be
microinjected directly into the plant embryo cells or introduced by
electroporation as
described in Fromm et al.,"Expression of Genes Transformed into Monocot and
Dicot
Plant Cells by Electroporation," Proc. Nat'1. Acad. Sci. U.S.A.82:5824-28
(1985) or it
can be introduced by direct precipitation using polyethylene glycol as
described in
Paszkowski et aI.,EMBO J. 3:2717-22 (1984). In the case of monocotyledonous
plants, pollen may be transformed with total DNA or an appropriate functional
clone
providing resistance, and the pollen then used to produce progeny by sexual
reproduction.
The Ti plasmid of Agrobacterium tumefaciens provides a means for
introducing DNA into plant cells (Caplan, A., et al., Science815-821 (1983);
Schell, J.
et al., Bio/Technology, April 1983, pp. 175-1980; Fraley, R. T., et al., Proc.
Nat'l.
Acad. Sci. U.S.A. 80:4803 (1983); (Hooykass, P. J. J. et al., In: Molecular
Form and
Function of the Plant Genome, Vlotan-Doltan, L. et al. (eds.), Plenum Press,
N.Y., pp.
655-667 (1984); Horsch, R. B. et al., In: Current Communications in Molecular
Biology, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., pp. 13-19 (1988);
Horsch et al., Science 233:496-498 (1984); all herein incorporated by
reference). As
such, it provides a highly preferred method for introducing the
polynucleotides of the
present invention into plant cells
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Ti plasmids contain two regions essential for the production of transformed
cells. One of these, termed "transfer DNA" (TDNA), induces tumor formation.
The
other, termed "virulent region," is essential for the formation but not
maintenance of
tumors. It is possible to insert the polynucleotides of the present invention
into the T
DNA region without affecting its transfer function. By removing the tumor-
causing
genes so that they no longer interfere, the modified Ti plasmid can then be
used as a
vector for the transfer of the gene constructs of the invention into an
appropriate plant
cell. The polynucleotides of the present invention are preferably inserted
between the
terminal sequences that flank the T-DNA.
A particularly useful Ti plasmid vector is pGV3850, a non-oncogenic
derivative of the nopaline Ti plasmid C58 (Caplan, A., et al., Science 815-821
( 1983)). This vector utilizes the natural transfer properties of the Ti
plasmid. The
internal T DNA genes that determine the undifferentiated crown gall phenotype
have
been deleted and are replaced by any commonly used cloning vehicle (such as
pBR322). The cloning vehicle sequence contained between T DNA border regions
serves as a region of homology for recombination to reintroduce foreign DNA
cloned
in a derivative of the same cloning vehicle. Any polynucleotide of the present
invention cloned in such plasmid can thus be inserted into pGV3850 by a single
recombination of the homologous sequences. Antibiotic resistance markers can
be
added to the plasmid to select for the recombination event. The presence of
thenopaline synthase (nos) gene in pGV3850 facilitates the monitoring of the
transformation.
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The introduction of the Ti plasmid is typically accomplished by infecting a
wounded leaf of the plant with Agrobacterium tumefaciens bacteria that
contains the
plasmid. Under appropriate growth conditions, a ring of calli forms around the
wound(Hooykass, P. J. J. et al., In: Molecular Form and Function of the Plant
Genome, Vlotan-Doltan, L. et al. (eds.), Plenum Press, N.Y., pp. 655-667
(1984)).
The calli are then transferred to growth medium, allowed to form shoots, roots
and
develop further into plants.
The procedure can alternatively be performed in tissue culture. All plants
from
which protoplasts can be isolated and cultured to give whole regenerated
plants can be
transformed by the present invention so that whole plants are recovered which
contain
the introduced polynucleotide. There is an increasing body of evidence that
practically
all plants can be regenerated from cultured cells or tissues, including but
not limited to
all major cereal crop species, sugarcane, sugar beet, cotton, fruit and other
trees,
legumes and vegetables (Hooykass, P. J. J. et al., In: Molecular Form and
Function of
the Plant Genome, Vlotan-Doltan, L. et al. (eds.), Plenum Press, N.Y., pp. 655-
667
(1984); Horsch, R. B. et al., In: Current Communications in Molecular Biology,
Cold
Spring Harbor Press, Cold Spring Harbor, N.Y., pp. 13-19 (1988)). Methods for
regenerating plants from cultural protoplasts are described by Evans et al.
(Handbook
of Plant Cell Culture 1:124-176; by Davey, M. R., In: Protoplasts1983--Lecture
Proceedings, pp. 19-29, Birkhauser, Basel (1983)); Dale, P. J. (In:
Protoplasts
1983--Lecture Proceedings, pp. 31-41, Birkhauser, Basel (1983)); Binding, H.
(In:
Plant Protoplasts, CRC Press, Boca Raton, pp. 21-37 (1985)) and Cooking, E. C.
In:
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Molecular Form and Function of the Plant Genome, Vlotan-Doltan, L. et al.
(eds.),
Plenum Press, N.Y., pp.27-32 ( 1984)).
Regeneration efficiency varies from species to species of plants, but
generally
a suspension of transformed protoplasts containing the introduced gene
sequence is
formed. Embryo formation can then be induced from the protoplast suspensions,
to
the stage of ripening and germination as natural embryos. The culture media
will
generally contain various amino acids and hormones, such as auxin and
cytokinins. It
is also advantageous to add glutamic acid and proline to the medium,
especially for
such species as corn and alfalfa. Shoots and roots normally develop
simultaneously.
Efficient regeneration will depend on the medium, on the genotype, and on the
history
of the culture. If these three variables are controlled, then regeneration is
fully
reproducible and repeatable.
Other systems, such as cauliflower mosaic virus, CaMV (Hohn, B., et al., In
"Molecular Biology of Plant Tumors," Academic Press, New York, pp. 549-560;
and
Howell, U.S. Pat. No. 4,407,956) can also be used to introduce the OPT1
polynucleotide of the present invention into plant cells. In accordance with
such
methods, the entire CaMV viral DNA genome is inserted into apparent bacterial
plasmid thus creating a recombinant DNA molecule which can be propagated in
bacteria. After cloning, the recombinant plasmid is cleaved with restriction
enzymes
either at random or at unique sites in the viral portion of the recombinant
plasmid for
insertion of the polynucleotides of the present invention. The modified viral
portion of
the recombinant plasmid is then excised from the parent bacterial plasmid, and
used to
inoculate the plant cells or plants.
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After transformation of the plant cell or plant, the same may be selected by
aid
of an appropriate marker, such as antibiotic resistance, and then assessed to
determine
whether it contains the desired polynucleotide of the invention. The mature
plants,
grown from the transformed plant cells, can be selfed to produce an inbred
plant
whose seeds will contain the introduced polynucleotides of the present
invention.
These seeds can be grown to produce plants that exhibit any of a set of
desired
properties.
In one embodiment of the present invention, the exhibited property will be an
increased facility to transport peptides, particularly, enkephalin. In this
embodiment,
the OPT1 polynucleotide of the invention are provided to the plant or plant
cells along
with transcriptional regulatory sequences, such that an overexpression of the
plant's
peptide transport gene occurs. Such plants are desirable in that their
enhanced peptide
transport system can be used to facilitate the up take of peptide-associated
molecules.
The invention, therefore, also contemplates a method for growing plants,
particularly crops such as those mentioned above, by providing said crops with
the
OPTIp encoding polynucleotide to allow for expression of OPTIp in the plant
and
providing the plant with a growth enhancement amount of an enkephalin to
promote
growth of the plant.
In all the embodiments of the invention, the polynucleotide of OPT1 that is
suitable is at least the nucleotide sequence encoding the protein of SEQ ID
N0:2.
EXPERIMENTS
Growth on Leu-Enkephalin-- An experiment was designed to determine whether
members of the OPT family could transport leucine enkephalin (Leu-enkephalin;
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YGGFL) to satisfy an auxotrophic requirement for leucine. For this study, a
strain of
S. cerevisiae auxotrophic for methionine and leucine (BY4730) along with the
prototrophic parental strain (BY4700) were selected for use. Fig. 1 shows the
growth
of S. cerevisiae BY4730 expressing OPT family members. Cells were transformed
with pDB20 (empty vector), pCaOPTI (C. albicans OPT1) under its endogenous
promoter, pADH 194C (S. cerevisiae ORF 194C under the S. cerevisiae ADH
promoter), and pADHOPTl (S. cerevisiae OPT1 under the S. cerevisiae ADH
promoter). Cells were spotted onto proline medium supplemented with various
sources of leucine, as indicated on the figure, to meet auxotrophic
requirements and
were grown for 72 h at 30 °C. S. cerevisiae BY4730 transformed with the
vector
(pDB20) and transformants expressing three members of the OPT family were able
to
use either leucine or leucyl-leucine for growth as shown in Fig. 1. In
contrast, only
cells transformed with YJL212C, expressing OPT1 (pADHOPTl), could grow on
Leu-enkephalin as a sole source of leucine. The parental strain BY4700
transformed
with an empty vector (pDB20) or three members of the OPT family (pCaOPTI,
pADH 194C, or pADHOPTl ) grew well in the presence of Leu-enkephalin at all
concentrations (10-1000 ~,M), indicating this peptide was not toxic. Growth on
Leu-
enkephalin in cells expressing OPT 1 was concentration-dependent, with the
most
robust growth seen at the highest concentrations. In a similar experiment, it
was
determined that cells expressing OPT1 could grow on methionine enkephalin (Met-
enkephalin) as a sole source of methionine.
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Transport of Radiolabeled Leu-Enkephalin-- To further explore the possibility
that
Leu-enkephalin transport was carrier-mediated, transport was measured directly
using
radiolabeled Leu-enkephalin ([3H]YGGFL). Fig. 2 shows uptake of [3H]Leu-
enkephalin by S. cerevisiae BY4730 transformants. Part A shows uptake versus
time
at 30 °C (~) or 4 °C (o) for cells transformed with pADHOPTI and
at 30°C for the
empty vector pDB20 (X). The inset iri part A shows Leu-enkephalin uptake at
30°C
versus concentration of Leu-enkephalin for cells transformed with pADHOPT 1.
Part
B shows uptake versus pH for cells transformed with pADHOPT 1.
Leu-enkephalin was transported into cells expressing OPT1 (Fig. 2A) in a
time- and temperature-dependent manner. In contrast, cells transformed with
the
vector pDB20 did not accumulate enkephalin. The uptake of Leu-enkephalin was
pH-
dependent. Transport of the substrate was highest at pH 5.5 and declined
sharply as
the proton concentration was raised or lowered (Fig. 2B). This pH optimum is
similar
to those reported for the eukaryotic di- and tripeptide transport systems, as
well as that
for peptide transport in the prokaryote Lactococcus lactis. Treatment of cells
with the
metabolic uncouplers 2,4-dinitrophenol, CCCP, or sodium azide, all of which
deplete
intracellular ATP and collapse the proton gradient, or treatment with the
sulfhydryl
reagent pCMBS substantially reduced enkephalin uptake (Table I). These data
are
consistent with a carrier-mediated uptake system for Leu-enkephalin encoded by
OPT 1.
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Table I
Leu-enkephalin uptake in the presence of various compounds
The uptake ofLeu-enkephalin (250 ~M) was measured over a 12-min time course
in the presence of the compounds indicated. Each measurement was completed a
minimum of four times. The results were normalized to uptake after 12 min of
incubation
measured in the absence of any other compound (none, 100%) and are reported as
mean
~ standard deviation.
Compound Percent of control
None 100%
Leucine Enkephalin (YGGFL)a 12 ~
1
Methionine Enkephalin (YGGFM)a 25 ~
4%
Tyrosine a 95 ~
12%
Leu-Leu a 97 ~
12%
Gly-Gly-Phe a 99 ~
5%
Gly-Gly-Phe-Leu a 41 ~
8%
Lys-Leu-Gly-Leu a 31 ~
14%
MIF-1 (PLG-NHZ)a 95 ~
7%
Tyr-MIF-1 (YPLG-NHZ)a 7g ~
9%
Tyr-Gly-Gly-Phe-Leu-NHz a 71 ~
5%
DPDPE (Y-D-Pen-GF-D-Pen)a 69 ~
11
DADLE (Y-D-AGF-D-L)a 58 ~
5%
Sodium Azideb 21 ~
2%
2,4-Dinitrophenolb 17 ~
2%
CCCPb 3 8 ~
6%
pCMBSb 55 ~ 5%
a All competitors were at a final concentration of 2.5 mM and added
simultaneously with
[3H]Leu-enkephalin in the uptake medium.
b Cells were pre-incubated with sodium azide ( 1 mM), CCCP (0.1 mM) 2,4-
dinitrophenol
( 1 mM), or pCMBS (0.2 mM) for 30 min prior to addition of the uptake medium.
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Table I shows Leu-enkephalin uptake in the presence of various compounds. The
uptake of Leu-enkephalin (250 ~M) was measured over a 12-min time course in
the
presence of the compounds indicated. Each measurement was completed a minimum
of
four times. The results were normalized to uptake after 12 min of incubation
measured
in the absence of any other compound (none, 100%) and are reported as mean ~
standard
deviation.
Fig. 3 shows a chromatographic analysis of Leu-enkephalin. Arrows indicate the
RF values for tyrosine and intact Leu-enkephalin. Part A shows an analysis of
uptake
assay medium after 2-min incubation with BY4730 transformed with pADHOPTl.
Similar analysis of medium prior to incubation with cells produced identical
results. Part
B shows an analysis of material extracted from cells after 12-min incubation
interval.
As shown in Figure 3, the rate of Leu-enkephalin uptake remained relatively
constant over a 12-min time course, suggesting that the opioid does not remain
intact
upon entering the cell. Chromatographic analysis of radiolabeled material
extracted from
cells indicated that the enkephalin was degraded, with virtually all
radioactivity
associated with free tyrosine. In contrast, analysis of an aliquot of medium
from which
cells were removed after 12 min of incubation at 30 °C revealed that no
extracellular
hydrolysis of the peptide had occurred. All radioactivity was still associated
with intact
Leu-enkephalin. If it is assumed that translocation of the substrate, rather
than its
hydrolysis, is rate-limiting, then an apparent Km for transport can be
determined by
measuring the rate of transport as a function of substrate concentration.
Transformation
of these data give an apparent Km of 310 qM for the uptake of Leu-enkephalin
by
transporter (Fig. 2A, inset)
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From Table I, the transport protein encoded by OPT 1 has a strong preference
for
both Leu-enkephalin and Met-enkephalin and does not appear to transport amino
acids
or di- or tripeptides. Accumulation of Leu-enkephalin was not affected by the
presence
of tyrosine or the di- and tripeptides tested, suggesting that the OPTI
protein does not
recognize these compounds. The uptake of radiolabeled Leu-enkephalin decreased
by 75-
88% in the presence of a 10-fold molar excess of Met-enkephalin or Leu-
enkephalin,
respectively. The tetrapeptide Lys-Leu-Gly-Leu (KLGL), a known substrate for
other
oligopeptide transporters was also an effective competitor. The amidated form
of Leu-
enkephalin (Tyr-Gly-Gly-Phe-Leu-NHZ) showed weak inhibition of enkephalin
uptake
in yeast.
Inhibition of Uptake by Enkephalin Analogs-- The nonmetabolized pentapeptide
enkephalin analogues DADLE and DPDPE were somewhat effective competitors,
blocking 30-40% of the uptake (Table I). The amidated tetrapeptide Tyr-MIF-1
(Tyr-Pro-
Leu-Gly-NHZ), a substrate for the previously described PTS-1 whole brain Met-
enkephalin transport system was a poor competitor, reducing Leu-enkephalin
uptake by
only 20%. The tripeptide MIF-1 did not cause a significant reduction in the
uptake of
Leu-enkephalin, further emphasizing the preference of the OPT1 system for
tetra- and
pentapeptides.
Fig. 4 shows the effects of naloxone and naltrexone on the uptake of [3H]Leu-
enkephalin and [3H]leucyl-leucine. Part A shows the uptake of Leu-enkephalin
(250 ~M)
as measured over a 12-min time course in the presence of naloxone (black bars)
or
naltrexone (shaded bars) at the concentrations indicated. The results were
normalized to
uptake of Leu-enkephalin (control, open bar) measured in the absence of either
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compound and are reported as mean ~ standard deviation. Part B shows the
uptake of
leucyl-leucine ( 160 ~M) as measured over a 12-min time course in the presence
and
absence of naloxone (black bar) or naltrexone (shaded bar) at the
concentrations
indicated. Results were normalized to control and reported as described for
part A.
Naloxone and naltrexone antagonize the binding of enkephalin to the opioid
receptor. It was found that these compounds also inhibit the transport of Leu-
enkephalin
across Optlp (Fig. 4A). In a similar experiment, the presence of these
compounds did not
inhibit the transport of leucyl-leucine, a substrate for the di- and
tripeptide transport
system Ptr2p (Fig. 4B).
We have discovered a function of the previously unknown open reading frame
YJL212C in the yeast S. cerevisiae. This gene is OPTI (SEQ ID NO. 1). The
protein
encoded by OPTI (SEQ ID NO. 2) consists of 799 amino acids, and based on the
amino
acid sequence the predicted protein structure suggests an integral membrane
protein
containing 12-14 putative membrane-spanning domains. In addition, the protein
contains
several motifs unique to the OPT family, the largest of which consists of 10
invariable
residues (SPYXEVRXXVXX~~DDP) located before the first hydrophobic domain.
OPT1, like other members of the OPT family, encodes a functional oligopeptide
transporter.
Because Optlp exhibited all the molecular characteristics of an OPT family
member, it was hypothesized that this protein was an oligopeptide transporter,
even
though it was known that S. cerevisiae could not utilize any tetra- or
pentapeptides tested
to date to satisfy auxotrophic requirements under routine growth conditions.
To see
activity of Optl, it was necessary to express OPTl under the control of the
ADH
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promoter, a strong, constitutive promoter which would presumably result in
high
expression of the gene product. Northern blot analysis confirmed that OPT 1
was not
expressed at detectable levels under routine conditions of logarithmic growth.
These
results were independently confirmed by serial analysis of gene expression
(SAGE)
which revealed that OPT1 is only expressed at a low level (~1 copy per cell)
following
nocodazole arrest in the G2/M phase of the cell cycle. Additional analysis of
sporulating
yeast cells by DNA microarray analysis indicated that OPT1 was expressed
during the
late stages of sporulation. In light of these observations, OPTlgene
expression must be
ectopically induced under the control of a heterologous promoter to enable
study of
Optlp function in log phase cells.
The product of OPT1 is the oligopeptide transporter Optlp, which translocates
pentapeptides, including both Met- and Leu-enkephalin. In BY4730, a strain of
S.
cerevisiae auxotrophic for leucine and methionine, only cells expressing OPT 1
could
grow on Leu-enkephalin in the absence of exogenous leucine. This indicates
that
enkephalins are transported intact into the cell and then hydrolyzed. If
oligopeptides were
hydrolyzed by an extracellular protease prior to transport, then the isogenic
control strain
(BY4730 transformed with the empty vector pDB20), as well as yeast cells
transformed
with plasmids encoding other OPT family members (CaOPT l, YPR194C) should be
able
to utilize the hydrolysis products for growth. This was not the case.
Chromatographic
analysis supports conclusion. No evidence for degraded forms of Leu-enkephalin
could
be found in the extracellular medium. In addition, a large body of work exists
which
demonstrates that di- and tripeptides enter the cell intact and are then
rapidly hydrolyzed
by intracellular peptidases.
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Transport of Leu-enkephalin is pH- and temperature-dependent, suggesting that
this is a proton-coupled, energy-dependent process. These observations are
supported by
the sensitivity of the transporter to agents which disrupt the proton gradient
or deplete
intracellular ATP. Utilization of the transmembrane proton gradient to
energize active
transport has been demonstrated for the PTR family of di- and tripeptide
transporters.
Uptake of radiolabeled Leu-enkephalin was inhibited in the presence of excess
unlabeled
Met- or Leu-enkephalin; amidated Leu-enkephalin was an ineffective competitor.
Tyr-
MIF-1 is an amidated tetrapeptide with opiate and anti-opiate activity. This
peptide is a
substrate for the previously described blood-brain barrier PTS-1 enkephalin
transport
activity but, like the amidated form of authentic Leu-enkephalin, was not an
effective
competitor for yeast Optlp. This observation is consistent with the need for a
free
carboxyl terminus for substrate recognition by Optlp. Tetrapeptides were
effective
inhibitors, with Lys-Leu-Gly-Leu and des-Tyrl Leu-enkephalin (Gly-Gly-Phe-Leu)
eliminating over 50% ofradiolabeled enkephalin accumulation, suggesting that
an amino-
terminal tyrosine is not essential for substrate recognition. Neither the
tripeptide
enkephalin fragment Gly-Gly-Phe nor the dipeptide Leu-Leu could inhibit
uptake,
indicating that this system is distinct from Ptr2p and is selective for tetra-
and
pentapeptides. These data show that intact oligopeptides are gaining access to
the cell via
a carrier-mediated process, and the discrete carrier is the gene product of
OPTI. If
enkephalins were entering by a nonspecific mechanism such as simple diffusion
or
endocytosis, then all strains, not just those expressing OPT1, should be able
to utilize this
substrate.
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Several enkephalin antagonists were assayed in this study for their effect on
enkephalin transport across Optlp. DADLE and DPDPE are enzymatically stable
delta
opioid receptor antagonists that are pentapeptide mimetics. Previous reports
indicated
that DPDPE gained access to the brain by a saturable, carrier-mediated
mechanism in the
blood-brain barrier, which has yet to be defined. Interestingly, transport of
DPDPE was
not inhibited by Leu-enkephalin in those studies, suggesting either the
existence of
separate transport systems or a common system with different affinities for
these two
substrates. A recent report suggests that DPDPE crosses the blood-brain
barrier by a
phenylarsine oxide-sensitive pathway, suggesting a role for a saturable
endocytic
mechanism in the in vitro and in situ models studied. It was found that DPDPE
and
DADLE were weak competitors for Leu-enkephalin transport, indicating that
Optlp
interacts with the stable antagonists with differential affinities compared
with authentic
Leu-enkephalin.
Naloxone and naltrexone are synthetic opioid receptor antagonists classically
used
to reverse the effects of opiate overdose. Naltrexone is also used clinically
in the
treatment of alcoholism. Despite the fact that these compounds are similar in
structure
to morphine, rather than resembling a peptide, they were effective competitors
for Leu-
enkephalin transport. The effect appears to be specific for the Optlp
transporter because
the presence of the morphine analogs did not influence the activity of the
unrelated di-
and tripeptide transporter Ptr2p. The nature of the inhibition of Leu-
enkephalin transport
by naloxone and naltrexone is currently under investigation. Specifically, it
would be of
interest to determine whether these compounds are substrates for transport or
are
nonsubstrate competitors for Optlp.
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There is increasing evidence that opioids and their analogues enter the
central
nervous system by carrier-mediated transport across the blood-brain barrier.
Evidence
also exists to suggest that the clearance of the enkephalin analogue DPDPE
occurs by
saturable efflux from the brain and systemic elimination of intact DPDPE via
biliary
excretion. Furthermore, it is possible that neuronal re-uptake systems exist
for enkephalin
similar to the well studied transport systems for neurotransmitters such as
serotonin and
y-aminobutyric acid. Previously, none of the putative transporters for
enkephalin have
been cloned or characterized at a molecular level. The present invention
presents the first
evidence for a genetically defined eukaryotic transport protein, Optlp, which
recognizes
and translocates both Met- and Leu-enkephalin into an intact eukaryotic cell.
The
identification of this transporter in Saccharomyces may facilitate the
discovery of
mammalian homologues, thus providing greater insight into the process of pain
and its
mediation. These mammalian homologues may aid in transporting opiates across
the
blood-brain barrier, and mediation of the homologues could allow pain
mediation.
Similarly, the homologues may be helpful in substance abuse treatment or in
fording
competitors for opiate transport mechanism to aid such treatment.
EXAMPLES
Strains, Media, and Vectors-- BY4700 (Mata ura300) and BY4730 (Mata 1eu200
met1500 ura300) were grown routinely on YEPD medium (1% yeast extract, 2%
peptone, 2% glucose, 2% agar). Strains transformed with a plasmid were
cultured on
minimal medium lacking uracil (0.67% Difco yeast nitrogen base with ammonium
sulfate, without amino acids, 2% glucose, 0.2% casamino acids). For growth
assays, cells
were inoculated into medium lacking uracil and ammonium sulfate (0.67% Difco
yeast
CA 02364584 2001-08-31
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WO 00/52162 PCT/US00/05158
-33-
nitrogen base without amino acids and ammonium sulfate, 2% glucose)
supplemented
with 0.1% proline as anitrogen source, 228 ~M leucine, and 191 pM methionine
(proline
medium). The plasmids pADH212C and pADH 194C were created by polymerase chain
reaction amplification of the appropriate ORFs (YJL212C and YPR194C,
respectively)
and cloning the resultant products into the URA3/2p-based vector pDB20 such
that the
genes were under the control of an ADH promoter. The plasmid pCaOPTI consists
of a
3.8-kilobase genomic fragment from C. albicans which contains the CaOPT 1 gene
cloned
into pRS202, a URA3/2 ~-based plasmid. Plasmids were transformed into yeast by
the
method of Geitz, and transformants were selected by growth on minimal medium
lacking
uracil.
Growth and Uptake Assa,~-- Transformed cells were grown overnight to mid-
exponential
phase in proline medium. For growth assays, cells were harvested, washed, and
adjusted
to a final concentration of 2 X 10' cells/ml in water. Five microliters (= 1 X
105 cells) of
each sample was spotted onto proline medium plus 2% agar, supplemented with
amino
acids or peptides, as indicated in the text and Fig. 1. Plates were incubated
at 30 °C for
72 h and observed for growth. For uptake assays, cells were harvested and
washed with
2% glucose and adjusted to a final concentration of 2 X 108 cells/ml. The
uptake assay
was initiated by combining equal volumes of pre-warmed (30 °C) cells
and 2X uptake
assay mixture (2% glucose, 20 mM sodium citrate/potassium phosphate, pH 5.5,
500 pM
Leu-enkephalin (Sigma), 1 gCi/ml [3H]leucine enkephalin (50 Ci/mmol, American
Radiolabeled Chemicals), and incubating at 30 °C. For determination of
leucyl-leucine
accumulation, 320 pM L-leucyl-L-[3H]leucine ( 16 mM,10 mCi/mmol) was used in
place
of Leu-enkephalin. L-Leucyl-L-[3H]leucine was synthesized by standard solution-
phase
WO 00/52162 PCT/LTS00/05158
-34-
techniques. For assays done in the presence of competitors, the 2 X uptake
assay mixture
was supplemented with competitor (2X final concentration) prior to combining
with the
cells. A concentrated stock of carbomyl cyanide 3-chlorophenylhydrazone (CCCP)
(Sigma) was prepared in Me2S0; naloxone and naltrexone (Sigma) were dissolved
in
methanol. The compounds were diluted such that the solvent was present at a
final
concentration of 5% in the uptake medium. All other compounds were dissolved
in either
water or sodium citrate/potassium phosphate buffer (pH 5.5). At the
appropriate time,
aliquots (90 pl) were removed and washed by vacuum filtration with 4 X 1 ml
ice cold
water onto a membrane filter (HAWP, Millipore). The membranes were counted by
liquid scintillation spectrometry, and results were reported as nmol/mg dry
weight. Data
points reflect the mean and standard deviation of a minimum of four
independent
measurements.
ChromatographX-- Cells were incubated with uptake medium for 12 min,
harvested, and
washed four times with ice-cold water. The cell pellet was extracted by
boiling in 50%
methanol. The methanol extracts, along with control samples, were spotted onto
silica
plates and developed by ascending chromatography using butanol:glacial acetic
acid:water solvent system (9:1:2.5). The chromatograms were sprayed with
ninhydrin
(0.1% in 95% ethanol) to visualize the nonradioactive standards. Lanes
containing
radioactive samples were scraped in 0.8-cm intervals and counted for retained
radioactivity.
CA 02364584 2001-08-31
