Title: Gene encoding organic anion transporter
United States Patent: 6,432,631
Issued: August 13, 2002
Inventors: Cihlar; Tomas (San Mateo, CA)
Assignee: Gilead Sciences, Inc. (Foster City, CA)
Appl. No.: 330245
Filed: June 10, 1999
This invention is concerned with human organic anion transporter ("hOAT").
Isolated nucleic acid encoding hOAT is provided, along with isolated hOAT
polypeptide. hOAT nucleic acid and/or hOAT polypeptide are employed in
transgenic animals, recombinant cells, replicable vectors and analytical
procedures for identifying hOAT agonists or antagonists, assays for
identifying hOAT alleles and/or isotypes, screening tests for nephrotoxic or
neurologically active compounds, and determination of drug-drug interactions
within the kidney or brain.
DETAILED DESCRIPTION OF THE INVENTION
hOAT polypeptide is defined as a polypeptide sequence that is at least
about 85% homologous by amino acid sequence (ordinarily at least about
90%, and preferably at least about 95%).
An expressed sequence tag (EST) of approximately 200 bp having high
homology to a segment of hOAT is found in the GenBank EST entries under
accession no. R25797. Invention hOAT nucleic acids per se as defined
herein exclude any expressed sequence tag (EST) or other nucleic acid
sequences found in public databases on the filing date (such databases
being expressly incorporated by reference), including the sequence of
accession no. R25797 as well as the rat ROAT1 and OAT1, and mouse NKT,
sequences of the prior art. However, other inventive subject matter such
as isolated protein, methods for screening and the like as set forth above
do not (unless expressly stated to the contrary) exclude the use of the
R25797 sequence or its expression product.
"Homology" is defined as the percentage of residues in a candidate amino
acid sequence that is identical with the residues in the reference
sequence hOAT after aligning the two sequences and introducing gaps, if
necessary, to achieve the maximum percent homology. Methods and computer
programs for the alignment are well know in the art. One computer program
which may be used or adapted for purposes of determining whether a
candidate sequence falls within this definition is "Align 2", authored by
Genentech, Inc., which was filed with user documentation in the United
States Copyright Office, Washington, D.C. 20559, on Dec. 10, 1991.
"Isolated" hOAT nucleic acid is one that has been separated from its
environment as it is found in nature, i.e., from the genome in the case of
DNA or from cellular environment in the instance of RNA.
"Isolated" hOAT polypeptide is one that has been separated from its normal
cellular environment, and includes hOAT protein that is homogeneous by SDS-PAGE
using silver stain.
In calculating amino acid sequence homology the candidate and reference
sequences are aligned in the fashion that produces the maximum number of
aligned residues, with insertions and deletions of residues represented by
gaps in the aligned sequences. For example, a 120 residue polypeptide
containing a 100 residue reference sequence fragment fused at its
N-terminus to a 6 residue polyhistidine affinity tag, but with a single
substitution in the hOAT domain, is calculated to be 99% homologous to the
hOAT reference sequence since the sequence of the fragment corresponds
exactly to the maximally aligned hOAT reference sequence except for a
single residue substitution and the 6 residue N-terminal fusion. Thus, if
the alignment-maximizing comparison of the candidate and reference
sequences reveals an insertion (or deletion) of one or more amino acid
residues, then these residues are ignored for the purposes of making the
homology calculation. Applicant recognizes that this convention gives rise
to theoretical 100% homology between 2 differing sequences, but has chosen
to establish his own definition for the purposes of this filing.
Analysis of homology is based on any one or more of the sequences imputed
from the nucleic acid used to express the hOAT, the sequence of the
product as first produced in vitro, or the sequence after any
post-translational modification. Thus, if the reference and candidate
sequences are identical when expressed, but a glutamine residue is later
deaminated to glutamic acid, the first candidate is 100% homologous, but
the deaminated sequence is not.
For the purposes herein "hOAT activity" means any one or more of the
functions performed by hOAT in the human, including in particular the
transport of organic anions.
It is not necessary for a polypeptide to have anion transport activity in
order to fall under the definition of hOAT herein. For example, in some
embodiments hOAT polypeptides possess at least one immune epitope that is
capable of substantial cross-reaction with an antibody raised against
reference sequence hOAT, and thus are useful in immunoassays for hOAT, but
may possess mutations that render the polypeptide incapable of anion
The hOAT polypeptides of this invention comprise substitutions for,
deletions of, or insertions of any amino acid residue adjacent to any of
the reference sequence amino acid residue sites. Substitutional
hOATs are those in which at least one amino acid residue in the reference
sequence has been removed and a different amino acid inserted in its place
at the same position. One or more residues are substituted.
Alanine is a common substitution for any residue, and is commonly used in
alanine scanning to identify functional residues, but it is within the
scope of this invention to substitute other residues into the hOAT
reference sequence. The introduced residues generally are naturally
occurring amino acids, commonly G, A, Y, V, L, I, S, T, D, E, Q, C, M, N,
F, P, W, K, R or H (using conventional single letter code; EP 323,149).
Suitable residues also include hydroxyproline, beta-hydroxyaspartic acid,
gamma-carboxyglutamic acid, hydroxylysine or norleucine, to be employed as
alternatives to their namesakes.
These substitutions may be conservative, i.e., the substituting residue is
structurally or functionally similar to the substituted residue. Other
substitutions will be less conservative in that they constitute an
exchange between different structural or functional classes of residues.
For the purposes herein, these classes are as follows: 1. Electropositive:
R, K, H; 2. Electronegative: D, E; 3. Aliphatic: V, L, I, M; 4. Aromatic:
F, Y, W; 5. Small: A, S, T, G, P, C; 6. Charged: R, K, D, E, H; 7. Polar:
S, T, Q, N, Y, H, W; and 8. Small Hydrophilic: C, S, T. Intergroup
substitutions generally will have greater effects on protein function than
conservative (intraclass) substitutions. Thus, it is particularly within
the scope of this invention to introduce conservative substitutions into
hOAT and, if the results are not satisfactory, to introduce
non-conservative substitutions at the sites. Typically, however, proline,
glycine, and cysteine substitutions or insertions into the sequence are
not favored. An example of an expressed variant is a change at codon 498
from AGC to ATC, resulting in expression of isoleucine in place of serine.
Other variants are introduced into DNA encoding hOAT without resulting in
a change in protein sequence, e.g. from ATC to ATT at codon 453 or from
GGG to GGT at codon 491.
hOAT variants are readily identified by methods apparent to the ordinary
artisan. For example, sites shown by alanine scanning to influence
selected biological activity are subjected to saturation mutagenesis to
identify the optimal modification for the activity in question, e.g.
selectivity for transport of a particular anion.
hOATs representing combinations of sequence variants are within the scope
of this invention. 2, 3, 4, 5, or more substitutions, deletions or
insertions are introduced into hOAT as defined herein. Typically, a
deletion of a single residue will be accompanied by an insertion within 1
to about 3 residues of the deletion site. Generally, deletions of larger
domains unnecessary for anion transport activity need not be accompanied
by an insertion. The results of individual amino acid substitutions are
generally additive except when the residues interact with each other
directly or indirectly. They are readily screened using the same
procedures described in Sweet et al. or Sekine et al. (supra) in order to
identify those having the properties of reference sequence hOAT or the
desired modified properties.
Included within the scope of this invention are hOATs having one or more
amino acids inserted immediately adjacent to a hOAT amino acid at any
position in the reference sequence. Insertional hOATs generally will have
a polypeptide structure comprising the sequence NH2
-PP-A-(X)nl -B-PP-COOH, wherein X is the inserted residue(s) (which
may be the same or different), n1 is an integer (generally 1-30, typically
1 or 2), either A or B are the designated residue sites for insertion and
PP represents the remainder of the hOAT or a bond at the hOAT N or C
The invention includes fusions of hOAT and selected antibody recognition
sequences (heterologous polypeptides) for immunoaffinity purification of
hOAT from cell culture, fusions of hOAT sequences with affinity tags such
as FLAG or polyhistidine, and chimeric sequences (particularly fusions of
hOAT sequence fragments with fragments of other receptors of the
12-transmembrane spanning region class).
Also included within the scope of this invention are hOATs in which a
glycosylation site is introduced or removed from the reference sequence,
whether by substitution, insertion or deletion of one or more amino acid
residues. Such changes will result in the installation or removal of the
sequence NXS or NXT, where X can be any residue. Thus, asparagine can be
substituted for any residue located 2 residues N-terminal to serine or
threonine to introduce a glycosylation site. Alternatively, single
glycosylation can be omitted by substituting glycosylated asp with any
residue, deleting site-adjacent serine or threonine substituting any
residue into the glycosylation site to perturb the NXS or NST sequence.
Also included within the scope of this invention are deletional hOATs,
i.e., hOATs in which one or more amino acid residues of the reference
sequence have been removed at a designated site, whereby flanking residues
are now joined by a peptide bond in the ordinary fashion. It generally is
not preferred to delete P, C or G residues.
Typically, deletions or insertions are relatively small, on the order of 1
to 10 residues and generally no more than 2, although deletions or
insertions can be quite large if they are not in critical portions of the
reference sequence, or the additional sequence is to be removed at some
point during post-translational or post-recovery processing. The number of
residues that are deleted or inserted in part will depend upon whether or
not they are found in secondary structural components such as helices or
sheets (whereupon only 1 or, preferably 2 residues are inserted or
deleted), or are in less structurally confined domains such as loops,
where larger numbers of residues may be deleted or inserted without unduly
perturbing the structure of hOAT.
The hOATs of this invention may be subject to post-translational covalent
modification, e.g. deamidation of asparagine or glutamine, or oxidation of
cysteine residues. Glycosylation can be variant or absent depending upon
the host cell used to express the variant or absent such modifications are
included within the scope of this invention. If hOAT is glycosylated in
recombinant cell culture, it preferably is glycosylated with carbohydrates
characteristic of mammalian cells, although it also may bear fungal (such
as yeast) glycosylation patterns. Glycosylation is acceptable which is
characteristic of expression of hOAT from one or more of fibroblast,
kidney, brain, lung, skin, neural liver or bone marrow cells or cell
lines, or from any mammalian cell line such as CHO or embryonic kidney
Naturally occurring human alleles are included within the scope of this
invention. The readily are identified by obtaining nucleic acid samples
from individuals in a population, sequencing hOAT from such individuals
and determining residues at which variation is found. Once each variation
is identified, it is straight-forward to determine the frequency of the
putative allele in other individuals by PCR using primers specific for the
domain in question, or such other methods as are conventional in the field
for determining proportions of alleles in human populations.
Claim 1 of 5 Claims
What is claimed is:
1. A method for identifying an inhibitor of the hOAT mediated transport
process comprising (a) providing a recombinant cell culture expressing
biologically active hOAT having the amino acid sequence of SEQ ID No. 2,
and providing a covalently modified form of a nucleotide phosphonate
analogue a candidate inhibitor and (b) determining whether or not the
analogue is transported by hOAT in the recombinant cell culture.
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