# Patent

## Title
Compositions of the SOC/CRAC calcium channel protein family

## Abstract

Nucleic acids encoding SOC/CRAC calcium channel polypeptides, including fragments and biologically functional variants thereof and encoded polypeptides are provided. The nucleic acids and polypeptides disclosed herein are useful as therapeutic and diagnostic agents. Agents that selectively bind to the foregoing polypeptides and genes also are provided.

## Background
RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. §120 or 35 U.S.C. §365(c) of PCT International application PCT/US99/29996 designating the United States of America, and filed Dec. 20, 1999, of which this application is a national stage filing under 35 U.S.C. §371, was published under PCT Article 21(2) in English.
Application number PCT/US99/29996 claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application 60/114,220, filed Dec. 30, 1998; 60/120,018, filed Jan. 29, 1999; and 60/140,415, filed Jun. 22, 1999.


FIELD OF THE INVENTION
This invention relates to nucleic acids coding for a novel family of calcium channel polypeptides, the encoded polypeptides, unique fragments of the foregoing, and methods of making and using same.
BACKGROUND OF THE INVENTION
Calcium channels are membrane-spanning, multi-subunit proteins that facilitate the controlled transport (“flux”) of Ca2+ ions into and out of cells. Cells throughout the animal kingdom, and at least some bacterial, fungal and plant cells, possess one or more types of calcium channels. In general, “excitable” cells, such as neurons of the central nervous system, peripheral nerve cells, and muscle cells, including those of skeletal muscles, cardiac muscles, and venous and arterial smooth muscles, possess voltage-dependent calcium channels. In a voltage-dependent calcium channel, the transport of Ca2+ ions into and out of the cells requires a certain minimal level of depolarization (the difference in potential between the inside of the cell bearing the channel and the extracellular environment) with the rate of Ca2+ cell flux dependent on the difference in potential. In “non-excitable” cells, calcium influx is thought to occur predominantly in response to stimuli which cause the release of calcium from intracellular stores. This process, termed store operated calcium influx, is not well understood.
Characterization of a particular type of calcium channel by analysis of whole cells is complicated by the presence of mixed populations of different types of calcium channels in the majority of cells. Although single-channel recording methods can be used to examine individual calcium channels, such analysis does not reveal information related to the molecular structure or biochemical composition of the channel. Furthermore, in this type of analysis, the channel is isolated from other cellular constituents that might be important for the channel's natural functions and pharmacological interactions. To study the calcium channel structure-function relationship, large amounts of pure channel protein are needed. However, acquiring large amounts of pure protein is difficult in view of the complex nature of these multisubunit proteins, the varying concentrations of calcium channel proteins in tissue sources, the presence of mixed populations of calcium channel proteins in tissues, and the modifications of the native protein that can occur during the isolation procedure.
SUMMARY OF THE INVENTION
The invention is based on the identification of a novel family of calcium channel polypeptides and the molecular cloning and partial characterization of a novel member of this family that is expressed predominantly in human hematopoietic cells, liver, and kidney. This newly identified family of calcium channel polypeptides is designated, “SOC” or “CRAC” or “ICRAC”, for Store Operated Channels or Calcium Release Activated Channels. Although not wishing to be bound to any particular theory or mechanism, it is believed that the SOC/CRAC calcium channel polypeptides are transmembrane polypeptides that modulate Ca2+ flux “into” and “out of” a cell, for example, in certain instances they may be activated upon depletion of Ca2+ from intracellular calcium stores, allowing Ca2+ influx into the cell. Accordingly, the compositions disclosed herein are believed to be useful for modulating calcium transport into and out of such intracellular stores and for the treatment of disorders that are characterized by aberrant calcium transport into and out of such intracellular stores. In particular, we believe that the SOC/CRAC calcium channel polypeptides disclosed herein play an important role in the influx of extracellular calcium by mediating the refilling of intracellular calcium stores following their depletion. Accordingly, we believe that the compositions for expressing functional SOC/CRAC calcium channel polypeptides in cells, as disclosed herein, are useful for treating patients having conditions that are characterized by reduced extracellular calcium influx into their SOC/CRAC-expressing cells. Additionally, the compositions of the invention are useful for delivering therapeutic and/or imaging agents to cells which preferentially express SOC/CRAC calcium channel polypeptides and, in particular, for delivering such agents to hematopoietic cells, liver, heart, spleen, and kidney to modulate proliferation and growth of these cells. Moreover, in view of the importance of cellular calcium levels to cell viability, we believe that SOC-2/CRAC-1, SOC-3/CRAC-2, and SOC-4/CRAC-3 as disclosed herein, and/or other members of the SOC/CRAC family of calcium channel polypeptides, represent an ideal target for designing and/or identifying (e.g., from molecular libraries) small molecule inhibitors that block lymphocyte proliferation, as well as other binding agents that selectively bind to SOC/CRAC polypeptides to which drugs or toxins can be conjugated for delivery to SOC/CRAC polypeptide expressing cells.
The invention is based, in part, on the molecular cloning and sequence analysis of the novel SOC/CRAC calcium channel molecules disclosed herein (also referred to as a “SOC-2/CRAC-1 molecule,” a “SOC-3/CRAC-2 molecule,” and/or “SOC-4/CRAC-3 molecule”) that are predominantly expressed in human hematopoietic cells, liver, spleen, heart, and kidney (SOC-2/CRAC-1), kidney and colon (SOC-3/CRAC-2), and prostate (SOC-4/CRAC-3 molecule). As used herein, a “SOC/CRAC molecule” embraces a “SOC/CRAC calcium channel nucleic acid” (or “SOC/CRAC nucleic acid”) and a “SOC/CRAC calcium channel polypeptide” (or “SOC/CRAC polypeptide”). Homologs and alleles also are embraced within the meaning of a SOC/CRAC calcium channel molecule.
According to one aspect of the invention, isolated SOC/CRAC nucleic acids which code for one or more member(s) of the SOC/CRAC family of calcium channel polypeptides or unique fragments thereof are provided. The isolated nucleic acids refer to one or more of the following:
(a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31, and which code for a SOC/CRAC polypeptide;
(b) deletions, additions and substitutions of (a) which code for a respective SOC/CRAC polypeptide;
(c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code, and
(d) complements of (a), (b) or (c).
The invention in another aspect provides an isolated nucleic acid molecule selected from the group consisting of (a) a unique fragment of a nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, and SEQ ID NO:31, (b) complements of (a), provided that the unique fragment includes a sequence of contiguous nucleotides which is not identical to any sequence selected from a sequence group consisting of (1) sequences having the SEQ ID NOs or GenBank accession numbers of Table I, (2) complements of (1), and (3) fragments of (1) and (2).
According to yet another aspect of the invention, isolated SOC/CRAC polypeptides are provided. The isolated SOC/CRAC polypeptide molecules are encoded by one or more SOC/CRAC nucleic acid molecules of the invention. Preferably, the SOC/CRAC polypeptide contains one or more polypeptides selected from the group consisting of the polypeptides having SEQ ID NOs 2, 4, 6, 8, 24, 26, 28, 30, and 32. In other embodiments, the isolated polypeptide may be a fragment or variant of the foregoing SOC/CRAC polypeptide molecules of sufficient length to represent a sequence unique within the human genome, and identifying with a polypeptide that functions as a calcium channel, provided that the fragment excludes a sequence of contiguous amino acids identified in Table II, and/or excludes a sequence of contiguous amino acids encoded for by a nucleic acid sequence identified in Table I. In another embodiment, immunogenic fragments of the polypeptide molecules described above are provided.
According to another aspect of the invention, isolated SOC/CRAC binding agents (e.g., polypeptides) are provided which selectively bind to a SOC/CRAC molecule (e.g., a SOC/CRAC polypeptide encoded by the isolated nucleic acid molecules of the invention). Preferably, the isolated binding agents selectively bind to a polypeptide which comprises the sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32, or unique fragments thereof. In the preferred embodiments, the isolated binding polypeptides include antibodies and fragments of antibodies (e.g., Fab, F(ab)2, Fd and antibody fragments which include a CDR3 region which binds selectively to a SOC/CRAC polypeptide). Preferably, the antibodies for human therapeutic applications are human antibodies.
According to another aspect of the invention, a pharmaceutical composition containing a pharmaceutically effective amount of an isolated SOC/CRAC nucleic acid, an isolated SOC/CRAC polypeptide, or an isolated SOC/CRAC binding polypeptide in a pharmaceutically acceptable carrier also is provided. The pharmaceutical compositions are useful in accordance with therapeutic methods disclosed herein.
According to yet another aspect of the invention, a method for isolating a SOC/CRAC molecule is provided. The method involves:
a) contacting a SOC/CRAC nucleic acid or a SOC/CRAC binding polypeptide with a sample that is believed to contain one or more SOC/CRAC molecules, under conditions to form a complex of the SOC/CRAC nucleic acid or the SOC/CRAC binding polypeptide and the SOC/CRAC molecule;
b) detecting the presence of the complex;
c) isolating the SOC/CRAC molecule from the complex; and
d) determining whether the isolated SOC/CRAC molecule has SOC/CRAC calcium channel activity. As used herein “SOC/CRAC calcium channel activity” refers to the transport of Ca2+ into and out of intracellular stores that is mediated by a SOC/CRAC polypeptide. In general, the SOC/CRAC calcium channel activity is initiated by a reduction or depletion of intracellular calcium stores.
In certain embodiments, the SOC/CRAC nucleic acid is a SOC-2/CRAC-1 nucleic acid (e.g., a nucleic acid having SEQ ID NO:27, or complements thereof); in certain other embodiments, the SOC/CRAC nucleic acid is a SOC-3/CRAC-2 nucleic acid (e.g., a nucleic acid having SEQ ID NO:29, or complements thereof); in further embodiments, the SOC/CRAC nucleic acid is a SOC-4/CRAC-3 nucleic acid (e.g., a nucleic acid having SEQ ID NO:31, or complements thereof). In yet other embodiments, the SOC/CRAC polypeptide is a SOC-2/CRAC-1 binding polypeptide (e.g., an antibody that selectively binds to a SOC-2/CRAC-1 polypeptide). In yet further embodiments, the SOC/CRAC polypeptide is a SOC-3/CRAC-2 binding polypeptide (e.g., an antibody that selectively binds to a SOC-3/CRAC-2 polypeptide). In some embodiments, the SOC/CRAC polypeptide is a SOC-4/CRAC-3 binding polypeptide (e.g., an antibody that selectively binds to a SOC-4/CRAC-3 polypeptide). In the preferred embodiments, the isolated binding polypeptides include antibodies and fragments of antibodies (e.g., Fab, F(ab)2, Fd and antibody fragments which include a CDR3 region which binds selectively to a SOC-2/CRAC-1, to a SOC-3/CRAC-2, and/or to a SOC-4/CRAC-3 polypeptide). Preferably the isolated binding polypeptides or other binding agents selectively bind to a single SOC/CRAC molecule, i.e., are capable of distinguishing between different members of the SOC/CRAC family. Accordingly, one or more SOC/CRAC binding agents can be contained in a single composition (e.g., a pharmaceutical composition) to identify multiple SOC/CRAC molecules in vivo or in vitro.
According to yet another aspect of the invention, a method for identifying agents useful in the modulation of SOC/CRAC calcium channel activity is provided. The method involves:
a) contacting a SOC/CRAC polypeptide with a candidate agent suspected of modulating SOC/CRAC calcium channel activity, under conditions sufficient to allow the candidate agent to interact selectively with (e.g. bind to) the SOC/CRAC polypeptide;
b) detecting a Ca2+ concentration of step (b) associated with the SOC/CRAC calcium channel activity of the SOC/CRAC polypeptide in the presence of the candidate agent; and
c) comparing the Ca2+ concentration of step (b) with a control Ca2+ concentration of a SOC/CRAC polypeptide in the absence of the candidate agent to determine whether the candidate agent modulates (increases or decreases) SOC/CRAC calcium channel activity.
According to another aspect of the invention, a method for identifying agents useful in the modulation of a SOC/CRAC polypeptide kinase activity is provided. The method involves:
a) contacting a SOC/CRAC polypeptide with kinase activity with a candidate agent suspected of modulating SOC/CRAC kinase activity, under conditions sufficient to allow the candidate agent to interact with the SOC/CRAC polypeptide and modulate its kinase activity;
b) detecting a kinase activity associated with the SOC/CRAC polypeptide in the presence of the candidate agent; and
c) comparing the kinase activity of step (b) with a control kinase activity of a SOC/CRAC polypeptide in the absence of the candidate agent to determine whether the candidate agent modulates (increases or decreases) SOC/CRAC kinase activity. In some embodiments the SOC/CRAC polypeptide comprises amino acids 999–1180 of the SOC-2/CRAC-1 polypeptide (SEQ ID NO:24), or a fragment thereof that retains the kinase activity.
According to yet another aspect of the invention, a method for determining the level of expression of a SOC/CRAC polypeptide in a subject is provided. The method involves:
a) measuring the expression of a SOC/CRAC polypeptide in a test sample, and
b) comparing the measured expression of the SOC/CRAC polypeptide in the test sample to the expression of a SOC/CRAC polypeptide in a control containing a known level of expression to determine the level of SOC/CRAC expression in the subject. Expression is defined as SOC/CRAC mRNA expression or SOC/CRAC polypeptide expression. Various methods can be used to measure expression. The preferred embodiments of the invention utilize PCR and Northern blotting for measuring mRNA expression, and monoclonal or polyclonal SOC/CRAC antisera as reagents for measuring SOC/CRAC polypeptide expression. In preferred embodiments, the SOC/CRAC molecule (nucleic acid and/or polypeptide) is SOC-2/CRAC-1. In other preferred embodiments, the SOC/CRAC molecule is SOC-3/CRAC-2. In yet further preferred embodiments, the SOC/CRAC molecule is SOC-4/CRAC-3. In certain embodiments, the test samples include biopsy samples and biological fluids such as blood. The method is useful, e.g., for assessing the presence or absence or stage of a proliferative disorder in a subject.
The invention also contemplates kits comprising a package including assays for SOC/CRAC epitopes, SOC/CRAC nucleic acids, and instructions, and optionally related materials such as controls, for example, a number, color chart, or an epitope of the expression product of the foregoing isolated nucleic acid molecules of the invention for comparing, for example, the level of SOC/CRAC polypeptides or SOC/CRAC nucleic acid forms (wild-type or mutant) in a test sample to the level in a control sample having a known amount of a SOC/CRAC nucleic acid or SOC/CRAC polypeptide. This comparison can be used to assess in a subject a risk of developing a cancer or the progression of a cancer. The kits may also include assays for other known genes, and expression products thereof, associated with, for example, proliferative disorders (e.g., BRCA, p53, etc.). In a preferred embodiment, the kit comprises a package containing: (a) a binding agent that selectively binds to an isolated nucleic acid of the invention or an expression product thereof to obtain a measured test value, (b) a control containing a known amount of a SOC/CRAC nucleic acid or a SOC/CRAC polypeptide to obtain a measured control value, and (c) instructions for comparing the measured test value to the measured control value to determine the amount of SOC/CRAC nucleic acid or expression product thereof in a sample.
The invention provides isolated nucleic acid molecules, unique fragments thereof, expression vectors containing the foregoing, and host cells containing the foregoing. The invention also provides isolated binding polypeptides and binding agents which bind such polypeptides, including antibodies, and pharmaceutical compositions containing any of the compositions of the invention. The foregoing can be used, inter alia, in the diagnosis or treatment of conditions characterized by the aberrant expression levels and/or the presence of mutant forms of a SOC/CRAC nucleic acid or polypeptide. The invention also provides methods for identifying agents that alter the function of the SOC/CRAC polypeptide.
These and other aspects of the invention, as well as various advantages and utilities, will be more apparent with reference to the detailed description of the preferred embodiments.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO:1 is a partial nucleotide sequence of the human SOC-2/CRAC-1 cDNA.
SEQ ID NO:2 is the predicted amino acid sequence of the translation product of human SOC-2/CRAC-1 cDNA (SEQ ID NO:1).
SEQ ID NO:3 is a partial nucleotide sequence of the human SOC-2/CRAC-1 cDNA.
SEQ ID NO:4 is the predicted amino acid sequence of the translation product of human SOC-2/CRAC-1 cDNA (SEQ ID NO:3).
SEQ ID NO:5 is a partial nucleotide sequence of the human SOC-2/CRAC-1 cDNA.
SEQ ID NO:6 is the predicted amino acid sequence of the translation product of human SOC-2/CRAC-1 cDNA (SEQ ID NO:5).
SEQ ID NO:7 is a partial nucleotide sequence of the mouse homologue (mSOC-2/CRAC-1) of the human SOC-2/CRAC-1 cDNA.
SEQ ID NO:8 is the predicted amino acid sequence of the translation product of the mSOC-2/CRAC-1 cDNA (SEQ ID NO:7).
SEQ ID NO:9 is the nucleotide sequence of the mouse MLSN-1 (SOC-1) cDNA.
SEQ ID NO:10 is the predicted amino acid sequence of the translation product of the mouse MLSN-1 (SOC-1) cDNA (SEQ ID NO:9).
SEQ ID NO:11 is the nucleotide sequence of a human calcium channel cDNA with GenBank Acc. no.: AB001535.
SEQ ID NO:12 is the predicted amino acid sequence of the translation product of the human calcium channel cDNA with GenBank Acc. no.: AB001535 (SEQ ID NO:11).
SEQ ID NO:13 is the amino acid sequence of a C. Elegans polypeptide at the c05c12.3 locus.
SEQ ID NO:14 is the amino acid sequence of a C. Elegans polypeptide at the F54D1 locus.
SEQ ID NO:15 is the amino acid sequence of a C. Elegans polypeptide at the t01H8 locus.
SEQ ID NO:16 is the nucleotide sequence of a mouse kidney cDNA with GenBank Acc. no.: A1226731.
SEQ ID NO:17 is the predicted amino acid sequence of the translation product of the mouse kidney cDNA with GenBank Acc. no.: AI226731 (SEQ ID NO:16).
SEQ ID NO:18 is the nucleotide sequence of a human brain cDNA with GenBank Acc. no.: H18835.
SEQ ID NO:19 is the predicted amino acid sequence of the translation product of the human brain cDNA with GenBank Acc. no.: H18835 (SEQ ID NO:18).
SEQ ID NO:20 is the nucleotide sequence of the human EST with GenBank Acc. no.: AA419592.
SEQ ID NO:21 is the nucleotide sequence of the human EST with GenBank Acc. no.: AA419407.
SEQ ID NO:22 is the nucleotide sequence of the mouse EST with GenBank Acc. no.: AI098310.
SEQ ID NO:23 is a partial nucleotide sequence of the human SOC-2/CRAC-1 cDNA that contains the SOC-2/CRAC-1 sequences of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.
SEQ ID NO:24 is the predicted amino acid sequence of the translation product of human SOC-2/CRAC-1 cDNA (SEQ ID NO:23).
SEQ ID NO:25 is a partial nucleotide sequence of the human SOC-3/CRAC-2 cDNA.
SEQ ID NO:26 is the predicted amino acid sequence of the translation product of human SOC-3/CRAC-2 cDNA (SEQ ID NO:25).
SEQ ID NO:27 is the full nucleotide sequence of the human SOC-2/CRAC-1 cDNA.
SEQ ID NO:28 is the predicted amino acid sequence of the translation product of human SOC-2/CRAC-1 cDNA (SEQ ID NO:27).
SEQ ID NO:29 is the full nucleotide sequence of the human SOC-3/CRAC-2 cDNA.
SEQ ID NO:30 is the predicted amino acid sequence of the translation product of human SOC-3/CRAC-2 cDNA (SEQ ID NO:29).
SEQ ID NO:31 is the full nucleotide sequence of the human SOC-4/CRAC-3 cDNA.
SEQ ID NO:32 is the predicted amino acid sequence of the translation product of human SOC-4/CRAC-3 cDNA (SEQ ID NO:31).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic depicting the intron/exon organization of the chicken SOC-2/CRAC-1 genomic sequence, as well as the putative transmembrane (TM) domains, and the targeting constructs utilized in the knockout experiments.



DETAILED DESCRIPTION OF THE INVENTION
One aspect of the invention involves the partial cloning of cDNAs encoding members of a novel family of calcium channel polypeptides, referred to herein as “SOC/CRAC” (designated “SOC” or “CRAC” or “ICRAC”, for Store Operated Channels or Calcium Release Activated Channels, or CECH). Although not intending to be bound to any particular mechanism or theory, we believe that a SOC/CRAC family member is a transmembrane calcium channel that modulates Ca2+ flux “into” and “out of” a cell; in certain instances it may be activated upon depletion of Ca2+ from intracellular calcium stores, allowing Ca2+ influx into the cell.
The first three isolated SOC/CRAC members disclosed herein, define a new family of calcium channels which is distinct from previously described calcium channels, such as voltage gated calcium channels, ryanodine receptor/inositol-1,4,5-triphosphate receptor channels, and Transient Receptor Potential (TRP) channels. The SOC/CRAC family of calcium channels exhibits high selectivity (with a PCa/PNa ratio near 1000), a unitary conductance below the detection level of the patch clamp method (the conductance estimated at approximately 0.2 picosiemens), and are subject to inhibition by high intracellular calcium levels. Although not intending to be bound to any particular mechanism or theory, we believe that SOC/CRAC calcium channels are responsible for the majority of, for example, calcium entry which occurs when intracellular calcium stores are depleted, and that SOC/CRAC currents are important for initiating various types of calcium-dependent processes. Thus, we believe that SOC/CRAC calcium channels play an important role in cellular calcium homeostasis by, e.g., modulating the supply of calcium to refill intracellular stores when depleted.
The isolated full-length sequence of a representative, first member of the SOC/CRAC family, human SOC/CRAC nucleic acid (cDNA), SOC-2/CRAC-1, is represented as the nucleic acid of SEQ ID NO:27. This nucleic acid sequence codes for the SOC-2/CRAC-1 polypeptide with the predicted amino acid sequence disclosed herein as SEQ ID NO:28. A. homologous mouse cDNA sequence (>90% identity to the human at the nucleotide level) is represented as the nucleic acid of SEQ ID NO:7, and codes for a unique fragment of a mouse SOC-2/CRAC-1 polypeptide having the predicted, partial amino acid sequence represented as SEQ ID NO:8. Analysis of the SOC-2/CRAC-1 partial sequence by comparison to nucleic acid and protein databases show that SOC-2/CRAC-1 shares a limited homology to mouse MLSN-1 (SOC-1, SEQ ID NOs: 9 and 10). Limited homology is also shared between SOC-2/CRAC-1 and three C. Elegans polypeptides (SEQ ID NOs:13, 14, and 15). We further believe that SOC-2/CRAC-1 plays a role in the regulation of cellular Ca2+ fluxing and, in particular, lymphocyte Ca2+ fluxing.
A second member of the human SOC/CRAC family of calcium channels, SOC-3/CRAC-2, is represented as the nucleic acid of SEQ ID NO:29, and codes for the human SOC-3/CRAC-2 polypeptide having the predicted amino acid sequence represented as SEQ ID NO:30 (this molecule may also be referred to as CECH2). SOC-3/CRAC-2 is predominantly expressed in human hematopoietic cells (including peripheral blood lymphocytes, liver, bone marrow, spleen, thymus, lymph nodes, heart, and kidney. Expression can also be detected (at lesser levels) in brain, skeletal muscle colon, small intestine, placenta, lung, and cells (cell lines) such as HL-60, HeLa, K562, MOLT-4, SW480, A459, and G361.
A third member of the human SOC/CRAC family of calcium channels, SOC-4/CRAC-3, is represented as the nucleic acid of SEQ ID NO:31, and codes for the human SOC-4/CRAC-3 polypeptide having the predicted amino acid sequence represented as SEQ ID NO:32 (this molecule may also be referred to as CECH6). It specifically expressed in the prostate gland/cells.
As used herein, a SOC/CRAC calcium channel nucleic acid (also referred to herein as a “SOC/CRAC nucleic acid” refers to a nucleic acid molecule which: (1) hybridizes under stringent conditions to one or more of the nucleic acids having the sequences of SEQ ID NOs 7, 27, 29, and/or 31 (sequences of the mouse and human SOC-2/CRAC-1, human SOC-3/CRAC-2, and human SOC-4/CRAC-3 nucleic acids), and (2) codes for a SOC-2/CRAC-1, a SOC-3/CRAC-2 or a SOC-4/CRAC-3 calcium channel polypeptide, respectively, or unique fragments of said SOC-2/CRAC-1, SOC-3/CRAC-2, or SOC-4/CRAC-3 polypeptide.
As used herein, a SOC/CRAC calcium channel polypeptide (also referred to herein as a “SOC/CRAC polypeptide”) refers to a polypeptide that is coded for by a SOC-2/CRAC-1, a SOC-3/CRAC-2, and/or a SOC-4/CRAC-3 nucleic acid. Preferably, the above-identified SOC/CRAC polypeptides mediate transport of calcium into and out of a cell.
SOC/CRAC polypeptides also are useful as immunogenic molecules for the generation of binding polypeptides (e.g., antibodies) which bind selectively to SOC/CRAC (e.g., SOC-2/CRAC-1, SOC-3/CRAC-2, and/or SOC-4/CRAC-3) polypeptides. Such antibodies can be used in diagnostic assays to identify and/or quantify the presence of a SOC/CRAC polypeptide in a sample, such as a biological fluid or biopsy sample. SOC/CRAC polypeptides further embrace functionally equivalent fragments, variants, and analogs of the preferred SOC/CRAC polypeptides, provided that the fragments, variants, and analogs also are useful in mediating calcium transport into and out of intracellular calcium stores.
As used herein, “SOC/CRAC calcium channel activity” refers to Ca2+ transport (“Ca2+ fluxing”) across the plasma membrane that is mediated by a SOC/CRAC calcium channel polypeptide. The SOC/CRAC calcium channel polypeptide typically has one or more of the following properties: high selectivity, a unitary conductance below the detection level of the patch clamp method, and are subject to inhibition by high intracellular calcium levels. Such activity can be easily detected using standard methodology well known in the art. See, e.g., the Examples and Neher, E., “Ion channels for communication between and within cells”, Science, 1992; 256:498–502; and Hoth, M., and Penner, R., “Depletion of intracellular calcium stores activates a calcium current in mast cells”, Nature, 1992; 355 (6358):353–6.
According to one aspect of the invention, isolated nucleic acid molecules which code for one or more member(s) of the SOC/CRAC family of calcium channel polypeptides are provided. The isolated nucleic acid molecules are selected from the following groups:
(a) nucleic acid molecules which hybridize under stringent conditions to one or more nucleic acid molecules selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31, and which code for a SOC/CRAC polypeptide;
(b) deletions, additions and substitutions of (a) which code for a respective SOC/CRAC polypeptide;
(c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code, and
(d) complements of (a), (b) or (c).
In certain embodiments, the isolated nucleic acid molecule comprises one or more of nucleotides 1–1212 of SEQ ID NO:1; nucleotides 1–739 of SEQ ID NO:3; nucleotides 1–1579 of SEQ ID NO:5; nucleotides 1–5117 of SEQ ID NO:23; the mouse homolog for SOC-2/CRAC-1 corresponding to SEQ ID NO:7; nucleotides 1–2180 of SEQ ID NO:25; nucleotides 382–5976 of SEQ ID NO:27; nucleotides 73–3714 of SEQ ID NO:29; and nucleotides 23–3434 of SEQ ID NO:31. In yet other embodiments, the isolated nucleic acid molecule comprises a molecule which encodes a polypeptide having one or more sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32.
According to yet another aspect of the invention, an isolated nucleic acid molecule is provided which is selected from the group consisting of:
(a) a unique fragment of a nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31, (of sufficient length to represent a sequence unique within the human genome); and (b) complements of (a), provided that the unique fragment includes a sequence of contiguous nucleotides which is not identical to a sequence in the prior art as represented by the sequence group consisting of: (1) sequences having the SEQ ID NOs or GenBank accession numbers of Table I, (2) complements of (1), and (3) fragments of (1) and (2).
In some embodiments, the sequence of contiguous nucleotides is selected from the group consisting of (1) at least two contiguous nucleotides nonidentical to the sequence group, (2) at least three contiguous nucleotides nonidentical to the sequence group, (3) at least four contiguous nucleotides nonidentical to the sequence group, (4) at least five contiguous nucleotides nonidentical to the sequence group, (5) at least six contiguous nucleotides nonidentical to the sequence group, (6) at least seven contiguous nucleotides nonidentical to the sequence group.
In other embodiments, the unique fragment has a size selected from the group consisting of at least: 8 nucleotides, 10 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides, 20, nucleotides, 22 nucleotides, 24 nucleotides, 26 nucleotides, 28 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 200 nucleotides, 1000 nucleotides and every integer length therebetween.
According to another aspect of the invention, expression vectors and host cells containing (e.g., transformed or transfected with) expression vectors comprising the nucleic acid molecules disclosed herein operably linked to a promoter are provided. In certain preferred embodiments, the host cells are eukaryotic cells.
The isolated nucleic acid molecules disclosed herein have various utilities, including their use as probes and primers to identify additional members of the SOC/CRAC family of calcium channels, as diagnostic reagents for identifying the presence of SOC/CRAC polypeptides in biological or other samples, and as agents for generating SOC/CRAC binding polypeptides (e.g., antibodies) that can be used as reagents in diagnostic and therapeutic assays to identify the presence, absence, and/or amounts of a SOC/CRAC nucleic acid or polypeptide in a biological or other sample.
As used herein with respect to nucleic acids, the term “isolated” means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one which is readily manipulatable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5′ and 3′ restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulatable by standard techniques known to those of ordinary skill in the art.
As used herein with respect to polypeptides (discussed below), the term “isolated” means separated from its native environment in sufficiently pure form so that it can be manipulated or used for any one of the purposes of the invention. Thus, isolated means sufficiently pure to be used (i) to raise and/or isolate antibodies, (ii) as a reagent in an assay, or (iii) for sequencing, etc.
Homologs and alleles of the SOC/CRAC nucleic acids of the invention can be identified by conventional techniques. Thus, an aspect of the invention is those nucleic acid sequences which code for SOC/CRAC polypeptides and which hybridize to a nucleic acid molecules selected from a group consisting of the nucleic acid of SEQ ID NO: 1, the nucleic acid of SEQ ID NO:3, the nucleic acid of SEQ ID NO:5, the nucleic acid of SEQ ID NO:7, the nucleic acid of SEQ ID NO:23, the nucleic acid of SEQ ID NO:25, the nucleic acid of SEQ ID NO:27, the nucleic acid of SEQ ID NO:29, and the nucleic acid of SEQ ID NO:31, under stringent conditions. The term “stringent conditions” as used herein refers to parameters with which the art is familiar. Nucleic acid hybridization parameters may be found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. More specifically, stringent conditions, as used herein, refers, for example, to hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% Bovine Serum Albumin, 2.5 mMNaH2PO4(pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.015M sodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid. After hybridization, the membrane upon which the DNA is transferred is washed at 2×SSC at room temperature and then at 0.1×SSC/0.1×SDS at temperatures up to 68° C.
There are other conditions, reagents, and so forth which can be used, and would result in a similar degree of stringency. The skilled artisan will be familiar with such conditions, and thus they are not given here. It will be understood, however, that the skilled artisan will be able to manipulate the conditions in a manner to permit the clear identification of homologs and alleles of the SOC/CRAC nucleic acids of the invention. The skilled artisan also is familiar with the methodology for screening cells and libraries for expression of such molecules which then are routinely isolated, followed by isolation of the pertinent nucleic acid molecule and sequencing.
In general homologs and alleles typically will share at least 40% nucleotide identity and/or at least 50% amino acid identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and/or SEQ ID NO:31, and SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and/or SEQ ID NO:32, respectively. In some instances sequences will share at least 50% nucleotide identity and/or at least 65% amino acid identity and in still other instances sequences will share at least 60% nucleotide identity and/or at least 75% amino acid identity. The homology can be calculated using various, publicly available software tools developed by NCBI (Bethesda, Md.) that can be obtained through the internet (ncbi.nlm.nih.gov/pub/). Exemplary tools include the BLAST system available at ncbi.nlm.nih.gov. Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysis can be obtained using the MacVector sequence analysis software (Oxford Molecular Group). Watson-Crick complements of the foregoing nucleic acids also are embraced by the invention.
In screening for SOC/CRAC related genes, such as homologs and alleles of SOC-2/CRAC-1 and/or SOC-3/CRAC-2, a Southern blot may be performed using the foregoing conditions, together with a radioactive probe. After washing the membrane to which the DNA is finally transferred, the membrane can be placed against X-ray film or a phosphoimager plate to detect the radioactive signal.
Given that the expression of the SOC/CRAC gene is prominent in certain human tissues (e.g., SOC-2/CRAC-1: lymphoid tissue/heart, SOC-3/CRAC-2: kidney/colon, SOC-4/CRAC-3: prostate), and given the teachings herein of partial human SOC/CRAC cDNA clones, full-length and other mammalian sequences corresponding to the human SOC/CRAC partial nucleic acid sequences can be isolated from, for example, a cDNA library prepared from one or more of the tissues in which SOC-2/CRAC-1 expression is prominent, SOC-3/CRAC-2 is prominent, and/or SOC-4/CRAC-3 expression is prominent, using standard colony hybridization techniques.
The invention also includes degenerate nucleic acids which include alternative codons to those present in the native materials. For example, serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is equivalent for the purposes of encoding a serine residue. Thus, it will be apparent to one of ordinary skill in the art that any of the serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine residue into an elongating SOC/CRAC polypeptide. Similarly, nucleotide sequence triplets which encode other amino acid residues include, but are not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons). Other amino acid residues may be encoded similarly by multiple nucleotide sequences. Thus, the invention embraces degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy of the genetic code.
The invention also provides isolated unique fragments of an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31. A unique fragment is one that is a ‘signature’ for the larger nucleic acid. For example, the unique fragment is long enough to assure that its precise sequence is not found in molecules within the human genome outside of the SOC/CRAC nucleic acids defined above (and human alleles). Those of ordinary skill in the art may apply no more than routine procedures to determine if a fragment is unique within the human genome.
Unique fragments, however, exclude fragments completely composed of the nucleotide sequences of any of GenBank accession numbers and SEQ ID NOs listed in Table I (SEQ ID NO:9, AB001535, A1226731, H18835, AA419592, AA261842, AA419407, A1098310, AA592910, D86107, AF071787, Z77132, Z83117, Z68333, AA708532, AA551759, AA932133, R47363, N31660, AC005538, AA654650, AA370110, AA313170, AA493512, A1670079, A1671853, AC005538, AA654650, AA370110, AA313170, AA493512, AI670079, AI671853), or other previously published sequences as of the filing date of this application.
A fragment which is completely composed of the sequence described in the foregoing GenBank deposits and SEQ ID NO:9, is one which does not include any of the nucleotides unique to the sequences of the invention. Thus, a unique fragment must contain a nucleotide sequence other than the exact sequence of those in GenBank or fragments thereof. The difference may be an addition, deletion or substitution with respect to the GenBank sequence or it may be a sequence wholly separate from the GenBank sequence.
Unique fragments can be used as probes in Southern and Northern blot assays to identify such nucleic acids, or can be used in amplification assays such as those employing PCR. As known to those skilled in the art, large probes such as 200, 250, 300 or more nucleotides are preferred for certain uses such as Southern and Northern blots, while smaller fragments will be preferred for uses such as PCR. Unique fragments also can be used to produce fusion proteins for generating antibodies or determining binding of the polypeptide fragments, as demonstrated in the Examples, or for generating immunoassay components. Likewise, unique fragments can be employed to produce nonfused fragments of the SOC/CRAC polypeptides, useful, for example, in the preparation of antibodies, immunoassays or therapeutic applications. Unique fragments further can be used as antisense molecules to inhibit the expression of SOC/CRAC nucleic acids and polypeptides, respectively.
As will be recognized by those skilled in the art, the size of the unique fragment will depend upon its conservancy in the genetic code. Thus, some regions of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31, and complements thereof, will require longer segments to be unique while others will require only short segments, typically between 12 and 32 nucleotides long (e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 bases) or more, up to the entire length of the disclosed sequence. As mentioned above, this disclosure intends to embrace each and every fragment of each sequence, beginning at the first nucleotide, the second nucleotide and so on, up to 8 nucleotides short of the end, and ending anywhere from nucleotide number 8, 9, 10 and so on for each sequence, up to the very last nucleotide, (provided the sequence is unique as described above). Virtually any segment of the region of SEQ ID NO:1 beginning at nucleotide 1 and ending at nucleotide 1212, or SEQ ID NO:3 beginning at nucleotide 1 and ending at nucleotide 739, or SEQ ID NO:5 beginning at nucleotide 1 and ending at nucleotide 1579, or SEQ ID NO:7 beginning at nucleotide 1 and ending at nucleotide 3532, or SEQ ID NO:23 beginning at nucleotide 1 and ending at nucleotide 5117, SEQ ID NO:25 beginning at nucleotide 1 and ending at nucleotide 2180, SEQ ID NO:27 beginning at nucleotide 1 and ending at nucleotide 7419, or SEQ ID NO:29 beginning at nucleotide 1 and ending at nucleotide 4061, or SEQ ID NO:31 beginning at nucleotide 1 and ending at nucleotide 4646, or complements thereof, that is 20 or more nucleotides in length will be unique. Those skilled in the art are well versed in methods for selecting such sequences, typically on the basis of the ability of the unique fragment to selectively distinguish the sequence of interest from other sequences in the human genome of the fragment to those on known databases typically is all that is necessary, although in vitro confirmatory hybridization and sequencing analysis may be performed.
As mentioned above, the invention embraces antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding a SOC/CRAC polypeptide, to decrease SOC/CRAC calcium channel activity. When using antisense preparations of the invention, slow intravenous administration is preferred.
As used herein, the term “antisense oligonucleotide” or “antisense” describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA. The antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene or transcript. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Based upon SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31, or upon allelic or homologous genomic and/or cDNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention. In order to be sufficiently selective and potent for inhibition, such antisense oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the target, although in certain cases modified oligonucleotides as short as 7 bases in length have been used successfully as antisense oligonucleotides (Wagner et al., Nat. Med. 1(11):1116–1118, 1995). Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20–30 bases. Although oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N-terminal or 5′ upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3′-untranslated regions may be targeted by antisense oligonucleotides. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs. In addition, the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol. Neurobiol. 14(5):439–457, 1994) and at which proteins are not expected to bind. Finally, although, SEQ ID No:1 discloses a cDNA sequence, one of ordinary skill in the art may easily derive the genomic DNA corresponding to this sequence. Thus, the present invention also provides for antisense oligonucleotides which are complementary to the genomic DNA corresponding to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, in SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31. Similarly, antisense to allelic or homologous SOC/CRAC cDNAs and genomic DNAs are enabled without undue experimentation.
In one set of embodiments, the antisense oligonucleotides of the invention may be composed of “natural” deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5′ end of one native nucleotide and the 3′ end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage. These oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors.
In preferred embodiments, however, the antisense oligonucleotides of the invention also may include “modified” oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness.
The term “modified oligonucleotide” as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5′ end of one nucleotide and the 3′ end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide. Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.
The term “modified oligonucleotide” also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3′ position and other than a phosphate group at the 5′ position. Thus modified oligonucleotides may include a 2′-O-alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. The present invention, thus, contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding SOC/CRAC polypeptides, together with pharmaceutically acceptable carriers. Antisense oligonucleotides may be administered as part of a pharmaceutical composition. Such a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art. The compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term “physiologically acceptable” refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
The invention also involves expression vectors coding for SOC/CRAC proteins and fragments and variants thereof and host cells containing those expression vectors. Virtually any cells, prokaryotic or eukaryotic, which can be transformed with heterologous DNA or RNA and which can be grown or maintained in culture, may be used in the practice of the invention. Examples include bacterial cells such as E. coli and eukaryotic cells such as mouse, hamster, pig, goat, primate, yeast, xenopous, etc. They may be of a wide variety of tissue types, including mast cells, fibroblasts, oocytes and lymphocytes, and they may be primary cells or cell lines. Specific examples include CHO cells and COS cells. Cell-free transcription systems also may be used in lieu of cells.
As used herein, a “vector” may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids and virus genomes. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein). Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
As used herein, a coding sequence and regulatory sequences are said to be “operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5′ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5′ non-transcribed and 5′ non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5′ non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5′ leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
According to yet another aspect of the invention, isolated SOC/CRAC polypeptides are provided. Preferably, the isolated SOC/CRAC polypeptides are encoded by the isolated SOC/CRAC nucleic acid molecules disclosed herein. More preferably, the isolated SOC/CRAC polypeptides of the invention are encoded by the nucleic acid molecules having SEQ ID Nos. 1, 3, 5, 7, 23, 25, 27, 29, and 31. In yet other embodiments, the isolated SOC/CRAC polypeptides of the invention have an amino acid sequence selected from the group consisting of SEQ ID Nos. 2, 4, 6, 8, 24, 26, 28, 30 and 32. Preferably, the isolated SOC/CRAC polypeptides are of sufficient length to represent a sequence unique within the human genome. Thus, the preferred embodiments include a sequence of contiguous amino acids which is not identical to a prior art sequence as represented by the sequence group consisting of the contiguous amino acids identified in Table II (SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 and GenBank Acc. Nos. AB001535, AA592910, D86107, AF071787, Z77132, Z83117, Z68333, AA708532, AA551759, AA932133, R47363, N31660, NP003298, CAB00861, NP002411, CAA92726, CAB05572).
In certain embodiments, the isolated SOC/CRAC polypeptides are immunogenic and can be used to generate binding polypeptides (e.g., antibodies) for use in diagnostic and therapeutic applications. Such binding polypeptides also are useful for detecting the presence, absence, and/or amounts of a SOC/CRAC nucleic acid or polypeptide in a sample such as a biological fluid or biopsy sample. Preferably, the SOC/CRAC polypeptides that are useful for generating binding polypeptides are unique polypeptides and, therefore, binding of the antibody to a SOC/CRAC polypeptide in a sample is selective for the SOC/CRAC polypeptide.
Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA) encoding a SOC/CRAC polypeptide or fragment or variant thereof. The heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.
Preferred systems for mRNA expression in mammalian cells are those such as pRc/CMV (available from Invitrogen, Carlsbad, Calif.) that contain a selectable marker such as a gene that confers G418 resistance (which facilitates the selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequences. Additionally, suitable for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen, Carlsbad, Calif.), which contains an Epstein Barr virus (EBV) origin of replication, facilitating the maintenance of plasmid as a multicopy extrachromosomal element. Another expression vector is the pEF-BOS plasmid containing the promoter of polypeptide Elongation Factor 1α, which stimulates efficiently transcription in vitro. The plasmid is described by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), and its use in transfection experiments is disclosed by, for example, Demoulin (Mol. Cell. Biol. 16:4710–4716, 1996). Still another preferred expression vector is an adenovirus, described by Stratford-Perricaudet, which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626–630, 1992). The use of the adenovirus as an Adeno.P1A recombinant is disclosed by Warnier et al., in intradermal injection in mice for immunization against P1A (Int. J. Cancer, 67:303–310, 1996).
The invention also embraces so-called expression kits, which allow the artisan to prepare a desired expression vector or vectors. Such expression kits include at least separate portions of each of the previously discussed coding sequences. Other components may be added, as desired, as long as the previously mentioned sequences, which are required, are included.
It will also be recognized that the invention embraces the use of the above described, SOC/CRAC cDNA sequence containing expression vectors, to transfect host cells and cell lines, by these prokaryotic (e.g., E. coli), or eukaryotic (e.g., CHO cells, COS cells, yeast expression systems and recombinant baculovirus expression in insect cells). Especially useful are mammalian cells such as mouse, hamster, pig, goat, primate, etc. They may be of a wide variety of tissue types, and include primary cells and cell lines. Specific examples include dendritic cells, U293 cells, peripheral blood leukocytes, bone marrow stem cells and embryonic stem cells. The invention also permits the construction of SOC/CRAC gene “knock-outs” in cells and in animals, providing materials for studying certain aspects of SOC/CRAC calcium channel activity.
The invention also provides isolated polypeptides (including whole proteins and partial proteins), encoded by the foregoing SOC/CRAC nucleic acids, and include the polypeptides of SEQ ID NO:2, 4, 6, 8, 24, 26, 28, 30, 32, and unique fragments thereof. Such polypeptides are useful, for example, to regulate calcium transport-mediated cell growth, differentiation and proliferation, to generate antibodies, as components of immunoassays, etc. Polypeptides can be isolated from biological samples including tissue or cell homogenates, and can also be expressed recombinantly in a variety of prokaryotic and eukaryotic expression systems by constructing an expression vector appropriate to the expression system, introducing the expression vector into the expression system, and isolating the recombinantly expressed protein. Short polypeptides, including antigenic peptides (such as are presented by MHC molecules on the surface of a cell for immune recognition) also can be synthesized chemically using well-established methods of peptide synthesis.
A unique fragment of a SOC/CRAC polypeptide, in general, has the features and characteristics of unique fragments as discussed above in connection with nucleic acids. As will be recognized by those skilled in the art, the size of the unique fragment will depend upon factors such as whether the fragment constitutes a portion of a conserved protein domain. Thus, some regions of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and/or SEQ ID NO:32, will require longer segments to be unique while others will require only short segments, typically between 5 and 12 amino acids (e.g. 5, 6, 7, 8, 9, 10, 11 and 12 amino acids long or more, including each integer up to the full length, >1,000 amino acids long). Virtually any segment of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and/or SEQ ID NO:32, excluding the ones that share identity with it (the polypeptides identified in Table II—SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, and GenBank Acc. Nos. AB001535, AA592910, D86107, AF071787, Z77132, Z83117, Z68333, AA708532, AA551759, AA932133, R47363, N31660, NP003298, CAB00861, NP002411, CAA92726, CAB05572) that is 9 or more amino acids in length will be unique.
Unique fragments of a polypeptide preferably are those fragments which retain a distinct functional capability of the polypeptide. Functional capabilities which can be retained in a unique fragment of a polypeptide include Ca2+ fluxing, high selectivity, a unitary conductance below the detection level of the patch clamp method, and/or and are subject to inhibition by high intracellular calcium levels.
One important aspect of a unique fragment is its ability to act as a signature for identifying the polypeptide. Optionally, another aspect of a unique fragment is its ability to provide an immune response in an animal. Those skilled in the art are well versed in methods for selecting unique amino acid sequences, typically on the basis of the ability of the unique fragment to selectively distinguish the sequence of interest from non-family members. A comparison of the sequence of the fragment to those on known databases typically is all that is necessary.
The invention embraces variants of the SOC/CRAC polypeptides described above. As used herein, a “variant” of a SOC/CRAC polypeptide is a polypeptide which contains one or more modifications to the primary amino acid sequence of a SOC/CRAC polypeptide. Modifications which create a SOC/CRAC polypeptide variant are typically made to the nucleic acid which encodes the SOC/CRAC polypeptide, and can include deletions, point mutations, truncations, amino acid substitutions and addition of amino acids or non-amino acid moieties to: 1) reduce or eliminate a calcium channel activity of a SOC/CRAC polypeptide; 2) enhance a property of a SOC/CRAC polypeptide, such as protein stability in an expression system or the stability of protein—protein binding; 3) provide a novel activity or property to a SOC/CRAC polypeptide, such as addition of an antigenic epitope or addition of a detectable moiety; or 4) to provide equivalent or better binding to a SOC/CRAC polypeptide receptor or other molecule. Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a fatty acid, and the like. Modifications also embrace fusion proteins comprising all or part of the SOC/CRAC amino acid sequence. One of skill in the art will be familiar with methods for predicting the effect on protein conformation of a change in protein sequence, and can thus “design” a variant SOC/CRAC polypeptide according to known methods. One example of such a method is described by Dahiyat and Mayo in Science 278:82–87, 1997, whereby proteins can be designed de novo. The method can be applied to a known protein to vary only a portion of the polypeptide sequence. By applying the computational methods of Dahiyat and Mayo, specific variants of a SOC/CRAC calcium channel polypeptide can be proposed and tested to determine whether the variant retains a desired conformation.
Variants can include SOC/CRAC polypeptides which are modified specifically to alter a feature of the polypeptide unrelated to its physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages. Similarly, certain amino acids can be changed to enhance expression of a SOC/CRAC polypeptide by eliminating proteolysis by proteases in an expression system (e.g., dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present).
Mutations of a nucleic acid which encodes a SOC/CRAC polypeptide preferably preserve the amino acid reading frame of the coding sequence and, preferably, do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such as hairpins or loops, which can be deleterious to expression of the variant polypeptide.
Mutations can be made by selecting an amino acid substitution, or by random mutagenesis of a selected site in a nucleic acid which encodes the polypeptide. Variant polypeptides are then expressed and tested for one or more activities to determine which mutation provides a variant polypeptide with the desired properties. Further mutations can be made to variants (or to non-variant SOC/CRAC polypeptides) which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli, are well known to those of ordinary skill in the art. Still other mutations can be made to the noncoding sequences of a SOC/CRAC gene or cDNA clone to enhance expression of the polypeptide.
The skilled artisan will realize that conservative amino acid substitutions may be made in SOC/CRAC polypeptides to provide functionally equivalent variants of the foregoing polypeptides, i.e, the variants retain the functional capabilities of the SOC/CRAC polypeptides. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution which does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning. A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Exemplary functionally equivalent variants of the SOC/CRAC polypeptides include conservative amino acid substitutions of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and/or SEQ ID NO:32. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) St T; (f) Q, N; and (g) E, D.
Thus functionally equivalent variants of SOC/CRAC polypeptides, i.e., variants of SOC/CRAC polypeptides which retain the function of the natural SOC/CRAC polypeptides, are contemplated by the invention. Conservative amino-acid substitutions in the amino acid sequence of SOC/CRAC polypeptides to produce functionally equivalent variants of SOC/CRAC polypeptides typically are made by alteration of a nucleic acid encoding SOC/CRAC polypeptides (e.g., SEQ ID NOs:1, 3, 5, 7, 23, 25, 27, 29, 31). Such substitutions can be made by a variety of methods known to one of ordinary skill in the art. For example, amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488–492, 1985), or by chemical synthesis of a gene encoding a SOC/CRAC polypeptide. The activity of functionally equivalent fragments of SOC/CRAC polypeptides can be tested by cloning the gene encoding the altered SOC/CRAC polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the altered SOC/CRAC polypeptide, and testing for a functional capability of the SOC/CRAC polypeptides as disclosed herein (e.g., SOC/CRAC calcium channel activity).
The invention as described herein has a number of uses, some of which are described elsewhere herein. First, the invention permits isolation of SOC/CRAC polypeptides, including the isolation of the complete SOC/CRAC polypeptide. A variety of methodologies well-known to the skilled practitioner can be utilized to obtain isolated SOC/CRAC molecules. The polypeptide may be purified from cells which naturally produce the polypeptide by chromatographic means or immunological recognition. Alternatively, an expression vector may be introduced into cells to cause production of the polypeptide. In another method, mRNA transcripts may be microinjected or otherwise introduced into cells to cause production of the encoded polypeptide. Translation of SOC/CRAC mRNA in cell-free extracts such as the reticulocyte lysate system also may be used to produce SOC/CRAC polypeptides. Those skilled in the art also can readily follow known methods for isolating SOC/CRAC polypeptides. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography and immune-affinity chromatography.
The invention also provides, in certain embodiments, “dominant negative” polypeptides derived from SOC/CRAC polypeptides. A dominant negative polypeptide is an inactive variant of a protein, which, by interacting with the cellular machinery, displaces an active protein from its interaction with the cellular machinery or competes with the active protein, thereby reducing the effect of the active protein. For example, a dominant negative receptor which binds a ligand but does not transmit a signal in response to binding of the ligand can reduce the biological effect of expression of the ligand. Likewise, a dominant negative inactive SOC/CRAC calcium channel which interacts normally with the cell membrane but which does not mediate calcium transport can reduce calcium transport in a cell. Similarly, a dominant negative transcription factor which binds to a promoter site in the control region of a gene but does not increase gene transcription can reduce the effect of a normal transcription factor by occupying promoter binding sites without increasing transcription.
The end result of the expression of a dominant negative polypeptide in a cell is a reduction in function of active proteins. One of ordinary skill in the art can assess the potential for a dominant negative variant of a protein, and using standard mutagenesis techniques to create one or more dominant negative variant polypeptides. See, e.g., U.S. Pat. No. 5,580,723 and Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press. The skilled artisan then can test the population of mutagenized polypeptides for diminution in a selected and/or for retention of such an activity. Other similar methods for creating and testing dominant negative variants of a protein will be apparent to one of ordinary skill in the art.
According to another aspect, the invention provides a method for isolating a SOC/CRAC molecule having SOC/CRAC calcium channel activity. The method involves contacting a binding molecule that is a SOC/CRAC nucleic acid or a SOC/CRAC binding polypeptide with a sample containing one or more SOC/CRAC molecules under conditions that allow such binding (see earlier discussion) to form a complex, detecting the presence of the complex, isolating the SOC/CRAC molecule from the complex, and determining whether the isolated SOC/CRAC molecule has SOC/CRAC calcium channel activity. Thus, the invention is useful for identifying and isolating full length complementary (cDNA) or genomic nucleic acids encoding SOC/CRAC polypeptides having SOC/CRAC calcium channel activity. Identification and isolation of such nucleic acids and polypeptides may be accomplished by hybridizing/binding, under appropriate conditions well known in the art, libraries and/or restriction enzyme-digested human nucleic acids, with a labeled SOC/CRAC molecular probe. As used herein, a “label” includes molecules that are incorporated into, for example, a SOC/CRAC molecule (nucleic acid or peptide), that can be directly or indirectly detected. A wide variety of detectable labels are well known in the art that can be used, and include labels that provide direct detection (e.g., radioactivity, luminescence, optical or electron density, etc), or indirect detection (e.g., epitope tag such as the FLAG epitope, enzyme tag such as horseseradish peroxidase, etc.). The label may be bound to a SOC/CRAC binding partner, or incorporated into the structure of the binding partner.
A variety of methods may be used to detect the label, depending on the nature of the label and other assay components. For example, the label may be detected while bound to the solid substrate or subsequent to separation from the solid substrate. Labels may be directly detected through optical or electron density, radioactive emissions, nonradioactive energy transfers, etc. or indirectly detected with antibody conjugates, strepavidin-biotin conjugates, etc. Methods for detecting the labels are well known in the art. Once a library clone or hybridizing fragment is identified in the hybridization/binding reaction, it can be further isolated by employing standard isolation/cloning techniques known to those of skill in the art. See, generally, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press. In addition, nucleic acid amplification techniques well known in the art, may also be used to locate splice variants of calcium channel (or calcium channel subunits) with SOC/CRAC calcium channel activity. Size and sequence determinations of the amplification products can reveal splice variants.
The foregoing isolated nucleic acids and polypeptides may then be compared to the nucleic acids and polypeptides of the present invention in order to identify homogeneity or divergence of the sequences, and be further characterized functionally to determine whether they belong to a family of molecules with SOC/CRAC calcium channel activity (for methodology see under the Examples section).
The isolation of the SOC/CRAC cDNA and/or partial sequences thereof also makes it possible for the artisan to diagnose a disorder characterized by an aberrant expression of SOC/CRAC. These methods involve determining expression of the SOC/CRAC gene, and/or SOC/CRAC polypeptides derived therefrom. In the former situation, such determinations can be carried out via any standard nucleic acid determination assay, including the polymerase chain reaction, or assaying with labeled hybridization probes as exemplified below. In the latter situation, such determination can be carried out via any standard immunological assay using, for example, antibodies which bind to the SOC/CRAC protein.
The invention also embraces isolated peptide binding agents which, for example, can be antibodies or fragments of antibodies (“binding polypeptides”), having the ability to selectively bind to SOC/CRAC polypeptides. Antibodies include polyclonal and monoclonal antibodies, prepared according to conventional methodology. In certain embodiments, the invention excludes binding agents (e.g., antibodies) that bind to the polypeptides encoded by the nucleic acids of SEQ ID NOs:10, 12, 13, 14, 15, 17, and 19.
Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The pFc′ and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc′ region has been enzymatically cleaved, or which has been produced without the pFc′ region, designated an F(ab′)2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.
It is now well-established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of “humanized” antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc′ regions to produce a functional antibody. Thus, for example, PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin. Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as “chimeric” antibodies.
Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab′)2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab′)2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes so-called single chain antibodies.
Thus, the invention involves binding polypeptides of numerous size and type that bind selectively to SOC/CRAC polypeptides, and complexes containing SOC/CRAC polypeptides. These binding polypeptides also may be derived also from sources other than antibody technology. For example, such polypeptide binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form, as bacterial flagella peptide display libraries or as phage display libraries. Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptides and non-peptide synthetic moieties.
Phage display can be particularly effective in identifying binding peptides useful according to the invention. Briefly, one prepares a phage library (using e.g. m13, fd, or lambda phage), displaying inserts from 4 to about 80 amino acid residues using conventional procedures. The inserts may represent, for example, a completely degenerate or biased array. One then can select phage-bearing inserts which bind to the SOC/CRAC polypeptide or a complex containing a SOC/CRAC polypeptide. This process can be repeated through several cycles of reselection of phage that bind to the SOC/CRAC polypeptide or complex. Repeated rounds lead to enrichment of phage bearing particular sequences. DNA sequence analysis can be conducted to identify the sequences of the expressed polypeptides. The minimal linear portion of the sequence that binds to the SOC/CRAC polypeptide or complex can be determined. One can repeat the procedure using a biased library containing inserts containing part or all of the minimal linear portion plus one or more additional degenerate residues upstream or downstream thereof. Yeast two-hybrid screening methods also may be used to identify polypeptides that bind to the SOC/CRAC polypeptides. Thus, the SOC/CRAC polypeptides of the invention, or a fragment thereof, or complexes of SOC/CRAC can be used to screen peptide libraries, including phage display libraries, to identify and select peptide binding polypeptides that selectively bind to the SOC/CRAC polypeptides of the invention. Such molecules can be used, as described, for screening assays, for purification protocols, for interfering directly with the functioning of SOC/CRAC and for other purposes that will be apparent to those of ordinary skill in the art.
A SOC/CRAC polypeptide, or a fragment thereof, also can be used to isolate naturally occurring, polypeptide binding partners which may associate with the SOC/CRAC polypeptide in the membrane of a cell. Isolation of binding partners may be performed according to well-known methods. For example, isolated SOC/CRAC polypeptides can be attached to a substrate, and then a solution suspected of containing an SOC/CRAC binding partner may be applied to the substrate. If the binding partner for SOC/CRAC polypeptides is present in the solution, then it will bind to the substrate-bound SOC/CRAC polypeptide. The binding partner then may be isolated. Other proteins which are binding partners for SOC/CRAC, may be isolated by similar methods without undue experimentation.
The invention also provides novel kits which could be used to measure the levels of the nucleic acids of the invention, expression products of the invention or anti-SOC/CRAC antibodies. In the case of nucleic acid detection, pairs of primers for amplifying SOC/CRAC nucleic acids can be included. The preferred kits would include controls such as known amounts of nucleic acid probes, SOC/CRAC epitopes (such as SOC/CRAC expression products) or anti-SOC/CRAC antibodies, as well as instructions or other printed material. In certain embodiments the printed material can characterize risk of developing a disorder that is characterized by aberrant SOC/CRAC polypeptide expression based upon the outcome of the assay. The reagents may be packaged in containers and/or coated on wells in predetermined amounts, and the kits may include standard materials such as labeled immunological reagents (such as labeled anti-IgG antibodies) and the like. One kit is a packaged polystyrene microtiter plate coated with a SOC/CRAC polypeptide and a container containing labeled anti-human IgG antibodies. A well of the plate is contacted with, for example, serum, washed and then contacted with the anti-IgG antibody. The label is then detected. A kit embodying features of the present invention is comprised of the following major elements: packaging an agent of the invention, a control agent, and instructions. Packaging is a box-like structure for holding a vial (or number of vials) containing an agent of the invention, a vial (or number of vials) containing a control agent, and instructions. Individuals skilled in the art can readily modify packaging to suit individual needs.
Another aspect of the invention is a method for determining the level of SOC/CRAC expression in a subject. As used herein, a subject is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent. In all embodiments, human subjects are preferred. Expression is defined either as SOC/CRAC mRNA expression or SOC/CRAC polypeptide expression. Various methods can be used to measure expression. Preferred embodiments of the invention include PCR and Northern blotting for measuring mRNA expression, and monoclonal or polyclonal SOC/CRAC antisera as reagents to measure SOC/CRAC polypeptide expression. In certain embodiments, test samples such as biopsy samples, and biological fluids such as blood, are used as test samples. SOC/CRAC expression in a test sample of a subject is compared to SOC/CRAC expression in control sample to, e.g., assess the presence or absence or stage of a proliferative disorder (e.g., a lymphocyte proliferative disorder) in a subject.
SOC/CRAC polypeptides preferably are produced recombinantly, although such polypeptides may be isolated from biological extracts. Recombinantly produced SOC/CRAC polypeptides include chimeric proteins comprising a fusion of a SOC/CRAC protein with another polypeptide, e.g., a polypeptide capable of providing or enhancing protein—protein binding, sequence specific nucleic acid binding (such as GAL4), enhancing stability of the SOC/CRAC polypeptide under assay conditions, or providing a detectable moiety, such as green fluorescent protein. A polypeptide fused to a SOC/CRAC polypeptide or fragment may also provide means of readily detecting the fusion protein, e.g., by immunological recognition or by fluorescent labeling.
The invention is also useful in the generation of transgenic non-human animals. As used herein, “transgenic non-human animals” includes non-human animals having one or more exogenous nucleic acid molecules incorporated in germ line cells and/or somatic cells. Thus the transgenic animal include “knockout” animals having a homozygous or heterozygous gene disruption by homologous recombination, animals having episomal or chromosomally incorporated expression vectors, etc. Knockout animals can be prepared by homologous recombination using embryonic stem cells as is well known in the art. The recombination may be facilitated using, for example, the cre/lox system or other recombinase systems known to one of ordinary skill in the art. In certain embodiments, the recombinase system itself is expressed conditionally, for example, in certain tissues or cell types, at certain embryonic or post-embryonic developmental stages, inducibly by the addition of a compound which increases or decreases expression, and the like. In general, the conditional expression vectors used in such systems use a variety of promoters which confer the desired gene expression pattern (e.g., temporal or spatial). Conditional promoters also can be operably linked to SOC/CRAC nucleic acid molecules to increase expression of SOC/CRAC in a regulated or conditional manner. Trans-acting negative regulators of SOC/CRAC calcium channel activity or expression also can be operably linked to a conditional promoter as described above. Such trans-acting regulators include antisense SOC/CRAC nucleic acids molecules, nucleic acid molecules which encode dominant negative SOC/CRAC molecules, ribozyme molecules specific for SOC/CRAC nucleic acids, and the like. The transgenic non-human animals are useful in experiments directed toward testing biochemical or physiological effects of diagnostics or therapeutics for conditions characterized by increased or decreased SOC/CRAC expression. Other uses will be apparent to one of ordinary skill in the art.
The invention further provides efficient methods of identifying agents or lead compounds for agents active at the level of a SOC/CRAC polypeptide (e.g., a SOC/CRAC polypeptide) or SOC/CRAC fragment dependent cellular function. In particular, such functions include interaction with other polypeptides or fragments thereof, and selective binding to certain molecules (e.g., agonists and antagonists). Generally, the screening methods involve assaying for compounds which interfere with SOC/CRAC calcium channel activity, although compounds which enhance SOC/CRAC calcium channel activity also can be assayed using the screening methods. Such methods are adaptable to automated, high throughput screening of compounds. The target therapeutic indications for pharmacological agents detected by the screening methods are limited only in that the target cellular function be subject to modulation by alteration of the formation of a complex comprising a SOC/CRAC polypeptide or fragment thereof and one or more SOC/CRAC binding targets. Target indications include cellular processes modulated by SOC/CRAC such as Ca2+ fluxing, and affected by SOC/CRAC ability to form complexes with other molecules and polypeptides as, for example, may be present in the cell membrane.
A wide variety of assays for pharmacological agents are provided, including, expression assays, labeled in vitro protein—protein binding assays, electrophoretic mobility shift assays, immunoassays, cell-based assays such as calcium transport assays, etc. For example, two-hybrid screens are used to rapidly examine the effect of transfected nucleic acids on the intracellular binding of SOC/CRAC or SOC/CRAC fragments to specific intracellular targets (e.g. a tyrosine kinase). The transfected nucleic acids can encode, for example, combinatorial peptide libraries or cDNA libraries. Convenient reagents for such assays, e.g., GAL4 fusion proteins, are known in the art. An exemplary cell-based assay involves transfecting a cell with a nucleic acid encoding a SOC/CRAC polypeptide fused to a GAL4 DNA binding domain and a nucleic acid encoding a reporter gene operably linked to a gene expression regulatory region, such as one or more GAL4 binding sites. Activation of reporter gene transcription occurs when the SOC/CRAC and reporter fusion polypeptides bind such as to enable transcription of the reporter gene. Agents which modulate a SOC/CRAC polypeptide mediated cell function are then detected through a change in the expression of reporter gene. Methods for determining changes in the expression of a reporter gene are known in the art.
In an expression system, for example, a SOC/CRAC polypeptide is attached to a membrane, the membrane preferably separating two fluid environments and being otherwise not permeable to Ca2+. Such separation is preferred so that a change in Ca2+ concentration on either side of the membrane is mediated only through the attached SOC/CRAC polypeptide. Preferably, a SOC/CRAC polypeptide is expressed in an intact cell and is present on the cell-membrane (as in physiologic conditions). The cell expressing the SOC/CRAC polypeptide is preferably a eukaryotic cell, and the SOC/CRAC polypeptide is preferably recombinantly expressed, although cells naturally expressing a SOC/CRAC polypeptide may also be used. Synthetic membranes, however, containing SOC/CRAC polypeptides may also be used. See, e.g., K. Kiselyov, et al., Functional interaction between InsP3 receptors and store-operated Htrp3 channels, Nature 396, 478–82 (1998).
The cell expressing the SOC/CRAC polypeptide is incubated under conditions which, in the absence of the candidate agent, permit calcium flux into the cell and allow detection of a reference calcium concentration. For example, depletion of intracellular calcium stores with thapsigargin or other agents (Putney, J. W. Jr., in Capacitative Calcium Entry, R.G. Landes Co. and Chapman & Hall, 1997) would produce a given level of SOC/CRAC channel activation and a given reference calcium concentration. Detection of a decrease in the foregoing activities (i.e., a decrease in the intracellular calcium concentration) relative to the reference calcium concentration indicates that the candidate agent is a lead compound for an agent to inhibit SOC/CRAC calcium channel activity. Preferred SOC/CRAC polypeptides include the polypeptides of claim 15.
SOC/CRAC fragments used in the methods, when not produced by a transfected nucleic acid are added to an assay mixture as an isolated polypeptide. SOC/CRAC polypeptides preferably are produced recombinantly, although such polypeptides may be isolated from biological extracts or chemically synthesized. Recombinantly produced SOC/CRAC polypeptides include chimeric proteins comprising a fusion of a SOC/CRAC protein with another polypeptide, e.g., a polypeptide capable of providing or enhancing protein—protein binding, sequence specific nucleic acid binding (such as GAL4), enhancing stability of the SOC/CRAC polypeptide under assay conditions, or providing a detectable moiety, such as green fluorescent protein or Flag epitope.
The assay mixture is comprised of a SOC/CRAC polypeptide binding target (candidate agent) capable of interacting with a SOC/CRAC polypeptide. While natural SOC/CRAC binding targets may be used, it is frequently preferred to use portions (e.g., peptides or nucleic acid fragments) or analogs (i.e., agents which mimic the SOC/CRAC binding properties of the natural binding target for purposes of the assay) of the SOC/CRAC binding target so long as the portion or analog provides binding affinity and avidity to the SOC/CRAC polypeptide (or fragment thereof) measurable in the assay.
The assay mixture also comprises a candidate agent (binding target, e.g., agonist/antagonist). Typically, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection. Candidate agents encompass numerous chemical classes, although typically they are organic compounds. Preferably, the candidate agents are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500. Candidate agents comprise functional chemical groups necessary for structural interactions with polypeptides and/or nucleic acids, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups. The candidate agents can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Candidate agents also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the agent is a nucleic acid, the agent typically is a DNA or RNA molecule, although modified nucleic acids as defined herein are also contemplated.
Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means. Further, known agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the agents. Non-SOC/CRAC calcium channel agonists and antagonists, for example, include agents such as dihydropyridines (DHPs), phenylalkylamines, omega conotoxin (omega.-CgTx) and pyrazonoylguanidines.
A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein—protein, protein-nucleic acid, and/or protein/membrane component binding association. Such a reagent may also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease, inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.
The mixture of the foregoing assay materials is incubated under conditions whereby, but for the presence of the candidate agent, the SOC/CRAC polypeptide specifically binds the cellular binding target, a portion thereof or analog thereof. The order of addition of components, incubation temperature, time of incubation, and other perimeters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4° C. and 40° C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 0.1 and 10 hours.
After incubation, the presence or absence of specific binding between the SOC/CRAC polypeptide and one or more binding targets is detected by any convenient method available to the user. For cell free binding type assays, a separation step is often used to separate bound from unbound components. The separation step may be accomplished in a variety of ways. Conveniently, at least one of the components is immobilized on a solid substrate, from which the unbound components may be easily separated. The solid substrate can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc. The substrate preferably is chosen to maximum signal to noise ratios, primarily to minimize background binding, as well as for ease of separation and cost.
Separation may be effected for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, rinsing a bead, particle, chromotograpic column or filter with a wash solution or solvent. The separation step preferably includes multiple rinses or washes. For example, when the solid substrate is a microtiter plate, the wells may be washed several times with a washing solution, which typically includes those components of the incubation mixture that do not participate in specific bindings such as salts, buffer, detergent, non-specific protein, etc. Where the solid substrate is a magnetic bead, the beads may be washed one or more times with a washing solution and isolated using a magnet.
Detection may be effected in any convenient way for cell-based assays such as two- or three-hybrid screens. The transcript resulting from a reporter gene transcription assay of SOC/CRAC polypeptide interacting with a target molecule typically encodes a directly or indirectly detectable product, e.g., β-galactosidase activity, luciferase activity, and the like. For cell-free binding assays, one of the components usually comprises, or is coupled to, a detectable label. A wide variety of labels can be used, such as those that provide direct detection (e.g., radioactivity, luminescence, optical or electron density, etc.) or indirect detection (e.g., epitope tag such as the FLAG epitope, enzyme tag such as horseseradish peroxidase, etc.). The label may be bound to a SOC/CRAC binding partner, or incorporated into the structure of the binding partner.
A variety of methods may be used to detect the label, depending on the nature of the label and other assay components. For example, the label may be detected while bound to the solid substrate or subsequent to separation from the solid substrate. Labels may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, strepavidin-biotin conjugates, etc. Methods for detecting the labels are well known in the art.
Of particular importance in any of the foregoing assays and binding studies is the use of a specific sequence motif identified in the SOC-2/CRAC-1 polypeptide sequence as a kinase catalytic domain. According to the invention, amino acids 999–1180 of the SOC-2/CRAC-1 polypeptide (SEQ ID NO:24) (or a fragment thereof), show a localized homology with the catalytic domains of eukaryotic elongation factor-2 kinase (eEF-2 kinase, GenBank Acc. no. U93850) and Dictyostelium myocin heavy chain kinase A (MHCK A, GenBank Acc. no. U16856), as disclosed in Ryazanov AG, et al., Proc Natl Acad Sci USA, 1997, 94(10):4884–4889. Therefore, according to the invention, a method for identifying agents useful in the modulation of SOC/CRAC polypeptide kinase activity is provided. The method involves contacting a SOC/CRAC polypeptide with kinase activity, that includes, for example, amino acids 999–1180 of the SOC-2/CRAC-1 polypeptide (SEQ ID NO:24) with a candidate agent suspected of modulating SOC/CRAC kinase activity, under conditions sufficient to allow the candidate agent to interact with the SOC/CRAC polypeptide and modulate its kinase activity; detecting a kinase activity associated with the SOC/CRAC polypeptide in the presence of the candidate agent; and comparing the kinase activity in the previous step with a control kinase activity of a SOC/CRAC polypeptide in the absence of the candidate agent to determine whether the candidate agent modulates (increases or decreases) SOC/CRAC kinase activity. Other controls for kinase activity can also be performed at the same time, for example, by utilizing eEF-2 kinase and/or Dictyostelium MHC Kinase A, in a similar manner to the SOC/CRAC member. Methods for performing such kinase activity assays are well known in the art.
The invention thus provides SOC/CRAC-specific binding agents, methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. For example, SOC/CRAC-specific agents are useful in a variety of diagnostic and therapeutic applications, especially where disease or disease prognosis is associated with altered SOC/CRAC and SOC/CRAC calcium channel fluxing characteristics. Novel SOC/CRAC-specific binding agents include SOC/CRAC-specific antibodies and other natural intracellular and extracellular binding agents identified with assays such as two hybrid screens, and non-natural intracellular and extracellular binding agents identified in screens of chemical libraries and the like.
In general, the specificity of SOC/CRAC binding to a specific molecule is determined by binding equilibrium constants. Targets which are capable of selectively binding a SOC/CRAC polypeptide preferably have binding equilibrium constants of at least about 107 M−1, more preferably at least about 108 M−1, and most preferably at least about 109 M−1. The wide variety of cell based and cell free assays may be used to demonstrate SOC/CRAC-specific binding. Cell based assays include one, two and three hybrid screens, assays in which SOC/CRAC-mediated transcription is inhibited or increased, etc. Cell free assays include SOC/CRAC-protein binding assays, immunoassays, etc. Other assays useful for screening agents which bind SOC/CRAC polypeptides include fluorescence resonance energy transfer (FRET), and electrophoretic mobility shift analysis (EMSA).
Various techniques may be employed for introducing nucleic acids of the invention into cells, depending on whether the nucleic acids are introduced in vitro or in vivo in a host. Such techniques include transfection of nucleic acid-CaPO4 precipitates, transfection of nucleic acids associated with DEAE, transfection with a retrovirus including the nucleic acid of interest, liposome mediated transfection, and the like. For certain uses, it is preferred to target the nucleic acid to particular cells. In such instances, a vehicle used for delivering a nucleic acid of the invention into a cell (e.g., a retrovirus, or other virus; a liposome) can have a targeting molecule attached thereto. For example, a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid delivery vehicle. For example, where liposomes are employed to deliver the nucleic acids of the invention, proteins which bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake. Such proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like. Polymeric delivery systems also have been used successfully to deliver nucleic acids into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acids.
Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the anti-inflammatory agent, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. Long-term release, are used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
The invention also contemplates gene therapy. The procedure for performing ex vivo gene therapy is outlined in U.S. Pat. No. 5,399,346 and in exhibits submitted in the file history of that patent, all of which are publicly available documents. In general, it involves introduction in vitro of a functional copy of a gene into a cell(s) of a subject which contains a defective copy of the gene, and returning the genetically engineered cell(s) to the subject. The functional copy of the gene is under operable control of regulatory elements which permit expression of the gene in the genetically engineered cell(s). Numerous transfection and transduction techniques as well as appropriate expression vectors are well known to those of ordinary skill in the art, some of which are described in PCT application WO95/00654. In vivo gene therapy using vectors such as adenovirus, retroviruses, herpes virus, and targeted liposomes also is contemplated according to the invention. See, e.g., U.S. Pat. No. 5,670,488, entitled “Adenovirus Vector for Gene Therapy”, issued to Gregory et al., and U.S. Pat. No. 5,672,344, entitled “Viral-Mediated Gene Transfer System”, issued to Kelley et al.
The invention will be more fully understood by reference to the following examples. These examples, however, are merely intended to illustrate the embodiments of the invention and are not to be construed to limit the scope of the invention.
EXAMPLES
As an initial approach to identifying SOC/CRAC channels, we considered publicly available data and hypothesized that the following characteristics are likely to be exhibited by SOC/CRAC calcium channels: i) SOC/CRAC calcium channels would be integral membrane proteins related (probably distantly) to one of the known calcium channel families (e.g. voltage gated, ligand gated, Trp), and therefore should have a pore region formed by a tetramer of 6–7 transmembrane (TM) regions; ii) high calcium selectivity was likely to come at the price of complexity, and therefore these were likely to be large proteins; iii) the high calcium selectivity of this type of channel was likely to be useful and, therefore, highly conserved; and iv) these channels should be expressed in one or more types of lymphocytes, since ICRAC is best defined in those cell types. Since the full genome of the nematode C. elegans is nearing completion, and IP3-dependent calcium signals have recently been shown to be required for one or more aspects of C. elegans development, we took the set of proteins encoded by this genome (at the time this search was initiated WORMPEP14 was the available predicted protein set) and began searching for proteins which fit the criteria above. This search began by proceeding in alphabetical order through WORMPEP14 and arbitrarily excluding all proteins below approximately 1000 amino acids in size, followed by focusing on remaining proteins with clear TM spanning regions similar to those of other calcium channels. We stopped this screen on encountering a protein designated C05C12.3, a predicted protein of 1816 amino acids (SEQ ID NO:13). C05C12.3 was noteable because its central pore region had some sequence similarity to but was clearly distinct from members of the Trp family of calcium channels, and the hydrophobicity plot of this region showed a characteristically wide spacing between the fifth and sixth TM regions for the amino acid residues which are thought to line the channel pore region and mediate the calcium selectivity of the channels. In addition, it lacked any ankyrin repeats in the region amino-terminal to its pore region, further distinguishing it from other Trp family proteins.
We then used C05C12.3 for BLAST alignment screening of the rest of the C. elegans genome and also mammalian databases for homologous proteins, revealing two other C. elegans homologues (SEQ ID NO:14 and SEQ ID NO:15), and also a recently cloned mammalian protein named melastatin-1 (MLSN-1/SOC-1, SEQ ID NOs:9 and 10, and GenBank Acc. No. AF071787). Using these sequences, we subsequently performed an exhaustive screening of publicly accessible EST databases in search of lymphocyte homologues, but were unsuccessful in detecting any homologous transcripts in any lymphocyte lines. Since MLSN-1 (SEQ ID NOs:9 and 10) was expressed exclusively in melanocytes and retina by Northern blot hybridization and by EST database searching, there was no evidence that this type of channel was expressed in the type of cell in which ICRAC-like currents were best defined. Subsequent BLAST searches picked up mouse EST sequence A1098310 (SEQ ID NO:22) from a monocyte cell line. The I.M.A.G.E. consortium clone containing the above-identified EST was then purchased from ATCC (clone ID. 1312756, Manassas, Va.) and was further characterized. Using other portions of this sequence in EST searches, we subsequently picked up similar sequences in human B-cells (SEQ ID NOs:20 and 21), and other cell types as well (SEQ ID NOs: 11, 12, 16, 17, 18, and 19). Most of these sequences were subsequently identified to be part of the 3′-UTR or of the carboxy terminal region of the proteins, which are not readily identifiable as Trp channels, providing an explanation for the art's inability to detect any type of Trp related transcripts in lymphocytes. Partial sequences from the 5′ and/or 3′ ends of the above identified clones were then used to screen leukocyte and kidney cDNA libraries to extend the original sequences more toward the 5′ and/or 3′ ends.
In view of the foregoing, it was concluded that channels of this type were expressed in many types of lymphocytes, and therefore were members of a new family of SOC/CRAC calcium channels.
Experimental Procedures
Screening of the cDNA Libraries
Leukocyte and kidney cDNA libraries from Life Technologies (Gaithersburg, Md.) were screened using the Gene Trapper II methodology (Life Technologies) according to manufacturer's recommendation, using the inserts of I.M.A.G.E. clone ID nos. 1312756 and 1076485 from ATCC (Manassas, Va.), under stringent hybridization conditions. Using standard methodology (Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York), individual cDNA clones were subjected to 3–4 rounds of amplification and purification under the same hybridization conditions.
After excision from the vector and subcloning of inserts into the plasmid forms, several clones were sequenced by the Beth Israel Deaconess Medical Center's Automated Sequencing Facility. Molecular biological techniques such as restriction enzyme treatment, subcloning, DNA extraction, bacterial culture and purification of DNA fragments were performed according to methods well known in the art. Computer analyses of protein and DNA sequences was done using “Assemblylign” (Oxford Molecular, Cambell, Calif.). Multiple alignments of the SOC/CRAC family members were produced using the CLUSTAL facility of the MacVector program. Restriction endonucleases, expression vectors, and modifying enzymes were purchased from commercial sources (Gibco-BRL). Sequencing vectors for DNA were purchased from Stratagene (La Jolla, Calif.).
Once the first members of what appeared to be a novel family of calcium channel receptors were identified and characterized, additional BLAST alignments were performed with the newly characterized nucleic acid sequences. An initial match was with genomic DNA fragment NH0332L11 (Genbank Acc. No. AC005538). Using this genomic sequence, promers were designed and a number of cDNA libraries was surveyed by PCR. A prostate specific message was identified and characterized, leading to the isolation and characterization of SOC-4/CRAC-3 (SEQ ID NOs: 31 and 32).
Functional Assays
Transient Expression of SOC/CRAC
In our initial transient expression experiments, we expressed or expect to express a SOC/CRAC molecule transiently in RBL-2H3 mast cells, Jurkat T cells, and A20 B-lymphocytes using both electroporation and vaccinia virus-driven expression, and measured the calcium influx produced by depletion of intracellular calcium stores with thapsigargin. Each of the foregoing techniques is well known to those of ordinary skill in the art and can be performed using various methods (see, e.g., Current Methods in Molecular Biology, eds. Ausubal, F. M., et al. 1987, Green Publishers and Wiley Interscience, N.Y., N.Y.). Exemplary methods are described herein.
Depletion of intracellular calcium stores is accomplished by treating the cells with 1 micromolar thapsigargin; alternative agents which function to deplete intracellular stores are described in by Putney, J. W. Jr., in Capacitative Calcium Entry, R.G. Landes Co. and Chapman & Hall, 1997 and include, for example, ionomycin, cyclopiazonic acid, and DBHQ.
Calcium influx is determined by measuring cytoplasmic calcium as indicated using the fura-2 fluorescent calcium indicator (see, e.g., G. Grynkiewicz, M. Poenie, R. Y. Tsien, A new generation of Ca2+ indicators with greatly improved fluorescence properties, J. Biol Chem 260, 3440–50 (1985), and M. Poenie, R. Tsien, Fura-2: a powerful new tool for measuring and imaging [Ca2+]i in single cells, Prog Clin Biol Res 210, 53–6 (1986)).
Patch Clamp Analysis and Determining Selectivity of SOC/CRAC
Patch clamp analysis of cells injected with SOC/CRAC cRNA is performed by using the general patch technique as described in Neher, E., “Ion channels for communication between and within cells”, Science, 1992; 256:498–502. Specific techniques for applying the patch clamp analysis to RBL cells are described in Hoth, M., and Penner, R., “Depletion of intracellular calcium stores activates a calcium current in mast cells”, Nature, 1992; 355:3535–355. Additional protocols for applying the patch clamp technique to other cell types are described in Putney, J. W. Jr., in Capacitative Calcium Entry, R.G. Landes Co. and Chapman & Hall, 1997
An exemplary protocol for patch clamp analysis of SOC/CRAC molecule expressed in RBL-2H3 mast cells using a recombinant vaccinia virus is as follows. The currents elicited by store depletion are determined using the whole cell configuration (Neher, E., Science 1992; 256:498–502). Currents in SOC/CRAC expressing cells are compared to currents in control cells expressing an irrelevant protein or a classic Trp family calcium channel known as VR1 (M. J. Caterina, et al., The capsaicin receptor: a heat-activated ion channel in the pain pathway [see comments], Nature 389, 816–24 (1997)) in order to assess the contribution of SOC/CRAC expression. In addition, the magnitude of whole cell currents in the presence of extracellular calcium (10 mM), barium (10 mM), or magnesium (10 mM) are compared to determine the relative permeability of the channels to each of these ions (Hoth, M., and Penner, R., Nature, 1992; 355:3535–355) and, thereby, determine the ionic selectivity.
Pharmacologic Behavior of SOC/CRAC
For analysis of the pharmacologic behavior of a SOC/CRAC molecule, a SOC/CRAC molecule is expressed in RBL-2H3 mast cells using a recombinant vaccinia virus, and the degree of calcium influx elicited by store depletion is monitored using a bulk spectrofluorimeter or a fluorescence microscope and the calcium sensitive dye fura-2 (G. Grynkiewicz, M. Poenie, R. Y. Tsien, A new generation of Ca2+ indicators with greatly improved fluorescence properties, J Biol Chem 260, 3440–50 (1985) and M. Poenie, R. Tsien, Fura-2: a powerful new tool for measuring and imaging [Ca2+]i in single cells, Prog Clin Biol Res 210, 53–6 (1986)). The level of cytoplasmic calcium in SOC/CRAC expressing cells is compared to the level achieved in control cells expressing an irrelevant protein or a classic Trp. family calcium channels known as VR1 (M. J. Caterina, et al., The capsaicin receptor: a heat-activated ion channel in the pain pathway [see comments], Nature 389, 816–24 (1997)). These cells then are pre-incubated with the desired pharmacologic reagent, and again the response to store depletion is monitored. Comparison of the effect of depleting stores in SOC/CRAC expressing cells relative to controls in the presence or absence of the pharmacologic reagent is used to assess the ability of that reagent to modulate SOC/CRAC activity. Sphingosine is an exemplary molecule that can be used as pharmacologic reagents for pharmacologic characterization of SOC/CRAC calcium channels. See, e.g., Mathes, C., et al., Calcium release activated calcium current as a direct target for sphingosine, J Biol Chem 273(39):25020–25030 (1998). Other non-specific calcium channel inhibitors that can be used for this purpose include SKR96365 (Calbiochem) and Lanthanum.
Bulk Calcium Assays
Bulk calcium assays can be performed in a PTI Deltascan bulk spectrofluorometer using fura-2 as described in Scharenberg A M, et al., EMBO J, 1995, 14(14):3385–94.
Gene Targeting
The method (and reagents) described by Buerstedde μM et al, (Cell, 1991, October 4;67(1):179–88), was used to generate “knockouts” in cells. Briefly, part of the chicken SOC-2/CRAC-1 genomic sequence coding for the transmembrane region was cloned utilizing the human sequence as the probe in a chicken library screen. Chicken SOC-2/CRAC-1 clones were isolated and characterized using standard methodology. The putative exon and domain arrangement of the chicken SOC-2/CRAC-1, is depicted in FIG. 1. The exons coding for TM5 (pore region) and TM6, were replaced with promoter/antibiotic cassettes (see FIG. 1). These targeting vectors were then used to target (and replace) the endogenous gene in DT-40 cells (chicken B lymphocyte cells).
Results
Example 1
Transient Expression of SOC/CRAC
In the above-identified cell lines and using both of the foregoing expression techniques, SOC/CRAC expression enhances thapsigargin-dependent influx. In addition, SOC/CRAC expression also enhances the amount of intracellular calcium stores. That this effect is likely due to SOC/CRAC acting as a plasma membrane calcium channel can be confirmed by producing an in-frame carboxy-terminal translational fusion with green fluorescent protein followed by confocal microscopy, revealing that SOC/CRAC is expressed predominantly as a plasma membrane calcium channel.
Example 2
Patch Clamp Analysis
The biophysical characteristics of SOC/CRAC enhanced currents when expressed in Xenopus oocytes are determined. SOC/CRAC cRNA injection is able to enhance thapsigargin-dependent whole cell currents. In addition. SOC/CRAC does not alter the reversal potential of these currents and the determination of the Pca/PNa ratio shows that SOC/CRAC channels are highly calcium selective.
Example 3
Pharmacologic Behavior of SOC/CRAC
The pharmacologic behavior of SOC/CRAC is evaluated as described above. SOC/CRAC-enhanced influx is inhibited by sphingosine in a manner that is substantially the same as that of endogenous thapsigargin-dependent calcium influx.
Example 4
Gene Targeting
Transfection of DT-40 cells with the foregoing targeting vectors, selection for antibiotic resistance, and screening, is collectivelly refered to, herein, as a round of targeting. For the first round of targeting SOC-2/CRAC-1, 18/24 clones with homologous recombination of the targeting construct into one of the endogenous SOC-2/CRAC-1 alleles were obtained. On the second round of targeting (in order to target the second allele and therefore generate a homozygous SOC-2/CRAC-1 mutant cell), 0/48 clones were obtained. These results indicate that a “null” SOC-2/CRAC-1 mutation is detrimental to DT-40 cells, and that SOC-2/CRAC-1 is required for cell viability.







TABLE I







Nucleotide Sequences with homologies to SOC/CRAC nucleic acids







Sequences with SEQ ID NOs and GenBank accession numbers:





SEQ ID NO:9, AB001535, AI226731, H18835, AA419592, AA261842,


AA419407, AA592910, D86107, AI098310, AF071787, Z77132, Z83117,


Z68333, AA708532, AA551759, AA932133, R47363, N31660,


AC005538, AA654650, AA370110, AA313170, AA493512, AI670079,


AI671853.
















TABLE II







Amino Acid Sequences with homologies to SOC/CRAC polypeptides







Sequences with SEQ ID NOs and GenBank accession numbers:





SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID


NO:17, SEQ ID NO:19, AB001535, AA592910, D86107, AF071787,


Z77132, Z83117, Z68333, AA708532, AA551759, AA932133, R47363,


N31660, NP003298, CAB00861, NP002411, CAA92726, CAB05572.









All references, patents, and patent documents disclosed herein are incorporated by reference herein in their entirety.
What is claimed is presented below and is followed by a Sequence Listing.

## Claims

1. An isolated nucleic acid molecule, comprising:
a nucleic acid sequence that encodes SEQ ID NO: 30, or a nucleic acid sequence that is fully complementary to the nucleic acid sequence that encodes SEQ ID NO: 30.

2. An isolated nucleic acid molecule comprising SEQ ID NO: 29, or a nucleic acid molecule that is fully complementary to the nucleic acid molecule comprising SEQ ID NO: 29.

3. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:30.

4. A pharmaceutical composition comprising:
an agent comprising the isolated polypeptide of claim 3, and a pharmaceutically acceptable carrier.

5. An isolated nucleic acid molecule comprising nucleotides 73–3714 of SEQ ID NO: 29, or a nucleic acid molecule that is fully complementary to the nucleic acid molecule comprising nucleotides 73–3714 of SEQ ID NO: 29.

6. An isolated nucleic acid molecule comprising SEQ ID NO: 25, or a nucleic acid molecule that is fully complementary to the nucleic acid molecule comprising SEQ ID NO: 25.