JP4451497B2 - Nucleic acid encoding acetyl coenzyme A transporter protein - Google Patents

Description

（2）配列番号２の情報：
（i）配列の特徴：
（A）長さ：549アミノ酸
（B）型：アミノ酸
（D）トポロジー：直鎖状
（ii）配列の種類：タンパク質
（xi）配列：配列番号２：

不利益にならない開示または新規性喪失の除外に関する陳述
「アセチル基によるシアル酸の修飾によるシアロスフィンゴ脂質の多様性およびアセチル化を担う新規の膜タンパク質のcＤＮＡクローニング（Diversity of Sialosphingolipids With Modification Of Sialic Acid By Acetyl Group And Molecular Cloning Of A cDNA Encoding A Novel Membrane Protein Responsible For Acetylation）」と題された抄録は、1996年８月13〜16日の複合糖質発現についての分子細胞生物学国際シンポジウム（International Symposium on Molecular And Cell Biology Of Glycoconjugate Expression）での登録後すぐに配布された非公式の抄録小冊子に取り込まれた。This invention was made with government support under grant numbers R01 CA 48737 and P01 CA 71932 awarded by the National Cancer Institute of the National Laboratory. The government has certain rights in the invention.
Field of Invention
The present invention relates generally to molecular biology and enzymology, and more specifically to nucleic acids encoding transporter proteins that regulate the transport of acetyl coenzyme A (Ac-CoA) across the membrane.
Background of the Invention
The diversity and complexity of the sugar chain structure in membrane gangliosides is partly caused by the appearance of several different species of sialic acid molecules. N-acetylneuraminic acid and N-glycolylneuraminic acid are the most ubiquitous sialic acids in gangliosides in the brain, but their O-acetylated forms are also LD1, B-series gangliosides (GD3 And GT1b), as well as C-series (NeuAca2-8NeuAca2-8NeuAca2-3Gal-R) gangliosides are found as minor components. Some biological properties are postulated to be related to the modification of sialic acid by O-acetylation. For example, the expression of 9-O-acetylated ganglioside is clearly associated with neuronal differentiation and migration (Stallcup et al.,Cold Spring Harbor Symp. Quant. Biol.48: 761-773 (1983)). 9-O-acetylated GD3, detected by D1.1 antibody in rat brain, was found to be localized in the germ cell region but disappear from post-mitotic cells. This is absent in normal adult brain and migration (Stallcup et al.,Cold Spring Harbor Symp. Quant. Biol.48: 761-773 (1983)). In rat brain, 9-O-acetylated GD3 detected by D1.1 antibody was found to be localized in the germ cell region but disappear from post-mitotic cells. This is absent in the normal adult brain (Levine et al.,J. Neuros.4: 820-831 (1984)). However, in weaver mice, sustained expression of 9-O-acetylated gangliosides in the adult brain was associated with a deletion in cerebellar granule cell migration (Johnstone et al.,J. Neurochem.51: 1655-1657 (1988)). Attachment of the O-acetyl group at the sialic acid residue causes a significant effect on enzymes of sialic acid metabolism such as sialidase. The effect is also seen on virus binding, cell adhesion, and immunogenicity of ganglioside sialic acid residues (for review, Varki,Glycobiology2: 25-40 (1992)).
Despite its importance, the O-acetylation mechanism is not well understood at the molecular and genetic level. A series of studies conducted by Varki's group showed that the generation of O-acetylated gangliosides is not a simple process, but in the same Golgi segment, acceptor ganglioside GD3, acetyl-CoA (Ac-CoA) transporter, acetyltransferase Show that co-localization is required (Varki,Glycobiology2: 25-40 (1992)). In fact, the detection of in vitro acetyltransferase activity is extremely difficult and once the intact cell membrane preparation has been treated with detergents, it has been shown that transfer activity is rapidly lost (Varki and Diaz). ,J. Biol. Chem.260: 6600-6608 (1985)).
Thus, there is a need to isolate and characterize other protein factors involved in the formation of O-acetylated gangliosides. The present invention fulfills this need and also provides related advantages.
Summary of the Invention
According to the present invention, an isolated mammalian acetyl coenzyme A transporter (AT) protein is provided. These AT proteins, or fragments thereof, are useful as immunogens to generate anti-AT antibodies or in therapeutic compositions containing such proteins and / or antibodies. The AT proteins of the present invention are also useful in bioassays to identify agonists and antagonists thereto.
According to the present invention, an isolated nucleic acid encoding a novel AT protein is also provided. Further provided are vectors comprising the nucleic acids of the invention, probes that hybridize to the nucleic acids of the invention, host cells transformed with the nucleic acids of the invention, antisense oligonucleotides to the nucleic acids of the invention, and related compositions. Is done. The nucleic acid molecules described herein can be incorporated into a variety of recombinant expression systems known to those of skill in the art to readily produce isolated recombinant AT protein. In addition, the nucleic acid molecules of the invention are useful as probes for assaying for the presence and / or amount of an AT gene or mRNA transcript in a given sample. The nucleic acid molecules described herein, and oligonucleotide fragments thereof, are also useful as primers and / or templates in PCR reactions for amplifying nucleic acids encoding AT proteins. Also provided are transgenic non-human mammals that express the proteins of the invention.
Antibodies that are immunoreactive with the AT protein of the invention are also provided. These antibodies are useful in diagnostic assays to determine the level of AT protein present in a given sample (eg, tissue sample, western blot, etc.). Antibodies can also be used to purify AT protein from crude cell extracts and the like. Furthermore, these antibodies are believed to be therapeutically useful in vivo to modulate the biological effects of AT proteins.
Also provided are methods and diagnostic systems for determining the level of AT protein in various tissue samples. These diagnostic methods can be used to monitor the level of therapeutically administered AT protein or fragment thereof to facilitate maintenance of a therapeutically effective amount. These diagnostic methods can also be used to diagnose physiological disorders resulting from abnormal levels or abnormally structured AT proteins.
[Brief description of the drawings]
FIG. 1 shows the hydrophobic hydrophilicity plot of SEQ ID NO: 2 (encoding AT-1), analyzed using the Kyte and Doolitte method with a window size of 10. Shaded and black squares indicate the transmembrane domains predicted by the Tmpred and Psort programs, respectively.
FIG. 2A shows [Ac-¹⁴C] Time-dependent uptake of radioactivity from Ac-CoA into semi-intact cells. White circle is HeLa / GT3⁺/ pcDNAI and black circle are HeLa / GT3⁺/ AT-1 is shown.
FIG. 2B shows [Ac-¹⁴C] shows the effect of CoA on time-dependent uptake of radioactivity from semi-intact cells from Ac-CoA.
Detailed Description of the Invention
According to the present invention, isolated mammalian acetyl coenzyme A transporter (AT) proteins, polypeptides, and fragments thereof encoded by the nucleic acids of the present invention are provided. As used herein, the phrase “AT” refers to a mammalian family, preferably human, isolated and / or capable of transporting acetyl coenzyme A (Ac-CoA) across a membrane. Refers to substantially pure protein. The AT proteins of the present invention are further characterized by having the ability to indirectly promote acetylation of sialic acid residues of gangliosides GD3 and GT3. The AT proteins of the present invention include naturally occurring allelic variants thereof encoded by mRNA produced by alternative splicing of the primary transcript, and further include at least one such as, for example, immunogenicity Including active fragments thereof that retain one natural biological activity.
In another embodiment of the invention, the AT proteins referred to herein are those polypeptides that are specifically recognized by antibodies that also specifically recognize the AT protein (preferably human); This includes the sequence set forth in SEQ ID NO: 2. The isolated AT protein of the present invention is free of cellular components and / or contaminants normally associated with the natural in vivo environment.
The proteins of the invention are further characterized by being expressed in at least the following cells: heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. The highest level of expression was detected in pancreatic tissue. Two major mRNA transcripts of AT protein with a size of 3.3 and 4.3 kb were detected in all tissues tested (observed by Northern blot assay). Two mRNA transcripts with a size of 2.7 and 3.4 kb were strongly expressed in AT-1 transfected HeLa cells and were hardly detected in mock transfected HeLa cells. Thus, splice variant cDNA transcripts encoding AT family proteins are clearly contemplated by the present invention.
The predicted amino acid sequence of AT-1 (SEQ ID NO: 2) is a neurotransmitter (for review see Hucho and Tsetlin,J. Neurochem.66: 1781-1792 (1996)), glucose (Davies et al.,Biochem. J.266: 799-808 (1990)), ammonia (Marini et al.,EMBO J.12: 3456-3463 (1994)), and nucleotide-sugars (Eckhardt et al.,Proc. Natl. Acad. Sci. USA93: 7572-7576 (1996); Miura et al.J. Biochem.120: 236-241 (1996); and Abeijon et al.,Proc. Natl. Acad. Sci. USA93: 5963-5968 (1996)), with multiple transmembrane domains typically observed in many transporters. Most transporter proteins contain 6 to 12 hydrophobic sequences that are thought to form a transmembrane α-helix. In the case of AT-1 protein, the presence of at least 6 transmembrane domains is predicted by Psort analysis.
In addition, AT-1 protein was originally found to be involved in dimerization of transcription factors (Landschulz et al.,Science240: 1759-1964 (1988)), including the leucine zipper motif. A very recent study is CMP-NeuAc (Eckhardt et al.,Proc. Natl. Acad. Sci. USA93: 7572-7576 (1996)), UDP-Gal (Miura et al.,J. Biochem.120: 236-241 (1996)), and UDP-GlcNAc (Abeijon et al.,Proc. Natl. Acad. Sci. USA93: 5963-5968 (1996)), nucleotide-sugar transporter proteins such as transporters also have this structure, indicating that these proteins are homodimers in the Golgi membrane. Suggest that it exists. Recently, rat liver Golgi PAPS transporter (Mandon et al.,Biochem. Biophy. Res. Commun.225: 932-938 (1996)) have also been shown to be homodimers, but it is not known whether a leucine zipper structure is present in this particular transporter protein. Thus, it is contemplated herein that AT-1 protein can function as a homodimer with multiple transmembrane domains. However, the molecular weight of AT-1 protein is substantially larger than that of transporters for CMP-NeuAc, UDP-Ga1, and UDP-GlcNAc, indicating that AT-1 protein is a novel AT transporter. It suggests that it may mean a member of the family.
All nucleotide-sugar transporters identified so far have been found to be localized to the Golgi membrane (Miura et al.,J. Biochem.120: 236-241 (1996); Abeijon et al.,Proc. Natl. Acad. Sci. USA93: 5963-5968 (1996); and Bhakdi et al.Infect. Immun.47: 52-60 (1985)). Immunohistochemical studies using antibodies specific for AT-1 have demonstrated that AT-1 protein is diffusely present in the cytoplasm, including the endoplasmic reticulum, Golgi, and possibly the mitochondrial membrane. This observation shows that the AT protein family does not function as an acetyltransferase, since acetyltransferase has been shown to be enriched in the Golgi membrane (Varki,Glycobiology, 2: 25-40 (1992) and Varki and Diaz,J. Biol. Chem.260: 6600-6608 (1985)). Furthermore, the results of in vitro assays using semi-intact cells (see, eg, Example VI and FIG. 2) indicate that AT-1 functions to transport Ac-CoA across the membrane. Indicates a member of a protein family (eg, Ac-CoA transporter).
Homology searches against protein and nucleic acid databases (GenBank, EMBL and Swiss-Prot) were performed on the nucleic acid and amino acid sequences homologous to the AT-1 and amino acid sequences set forth in SEQ ID NO: 1. Although no significant homology with known proteins was detected, the homology search identified two putative proteins with a high degree of homology: having 560 amino acids with 35% amino acid sequence identity. Caenorhabditis elegans T26C5.3 [EMBL, accession number, having 632 amino acids with 47% amino acid sequence identity; and the putative protein of S. cerevisiae [accession numbers Z36088 (EMBL) and P38318 (Swiss Prot)]; Z50859]. Thus, since the AT-1 protein appears to be universal and evolutionarily conserved, and the appearance of sialic acid has not been reported in these two organisms, the AT protein family is It does not appear to function as a sialic acid acetyltransferase. AT-1 transcripts are more widely distributed than O-acetylated gangliosides, supporting the assumption that molecules other than AT-1 protein direct regulation of O-acetylated GD3 expression. The results described herein show that the AT-1 protein functions as an Ac-CoA transporter, and this plays an important role in acetylation processes other than sialic acid acetylation processes in gangliosides. It strongly suggests that it can be achieved.
The use of the terms “isolated” and / or “purified” as a modifier of DNA, RNA, polypeptide, or protein herein and in the claims is the DNA so designed. It is meant that RNA, polypeptide, or protein is produced in such a form by the human hand and is thus separated from their natural in vivo cellular environment. As a result of this human intervention, it is herein described that the recombinant DNAs, RNAs, polypeptides, and proteins of the present invention are not DNAs, RNAs, polypeptides, or proteins as they naturally occur. In the method described in 1. above.
As used herein, “mammal” refers to various species from which the AT protein of the invention is derived (eg, human, rat, mouse, rabbit, monkey, baboon, cow, pig, sheep, dog). , Cats, etc.).
The presently preferred AT protein of the present invention comprises an amino acid sequence that is substantially the same as the protein sequence set forth in SEQ ID NO: 2, as well as a biologically active modified form thereof. One skilled in the art will recognize that multiple residues of the above sequences will leave other chemically, sterically and / or electronically similar residues without substantially altering the biological activity of the resulting protein species. Recognize that a group can be substituted. In addition, larger or smaller polypeptide sequences (eg, splice variants, active fragments of AT, etc.) comprising substantially the same sequence as SEQ ID NO: 2 herein are contemplated.
As used herein, the term “substantially the same amino acid sequence” is at least about 70% identical to a reference amino acid sequence and is comparable in the proteins defined by the reference amino acid sequence. An amino acid sequence that retains the characteristics of functional and biological activity. Preferably, proteins having “substantially the same amino acid sequence” have at least about 80%, more preferably 90% amino acid identity with respect to the reference amino acid sequence; more than about 95% amino acid sequence identity is particularly preferred . However, polypeptides (or as used herein) that occur as splice variants that contain less than the stated level of sequence identity or are modified by conservative amino acid substitutions or by degenerate codon substitutions. The previously mentioned nucleic acids) are also included within the scope of the present invention. A preferred AT protein disclosed herein is human AT-1 (SEQ ID NO: 2).
The term “biologically active” or “functional” as used herein as a modifier of an AT protein of the invention, or a polypeptide fragment thereof, is a functional attribute attributable to AT. A polypeptide that exhibits at least one of the characteristics. For example, one biological activity of AT is the ability to transport acetyl coenzyme A (Ac-CoA) across cell membranes, preferably intracellular organelle membranes (eg, Golgi membrane, endoplasmic reticulum membrane, mitochondrial membrane, etc.). It is. Yet another biological activity of AT is the ability to indirectly promote acetylation of sialic acid residues of gangliosides GD3 and GT3.
Another biological activity of AT is the ability to act as an immunogen for the production of polyclonal and monoclonal antibodies that specifically bind to AT. Therefore, the nucleic acid of the present invention encoding AT is specifically recognized by an antibody that also specifically recognizes an AT protein (preferably human) containing the sequence (At-1) set forth in SEQ ID NO: 2. Encodes a polypeptide. Such activity can be assayed by any method known to those of skill in the art. For example, test polypeptides encoded by AT cDNA can be used to generate antibodies, which are then assayed for their ability to bind to a protein comprising the sequence set forth in SEQ ID NO: 2. If the antibody binds to the test polypeptide and a protein comprising the sequence set forth in SEQ ID NO: 2 with substantially the same affinity, the polypeptide retains the required biological activity.
The AT protein of the present invention can be obtained by a variety of methods well known in the art (eg, recombinant expression systems, precipitation, gel filtration, ion exchange chromatography, reverse phase chromatography, and affinity chromatography described herein). Etc.). Other well-known methods are Deutscher et al.Guide to Protein Purification: Methods in Enzymology182 (Academic Press, (1990)), which is incorporated herein by reference. Alternatively, an isolated polypeptide of the invention can be obtained using well-known recombinant methods, for example, as described in Sambrook et al. (Supra, 1989).
Examples of means for preparing the polypeptides of the present invention include suitable host cells (eg, bacterial cells, yeast cells, amphibian cells (ie, oocytes), or mammals) using methods well known in the art. Expressing nucleic acids encoding AT in animal cells) and again recovering the expressed polypeptide using well-known methods. The AT polypeptides of the invention can be isolated directly from cells transformed with an expression vector as described herein. The polypeptides, biologically active fragments, and functional equivalents thereof of the present invention can also be produced by chemical synthesis. For example, synthetic polypeptides can be generated using Applied Biosystems, Inc. model 430A or model 431A automated peptide synthesizer (Foster City, Calif.) Using chemical products provided by the manufacturer.
Also encompassed by the term AT is an active fragment or polypeptide analog thereof. The term “active fragment” refers to a peptide fragment that is part of a full-length AT protein, provided that this part has an activity that is characteristic of the corresponding full-length protein. For example, an active fragment of an AT protein (eg, a cytoplasmic domain) can have an activity such as the ability to bind Ac-CoA or mediate membrane transport of Ac-CoA after binding to Ac-CoA. . The characteristics of active fragments of AT proteins that elicit an immune response are useful for obtaining anti-AT antibodies. Thus, the present invention also provides active fragments of the AT protein of the present invention, which fragments can be identified using the assays described herein.
The term “polypeptide analog” refers to an amino acid substantially identical to a sequence specifically set forth herein, wherein one or more residues thereof is conservatively substituted with a functionally similar residue. Any polypeptide having a residue sequence and exhibiting the ability to mimic AT as described herein is included. Examples of conservative substitutions include substitution of another nonpolar residue with one nonpolar (hydrophobic) residue (eg, isoleucine, valine, leucine, or methionine), with one polar (hydrophilic) residue Substitution with another polar residue (eg, between arginine and lysine, between glutamine and asparagine, between glycine and serine), another basic residue (eg, lysine, arginine, or histidine) Substitution of one basic residue, or substitution of another acidic residue with one acidic residue (eg, aspartic acid or glutamic acid).
As used herein, the phrase “conservative substitution” also includes the use of chemically derived residues in place of non-derivatized residues, provided that such polypeptides are: Shows the required binding activity. The phrase “chemical derivative” refers to a polypeptide of interest having one or more residues chemically derivatized by reaction of a functional side group. Such a derivatized molecule is, for example, a free amino group that is derivatized to form an amine hydrochloride, p-toluenesulfonyl group, carbobenzoxy group, t-butyloxycarbonyl group, chloroacetyl group, or formyl. Including those molecules that form groups. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters, or other types of esters or hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzyl histidine. Also included as chemical derivatives are those peptides that include one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine; and ornithine can , Lysine may be substituted. The polypeptides of the present invention also have one or more additions and / or deletions of residues as compared to the sequences of the polypeptides set forth herein as long as the required activity is maintained. Any polypeptide having is included.
The present invention also provides an acceptable carrier and any isolated, purified AT polypeptide, active fragment or polypeptide analog thereof, or purified mature protein and active fragment thereof alone. Alternatively, a composition is provided that includes each in combination. These polypeptides or proteins can be obtained recombinantly, chemically synthesized, or purified from natural sources. As used herein, the term “acceptable carrier” refers to any standard pharmaceutical carrier (eg, phosphate buffered saline solution, water, and emulsion (eg, oil / Water, or water / oil emulsions), as well as various types of wetting agents).
According to another embodiment of the present invention, there are provided isolated nucleic acids encoding the AT (acetyl-coenzyme A transporter) protein of the present invention, and fragments thereof. The nucleic acid molecules described herein are useful for producing the proteins of the present invention when such nucleic acids are incorporated into a variety of protein expression systems known to those skilled in the art. Furthermore, such nucleic acid molecules or fragments thereof can be labeled with easily detectable substituents to assay the presence and / or amount of an AT gene or mRNA transcript in a given sample, and hybridization probes Can be used as The nucleic acid molecules described herein, and fragments thereof, are also useful as primers and / or templates in PCR reactions for amplifying genes encoding the proteins of the invention described herein. is there.
The term “nucleic acid” (also referred to as a polynucleotide) includes ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), probes, oligonucleotides, and primers. The DNA can be either complementary DNA (cDNA) or genomic DNA (eg, a gene encoding an AT protein). One means of isolating nucleic acids encoding AT polypeptides is to probe mammalian genomic libraries with natural or artificially designed DNA probes using methods well known in the art. That is. DNA probes derived from the AT gene are particularly useful for this purpose. DNA and cDNA molecules encoding AT polypeptides can be obtained from complementary genomic DNA from mammals (eg, humans, mice, rats, rabbits, pigs, etc.) or other animal sources by methods described in more detail below. Can be used to obtain cDNA, or RNA, or to isolate related cDNA or genomic clones by screening cDNA or genomic libraries. Examples of nucleic acids are RNA, cDNA, or isolated genomic DNA that encodes an AT polypeptide. Such nucleic acids can include, but are not limited to, nucleic acids having substantially the same nucleotide sequence as set forth in SEQ ID NO: 1 (at least nucleotides 388-2034 of SEQ ID NO: 1), or splice variant cDNA sequences thereof. Not.
As used herein, the phrase “splice variant” or “alternatively spliced” when used to describe a particular nucleotide sequence encoding a receptor of the present invention means A cDNA sequence that results from the well-known eukaryotic RNA splicing process. The RNA splicing process involves the removal of introns from eukaryotic primary RNA transcripts and the binding of exons to produce cytoplasmic mature RNA molecules. Methods for isolating splice variant nucleotide sequences are well known in the art. For example, one of skill in the art can use a nucleotide probe derived from a cDNA encoding the AT of SEQ ID NO: 1 to screen a cDNA library or a genomic library as described herein.
In one embodiment of the invention, the cDNA encoding the AT protein of the invention disclosed herein comprises substantially the same nucleotide sequence as set forth in SEQ ID NO: 1. In another embodiment of the invention, the cDNA molecule encoding the protein of the invention comprises the same nucleotide sequence as nucleotides 388-2034 of SEQ ID NO: 1.
As used herein, the term “substantially the same nucleotide sequence” refers to a reference polynucleotide that has sufficient identity to hybridize to a reference nucleotide under moderately stringent hybridization conditions. It has DNA. In one embodiment, the DNA having substantially the same nucleotide sequence as the reference nucleotide sequence encodes an amino acid sequence substantially the same as the amino acid sequence set forth in SEQ ID NO: 2, or a larger amino acid sequence comprising SEQ ID NO: 2. To do. In another embodiment, a DNA having “substantially the same nucleotide sequence” as the reference nucleotide sequence has at least 60% identity with respect to the reference nucleotide sequence. DNA having at least 70% identity, more preferably at least about 90%, even more preferably at least 95% identity to the reference nucleotide sequence is preferred.
The present invention also includes nucleic acids that differ from the nucleic acid set forth in SEQ ID NO: 1 but have the same phenotype. Nucleic acids with similar phenotypes are also referred to as “functionally equivalent nucleic acids”. As used herein, the phrase “functionally equivalent nucleic acid” functions in substantially the same manner and produces the same protein product as the nucleic acids disclosed herein. Includes nucleic acids characterized by significant and unimportant sequence modifications. In particular, a functionally equivalent nucleic acid encodes a polypeptide that is the same as a polypeptide disclosed herein, or a polypeptide having a conservative amino acid modification, or a larger polypeptide comprising SEQ ID NO: 2. Encodes the peptide. For example, conservative modifications include the replacement of a nonpolar residue with another nonpolar residue, or the replacement of a charged residue with a similarly charged residue. These modifications include those recognized by those skilled in the art as modifications that do not substantially alter the tertiary structure of the protein.
Further provided is a nucleic acid encoding an AT polypeptide that does not necessarily hybridize to the nucleic acid of the invention under the specified hybridization conditions due to the degeneracy of the genetic code. A preferred nucleic acid encoding an AT polypeptide of the present invention is comprised of nucleotides that encode substantially the same amino acid sequence as set forth in SEQ ID NO: 2 (ie, AT-1).
Thus, exemplary nucleic acids encoding the AT protein of the present invention can be selected from:
(A) DNA encoding the amino acid sequence set forth in SEQ ID NO: 2;
(B) DNA that hybridizes to the DNA of (a) under moderately stringent conditions, wherein the DNA encodes biologically active AT; or
(C) a DNA degenerate with respect to either (a) or (b) above, wherein the DNA encodes a biologically active AT.
Hybridization refers to the binding of complementary strands of nucleic acids (ie, sense: antisense strand, or probe: target-DNA) to each other via hydrogen bonding, similar to the naturally occurring binding in chromosomal DNA. . The level of stringency used to hybridize a given probe with target-DNA can be readily varied by those skilled in the art.
The phrase “stringent hybridization” is used herein to refer to conditions under which a polynucleic acid sequence hybrid is stable. As is known to those skilled in the art, the stability of a hybrid depends on the melting point of the hybrid (T_m). In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridization reaction is performed under conditions of lower stringency and then varies but performs a higher stringency wash. Reference to hybridization stringency relates to such washing conditions.
As used herein, the phrase “moderately stringent hybridization” refers to about 60% identity, preferably about 75% identity, more preferably about 85% identity to the target DNA. Refers to conditions that allow target-DNA to bind to complementary nucleic acids having identity; greater than about 90% identity to target-DNA is particularly preferred. Preferably, moderately stringent conditions include: hybridization at 42 ° C. in 50% formamide, 5 × Denhardt's solution, 5 × SSPE, 0.2% SDS, followed by washing at 42 ° C. in 0.5 × SSPE, 0.2% SDS Is a condition that is equivalent to.
The phrase “high stringency hybridization” refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids at 0.018 M NaCl at 65 ° C. (ie, the hybrid is 0.018 at 65 ° C. If not stable in M NaCl, it is not stable under high stringency conditions as intended herein. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5 × Denhardt's solution, 5 × SSPE, 0.2% SDS at 42 ° C., followed by washing in 0.1 × SSPE and 0.1% SDS at 65 ° C. .
The phrase “low stringency hybridization” refers to hybridization in 10% formamide at 37 ° C., 5 × Denhardt's solution, 6 × SSPE, 0.2% SDS, followed by washing in 1 × SSPE, 0.2% SDS at 50 ° C. An equivalent condition. Denhardt's solution and SSPE (eg, Sambrook et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989), as well as other suitable hybridization buffers, are well known to those skilled in the art.
As used herein, the term “degenerate” refers to a codon that differs from a reference nucleic acid (eg, SEQ ID NO: 1) by at least one nucleotide, but encodes the same amino acid as the reference nucleic acid. For example, the three nucleotides “UCU”, “UCC”, “UCA”, and “UCG” are degenerate with respect to each other because all four of these codons encode the amino acid serine.
Preferred nucleic acids encoding the polypeptides of the invention will hybridize to substantially the entire sequence of the nucleic acid sequence set forth in SEQ ID NO: 1 under moderately stringent, preferably high stringency conditions.
Site-directed mutagenesis of any region of AT cDNA is contemplated herein for the production of mutant AT cDNA. For example, the Transformer Mutagenesis Kit (available from Clontech) can be used to construct various missense and / or nonsense mutations on AT cDNA, and so forth.
Nucleic acids of the invention can be obtained by various methods well known in the art, such as those described herein using PCR amplification using oligonucleotide primers from various regions of SEQ ID NO: 1. Can be generated.
According to a further embodiment of the present invention, an optionally labeled AT-encoding cDNA or fragment thereof is stored in a library (eg, cDNA, genome, etc.) for additional nucleic acid sequences encoding related novel mammalian AT proteins. ). The construction of mammalian cDNA and genomic libraries, preferably human libraries, is well known in the art. Screening of such cDNA or genomic libraries is first initiated under low stringency conditions, including adjustments to temperatures below about 42 ° C, formamide concentrations below about 50%, and low salt concentrations. The
In one embodiment, the probe-based screening conditions comprise a temperature of about 37 ° C., a formamide concentration of about 20%, and about 5 × standard saline citric acid (SSC; 3M sodium chloride, 0.3M sodium citrate). 20 × SSC, pH 7.0). Such conditions allow the identification of sequences that have a substantial degree of similarity to the probe sequence without requiring complete homology. The phrase “substantial similarity” refers to sequences that share at least 50% homology. Preferably, hybridization conditions are selected that allow identification of sequences having at least about 70% homology with the probe, but distinguish against sequences having a low degree of homology with the probe. As a result, a nucleic acid is obtained having a nucleotide sequence substantially the same (ie, similar) as nucleotides 388-2034 of SEQ ID NO: 1.
As used herein, a nucleic acid “oligonucleotide”, also referred to herein as a probe or primer, is a single-stranded DNA or RNA, or analog thereof, which is SEQ ID NO: At least 14, preferably at least 20, more preferably at least 50 consecutive, the same as (or complementary to) any 14 or more consecutive bases optionally described It has a nucleotide sequence including a base. Preferred regions for constructing the probe include the 5 'and / or 3' coding region of SEQ ID NO: 1. Furthermore, the complete cDNA encoding a region of the AT protein of the present invention, or the complete sequence corresponding to SEQ ID NO: 1 (AT) can be used as a probe. Probes can be labeled by methods well known in the art and used in various diagnostic kits, as described hereinafter.
As used herein, the terms “label” and “indicating means” in various grammatical forms are either directly or indirectly involved in the production of a detectable signal. Refers to single atoms and molecules. Any label or indicating means may be linked to the nucleic acid probe, expressed protein, polypeptide fragment, or antibody molecule of the invention. These atoms or molecules can be used alone or in combination with further reagents. Such labels are well known per se in clinical diagnostic chemistry.
The labeling means can be a fluorescent labeling agent that chemically binds to the antibody or antigen without denaturation to form a fluorescent dye (dye) that is a useful immunofluorescent tracer. For the description of immunofluorescence analysis technology, see DeLuca, “Immunofluorescence Analysis”,Antibody As a Tool, Edited by Marchalonis et al., John Wiley & Sons, Ltd., 189-231 (1982), which is incorporated herein by reference.
In one embodiment, the indicating group is an enzyme (eg, horseradish peroxidase (HRP), glucose oxidase, etc.). In another embodiment, the radioactive element uses a labeling agent. Linkage of labels to substrates (ie, nucleic acid probes, antibodies, polypeptides, and protein labels) is well known in the art. For example, the antibodies of the invention can be labeled by metabolic uptake of a radiolabeled amino acid provided in the culture medium. For example, Galfre et al.Meth. Enzymol.73: 3-46 (1981). Conventional means of coupling by protein binding or activated functional groups are particularly applicable. For example, Aurameas et al.Scand. J. Immunol.8: Addendum 7: 7-23 (1978), Rodwell et al.Biotech.3: 889-894 (1984) and U.S. Pat. No. 4,493,795.
Also provided are antisense oligonucleotides having sequences that can specifically bind to any portion of mRNA encoding an AT polypeptide so as to prevent translation of the mRNA. An antisense oligonucleotide can have a sequence that can specifically bind to any portion of the sequence of a cDNA encoding an AT polypeptide. As used herein, the phrase “specifically binds” refers to complementary nucleic acid sequences through the recognition of complementary nucleic acid sequences and through the formation of hydrogen bonds between complementary base pairs. Includes the ability of nucleic acid sequences to form double helix segments. An example of an antisense oligonucleotide is an antisense oligonucleotide comprising a chemical analog of nucleotides.
An amount of the above-described antisense oligonucleotide that is effective to reduce expression of the AT polypeptide by specifically binding to mRNA encoding the AT polypeptide so as to cross the cell membrane and prevent translation , As well as compositions comprising an acceptable hydrophobic carrier that can pass across the cell membrane are also provided herein. Suitable hydrophobic carriers are described, for example, in US Pat. Nos. 5,334,761; 4,889,953; 4,897,355, and the like. Acceptable hydrophobic carriers that can pass across the cell membrane may also include structures that bind to receptors specific for the selected cell type and are thereby taken up by cells of the selected cell type. The structure can be part of a protein known to bind to a cell type specific receptor.
Antisense oligonucleotide compositions are useful for inhibiting translation of mRNA encoding the polypeptides of the present invention. Synthetic oligonucleotides, or other antisense chemical structures, are designed to bind to and inhibit mRNA translation encoding AT polypeptides, and to express AT-related genes in tissue samples or subjects Useful as a composition to inhibit.
According to another embodiment of the present invention, a kit for detecting the presence of an AT nucleic acid sequence is provided, comprising at least one oligonucleotide (eg, a probe or antisense oligonucleotide according to the present invention). Such kits can be used to detect mutations, duplications, deletions, rearrangements, or aneuploidies in the AT gene.
The present invention provides a means for regulating the level of expression of these polypeptides by using synthetic antisense oligonucleotide compositions (hereinafter referred to as SAOCs) that inhibit the translation of mRNAs encoding AT polypeptides. I will provide a. Synthetic oligonucleotides designed to recognize and selectively bind to mRNA, or other antisense chemical structures, are complementary to a portion of the AT coding strand or the nucleotide sequence shown in SEQ ID NO: 1 To be built. SAOCs are designed to be stable in the bloodstream or stable in laboratory cell culture conditions for administration to a subject by injection or by direct tumor site uptake. SAOC recognizes SAOC by, for example, designing the small, hydrophobic SAOC chemical structure, allowing SAOC to cross the cell membrane, or by the physical and chemical properties of SAOC It is designed to be able to cross the cell membrane to enter the cytoplasm of the cell by a specific transport system in the cell that transports to the cell. In addition, SAOCs only bind to and take up SAOCs within selected cell populations, targeting them to specific selected cell populations by targeting them as recognized by specific cell uptake mechanisms. Can be designed for administration to.
For example, SAOCs can be designed to bind to receptors found only in certain cell types, as described above. SAOC is also designed to recognize and selectively bind to a target mRNA sequence that may correspond to a sequence contained within the sequence shown in SEQ ID NO: 1. SAOC binds to the target mRNA sequence and inhibits translation of the mRNA target sequence, for example, by inducing the digestion of mRNA by RNase I digestion, or by preventing the binding of translational regulators or ribosomes, or Inactivate the target mRNA sequence either by degradation of the target mRNA or by chemical modification, either by inclusion of other chemical structures (eg ribozyme sequences or reactive chemical groups) Designed. SAOC has been shown to be capable of such properties when directed against mRNA targets (Cohen et al.,TIPS, 10: 435 (1989) and Weintraub,Sci. American, January (1990), page 40; both of which are incorporated herein by reference).
According to yet another embodiment of the present invention, there is provided a method for recombinantly producing the AT protein of the present invention by expressing the above nucleic acid sequence in a suitable host cell. Recombinant DNA expression systems that are suitable for producing the AT proteins described herein are well known in the art. For example, the above nucleotide sequence can be incorporated into a vector for further manipulation. As used herein, a vector (or plasmid) is a discrete element used to introduce heterologous DNA into cells for either heterologous DNA expression or replication. Say.
Suitable expression vectors are well known in the art and include vectors capable of expressing such DNA operably linked to regulatory sequences (eg, promoter regions capable of controlling the expression of the DNA). Thus, an expression vector refers to a recombinant DNA or RNA construct (eg, a plasmid, phage, recombinant virus, or other vector that results in expression of the inserted DNA upon incorporation into a suitable host cell). Suitable expression vectors are well known to those of skill in the art and include vectors that are replicable in eukaryotic and / or prokaryotic cells as well as vectors that remain episomal or integrate into the host cell genome. In addition, the vector may include appropriate packaging signals that allow the vector to be packaged by many viral virions (eg, retroviruses, herpesviruses, adenoviruses) resulting in the formation of a “viral vector”. .
As used herein, a promoter region refers to a segment of DNA that controls transcription of DNA to which the DNA is operably linked. The promoter region contains specific sequences that are sufficient for RNA polymerase recognition, binding, and transcription initiation. In addition, the promoter region includes sequences that regulate this recognition, binding, and transcription initiation activity of RNA polymerase. These sequences can be cis-acting or responsive to trans-acting factors. A promoter can be constitutive or regulated depending on the nature of the regulation. Exemplary promoters contemplated for use in the practice of the present invention include the SV40 early promoter, cytomegalovirus (CMV) promoter, mouse mammary tumor virus (MMTV) steroid inducible promoter, Moloney murine leukemia virus (MMLV) promoter Etc.
As used herein, the term “operably linked” refers to DNA and regulatory and effector nucleotide sequences (eg, promoters, enhancers, transcription and translation stop sites, and other signal sequences). A functional relationship with For example, an operable linkage of DNA to a promoter refers to a physical and functional relationship between the DNA and the promoter, whereby transcription of such DNA specifically recognizes and binds to the DNA. And initiated from the promoter by the RNA polymerase that transcribes.
As used herein, expression refers to the process by which polynucleic acids are transcribed into mRNA and translated into peptides, polypeptides, or proteins. Where the polynucleic acid is derived from genomic DNA, expression can include splicing of mRNA when an appropriate eukaryotic host cell or organism is selected.
Prokaryotic transformation vectors are well known in the art and include pBlueskript and phage λZAP vectors (Stratagene, La Jolla, Calif.) And the like. Other suitable vectors and promoters are disclosed in detail in US Pat. No. 4,798,885, issued Jan. 17, 1989, the disclosure of which is hereby incorporated by reference in its entirety.
Other suitable vectors for transformation of E. coli cells include the pET expression vector (see Novagen, US Pat. No. 4,952,496) (eg, pETlla, which is a T7 promoter, T7 terminator, inducible E. coli). E. coli lac operator, and lac repressor gene; and pET12a-c, which contains the T7 promoter, T7 terminator, and E. coli ompT secretion signal). Another suitable vector is pIN-IIIompA2 (Duffaud et al.,Meth. In Enzymology153: 492-507, 1987), which includes the lpp promoter, the lacUV5 promoter operator, the ompA secretion signal, and the lac repressor gene.
Exemplary eukaryotic transformation vectors include a cloned bovine papillomavirus genome, a murine retrovirus cloned genome, and a eukaryotic cassette (eg, the pSV-2 gpt system (Mulligan and Berg ,Nature277: 108-114 (1979)), Okayama-Berg cloning system (Mol. Cell Biol., 2: 161-170, (1982)), as well as the Genetics Institute (Science228: 810-815 (1985)) is available, which provides substantial assurance of the expression of at least some of the proteins of interest in transformed eukaryotic cell lines. Provide sex.
For transfection of mammalian cells, particularly preferred base vectors containing regulatory elements that can be ligated to DNA encoding the AT of the present invention are cytomegalovirus (CMV) promoter-based vectors (eg, pcDNA1 (Invitrogen , San Diego, CA)), MMTV promoter-based vectors (eg, pMAMNeo (Clontech, Palo Alto, Calif.), And pMSG (Pharmacia, Piscataway, NJ), and SV40 promoter-based vectors (eg, pSVβ (Clontech, Palo Alto, CA).
According to another embodiment of the present invention, there is provided a “recombinant cell” comprising a nucleic acid molecule (ie, DNA or mRNA) of the present invention. Methods for transforming suitable host cells, preferably bacterial cells, and more preferably E. coli cells, and methods applicable for culturing this cell containing a gene encoding a heterologous protein are generally described in the art. Are known. For example, Sambrook et al.Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1989).
An exemplary method for introducing (transducing) an expression vector containing a nucleic acid of the invention into a host cell to produce a transduced recombinant cell (ie, a cell containing a recombinant heterologous nucleic acid) is described in the art. (For review, Friedmann,Science244: 1275-1281 (1989); Mulligan,Science260: 926-932 (1993), each of which is hereby incorporated by reference in its entirety). Exemplary methods of transduction include, for example, infection with viral vectors (see, eg, US Pat. Nos. 4,405,712 and 4,650,764), calcium phosphate transfection (US Pat. Nos. 4,399,216 and 4,634,665). Dextran sulfate transfection, electroporation, lipofection (see, eg, US Pat. Nos. 4,394,448 and 4,619,794), cytofection, particle bead bombardment, and the like. The heterologous nucleic acid can optionally contain sequences that permit its extrachromosomal (ie, episomal) maintenance, or the heterologous DNA can be caused to integrate into the host genome (stable maintenance in the host). As an alternative means to ensure).
Host organisms intended for use in the practice of the present invention include organisms in which recombinant production of heterologous proteins has been performed. Examples of such host organisms include bacteria (eg, E. coli), yeasts (eg, Saccharomyces cerevisiae, Candida tropicalis, Hansenula polymorpha, and P. pastoris; eg, US Pat. Nos. 4,882,279, 4,837,184, 4,929,555, and 4,855,231), mammalian cells (eg, HEK293, CHO, and Ltk)^-Cell) and insect cells. A presently preferred host organism is a bacterium. The most preferred bacterium is E. coli.
In one embodiment, the nucleic acid encoding the AT protein of the invention is transferred to mammalian cells in vivo or in vitro using suitable viral vectors well known in the art (eg, retroviral vectors, adenoviral vectors, etc.). Can be delivered either. Furthermore, where it is desired to limit or reduce the in vivo expression of the AT of the invention, the introduction of the antisense strand of the nucleic acid of the invention is contemplated.
Virus-based systems offer the advantage that relatively high levels of heterologous nucleic acid can be introduced into various cells. Suitable viral vectors for introducing AT nucleic acids of the invention encoding AT proteins into mammalian cells (eg, vascular tissue zones) are well known in the art. Examples of these viral vectors include herpes simplex virus vectors (eg, Geller et al., Science, 241: 1667-1669 (1988)), vaccinia virus vectors (eg, Piccini et al., Meth. In Enzymology, 153: 545-563 ( 1987); cytomegalovirus vectors (Mocarski et al., Viral Vectors, Y. Gluzman and SH Hughes, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988, pp. 78-84), Moloney murine leukemia virus vectors (Danos et al., PNAS, USA, 85: 6469 (1980)), adenoviral vectors (eg, Logan et al., PNAS, USA, 81: 3655-3659 (1984); Jones et al., Cell, 17: 683-689 (1979); Berkner, Biotechniques, 6: 616-626 (1988); Cotten et al., PNAS, USA 89,: 6094-6098 (1992); Graham et al., Meth. Mol. Biol., 7: 109-127 (1991)), adeno-associated virus. Vectors, retroviral vectors (eg, US Pat. Nos. 4,405,712 and 4 , 650,764) etc. Particularly preferred viral vectors are adenoviral vectors and retroviral vectors.
For example, in one embodiment of the invention, an adenovirus-transferrin / polylysine-DNA (TfAdpl-DNA) vector complex (Wagner et al., PNAS, USA, 89: 6099-6103 (1992); Curiel et al., Hum. Gene Therapy, 3: 147-154 (1992); Gao et al., Hum. Gene Ther., 4: 14-24 (1993)) is used to introduce heterologous AT nucleic acids into mammalian cells. Any plasmid expression vector described herein can be used in the TfAdpl-DNA complex.
As used herein, a “retroviral vector” refers to a well-known gene transfer plasmid having an expression cassette that encodes a heterologous gene present between two retroviral LTRs. Retroviral vectors typically allow retroviral vectors or RNA to be transcribed using a retroviral vector as a template that is packaged into a viral virion in an appropriate packaging cell line. Including a suitable packaging signal (see, eg, US Pat. No. 4,650,764).
Retroviral vectors suitable for use herein include, for example, US Pat. No. 5,252,479, and WIPO publications WO 92/07573, WO 90/06997, WO 89/05345, WO 92/05266, and WO 92 / 14829 (incorporated herein by reference). These provide a description of methods for efficiently introducing nucleic acids into human cells using such retroviral vectors. Examples of other retrovirus vectors include mouse mammary tumor virus vectors (for example, Shackleford et al., PNAS, USA, 85: 9655-9659 (1988)).
In accordance with yet another embodiment of the present invention, there are provided anti-AT antibodies having specific reactivity with the AT polypeptides of the present invention. Active fragments of antibodies are included within the definition of “antibody”. The antibodies of the present invention can be produced by methods known in the art using AT polypeptides, proteins, or portions thereof as antigens. For example, polyclonal and monoclonal antibodies are described in the art, for example, as described in Harlow and Lane, Antibodies; A Laboratory Manual (Cold Spring Harbor Laboratory (1988)), incorporated herein by reference. It can be produced by known methods. The AT polypeptides of the present invention can be used as immunogens in generating such antibodies. Alternatively, synthetic peptides can be prepared (using commercially available synthesizers) and used as immunogens. Amino acid sequences can be analyzed by methods well known in the art to determine whether they encode the hydrophobic or hydrophilic domains of the corresponding polypeptide. Altered antibodies (eg, chimeric, humanized, CDR-grafted, or bifunctional antibodies) can also be produced by methods well known in the art. Such antibodies can also be produced by hybridoma, chemical synthesis, or recombinant methods, for example, as described in Sambrook et al., Supra, and Harlow and Lane, supra. Both anti-peptide and anti-fusion protein antibodies can be used. (See, eg, Bahouth et al., Trends Pharmacol. Sci. 12: 338 (1991); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989), incorporated herein by reference). )
The antibodies thus produced are used, inter alia, in diagnostic methods and systems for detecting the level of AT protein present in mammals (preferably humans, body samples (eg tissue or blood)). obtain. Such antibodies can also be used for immunoaffinity purification or affinity chromatography purification of the AT protein of the invention. In addition, kono methods are contemplated herein for detecting the presence of AT polypeptides either on the surface of cells or intracellularly (eg, in the nucleus). The method comprises contacting a cell with an antibody that specifically binds to an AT polypeptide under conditions that allow binding of the antibody to the AT polypeptide, detecting the presence of an antibody bound to AT, and Thereby detecting the presence of a polypeptide of the invention on the surface or in the cell. For detection of such polypeptides, the antibodies can be used for in vitro diagnostic methods or in vivo diagnostic imaging methods.
Useful immunological procedures for in vitro detection of target AT polypeptides in a sample include immunoassays using detectable antibodies. Such immunoassays include, for example, ELISA, Pandex microfluorimetric assay, agglutination assay, flow cytometry, serum diagnostic assay, and immunohistochemical staining procedures well known in the art. The antibody can be made detectable by various means well known in the art. For example, a detectable marker can be directly or indirectly attached to the antibody. Useful markers include, for example, radioactive nucleotides, enzymes, fluorophores, chromogens, and chemiluminescent labels.
The anti-AT antibodies of the present invention may be used herein to modulate the activity of AT polypeptides in living animals, humans, or biological tissues or fluids isolated therefrom. Intended. The term “modulation” refers to the ability of a compound to increase (eg, via an agonist) or inhibit (eg, via an antagonist) the biological activity of an AT protein (eg, the Ac-CoA transport activity of AT). . Accordingly, the carrier and a naturally occurring ligand or other AT binding protein contain an amount of antibody having specificity for an AT polypeptide effective to block binding to the AT polypeptide of the invention. Compositions are contemplated herein. For example, a monoclonal antibody directed to an epitope of an AT polypeptide molecule that is present on the surface of a cell and has an amino acid sequence substantially the same as the amino acid sequence of a cell surface epitope of an AT polypeptide comprising the amino acid sequence set forth in SEQ ID NO: Can be useful for this purpose.
The invention further provides a transgenic non-human mammal capable of expressing an exogenous nucleic acid encoding an AT polypeptide. As used herein, the phrase “exogenous nucleic acid” is not native to the host or is present in a host other than its natural environment (eg, one of the genetically engineered DNA constructs). Part) refers to a nucleic acid sequence. In addition to naturally occurring levels of AT, the AT proteins of the invention can be overexpressed, underexpressed, or expressed in an inactive mutant form in a transgenic mammal (eg, Any of the known knockout transgenics).
Also provided is a transgenic non-human mammal capable of expressing a nucleic acid encoding an AT polypeptide that has been mutated so that it does not have normal activity (ie, does not express native AT). The invention also includes a genome comprising an antisense nucleic acid complementary to a nucleic acid encoding an AT polypeptide, wherein the genome is arranged to be transcribed into an antisense mRNA complementary to the mRNA encoding the AT polypeptide. A transgenic non-human mammal is provided. This antisense nucleic acid hybridizes to the mRNA, thereby reducing its translation. The nucleic acid can further include an inducible promoter and / or tissue specific regulatory elements so that expression can be induced or restricted to specific cell types. An example of a nucleic acid is DNA or cDNA having a coding sequence substantially the same as the coding sequence shown in SEQ ID NO: 1. An example of a non-human transgenic mammal is a transgenic mouse.
Animal model systems that illustrate the physiological and functional roles of AT polypeptides are also provided and are produced by creating transgenic animals. Here, the expression of the AT polypeptide is altered using various techniques. Examples of such techniques include the production of a normal or mutant version of a nucleic acid encoding an AT polypeptide by microinjection, retroviral infection, or other means well known to those skilled in the art. Insertion into an appropriate fertilized embryo. (See, eg, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory, (1986)).
Also contemplated herein is the use of a variant or normal version of homologous recombination of an AT gene with a natural locus in a transgenic animal to modulate the regulation or structure of an AT polypeptide. (See Capecchi et al., Science, 244: 1288 (1989); Zimmer et al., Nature, 338: 150 (1989); these are incorporated herein by reference). Homologous recombination techniques are well known in the art. Homologous recombination cannot replace a natural (endogenous) gene with a recombinant or mutated gene to express a natural (endogenous) protein, but for example, altered expression of an AT polypeptide. The resulting animal is capable of expressing the mutant protein.
In contrast to homologous recombination, microinjection adds a gene to the host genome without removing the host gene. Microinjection can produce transgenic animals that can express both endogenous and exogenous AT proteins. Inducible promoters can be linked to the coding region of the nucleic acid to provide a means to regulate the expression of the transgene. Tissue specific regulatory elements can be linked to the coding region to allow tissue specific expression of the transgene. Transgenic animal model systems are useful for in vivo screening of compounds for the identification of specific reagents (ie, agonists and antagonists that activate or inhibit protein responses).
The nucleic acids, oligonucleotides (including antisense) of the present invention, vectors comprising the same, transformed host cells, polypeptides, and combinations thereof, as well as the antibodies of the present invention, the compounds of the AT polypeptides of the present invention Can be used to screen compounds in vitro to determine whether they act as potential agonists or antagonists. These in vitro screening assays provide information regarding the function and activity of the AT polypeptides of the present invention. This can lead to the identification and design of compounds that can specifically interact with one or more types of polypeptides, peptides, or proteins.
In accordance with yet another embodiment of the invention, a method is provided for identifying compounds (eg, antibodies, binding reagents, etc.) that bind to an AT polypeptide. For example, the AT proteins of the present invention can be used in competitive binding assays. Such an assay can apply a large number of compounds to rapid screening to determine which compounds, if any, can bind to the AT protein. Subsequently, more detailed assays have been found to bind to further determine whether such compounds act as modulators (eg, agonists or antagonists) of the proteins of the invention. Can be used.
AT is considered a gene that, when deficient, responds to abnormal neuronal differentiation and / or migration. In addition, abnormal levels (eg, higher or lower levels) of AT protein of the present invention, or abnormally acting AT protein, may prevent breast cancer and cancers from ectoderm origin such as neuroblastoma and melanoma. It is thought to be related to human tumors including.
Accordingly, in another embodiment of the invention, a bioassay for identifying a compound that modulates the activity of an AT polypeptide of the invention is provided. According to this method, a membrane bound to an AT polypeptide of the invention is contacted with Ac-CoA in the presence and absence of a test compound; the activity of the AT protein is monitored following contact with the test compound. , And those substances that cause either increased or decreased transport of Ac-CoA across membranes with AT proteins are identified as functional reagents for modulating AT polypeptides.
In accordance with another embodiment of the present invention, a transformed host cell (either intact or semi-intact cell) that recombinantly expresses an AT polypeptide of the present invention is combined with a test compound. And then its modulating effect is mediated by AT of Ac-CoA (eg, radiolabeled Ac-CoA as described in Example VI) in the presence and absence of the test compound. It can be assessed against the presence of the compound by comparing transport or by comparing the response of a test cell or control cell (ie, a cell that does not express an AT polypeptide).
As used herein, a compound or signal that “modulates” an activity of an AT polypeptide of the invention alters the activity of the AT polypeptide, so that the activity of the AT polypeptide of the invention is A compound or signal that differs in the presence of a compound or signal than in the absence of the compound or signal. In particular, such compounds or signals include agonists or antagonists. Agonists include compounds or signals that activate or increase the function of the AT protein. Alternatively, an antagonist includes a compound or signal that inhibits or otherwise reduces and interferes with the function of the AT protein. Typically, the effect of an antagonist is observed as a block of protein activity induced by an agonist. Antagonists include competing and non-competitive antagonists. Competitive antagonists (or competitive blockers) interact with or near sites specific for Ac-CoA transport. A non-competitive antagonist or blocker inactivates the function of the polypeptide by interacting at a site different from the Ac-CoA transport region of the AT protein of the present invention.
As will be appreciated by those skilled in the art, assay methods for identifying compounds that modulate AT activity generally require comparison with controls. One type of “control” is a cell or culture that has undergone substantially the same treatment as a test cell or test culture exposed to a compound, characterized in that the “control” cell or culture is not exposed to the compound. It is. For example, the type of “control” cell or culture can be a cell or culture that is identical to the transfected cell, except for a “control” cell or culture that does not express the native protein. Thus, the response of the transfected cell to the compound is compared to the response (or lack thereof) of the “control” cell or culture to the same compound under the same reaction conditions.
Thus, according to another embodiment of the present invention, a bioassay is provided for assessing whether a test compound can act as an agonist or antagonist of an AT protein. Here, the bioassay described above includes:
(A) culturing cells comprising:
DNA expressing AT protein or functionally modified form thereof,
Here, the above culturing step is carried out in the presence of at least one compound whose ability to modulate the Ac-CoA transport activity of the AT protein has been determined, and thereafter
(B) monitoring the cells for any increase or decrease in the level of Ac-CoA.
Methods well known in the art for measuring uptake of Ac-CoA, such as the transport of radiolabeled Ac-CoA into intracellular organelles, are used herein to identify agonists and antagonists of AT proteins. Can be used in the bioassays described in. For example, the method described in Example VI can be used to assess the Ac-CoA transport activity of a recombinant AT protein, or a variant and / or analog thereof, expressed in a mammalian host cell. .
As used herein, “ability to modulate Ac protein's Ac-CoA transport activity” induces (agonist) or inhibits AT protein's Ac-CoA transport activity across the intracellular membrane. Refers to a compound that has either ability to (antagonist).
In another embodiment of the invention, a bioassay for assessing whether a test compound can act as an antagonist of the AT protein of the invention or is a functionally modified form of the AT protein described above. Includes the following steps:
(A) culturing cells comprising:
DNA expressing AT protein or functionally modified form thereof,
Here, the above culture step is carried out in the presence of:
Increasing concentrations of at least one compound that is sought to determine its ability to inhibit the Ac-CoA transport ability of an AT protein; and
A fixed concentration of Ac-CoA;
And then
(B) monitoring the level of Ac-CoA transported into the organelle as a function of the concentration of the compound in the cell, thereby indicating the ability of the compound to inhibit AT transport activity.
In step (a) of the above antagonist bioassay, the culturing step is also performed in the presence of:
At a fixed concentration of at least one compound that is required to determine its ability to inhibit the Ac-CoA transport activity of an AT protein;
Increasing concentrations of Ac-CoA.
As used herein, the phrase “intracellular organelle” refers to, for example, the Golgi apparatus, endoplasmic reticulum, mitochondria and the like. Host cells intended for use in the bioassay of the present invention include CV-1 cells, COS cells, HeLa cells and the like. Currently preferred host cells for performing the bioassays of the present invention are HeLa cells as described in Example VI.
In yet another embodiment of the invention, a method for modulating Ac protein-mediated transport activity mediated by AT protein is also contemplated. The above method includes the following steps:
Contacting the AT protein with an effective modulating amount of an agonist or antagonist identified by the bioassay described above.
Also contemplated herein are methods for treating abnormal neuronal differentiation, abnormal neuronal migration, neuroblastoma, or melanoma. The above methods involve administering an effective amount of a compound (agonist or antagonist) identified by the methods described herein. Such compounds are typically administered in a biologically acceptable composition.
Accordingly, the present invention contemplates therapeutic compositions useful for practicing the therapeutic methods described herein. The therapeutic composition of the present invention is a physiologically compatible carrier with an AT-1 modulator, or anti-AT as described herein dissolved or dispersed therein as an active ingredient. Includes -1 antibody. In preferred embodiments, the therapeutic composition is not immunogenic when administered to a mammalian or human patient for therapeutic purposes.
As used herein, the terms “pharmaceutically acceptable”, “physiologically acceptable”, and grammatical variations thereof refer to compositions, carriers, diluents, and reagents. When used, it is used interchangeably and indicates that it can be administered to a mammal without producing unwanted physiological effects such as nausea, dizziness, stomach upset and the like.
The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well known in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; however, suitable for solutions or suspensions in liquid prior to use. Solid forms can also be prepared. The preparation can also be an emulsion.
The active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations of any two or more thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, etc. that enhance the effectiveness of the active ingredient.
The therapeutic composition of the present invention may include pharmaceutically acceptable salts of the components herein. Pharmaceutically acceptable non-toxic salts include, for example, acid addition salts (formed using the free amino group of the polypeptide), formed using inorganic acids such as the following: hydrochloric acid , Hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid Cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like.
Salts formed using free carboxyl groups can also be derived from inorganic bases such as, for example: sodium hydroxide, ammonium hydroxide, potassium hydroxide, etc .; and derived from organic bases such as, for example: Mono-, di-, and tri-alkylamines and tri-arylamines (eg, triethylamine, diisopropylamine, methylamine, dimethylamine, and the like), and optionally ethanolamines (eg, ethanolamine, diethanolamine, etc.) Is replaced by
Physiologically tolerant carriers are well known in the art. Exemplary liquid carriers are free of other ingredients in addition to the active ingredient and water, or are buffered (eg, sodium phosphate at physiological pH, physiological saline, or both (eg, A sterile aqueous solution containing phosphate buffered saline)). Still further, the water-soluble carrier can include one or more buffer salts, and salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes.
Liquid compositions may also include a liquid layer in addition to water, and may include a liquid layer to exclude water. Exemplary additional liquid layers include glycerin, vegetable oils (eg, cottonseed oil), and water-oil emulsions.
As described herein, an “effective amount” is pre-calculated to achieve a desired therapeutic effect, eg, to modulate the Ac-CoA transport activity of an AT protein of the invention. It is a determined amount. The required dosage will vary with the particular treatment and with the duration of treatment desired; however, dosages of between about 10 μg to about 1 mg / kg body weight / day are used for therapeutic treatment. Is predicted. It may be particularly useful to administer such compounds in a depot or long-lasting form, as discussed herein below. A therapeutically effective amount, when administered in a physiologically acceptable composition, is typically the amount of an AT modulator or a compound identified herein. And achieve a plasma concentration of about 0.1 μg / ml to about 100 μg / ml, preferably about 1.0 μg / ml to about 50 μg / ml, more preferably at least about 2 μg / ml, and usually 5 to 10 μg / ml. Enough for. The therapeutic anti-AT antibodies of the present invention can be administered in proportionally appropriate amounts according to the knowledge of the skilled artisan.
All US patents and all publications mentioned in this specification are incorporated by reference in their entirety. The invention will now be described in further detail with reference to the following non-limiting examples.
Example
Unless stated otherwise, the invention was performed using standard procedures such as those described below: Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. New York, USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1989); Davis et al., Basic Method in Molecular Biology. Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques vol 152, edited by SL Berger and AR Kimmerl, Academic Press Inc., San Diego, USA (1987).
Example I
Description of antibodies used in this specification
Monoclonal antibody (mAb) M6703 specific for GT3 having the structure of the C-series polysialoganglioside NeuNAcα2-8NeuNAcα2-8NeuNAcα2-3Galβ1-R was prepared as described in Hirabayashi et al., 1988, J. Biochem., 104: 973-979 Which was incorporated by reference in its entirety). MAb R24 (Pukel et al., 1982, J. Exp. Med., 155: 1133-1147, incorporated herein by reference), which was shown to react with GD3, was obtained from the American Type Culture Collection ( ATCC # HB-8445). MAb D1.1 that recognizes 9-O-acetylated dysialoglioside is described in Levine et al., 1984, J. Neuroscience, 4: 820-831 (incorporated herein by reference). MAB 493D4 established and reacted with 9-O-acetylated GT3 was provided by Dr. S. Fujita (Mitsubishi Kasei Institute of Life Sciences). Polyclonal antibodies against the N-terminal 14 amino acid peptide of the synthesis of AT-1 protein (amino acids 1-14 of SEQ ID NO: 2) were prepared using well-known methods.
Construction of stably transfected recipient cells
This example provides a method for obtaining transfected cell lines that produce gangliosides but not their 9-O-Ac derivatives. The purpose of this procedure is to produce cell lines that express gangliosides GD3 and GT3 but not their 9-O-acetylated derivatives. To clone the novel factors required for the expression of 9-O-acetylated gangliosides, use recipient cells that express the precursor ganglioside but do not express the corresponding 9-O-acetylated ganglioside It was necessary to do. The specific gangliosides studied were NeuAcα2-8NeuAcα2-3Galβ1-4Glcβ1-1'Cer (hereinafter referred to as “GD3”), 9-O-Ac-GD3 (terminal 9-position of neuraminic acid). NeuAcα2-8NeuAcα2-8NeuAcα2-3Galβ1-4Glcβ1-1'Cer (hereinafter referred to as “GT3”), and 9-O-Ac-GT3 (which is acetylated at the 9-position of terminal neuraminic acid) )Met.
Cell line COS-1 / GD3⁺(Which stably expresses ganglioside GD3, but does not express 9-O-Ac-GD3) was developed by Nakayama et al. (J. Biol. Chem., 271: 3684-3691 (incorporated herein by reference)). 1996)). And HeLa / GT3⁺(Stable expression of gangliosides GD3 and GT3 but not 9-O-Ac-GD3 or 9-O-Ac-GT3) as previously described (Nakayama et al., Supra, 1996) Prepared.
Example II
Isolation of cDNA encoding AT-1
CDNA library based on mammalian expression vector pcDNAI-SK-MEL-28 (9-O-AD-GD3⁺(9-O-Ac-GD3⁺) Isolated from human melanoma SK-MEL-28 cells expressing poly (A)⁺(Built using RNA) was purchased from Invitrogen (San Diego, Calif.) (US Pat. No. 5,484,590, issued January 16, 1996, incorporated herein by reference). See; Bierhuzen and Fukuda, Proc. Natl. Acad. Sci., USA, 89: 9326-9330 (1992), incorporated herein by reference).
1.2 × 10⁷COS-1 / GD3⁺Cells were transfected with 20 μg pcDNAI-SK-MEL-28 using Lipofectamine (Life Technologies, Inc.). After 60 hours, the transfected cells producing 9-O-Ac-ganglioside are treated with monoclonal antibody D1.1 that specifically binds to 9-O-Ac-GD3 (incorporated herein by reference, Levine et al., J. Neuros., 4: 820-831 (1984)) and stained by indirect immunofluorescence. Fluorescently labeled cells were isolated using fluorescence activated cell sorting using a FACStar cell sorter (Becton Dickinson).
COS-1 / GD3 stained positive for 9-O-Ac-GD3⁺Cells were harvested and plasmid DNA was isolated using the Hirt procedure (Hirt, J. Mol. Biol., 26: 365-396 (1967), incorporated herein by reference). Plasmid DNA is transformed into the host bacterium MC1061 / P3 in the presence of ampicillin and tetracycline and is well known as described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989)). The method was used to isolate from a bacterial host.
Isolated and pooled plasmids were subjected to sibling selection (see US Pat. No. 5,484,590). Briefly, isolated plasmids are transformed into COS-1 / GD3, where the active pool is continuously reduced.⁺The cells were subjected to sister selection using cells. Transfected cells were screened by indirect immunofluorescence microscopy using monoclonal antibody D1.1. Positively stained cells were collected and plasmid DNA was extracted. After subsequent rounds of sister selection, a single plasmid pcDNAI-AT-1 encoding the factor required for 9-O-Ac-GD3 expression was isolated. This factor was named AT-1 (SEQ ID NO: 1).
Plasmids from selected cloned cells were isolated and the nucleotide sequence of the insert was determined in both directions. The nucleotide sequence is shown in SEQ ID NO: 1 (GenBank accession number D88152).
The cDNA encoding AT-1 contains an open reading frame having 1650 base pairs encoding a 549 amino acid polypeptide (SEQ ID NO: 2) having a calculated molecular weight of 60,906 Da. Homology searches against protein and nucleic acid databases (GenBank, EMBL and Swiss-Prot) showed no significant homology with any known protein. This indicates that the protein encoded by AT-1 is novel. Hydropathy analysis of the AT-1 polypeptide sequence (Kyte and Doolittle (J. Mol. Biol., 157: 105-132 (1982)) has shown that this polypeptide contains several possible transmembrane domains. (See FIG. 1.) Similar analyzes were performed for programs Psort (Nakai et al., Genomics, 14: 897-911 (1992) and Tmpred (Hofmann and Stoffel, Biol. Chem. Hoppe-Seyler, 347: 166 (1993)) These two analyzes showed that the AT-1 polypeptide has a type IIIa membrane protein structure with 6 to 10 transmembrane domains. One polypeptide contains a leucine zipper motif in the transmembrane domain at amino acid residues 144 to 165. It should be noted that leucine zipper motifs are often found in other transporter proteins (eg, Eckhardt 1996, PNAS, USA, 93 7572-7576; Miura et al., 1996, J Biochem (Tokyo), 120:. 236-241; Abeijon et al, 1996, PNAS, USA, 93: 5963-5968 see).
Example III
Expression assay characterizing the effect of AT-1 on O-acetylation of gangliosides
COS-1 / GD3 ⁺ cell
COS-1 / GD3⁺Cells were transiently transfected with pcDNAI (mock transfection) or pcDNAI-AT-1. Sixty hours after transfection, the transfected cells are fixed and immunostained separately by incubation with mAb R24 (anti-GD3) or mAb D1.1 (anti-9-O-acetylated dicialogangioside) This was followed by incubation with rhodamine-conjugated anti-mouse IgG for mAb R24 or fluorescein isothiocyanate-conjugated anti-mouse IgM for mAb D1.1. Although there was no significant difference in R24 reactivity between pseudotransfectants and AT-1 transfectants, the expression of 9-O-acetylated GD3 was found in AT-1 transfectants Strongly detected with mAb D1.1. Only weak puncture staining was detected in pseudo-transfectants. These results indicate that the AT-1 protein of the present invention mediates (influences) 9-O-acetylation of GD3.
HeLa / GT3 + cells
HeLa / GT3⁺Cells were transfected with either pcDNAI (control) or pcDNAI-AT-1 and subsequently immunostained with mAb 493D4 binding to 9-O-Ac-GT3. HeLa / GT3⁺Cells transfected with / AT-1 stained positive for 9-O-Ac-GT3, but HeLa / GT3⁺The / pcDNAI control cells showed no detectable staining. HeLa / GT3⁺The staining of / AT-1 cells was eliminated by alkaline treatment (0.1N NaOH for 15 minutes at room temperature). These results indicate that the AT-1 protein of the present invention mediates (influences) 9-O-acetylation of GT3.
Stablely transfected HeLa / GT3 ⁺ / AT-1 cell establishment
HeLa / GT3⁺Cells were co-transfected with pcDNA-I-AT-1 and pSV2HyB (10: 1) (the latter expression confers resistance to hygromycin B) and then selected for hygromycin B resistance. Transfected cells were screened for the presence of 9-O-Ac-GT3 by immunofluorescence staining with mAb 493D4 as discussed above. One stably transfected clone was isolated and HeLa / GT3⁺It was named / AT-1.
Quantification of total intracellular gangliosides
To confirm that cells transfected with AT-1 synthesize O-acetylated gangliosides, all gangliosides were HeLa / GT3⁺Isolated from cells and from their transient and stable AT-1 transfectants. HeLa / GT3⁺We found that the cells contained only simple gangliosides, GM3, GD3, GT3, and a small amount of putative GQ3, which allowed them to be combined with ganglioside composition after transfection with AT-1 using thin layer chromatography. Ideal for testing.
Analytical thin layer chromatography was performed on precoated thin layer chromatography plates (Kieselgel 60, Merck). The solvent system used was chloroform, methanol, 12 mM MgCl.₂(5: 4: 1, volume ratio). Gangliosides were visualized with resorcinol / HCl reagent. 5 × 10^FiveIsolation of gangliosides from individual transfected cells and TLC-immunostaining was performed as described in Hirabayashi et al., J. Biol. Chem. 104: 973-979 (1988). Ganglioside purified from cells was mounted on plastic plates (Polygram Sil G, Nagel, Doren, Germany) and developed under the same conditions as described above. Plates were subjected to immunostaining with mAb followed by peroxidase-conjugated goat anti-mouse IgG antibody. Peroxidase activity was measured using 4-chloro-1-naphthol / H₂O₂Visualized with. Gangliosides were analyzed simultaneously after deacetylation by alkali treatment.
The results show that the expression of O-Ac-GT3 and putative O-Ac-GQ3 was strongly positive in cells stably transfected with AT-1. Again, no positive spots were observed when gangliosides were subjected to alkaline hydrolysis. HeLa / GT3⁺And HeLa / GT3⁺It is important to note that / AT-1 cells contain similar total ganglioside content. In addition, no visible differences in the composition of neutral and non-acetylated gangliosides were detected using thin layer chromatographic analysis.
Example IV
Northern blot analysis of AT-1 mRNA
The expression of AT-1 mRNA in various human tissues was examined by Northern blot analysis. Poly (A) prepared from stably transfected HeLa cells⁺Samples of RNA were electrophoresed in an agarose gel containing 2.2M formaldehyde and transferred to a nylon filter (Hybond ™ -N, Amersham). Hybridization with human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA was performed as a control experiment. These blots were analyzed with [α- by multiprime DNA labeling system (Amersham)³²After labeling with P] dCTP, it was hybridized with a gel-purified cDNA insert of AT-1.
Two major transcripts of AT-1 with a size of 3.3 and 4.3 kb were detected in all tissues tested, including heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas . Interestingly, pancreatic tissue developed the strongest signal among the tissues tested. In the pancreas, islets of Langerhans were shown by immunohistochemical staining to express O-acetylated sialoglycoconjugates using mAb 493D4 and mAb D1.1. Two transcripts of AT-1 having a size of 2.7 and 3.4 kb were strongly expressed in AT-1 transfected HeLa cells and barely detected in mock transfected HeLa cells.
Example V
Immunohistochemical localization of AT-1 protein
HeLa / GT3⁺In order to study the intracellular localization of AT-1 protein in / AT-1 cells, the inventors have synthesized amino acids 1 to 2 of SEQ ID NO: 2 corresponding to the N-terminus of AT-1. Specific antibodies against 14) were prepared. Stably transfected cells HeLa / GT3⁺/ AT-1 and HeLa / GT3⁺/ pcDNAI was immobilized, permeabilized with saponin, and incubated with an affinity purified rabbit anti-AT-1 antibody specific for AT-1 protein. Subsequently, it was incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG.
HeLa / GT3 using anti-AT-1 antibody⁺Western blot analysis of / AT-1 cells revealed a major positive band of 54 kd and a weak reactive spot of 58 kd. Immunofluorescence analysis using affinity-purified anti-AT-1 peptide showed that this AT-1 protein was localized in the cytoplasm (including endoplasmic reticulum, Golgi apparatus, and mitochondria). This specific staining was not observed with preimmune IgG or in the presence of peptide antigen.
Example VI
Ac-CoA incorporation into semi-intact cells
Since it was difficult to prepare endoplasmic reticulum or Golgi membrane fractions from cultured cells, the Ac-CoA transporter activity in the cells was determined by reference to Bhakdi et al. (Incorporated herein by reference, Infect. Immun., 47: 52-60 (1985)); Kuncan and Schlegel (J. Cell Biol., 67: 160-173 (1993), incorporated herein by reference); Using semi-intact, permeabilized HeLa cells using the method of Kain et al. (J. Biol. Chem., 268: 19640-19649 (1993)), incorporated herein by reference. Treatment with streptolysin O (hereinafter “SLO”, GIBCO BRL) produces pores in the plasma membrane of the cell without any damage to the intracellular membrane. This allows the passage of large molecules such as Ac-CoA through the plasma membrane and then their intracellular The results may be as follows.
Cultured cells were harvested by trypsinization and PBS and calcium-free magnesium-free Hepes (HCMF: 0.14 M NaCl-5 mM KCl-6 mM Glc-0.3 mM Na₂HPO_FourWashed continuously with -10 mM Hepes, pH 7.4). After treatment with activated streptolysin O (SLO, GIBCO BRL) diluted in HCMF for 20 minutes at 28 ° C., the cells were washed and transporter buffer (TB: 25 mM Hepes-KOH-75 mM KOAc-2.5 mM MgOAc-5 mM EGTA-1.8 mM CaCl₂[Ac- in pH 7.2)₁₄C] Ac-CoA was incubated at room temperature for various times. The reaction was quenched by the addition of ice cold TB. After removing the supernatant by centrifugation, the pellet was washed 3 times with TB, solubilized with 1% SDS, and radioactivity was counted.
As shown in FIG. 2, semi-intact cells are expressed as [Ac-¹⁴When incubated with C] Ac-CoA (35 μM), increased radioactivity uptake was clearly demonstrated in cells transfected with AT-1. This effect was not seen with pseudo-transfectants. Upon addition of transporter CoA (350 μM) inhibitor radioactivity uptake was reduced to 60% of control. These results strongly suggest that AT-1 encodes an Ac-CoA transporter.
Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the claims.
Sequence listing
(1) General information:
(I) Applicant: The Burnham Institute and RIKEN (RIKEN)
(Ii) Title of invention: Nucleic acids encoding a family of acetyl coenzyme A transporter proteins, and related products
(Iii) Number of sequences: 2
(Iv) Contact address:
(A) Name: Campbell and Flora LLP
(B) Address: La Jolla Village Drive 4370, Suite 700
(C) City: San Diego
(D) State: California
(E) Country: United States
(F) Zip code: 92122
(V) Computer readout form:
(A) Media type: floppy disk
(B) Computer: IBM PC compatible
(C) OS: PC-DOS / MS-DOS
(D) Software: Patent In Release # 1.0, Version # 1.30
(Vi) Current application data:
(A) Application number:
(B) Application date:
(C) Classification:
(Viii) Agent / office information:
(A) Name: Ramos, Robert Tee.
(B) Registration number: 37,915
(C) Inquiry / Record number: FP-LJ 2436
(Ix) Telephone line information:
(A) Phone: (619) 535-9001
(B) Telefax: (619) 535-8949
(2) Information of SEQ ID NO: 1:
(I) Sequence features:
(A) Length: 2682 base pairs
(B) Type: Nucleic acid
(C) Number of chains: both forms
(D) Topology: Both forms
(Ii) Sequence type: cDNA
(Ix) Sequence features:
(A) Symbols representing features: CDS
(B) Location: 388..2035
(Xi) Sequence: SEQ ID NO: 1:

(2) Information of SEQ ID NO: 2:
(I) Sequence features:
(A) Length: 549 amino acids
(B) Type: Amino acid
(D) Topology: Linear
(Ii) Sequence type: protein
(Xi) Sequence: SEQ ID NO: 2:

Statement regarding exclusion of disclosure or loss of novelty that is not disadvantageous
“Diversity of Sialosphingolipids With Modification Of Sialic Acid By Acetyl Group And Molecular Cloning Of A cDNA Encoding A Novel Membrane Protein The abstract entitled “Responsible For Acetylation” is shortly after registration at the International Symposium on Molecular And Cell Biology Of Glycoconjugate Expression on August 13-16, 1996. Incorporated in an unofficial abstract booklet distributed to.

Claims (31)

An isolated nucleic acid encoding a human acetyl coenzyme A transporter (AT) protein, the nucleic acid comprising:
(A) DNA encoding the amino acid sequence shown in SEQ ID NO: 1;
(B) DNA that hybridizes to the DNA of (a) under highly stringent conditions, wherein the DNA of (b) encodes a biologically active AT;
(C) encodes a protein derived from the amino acid sequence of SEQ ID NO: 2 having one or several amino acid substitutions, additions or deletions in the protein of the amino acid sequence of SEQ ID NO: 2, and the biological of AT Active DNA,
A nucleic acid selected from the group consisting of: The nucleic acid according to claim 1, wherein the nucleic acid encodes a human protein that hybridizes to the AT coding portion of nucleotides 388 to 2034 of SEQ ID NO: 1 under conditions of high stringency and has AT activity. Nucleic acid. 2. The nucleic acid of claim 1, wherein the nucleic acid sequence encodes AT-1 and is the same as nucleotides 388-2034 of SEQ ID NO: 1. The nucleic acid according to claim 1, wherein the nucleic acid is cDNA. A vector comprising the nucleic acid of claim 1. A recombinant cell comprising the nucleic acid of claim 1. An oligonucleotide, the sequence of nucleotides in a nucleic acid sequence set forth in SEQ ID NO: 1, comprising at least 50 contiguous nucleotides capable of specifically hybridizing an oligonucleotide. 8. The oligonucleotide of claim 7, wherein the oligonucleotide is labeled with a detectable marker. A kit for detecting the presence of an AT cDNA sequence, comprising at least one oligonucleotide according to claim 8. An isolated acetyl coenzyme A transporter (AT) protein comprising:
(A) a protein having the amino acid sequence shown in SEQ ID NO: 2 and characterized by the ability to transport acetyl CoA across the membrane; or (b) substitution of one or several amino acids in the amino acid sequence of SEQ ID NO: 2 A protein which is a protein derived from the protein of (a) having an addition or deletion and having the activity of transporting acetyl-CoA across the membrane. 11. The AT protein according to claim 10, wherein the amino acid sequence of the protein has one or several amino acid substitutions, additions or deletions in the protein sequence of SEQ ID NO: 2 and crosses the membrane. A protein derived from the protein (a) having an activity of transporting acetyl CoA. 12. The AT protein of claim 11, wherein the protein has the same amino acid as the human AT-1 protein sequence of SEQ ID NO: 2. 11. The AT protein of claim 10, wherein the protein is:
(A) a DNA having the nucleotide sequence shown in SEQ ID NO: 1; or (b) a DNA that hybridizes under highly stringent conditions to the DNA of (a), the DNA crossing the membrane A protein encoded by a nucleotide sequence that is human AT-1, selected from the group consisting of DNA, that encodes an AT protein having activity to transport acetyl-CoA. 14. The AT protein of claim 13, wherein the protein is human AT and is encoded by the nucleotide sequence set forth in SEQ ID NO: 1. The AT protein according to claim 10, wherein the protein is
A nucleotide sequence that hybridizes under highly stringent conditions to the nucleotide sequence of (a) nucleotides 388-2034 set forth in SEQ ID NO: 1; and (b) (a), the nucleotide sequence comprising: Selected from the group consisting of nucleotide sequences encoding an AT protein having activity of transporting acetyl CoA across the membrane,
A protein encoded by a nucleotide sequence. 2. The AT protein of claim 1, wherein the protein is encoded by the same nucleotide sequence as nucleotides 388-2034 set forth in SEQ ID NO: 1. A method for expression of an AT protein, the method comprising culturing the cell of claim 6 under conditions suitable for the expression of the AT protein. An isolated anti-AT protein antibody having specific reactivity with the AT protein according to claim 10. The antibody according to claim 18, wherein the antibody is a monoclonal antibody. The antibody according to claim 18, wherein the antibody is a polyclonal antibody. A transgenic non-human mammal expressing an exogenous nucleic acid encoding an AT protein, wherein the exogenous nucleic acid is
(A) DNA encoding the amino acid sequence shown in SEQ ID NO: 1;
(B) DNA that hybridizes to the DNA of (a) under highly stringent conditions, wherein the DNA of (b) encodes a biologically active AT;
(C) encodes a protein derived from the amino acid sequence of SEQ ID NO: 2 having one or several amino acid substitutions, additions or deletions in the protein of the amino acid sequence of SEQ ID NO: 2, and the biological of AT Active DNA,
A transgenic non-human animal that is a nucleic acid selected from the group consisting of: The transgenic non-human mammal according to claim 21, wherein the nucleic acid encoding the AT protein hybridizes to the nucleotide sequence shown in SEQ ID NO: 1 under highly stringent conditions, A transgenic non-human mammal having the activity of and wherein the AT protein so expressed is not native AT. The transgenic non-human mammal according to claim 21, wherein the transgenic non-human mammal is a mouse. A method for identifying a nucleic acid encoding a mammalian AT protein, the method comprising the following steps:
8. The step of contacting the oligonucleotide of claim 7 with a sample containing nucleic acid, wherein the contacting step is achieved under high stringency hybridization conditions and hybridizes thereto. Identifying a compound;
Including the method. A method for detecting the presence of a human AT protein in a sample, the method comprising contacting the antibody of claim 18 with a test sample, detecting the presence of an antibody-AT complex. And thereby detecting the presence of human AT protein in the test sample. A primer which is a single-stranded DNA primer for amplification of AT nucleic acid, the primer consisting of at least 14 consecutive nucleotides of the nucleic acid sequence shown in SEQ ID NO: 1 and not exceeding 50 consecutive nucleotides. A bioassay method for assessing whether a test compound can act as an agonist or antagonist for the AT protein according to claim 10, wherein the bioassay method comprises the following steps:
(A) culturing a cell containing DNA expressing an AT protein or a functionally modified form thereof,
Wherein the culturing step is performed in the presence of at least one compound to which the ability to modulate the Ac-CoA transport activity of the AT protein is to be determined, and then (b) the level of Ac-CoA Monitoring the cells for either an increase or a decrease in
A bioassay method . Can the test compound act as an antagonist for the AT protein of claim 10, or a functionally modified form of the AT protein, characterized by having the ability to transport acetyl CoA across the membrane? A bioassay method for evaluating whether the bioassay method comprises the following steps:
(A) culturing a cell containing DNA expressing an AT protein or a functionally modified form thereof,
Where the culturing step is:
Increasing concentrations of at least one compound to which the ability of AT protein to inhibit Ac-CoA transport activity is to be determined, and a constant concentration of Ac-CoA
Carried out in the presence of; and thereafter
(B) monitoring the level of Ac-CoA transported to intracellular organelles as a function of the concentration of the compound in the cell, thereby indicating the ability of the compound to inhibit AT transport activity;
A bioassay method . Can the test compound act as an antagonist for the AT protein of claim 10, or a functionally modified form of the AT protein, characterized by having the ability to transport acetyl CoA across the membrane? A bioassay method for evaluating whether the bioassay method comprises the following steps:
(A) culturing a cell containing DNA expressing an AT protein or a functionally modified form thereof,
Here, the step of culturing comprises
A fixed concentration of at least one compound, and increasing concentrations of Ac-CoA, whose ability to inhibit the Ac-CoA transport activity of an AT protein is to be determined
Carried out in the presence of; and thereafter
(B) monitoring the level of Ac-CoA transported to intracellular organelles as a function of the concentration of the compound in the cell, thereby indicating the ability of the compound to inhibit AT transport activity;
A bioassay method . Can the test compound act as an agonist for the AT protein of claim 10, or a functionally modified form of the AT protein, characterized by having the ability to transport acetyl CoA across the membrane? A bioassay method for evaluating whether the bioassay method comprises the following steps:
(A) culturing a cell containing DNA expressing an AT protein or a functionally modified form thereof,
Here, the step of culturing comprises
Increasing concentrations of at least one compound to which the ability of AT protein to increase Ac-CoA transport activity is to be determined, and a constant concentration of Ac-CoA
Carried out in the presence of; and thereafter
(B) monitoring the level of Ac-CoA transported to intracellular organelles as a function of the concentration of the compound in the cell, thereby indicating the ability of the compound to increase AT transport activity;
A bioassay method . Can the test compound act as an agonist for the AT protein of claim 10, or a functionally modified form of the AT protein, characterized by having the ability to transport acetyl CoA across the membrane? A bioassay method for evaluating whether the bioassay method comprises the following steps:
(A) culturing a cell containing DNA expressing an AT protein or a functionally modified form thereof,
Here, the step of culturing comprises
A fixed concentration of at least one compound, and increasing concentrations of Ac-CoA, whose ability to increase the Ac protein's Ac-CoA transport activity is to be determined
Carried out in the presence of; and thereafter
(B) monitoring the level of Ac-CoA transported to intracellular organelles as a function of the concentration of the compound in the cell, thereby indicating the ability of the compound to increase AT transport activity;
A bioassay method .

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