Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Utility Patent
1999-06-08
2001-01-02
Sisson, Bradley L. (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091200, C435S069100, C536S023100, C436S094000
Utility Patent
active
06168933
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid molecules encoding a new mammalian protein and to the use of these molecules in the characterization, diagnosis, prevention, and treatment of conditions such as disorders associated with cell proliferation, lipid metabolism and transport.
BACKGROUND OF THE INVENTION
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of biochemical and physiological mechanisms and metabolic pathways. Despite different evolutionary pressures, proteins that regulate the cell cycle in yeast, nematode, fly, rat, and man have common chemical or structural features and modulate the same general activity. Comparisons of human gene sequences with those from other organisms where structure and/or function are known allow researchers to draw analogies and to develop model systems for testing hypotheses. These model systems are of great importance in developing and testing diagnostic and therapeutic agents for human conditions, diseases, and disorders.
The phospholipid transfer protein family, important in phospholipid trafficking, includes phosphatidylinositol transfer protein (PI-TP, identical to yeast SEC14p), non-specific lipid transfer protein (nsL-TP, identical to sterol carrier protein 2), and phosphatidylcholine transfer protein (PC-TP). The sequence of PC-TP is unique and unrelated to other cytosolic lipid transfer proteins. It is encoded by a single gene and appears to be present in all eukaryotes (Geijtenbeek et al. (1996) Biochem. J. 316:49-55). Although amino acid compositions, molecular weights, and elution profiles of bovine and porcine PC-TP differ markedly, they retain similar transfer activity levels suggesting that amino acid composition of PC-TP can be altered without changing activity and specificity (Chen et al. (1991) Biochem. Int. 23:377-382).
PC-TP catalyzes PC intermembrane transfer (Feng and Cohen (1998) J. Lipid Res. 39:1862-1869), and certain PC molecular species are secreted preferentially into bile. Using multivariate analysis, LaMorte et al. (1998; Hepatology 28:631-637) examined the relationship between PC structure and the probability that individual PC species are secreted into the bile. They suggest that the likelihood of a PC being secreted into bile is closely related to its binding affinity for PC-TP.
Bovine liver PC-TP binds one molecule of PC non-covalently. Bound PC is not the predominant molecular species representative of bovine liver. Although PC species carrying a palmitoyl chain at the sn-1 position are abundant in bovine liver, they are rarely found bound as PC-TP. These findings led Geijtenbeek et al. (1996; FEBS Lett. 391:333-335) to suggest that PC-TP may have a role in the metabolism of highly unsaturated, stearoyl-containing PC molecular species.
PC is implicated in many different diseases relating to phospholipid metabolism, including neutral lipid storage disease (NLSD), lung diseases such as atelectasis, edema, and cystic fibrosis, and cholecystitis (Nicholas (1996) Respirology 1:247-257; Griese, et al. (1997) Eur. Respir. J. 10:1983-1988; and Venkataramani, et al. (1998) Am. J. Gastroenterol. 93:434-441). NLSD is a genetic disorder causing abnormalities in the regulation of phospholipid metabolism. Igal and Coleman (1998; J. Lipid Res. 39:31-43) suggest that an underlying regulatory defect in NLSD alters the rates of synthesis and degradation of the major cellular phospholipids including phosphatidylcholine and phosphatidyletha-nolamine.
Phosphatidylinositol-transfer proteins are important in vesicle-trafficking and signal-transduction (Cockcroft (1998) Bioessays 20:423-432; Alb, et al. (1996) Curr. Opin. Cell Biol. 8:534-541). Because PC-TP is a member of the family containing PI-TP, catalyzes PC intermembrane transfer (Feng and Cohen, supra), and is found in many organs (lung, kidney, testis, liver, etc.); PC is implicated in vesicle trafficking disorders and transport disorders. Therefore, the ability to control PC-TP provides the ability to intercede in disease processes.
The discovery of a nucleic acid molecule encoding a phospholipid transfer protein provides new compositions which are useful in the characterization, diagnosis, prevention, and treatment of disorders associated with cell proliferation, lipid metabolism and transport.
SUMMARY OF THE INVENTION
The invention is based on the discovery of a nucleic acid molecule encoding a mammalian protein, phospholipid transfer protein (PTP), which satisfies a need in the art by providing new compositions useful in the characterization, diagnosis, prevention, and treatment of conditions such as disorders associated with cell proliferation, lipid metabolism and transport.
The invention provides an isolated and purified mammalian nucleic acid molecule comprising SEQ ID NO:1 or a fragment thereof (SEQ ID NOs:3-11). The invention further provides fragments homologous to the mammalian nucleic acid molecules from rat identified as SEQ ID NOs:12-16 in the Sequence Listing. The invention also provides a substantially purified mammalian nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:2 or a portion thereof.
The invention further provides an isolated and purified nucleic acid molecule or a fragment thereof which hybridizes under high stringency conditions to the nucleic acid sequence of SEQ ID NO:1. The invention also provides an isolated and purified nucleic acid molecule or a fragment thereof which is complementary to the nucleic acid sequence of SEQ ID NO:1. In one aspect, a single stranded complementary RNA or DNA molecule is used as a probe and hybridizes under high stringency conditions to the mammalian nucleic acid sequence or a fragment thereof.
The invention further provides a method for detecting a nucleic acid molecule in a sample, the method comprising the steps of hybridizing the probe to at least one nucleic acid sequence of the sample, thereby forming a hybridization complex; and detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a nucleic acid sequence in the sample. In one aspect, the method further comprises amplifying the nucleic acid sequence prior to hybridization. The nucleic acid molecule or fragment thereof may comprise either an element or a target on a microarray. The invention also provides a method for using a nucleic acid sequence or a fragment thereof to screen a library of molecules to identify at least one molecule which specifically binds the nucleic acid sequence, the method comprising providing a library of molecules, combining the nucleic acid sequence with the library of molecules under conditions allowing specific binding, and detecting specific binding, thereby identifying a molecule which specifically binds the nucleic acid sequence. Such libraries include DNA and RNA molecules, peptides, PNAs, proteins, and the like which are potential regulators of replication, transcription, and translation. In an analogous method, the nucleic acid molecule or a fragment thereof is used to purify a ligand.
The invention also provides an expression vector containing at least a fragment of the nucleic acid molecule of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell. The invention further provides a method for producing a protein, the method comprising the steps of culturing the host cell under conditions for the expression of the protein and recovering the protein from the host cell culture. The invention also provides an isolated and purified protein comprising the amino acid sequence of SEQ ID NO:2 or a portion thereof. Additionally, the invention provides a pharmaceutical composition comprising a substantially purified protein having the amino acid sequence of SEQ ID NO:2 or a portion thereof in conjunction with a pharmaceutical carrier.
The invention further provides a method for using a portion of the protein to produce antibodies. The invention also provides
Baughn Mariah R.
Hillman Jennifer L.
Kaser Matthew R.
Incyte Genomics, Inc.
Incyte Pharmaceuticals Inc.
Lu Frank
Murry Lynn E.
Sisson Bradley L.
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