47174, a novel human glycosyltransferase and uses thereof

Details 47174, a novel human glycosyltransferase and uses thereof 47174, a novel human glycosyltransferase and uses thereof

2001-10-09

2004-03-09

Rao, Manjunath (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S004000, C435S015000, C435S069100, C435S183000, C435S193000, C435S226000, C536S023200, C514S789000, C530S350000

Reexamination Certificate

active

06703230

ABSTRACT:

BACKGROUND OF THE INVENTION
A great diversity of oligosaccharide structures and types of glycoconjugates is found in nature, and these are synthesized by a large number of glycosyltransferases. Glycosyltransferases catalyze the synthesis of glycoconjugates, including glycolipids, glycoproteins, and polysaccharides, by transferring an activated mono- or oligosaccharide residue to an existing acceptor molecule for the initiation or elongation of the carbohydrate chain. A catalytic reaction is believed to involve the recognition of both the donor and acceptor by suitable domains, as well as the catalytic site of the enzyme (Amado et al. (1999)
Biochim Biophys Acta
1473:35-53; Kapitonov et al. (1999)
Glycobiology
9:961-78).
Because the glycosylation reaction is highly specific with respect to both the configuration of the sugar residue and the site of the addition, it is expected that unique domain structures for substrate recognition and nucleotide-sugar binding are located within the enzyme molecule. Evidence indicates that formation of many glycosidic linkages is covered by large homologous glycosyltransferase gene families, and that the existence of multiple enzyme isoforms provides a degree of redundancy as well as a higher level of regulation of the glycoforms synthesized (Kapitonov et al. (1999)
Glycobiology
9:961-78).
Glycosylation is the principal chemical modification to proteins as they pass through Golgi vesicles. Glycosyltransferases of the Golgi do not possess an obvious sequence homology which would suggest a common Golgi retention signal. However, they are all membrane proteins and share type II topology, consisting of an amino terminal cytoplasmic tail, a signal anchor transmembrane domain, a stem region, and a large luminal catalyitc domain. The membrane-spanning domain and its flanking regions contain necessary and sufficient information for Golgi retention of these enzymes (Jaskiewicz (1997)
Acta Biochim Pol
44:173-9). ER localized glycosyltransferases can have either a type II topology, like the Golgi glycosyltransferases, or a type I topolgy, e.g., the N-terminus and catalytic domain inside the ER (Kapitonov et al. (1999)
Glycobiology
9:961-78). Some glycosyltransferases are present on the cell surface and are thought to function as cell adhesion molecules by binding oligosaccharide substrates on adjacent cell surfaces or in the extracellular matrix. The best studied of these is beta 1,4-galactosyltransferase, which mediates sperm binding to the egg coat and selected cell interactions with the basal lamina (Shur (1993)
Curr Opin Cell Biol
5:854-63).
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery of a novel glycosyltransferase family member, referred to herein as “47174.” The nucleotide sequence of a cDNA encoding 47174 is shown in SEQ ID NO:1, and the amino acid sequence of a 47174 polypeptide is shown in SEQ ID NO:2. In addition, the nucleotide sequence of the coding region is depicted in SEQ ID NO:3.
Accordingly, in one aspect, the invention features a nucleic acid molecule which encodes a 47174 protein or polypeptide, e.g., a biologically active portion of a 47174 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2. In other embodiments, the invention provides isolated 47174 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:3 In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringent hybridization condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:3, wherein the nucleic acid encodes a full length 47174 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs that include a 47174 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 47174 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 47174 nucleic acid molecules and polypeptides. The invention thus also provides vectors and host cells that express the 47174 nucleic acid molecules and polypeptides of the invention. Transgenic animals expressing 47174 nucleic acid molecules and polypeptides of the invention also are provided.
In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 47174-encoding nucleic acids.
In still another related aspect, isolated nucleic acid molecules that are antisense to a 47174 encoding nucleic acid molecule are provided.
In a preferred embodiment, the 47174 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:1 or 3. In other embodiments, the 47174 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 1500, 2000, 2500, 2550, or more contiguous nucleotides of SEQ ID NO:1 or 3. In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 47174 polypeptides.
In another embodiment, the invention provides 47174 polypeptides. Preferred polypeptides are 47174 proteins having a 47174-associated activity, e.g., a glycosyltransferase activity as described herein. In another aspect, the invention features, 47174 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 47174 mediated or related disorders.
In other embodiments, the invention provides 47174 polypeptides, e.g., a 47174 polypeptide having the amino acid sequence shown in SEQ ID NO:2; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringent hybridization condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, wherein the nucleic acid encodes a full length 47174 protein or an active fragment thereof.
In a preferred embodiment, the 47174 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2. In other embodiments, the 47174 polypeptide is a fragment of at least 15, 20, 50, 100, 150, 200, 300, 400, 500, 600, or more contiguous amino acids of SEQ ID NO:2.
In a related aspect, the invention provides 47174 polypeptides or fragments operatively linked to non-47174 polypeptides to form fusion proteins.
In another aspect, the invention provides methods of screening for agents, e.g., compounds, that modulate the expression or activity of the 47174 polypeptides or nucleic acids, e.g., compounds that modulate neurological activity or function, e.g., central nervous system (CNS) development or function, or that modulate the normal, or aberrant or altered pain response.
In a preferred embodiment, the effect of an agent, e.g., a compound, on the pain response is evaluated by an analgesic test, e.g., the hot plate test, tail flick test, writhing test, paw pressure test, all electric stimulation test, tail withdrawal test, or formalin test.
In a preferred embodiment, the agent, e.g., compound, inhibits 47174 activity.
In a preferred embodiment, the agent, e.g., compound, increases endogenous levels of a 47174 substrate, e.g., glycoconjugates, including glycolipids, glycoproteins, and polysaccharides.
In still another aspect, the invention provides a process for modulating 47174 polypeptide or nucleic acid expression or activity, e.g. using the screened compound

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