33167, a novel human hydrolase and uses therefor

Details 33167, a novel human hydrolase and uses therefor 33167, a novel human hydrolase and uses therefor

2000-05-31

2002-12-31

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S006120, C435S252300, C435S325000, C435S320100, C536S023200

Reexamination Certificate

active

06500657

ABSTRACT:

BACKGROUND OF THE INVENTION
The hydrolysis of chemical bonds within molecules is of critical importance in most metabolic (e.g., catabolic and anabolic) pathways in cells. A large family of enzymes which catalyze the cleavage of a bond with the addition of water, termed hydrolases, has been identified. Members of the hydrolase family are found in nearly all organisms, from microbes to plants to humans. Different classes of hydrolases are specific for an array of biological and chemical substrates. Members of the hydrolase family of enzymes include enzymes that hydrolyze ester bonds (e.g., phosphatases, sulfatases, exonucleases, and endonucleases), glycosidases, enzymes that act on ether bonds, peptidases (e.g., exopeptidases and endopeptidases), as well as enzymes that hydrolyze carbon-nitrogen bonds, acid anhydrides, carbon-carbon bonds, halide bonds, phosphorous-nitrogen bonds, sulfur-nitrogen bonds, carbon-phosphorous bonds, and sulfur-sulfur bonds (E. C. Webb ed.,
Enzyme Nomenclature
, pp. 306-450, ©1992 Academic Press, Inc. San Diego, Calif.).
Hydrolases vary widely in primary sequence, substrate specificity, and physical properties. However, despite the lack of sequence homology, hydrolase family members display structural similarities, e.g., conservation of a catalytic site framework. For example, the alpha/beta hydrolase fold is a structural motif that is common to a variety of hydrolytic enzymes including, lipases, e.g., fungal, bacterial and pancreatic lipase, acetylcholinesterases, serine carboxypeptidases, haloalkane dehalogenases, dienelactone hydrolases, A
2
bromoperoxidases, and thioesterases (Schrag, J. et al. (1997)
Meth. Enzymol
. 284:85-107). Enzymes possessing the alpha/beta hydrolase fold have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues (Ollis, D. et al. (1992)
Protein Eng
. 5:197-211). In particular, one conserved feature of the alpha/beta hydrolase fold is a nucleophile-histidine-acid catalytic triad. The identities of the triad residues in alpha/beta hydrolase fold enzymes are quite variable in that serine, aspartate, and cysteine have all been identified as catalytic nucleophiles (Schrag, J. et al. supra).
Hydrolases play important roles in the synthesis and breakdown of nearly all major metabolic intermediates, including polypeptides, nucleic acids, and lipids. As such, their activity contributes to the ability of the cell to grow and differentiate, to proliferate, to adhere and move, and to interact and communicate with other cells. Hydrolases also are important in the conversion of pro-proteins and pro-hormones to their active forms, the inactivation of peptides, the biotransformation of compounds (e.g., a toxin or carcinogen), antigen presentation, and the regulation of synaptic transmission.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of novel members of the family of hydrolase molecules, referred to herein as “hydrolase-1” or “HYDL-1” nucleic acid and protein molecules. The HYDL-1 nucleic acid and protein molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes, e.g., cellular proliferation, growth, differentiation, or migration. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding HYDL-1 proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of HYDL-1-encoding nucleic acids.
In one embodiment, an HYDL-1 nucleic acid molecule of the invention is at least 50%, 55%, 60%, 65%, 70%, 74%, 75%, 80%, 85%, 89%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:1 or 3.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO:1 or 3, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-46 of SEQ ID NO:1. In yet a further embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 995-1332 of SEQ ID NO:1. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO: 1 or 3.
In another embodiment, an HYDL-1 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:2. In a preferred embodiment, an HYDL-1 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 74%, 75%, 80%, 85%, 89%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire length of the amino acid sequence of SEQ ID NO:2.
In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human HYDL- 1. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2. In yet another preferred embodiment, the nucleic acid molecule is at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 653, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300 or more nucleotides in length. In a further preferred embodiment, the nucleic acid molecule is at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 653, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300 or more nucleotides in length and encodes a protein having an HYDL-1 activity (as described herein).
Another embodiment of the invention features nucleic acid molecules, preferably HYDL-1 nucleic acid molecules, which specifically detect HYDL-1 nucleic acid molecules relative to nucleic acid molecules encoding non-HYDL-1 proteins. For example, in one embodiment, such a nucleic acid molecule is at least 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 653, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1 or 3.
In preferred embodiments, the nucleic acid molecules are at least 15 nucleotides (e.g., 15 contiguous nucleotides) in length and hybridize under stringent conditions to the nucleotide molecules set forth in SEQ ID NO:1 or 3 or a complement thereof. In certain embodiments, the nucleic acid molecules are at least 15 nucleotides in length and hybridize under stringent conditions to nucleotides 1-23 and 1002-1332 of SEQ ID NO:1. In another embodiment, the nucleic acid molecules comprise nucleotides 1-23 and 1002-1332 of SEQ ID NO:1. In yet another embodiment, the nucleic acid molecules consist of nucleotides 1-23 and 1002-1332 of SEQ ID NO:1.
In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2., wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:1 or 3 under stringent conditions.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a HYDL-1 nucleic acid molecule, e.g., the coding strand of an HYDL-1 nucleic acid molecule.
Another aspect of the invention provides a vector comprising an HYDL-1 nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. In yet another embodiment, the invention provides a host cell containing a nucleic acid molecule of the invention. The invention also provides a method for producing a protein, preferably an HYDL-1 protein, by culturing in a suitable medium, a host cell, e.g., a mammalian host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.
Another aspect of this invention features isolated or recombinant HY

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