Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-10-01
2003-05-06
Monshipouri, M. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252300, C435S325000, C435S320100, C435S006120, C536S023200
Reexamination Certificate
active
06558936
ABSTRACT:
BACKGROUND OF THE INVENTION
Lipids are esters of long chain fatty acids (generally C
14
to C
24
saturated and unsaturated fatty acids in animal fats) and polyols such as glycerol, glycerol phosphates, alkyl glyceryl ethers, glycerol phosphoryl-choline, glycerol phosphoryl-serine, glycerol phosphoryl-ethanolamine, and the like. Lipids, in the form of cell membranes and fats, for example, constitute a significant proportion of animal body weight (e.g., about 5% to 25% of body weight in normal humans).
Lipids are not water-soluble, and generally do not cross biological membranes efficiently by simple diffusion. Dietary lipids are taken up primarily by hydrolysis of fatty acyl moieties from their corresponding polyol moiety and diffusion of the two moieties across the gut wall (although limited uptake of intact lipids occurs). Following absorption, lipids are reformed by reestablishment of ester bonds between polyol and fatty acyl moieties, and lipids are delivered throughout the body in esterified form (generally in lipoprotein-containing particles such as chylomicrons, very low, intermediate, low, and high density lipoprotein particles, and the like). Prior to uptake by cells (either for storage or for metabolism), lipids must again be hydrolyzed in order to facilitate passage across the cell membrane. Thus, enzymes which catalyze formation and hydrolysis of the ester bonds between fatty acyl moieties and polyol moieties of lipids must be present at several physiological locations, and the particular activities catalyzed by these enzymes (‘lipases’ ) varies depending on the physiological location and function of the enzyme.
A number of lipase enzymes have been characterized in various organisms, including in humans. However, it is far from clear that all physiologically relevant lipases have been discovered or characterized. The present invention provides novel nucleotide and amino acid sequence information corresponding to one or more human lipases.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on discovery of human cDNA molecules which encode lipase proteins such as the one herein designated MLip-1. These proteins catalyze formation and cleavage of ester bonds between fatty acyl moieties and glyceride moieties. MLip-1 protein, fragments thereof, derivatives thereof, and variants thereof are collectively referred to herein as polypeptides of the invention or proteins of the invention. Nucleic acid molecules encoding polypeptides of the invention (i.e., nucleic acids encoding MLip-1 protein, fragments thereof, derivatives thereof, and variants thereof) are collectively referred to as nucleic acids of the invention.
The nucleic acids and polypeptides of the present invention are useful as modulating agents in regulating a variety of cellular processes, particularly including processes which involve lipid metabolism and pancreatic function. Accordingly, in one aspect, the present invention provides isolated nucleic acid molecules encoding a polypeptide of the invention or a biologically active portion thereof. The present invention also provides nucleic acid molecules which are suitable as primers or hybridization probes for detection of nucleic acids encoding a polypeptide of the invention.
The invention also includes nucleic acid molecules which are at least 40% (or, for example, 50%, 60%, 70%, 80%, 90%, 95%, or 98% or more) identical to the nucleotide sequence of either of SEQ ID NOs: 1 and 2, or a complement thereof.
The invention includes nucleic acid molecules which include a fragment of at least 56 (or, for example, 58, 60, 70, 80, 100, 125, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, or 2352) consecutive nucleotide residues of either of SEQ ID NOs: 1 and 2, or a complement thereof.
The invention also includes nucleic acid molecules which have a nucleotide sequence encoding a protein having an amino acid sequence that is at least 50% (or, for example, 60%, 70%, 80%, 90%, 95%, or 98% or more) identical to all or residues about 18-467 of the amino acid sequence SEQ ID NO: 3, or a complement thereof.
In certain embodiments, the nucleic acid molecules have the nucleotide sequence of either of SEQ ID NOs: 1 and 2.
Also within the invention are nucleic acid molecules which encode a fragment of a polypeptide having the amino acid sequence of SEQ ID NO: 3, the fragment including at least 17 (or, for example, 18, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 400, or 467) consecutive amino acid residues of SEQ ID NO: 3.
The invention includes nucleic acid molecules which encode a naturally-occurring allelic variant of a polypeptide having the amino acid sequence of SEQ ID NO: 3, wherein the nucleic acid molecule hybridizes under stringent conditions with a nucleic acid molecule having a nucleic acid sequence comprising either of SEQ ID NOs: 1 and 2, or a complement thereof.
The invention also includes nucleic acid molecules that hybridize under stringent conditions with a nucleic acid molecule having the nucleotide sequence of either of SEQ ID NOs: 1 and 2, or a complement thereof. In other embodiments, the nucleic acid molecules are at least 56 (or, for example, 58, 60, 70, 80, 100, 125, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, or 2352) nucleotides in length and hybridize under stringent conditions with a nucleic acid molecule having the nucleotide sequence of either of SEQ ID NOs: 1 and 2, or a complement thereof. In some embodiments, the isolated nucleic acid molecules encode an immature or mature form of a polypeptide of the invention. In other embodiments, the invention provides an isolated nucleic acid molecule which is antisense with respect to the coding strand of a nucleic acid of the invention.
Another aspect of the invention provides vectors, e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention. In a related aspect, the invention provides isolated host cells, e.g., mammalian and non-mammalian cells, containing such a vector or a nucleic acid of the invention. The invention also provides methods for producing a polypeptide of the invention by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector encoding a polypeptide of the invention such that the polypeptide of the invention is produced.
Another aspect of this invention includes isolated or recombinant proteins and polypeptides of the invention. Isolated polypeptides or proteins have an amino acid sequence that is at least about 50% (or, for example, 60%, 75%, 90%, 95%, or 98% or more) identical to all or a portion of the amino acid sequence of SEQ ID NO: 3. Exemplary polypeptides of the invention include a polypeptide having the amino acid sequence SEQ ID NO: 3, a polypeptide having the amino acid sequence of only residues 1 to about 17 of SEQ ID NO: 3 (i.e., the signal peptide of MLip-1), a polypeptide having the amino acid sequence of about residues 18 to 467 of SEQ ID NO: 3 (i.e., mature MLip-1 protein), and a polypeptide corresponding to a solvent-exposed portion of MLip-1 protein (e.g., about amino acid residues 80 to 105 of SEQ ID NO: 3).
Also within the invention are isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 40% (or, for example, 50%, 75%, 85%, or 95% or more) identical to the nucleic acid sequence of either of SEQ ID NOs: 1 and 2, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions with a nucleic acid molecule having the nucleotide sequence of either of SEQ ID NOs: 1 and 2.
Also within the invention are polypeptides which are naturally-occurring allelic variants of a polypeptide that has the amino acid sequence SEQ ID NO: 3, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes under stringent conditions with a nucleic acid molecule having the nucleotide
Kapeller-Libermann Rosana
Khodadoust Mehran
Millennium Pharmaceuticals Inc.
Millennium Pharmaceuticals Inc.
Monshipouri M.
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