Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-19
2001-06-19
Hauda, Karen M. (Department: 1633)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S023200, C530S350000
Reexamination Certificate
active
06248527
ABSTRACT:
FIELD OF THE INVENTION
The invention is directed to methods for diagnosing Type II diabetes or the risk for developing Type II diabetes by detecting alterations in expression, sequence, or function of carboxypeptidase E nucleic acid or protein. The invention is also directed to methods for preventing or treating Type II diabetes by modulating the levels, altering the sequence, or controlling the function of carboxypeptidase E nucleic acid or protein. The invention is also directed to methods for identifying agents that modulate the levels or affect the function of carboxypeptidase E nucleic acid or protein. The invention is also directed to methods using the agents to treat or diagnose Type II diabetes. The invention is also directed to animal models of Type II diabetes using the carboxypeptidase E gene. The invention is also directed to compositions based on carboxypeptidase E nucleic acid or protein useful for treating or diagnosing diabetes, identifying compounds for treating or diagnosing diabetes, and developing models of diabetes.
BACKGROUND OF THE INVENTION
Carboxypeptidase E
Carboxypeptidase E (CPE), known also as carboxypeptidase H and enkephalin convertase, is involved in the processing of various bioactive peptides including peptide hormones and neurotransmitters (Fricker, in
Peptide Biosynthesis and Processing
) Fricker, ed. (pages 199-230 CRC Press, Boca Raton, Fla.) (1991). Many peptide hormones and neurotransmitters are initially produced as precursors that are enzymatically processed into bioactive peptides (Fricker
J. Cell Biochem.
38:279-289 (1988)). Initially, endopeptidases cleave the prohormone precursor at multiple basic amino acid cleavage sites (Varlamov et al.
J. Biochem.
271:13981-13986) (1996)). Then a carboxypeptidase removes the basic amino acids from the C terminus of the peptide to generate either the bioactive product or a precursor to form the C-terminal amide group. This process is important for the production of bioactive peptides in many tissues.
CPE is present in many tissues where peptide biosynthesis occurs including brain, pituitary, and adrenal medulla (Fricker,
J. Cell. Biochem.,
cited above). The activity is localized to secretory granules where CPE exists in membrane and soluble forms (Manser et al.
Biochem. J.
267:517-525 (1990)). CPE does not appear to contain a transmembrane-spanning helical region, which suggests that CPE is membrane bound through another mechanism. A recent study has shown that the C-terminal region of CPE particularly the C-terminal 14 amino acids are required for membrane binding (Varlamov et al.
J. Biol. Chem.
271:6077-6083 (1996)). Using deletion mutation and fusion protein analysis to study membrane binding and targeting, the authors concluded that there were three separate functions within the C-terminal region of CPE. The 51 C-terminal amino acids appear to direct the sorting to the membrane. Another important region, located 23-33 amino acids from the C-terminus appear to be required for proper folding in that protein lacking this region is neither active nor secreted. A third domain, located within the predicted amphiphilic helix of the C-terminal 14 residues was involved with a binding of CPE to membranes.
A high degree of conservation of the C-terminal region among CPE from different species has been shown. The last exon which encodes the C-terminal 32 amino acids is a hundred percent identical in human, rat, mouse, and bovine CPE and contains only 4 conservative substitutions in angler fish CPE (Varlamov,
J. Biochem.
271, cited above).
Within secretory granules, CPE has been shown to be present in several forms having different solubility. Different forms of CPE have been purified to apparent homogeneity (Supattapone et al.
J. Neurochem.
42:1017 1984); and (Fricker et al.
J Biol. Chem.
258:10950 (1983)). Soluble and membrane associated forms have similar enzymatic and physical properties (Pricker,
J. Cell. Biochem.,
cited above). Both forms have the same amino acid sequence at the N-terminal region (Fricker et al.
Nature
323:461 (1986)). It has thus been suggested that differences between soluble and membrane forms may be the result of post-translational modifications of a single precursor protein (Fricker
J. Cell. Biochem.,
cited above). It has been shown that membrane and soluble forms of CPE are synthesized in the rough endoplasmic reticulum and apparently derived from a single mRNA species by post-translational processing. Synthesis of translation products from human CPE mRNA in a reticulocyte lysate in the presence of microsomal membranes produced 3 processed forms of CPE also showing differences in glycosylation (Manser et al., cited above).
Non-insulin-dependent diabetes mellitus
Diabetes mellitus is among the most common of all metabolic disorders, affecting up to 11% of the population by age 70. Type I diabetes (insulin-dependent diabetes mellitus or IDDM) represents about 5 to 10% of this group and is the result of progressive autoimmune destruction of the pancreatic &bgr;-cells with subsequent insulin deficiency.
Type II diabetes (non-insulin dependent diabetes mellitus, or type II diabetes) represents 90-95% of the affected population, more than 100 million people worldwide (King et al. (1988)
Wld. Hlth. Statist. Quart.
41:190-196; Harris et al. (1992)
Diabetes Care
15:815-819), and is associated with peripheral insulin resistance, elevated hepatic glucose production, and inappropriate insulin secretion (DeFronzo, R. A. (1988)
Diabetes
37:667-687). Family studies point to a major genetic component (Newman et al. (1987)
Diabetologia
30:763-768; Kobberling, J. (1971)
Diabetologia
7:46-49; Cook, J. T. E. (1994)
Diabetologia
37:1231-1240). However, few susceptibility genes have been identified.
Familial predisposition for obesity is the major phenotypic risk factor associated with development of type II diabetes in humans. This obesity is usually accompanied by the development of insulin resistance (Naggert et al.
Nature: Genetics
10:135-141 (1995)).
In mice, six different loci on five different chromosomes produce the obesity-diabetes syndrome. These mutations affect not only obesity and insulin resistance but also other neuroendocrine disturbances. One of these mutations (fat/fat) is associated with a lesion in the CPE gene. The fat mutation maps to mouse chromosome 8 close to the gene for CPE. It was first shown that in extracts of fat/fat pancreatic islets and pituitaries, proinsulin processing was severely reduced. This was associated with a ser202pro mutation in the CPE coding region. This mutation was shown to abolish enzymatic activity in vitro. Thus, this mutation was proposed to demonstrate an obesity-diabetes syndrome caused by a defect in a prohormone processing pathway, i.e., in CPE (Naggert et al. cited above). The importance of this mutation in CPE function in mice was further investigated by studying the effects on activity, amount, and properties of CPE with ser to pro, ala, gly, or phe substitutions at amino acid 202. In an in vitro system, phe and pro mutants were enzymatically inactive, could not bind to a substrate, and were not secreted. Ala or gly mutants, however, exhibited normal enzymatic activities. In a mouse pituitary derived cell line, pro and phe mutants were not secreted. Further, they were degraded within several hours. The analysis of CPE from pituitary cells derived from fat/fat mice showed that the natural pro mutant produced in these cells was not secreted but was degraded. These results provided further support for the hypothesis that fat/fat mice are defective in CPE activity because of the ser to pro substitution at amino acid 202. A subsequent study examined CPE activity and peptide processing in several tissues of fat/fat mice. The report found that there is no active CPE in these mice. It was concluded therefore that the ser to pro mutation causes the enzyme to be completely inactive. It was also concluded that the absence of active CPE causes a large decrease in the levels of fully processed peptides, such as th
Chen Hong
Meyer Joanne
Alston & Bird LLP
Chen Shin-Lin
Hauda Karen M.
Millennium Pharmaceuticals Inc.
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