Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-18
2002-10-15
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300, C536S024330, C536S024310
Reexamination Certificate
active
06465185
ABSTRACT:
Insulin resistance occurs in 25% of non-diabetic, non-obese, apparently healthy individuals, and predisposes them to both diabetes and coronary artery disease. Diabetes mellitus is a major health problem in the United States affecting approximately 7% of the population. The most common form of diabetes mellitus is non-insulin-dependent diabetes mellitus (NIDDM or type II diabetes). Hyperglycemia in type II diabetes is the result of both resistance to insulin in muscle and other key insulin target tissues, and decreased beta cell insulin secretion. Longitudinal studies of individuals with a strong family history of diabetes indicate that the insulin resistance precedes the secretory abnormalities. Prior to developing diabetes these individuals compensate for their insulin resistance by secreting extra insulin. Diabetes results when the compensatory hyperinsulinemia fails. The secretory deficiency of pancreatic beta cells then plays a major role in the severity of the diabetes.
Reaven (1988)
Diabetes
37:1595-607 were the first to have investigated insulin resistant, non-diabetic, healthy individuals from the general population who are non-obese. Strikingly, they observed that 25% of them have insulin resistance that is of a similar magnitude to that seen in type II diabetes patients. These individuals compensate by having insulin levels that are 3-4 times higher than normal. These elevated insulin levels are sufficient to maintain normoglycemia. Others have also confirmed that a large proportion of the non-diabetic population is insulin resistant. These insulin resistant, non-diabetic individuals have a much higher risk for developing type II diabetes than insulin sensitive subjects.
However, even without developing hyperglycemia and diabetes, these insulin resistant individuals pay a significant price in terms of general health. Insulin resistance results in an increased risk for having elevated plasma triglycerides (TG), lower high density lipoproteins (HDL), and high blood pressure, a cluster of abnormalities that have been termed by different investigators as either Syndrome X, the insulin resistance syndrome, or the metabolic syndrome. It is believed that either the hyperinsulinemia, insulin resistance, or both play a direct role in causing these abnormalities. Data from ethnic, family, and longitudinal studies suggest that a major component of resistance is inherited.
The cellular response to insulin is mediated through the insulin receptor (IR), which is a tetrameric protein consisting of two identical extracellular alpha subunits that bind the hormone and two identical transmembrane beta subunits that have intracellular tyrosine kinase activity. When insulin binds to the IR alpha subunit, the beta subunit tyrosine kinase domain is activated, and insulin action ensues. When insulin activates the receptor, the beta subunit is autophosphorylated at the juxtamembrane domain, the tyrosine kinase domain and the C-terminal domain. Subsequently, endogenous substrates including IRS-1, IRS-2 and SHC are tyrosine phosphorylated. These phosphorylated substrates act as docking molecules to activate SH2 domain molecules including: GRB-2 which activates the ras pathway; the p85 subunit of PI-3-kinase; protein tyrosine phosphatase PTP2/SYP; PLC&ggr;/NCK; AKT and others.
PC-1 is a class II transmembrane glycoprotein that is located both on plasma membranes and in the endoplasmic reticulum. PC-1 is the same protein as liver nucleotide pyrophosphatase/alkaline phosphodiesterase I. In addition to muscle tissue, PC-1 has been reported to be expressed in plasma and intracellular membranes of plasma cells, placenta, the distal convoluted tubule of the kidney, ducts of the salivary gland, epididymis, proximal part of the vas deferens, chondrocytes and dermal fibroblasts. PC-1 exists as a disulfide linked homodimer of 230-260 kDa; the reduced form of the protein has a molecular size of 115-135 kDa, depending on the cell type. Human PC-1 is predicted to have 873 amino acids.
PC-1 is inserted into the plasma membrane such that there is a small cytoplasmic amino terminus, and a larger extracellular carboxyl terminus. The extracellular domain of PC-1 has a high cysteine region that is involved in dimer formation, an ATP binding site and enzymatic activity which cleaves sugar-phosphate, phosphosulfate, pyrophosphate, and phosphodiesterase linkages. The active enzyme site for phosphodiesterase and pyrophosphatase contains a key threonine residue, however a mutation of this residue does not impair the ability of PC-1 to inhibit IR function.
Belli et al. (1993)
Eur J Biochem
217(1):421-8 discloses the existence of enzymatically active water-soluble forms of PC-1. Biosynthetic studies revealed a single, monomeric, endoglycosidase-H-sensitive membrane PC-1 precursor, which was gradually converted to a disulphide-bonded, endoglycosidase-H-resistant form. The soluble form of PC-1 does not appear to arise by proteolytic cleavage from the cell surface, although cleavage inside the cell remains a possibility. The data suggest that the most likely site of cleavage is between Pro 152 and Ala 153.
PC-1 levels are increased in fibroblasts from most patients with typical NIDDM and insulin resistance. In addition, overexpression of PC-1 in transfected cultured cells reduces insulin-stimulated tyrosine kinase activity (Goldfine et al. (1998)
Mol Cell Biochem
182:177-184). PC-1 content in fibroblasts negatively correlates with both decreased in vivo insulin sensitivity and decreased in vitro IR autophosphorylation (Frittitta et al. (1998)
Diabetes
47:1095-1100).
In cells from insulin-resistant subjects, insulin stimulation of glycogen synthetase was decreased. PC-1 content is also elevated in fibroblats, muscle and fat of non-diabetic insulin resistant subjects. The elevation of PC-1 content may be a primary factor in the cause of insulin resistance, although the mechanism by which PC-1 inhibits insulin receptor activity is unknown.
Many mechanisms may potentially contribute to insulin resistance. One major mechanism is the impairment of insulin receptor tyrosine kinase (IR-TK) activity, a key step in insulin receptor signalling. Several inhibitors of IR-TK have been associated to insulin resistance. Among them is PC-1, a class II transmembrane glycoprotein that is overexpressed in tissues of insulin resistant subjects. The human PC-1 gene has been assigned to the same chromosomal region (6q22-q23) where both STS D6S290 (which has been linked to type 2 diabetes in Mexican-Americans), and the gene responsible for transient neonatal diabetes map. The identification and characterization of genetic sequences involved in insulin resistance is of great medical interest.
Database References for Genetic Sequences
The human cDNA and encoded amino acid sequence for PC-1 may be accessed in Genbank, M57736 J05654. As a reference, the “K” allele is provided herein as SEQ ID NO:1, and the encoded polypeptide as SEQ ID NO:2. The “Q” allele is provided as SEQ ID NO:3, and the encoded polypeptide as SEQ ID NO:4.
SUMMARY OF THE INVENTION
Human PC-1 nucleic acids and polypeptides are provided, including promoter and intron-exon boundaries. Polymorphic sequences are provided that encode a form of the protein associated with increased insulin resistance, where a naturally occurring polymorphism of interest comprises a lys→glu substitution at position 121 of the protein, in the high cysteine region. Also provided are polymorphisms in the 3′ untranslated region of PC-1. The subject nucleic acids and fragments thereof, encoded polypeptides, and antibodies specific for the polymorphic amino acid sequence are useful in determining a genetic predisposition to insulin resistance. The encoded protein is useful in drug screening for compositions that affect the activity of PC-1 and insulin receptor activity or expression. Screening methods that analyze plasma levels of soluble PC-1 are also provided, where convenient quantitation of PC-1 content is used in diagnosis of insulin resistance.
REFERENCES:
Buckley et al. Genbank Ac
Frittitta Lucia A.
Goldfine Ira
Pizzuti Antonio
Trischitta Vincenzo
Vigneri Riccardo
Bozicevic Field & Francis LLP
Goldberg Jeanine
Instituto di Ricovero e Cura a Carattere Scientifico
Jones W. Gary
Keddie James S.
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