Pancreas-derived serpin

Details Pancreas-derived serpin Pancreas-derived serpin

1999-07-27

2001-03-06

Campbell, Eggerton A. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C530S350000

Reexamination Certificate

active

06197519

ABSTRACT:

FIELD OF THE INVENTION
The present invention is in the field of molecular biology; more particularly, the present invention describes the nucleic acid and amino acid sequences of a novel pancreas-derived serpin.
BACKGROUND OF THE INVENTION
Serpins
Serpins are extracellular, irreversible serine protease inhibitors. As a group, they are defined on the basis of their structural and functional characteristics—high molecular weight, 370-420 amino acid residues, and C-terminal reactive region. Proteins which have been assigned to the serpin family Include the following: &agr;-1 protease inhibitor, &agr;-1-antichymotrypsin, antithrombin III, &agr;-2-antiplasmin, heparin cofactor II, complement C1 inhibitor, plasminogen activator inhibitors 1 and 2, glia derived nexin, protein C inhibitor, rat hepatocyte inhibitors, crmA (a viral serpin which inhibits interleukin 1-&bgr; cleavage enzyme), human squamous cell carcinoma antigen (which may modulate the host immune response against tumor cells), human maspin (which seems to function as a tumor supressor; Zou Z et al (1994) Science 263:526-529), lepidopterian protease inhibitor, leukocyte elastase inhibitor (the only known intracellular serpin), and three orthopoxviruses (which may be involved in the regulation of the blood clotting cascade and/or of the complement cascade in the mammalian host).
In addition, a number of proteins with no known inhibitory activity are also categorized as serpins on the basis of strong sequence and structural similarities. They include bird ovalbumin, angiotensinogen, barley protein Z, corticosteroid binding globulin, thyroxine binding globulin, sheep uterine milk protein, pig uteroferrin-associated protein, an endoplasmic reticulum heat-shock protein (which binds strongly to collagen and could act as a chaperone), pigment epithelium-derived factor, and an estrogen-regulated protein from Xenopus.
The signature pattern for the serpins is based on a well conserved pro-phe sequence which is located ten to fifteen residues C-terminal to the reactive site loop (RSL). The serpin consensus pattern is [LIVMFY]-x-[LIVMFYAC]-[DNQ]-[RKHQS]-[PST]-F-[LIVMFY] [LIVMFYC]-x-[LIVMFAH], and P is found In position 6 of the pattern in most serpins.
Serpins are defined and described in Carrell R and Travis J (1985) Trends Biochem Sci 10:20-24; Carrell R et al (1987) Cold Spring Harbor Symp Quant Biol 52:527-535; Huber R and Carrell RW (1989) Biochemistry 28:8951-8966; and Remold-O'Donneel E (1993) FEBS Lett 315:105-108.
Mode of Action
Protease inhibitors form tight complexes with their target proteases. For instance, small molecule Inhibitors such as tetrapeptide keto esters form a covalent bond with the catalytic site of serine proteases and also interact with substrate-binding subsites. For the Kunitz family of protease inhibitors, extended interactions involving the entire substrate binding surface on both sides of the reactive site are utilized.
The region of a serpin which binds to the target protease is an exposed reactive site loop (RSL). In contrast to the above inhibitors, serpins have mobile RSLs. The RSL sequence from P17 to P8 is highly conserved, and small amino acid with side chains are found at positions P9, P10, P11, P12, and P15 in active inhibitors. Sequence divergence in the hinge region is usually associated with conversion of the molecule from an inhibitor to a substrate. In fact, proteolytic cleavage near the reactive site results in profound structural changes. Cleavage of the characteristic serpin P1-P1′ bond of &agr;1-proteinase inhibitor results in a separation of about 69 Å between the two residues (Loebermann H et al (1984) J Mol Biol 177:531-556). In addition, the peptide loop from P14-P2 (numbering from the active site P1-P1′) is inserted into the middle of the A-sheet. These structural changes are accompanied by pronounced increase in stability to heat- or guanidine-induced denaturation and this change is referred to as the stressed-to-relaxed (S->R) transition. The ability of a serpin to function as an inhibitor may be directly related to its ability to undergo this S->R transition (Bruch M et al (1988) J Biol Chem 263:1662 6-30; Carrell RW et al (1992) Curr Opin Struct Biol 2:438-446). Ovalbumin, a noninhibitor of the serpin family, is unable to undergo this S->R transition.
To determine the role of small amino acids In the hinge region of protease nexin-1, Braxton SM et al (Keystone Symposium, Mar. 11, 1994) replaced glycine at position 331 (P15) with serine, alanine, proline and valine. The G
331
->V mutation was nearly inactive, the G
331
->P was completely inactive, and replacement of G
331
with S and A had a smaller effect on inhibition. P12 (A
334
>V) and P10 (A
336
->V) mutations also significantly reduced activity. These mutagenesis experiments indicate that a portion of the RSL, up to at least P10, must incorporate into the A-sheet in order for PN-1 to act as an inhibitor, and mutations which hinder this structural transition cause PN-1 to act as a substrate.
Discovery
The serpin molecule which is the subject of this application was identified among the cDNAs of a normal pancreas library. The exocrine pancreas produces an abundance of proteolytic enzymes such as trypsin, chymotrypsin, carboxypeptidase and the serine proteases which split whole and partially-digested proteins into polypeptides and smaller moieties. Several elastases and nucleases are also found in the pancreatic juice. Other digestive enzymes produced by the pancreas include pancreatic amylase which digests carbohydrates, and pancreatic lipase, cholesterol esterase, and phospholipase which hydrolyze lipids and fats.
The four molecules which control pancreatic secretion are acetylcholine and the hormones, gastrin, cholecystokinin (CCK), and secretin. Acetylcholine is released from the parasympathetic vagus and other cholinergic nerve endings, gastrin is secreted by cells of the stomach, and CCK and secretin are secreted by the upper small intestine. The gastrointestinal (GI) hormones are absorbed into the blood and transported to the pancreas where they stimulate the secretion of enzymes and of sodium bicarbonate and water (which wash the pancreatic enzymes into the duodenum).
The endocrine pancreas consists of islets of Langerhans, whose cells are separated from the exocrine lobules and are distributed throughout the pancreas. The endocrine cells of the islets secrete hormones which participate in the metabolism of proteins, carbohydrates, and fats.
The major endocrine cells are &agr;, &bgr;, and &dgr; cells; the minor cells are C cells, EC cells, and PP cells. About 15% of the islet cell population are a cells which are located along the periphery of islets and secrete the hormone glucagon. &bgr; cells comprise about 70% of the islet cell population, are located around the center of the islets, and secrete the hormone insulin. &dgr; cells comprise about 10% of the population, are located close to &agr; cells and secrete two different hormones, somatostatin and vasoactive intestinal peptide (VIP). C, EC, and PP cells make up the final 5% of the islet cell population. Although the function of C cells is unknown, EC and PP cells secrete seratonin and pancreatic polypeptide, respectively.
Inflammation of the pancreas or pancreatitis may be classified as either acute or chronic by clinical criteria. With treatment, acute pancreatitis can often be cured and normal function restored. Chronic pancreatitis often results in permanent damage. The precise mechanisms which trigger acute inflammation are not understood. However, some causes in the order of their importance are alcohol ingestion, binary tract disease, post-operative trauma, and hereditary pancreatitis. One theory provides that autodigestion, the premature activation of proteolytic enzymes in the pancreas rather than in the duodenum, causes acute pancreatitis. Any number of other factors including endotoxins, exotoxins, viral infections, ischemia,

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