Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-01-04
2003-10-07
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Reexamination Certificate
active
06630295
ABSTRACT:
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to a novel high throughput assay for monitoring polyion (polycation or polyanion) degradation or polymerization and for determining a molecular weight of a polyion. More particularly, the present invention relates to a high throughput assay for monitoring the activity of enzymes which either degrade or synthesize polyions and for screening for potential modulators (inhibitors or activators) of such enzymes, however, physical and chemical degradation/polymerization of polyions and modulators thereof can also be monitored by the method of the present invention. Most, particularly, the present invention relates to a high throughput assay for monitoring the catalytic activity of glycosaminoglycans (GAGs) degrading enzymes and for screening of modulators, especially inhibitors, thereof.
Proteoglycans (PGs):
Proteoglycans (previously named mucopolysaccharides) are remarkably complex molecules and are found in every tissue of the body. They are associated with each other and also with other major structural components, such as collagen and elastin. Some PGs interact with certain adhesive proteins, such as fibronectin and laminin.
Glycosaminoglycans (GAGs):
Glycosaminoglycans (GAGs) proteoglycans are polyanions and hence bind polycations and cations, such as Na
+
and K
+
. This latter ability attracts water by osmotic pressure into the extracellular matrix and contributes to its turgor. GAGs also gel at relatively low concentrations. The long extended nature of the polysaccharide chains of GAGs and their ability to gel, allow relatively free diffusion of small molecules, but restrict the passage of large macromolecules. Because of their extended structures and the huge macromolecular aggregates they often form, they occupy a large volume of the extracellular matrix relative to proteins. Murry R K and Keeley F W; Harper's Biochemistry, 24th Ed. Ch. 57. pp. 667-85.
Heparan sulfate (HS) proteoglycans:
Heparan sulfate (HS) proteoglycans are acidic polysaccharide-protein conjugates associated with cell membranes and extracellular matrices. They bind avidly to a variety of biologic effector molecules, including extracellular matrix components, growth factor, growth factor binding proteins, cytokines, cell adhesion molecules, proteins of lipid metabolism, degradative enzymes, and protease inhibitors. Owing to these interactions, heparan sulfate proteoglycans play a dynamic role in biology, in fact most functions of the proteoglycans are attributable to the heparan sulfate chains, contributing to cell-cell interactions and cell growth and differentiation in a number of systems. It maintains tissue integrity and endothelial cell function. It serves as an adhesion molecule and presents adhesion-inducing cytokines (especially chemokines), facilitating localization and activation of leukocytes. The adhesive effect of heparan sulfate-bound chemokines can be abrogated by exposing the extracellular matrices to heparanase before or after the addition of chemokines. Heparan sulfate modulates the activation and the action of enzymes secreted by inflammatory cells. The function of heparan sulfate changes during the course of the immune response are due to changes in the metabolism of heparan sulfate and to the differential expression of and competition between heparan sulfate-binding molecules. Selvan R S et al.; Ann. NY Acad. Sci. 1996; 797:127-139.
Other PGs and GAGs, such as hyaluronic acid, chondroitin sulfates, keratan sulfates I, II, dermatan sulfate and heparin have also important physiological functions.
GAG degrading enzymes:
Degradation of GAGs is carried out by a battery of lysosomal hydrolases. These include certain endoglycosidases, such as, but not limited to, mammal heparanase (U.S. Pat. No. 5,968,822 for recombinant and WO91/02977 for native human heparanase) and connective tissue activating peptide III (CTAP, WO95/04158 for native and U.S. Pat. No. 4,897,348 for recombinant CTAP) which degrade heparan sulfate and to a lesser extent heparin; heparinase I, II and III (U.S. Pat No. 5,389,539 for the native form and WO95/34635 A1, U.S. Pat. No. 5,714,376 and U.S. Pat. No. 5,681,733 for the recombinant form), e.g., from
Flavobacterium heparinum
and Bacillus sp., which cleave heparin-like molecules; heparitinase T-I, T-II, T-III and T-VI from
Bacillus circulans
(U.S. Pat. No. 5,405,759, JO 4278087 and JP04-278087); &bgr;-glucoronidase; chondroitinase ABC (EC 4.2.2.4) from
Proteus vulgaris,
AC (EC 4.2.2.5) from
Arthrobacter aurescens
or
Flavobacterium heparinum,
B and C (EC 4.2.2) from
Flavobacterium heparinum
which degrade chondroitin sulfate; hyaluronidase from sheep or bovine testes which degrade hyaluronidase and chondroitin sulfate; various exoglycosidases (e.g., &bgr;-glucoronidase EC 3.2.1.31) from bovine liver, mollusks and various bacteria; and sulfatases (e.g., iduronate sulfatase) EC 3.1.6.1 from limpets (
Patella vulgaris
),
Aerobacter aerogens, Abalone entrails
and
Helix pomatia,
generally acting in sequence to degrade the various GAGs.
Heparanase:
One important enzyme involved in the catabolism of certain GAGs is heparanase. It is an endo-&bgr;-glucuronidase that cleaves heparan sulfate at specific interchain sites. Interaction of T and B lymphocytes, platelets, granulocytes, macrophages and mast cells with the subendothelial extracellular matrix (ECM) is associated with degradation of heparan sulfate by heparanase activity. The enzyme is released from intracellular compartments (e.g., lysosomes or specific granules) in response to various activation signals (e.g., thrombin, calcium ionophore, immune complexes, antigens and mitogens), suggesting its regulated involvement in inflammation and cellular immunity. Vlodavsky I et al.; Invasion Metas. 1992; 12(2):112-27.
Cloning and expression of the heparanase gene:
A purified fraction of heparanase isolated from human hepatoma cells was subjected to tryptic digestion. Peptides were separated by high pressure liquid chromatography and micro sequenced. The sequence of one of the peptides was used to screen data bases for homology to the corresponding back translated DNA sequence. This procedure led to the identification of a clone containing an insert of 1020 base pairs (bp) which included an open reading frame of 963 bp followed by 27 bp of 3′ untranslated region and a poly A tail. The new gene was designated hpa. Cloning of the missing 5′ end of hpa was performed by PCR amplification of DNA from placenta cDNA composite. The entire heparanase cDNA was designated phpa. The joined cDNA fragment contained an open reading frame which encodes a polypeptide of 543 amino acids with a calculated molecular weight of 61,192 daltons. Cloning an extended 5′ sequence was enabled from the human SK-hep1 cell line by PCR amplification using the Marathon RACE system. The 5′ extended sequence of the SK-hep1 hpa cDNA was assembled with the sequence of the hpa cDNA isolated from human placenta. The assembled sequence contained an open reading frame which encodes a polypeptide of 592 amino acids with a calculated molecular weight of 66,407 daltons. The cloning procedures are described in length in U.S. Pat. No. 5,968,822, PCT Application No. U.S. Ser. No. 98/17954 and U.S. patent application Ser. Nos. 09/109,386 now abandoned and 09/258,892 now abandoned.
The ability of the hpa gene product to catalyze degradation of heparan sulfate (HS) in vitro was examined by expressing the entire open reading frame of hpa in High five and Sf21 insect cells, and the mammalian human 293 embryonic kidney cell line expression systems. Extracts of infected cells were assayed for heparanase catalytic activity. For this purpose, cell lysates were incubated with sulfate labeled, ECM-derived HSPG (peak I), followed by gel filtration analysis (Sepharose 6B) of the reaction mixture. While the substrate alone consisted of high molecular weight material, incubation of the HSPG substrate with lysates of cells infected with hpa containing virus resulted in a co
Ayal-Hershkovitz Maty
Mayer Raphael
Shemesh Simha
G. E. Ehrlich Ltd.
Insight Strategy & Marketing Ltd.
Sandals William
Yucel Remy
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