Methods and compositions for glycosidase assays

Details Methods and compositions for glycosidase assays Methods and compositions for glycosidase assays

2001-09-14

2003-12-02

Leary, Louise N. (Department: 1654)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S013000, C435S004000, C435S007720, C435S007720, C435S007100

Reexamination Certificate

active

06656699

ABSTRACT:

TECHNICAL FIELD
This application relates to compositions and methods for measurement of glycosidase activity. Additionally, the present invention provides for compositions and methods for determining metastatic and inflammatory states. The present invention further provides compositions and methods for high throughput assays for compounds that affect glycosidase activity.
BACKGROUND OF THE INVENTION
Proteoglycans (PG) are complex macromolecules present on the cell surfaces and in the extracellular matrices of a wide range of cells (
1
-
3
). They are thought to play a major part in chemical signaling between cells. They bind secreted signaling molecules, which can enhance or inhibit the activity of the signaling molecule. Proteoglycans can also bind and regulate the activity of secreted proteins by immobilizing the protein, sterically blocking the activity of the protein, providing a reservoir for delayed release, protecting the protein from proteolytic degradation, or altering the protein for more effective presentation to cell surface receptors.
Proteoglycans are polyanionic substances of high molecular weight and contain many different types of heteropolysaccharide side chains covalently linked to a polypeptide backbone. PG consists of a protein core to which long carbohydrate chains termed glycosaminoglycans (GAG) are covalently attached. GAGs are linear, highly charged polysaccharides composed of a repeating pair of sugars, one of which is always an amino sugar. Formerly, these carbohydrate groups were called mucopolysaccharides, but they are now termed glycosaminoglycans because they can contain derivatives of glucosamine or galactosamine. In principle, proteoglycans have the potential for almost limitless heterogeneity. The underlying repeating pattern of disaccharides in each GAG can be modified by patterns of sulfate groups.
The three major types of GAG found in PG are: 1) hyaluronan (HA), 2) glucosaminoglycans (heparan sulfate (HS), heparin, and keratan sulfate (KS)), and 3) galactosaminoglycans (chondroitin sulfate (CS) and dermatan sulfate (DS)). Approximately 25% of heparan sulfate linear polysaccharides consist of alternating N-acetylated disaccharide units [→4) &agr;-D-GlcNpAc-(1→4)-&bgr;-D-GlcAp(1→] and N-sulfated disaccharides →[4)&agr;-D-GlcNpS-(1→4)-&bgr;-D-GlcAp or &agr;-L-IdoAp(1→]. These polymers are formed by the attachment of a repeating →[4)&agr;-D-GlcNpAc(1→4)-&bgr;-D-GlcAp(1→] disaccharide sequence to a serine residue of a core protein through a tetrasaccharide, glucuronosyl-galactosyl-galactosyl-xylosyl, linkage region. This molecule then undergoes partial N-deacetylation followed by N-sulfation of the newly exposed amino groups, followed by partial C-5 epimerization of D-GlcAp to L-IdoAp, and finally O-sulfation. O-sulfates are always found in proximity to N-sulfates, which enhances the clustering of the sulfate residues and the heterogeneity in chemical composition and charge density of heparan sulfate. A typical HS chain consists of a repeating disaccharide unit of hexuronic acid and D-glucosamine. Heparan sulfate proteoglycans are involved in many biological events such as angiogenesis, blood coagulation, cell adhesion, lipid metabolism, tissue morphogenesis, cell differentiation, and regulation of various growth factors and cytokine activities.
Heparan sulfate proteoglycans are important components of the subendothelial extracellular matrix and the basement membrane of blood vessels (
2
). Basement membranes are continuous sheets of extracellular matrix composed of collagenous and noncollagenous proteins and proteoglycans that separate parenchymal cells from underlying interstitial connective tissue. They have characteristic permeabilities and play a role in maintaining tissue architecture.
In addition to heparan sulfate proteoglycan (HSPG), the basal lamina consists predominantly of a complex network of adhesion proteins, fibronectin, laminin, collagen and vitronectin (
6
). Heparan sulfate (HS) is an important structural component of the basal lamina. Each of the adhesion proteins interacts with HS side chains of HSPG within the matrix. Thus, HSPG functions as a barrier to the extravasation of metastatic and inflammatory cells. Cleavage of HS by the endoglycosidase heparanase produced by metastatic tumor cells and inflammatory cells destroys the filtering properties of the lamina. In addition, the degradation of the HS may assist in the disassembly of the extracellular matrix and thereby facilitate cell migration (
5
) by allowing blood borne cells to escape into the bloodstream.
Heparanase activity has been described in a number of tissues and cell types including liver, placenta, platelets, fibroblasts, neutrophils, activated T and B-lymphocytes, monocytes, and endothelial cells (
7
-
16
).
No sensitive non-radioactive method is currently available for determination of heparanase activity in tissue or biological fluids. There is currently a need for the development of compositions and methods for simple, rapid, and non-radioactive quantitative assays for the detection of glycosidase activity, particularly heparanase activity. There is also a need for treatments and therapeutic compositions for diseases associated with heparanse activities.
SUMMARY OF THE INVENTION
The present invention is directed to methods for the measurement of cellular activities. Additionally, the present invention comprises compositions and methods for diagnosing diseases, preferably the presence of metastases or neoplastic growth, and for determining the metastatic potential for tumors. The present invention furthers comprises compositions and methods for the diagnosis of inflammatory states in vitro and in vivo.
An aspect of the present invention comprises quantitative measurements of glycosidase activity. A preferred method of the present invention comprises assays for glycosidase activity, more preferably endoglycosidase activity, most preferably determination of heparanase activity. The present invention provides for assays which comprise biotinylated HS bound on streptavidin-coated wells binding additional streptavidin molecules provided in a solution, and this binding is inversely proportional to the extent of digestion of the HS (see FIG.
1
). Thus, after digestion by heparanase, HS retains its ability to bind the streptavidin coated in the wells, but HS loses its ability to bind additional streptavidin molecules in solution. By using enzyme-coupled streptavidin, the amount of streptavidin binding biotin-HS following heparanase digestion can be effectively determined, preferably by a color reaction.
The present invention also comprises compositions and methods for screening for compounds that are capable of inhibiting glycosidase activity, preferably heparanase activity. Additionally, the compositions and methods of the present invention may be used in high throughput assays for the identification of compounds capable of inhibiting such enzymatic activity. The present invention also comprises compositions and methods for determining compounds that are capable of inhibiting the metastatic potential of tumors or altered cells and compounds that are capable of inhibiting inflammatory states.
Accordingly, it is an object of the present invention to provide compositions and methods for assays of glycosidase activity that are rapid, simple, and non-radioactive.
Another object of the present invention is to provide compositions and methods for the quantitative measurement of glycosidase activity.
It is another object of the present invention to provide compositions and methods for the measurement of heparanase activity.
Yet another object of the present invention is to provide compositions and methods for screening compounds capable of inhibiting glycosidase activity, particularly heparanase activity.
It is a further object of the present invention to provide compositions and methods for determining the effect of certain compounds on various cellular and enzymatic activities.
It is another o

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