Delta 4, 5 glycuronidase and uses thereof

Inactive Publication Date: 2005-09-29
MASSACHUSETTS INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016] In other aspects the invention also provides a method of cleaving a glycosaminoglycan comprised of at least one disaccharide unit. The method may be performed by contacting the glycosaminoglycan with a glycuronidase of the invention in an effective amount to cleave the glycosaminoglycan. In some embodiments the glycosaminoglycan is a long chain saccharide. In other embodiments the glycosaminoglycan does not contain a 2-O sulfated uronidate or it does not contain N-substituted glycosamine. In yet another embodiment the glycosaminoglycan is 6-0 sulfated. The disaccharide units in some embodiments are ΔUHNAc; ΔUHNAc,6S; ΔUHNS,6S; or ΔUHNS. In another embodiment the invention also provides for the products of the cleavage of a glycosaminoglycan with the Δ4,5 glycuronidase. In some embodiments the glycuronidase is used to generate a LMWH.
[0017] The present invention also provides methods for the analysis of glycosaminoglycan. In one aspect the invention is a method of analyzing a glycosaminoglycan by contacting a glycosaminoglycan with the glycuronidase of the invention in an effective amount to analyze the glycosaminoglycan. In one embodiment the method is a method for identifying the presence of a particular glycosaminoglycan in a sample. In another embodiment the method is a method for determining the identity of a glycosaminoglycan in a sample. In yet another embodiment the method is a method for determining the purity of a glycosaminoglycan in a sample. In still a further embodiment the method is a method for de

Problems solved by technology

The high degree of complexity for HSGAGs arises not only from their polydispersity and the possibility of two different uronic acid components, but also from differential modification at four positions of the disaccharide unit.
The ability of HSGAGs to orchestrate multiple biological events is again likely a consequence of its structural complexity and information density [Sasisekharan, R. and Venkataraman, G.
Although the structure and chemistry of HSGAGs are fairly well understood, information on how specific HSGAG sequences modulate different biological processes

Method used

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  • Delta 4, 5 glycuronidase and uses thereof
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  • Delta 4, 5 glycuronidase and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Molecular Cloning of Δ4, 5 Glycuronidase Gene from F. heparinum Genome

[0162] To clone the Δ4, 5 glycuronidase gene, we isolated a series of Δ4, 5 glycuronidase-derived peptides after protease treatment of the purified enzyme. The native enzyme was directly purified from fermentation cultures of F. heparinum using a 5-step chromatography scheme as previously described [McLean, M. W., Bruce, J. S., Long, W. F., and Williamson, F. B., 1984, Eur J Biochem 145, 607-15]. The extent of purity was ultimately characterized by reverse phase chromatography, which indicated a single major peak (FIG. 1A). We were able to generate a number of peptides by a limited trypsin digestion of the purified enzyme. 26 peptide fragments were resolved by reverse phase chromatography (FIG. 1B). From these 26, at least eight peptides (corresponding to major peaks 8, 12, 13, 19, 24, and 26) were of sufficient yield and purity and were selected for protein sequence determination (FIG. 1C).

[0163] Based on this ...

example 2

Recombinant Expression and Purification of the Δ4, 5 Glycuronidase

[0167] Using PCR, we cloned from the F. heparinum genome both the full-length enzyme and the “mature” enzyme lacking the N-terminal 20 amino acid signal sequence (Δ4,5Δ20) into a T7-based expression plasmid. Cloning into pET28a permitted the expression of the glycuronidase as an N-terminal 6×His-tag fusion protein. Pilot expression studies focused on the full-length enzyme. In these initial experiments, we examined several different induction conditions such as temperature, time and length of induction, and even IPTG concentrations. In every case, the full-length enzyme was present nearly exclusively as an insoluble fraction. Attempts to purify the enzyme directly from inclusion bodies and then refold the apparently mis-folded protein were initially not successful; while solubility was partially achieved by a combined use of detergents (e.g., CHAPS), increasing salt concentrations, and the presence of glycerol, the p...

example 3

Biochemical Conditions for Optimal Enzyme Activity

[0171] To determine the optimal reaction conditions for Δ4, 5 glycuronidase activity, we analyzed initial reaction rates as a function of buffer, pH, temperature, and ionic strength (FIG. 6). For these experiments, we used the disulfated heparin disaccharide substrate ΔUHNS,6S. Based on what is known about the degradation of heparin / heparan sulfate-like glycosaminoglycans byflavobacteria as well as initial biochemical characterization of this and related enzymes [Warnick, C. T. and Linker, A., 1972, Biochemisiry 11, 568-72], we hypothesized that a heparin disaccharide would be an optimal substrate for the Δ4, 5 glycuronidase. Enzyme activity was routinely monitored by a loss of absorbance at 232 nm, corresponding indirectly to the hydrolysis of the uronic acid from the non-reducing end.

Results

[0172] Under these conditions, we observed a NaCl concentration-activity dependence that was optimal between 50 and 100 mM. NaCl concentrat...

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Abstract

The invention relates to Δ4,5 glycuronidase, related compositions, and methods of use thereof.

Description

RELATED APPLICATIONS [0001] This application claims priority under 35 U.S.C. §119 from U.S. provisional application serial No. 60 / 377,488 filed May 3, 2002, the entire contents of which is incorporated by reference.GOVERNMENT SUPPORT [0002] Aspects of the invention may have been made using funding from National Institutes of Health Grant number NIHGM57073 and CA090940. Accordingly, the Government may have rights in the invention.FIELD OF THE INVENTION [0003] The invention relates to Δ4, 5 glycuronidase and uses thereof. In particular, the invention relates to substantially pure Δ4, 5 glycuronidase which is useful for a variety of purposes, including analysis of glycosaminoglycans (GAGs), sequencing, identifying, quantifying and purifying glycosaminoglycans present in a sample, removing glycosaminoglycans, such as heparin, from a solution and inhibiting angiogenesis, controlling coagulation, etc. The invention also relates to methods of treating cancer and inhibiting cellular prolife...

Claims

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Application Information

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IPC IPC(8): A61K38/00C07H21/04C12N9/24C12Q1/34
CPCA61K38/00C07H21/04C12N9/2402C12Y302/01056C12Y302/01166C12Q1/34A61P35/00A61P43/00A61P7/00A61P7/04A61P9/00Y02A50/30
Inventor MYETTE, JAMESSHRIVER, ZACHARYVENKATARAMAN, GANESHSASISEKHARAN, RAMMCLEAN, MAITLAND
Owner MASSACHUSETTS INST OF TECH
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