Delta 4,5 glycuronidase and methods of analyzing therewith

a technology of glycuronidase and glycuronidase, applied in the field of glycuronidase, can solve the problems of difficult determination of hsgag sequences, difficult to obtain information on how specific hsgag sequences modulate different biological processes, and high degree of complexity of hsgags

Inactive Publication Date: 2006-08-10
MASSACHUSETTS INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 has proven harder to obtain.
Determination of these HSGAG sequence has been technically challenging.
HSGAGs are naturally present in very limited quantities, which, unlike other biopolymers such as proteins and nucleic acids, cannot be readily amplified.
Second, due to their highly charged character and structural heterogeneity, HSGAGs are not easily isolated from biological sources in a highly purified state.
Additionally, the lack of sequence-specific tools to cleave HSGAGs in a manner analogous to DNA sequencing or restriction mapping has made sequencing a challenge.

Method used

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  • Delta 4,5 glycuronidase and methods of analyzing therewith
  • Delta 4,5 glycuronidase and methods of analyzing therewith
  • Delta 4,5 glycuronidase and methods of analyzing therewith

Examples

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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 inf...

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, Biochemistry 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 concentratio...

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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 is a divisional of U.S. patent application Ser. No. 10 / 429921, filed on May 5, 2003 and currently pending, which claims priority under 35 U.S.C. §119 from U.S. provisional application Ser. No. 60 / 377,488 filed May 3, 2002, the entire contents of each of which are incorporated herein 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 angioge...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/34C12P21/06C12N9/24A61K38/00C07H21/04
CPCA61K38/00C07H21/04C12Q1/34C12Y302/01056C12Y302/01166C12N9/2402A61P35/00A61P43/00A61P7/00A61P7/04A61P9/00Y02A50/30
Inventor MYETTE, JAMESSHRIVER, ZACHARYVENKATARAMAN, GANESHSASISEKHARAN, RAMMCLEAN, MAITLAND
Owner MASSACHUSETTS INST OF TECH
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