Enzymatic and analytical tools for the characterization of chondroitin sulfate and dermatan sulfate glycosaminoglycans

Glycosaminoglycans (GAGs) are complex polysaccharides that reside in the extracellular matrix and on the surfaces of all cells. The same complexity that contributes to the diversity of GAG function has also hindered their chemical characterization. Recent progress in coupling bacterial GAG-degrading enzymes with sensitive analytical techniques has led to a revolution in understanding the structure-function relationship for an important subset of GAGs, namely heparin/heparan sulfate-like glycosaminoglycans (HSGAGs). The study of chondroitin sulfate and dermatan sulfate (CS/DS), an equally important subset of GAGs, has lagged behind partially due to a lack of enzymatic and analytical tools akin to those developed for HSGAGs. The Flavobacterial heparinases have proven indispensable in characterizing the fine structure of HSGAGs responsible for their different biological functions. As a continuation of ongoing research, a combination of chemical modification, peptide mapping, and site-directed mutagenesis was employed to explore the role of histidine in the activity of heparinase III. Of the thirteen histidines in the enzyme, His295 and His510 were found to be critical for the degradation of heparan sulfate by heparinase III. As a first step to developing the chondroitinases as enzymatic tools for the characterization of CS/DS oligosaccharides, recombinant expression and purification schemes were developed for chondroitinase AC and B from Flactobacterium heparinum. The recombinant enzymes were characterized using biochemical techniques and kinetic parameters were determined for their respective CS/DS substrates.

Department dc:contributor.department

Massachusetts Institute of Technology. Biological Engineering Division.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2003

Author dc:creator

• Pojasek, Kevin R. (Kevin Robert), 1976-

Advisor dc:contributor.advisor

• Ram Sasisekharan.

• Biological Engineering Division.

Statement dc:rights

• MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/7984

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/7984