Author ORCID Identifier

https://orcid.org/0000-0001-9353-6653

Defense Date

2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Pharmaceutical Sciences

First Advisor

Umesh R. Desai, Ph.D.

Abstract

Glycosaminoglycans are heterogeneous polysaccharides that mediate important biological functions. There has been considerable interest in deciphering the precise GAG sequences that are responsible for protein interactions. In fact, several GAG oligosaccharides have been discovered to date as targeting proteins with higher level of specificity. Yet, it has been difficult to develop GAG oligosaccharides as drugs. One of the key reasons for this state of art is that GAG synthesis is extremely challenging and is highly structure-specific. Thus, much of the biology and pharmacology of GAG remains unknown and unexploited to date.

An alternative approach is to prepare GAG oligosaccharides using enzymatic depolymerization of polymeric GAGs. GAG lyases, including heparinases and chondritinases represent powerful tools that can theoretically generate multiple oligosaccharides in parallel. However, it is difficult to implement such procedures with high consistency. Moreover, GAG lyases can digest GAGs down to disaccharides. A priori, non-polymeric GAGs, or alternatively GAG oligosaccharides containing 4 to 10 residues, would be expected to function better as therapeutic agents because they would be more homogeneous and less non-specific than their polymeric precursors.

Thus, we reasoned that immobilization of these enzymes may engineer altered biopolymer processing, which may afford longer oligosaccharides in higher proportions and greater consistency. Heparinase-I and chondroitinase ABC were immobilized on CNBr-activated Sepharose and compared with the free form of the enzyme. Immobilized GAG lyases retained high efficiency of depolymerization over a wide range of pH, temperature and reusability. Most importantly, the immobilized enzyme was found to produce larger proportions of oligosaccharides longer than di- and tetra-saccharides as compared to lyases in the free form.

A two dimensional separation involves size exclusion chromatography followed by reversed phase ion-pairing ultra performance liquid chromatography coupled to electrospray ionization mass spectrometry was employed to separate and characterize oligosaccharide structures. We have identified 40 heparin oligosaccharides, including regular and rare structures ranging from dp4 to dp10 and 39 chondroitin sulfate oligosaccharides in high homogeneity and significant yields. Overall, this technology is likely to offer a simple and cost effective route to preparation of larger amounts of sequences that can be expected to bind and modulate protein function.

Rights

© Alhumaidi B. Alabbas

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

4-9-2018

Available for download on Sunday, April 09, 2023

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