DOI
https://doi.org/10.25772/WH6K-3Q50
Author ORCID Identifier
0000-0002-2861-6646
Defense Date
2021
Document Type
Thesis
Degree Name
Master of Science
Department
Pharmacy - Dean's Office
First Advisor
Umesh Desai
Second Advisor
Shijun Zhang
Third Advisor
Mary Peace McRae
Abstract
Glycosaminoglycans (GAGs) are linear polysaccharides whose disaccharide building blocks consist of an amino sugar (D-glucosamine) and either uronic acid (D-glucuronic acid or L-iduronic acid) or galactose. Nearly all GAGs (with the exception of hyaluronic acid) are covalently attached to proteins, forming proteoglycans, and are ubiquitous in nature. The monomeric units, chain length, degree and pattern of sulfation, and epimerization are important properties that determine the influence of these molecules. Through Coulombic forces, hydrogen bonds and hydrophobic interactions, they regulate many physiological and pathophysiological processes such as blood coagulation, cell growth and differentiation, inflammatory processes, host defense and viral infection mechanisms.
Alzheimer’s Disease (AD) is a chronic neurodegenerative disorder, and the most common form of dementia, characterized by a loss in memory and other cognitive abilities. It represents memory impairment, prominent psychiatric symptoms, and as the dementia progresses, language dysfunction, visuospatial difficulty, loss of insight, and personality changes (withdrawal, decreased initiative, and occasionally, depression) are frequently apparent.
At the microscopic level, AD presents with extensive neuronal loss, neurofibrillary tangles (NFTs), and amyloid plaques. Amyloid precursor protein (APP) is processed by beta secretase (BACE1) to form amyloid beta (Aβ) – the primary component of amyloid plaques. Tau, a microtubule associated protein, is hyperphosphorylated, leading to destabilized microtubules and the formation of NFTs. Cathepsin D (catD) is a lysosomal aspartyl protease that has been shown to break down tau tangles and has been isolated with amyloid plaques. Interestingly, GAGs are known to bind to all key proteins involved AD pathogenesis, i.e. APP, Aβ, BACE1, tau, phosphorylated tau and cathepsin D and while their functions are not well understood, their influence is irrefutable.
It is estimated that 6.2 million Americans aged 65 and older are living with AD in 2021. As the number of older Americans grows rapidly, that number is projected to increase to 12.7 million by 2050. One in three seniors dies with Alzheimer’s or another dementia. Since its first report in 1906, much has been learnt about AD, but we are yet to uncover many mysteries about this elusive disease. As the understanding of GAGs and their many roles grows, the work described here is a step forward in our path.
CatD is synthesized in the endoplasmic reticulum as preprocatD along with a signal peptide and prodomain, and undergoes a series of biochemical changes before it is activated. Its mechanism of activation is still a topic that requires clarity. Studies have shown that heparin, an extensively explored GAG, has a stimulatory effect on the autocatalytic cleavage of procatD as well as proBACE1. Thus, attempts to understand GAG involvement would be play a key role in catD’s activation. However, no crystal structure or homology model of the zymogenic form of catD has been published to date. The construction of a model of the procatD structure would greatly accelerate our understanding of catD physiology and pathology in AD.
The crystal structure of mature catD has been deposited in the Protein Data Bank (PDB) with good resolution. Thus, we first attempted to model the prodomain consisting of 44 amino acids alone, but no reliable model was built. The manipulation of well-established homology modelling tools available from the MODELLER website allowed the generation of models using crystal structures of related proteases from other organisms. A Ramachandran plot (RP) showing the allowed torsional angles, offers insight into stereochemical quality of the protein structures. The RP showed that percentage of residues in the final proposed model was 94.6%. However, upon closer visual examination of the model, we observe a large shift in N-terminal residues and alterations in the secondary structural features when compared to mature catD. While some speculation may be warranted, previous literature suggests that these changes are expected and possibly contribute greatly to the mechanism of GAG modulation.
Understanding catD’s role in neurodegenerative diseases may serve as a starting point to design therapeutic agents in order to modulate its activity. A lack of consensus exists regarding the effects of GAGs on catD. One study exploring the effects of GAGs on lysosomal enzymes found them to have an inhibitory effect on catD. However, Beckman et al. found that GAGs stimulate the activity of mature catD. These conflicting discoveries led us to conduct molecular docking studies to first understand which GAGs bind and in what manner.
The screening of commercially available GAGs that can be purchased and evaluated in vitro in biophysical studies serves advantageous over a library of natural and unnatural sequences. Upon generating the electrostatic potential surface, two putative GAG-binding sites were selected. The oligosaccharide library was subjected to triplicate docking using GOLD Docking Software. The screening process has allowed us to determine a preferred GAG-binding site and binding mode. The binding affinity does not appear to be a depend upon chain length or degree of sulfation, however, the pattern of sulfation of some GAGs informs us that the unique 3-O-sulfation may play an important role but has yet to be elucidated conclusively. A single best sequence might be inferred from the high affinity and high consistency seen by a hexasaccharide at both binding sites.
Future studies that allow us to explore the binding studies with both, procatD as well as mature catD will serve as crucial steps in the effort to fully describe GAG binding and modulation of this key enzyme in the elusive neurodegenerative AD.
Rights
© Tamim Chiba
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
8-12-2021
Included in
Medicinal and Pharmaceutical Chemistry Commons, Molecular Biology Commons, Neurosciences Commons