Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
Food Science & Technology
Non Technical Summary
Non-Technical Summary:Prion protein is a naturally occurring protein found in mammals. When the protein is folded into its normal structure, it may play several beneficial roles. When the protein becomes improperly folded and aggregates with other misfolded prion proteins, this can lead to the formation of an infectious and toxic form of the protein referred to as 'prion'. Prions cause diseases that affect several species of mammals, including humans (e.g., CJD, Kuru), cattle (mad cow disease), sheep (scrapie), moose, elk and deer (chronic wasting diseases). These diseases are all invariably fatal and are currently incurable. To date, there is no evidence that humans can contract prion disease by eating contaminated sheep or cervids (moose, elk, and deer), although there is evidence that humans can contract prion disease by eating contaminated beef. Basic details about prion disease remain unknown, such as the structure of 'prion' and how prions convert healthy native prion protein into more prions. One important factor that requires further study is the role that glycosylation may play in prion protein aggregation. Native prion protein is glycosylated--linked with carbohydrate molecules--at two positions on the protein. Previous biophysical studies have largely ignored glycosylation and have focused on the carbohydrate-free form of the protein, owing mainly to the difficulty in preparing glycosylated prion protein. In this study, we will use a recently developed method for producing proteins from bacteria and labeling the proteins at specific sites using 'click' chemistry. This will allow us to insert special chemical groups at specific locations in the prion protein that can then be used to attach carbohydrate groups at these sites. We can add various different carbohydrates to examine the effect of glycosylation on prion protein aggregation and the formation of prion-like conformations. These effects will be examined using hamster prion protein and measured in vitro using biophysical tools. With this approach, we can better understand how glycosylation can impact the stability of native prion protein and the propensity to form disease-causing prions. This knowledge could be used to inform the development of treatments (e.g., drugs, antibodies) for prion disease.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Goals / Objectives:The goal of this work is to understand how glycosylation of prion protein may affect the conversion of native prion protein into the toxic/infectious prion form. Native prion protein will be labeled with different sugars at the two naturally occurring N-linked glycosylation sites and biophysical tools will be used to determine the effects of glycosylation on the structure, stability, and aggregation behavior of this protein. Specific objectives include:Prepare various glycated forms of prion proteinUse unnatural amino acid mutagenesis to insert azido-phenylalanine into prion protein at each glycosylation site (N181 and N197)Recombinantly express and purify wild-type and mutant forms of prion proteinChemically label mutant prion protein with carbohydrate molecules targeting the azide functional groupCompare prion protein labeled with different mono- and oligosaccharides that mimic natural glycoformsUse biophysical tools (e.g., CD, FTIR, DSC, DLS, Fluorescence, TEM, and AFM) to characterize the structure, stability and aggregation kinetics of the various prion protein samplesExamine the effect of glycosylation on prion-like properties such as cell toxicity and protease resistance, used (along with structure) to define strainsAnalyze the data to map out how different glycoforms affect the aggregation pathways of prion proteinRelate specific glycation types to specific strainsExamine glycosylation effects on strain propagation
Project Methods
Methods:Hamster Prion Protein Expression and Purification: Syrian hamster prion protein (ShaPrP) will be used as the model system as a bacterial expression system has been optimized for producing the recombinant protein and, critically, hamster prion is not known to pose a risk to human or livestock health. Wild-type ShaPrP is N-glycosylated at two positions: N181 and N197. The native truncated protein (ShaPrP 90-242) will be expressed and purified from E. coli as described previously (Dee, 2012). Briefly, ShaPrP will be expressed from E. coli strain BL21(DE3) using a pET15b expression vector containing an N-terminal His-6 tag and a thrombin cleavage sequence. Following three hours of expression at 37°C, the cells will be harvested and sonicated to release the ShaPrP protein from inclusion bodies (in a 6 M GdnHCl solution). The denatured protein will be purified using an immobilized metal affinity column and refolded by dialysis into water overnight at 4°C.Expression of ShaPrP Containing Non-Standard Amino Acids: ShaPrP will be engineered to contain non-standard amino acids at positions 181 and/or 197 by replacing each targeted codon of the protein gene with an amber codon (TAG) that normally acts as a translation stop codon. Instead of stopping translation, however, a tRNA/RNA-synthetase pair is added that have been designed to recognize the amber codon and insert the non-standard amino acid instead. To do so, ShaPrP will be expressed and purified from E. coli as described above, except that the TAG-mutated gene for ShaPrP will be co-expressed along with a vector containing the tRNA/RNA-synthetase pair, and the non-standard amino acid (p-azidophenylalanine) will be added to the growth media at 1 mM concentration. The plasmid encoding the tRNA/RNA-synthetase pair designed to incorporate a non-standard amino acid bearing a tetrazine 'click' chemistry group will be purchased from Addgene (Chin, 2002).Click Chemistry Approach: A generic class of reactants termed "click chemistries" meet the criteria of being highly specific, selective, efficient and biocompatible (Patterson, 2014). A prominent example is the strain-promoted cycloaddition reaction between an azide and cyclooctyne reactive pair (Jewett, 2010). These click chemistries will be used to add various glycans at position 181 and 197 in ShaPrP. When extracted from animal tissues, both native prion protein and the infectious prion form are found to contain a variety of glycans (> 52 forms) (Rudd, 2001; Lawson, 2005). As such, several different glycans will be examined here, which are available as synthetic compounds. As a first approach, oligomers comprised of N-acetylglucosamine, mannose, galactose and fucose will be examined. These can be labeled to contain either an azide or cyclooctyne (or other functional group) as needed to covalently attach to the mutant ShaPrP.Inducing Fibril Formation: Prion protein is known to aggregate and form amyloid fibrils under various conditions, which are generally destabilizing to the native conformation. These include using chemical denaturant, shaking, reducing agents and low pH (Colby, 2007; Bjorndahl, 2011). These conditions will be used to induce aggregation/amyloid formation and to compare the effects of glycosylation on aggregation kinetics and structure.Biophysical Tools: The primary structure and presence of chemical modifications will be measured using mass-spectrometry. Protein secondary structure will be measured using circular dichroism and FTIR spectroscopy. Tertiary structure will be probed using the fluorescence of tryptophan residues to monitor conformational changes and stability of monomers. Quaternary structure will be measured using electron microscopy (TEM) and atomic-force microscopy (AFM) to directly visualize and characterize prion protein aggregates. Size-exclusion chromatography combined with multi-angle-light scattering and refractive index detection (SEC-MALS-RI) will be used to examine the early stages of oligomerization (Mw-analysis) and kinetics.Kinetics of amyloid fibril formation: The increase in fluorescence intensity of the compound thioflavin T (ThT) as a function of time will be used to measure the kinetics of fibril formation. ThT gives off a much higher fluorescence intensity upon binding and intercalating into protein β-sheet structures--particularly amyloid-fibril like structures--and this method is widely used to study amyloid fibril formation (Nilsson, 2004).Cell Toxicity and Protease Resistance: Toxicity against cultured cells and the ability of proteases such as pepsin, pancreatin, and proteinase K to hydrolyze the various glycoforms of ShaPrP will be compared using SDS-PAGE, as described (Lasse, 2016).