The biophysical characterization of a novel amyloidogenic BETA2-microglobulin variant, P32L

• Main Author: Gibson, Victoria M.
• Other Authors: Connors, Lawreen H.
• Language: en_US
• Published: 2021
• Subjects: Biochemistry
• Online Access: https://hdl.handle.net/2144/43371

Amyloidosis encompasses a group of diseases that are characterized by protein misfolding and irregular extracellular deposits formed by the buildup of non-native protein structures and amyloid fibrils in visceral organs and tissues. There are over 35 proteins having the potential to form amyloid deposits and fibrils in humans. Amyloid pathologies are hereditary or can develop sporadically and exist in local and systemic forms. In localized forms of the disease, amyloid deposits are limited to one tissue area or organ in the body, whereas the systemic types feature multiple sites of infiltration. There are many different systemic amyloid disease types, but this study focuses on a rare form of systemic amyloidosis known as beta2-microglobulin (ß2-m)-related amyloidosis. The ß2-m protein, it is a 99-residue protein, that makes up the light chain portion of the class 1 major histocompatibility complex (MHCI) which is present of the surface of all nucleated cells. ß2-m is usually cleared through the kidneys upon dissociation from MHCI. However, patients with end stage renal failure can develop hemodialysis-related systemic amyloidosis when dialysis fails to remove ß2-m from the circulation and elevating concentrations of the protein lead to formation of amyloid fibrils. Alternatively, a hereditary form of ß2-m amyloidosis was identified in 2012 and reported to be associated with the gene mutation encoding the ß2-m variant, D76N. The phenotypic expression in this type of ß2-m amyloidosis was very different from the dialysis-related disease associated with the wild-type protein. The present study focuses on a newly identified and unique amyloid-forming ß2-m variant protein associated with a clinical presentation unlike the dialysis-related or previously reported hereditary form of ß2-m amyloidosis. In 2019, a novel ß2-m variant was discovered at the Amyloidosis Center at Boston Medical Center. A 59-year-old woman of Portuguese descent with a family history of cardiac amyloidosis presented with amyloid deposits in her fat tissue along with indications of cardiac involvement; the combination of these features led to a diagnosis of systemic amyloidosis. DNA sequencing and mass spectroscopy revealed that the patient had a mutation in her ß2-m gene resulting in the replacement of leucine for proline at the 32nd residue (P32L) of the ß2-m protein. The aim of this research project was to understand the amyloid-forming nature of the P32L variant. To address this goal, we characterized the biophysical properties of P32L ß2-m. The structural stability of P32L was compared to results for wild-type ß2-m, the previously described D76N amyloidogenic protein, and a synthetic variant with an alternative amino acid replacement at residue 32, P32G. This latter protein was chosen as it has been previously studied and shown to have amyloidogenic properties in vitro. Using a pQE-1 plasmid optimized for expression in E. coli, N-terminal MKH6 tagged wild-type ß2-m, was produced and subsequently modified, to individually produce P32L, D76N, and P32G. Variant plasmids were transformed in Rosetta-gami 2 cells for expression and purification. Circular dichroism, chemical denaturation, and limited proteolysis were used to assess protein secondary structure and stability. Aggregation of variants was evaluated using Thioflavin T fluorescence and ultrastructural imaging of aggregates was accomplished with electron microscopy. Results displayed that wild-type and variant ß2-m proteins showed similar secondary structures, all mainly comprised of beta-sheets. Based on high percentages of beta-sheets and low amounts of unordered secondary structure, wild-type ß2-m was the most stable of the proteins and D76N was the least. Moreover, wild-type had the highest apparent melting temperature followed by P32L, P32G and D76N. In thermal unfolding and refolding experiments, wild-type exhibited an irreversible mechanism, while P32L was semi-reversible upon heating and cooling. Chemical denaturation showed that D76N required the lowest concentration of guanidine hydrochloride to denature and wild-type the highest amount. Aggregation of digestion products was apparent in both trypsin and thrombin reactions; electron microscopy demonstrated that trypsin treatment of P32L yielded pre-fibril oligomeric structures. Overall, our data suggest that P32L and all variants were less stable than the wild-type protein. D76N appeared to be the least stable of the variants; P32L and P32G exhibited similar characteristics, both more stable than D76N and less stable than wild-type. The amyloid-forming nature of P32L ß2-m may be a result of the peptidyl-prolyl bond at P32 presenting in a trans conformation. When P32 is in the thermodynamically favorable trans conformation, it has been shown to trigger amyloid formation. This case was heterozygous for the mutation and the mixture of variant and wild-type proteins may destabilize the P32 isomer.