The Effects of Trypsin on the Aggregation of Lysozyme and its Aggregates

The proteins are essential macromolecules which play different functions in the body and can form supramolecular structures. They can cause neurodegenerative diseases such as Alzheimer s and Parkinson s diseases and ALS, if their function become disordered by aggregation. Lysozyme is a model protein for protein aggregation and mutations in lysozyme gene may cause lysozyme systemic amyloidosis disease. The proteolytic enzymes are known to be capable to degrade amyloid structures, and in the body, they degrade the amyloid precursors and balance their level. In this study, we investigated the effect of trypsin on lysozyme aggregation and also on its aggregates. We incubated the 1 mL of lysozyme solutions (2mg/mL) in aggregation conditions (24 hours at 57 with 300 RPM rotation). They were then exposed to 20 μL of the trypsin solution (5.3mM) for 15 minutes. In another experimentation lysozyme solutions were first exposed to the enzyme then incubated in aggregation conditions. The aggregates were detected by atomic force microscopy (AFM), fluorescence spectroscopy and SDS-PAGE. The results showed that hydrolysis of lysozyme by trypsin couldn t reduce the aggregate formation but it could degrade the lysozyme aggregates. These results indicated that trypsin either couldn t cleave the susceptible regions of lysozyme, or the hydrolysis products might retain the ability to aggregate. From the observation that aggregated molecules could be hydrolyzed by trypsin shows that the susceptible peptide bonds are still exposed in the aggregates to be cleaved by the enzyme. These results emphasize the role of proteolytic enzymes in the clearance of toxic protein and peptide aggregates and provide another reason to consider the use of proteolytic enzymes in clearance of amyloid aggregates as a therapeutic approach.