Proteolytic Activity and Substrate Specificity of Lake Geneva

Heterotrophic microorganisms in lakewater secrete proteases which contribute to the turnover of dissolved organic matter and the degradation of peptidic contaminants. However, little is known about the identities and substrate specificities of these proteases. Herein, we sought to characterize the global proteolytic fingerprint of the extracellular proteases present in Lake Geneva, the largest freshwater body in Central Europe. Using Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS), we identified preferred enzymatic cleavage next to positively charged and certain non-polar amino acids, while cleavage next to negatively charged residues was disfavored. Specifically, many of the detected cleavage sites were predominantly surrounded by arginine and lysine, consistent with a trypsin-like substrate specificity. This pattern was conserved across seasons and water depths and were shared with two other Swiss lakes. In contrast, we observed variability in the number and types of cleavage sites across samples, suggesting spatial and temporal differences in overall protease diversity. Using class-specific inhibitors, we found that serine and metalloproteases contribute to both exo- and endo-proteolytic activity in lakewater. Our findings expand our understanding of protein stability in lake ecosystems and may be used to predict the fate of peptidic contaminants in the environment.