Proteomic characterization of serine hydrolase activity and composition in normal urine

Abstract

Background: Serine hydrolases constitute a large enzyme family involved in a diversity of proteolytic and metabolic processes which are essential for many aspects of normal physiology. The roles of serine hydrolases in renal function are largely unknown and monitoring their activity may provide important insights into renal physiology. The goal of this study was to profile urinary serine hydrolases with activity-based protein profiling (ABPP) and to perform an in-depth compositional analysis.

Methods: Eighteen healthy individuals provided random, mid-stream urine samples. ABPP was performed by reacting urines (n = 18) with a rhodamine-tagged fluorophosphonate probe and visualizing on SDS-PAGE. Active serine hydrolases were isolated with affinity purification and identified on MS-MS. Enzyme activity was confirmed with substrate specific assays. A complementary 2D LC/MS-MS analysis was performed to evaluate the composition of serine hydrolases in urine.

Results: Enzyme activity was closely, but not exclusively, correlated with protein quantity. Affinity purification and MS/MS identified 13 active serine hydrolases. The epithelial sodium channel (ENaC) and calcium channel (TRPV5) regulators, tissue kallikrein and plasmin were identified in active forms, suggesting a potential role in regulating sodium and calcium reabsorption in a healthy human model. Complement C1r subcomponent-like protein, mannan binding lectin serine protease 2 and myeloblastin (proteinase 3) were also identified in active forms. The in-depth compositional analysis identified 62 serine hydrolases in urine independent of activity state.

Conclusions: This study identified luminal regulators of electrolyte homeostasis in an active state in the urine, which suggests tissue kallikrein and plasmin may be functionally relevant in healthy individuals. Additional serine hydrolases were identified in an active form that may contribute to regulating innate immunity of the urinary tract. Finally, the optimized ABPP technique in urine demonstrates its feasibility, reproducibility and potential applicability to profiling urinary enzyme activity in different renal physiological and pathophysiological conditions.

Figure 1

Overview of activity-based protein profiling.

Figure 2

Activity based protein profiling demonstrates consistent patterns of serine hydrolase activity in normal male (n = 9) and female urine (n = 9).(A) Activity-based protein profiling (ABPP, green). (B) Total protein (Sypro Ruby, red). Although serine hydrolase activity tended to correlate with total protein, activity was sometimes independent of total protein. Example: Female 14, activity and protein content highlighted in panel A and B, respectively.

Figure 3

Immunoprecipitation of active serine hydrolases from normal urine. Affinity purification demonstrated enrichment of labeled bands (ABPP, green) relative to total protein content (Sypro Ruby, red). Active bands were cut from Lane 1 and in-gel digestion performed for enzyme identification with LC-MS/MS.

Figure 4

Quantitative enzyme activity assays demonstrating the range of normal urinary enzyme activity, both uncorrected (A) and corrected for urinary protein (B). Bile-salt activated lipase activity was demonstrated with the substrate palmitate, which releases 4-nitrophenol, measured in μmol/mL/min [U]. Tissue kallikrein and urokinase activity are demonstrated with the substrate [D] Val-Leu-Arg which releases paranitronilide, measured in μmol/mL/min [U].