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. 2015 May 18;10(5):e0126556.
doi: 10.1371/journal.pone.0126556. eCollection 2015.

Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13

Shobhit Gogia  1 , Chi Y Lo  1 , Sriram Neelamegham  1
Affiliations

Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13

Shobhit Gogia et al. PLoS One. .

Abstract

Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594-1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. ADAMTS13 substrate synthesis.
A. The 37.3 kDa fusion protein A2-77p-Venus, purified from E. coli, was labeled with fluorescein yielding A2-77p-Venus*. A2-77p-Venus* incubated with TEV protease resulted in two fragments A2-77p* and Venus*, which were separated using a HisTrap HP column. A2-77p* was coupled with a biotinylation linker that had either 2 or 77 PEG spacer units. The final product that is coupled to both fluorescein and biotin is called A2-77p-short (2 PEG units) or A2-77p-long (77 PEG units). A similar protocol was applied to create negative control proteins where residues Y1606 and M1605 were replaced by alanine. T—TEV cleavage site, H—7x His tag. B. Starting material and intermediates resolved using a 4–20% gradient gel under reducing conditions. Fluorescence is due to fluorescein alone since Venus signal is lost upon boiling. C. Silver stain of gel in panel B. Peptide of interest (A2-77p*) appears at 9.8 kDa (arrow). D. A2-77p-Venus (unlabeled) and A2-77p-Venus* (fluorescein labeled) resolved using a 4–20% gradient gel under non-reducing conditions. Fluorescence in the gel is due to both Venus and fluorescein. E. A2-77p-short was incubated with or without 4.4U/ml rADAMTS13 for 90min. Product was resolved using a tricine gel. Top panel shows fluorescence under UV illumination. Bottom panel shows western blot of the gel using anti-biotin antibody. As expected, the 9.8 kDa substrate is cleaved into 1.5 kDa fluorescent (top gel) and 8.3 kDa biotinylated product (bottom gel).
Fig 2
Fig 2. Specificity of the proteolysis assay and effect of calcium.
A. Fluorescent, biotinylated substrate was immobilized on streptavidin coated microspheres. N-terminal proteolysis by ADAMTS13 reduced microsphere fluorescence as detected using flow cytometry. B. Cleavage of A2-77p-short was detected upon addition of 4.4 U/mL rADAMTS13 (AD). Microsphere fluorescence was unchanged when either rADAMTS13 was absent, rADAMTS13 was heat inactivated (H.I. AD), or when alanine mutated substrate A2-77p(AA)-short was used. Reaction buffer is 50mM Tris pH8.0, 0mM calcium. Error bars are too small to see in some cases. C. Cleavage of A2-77p-short by 0.55U/ml rADAMTS13 increased upon decreasing buffer calcium concentration. 10 mM EDTA in buffer abrogated substrate cleavage due to ADAMTS13 inactivation.
Fig 3
Fig 3. Dose-dependent proteolysis by rADAMTS13.
A. Streptavidin coated microspheres bearing A2-77p-short were incubated with varying concentrations of rADAMTS13. Proteolysis was detected within 30min. even when protease concentration was 3.4% of normal plasma levels. B. Rate of proteolysis varied linearly with rADAMTS13 activity (R2 = 0.99).
Fig 4
Fig 4. Effect of spacer length on proteolysis.
A. A2-77p-short microspheres were incubated with varying concentrations of rADAMTS13 in the presence or absence of 25% plasma. Plasma failed to cleave A2-77p-short and also inhibited proteolysis of A2-77p-short by rADAMTS13. B. Experiments identical to panel A were performed, only using A2-77p-long. Here, plasma acts in synergy with rADAMTS13 to promote substrate cleavage. C. Direct comparison of the cleavage rates between A2-77p-short and A2-77p-long upon varying rADAMTS13 concentration. N.D. = cleavage not detected. *P<0.05 for indicated comparisons.
Fig 5
Fig 5. Dose-dependent proteolysis in runs containing plasma.
A. Plasma was prepared from blood drawn using heparin or sodium citrate as anti-coagulant. This was diluted at indicated concentrations, into either normal Tris cleavage buffer or Tris cleavage buffer containing 12.5mM CaCl2. Microspheres bearing A2-77p-long were added. At all plasma dilutions, the cleavage reaction was maximum when using heparin as anti-coagulant. B. Microspheres bearing A2-77p-long were incubated with 25% HIP prepared in sodium citrate and 12.5 mM calcium. Recombinant ADAMTS13 was titrated at indicated concentrations. Proteolysis varied linearly with rADAMTS13 activity (R2 = 0.99).
Fig 6
Fig 6. Effect of bilirubin on measurements of ADAMTS13 activity using XS-VWF and A2-77p-long.
A. XS-VWF consists of Venus and Cerulean flanking the VWF-A2 77 amino acid peptide (residues 1594–1670). Substrate FRET ratio increases upon proteolysis. Here, 0.5μM XS-VWF was incubated with 25% normal human plasma containing different concentrations of recombinant bilirubin (0–400 μM). Baseline FRET ratio at t = 0s decreased upon increasing bilirubin dose. B. XS-VWF samples obtained at 60min. in panel A were analyzed using western blots and anti-tetra His antibody to detect the ~36kDa substrate cleavage band. Equal substrate proteolysis was detected independent of bilirubin in plasma (top). No proteolysis occurred in runs with HIP (bottom). C. A2-77p-long microspheres were incubated with 25% plasma (or 25% HIP) at different bilirubin concentrations (0–400μM). Substrate cleavage measured using flow cytometry was unaffected by bilirubin.
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