Interaction of high-molecular-weight kininogen with endothelial cell binding proteins suPAR, gC1qR and cytokeratin 1 determined by surface plasmon resonance (BiaCore)

Abstract

The physiologic activation of the plasma kallikrein-kinin system requires the assembly of its constituents on a cell membrane. High- molecular-weight kininogen (HK) and cleaved HK (HKa) both interact with at least three endothelial cell binding proteins: urokinase plasminogen activator receptor (uPAR), globular C1q receptor (gC1qR,) and cytokeratin 1 (CK1). The affinity of HK and HKa for endothelial cells are KD=7-52 nM. The contribution of each protein is unknown. We examined the direct binding of HK and HKa to the soluble extracellular form of uPAR (suPAR), gC1qR and CK1 using surface plasmon resonance. Each binding protein linked to a CM-5 chip and the association, dissociation and KD (equilibrium constant) were measured. The interaction of HK and HKa with each binding protein was zinc-dependent. The affinity for HK and HKa was gC1qR>CK1>suPAR, indicating that gC1qR is dominant for binding. The affinity for HKa compared to HK was the same for gC1qR, 2.6-fold tighter for CK1 but 53-fold tighter for suPAR. Complex between binding proteins was only observed between gC1qR and CK1 indicating that a binary CK1-gC1qR complex can form independently of kininogen. Although suPAR has the weakest affinity of the three binding proteins, it is the only one that distinguished between HK and HKa. This finding indicates that uPAR may be a key membrane binding protein for differential binding and signalling between the cleaved and uncleaved forms of kininogen. The role of CK1 and gC1qR may be to initially bind HK to the membrane surface before productive cleavage to HKa.

Figure 1. SDS PAGE of purified suPAR, gC1qR, HK and HKa

SDS PAGE was performed on purified HK, HKa, suPAR and gC1qR under reduced (left) and non-reduced (right) conditions on using a 4–20% Bis/Tris gel using MOPS buffer. To the left of the gel are the positions of molecular standard markers.

Figure 2. Binding protein integrity sensorgram

In the presence of 10 μM ZnCl₂, HKa (100 nM) alone or preincubated with gC1qR (100 nM), cytokeratin-1 (100 nM) or suPAR (100 nM) was injected over a suPAR immobilised CM5 chip. All reagents were found to be functionally active by HKa competing with the soluble binding protein to prevent binding to the chip immobilised suPAR. HKa alone (solid line), HKa + gC1qR (dotted line), HKa + cytokeratin 1 (short dashed line), HKa + suPAR (long dashed line). Each line represents a mean of two similar injections. Analyte injection was terminated at 50 seconds.

Figure 3. Sensorgrams of HK and HKa binding to immobilised binding proteins in the presence and absence of 10 μM ZnCl₂

A) gC1qR in the presence and absence of 10 μM ZnCl₂. Top graphs:HKa (bottom sensorgrams to top 0,25,50,100, 150,200,250,300,350 nM) were injected over a gC1qR immobilised (base 600 RU) CM5 chip by an automated method file. Top left: in the presence of 10 μM ZnCl₂. Top right graph: sensorgram in the presence of 3 mM EDTA. Bottom graphs: HK (bottom sensorgrams to top 0, 50, 100, 200, 300, 400 nM) were injected. Bottom left graph: sensorgram in the presence of 10 μM ZnCl₂. Bottom right graph: sensorgram in the presence of 3 mM EDTA. Each sensorgram line is an average of three injection runs in the method. Analyte injection was terminated at 120 seconds. B) suPAR in the presence and absence of 10 μM ZnCl₂. Top graphs: HKa (bottom sensorgram to top 0,25,50,100, 150,200,250,300,350 nM) were injected over a suPAR immobilised CM5 chip (base 950 RU) by an automated method file. Top left graph: in the presence of 10 μM ZnCl₂. Top right graph: in the presence of 3 mM EDTA. Bottom graphs: HK (bottom sensorgram to top 0, 50, 100, 200, 300, 400 nM) were injected. Bottom left graph: sensorgram in the presence of 10 μM ZnCl₂. Bottom right graph: sensorgram in the presence of 3 mM EDTA. Each sensorgram line is an average of three injection runs. Analyte injection was terminated at 120 seconds. C) Cytokeratin 1 in the presence and absence of 10 μM ZnCl₂. Top graph: HKa (bottom sensorgram to top 0,25,50, 100, 150,200,250,300,350 nM) were injected over a cytokeratin 1 immobilised (base 750 RU) CM5 chip by an automated method file. Top left graph: sensorgram in the presence of 10 μM ZnCl₂. Top right graph: sensorgram in the presence of 3 mM EDTA. Bottom graph: HK (bottom sensorgram to top 0, 50, 100, 200, 300, 400 nM) were injected. Bottom left graph: sensorgram in the presence of 10 μM ZnCl₂. Bottom right graph: sensorgram in the presence of 3 mM EDTA. Each sensorgram line is an average of three injection runs in the method. Analyte injection was terminated at 120 seconds.

Figure 4. CK1-gC1qR interactions

Sensorgrams show comparative binding of purified binding proteins and HKa in the presence of 10 μM ZnCl₂ to immobilised cytokeratin 1 and gC1qR. HKa, cytokeratin 1, and gC1qR (all 100 nM) were injected in the sequence indicated in the figure. Injection times were adjusted, but the ΔRU were not adjusted, for comparative illustrative purposes. Arrows with vertical dotted line indicates the position where each analyte was injected. The chip-linked binding protein base is indicated on the right each line. Each analyte injection was for 50 seconds. A: peak of suPAR-HKa-gC1qR complex; B: peak of CK1-HKa-gC1qR complex; C: peak of CK1-gC1qR complex.

Figure 5. CK1, gC1qR, suPAR interactions

Sensorgram indicating lack of binding of CK1, gC1qR and suPAR to immobilised suPAR in the presence of 10μM ZnCl₂ (no binding was found in the absence of Zinc). 100 nM of HKa, gC1qR, CK1 and suPAR were each injected separately at zero time. Each plot represents the mean of duplicate runs. Each analyte was injected for 60 seconds.

Figure 6. HKa binding protein complexes on suPAR

Sensorgram indicating lack of binding of CK1, gC1qR and suPAR to suPAR bound HKa. HKa (100 nM) in the presence of 10 μM ZnCl₂ was first injected over an immobilised suPAR chip. Subsequent addition of 100 nM CK1, gC1qR, or suPAR did not increase the binding signal. Each analyte injection was for 50 seconds.