Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

Abstract

Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow on- and off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling.

Keywords: CD44; PSGL-1; cell adhesion; cell migration; glycobiology; glycoprotein; selectin; selectin ligand; sialyl Lewis x; surface plasmon resonance (SPR).

FIGURE 1.

Validation of CD44 binding to E-selectin using well established approaches. A, representative flow cytometry histograms of KG1a cells stained for the expression of CD44 (left) and the binding of dE-selectin (middle). A dot plot showing KG1a cells dually stained for CD44 and dE-selectin is shown on the right. Dotted curves show isotype (for CD44) and EDTA (20 mm; for dE-selectin) controls, and shaded curves show specific binding. B, KG1a cells were dually immunostained with CD44 mAb (red; left) and recombinant dE-selectin (green; middle), and in the merged image, colocalization of CD44 with dE-selectin is shown (orange; right). Cell surface labeling with isotype control or secondary antibody alone served as a background control (data not shown). C, CD44 was immunoprecipitated from KG1a cell lysate. The captured proteins were run on a 4–20% SDS-polyacrylamide gradient gel and transferred to a PVDF membrane for Western blot analysis. The membrane was blotted with either dE-selectin (left) or CD44 mAbs (clones 515 and 2C5; right) followed by isotype-matched HRP-conjugated antibody for visualization. D, interaction of CD44 with E-selectin by blot rolling assay was performed on immunoprecipitated CD44 from KG1a cell lysates. Immunoprecipitated protein was resolved by 4–20% SDS-PAGE and blotted for sLe^x binding using HECA-452 mAb. CHO-E cells were then perfused over the stained PVDF membrane at 0.25 dyne/cm². After cell perfusion, the numbers of rolling cells/field were counted from six experiments using four distinct fields of view in each experiment. As a control, 5 mm EDTA was added to the buffer containing CHO-E cells. CHO-Mock (CHO-M) cells were used to assess the nonspecific adhesion. Results shown reflect multiple assays (n = 4) on HECA-452 blots of multiple (n = 3) membrane preparations. Data are the mean ± S.E. (error bars).

FIGURE 2.

Optimization of the SPR-based IP in studying the interaction of E-selectin with CD44/HCELL. A, an experimental schematic diagram. Step 1, mAb immobilization on CM5 chip; step 2, lysate injection to capture CD44; step 3, injection of dE-selectin. B, raw data of KG1a lysate injection on surface-immobilized mAb. Three flow cells were used in this experiment: blank (dashed line), immobilized 515 mAb (5500 RU; gray line), and immobilized isotype control (5000 RU; black line); the mAb immobilization (step 1) is not shown. Arrows mark the start and end of the lysate injection followed by washing with a running buffer (step 2). The sensorgrams are uncorrected for the bulk refractive index of the buffer and therefore display a large increase in RU during the lysate injection. Inset, Western blot analysis of 515 mAb-captured proteins from crude KG1a lysate. Captured proteins were eluted (as described under “Experimental Procedures”) following a buffer wash of either 1 or 5 min. The recovered material was analyzed by Western blotting with CD44 mAb (left panels) and/or with PSGL-1 mAb (right panels). C, flow cytometry was used to determine the ability of three different clones of CD44 mAbs (515, Hermes-3, and IM7) to stain CD44 on KG1a cells. The dotted curves represent isotype controls, and the shaded curves represent specific binding. D, Western blot analysis of the efficiency of three different clones of CD44 mAbs (515, Hermes-3, and IM7) to each immunoprecipitate CD44 from KG1a lysate; blots were stained with CD44 (515, Hermes-3, and IM-7) and HECA-452 mAbs. E, CD44 mAb clones (515 (7200 RU; gray line), Hermes-3 (5740 RU; black line), and IM7 (4100 RU; dashed line)) or isotype (4600 RU) was immobilized to capture CD44 from KG1a lysate. Subsequently, dE-selectin binding to the captured CD44 was determined. The mAb immobilization step is omitted. The sensorgrams show the lysate injection followed by dE-selectin injection (177 nm), which are normalized after subtracting the bulk refractive index and nonspecific interaction using the control isotype flow cell. k_off is the dissociation rate constant for CD44 from Hermes-3 mAb or 515 mAb, and k_off-apparent is for dE-selectin or CD44·dE-selectin from Hermes-3 mAb or 515 mAb and dE-selectin from CD44·Hermes-3 mAb or CD44·515 mAb. F, using Hermes-3 mAb (3670 RU) to capture CD44 and isotype control (3600 RU), the Ca²⁺-dependent binding of CD44 to dE-selectin was measured as in E above. dE-selectin (177 nm) was injected first in the presence of 5 mm EDTA and then in the presence of 1 mm CaCl₂. This is representative of two independent experiments. G, purified CD44 from KG1a lysate (1 μm; according to “Experimental Procedures”) was injected for 200 s at 20 μl/min over either immobilized Hermes-3 mAb (left) or immobilized dE-selectin (right). 13,000 RU of dE-selectin was immobilized using an amine coupling kit on a CM5 chip. Inset, Western blot of purified CD44 from KG1a lysate stained for CD44 (using Hermes-3 mAb). All SPR binding was conducted in running buffer containing 50 mm NaCl. k_off is for CD44·dE-selectin complex. This is representative of n = 2 independent experiments. For all SPR experiments, k_off and k_off-apparent were calculated by fitting the stable phase in the buffer washing step using the Biacore evaluation software.

FIGURE 3.

Characterization of glycans essential for mediating binding of CD44/HCELL to E-selectin using SPR. A, CD44 mAb clones (515 (7000 RU; black line) and 2C5 (8600 RU; gray line)) or isotype control (8000 RU) was immobilized to capture rH-CD44. The mAb immobilization step is omitted. The sensorgrams show the lysate injection followed by dE-selectin injection (177 nm), which are normalized after subtracting the bulk refractive index and nonspecific interaction using the control isotype flow cell. No binding of rH-CD44 to dE-selectin was detected. This is representative of n = 2 independent experiments. B, rH-CD44 and CD44 immunoprecipitated from KG1a lysate was run on a 4–20% SDS-polyacrylamide gradient gel and transferred to a PVDF membrane for Western blot analysis. The membrane was blotted with anti-sLe^x mAb (HECA-452 clone) (top panel). To confirm the presence of CD44, rH-CD44 was blotted and stained with either 2C5 or 515 anti-CD44 mAb (bottom panels). Note that rH-CD44 could be blotted with 2C5 but not with 515 (bottom panel) but lacked sLe^x/a, which is required for E-selectin recognition (as evident by the absence of HECA-452 mAb binding) (top panel). C, Hermes-3 mAb (3270 RU) (isotype control, 2570 RU) was immobilized to capture CD44 from KG1a lysates that had been either treated with PNGase F (Treated; black line) or not (Control; gray line). Following CD44 capture, dE-selectin was injected at 354 nm. The same surface was used for both the treated and control binding studies with a regeneration step between the two runs. The normalized (dashed line) sensorgram is the same as control but normalized to the treated sensorgram based on the ratio of accumulated CD44 RU prior to dE-selectin injection. k_off and k_off-apparent were calculated as described in Fig. 2E. This is representative of n = 2 independent experiments. D, for Western blot analysis, CD44 was immunoprecipitated (with 515 or Hermes-3) from equivalent amounts of KG1a lysates either treated with PNGase F (+) or not (−) and blotted with either dE-selectin (top panel) or CD44 (middle panel). Note the disappearance of the QBend-10 signal due to the N-glycan removal, whereas equal amounts of CD34 protein were recognized by the CD34 mAb EP373Y (C-terminal domain of CD34; bottom panels). E, this SPR study was performed as in C except using 515 mAb (4000 RU) and its isotype (2570 RU) in place of Hermes-3 mAb. This is representative of n = 3 independent experiments. F and H, SPR analysis of OSGE treatment was performed as in C and E, respectively, using Hermes-3 mAb (8000 RU) (isotype control, 5000 RU) (F) or 515 mAb (5900 RU) (isotype control, 5000 RU) (H). These are representative of n = 2 independent experiments for Hermes-3 mAb and n = 3 independent experiments for 515 mAb. G, CD44 was immunoprecipitated following OSGE treatment as in D and subjected to Western blotting for CD44 (top panel). Because following OSGE treatment the Hermes-3 mAb does not immunoprecipitate CD44 as efficiently as untreated CD44, CD44 immunoprecipitations were performed on adjusted amounts of OSGE-treated samples (i.e. 3 times more OSGE-treated KG1a lysate) and subjected to Western blot analysis for CD44 and dE-selectin (middle panel). Note that this is consistent with the SPR results in F and H. Qbend-10 was used as an internal control to confirm O-glycan removal as in D (bottom panel). I and J, SPR analyses of sialidase treatment were performed as in C and E using Hermes-3 mAb (5440 RU) (isotype control, 4000 RU) (I) or 515 mAb (5461 RU) (isotype control, 4036 RU) (J). These are representative of n = 3 independent experiments. K, Western blot analysis of sialidase treatment was performed as in D. All SPR binding studies were conducted in a running buffer containing 50 mm NaCl. The treated and control bindings were performed on the same flow cell with a regeneration step between the two runs. dE-selectin was injected at 354 nm unless stated otherwise.

FIGURE 4.

Determination of the K_D for the binding of CD44/HCELL and PSGL-1 to dE-selectin at different salt concentrations. A, binding of different concentrations of dE-selectin to CD44 at 50 mm NaCl; the sensorgram shows binding of consecutive injections of dE-selectin at 30 μl/min for 130 s at concentrations of 0.78, 1.5, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400, and 800 nm that are spaced by a 60-s buffer washing step to CD44 captured from a KG1a lysate injection over surface-immobilized Hermes-3 mAb (7142 RU). The lysate injection is not shown. The sensorgrams are corrected for the bulk refractive index and nonspecific interactions using isotype control (6852 RU). K_D was determined by fitting the binding isotherm using a steady-state model and the RU_max values just prior to the start of the buffer injection where steady-state conditions were nearly met (inset). k_off-apparent was calculated as described in Fig. 2E, and k_on-apparent was calculated based on the determined K_D and k_off-apparent values. B, binding of different concentrations of dE-selectin to CD44 at 200 mm NaCl; the experimental conditions are similar to those in A with the following concentration range used: 25, 50, 100, 200, 400, 800, and 1600 nm dE-selectin. The RU value for Hermes-3 mAb was 4900, and that for the isotype control was 4280. C, binding of different concentrations of dE-selectin to PSGL-1 at 50 mm NaCl; the experimental conditions are similar to those in A with the same concentration range of dE-selectin used. The RU value for the KPL-1 mAb was 8200, and that for the isotype control was 6852. D, binding of different concentrations of dE-selectin to PSGL-1 at 200 mm NaCl; the experimental conditions are similar to those B with the same concentration range of dE-selectin used. The RU value for KPL-1 mAb was 5800, and that for the isotype control was 4280. E, summary of binding constants of CD44·dE-selectin and PSGL-1·dE-selectin at 50 and 200 mm NaCl concentration from three independent experiments of those shown in A–D. The binding study was performed and analyzed as described in A and B.

FIGURE 5.

Comparison between the binding of CD44 and PSGL-1 to CHO-E cells by blot rolling assay at various salt concentrations. A and B, adhesion bar graph for the blot rolling assay (rolling cells/mm²) for CHO-E cells perfused over SDS-PAGE immunoblots of HECA-452-reactive membrane glycoproteins of KG1a cells at 0.25 dyne/cm² and buffer NaCl concentrations of 50, 150, and 200 mm (black bars). Immunoprecipitates of CD44/HCELL (A) and PSGL-1 (B) from KG1a cells were resolved by SDS-PAGE and blotted for HECA-452 prior to performing the assay. To control for the specificity of CHO-E binding to membrane glycoproteins, EDTA was added to the buffer containing the CHO-E cells before use in adhesion assays (gray bars). After cell perfusion, the numbers of rolling cells/field were counted from n = 3 experiments using four distinct fields of view in each experiment. Data are reported as the mean ± S.E. (error bars). C, single cell tracking of CD44 and PSGL-1 binding to E-selectin by blot rolling assays. The experiments were performed as described in A and B. The images were acquired at one frame/s, subjected to background subtraction and contrast enhancement in NIH ImageJ, and tracked on Imaris V7.6.4 software as described under “Experimental Procedures.” The centroid position of the cells was determined with an accuracy of 10 ± 4 μm. The trajectories show the X-position of each rolling cell (parallel to the flow) versus time. Displacements that were greater than 4 μm within 4 s were considered as rolling events, and those that were less were considered as adhesion events. The slope of the rolling phase of the trajectory was fitted linearly using OriginPro (gray line), and the mean velocity, V_x, for each condition was calculated from the average of the slopes. The average velocity, percentage (%) of rolling cells that displayed adhesion behavior (as illustrated for the trajectories), and averaged traveling distance during the rolling phase of the trajectories under each condition are reported. Data are reported as the mean ± S.E.

FIGURE 6.

SPR binding of CD44 (black line) and PSGL-1 (dashed line) to mE-selectin. A, Hermes-3 mAb (for CD44; 3650 RU), KPL-1 mAb (for PSGL-1; 5000 RU), or isotype control (3400 RU) was used for real time immunoprecipitation from KG1a lysates. Following antigen capture, mE-selectin was injected at either 177 or 354 nm. The sensorgrams were corrected for the bulk refractive index and nonspecific interactions using the isotype control. This is a representative experiment of n = 2 independent experiments. B, native PAGE of E-selectin monomer and dimer was performed where 2 μg of mE-selectin and 4 μg of dE-selectin were diluted in 2× loading buffer (25% glycerol, 62.5 mm Tris base (pH 6.8), and 1% bromphenol blue) and then run on a 10% SDS-polyacrylamide gel without SDS in the running buffer. The gel was then visualized using SimplyBlue^TM safe stain. This assay is representative of two independent experiments.

FIGURE 7.

Comparison of the binding affinity of CD44 captured from different cell lysates to dE-selectin. A, flow cytometric analysis of CD44 expression on KG1a, HL-60, and THP-1 cells is shown as the average geometric mean fluorescence intensity (above the isotype control) of three independent experiments. Note that KG1a expresses the highest levels of CD44 followed by THP-1 and HL-60. B, CD44 was immunoprecipitated from KG1a, HL-60, or THP-1 cell lysate normalized for total CD44 protein. The captured proteins were run on a 4–20% SDS-polyacrylamide gradient gel and transferred to a PVDF membrane for Western blot analysis. The membrane was blotted with either dE-selectin, HECA-452 mAb, or CD44 mAbs (clones 515, Hermes-3, and 2C5) followed by isotype-matched HRP-conjugated antibody for visualization. NIH ImageJ was used to quantify the intensity of Western blot bands using the gel analyzer tool; the number displayed represents the density of each band related to the KG1a band as a standard. All results are representative of three independent experiments. C, binding of different concentrations of dE-selectin to CD44 at 50 mm NaCl; the sensorgram shows binding of consecutive injections of dE-selectin at 20 μl/min for 180 s at concentrations of 0.78, 1.5, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400, and 800 nm that are spaced by a 60-s buffer washing step to CD44 captured from a HL-60 (left) or THP-1 (right) lysate injection over surface-immobilized Hermes-3 mAb (7369 RU for HL-60 and 6808 RU for THP-1). The lysate injection is not shown. The sensorgrams are corrected for the bulk refractive index and nonspecific interactions using anti-MsIgG_2a isotype control (7834 RU for HL-60 and 6852 RU for THP-1). K_D, k_off-apparent, and k_on-apparent were determined as described in Fig. 4. D, summary of binding constants of CD44·dE-selectin at 50 mm NaCl captured from different cell lysates from four independent experiments. The binding study was performed and analyzed as described in Fig. 4A. E, adhesion bar graph for the blot rolling assay (rolling cells/mm²) for CHO-E cells perfused over SDS-PAGE immunoblots of HECA-452-reactive membrane glycoproteins of either KG1a, HL-60, or THP-1 cells at 0.25 dyne/cm² and buffer NaCl concentrations of 150 mm. Immunoprecipitates of CD44/HCELL from KG1a, HL-60, or THP-1 cells were resolved by SDS-PAGE and blotted for HECA-452 prior to performing the assay (as described for Fig. 5). To control for the specificity of CHO-E binding to membrane glycoproteins, EDTA was added to the buffer containing the CHO-E cells before use in adhesion assays (gray bars). After cell perfusion, the numbers of rolling cells/field were counted in four distinct fields of view in each experiment. The adhesion bar graph is representative of three independent experiment and data reported as the mean ± S.E. (error bars).