Effect of enzymatic desialylation on chemokine binding and function of human CXCR2. (A) Cell lysates of CXCR2-transfected and untransfected HEK293 cells were incubated with an mAb to CXCR2, followed by precipitation with protein A. Precipitates were treated with 20 U/ml neuraminidase for 60 min at 37°C, or were left untreated and analyzed by Western blotting for binding of biotinylated anti-CXCR2 antibody or biotinylated MAL-II, respectively. (B) Isolated human neutrophils were treated with the indicated dosages of neuraminidase for 30 min at 37°C, washed, and subsequently assayed for binding of ¹²⁵I-labeled CXCL7 and ¹²⁵I-labeled CXCL4. Specifically bound radioactivity was expressed as the percentage of WT control cells receiving no enzyme treatment. (C) Scatchard plot for CXCL7 binding to untreated and neuraminidase-treated (20 U/ml) human neutrophils. The binding data were curve fit to determine affinity constants for high and low affinity binding sites. (D) Neutrophils were treated with the indicated dosages of neuraminidase for 30 min at 37°C, washed, and subsequently assayed for elastase release in response to increasing concentrations of CXCL7, CXCL8, and fMLP. The residual responsiveness of neuraminidase-treated neutrophils was expressed as the percentage of that determined for untreated cells. Data in A–D represent means ± SD determined in three different experiments, each with blood from a different donor.