Fig. 5
Mac-1 αI domain interaction with FcγRIIA requires FcγRIIA sialylation and divalent cations. a A model of glycosylated FcγRIIA and bent Mac-1 with high rotational ability and flexibility of the αI domain is depicted. Spheres are Mg2+ (green) and Ca2+ (magenta). The electrostatic surface (−5 [red] to + 5 [blue] kT/e) was calculated using the Adaptive Poisson-Boltzmann Solver software plug-in for PyMOL. b Site-specific glycopeptide analysis of human neutrophil derived FcγRIIA. Average relative abundance for n = 5 donors are shown for both sites in the context of the crystal structure of the FcγRIIA-R131 variant. c Site-specific glycopeptide analysis of FcγRIIA-R131 variant and FcγRIIIB NA2 allele expressed in Jurkat cells. d Plates coated with GST-αI domain or GST were incubated with FcγRIIA or neuraminidase A treated FcγRIIA (asialylated). As indicated, EDTA was included in the buffer to chelate divalent cations. For experimental controls, GST was incubated with FcγRIIA and GST-αI domain was incubated with NIF to show αI-domain specificity. Lectin blots were performed to detect terminal α2,6 sialic acid on FcγRIIA in the absence (−) and presence ( + ) of neuraminidase (Neur). e J-IIA and J-IIA Mac1 cells were incubated with neuraminidase for 30 min at 37 °C. The cells were diluted 100-fold in flow buffer and drawn over IC-coated coverslips or TNF stimulated anti-endoglin (IC) coated HDMEC coverslips at 1.0 dynes/cm2 and the number of adherent cells were determined as in Fig. 1. Data is represented as fold change compared to J-IIA cells (-Neur) of an average of 3 experiments ± SEM; **p < 0.01 using Student’s unpaired t-test. f Indicated Jurkat cells were incubated ± neuraminidase as in e and seeded onto immobilized ICs for 30 min. Coverslips were fixed, permeabilized, and stained with Alexa 568 phalloidin to visualize the actin cytoskeleton. Bar graphs represent average adherent cell area. g Human neutrophils ± neuraminidase (Neur) were drawn across TNF-stimulated HDMEC coated with or without anti-endoglin (IC) at 1 dynes/cm2 and the number of adherent cells were evaluated. Data is average of 3 experiments ± SEM. Student’s unpaired t-test. For c–e), **p < 0.05; ***p < 0.001 using the One way ANOVA followed by Sidak’s Multiple comparison test
