SSL5 binds glycan dependently to the N-termini of GPCRs. (A,B) Neutrophils were treated with or without 10 μg/mL SSL5 and stimulated with 1 μM C5a C-terminal peptide (A) or 100 μg/mL PGP (B). Calcium mobilization was measured over time. (C-E) HEK cells were transfected with (C) the N-terminus of CXCR1, CXCR2, CXCR4, CCR1, CCR2, CCR5, C3aR, or C5aR, and with (E) FPR, FPRL1, P2Y2, LTB4R, or PAFR. Histograms depict binding of 30 μg/mL SSL5-FITC to untreated (gray) or neuraminidase-treated (thick line) cells. The thin line represents SSL5-FITC binding to untransfected cells, and black histograms symbolize background fluorescence of transfected cells. Results are representative plots of at least 3 independent experiments. (D) Amino acid sequence of the cloned and expressed N-termini with putative (gray) and described (black) glycosylation sites. References are mentioned between square brackets. (F,G) HEK cells transfected with the N-terminus of wild-type CXCR1 or CXCR2, and CXCR1 or CXCR2 glycosylation mutants were incubated with (line) or without (gray) 10 μg/mL SSL5 for 15 minutes before staining with anti-CXCR1 (F) or anti-CXCR2 (G). (H) Binding of 10 μg/mL HIS-SSL5 was measured to untreated (■) or neuraminidase-treated (□) N-terminal peptides of CXCR1, CXCR4, and C5aR fused to the C-terminus of GH. GH protein without a C-terminal GPCR sequence served as a control.
