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First published online May 24, 2004
doi: 10.1242/10.1242/jcs.01088

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Distinct kinetic and mechanical properties govern selectin-leukocyte interactions

¹ Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA
² Department of Materials Science and Engineering, The Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA

View larger version (30K): [in a new window]   Fig. 1. (A) Structural diagrams representing P-, E- and L-selectin. The selectins are rigid, asymmetric molecules that share structural similarities including the presence of the C-type lectin domain, followed by an epidermal-growth-factor-like (EGF) motif, a variable series of short consensus repeats (nine, six and two for P-, E- and L-selectins, respectively), a transmembrane domain (TM) and a cytoplasmic tail (cyto). There is also a high degree of homology between the three proteins within each region. (B) Selectin-mediated leukocyte recruitment. PSGL-1 on free-flowing leukocytes tethers to E- and/or P-selectin on activated endothelial cells, allowing leukocytes to roll to sites of infection or inflammation. E-Selectin might also bind to an as-yet-unidentified leukocyte glycosphingolipid. PSGL-1 on adherent leukocytes participates in secondary tethering by interacting with L-selectin on free-flowing leukocytes.

View larger version (16K): [in a new window]   Fig. 2. Force versus time traces for E-selectin binding to PMNs in the absence (A) and presence (B) of EDTA. Experimental conditions, including selectin concentration on the cantilever, contact force and dwell time, were optimized for both E-selectin and L-selectin experiments to result in 30% binding events (30 adhesion events per 100 contacts). The arrows indicate rupture events with magnitudes of about 100 pN taken from an experiment with a reproach velocity of 15 µm second–1. The presence of EDTA was consistently found to eliminate receptor-ligand binding. The linear increase in slope just before each rupture event is indicative of the loading rate exerted on the receptor-ligand bond (Benoit et al., 2000; Li et al., 2003; Wojcikiewicz et al., 2003).

View larger version (31K): [in a new window]   Fig. 3. Frequency of binding events for control and specificity experiments for L-selectin (A) and E-selectin (B). The presence of EDTA was found to abrogate binding. The presence of blocking antibodies for L-selectin (LAM1-116), E-selectin (ENA2) and PSGL-1 (KPL-1) dramatically reduced the frequency of binding. (C) Reduction in frequency of selectin binding to PMNs treated with OSGE. Reductions in the frequencies indicate that heavily O-linked glycoproteins are crucial for L- and P-selectin binding, and play a predominant role in E-selectin binding to PMNs. (D) Rupture-force histograms for E-selectin binding to control PMNs and OSGE-treated PMNs at a reproach velocity of 10 µm second–1. E-selectin binding to OSGE-treated PMNs resulted in a lower frequency of rupture events with a lower overall magnitude. As a result of the lower binding frequency, the area under the force histogram for OSGE-treated PMNs is about 40% that of the control. The less rigorous requirements for E-selectin binding might allow it to bind more easily to other glycosylated ligands (N-linked glycoproteins or glycosphingolipids) previously shielded by the heavily O-linked glycoproteins.

View larger version (18K): [in a new window]   Fig. 4. Representative force histogram and Monte Carlo simulations for L-selectin (A) and E-selectin (B) binding to human PMNs at a reproach velocity of 10 µm second–1. Simulations were conducted using the Bell model parameters extracted from the non-linear estimation and yielded most frequent and mean rupture forces in accord with those observed experimentally. The area under the force histograms represents the proportion of successful binding events (30% in these cases).

View larger version (25K): [in a new window]   Fig. 5. Rupture force plotted against the natural logarithm of loading rate for E-, P- and L-selectin binding to PMNs. Data comprise at least eight independent experiments and a range of reproach velocities for each selectin, conducted on separate days to validate reproducibility. Each force spectrum represents at least 2000 successful events. The superimposed solid lines indicate the non-linear least-squares fit over the entire range of experimental loading rates, found from the probability density function for bond rupture and corresponding Bell model parameters.