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Zhu, Cheng

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    P-Selectin Glycoprotein Ligand-1 Forms Dimeric Interactions with E-Selectin but Monomeric Interactions with L-Selectin on Cell Surfaces
    (Georgia Institute of Technology, 2013-02-25) Zhang, Yan ; Jiang, Ning ; Zarnitsyna, Veronika I. ; Klopocki, Arkadiusz G. ; McEver, Rodger P. ; Zhu, Cheng
    Interactions of selectins with cell surface glycoconjugates mediate the first step of the adhesion and signaling cascade that recruits circulating leukocytes to sites of infection or injury. P-selectin dimerizes on the surface of endothelial cells and forms dimeric bonds with P-selectin glycoprotein ligand-1 (PSGL-1), a homodimeric sialomucin on leukocytes. It is not known whether leukocyte L-selectin or endothelial cell E-selectin are monomeric or oligomeric. Here we used the micropipette technique to analyze two-dimensional binding of monomeric or dimeric L- and E-selectin with monomeric or dimeric PSGL- 1. Adhesion frequency analysis demonstrated that E-selectin on human aortic endothelial cells supported dimeric interactions with dimeric PSGL-1 and monomeric interactions with monomeric PSGL-1. In contrast, L-selectin on human neutrophils supported monomeric interactions with dimeric or monomeric PSGL-1. Our work provides a new method to analyze oligomeric cross-junctional molecular binding at the interface of two interacting cells.
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    Platelet glycoprotein Ibα forms catch bonds with human WT vWF but not with type 2B von Willebrand disease vWF
    (Georgia Institute of Technology, 2008-09) Yago, Tadayuki ; Lou, Jizhong ; Wu, Tao ; Yang, Jun ; Miner, Jonathan J. ; Coburn, Leslie ; López, José A. ; Cruz, Miguel A. ; Dong, Jing-Fei ; McIntire,Larry V. ; McEver, Rodger P. ; Zhu, Cheng
    Arterial blood flow enhances glycoprotein Ibα (GPIbα) binding to vWF, which initiates platelet adhesion to injured vessels. Mutations in the vWF A1 domain that cause type 2B von Willebrand disease (vWD) reduce the flow requirement for adhesion. Here we show that increasing force on GPIbα/vWF bonds first prolonged (“catch”) and then shortened (“slip”) bond lifetimes. Two type 2B vWD A1 domain mutants, R1306Q and R1450E, converted catch bonds to slip bonds by prolonging bond lifetimes at low forces. Steered molecular dynamics simulations of GPIbα dissociating from the A1 domain suggested mechanisms for catch bonds and their conversion by the A1 domain mutations. Catch bonds caused platelets and GPIbα-coated microspheres to roll more slowly on WT vWF and WT A1 domains as flow increased from suboptimal levels, explaining flowenhanced rolling. Longer bond lifetimes at low forces eliminated the flow requirement for rolling on R1306Q and R1450E mutant A1 domains. Flowing platelets agglutinated with microspheres bearing R1306Q or R1450E mutant A1 domains, but not WT A1 domains. Therefore, catch bonds may prevent vWF multimers from agglutinating platelets. A disintegrin and metalloproteinase with a thrombospondin type 1 motif–13 (ADAMTS-13) reduced platelet agglutination with microspheres bearing a tridomain A1A2A3 vWF fragment with the R1450E mutation in a shear-dependent manner. We conclude that in type 2B vWD, prolonged lifetimes of vWF bonds with GPIbα on circulating platelets may allow ADAMTS-13 to deplete large vWF multimers, causing bleeding.
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    Measuring Diffusion and Binding Kinetics by Contact Area FRAP
    (Georgia Institute of Technology, 2008-07) Tolentino, Timothy P. ; Wu, Jianhua ; Zarnitsyna, Veronika I. ; Fang, Ying ; Dustin, Michael L. ; Zhu, Cheng
    Binding of selectins to P-selectin glycoprotein ligand-1 (PSGL-1) mediates tethering and rolling of leukocytes on the endothelium during inflammation. Previous measurements obtained with a flow-chamber assay have shown that mutations of three tyrosines at the PSGL-1 N-terminus (Y46, Y48, and Y51) increase the reverse rates and their sensitivity to the force of bonds with P- and L-selectin. However, the effects of these mutations on the binding affinities and forward rates have not been studied. We quantified these effects by using an adhesion frequency assay to measure two-dimensional affinity and kinetic rates at zero force. Wild-type PSGL-1 has 2.2- to 8.5-fold higher binding affinities for P- and L-selectin than PSGL-1 mutants with two of three tyrosines substituted by phenylalanines, and 9.6- to 49-fold higher affinities than the PSGL-1 mutant with all three tyrosines replaced. In descending order, the affinity decreased from wild-type to Y48/51F, Y46/51F, Y46/48F, and Y46/48/51F. The affinity differences were attributed to major changes in the forward rate and minor changes in the reverse rate, suggesting that these tyrosines regulate the accessibility of PSGL-1 to P- and L-selectin via electrostatic interactions, which is supported by molecular-dynamics simulations. Our results provide insights into the structure-function relationship of receptor-ligand binding at a single-residue level.
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    Monitoring receptor-ligand interactions between surfaces by thermal fluctuations,
    (Georgia Institute of Technology, 2008-01) Chen, Wei ; Evans, Evan A. ; McEver, Rodger P. ; Zhu, Cheng
    We describe a new method for determining receptor-ligand association/dissociation events across the interface of two surfaces (two-dimensional binding) by monitoring abrupt decrease/resumption in thermal fluctuations of a biomembrane force probe. Our method has been validated by rigorous control experiments and kinetic experiments. We show that cellular on-rate of association can be measured by analysis of intervals from a dissociation event to the next association event (waiting times). Similarly, off-rate of molecular dissociation can be measured by analysis of intervals from an association event to the next dissociation event (bond lifetimes). Different types of molecular bonds could be distinguished by different levels of reduction in thermal fluctuations. This novel method provides a powerful tool to study cell adhesion and signaling mediated by single or multiple receptor-ligand species.
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    Flow-enhanced adhesion regulated by a selectin interdomain hinge
    (Georgia Institute of Technology, 2006-09) Lou, Jizhong ; Yago, Tadayuki ; Klopocki, Arkadiusz G. ; Mehta, Padmaja ; Chen, Wei ; Zarnitsyna, Veronika I. ; Bovin, Nicolai V. ; Zhu, Cheng ; McEver, Rodger P.
    L-selectin requires a threshold shear to enable leukocytes to tether to and roll on vascular surfaces. Transport mechanisms govern flow-enhanced tethering, whereas force governs fl ow-enhanced rolling by prolonging the lifetimes of L-selectin–ligand complexes (catch bonds). Using selectin crystal structures, molecular dynamics simulations, site-directed mutagenesis, single-molecule force and kinetics experiments, Monte Carlo modeling, and flow chamber adhesion studies, we show that eliminating a hydrogen bond to increase the fl exibility of an interdomain hinge in L-selectin reduced the shear threshold for adhesion via two mechanisms. One affects the on-rate by increasing tethering through greater rotational diffusion. The other affects the off-rate by strengthening rolling through augmented catch bonds with longer lifetimes at smaller forces. By forcing open the hinge angle, ligand may slide across its interface with L-selectin to promote rebinding, thereby providing a mechanism for catch bonds. Thus, allosteric changes remote from the ligand-binding interface regulate both bond formation and dissociation.
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    Catch bonds govern adhesion through L-selectin at threshold shear
    (Georgia Institute of Technology, 2004-09) Yago, Tadayuki ; Wu, Jianhua ; Wey, C. Diana ; Klopocki, Arkadiusz G. ; Zhu, Cheng ; McEver, Rodger P.
    Flow-enhanced cell adhesion is an unexplained phenomenon that might result from a transport-dependent increase in on-rates or a force-dependent decrease in off-rates of adhesive bonds. L-selectin requires a threshold shear to support leukocyte rolling on P-selectin glycoprotein ligand-1 (PSGL-1) and other vascular ligands. Low forces decrease L-selectin–PSGL-1 off-rates (catch bonds), whereas higher forces increase off-rates (slip bonds). We determined that a force-dependent decrease in off-rates dictated flowenhanced rolling of L-selectin–bearing microspheres or neutrophils on PSGL-1. Catch bonds enabled increasing force to convert short-lived tethers into longer-lived tethers, which decreased rolling velocities and increased the regularity of rolling steps as shear rose from the threshold to an optimal value. As shear increased above the optimum, transitions to slip bonds shortened tether lifetimes, which increased rolling velocities and decreased rolling regularity. Thus, force-dependent alterations of bond lifetimes govern L-selectin–dependent cell adhesion below and above the shear optimum. These findings establish the first biological function for catch bonds as a mechanism for flow-enhanced cell adhesion.