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Zhu, Cheng

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Author: Zarnitsyna, Veronika I. × search.filters.applied.f.isOrgUnitOfPublicationTitle: Wallace H. Coulter Department of Biomedical Engineering × search.filters.applied.f.resourcesubtype: Article ×

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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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    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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    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.