The structure of human CD23 and its interactions with IgE and CD21

The low-affinity immunoglobulin E (IgE) receptor, CD23 (FcɛRII), binds both IgE and CD21 and, through these interactions, regulates the synthesis of IgE, the antibody isotype that mediates the allergic response. We have determined the three-dimensional structure of the C-type lectin domain of CD23 in solution by nuclear magnetic resonance spectroscopy. An analysis of concentration-dependent chemical shift perturbations have allowed us to identify the residues engaged in self-association to the trimeric state, whereas ligand-induced changes have defined the binding sites for IgE and CD21. The results further reveal that CD23 can bind both ligands simultaneously. Despite the C-type lectin domain structure, none of the interactions require calcium. We also find that IgE and CD23 can interact to form high molecular mass multimeric complexes. The interactions that we have described provide a solution to the paradox that CD23 is involved in both up- and down-regulation of IgE and provide a structural basis for the development of inhibitors of allergic disease

Regulation of the Cardiac Na+/K+ ATPase by Phospholemman

Hansraj Dhayan, Rajender Kumar, Andreas Kukol, ‘Regulation of the Cardiac Na+/K+ ATPase by Phospholemman’, in Sajal Chakraborti, Naranjan Dhalla, eds., Regulation of Membrane Na+-K+ ATPase, (Switzerland: Springer, 2016), ISBN 978-3-319-24748-9, eISBN 978-3-319-24750-2.Peer reviewe

Soluble CD44 Interacts with Intermediate Filament Protein Vimentin on Endothelial Cell Surface

CD44 is a cell surface glycoprotein that functions as hyaluronan receptor. Mouse and human serum contain substantial amounts of soluble CD44, generated either by shedding or alternative splicing. During inflammation and in cancer patients serum levels of soluble CD44 are significantly increased. Experimentally, soluble CD44 overexpression blocks cancer cell adhesion to HA. We have previously found that recombinant CD44 hyaluronan binding domain (CD44HABD) and its non-HA-binding mutant inhibited tumor xenograft growth, angiogenesis, and endothelial cell proliferation. These data suggested an additional target other than HA for CD44HABD. By using non-HA-binding CD44HABD Arg41Ala, Arg78Ser, and Tyr79Ser-triple mutant (CD443MUT) we have identified intermediate filament protein vimentin as a novel interaction partner of CD44. We found that vimentin is expressed on the cell surface of human umbilical vein endothelial cells (HUVEC). Endogenous CD44 and vimentin coprecipitate from HUVECs, and when overexpressed in vimentin-negative MCF-7 cells. By using deletion mutants, we found that CD44HABD and CD443MUT bind vimentin N-terminal head domain. CD443MUT binds vimentin in solution with a Kd in range of 12–37 nM, and immobilised vimentin with Kd of 74 nM. CD443MUT binds to HUVEC and recombinant vimentin displaces CD443MUT from its binding sites. CD44HABD and CD443MUT were internalized by wild-type endothelial cells, but not by lung endothelial cells isolated from vimentin knock-out mice. Together, these data suggest that vimentin provides a specific binding site for soluble CD44 on endothelial cells

A single molecule assay to probe monovalent and multivalent bonds between hyaluronan and its key leukocyte receptor CD44 under force

Glycosaminoglycans (GAGs), a category of linear, anionic polysaccharides, are ubiquitous in the extracellular space, and important extrinsic regulators of cell function. Despite the recognized significance of mechanical stimuli in cellular communication, however, only few single molecule methods are currently available to study how monovalent and multivalent GAG•protein bonds respond to directed mechanical forces. Here, we have devised such a method, by combining purpose-designed surfaces that afford immobilization of GAGs and receptors at controlled nanoscale organizations with single molecule force spectroscopy (SMFS). We apply the method to study the interaction of the GAG polymer hyaluronan (HA) with CD44, its receptor in vascular endothelium. Individual bonds between HA and CD44 are remarkably resistant to rupture under force in comparison to their low binding affinity. Multiple bonds along a single HA chain rupture sequentially and independently under load. We also demonstrate how strong non-covalent bonds, which are versatile for controlled protein and GAG immobilization, can be effectively used as molecular anchors in SMFS. We thus establish a versatile method for analyzing the nanomechanics of GAG•protein interactions at the level of single GAG chains, which provides new molecular-level insight into the role of mechanical forces in the assembly and function of GAG-rich extracellular matrices

Surface Charges of the Membrane Crucially Affect Regulation of Na,K-ATPase by Phospholemman (FXYD1)

Abstract The human a1/His10-b1 isoform of Na,K-ATP-ase has been reconstituted as a complex with and without FXYD1 into proteoliposomes of various lipid compositions in order to study the effect of the regulatory subunit on the half-saturating Na? concentration (K1/2) of Na? ions for activation of the ion pump. It has been shown that the fraction of negatively charged lipid in the bilayer crucially affects the regulatory properties. At low concentrations of the nega-tively charged lipid DOPS (\10 %), FXYD1 increases K1/2 of Na? ions for activation of the ion pump. Phosphorylation of FXYD1 by protein kinase A at Ser68 abrogates this effect. Conversely, for proteoliposomes made with high concen-trations of DOPS ([10 %), little or no effect of FXYD1 on theK1/2 ofNa? ions is observed. Depending on ionic strength and lipid composition of the proteoliposomes, FXYD1 can alter the K1/2 of Na? ions by up to twofold. We propose possible molecular mechanisms to explain the regulatory effects of FXYD1 and the influence of charged lipid and protein phosphorylation. In particular, the positively charged C-terminal helix of FXYD1 appears to be highly mobile and may interactwith the cytoplasmicNdomain of thea-subunit, the interaction being strongly affected by phosphorylation at Ser68 and the surface charge of the membrane

Structural and functional characterization of a novel T cell receptor co-regulatory protein complex, CD97-CD55.

CD97, the archetypal member of the EGF-TM7 protein family, is constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells following activation. The key isoform of CD97 expressed on leukocytes binds the complement regulatory protein CD55 (also termed decay-accelerating factor). CD97 has been shown recently to mediate co-stimulation of T cells via CD55. Here, we demonstrate that blocking the interaction between CD55 on monocytes and CD97 on T cells leads to inhibition of proliferation and interferon-gamma secretion. This implies that bidirectional interactions between CD97 and CD55 are involved in T cell regulation. Structural studies presented here reveal the molecular basis for this activity. We have solved the structure of EMR2, a very close homolog of CD97, using x-ray crystallography. NMR-based chemical shift mapping of the EMR2-CD55 interaction has allowed us to generate a model for the CD97-CD55 complex. The structure of the complex reveals that the T cell and complement regulatory activities of CD55 occur on opposite faces of the molecule. This suggests that CD55 might simultaneously regulate both the innate and adaptive immune responses, and we have shown that CD55 can still regulate complement when bound to CD97