title: Membrane microdomains and glycosylation of activating receptors influence natural killer cell regulation
creator: Margraf-Schönfeld, Stefanie
subject: 570
subject: 570 Life sciences
description: Natural killer (NK) cells represent the first lymphoid subpopulation in the defense against tumors and viral infection. The activity of NK cells is regulated by the interplay of activating and inhibitory surface receptors. The mechanisms by which these receptors transduce their signals and the integration of positive and negative signals are not completely understood. 2B4 (CD244) is an important activating NK cell receptor. Ligand engagement results in the recruitment of 2B4 to membrane microdomains, which is essential for the function of this receptor. Here we successfully established a new approach to investigate membrane microdomains without destroying the membrane’s natural composition, as it has often been criticized for the classical cold non-ionic detergent lysis followed by sucrose density gradient centrifugation. NK cell receptors were stimulated by antibodies coupled to magnetic beads. NK cells were then physically disrupted in a nitrogen-cavitation bomb and membrane fragments surrounding the engaged receptor including the associated signaling machinery were immunoisolated by separation of the magnetic beads. By comparing bead contents of activating (2B4) with inhibitory (NKG2A) NK cell receptors using 2D-DIGE, several proteins with significant difference emerged, which were subsequently identified by mass spectrometry. Important candidates for cytoskeletal remodeling and signal transduction were identified to be specifically associated with 2B4, which gave us a first hint for the formation of multimolecular complexes and their role in the early steps of NK cell activation. In a second project we analyzed the glycosylation of 2B4. We demonstrated that 2B4 is heavily and differentially glycosylated in primary human NK cells and NK cell lines. The differential glycosylation of 2B4 could be attributed to sialic acid residues on N- and O-linked carbohydrates. Glycosylation plays an important role in receptor-ligand recognition and interaction but may also lead to repulsion between molecules. Using a recombinant fusion protein of the extracellular domain of 2B4 we demonstrated that N-linked glycosylation of 2B4 is essential for binding to its ligand CD48. In contrast, sialylation of 2B4 had a negative impact on ligand binding as the interaction between 2B4 and CD48 was increased after the removal of sialic acids. This was confirmed in a functional assay system, where the desialylation of NK cells or the inhibition of O-linked glycosylation resulted in increased 2B4-mediated lysis of CD48 expressing tumor target cells. These data demonstrate that glycosylation has an important impact on 2B4-mediated NK cell function and suggest that regulated changes in glycosylation during NK cell development and activation might be involved in the regulation of NK cell responses.
date: 2010
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/11531/1/Dokument_1.pdf
identifier: DOI:10.11588/heidok.00011531
identifier: urn:nbn:de:bsz:16-opus-115310
identifier:   Margraf-Schönfeld, Stefanie  (2010) Membrane microdomains and glycosylation of activating receptors influence natural killer cell regulation.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/11531/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng