Structure and Function of L-selectin
The aims of this project are:
- Analysis of the ligand binding to the lectin domain
- Influence of the EGF-like domain and the N-glycans in ligand binding
- Principles of the topology of L-selectin on microvilliIdentification and characterization of new ligands (extracellular and intracellular)
- Insights into the L-selectin-mediated signal transduction
Sie befinden sich hier:
The leukocytic cell adhesion receptor L-selectin mediates the initial step of the adhesion cascade, the capture and rolling of leukocytes on endothelial cells. This event enables leukocytes to migrate out of the vasculature into surrounding tissues during inflammation and immune surveillance.
The importance of L-selectin is shown in knock-out mice. These mice show a profound defect of lymphocyte accumulation in secondary lymphatic tissue and of neutrophils at sites of inflammation (Arbonés et al., 1994; Jung and Ley, 1999).
L-selectin is a type I transmembrane protein of the selectin family. It belongs to the c-type lectins, which binds Ca2+-dependent special carbohydrate structures, and consists N-terminal of a lectin domain, an epidermal growth factor (EGF)-like domain, two short consensus repeats (scr), a transmembrane domain and a cytoplasmatic tail (Bevilacqua et al., 1989; Siegelman and Weissman, 1989). Distinct domains of L-selectin contribute to proper leukocyte migration:
The Topology of L-selectin
Up to 80 % of cell surface L-selectin is localized on microvilli (MV) (Bruehl et al., 1996). These microvilli are membrane protrusions, which serve as active grouping sites for several receptors like P-selectin glycoprotein ligand-1 (PSGL-1) (Miner et al., 2008; Moore et al., 1995) and the α4-intergrins (Berlin et al., 1995). In contrast, the integrins Mac-1 (Erlandsen et al., 1993) and LFA-1 (Hocdé et al., 2009) and E-selectin ligand CD44 (von Andrian et al., 1995) are localized on the planar cell body (CB). Owing to the shear-dependent microenvironment in post-capillary venules, L-selectin is expressed on microvilli thereby enabling efficient leukocyte tethering. When expressed on the planar cell body using L-selectin/CD44 domain-swapped chimeras, transfectants showed a reduced tethering under flow (von Andrian et al., 1995). We could show, that the topology of L-selectin on MV depends on the transmembrane domain and that MV-based chimeras of L-selectin had a significant higher adhesion rate compared to cells with CB-expressed receptors in a flow chamber assay (Buscher et al., 2010). This study showed that the physiological function of L-selectin closely depends on this topography.
The N-Glycans of L-selectin
The extracellular domain (EC) of human L-selectin contains 7 potentail N-glycosylation sites (Lasky et al., 1989; Siegelman and Weissman, 1989). In the past, it could be shown, that the glycosylation of L-selectin exhibits cell type-specific differences. L-selectin from neutrophils had an apparent molecular mass of 95-105 kDa when separated by SDS-PAGE (Kishimoto et al., 1989; Berg and James, 1990; Griffin et al., 1990) while L-selectin from lymphocytes (Tedder et al., 1990) or from Jurkat T cells (Berg and James, 1990) migrated on SDS-polyacrylamid gels at ~74 kDa and between 50 and 70 kDa, respectively.
The observation that in patients with chronic lymphocytic leukemia the apparent molecular mass of L-selectin was decreased by ~3.7 kDa in comparison to samples from healthy controls (Prystas et al., 1993) had suggested that glycosylation of L-selectin might be related to certain types of disease. It was shown that L-selectin specific antibodies inhibited the binding of polymorphonuclear leukocytes to E-selectin transfectants by 70% (Picker et al., 1991). Similarly, recombinant E-selectin-IgG chimeras bound specifically to L-selectin on human neutrophils, but not on mouse neutrophils (Zollner et al., 1997). In conjunction with the finding that the binding was calcium dependent and was abolished after sialidase digestion, these data indicate that human L-selectin is glycosylated with glycan epitopes that serve as binding sites for E-selectin.
We performed a detailed per site N-glycan analysis for recombinant L-selectin variants which comprised the functionally essential lectin and EGF-like domain. These two domains contain three N-glycosylation sites, two in the lectin domain at position N22 and N66 and one in the EGF-like domain at N139. Mutant proteins with only one intact N-glycosylation site each were expressed in the human cell line HEK293F (Wedepohl et al., 2010). Analysis by sequential exoglycosidase digestions in combination with MALDI-TOF and MALDI-TOF/TOF mass spectrometry revealed three features characteristic for the N-glycosylation of human L-selectin.
First, comparison of the N-glycans of each of the three N-glycosylation sites of the lectin- and of the EGF-domain displayed a clear site heterogeneity of the N-glycan structures with the N-glycans at the first site (N22) being least heterogeneous. The N-glycans were mainly diantennary, highly fucosylated and terminating with unusual N-acetylgalactosamine (GalNAc) residues, partially replacing the more common galactose (Gal).
Second, the lectin and the EGF-like domain of L-selectin contained unusual N-glycan structures, i.e. diantennary N-glycans with antennae terminating with the motif GalNAc–GalNAc, which has not been described for N-glycans before. These unusual glycans were found especially at the first glycosylation site at position N22.
Third, the N-glycan structures of L-selectin were found to be sulfated at terminal GalNAc residues. Sulfated N-glycans have not been described for L-selectin so far.
The signaling of L-selectin
In conjunction with its adhesive function, L-selectin mediates outside-in and inside-out signaling in leukocytes. Ligation of L-selectin with antibody or ligand elicits a number of cellular events, including elevated Ca2+ and phosphotyrosine protein levels (Laudanna et al., 1994; Waddell et al., 1995), synthesis of reactive oxygen compounds (Waddell et al., 1994), activation of the mitogen-activated protein kinases extracellular regulated kinase and Jun N-terminal kinase (Brenner et al., 1996, 1997), and Rac-mediated rearrangements of the actin cytoskeleton (Brenner et al., 1997). Furthermore, L-selectin triggers the enhanced binding activity of both β1 and β2 integrins (Hwang et al., 1996; Giblin et al., 1997). In lymphocytes and neutrophils, L-selectin cross-linking can lead to activation of a number of intracellular signaling pathways that are mostly dependent on the src-family kinase p56lck. This kinase interacts via its SH2 domain with the cytoplasmic domain of L-selectin and phosphorylates the receptor on the C-terminal tyrosine residue (Brenner et al., 1996; Xu et al., 2008).
Apart from transducing signals received from the outside of the cell, L-selectin itself is subject to regulation by cellular signals. Treatment of leukocytes with lineage-specific stimuli (cross-linking of CD2 or CD3 for lymphocytes, granulocyte colonystimulating factor, granulocyte-macrophage colony-stimulating factor, or tumor necrosis factor alpha for neutrophils) led to an increased binding of L-selectin to the soluble ligand and of lymphocytes to high endothelial venule frozen sections (Spertini et al., 1991). This enhancement of binding activity is assumed to be a result of phosphorylation of the cytoplasmic domain of the receptor on serine residues, which occurs constitutively at low levels and is significantly increased by stimulation with phorbol ester or chemokines through co-transfected G-protein-coupled chemokine receptors (Haribabu et al., 1997).
We could show that phosphorylation of the cytoplasmic domain of L-selectin on serine residues at position 364 and 367 is catalyzed by two protein kinase C (PKC) isozymes, which both bind to the non-phosphorylated cytoplasmic domain (Kilian et al., 2004). Phosphorylation on serine residues led to the additional firm binding of PKC alpha. Binding of PKC alpha and theta to L-selectin can be induced by treatment of cells with phorbol esters and by CD3 cross-linking. This observation indicates a role of these kinases in the regulation of L-selectin through the T-cell receptor (TCR) complex. Functional cross-talk between l-selectin and the TCR complex has been indicated in a study where immobilized antibodies directed against L-selectin provided a co-stimulatory signal for lymphocyte activation via the TCR (Murakawa et al., 1992). Vice versa, stimulation of T-cells via CD3 increased the binding activity of L-selectin (Spertini et al., 1991).
L-selectin and allosteric antibodies
Conformation-specific Abs have been invaluable in deciphering the activation mechanism of integrins, but similar reagents are not available for selectins.
We could show that the anti-human L-selectin mAbs DREG-55 and LAM1-5 but not DREG-56, DREG-200, or LAM1-1 heterotropically modulate adhesion presumably by stabilizing the extended receptor conformation. Force-free affinity assays, flow chamber, and microkinetic studies reveal a ligand-specific modulation of L-selectin affinity by DREG-55 mAb, resulting in a dramatic decrease of rolling velocity under flow. Furthermore, secondary tethering of polymorphonuclear cells was blocked by DREG-200 but significantly boosted by DREG-55 mAb. The results emphasize the need for a new classification for selectin Abs and introduce the new concept of heterotropic modulation of receptor function (Riese et al., 2014).