Functional roles of selectin structural domains

The leukocyte homing receptor (HR), the endothelial leukocyte adhesion molecule, and gmp140/platelet activation-dependent granule membrane protein are three members of a family of adhesion molecules, termed the lectin cell adhesion molecules (LEC-CAMS); they are unified by a multi-domain structure containing a lectin motif, an epidermal growth factor-like (egf) motif, and variable numbers of a complement binding-like (CB) motif. The lectin motif in cell adhesion is directed by the LEC-CAMS, although the egf-like domain of the HR may also play a potential role in cell binding. While the role(s) of the CB domains in the LEC-CAMS is currently not understood, they have been hypothesized to act as rigid spacers or stalks for lectin and perhaps, egf domain presentation. The functional characteristics of murine HR-IgG chimeras containing lectin, lectin plus egf, and lectin plus egf plus CB domains were analyzed. The Mel 14 mAb, an adhesion blocking antibody that recognizes a conformational determinant in the N-terminus of the HR lectin domain, shows a significantly decreased affinity for HR construct that lacks the CB motifs, consistent with the possibility that the CB domains are involved with lectin domain structure. In agreement with this conjecture, HR mutants lacking the CB domains show a profound decrease in lectin-specific interaction with the carbohydrate polyphosphomannan ester, suggesting that the changes in Mel 14 affinity for the lectin domain are reflected in lectin functionality. Various assays investigating the interactions between the HR deletion mutants and the peripheral lymph node high endothelium, including cell blocking, immunohistochemical staining, and radioactively labeled ligand binding, all show that removal of the CB domains results in a lack of HR adhesive function. These results imply that the CB domains of the HR, and, by analogy, the other members of the LEC-CAM family, may play important structural roles involving induction of lectin domain conformation and resultant functionality (Watson, 1991).

The three-dimensional structure of the ligand-binding region of human E-selectin has been determined at 2.0 A resolution. The structure reveals limited contact between the two domains and a coordination of Ca2+ not predicted from other C-type lectins. Structure/function analysis indicates a defined region and specific amino-acid side chains that may be involved in ligand binding (Graves, 1994).

E-selectin is a member of the selectin family of proteins that recognize carbohydrate ligands in a Ca(2+)-dependent manner. In order to better understand the role of Ca2+ in E-selectin-ligand interactions, the E-selectin structure has been examined by limited proteolysis. A Ca(2+)-free form of soluble E-selectin containing the entire extracellular domain, is sensitive to limited proteolysis by endoproteinase. Amino-terminal sequencing analysis of the proteolytic fragments reveals that the major cleavage site is at Glu98, found in the loop (residues 94-103) adjacent to the Ca2+ binding region of the lectin domain. Upon Ca2+ binding Glu98 is protected from proteolysis. This Ca(2+)-dependent protection is further augmented upon sialyl Lewis x (sLex) ligand binding. These results imply that Ca2+ binding to E-selectin induces a conformational change and perhaps facilitates ligand binding. The sLex-bound complex in turn stabilizes Ca2+ binding. The lectin contains only one high-affinity Ca2+ site. Ba2+ is a potent antagonist in blocking lectin-mediated HL-60 cell adhesion. Ba2+ binds to lectin 5-fold tighter than Ca2+ and abolishes ligand binding activity. Sr2+ also binds tighter than Ca2+. However, Sr(2+)-regenerated lectin shows 50% ligand binding activity. Mg2+ binds with much weaker affinity than Ca2+ and the lecting does not show any activity (Anostario, 1995).

The selectin family of adhesion molecules mediates the initial interactions of leukocytes with endothelium. The extracellular region of each selectin contains an amino-terminal C-type lectin domain, followed by an EGF-like domain and multiple short consensus repeat units (SCRs). Previous studies have indirectly suggested a role for each of the extracellular domains of the selectins in cell adhesion. In this study, a panel of chimeric selectins created by exchange of domains between L- and P-selectin was used to directly examine the role of the extracellular domains in cell adhesion. Exchange of only the lectin domains between L- and P-selectin confers the adhesive and ligand recognition functions of the lectin domain of the parent molecule. However, chimeric selectins which contained both the lectin domain of L-selectin and the EGF-like domain of P-selectin exhibit dual ligand-binding specificity. These chimeric proteins supported adhesion both to myeloid cells and to high endothelial venules (HEV) of lymph nodes and mesenteric venules in vivo. Exchange of the SCR domains has no detectable effect on receptor function or specificity. Thus, the EGF-like domain of P-selectin may play a direct role in ligand recognition and leukocyte adhesion mediated by P-selectin, with the lectin plus EGF-like domains collectively forming a functional ligand recognition unit (Kansas, 1994).

To estimate the density of P-selectin in membranes necessary to support adhesion, purified P-selectin was incorporated at varying concentrations into phospholipid bilayers that encapsulated glass microspheres. Maximal binding of these lipospheres to HL60 cells (a P-selectin ligand-expressing cell line) is approached at a P-selectin density of about 100 molecules per square micron; half-maximal binding is observed at about 50 to 60 molecules per square micron. Compatible results are obtained with P-selectin expressed on Chinese hamster ovary cells. The P-selectin density on stimulated platelets is estimated to be 150 to 200 molecules/square micron. To identify the domains of P-selectin required for HL60 cell binding, chimeras of P-selectin and L-selectin were stably expressed in Chinese hamster ovary cells and clones that expressed the chimeras at the estimated physiologic density were selected. Chimeras containing the P-selectin lectin and epidermal growth factor (EGF) domains or the lectin, EGF, and short consensus repeats bind HL60 cells equivalently, but in comparison, a chimera containing the P-selectin lectin domain alone binds HL60 cells much less well. These results indicate that at a physiologically relevant P-selectin density on membrane surfaces, the lectin, and EGF domains of P-selectin are together required for optimal leukocyte binding (Gibson, 1995).

Selectin interaction with the cytoskeleton

The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Truncation of the L-selectin cytoplasmic tail by 11 amino acids abolishes binding to lymph node HEV and leukocyte rolling in vivo. The cytoplasmic domain of L-selectin interacts directly with the cytoplasmic actin-binding protein alpha-actinin and forms a complex with vinculin and possibly talin. Direct, specific, and saturable binding of purified alpha-actinin to L-selectin cytoplasmic tail has been detected, but no direct binding of purified talin or vinculin. Interestingly, talin potentiates binding of alpha-actinin to the L-selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin can not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide is detectable only in the presence of alpha-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins, including alpha-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that alpha-actinin binds directly to L-selectin and that vinculin associates by binding to alpha-actinin in vivo to link actin filaments to the L-selectin cytoplasmic domain. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain fails to coprecipitate with alpha-actinin or vinculin. Surprisingly, this mutant L-selectin localizes normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of alpha-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L-selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L-selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton (Pavalko, 1995).

Selectin mutation

Mice possessing a mutant L-selectin gene that results in the complete loss of cell surface receptor expression were generated by gene targeting. Lymphocytes from these mice do not bind to peripheral lymph node HEV; these mice show a severe reduction in the number of lymphocytes localized to peripheral lymph nodes. Short-term homing experiments demonstrate that L-selectin is also involved in lymphocyte migration to mucosal lymph nodes, Peyer's patches, and spleen. Furthermore, significant defects in leukocyte rolling and neutrophil migration into the peritoneum in response to an inflammatory stimulus are observed. Thus, L-selectin plays an essential role in leukocyte homing to lymphoid tissues and sites of inflammation (Arbones, 1994).

P selectin-deficient mice, generated by gene targeting in embryonic stem cells, exhibit a number of defects in leukocyte behavior, including elevated numbers of circulating neutrophils, virtually total absence of leukocyte rolling in mesenteric venules, and delayed recruitment of neutrophils to the peritoneal cavity upon experimentally induced inflammation. These results clearly demonstrate a role for P selectin in leukocyte interactions with the vessel wall and in the early steps of leukocyte recruitment at sites of inflammation (Mayadas, 1993).

The phenotype of mice lacking both endothelial selectins is described after sequential ablation of the genes encoding P- and E-selectins. In contrast with the rather mild phenotypes observed in mice deficient in a single selectin gene, the doubly deficient mice present extreme leukocytosis, elevated cytokine levels, and alterations in hematopoiesis. Granulocytopoiesis is increased both in bone marrow and spleen, while erythropoiesis is partially translocated to the spleen. Virtual lack of leukocyte rolling and low extravasation at sites of inflammation make these animals susceptible to opportunistic bacterial infections, to which they succumb. The absence of endothelial selectins severely affects leukocyte homeostasis and indicates that these two selectins are as important for normal leukocyte function as are the leukocyte beta2 integrins (Frenette, 1996).

Selectin ligand structure

The binding of L-selectin to endothelial-associated glycoprotein ligands GlyCAM-1 and CD34 requires oligosaccharide sialylation, sulfation, and probably fucosylation. A major capping group in GlyCAM-1 is 6' sulfated sialyl Lewis x, a novel structure that potentially satisfies all of these requirements. The complete structure of beta-eliminated chains of GlyCAM-1 has been determined. The majority of the O-glycans in GlyCAM-1 contain the T-antigen, i.e. Gal beta 1-->3GalNAc, which is incorporated into the core-2 structure, i.e. Gal beta 1-->3[GlcNAc beta 1-->6]GalNAc or larger core structures with additional GlcNAc residues (Hemmerich, 1995).

One of the endothelial-derived ligands for L-selectin is GlyCAM-1 (previously known as Sgp50), a mucin-like glycoprotein with sulfated, sialylated, and fucosylated O-linked oligosaccharide chains. Sialylation, sulfation, and fucosylation appear to be required for the avid interaction of this ligand with L-selectin, but the exact carbohydrate structures involved in recognition remain undefined. GlyCAM-1 was metabolically labeled in lymph node organ culture with 35SO4 and a panel of tritiated carbohydrate precursors. Mild hydrolysis conditions release sulfated oligosaccharides without cleavage of sulfate esters. Sulfated constituents of GlyCAM-1 are Gal-6-SO4, GlcNAc-6-SO4, (SO4-6)Gal beta 1-->4GlcNAc, and Gal beta 1-->4(SO4-6)GlcNAc. In the accompanying paper evidence is presented that (SO4-6)Gal beta 1-->4GlcNAc forms the core of a sulfated sialyl Lewis x structure that may comprise a recognition determinant on GlyCAM-1 (Hemmerich, 1994).

The mucin-type polypeptides GlyCAM-1, CD34, and MAdCAM-1 can function as ligands for L-selectin only when they are synthesized by the specialized high-endothelial venules (HEV) of lymph modes. Since sialylation, sulfation, and possibly fucosylation are required for generating recognition, it was reasoned that other mucins known to have such components might also bind L-selectin. Soluble mucins secreted by human colon carcinoma cells, as well as those derived from human bronchial mucus can bind to human L-selectin in a calcium-dependent manner. As with Gly-CAM-1 synthesized by lymph node HEV, alpha 2-3 linked sialic acids and sulfation seem to play a critical role in generating this L-selectin binding. In each case, only a subset of the mucin molecules is recognized by L-selectin. Binding is not destroyed by boiling, suggesting that recognition may be based primarily upon carbohydrate structures. Despite this, O-linked oligosaccharide chains released from these ligands by beta-elimination do not show any detectable binding to L-selectin. Following protease treatment of the ligands, binding persists in a subset of the resulting fragments, indicating that specific recognition is determined by certain regions of the original mucins. However, O-linked oligosaccharides released from the subset of non-binding mucin fragments do not show very different size and charge profiles when compared to those that do bind. Furthermore, studies with polylactosamine-degrading endoglycosidases suggest that the core structures involved in generating binding can vary among the different ligands. Taken together, these data indicate that a single unique oligosaccharide structure may not be responsible for high-affinity binding. Rather, diverse mucins with sialylated, sulfated, fucosylated lactosamine-type O-linked oligosaccharides can generate high-affinity L-selectin ligands, but only when they present these chains in unique spacing and/or clustered combinations, presumably dictated by the polypeptide backbone (Crottet, 1996).