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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 ofa 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 complementbinding-like (CB) motif. The lectinmotif in cell adhesion is directed by the LEC-CAMS, although the egf-like domain of theHR may also play a potential role in cell binding. While the role(s) of the CB domainsin the LEC-CAMS is currently not understood, they have been hypothesized to act asrigid 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, anadhesion blocking antibody that recognizes a conformational determinant in theN-terminus of the HR lectin domain, shows a significantly decreased affinity for HRconstruct that lacks the CB motifs, consistent with the possibility that the CBdomains are involved with lectin domain structure. In agreement with this conjecture,HR mutants lacking the CB domains show a profound decrease in lectin-specificinteraction with the carbohydrate polyphosphomannan ester, suggesting that thechanges in Mel 14 affinity for the lectin domain are reflected in lectin functionality.Various assays investigating the interactions between the HR deletion mutants and theperipheral lymph node high endothelium, including cell blocking, immunohistochemicalstaining, and radioactively labeled ligand binding, all show that removal of the CBdomains results in a lack of HR adhesive function. These results imply that the CBdomains of the HR, and, by analogy, the other members of the LEC-CAM family,may play important structural roles involving induction of lectin domain conformationand resultant functionality (Watson, 1991).

The three-dimensional structure of the ligand-binding region of human E-selectin hasbeen determined at 2.0 A resolution. The structure reveals limited contact betweenthe 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 sidechains that may be involved in ligand binding (Graves, 1994).

E-selectin is a member of the selectin family of proteins that recognize carbohydrateligands 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 limitedproteolysis. A Ca(2+)-free form of soluble E-selectin containingthe entire extracellular domain, is sensitive to limited proteolysis by endoproteinase. Amino-terminal sequencing analysis of the proteolytic fragmentsreveals 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 isfurther augmented upon sialyl Lewis x (sLex) ligand binding. These results imply that Ca2+ binding to E-selectin induces a conformational change and perhapsfacilitates 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 bindingactivity. 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 ofleukocytes with endothelium. The extracellular region of each selectin contains anamino-terminal C-type lectin domain, followed by an EGF-like domain and multipleshort consensus repeat units (SCRs). Previous studies have indirectly suggested a rolefor each of the extracellular domains of the selectins in cell adhesion. In this study, apanel of chimeric selectins created by exchange of domains between L- andP-selectin was used to directly examine the role of the extracellular domains in celladhesion. Exchange of only the lectin domains between L- and P-selectin confersthe adhesive and ligand recognition functions of the lectin domain of the parentmolecule. However, chimeric selectins which contained both the lectin domain ofL-selectin and the EGF-like domain of P-selectin exhibit dual ligand-bindingspecificity. These chimeric proteins supported adhesion both to myeloid cells and tohigh endothelial venules (HEV) of lymph nodes and mesenteric venules in vivo.Exchange of the SCR domains has no detectable effect on receptor function orspecificity. Thus, the EGF-like domain of P-selectin may play a direct role in ligandrecognition and leukocyte adhesion mediated by P-selectin, with the lectin plusEGF-like domains collectively forming a functional ligand recognition unit (Kansas, 1994).

To estimate the density of P-selectin inmembranes 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 cellline) 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 ovarycells. The P-selectin density on stimulated platelets is estimated to be 150 to 200molecules/square micron. To identify the domains of P-selectin required for HL60 cellbinding, chimeras of P-selectin and L-selectin were stably expressed in Chinesehamster ovary cells and clones that expressed the chimeras at the estimatedphysiologic density were selected. Chimeras containing the P-selectin lectin andepidermal growth factor (EGF) domains or the lectin, EGF, and short consensusrepeats bind HL60 cells equivalently, but in comparison, a chimera containing the P-selectin lectindomain alone binds HL60 cells much less well. These results indicate that at aphysiologically relevant P-selectin density on membrane surfaces, the lectin, and EGFdomains 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 highendothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sitesof inflammation. Truncation of the L-selectin cytoplasmictail by 11 amino acids abolishes binding to lymph node HEV and leukocyte rolling invivo. The cytoplasmic domain of L-selectin interacts directly with the cytoplasmicactin-binding protein alpha-actinin and forms a complex with vinculin and possiblytalin. Direct, specific, and saturable binding ofpurified alpha-actinin to L-selectin cytoplasmic tail has been detected, but no direct binding of purifiedtalin or vinculin. Interestingly, talin potentiates binding of alpha-actinin to the L-selectincytoplasmic domain peptide despite the fact that direct binding of talin to L-selectincan not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptideis detectable only in the presence of alpha-actinin. L-selectin coprecipitated with acomplex of cytoskeletal proteins, including alpha-actinin and vinculin from cellstransfected with L-selectin, consistent with the possibility that alpha-actinin bindsdirectly to L-selectin and that vinculin associates by binding to alpha-actinin in vivo tolink actin filaments to the L-selectin cytoplasmic domain. In contrast, a deletion mutantof L-selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domainfails to coprecipitate with alpha-actinin or vinculin. Surprisingly, this mutant L-selectinlocalizes normally to the microvillar projections on the cell surface. These data suggestthat the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain arerequired for mediating interactions with the actin cytoskeleton via a complex ofalpha-actinin and vinculin, but that this portion of the cytoplasmic domain is notnecessary for proper localization of L-selectin on the cell surface. Correct L-selectinreceptor positioning is therefore insufficient for leukocyte adhesion mediated byL-selectin, suggesting that this adhesion may also require direct interactions with thecytoskeleton (Pavalko, 1995).

Selectin mutation

Mice possessing a mutant L-selectin gene that results in the completeloss of cell surface receptor expression were generated by gene targeting.Lymphocytes from these mice do not bind to peripheral lymph node HEV; thesemice show a severe reduction in the number of lymphocytes localized to peripherallymph nodes. Short-term homing experiments demonstrate that L-selectin is alsoinvolved in lymphocyte migration to mucosal lymph nodes, Peyer's patches, andspleen. Furthermore, significant defects in leukocyte rolling and neutrophil migrationinto 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 ofinflammation (Arbones, 1994).

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

The phenotype of mice lacking both endothelial selectins is described after sequentialablation of the genes encoding P- and E-selectins. In contrast with the rather mildphenotypes observed in mice deficient in a single selectin gene, the doubly deficientmice present extreme leukocytosis, elevated cytokine levels, and alterations inhematopoiesis. Granulocytopoiesis is increased both in bone marrow and spleen, whileerythropoiesis is partially translocated to the spleen. Virtual lack of leukocyte rollingand low extravasation at sites of inflammation make these animals susceptible toopportunistic bacterial infections, to which they succumb. Theabsence of endothelial selectins severely affects leukocyte homeostasis and indicatesthat these two selectins are as important for normal leukocyte function as are theleukocyte 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 probablyfucosylation. A major capping group in GlyCAM-1 is 6'sulfated sialyl Lewis x, a novel structure that potentially satisfies all of theserequirements. The complete structure ofbeta-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. Galbeta 1-->3[GlcNAc beta 1-->6]GalNAc or larger core structures with additionalGlcNAc residues (Hemmerich, 1995).

One of theendothelial-derived ligands for L-selectin is GlyCAM-1 (previously known as Sgp50),a mucin-like glycoprotein with sulfated, sialylated, and fucosylated O-linkedoligosaccharide chains. Sialylation, sulfation, and fucosylation appear to be required forthe avid interaction of this ligand with L-selectin, but the exact carbohydrate structuresinvolved in recognition remain undefined. GlyCAM-1 was metabolically labeledin lymph node organ culture with 35SO4 and a panel of tritiated carbohydrateprecursors. Mild hydrolysis conditions release sulfatedoligosaccharides without cleavage of sulfate esters. Sulfatedconstituents of GlyCAM-1 are Gal-6-SO4, GlcNAc-6-SO4, (SO4-6)Galbeta 1-->4GlcNAc, and Gal beta 1-->4(SO4-6)GlcNAc. In the accompanying paper evidence ispresented that (SO4-6)Gal beta 1-->4GlcNAc forms the core of a sulfated sialylLewis x structure that may comprise a recognition determinant on GlyCAM-1 (Hemmerich, 1994).

The mucin-type polypeptides GlyCAM-1, CD34, andMAdCAM-1 can function as ligands for L-selectin only when they are synthesized bythe 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 bindL-selectin. Soluble mucins secreted by human colon carcinomacells, as well as those derived from human bronchial mucus can bind to humanL-selectin in a calcium-dependent manner. As with Gly-CAM-1 synthesized by lymphnode HEV, alpha 2-3 linked sialic acids and sulfation seem to play a critical role ingenerating this L-selectin binding. In each case, only a subset of the mucin moleculesis recognized by L-selectin. Binding is not destroyed by boiling, suggesting thatrecognition may be based primarily upon carbohydrate structures. Despite this,O-linked oligosaccharide chains released from these ligands by beta-elimination do notshow any detectable binding to L-selectin. Following protease treatment of the ligands,binding persists in a subset of the resulting fragments, indicating that specificrecognition is determined by certain regions of the original mucins. However, O-linkedoligosaccharides released from the subset of non-binding mucin fragments do notshow very different size and charge profiles when compared to those that do bind.Furthermore, studies with polylactosamine-degrading endoglycosidases suggest thatthe core structures involved in generating binding can vary among the differentligands. Taken together, these data indicate that a single unique oligosaccharidestructure may not be responsible for high-affinity binding. Rather, diverse mucins withsialylated, sulfated, fucosylated lactosamine-type O-linked oligosaccharides cangenerate high-affinity L-selectin ligands, but only when they present these chains inunique spacing and/or clustered combinations, presumably dictated by the polypeptidebackbone (Crottet, 1996).

furrowed: Biological Overview | Developmental Biology | Effects of Mutation | References

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