Identification of a missense mutation (G329A;Arg(110)--> GLN) in the human FUT7 gene

The human FUT7 gene codes for the alpha1,3-fucosyltransferase VII (Fuc-TVII), which is involved in the biosynthesis of the sialyl Lewis x (SLe(x)) epitope on human leukocytes. The FUT7 gene has so far been considered to be monomorphic. Neutrophils isolated from patients with ulcerative colitis were examined for apparent alterations in protein glycosylation patterns by Western blot analysis using monoclonal antibodies directed against SLe(x) and SLe(x)-related epitopes. One individual showed lower levels of SLe(x) expression and an elevated expression of CD65s compared to controls. The coding regions of the FUT7 gene from this individual were cloned, and a G329A point mutation (Arg(110) --> Gln) was found in one allele, whereas the other FUT7 allele was wild type. No Fuc-TVII enzyme activity was detected in COS-7 cells transiently transfected with the mutated FUT7 construct. The FUT7 Arg(110) is conserved in all previously cloned vertebrate alpha 1,3-fucosyltransferases. Polymerase chain reaction followed by restriction enzyme cleavage was used to screen 364 unselected Caucasians for the G329A mutation, and a frequency of < or =1% for this mutation was found (3 heterozygotes). Genetic characterization of the family members of one of the additional heterozygotes identified one individual carrying the G329A mutation in both FUT7 alleles. Peripheral blood neutrophils of this homozygously mutated individual showed a lowered expression of SLe(x) and an elevated expression of CD65s when analyzed by Western blot and flow cytometry. The homozygous individual was diagnosed with ulcer disease, non-insulin-dependent diabetes, osteoporosis, spondyloarthrosis, and Sj?gren's syndrome but had no history of recurrent bacterial infections or leukocytosis.     

Acquisition of P-selectin binding activity by en bloc transfer of sulfo Le(x) trisaccharide to the cell surface: comparison to a sialyl Le(x) tetrasaccharide transferred on the cell surface

Sialyl Le(x), NeuNAcalpha2 --> 3Galbeta1 --> 4(Fucalpha1 --> 3)GlcNAcbeta --> R, is known to be a ligand for E-selectin in various assays. The sulfated counterpart of sialyl Le(x), sulfo Le(x), (Sulfo --> 3) Galbeta1 --> 4 (Fucalpha1 --> 3) GlcNAcbeta --> R, was also shown to be a ligand for E-selectin in solid-phase assays employing immobilized oligosaccharides. In order to determine whether sulfo Le(x) structure on the cell surface also works as E-selectin or P-selectin ligand, a novel approach for in vitro transfer of oligosaccharides (S. Tsuboi, Y. Isogai, N. Hada, J. K. King, O. Hindsgaul, and M. Fukuda (1996) J. Biol. Chem. 271, 27213-27216) was utilized. A synthetic GDP-fucose harboring sialyl Le(x) or sulfo Le(x) oligosaccharide was enzymatically transferred to Chinese hamster ovary (CHO) cells with a milk fucosyltransferase. The resultant cells, CHO-sialyl Le(x) and CHO-sulfo Le(x) were tested for adhesion to E-selectin. IgG or P-selectin. IgG chimeric protein coated on plates. The results indicate that CHO-sialyl Le(x) adhered efficiently to E-selectin, while adhesion of CHO-sulfo Le(x) was very poor despite the fact that near equal number of the ligands had been attached to the cell surface. In contrast, CHO-sulfo Le(x) adhered efficiently to P-selectin, while CHO-sialyl Le(x) adhered modestly to P-selectin. These results demonstrate that sialyl Le(x) and sulfo Le(x) structures on the cell surface differ substantially in their ability to adhere to E- and P-selectin.     

P- and E-selectins recognize sialyl 6-sulfo Lewis X, the recently identified L-selectin ligand

Recently we identified sialyl 6-sulfo Le(x) as a major L-selectin ligand on high endothelial venules of human peripheral lymph nodes. In this study we investigated the ligand activity of sialyl 6-sulfo Le(x) to E- and P-selectins and compared it with the binding activity of conventional sialyl Le(x), by using cultured human lymphoid cells expressing both carbohydrate determinants. The results of the recombinant selectin binding studies and the nonstatic monolayer cell adhesion assays indicated that both sialyl 6-sulfo Le(x) and conventional sialyl Le(x) served as ligand for E- and P-selectins, while L-selectin was quite specific to sialyl 6-sulfo Le(x). Anti-PSGL-1 antibodies as well as O-sialoglycoprotein endopeptidase treatment almost completely abrogated the binding of P-selectin but barely affected the binding of E-selectin, indicating that these carbohydrate determinants carried by O-glycans of PSGL-1 selectively serves as a ligand for P-selectin, while the ligand for E-selectin is not restricted to PSGL-1 nor to O-sialoglycoprotein endopeptidase-sensitive glycans. The binding of L-selectin was markedly reduced by O-sialoglycoprotein endopeptidase treatment but only minimally affected by anti-PSGL-1 antibodies, indicating that O-glycans carrying sialyl 6-sulfo Le(x) were the major L-selectin ligands, while PSGL-1 was only a minor core protein for L-selectin in these cells. These results indicated that each member of the selectin family has a distinct ligand binding specificity.     

The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis(x) oligosaccharide

The selectins (lectin-EGF-complement binding-cell adhesion molecules [LEC-CAMs]) are a family of mammalian receptors implicated in the initial interactions between leukocytes and vascular endothelia, leading to lymphocyte homing, platelet binding, and neutrophil extravasation. The three known selectins, L-selectin (leukocyte adhesion molecule-1 [LECAM-1]), E-selectin (endothelial-leukocyte adhesion molecule-1 [ELAM-1]), and P-selectin (GMP-140) share structural features that include a calcium-dependent lectin domain. The sialyl Lewis(x) carbohydrate epitope has been reported as a ligand for both E- and P-selectins. Although L-selectin has been demonstrated to bind to carbohydrates, structural features of potential mammalian carbohydrate ligand(s) have not been well defined. Using an ELISA developed with a sialyl Lewis(x)-containing glycolipid and an E-selectin-IgG chimera, we have demonstrated the direct binding of the L-selectin-IgG chimera to sialyl Lewis(x). This recognition was calcium dependent, and could be blocked by Mel-14 antibody but not by other antibodies. Recognition was confirmed by the ability of cells expressing the native L-selectin to adhere to immobilized sialyl Lewis(x). These data suggest that the sialyl Lewis(x) oligosaccharide may form the basis of a recognition domain common to all three selectins.     

In vitro experimental studies of sialyl Lewis x and sialyl Lewis a on endothelial and carcinoma cells: crucial glycans on selectin ligands

Extravasation from the blood of malignant tumour cells that form metastasis and leukocytes that go into tissues require contact between selectins and their sialyl Lewis x and sialyl Lewis a (sLex and sLea respectively) decorated ligands. Endothelial cells have been shown to express sLex epitopes in lymph nodes and at sites of inflammation, and this is crucial for the selectin-dependent leukocyte traffic. Besides the ability to synthesize sLex on sialylated N-acetyllactosamine via the action of α(1,3)fucosyltransferase(s), endothelial cells can also degrade sLex to Lewis x through the action of α(2,3)sialidase(s). In addition, several epithelial tumors possess the machinery to synthesize sLex, which facilitates their adhesion to endothelial E- and P-selectin. This revised version was published online in November 2006 with corrections to the Cover Date.     

Reduction of rat myocardial ischemia and reperfusion injury by sialyl Lewis x oligosaccharide and anti-rat P-selectin antibodies

Polymorphonuclear leukocytes (PMN) are directly involved indevelopment of ischemic myocardial injury. Adhesion of PMN toendothelial cells is an initial step that triggers a sequentialprocess leading to acute inflammatory responses. Interactionbetween P-selectin and its oligosaccharide ligand, sialyl Lewisx (sLe^x), plays an important role in the early stage of theadhesion. To examine the role of P-selectin in various animaldisease models especially in rats, we have cloned rat E- andP-selectin cDNAs and established monoclonal antibodies againstthese rat selectins. In this report, we describe the generationand characterization of anti-rat P-selectin antibodies (ARPs).These antibodies detect cell surface P-selectin on thrombin-stimulatedrat platelets. More importantly, intravenous administrationof ARP2-4 reduced infarction developed after 30 min of ischemiafollowed by 24 h of reperfusion in a rat myocardial injury model.In addition, similar protective effect was also observed byadministration of a sLe^x- oligosaccharide. These results indicatethat cell adhesion mediated via P-selectin is involved in thedevelopment of ischemia and reperfusion injury in rat heart. ischemia and reperfusion injury monoclonal antibodies selectins sialyl Lewis x     

P-selectin and E-selectin. Distinct but overlapping leukocyte ligand specificities.

P-selectin on platelets and endothelial cells and E-selectin on endothelial cells are leukocyte receptors that recognize lineage-specific carbohydrates on neutrophils and monocytes. The proposed ligands for these receptors contain the Le(x) core and sialic acid. Since other investigators have shown that both E-selectin and P-selectin bind to sialylated Le(x), we evaluated whether E-selectin and P-selectin recognize the same counter-receptor on leukocytes. The interaction of HL60 cells with Chinese hamster ovary (CHO) cells expressing P-selectin or E-selectin was studied. To determine whether a protein component is required in addition to sialyl Le(x) for either P-selectin or E-selectin recognition, HL60 cells or neutrophils were digested with proteases, including chymotrypsin, elastase, proteinase Glu-C, ficin, papain, or thermolysin. Cells treated with these proteases bound E-selectin but not P-selectin. Fucosidase or neuraminidase treatment of HL60 cells markedly decreased binding to both E-selectin- and P-selectin-expressing CHO cells. Growth of HL60 cells in tunicamycin inhibited the ability of these cells to support P-selectin-mediated binding and, to a lesser extent, E-selectin-mediated binding. Purified P-selectin inhibited CHO:P-selectin binding to HL60 cells, but incompletely inhibited CHO:E-selectin binding to HL60 cells. However, purified soluble E-selectin inhibited CHO:P-selectin and CHO:E-selectin binding to HL60 cells equivalently and completely. COS cells, unable to bind to E-selectin or P-selectin, bound E-selectin but not P-selectin upon transfection with alpha-1,3-fucosyltransferase or alpha-1,3/1,4-fucosyltransferase. Similarly, LEC 11 cells expressing sialyl Le(x) bound E-selectin- but not P-selectin-expressing CHO cells. Sambucus nigra lectin, specific for the sialyl-2,6 beta Gal/GalNAc linkage, inhibited P-selectin but not E-selectin binding to HL60 cells. Although sialic acid and Le(x) are components of the P-selectin ligand and the E-selectin ligand, these results indicate that the ligands are related, having overlapping specificities, but are structurally distinct. A protein component containing sialyl Le(x) in proximity to sialyl-2,6 beta Gal structures on the P-selectin ligand may contribute to its specificity for P-selectin.     

Sialyl Lewis(x)-mediated, PSGL-1-independent rolling adhesion on P-selectin

Selectin-mediated cell adhesion is an essential component of the inflammatory response. In an attempt to unambiguously identify molecular features of ligands that are necessary to support rolling adhesion on P-selectin, we have used a reconstituted ("cell-free") system in which ligand-coated beads are perfused over soluble P-selectin surfaces. We find that beads coated with the saccharides sialyl Lewis(x) (sLe(x)), sialyl Lewis(a) (sLe(a)), and sulfated Lewis(x) (HSO(3)Le(x) support rolling adhesion on P-selectin surfaces. Although it has been suggested that glycosylation and sulfation of P-selectin glycoprotein ligand-1 (PSGL-1) is required for high-affinity binding and rolling on P-selectin, our findings indicate that sulfation of N-terminal tyrosine residues is not required for binding or rolling. However, beads coated with a tyrosine-sulfated, sLe(x)-modified, PSGL-1-Fc chimera support slower rolling on P-selectin than beads coated with sLe(x) alone, suggesting that sulfation improves rolling adhesion by modulating binding to P-selectin. In addition, we find it is not necessary that P-selectin carbohydrate ligands be multivalent for robust rolling to occur. Our results demonstrate that beads coated with monovalent sLe(x), exhibiting a more sparse distribution of carbohydrate than a similar amount of the multivalent form, are sufficient to yield rolling adhesion. The relative abilities of various ligands to support rolling on P-selectin are quantitatively examined among themselves and in comparison to human neutrophils. Using stop-time distributions, rolling dynamics at video frame rate resolution, and the average and variance of the rolling velocity, we find that P-selectin ligands display the following quantitative trend, in order of decreasing ability to support rolling adhesion on P-selectin: PSGL-1-Fc > sLe(a) approximately sLe(x) > HSO(3)Le(x).     

Expression of human alpha-l-fucosyltransferase gene homologs in monkey kidney COS cells and modification of potential fucosyltransferase acceptor substrates by an endogenous glycosidase

Previous investigations on the monkey kidney COS cell line demonstrated the weak expression of fucosylated cell surface antigens and presence of endogenous fucosyltransferase activities in cell extracts. RT-PCR analyses have now revealed expression of five homologs of human fucosyltransferase genes, FUT1, FUT4, FUT5, FUT7, and FUT8, in COS cell mRNA. The enzyme in COS cell extracts acting on unsialylated Type 2 structures is closely similar in its properties to the alpha1,3- fucosyltransferase encoded by human FUT4 gene and does not resemble the product of the FUT5 gene. Although FUT1 is expressed in the COS cell mRNA, it has not been possible to demonstrate alpha1,2- fucosyltransferase activity in cell extracts but the presence of Le(y) and blood-group A antigenic determinants on the cell surface imply the formation of H-precursor structures at some stage. The most strongly expressed fucosyltransferase in the COS cells is the alpha1,6-enzyme transferring fucose to the innermost N -acetylglucosamine unit in N - glycan chains; this enzyme is similar in its properties to the product of the human FUT8 gene. The enzymes resembling the human FUT4 and FUT8 gene products both had pH optima of 7.0 and were resistant to 10 mM NEM. The incorporation of fucose into asialo-fetuin was optimal at 5.5 and was inhibited by 10 mM NEM. This result initially suggested the presence of a third fucosyltransferase expressed in the COS cells but we have now shown that triantennary N- glycans with terminal nonreducing galactose units, similar to those present in asialo-fetuin, are modified by a weak endogenous beta-galactosidase in the COS cell extracts and thereby rendered suitable substrates for the alpha1,6- fucosyltransferase.      

Regulation of PSGL-1 interactions with L-selectin, P-selectin, and E-selectin: role of human fucosyltransferase-IV and -VII

P-selectin glycoprotein ligand-1 (PSGL-1) interactions with selectins regulate leukocyte migration in inflammatory lesions. In mice, selectin ligand activity regulating leukocyte recruitment and lymphocyte homing into lymph nodes results from the sum of unequal contributions of fucosyltransferase (FucT)-IV and FucT-VII, with FucT-VII playing a predominant role. Here we have examined the role of human FucT-IV and -VII in conferring L-selectin, P-selectin, and E-selectin binding activities to PSGL-1. Lewis x (Le(x)) carbohydrate was generated at the CHO(dhfr)(-) cell surface by FucT-IV expression, whereas sialyl Le(x) (sLe(x)) was synthesized by FucT-VII. Both human FucT-IV and -VII had the ability to generate carbohydrate ligands that support L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a major role. Cooperation was observed between FucT-IV and -VII in recruiting L-, P-, or E-selectin-expressing cells on PSGL-1 and in regulating cell rolling velocity and stability. Additional rolling adhesion assays were performed to assess the role of Thr-57-linked core-2 O-glycans in supporting L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1. These studies confirmed that core-2 O-glycans attached to Thr-57 play a critical role in supporting L- and P-selectin-dependent rolling and revealed that additional binding sites support >75% of E-selectin-mediated rolling. The observations presented here indicate that human FucT-IV and -VII both contribute and cooperate in regulating L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a predominant role in conferring selectin binding activity to PSGL-1.     

sLeX/L-selectin mediates adhesion in vitro implantation model

The complex implantation process is initiated by the recognition and adhesion between the embryo and uterine endometrial epithelium. The expression and interactions between the adhesive molecules from both fetal and maternal sides are crucial for the successful implantation. In this study, we aimed to investigate the expression and adhesive function of sLeX on the trophoblasts and L-selectin on uterine epithelial cells mediated the adhesion at the fetal-maternal interface, and to further explore whether this adhesion system could induce endometrial apoptosis, using in vitro implantation model consisting of the human trophoblast cell line (JAR) and human uterine epithelial cell line (RL95-2). The results showed that sLeX was expressed on JAR cells by indirect immunofluorescence staining. After transfection of JAR cells with fucosyltransferase VII (FUT7) which is the key enzyme for sLeX synthesis, the expression of FUT7 and sLeX synthesis were increased, and the percent adhesion of trophoblast cells to RL95-2 cell monolayer was significantly increased (P?相似文献   

Expression of cell surface Lewis X and Y antigens and FUT4 mRNA is increased in Jurkat cells undergoing apoptosis

Cell surface molecules undergo specific changes during apoptosis, including the expression of phosphatidylserine (PS) and some proteins and alterations in sugar chains. Among the various sugar chains on the cell surface, Lewis X (Le(X)) and Lewis Y (Le(Y)) antigens are key determinants for a variety of biological processes. We studied the changes in Le(X) and Le(Y) expression in Jurkat cells, a human T cell line, during apoptosis. Flow cytometry showed that Le(X) and Le(Y) antigen expression was enhanced on the cell surface during apoptosis induced by anti-Fas antibody. To clarify the mechanism of enhanced Le(X) and Le(Y) expression, we assessed the expression levels of fucosyltransferase (FUT1, 2, 3-5-6, 4, and 9) mRNAs that are predominantly expressed in Jurkat cells and which are considered to form Le(X) and Le(Y). The expression of FUT4 mRNA was up-regulated after exposing cells to anti-Fas antibody. Moreover, the increase in Le(X) and Le(Y) antigen levels was significantly suppressed by caspase 3 or 8 inhibitors. These results indicated that the induction of FUT (mainly FUT4), the gene expression of which is mediated by signals downstream of caspase 3, increases Le(X) and Le(Y) expression in apoptotic cells.     

Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLe(X) and PSGL-1

P-, E- and L-selectin constitute a family of cell adhesion receptors that mediate the initial tethering and rolling of leukocytes on inflamed endothelium as a prelude to their firm attachment and extravasation into tissues. The selectins bind weakly to sialyl Lewisx (SLe(X))-like glycans, but with high-affinity to specific glycoprotein counterreceptors, including PSGL-1. Here, we report crystal structures of human P- and E-selectin constructs containing the lectin and EGF (LE) domains co-complexed with SLe(X). We also present the crystal structure of P-selectin LE co-complexed with the N-terminal domain of human PSGL-1 modified by both tyrosine sulfation and SLe(X). These structures reveal differences in how E- and P-selectin bind SLe(X) and the molecular basis of the high-affinity interaction between P-selectin and PSGL-1.     

Deciphering the catalytic machinery in a universally conserved ribosome binding ATPase YchF

The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewis(x) (sLe(x)) in selectin-dependent mo-DC binding. Our data reveal that sLe(x) is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLe(x)-dependent binding of mo-DC to selectins was further substantiated by using sLe(x) free sugar and anti-sLe(x) antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLe(x) expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols.     

Significance of each of three missense mutations, G484A, G667A, and G808A, present in an inactive allele of the human Lewis gene (FUT3) for α(1,3/1,4)fucosyltransferase inactivation

Recently, we found three novel missense mutations, G484A (Asp162Asn), G667A (Gly223Arg), and G808A (Val270Met), present in a Lewis-negative allele (le484,667,808) from an African (Xhosa) population. To define the relative contribution of each of the three mutations in the le484,667,808 allele for inactivation of the FUT3-encoded enzyme, we made chimeric FUT3 containing each of the three mutations. A transient expression study indicated that COS7 cells transfected with the FUT3 construct containing the G484A mutation expressed the Lewis antigen and had about 20% enzyme activity as compared with COS7 cells transfected with the wild type FUT3 allele, whereas COS7 cells transfected with the FUT3 construct containing either the G667A mutation or the G808A mutation did not express the Lewis antigen and showed no detectable (1,3/1,4)fucosyltransferase activity. These results suggest that the G667A and/or the G808A missense mutations of FUT3 alleles are responsible for the inactivation of the FUT3-encoded enzyme.     

The selectin family of carbohydrate-binding proteins: structure and importance of carbohydrate ligands for cell adhesion.

Protein-carbohydrate interactions have been found to be important in many steps in lymphocyte recirculation and inflammatory responses. A family of carbohydrate-binding proteins or lectins, termed selectins, has been discovered and shown to be involved directly in these processes. The three known selectins, termed L-, E- and P-selectins, have domains homologous to other Ca(2+)-dependent (C-type) lectins. L-selectin is expressed constitutively on lymphocytes, E-selectin is expressed by activated endothelial cells, and P-selectin is expressed by activated platelets and endothelial cells. Here, we review the nature of the carbohydrate determinants in tissues recognized by these selectins. The expression of specific sialylated, fucosylated and sulfated carbohydrates in activated endothelium and high endothelial venules promotes interactions with L-selectin on leukocyte surfaces. In contrast, E- and P-selectins recognize specific carbohydrate determinants related to sialyl Le(x) antigen on neutrophil and monocyte surfaces. The discovery of the selectins has generated excitement among glycoconjugate researchers that other carbohydrate-binding proteins and their cognate ligands will be found to function in regulating many types of cellular interactions.