A cholesterol-independent membrane microdomain serves as a functional counter-receptor for E-selectin at the Colo201 cell surface and initiates signalling on E-selectin binding

The present study demonstrates that the functional counter-receptors for E-selectin at the cell surface of Colo201 human colon cancer cells are localized in detergent-insoluble membrane microdomains (DIM). Following isolation of counter-receptors from whole cell lysates using E-selectin-coupled magnetic beads followed by sucrose density gradient separation, both sialyl Lewis a (SLe(a))- and sialyl Lewis x (SLe(x))-carrying glycoproteins which had bound to the E-selectin-beads were distributed in detergent-soluble fractions as well as DIM. In contrast, following isolation of counter-receptors directly from the cell surface, SLe(a)-carrying glycoproteins which had bound to E-selectin-beads at the cell surface were localized only in DIM, together with a Src family kinase, Lyn, while SLe(x)-carrying glycoproteins were not detected in any fraction. The counter-receptors were distributed in a diffuse pattern on the cell surface but clustered following E-selectin binding, leading to the subsequent phosphorylation of extracellular signal-regulated kinase (ERK). Treatment of the cells with methyl-beta-cyclodextrin, a cholesterol-depleting drug, had little effect on either the association of SLe(a)-carrying glycoproteins and Lyn with the domain or ERK phosphorylation. Thus, the functional counter-receptors and Lyn are co-localized in a cholesterol-independent microdomain and create a physiological domain ('glycosynapse') at the cell surface that initiates signalling in cancer cells upon binding to E-selectin.     

Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ proximity ligation

Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification of these biomarkers has been based on the detection of either the protein or the O-glycan modifications. We therefore aimed to identify the combined mucin and O-glycan features, that is, specific glycoforms, in an attempt to increase specificity of these cancer biomarkers. Using in situ proximity ligation assays (PLA) based on existing monoclonal antibodies directed to MUC1, MUC2, MUC5AC and MUC6 mucins and to cancer-associated carbohydrate antigens Tn, Sialyl-Tn (STn), T, Sialyl-Le(a) (SLe(a)) and Sialyl-Le(x) (SLe(x)) we screened a series of 28 mucinous adenocarcinomas from different locations (stomach, ampulla of Vater, colon, lung, breast and ovary) to detect specific mucin glycoforms. We detected Tn/STn/SLe(a)/SLe(x)-MUC1 and STn/SLe(a)/SLe(x)-MUC2 glycoforms in ≥50% of the cases, with a variable distribution among organs. Some new glycoforms-T/SLe(a)-MUC2, STn/T/SLe(a) SLe(x)-MUC5AC and STn/T/SLe(a)/SLe(x)-MUC6-were identified for the first time in the present study in a variable percentage of cases from different organs. In conclusion, application of the PLA technique allowed sensitive detection of specific aberrant mucin glycoforms in cancer, increasing specificity to the use of antibodies either to the mucin protein backbone or to the O-glycan haptens alone.     

PSGL-1 from the murine leukocytic cell line WEHI-3 is enriched for core 2-based O-glycans with sialyl Lewis x antigen

Leukocyte trafficking involves specific recognition between P-selectin and L-selectin and PSGL-1 containing core 2-based O-glycans expressing sialyl Lewis x (SLe(x)) antigen. However, the structural identity of the glycan component(s) displayed by murine neutrophil PSGL-1 that contributes to its P-selectin counter-receptor activity has been uncertain, since these cells express little if any SLe(x) antigen, and because there have been no direct studies to examine murine PSGL-1 glycosylation. To address this uncertainty, we studied PSGL-1 glycosylation in the murine cell line WEHI-3 using metabolic-radiolabeling with (3)H-monosaccharide precursors to detect low-abundance O-glycan structures. We report that PSGL-1 from WEHI-3 cells expresses a di-sialylated core 2 O-glycan containing the SLe(x) antigen. This fucosylated O-glycan is scarce on PSGL-1 and essentially undetectable in total leukocyte glycoproteins from WEHI-3 cells. These results demonstrate that WEHI-3 cells selectively fucosylate PSGL-1 to generate functionally important core 2-based O-glycans containing the SLe(x) antigen.     

Multi-recognition capability of E-selectin in a dynamic flow system, as evidenced by differential effects of sialidases and anti-carbohydrate antibodies on selectin-mediated cell adhesion at low vs. high wall shear stress: a preliminary note.

E-selectin has a "multi-recognition" capability in terms of epitope binding specificity, depending on adhesion conditions (static vs. low- or high-shear stress dynamic systems). Specifically, (i) adhesion based on expression of alpha 2-->3 sialylated Le(x) (SLe(x)) is prominent under static or low shear stress dynamic conditions; (ii) adhesion under high shear stress dynamic conditions does not depend on the known SLe(x) species, but rather on Lex with an adjacent unidentified sialosyl substitution, which shows different susceptibility to sialidases and antibodies compared to known SLe(x).     

CD66 nonspecific cross-reacting antigens are involved in neutrophil adherence to cytokine-activated endothelial cells

Neutrophil adherence to cytokine-activated endothelial cell (EC) monolayers depends on the expression of the endothelial leukocyte adhesion molecule-1 (ELAM-1). The ligand for ELAM-1 is the sialylated Lewis-x antigen (SLe(x)) structure. The selectin LAM-1 (or LECAM-1) has been described as one of the SLe(x)-presenting glycoproteins involved in neutrophil binding to ELAM-1. Other presenter molecules have not yet been described. Our data demonstrate that the carcinoembryonic antigen (CEA)-like surface molecules on neutrophils--known as the nonspecific cross-reacting antigens (NCAs)--are involved in neutrophil adherence to monolayers of IL-1-beta-activated EC. The NCAs are recognized by CD66 (NCA-160 and NCA-90) and CD67 (NCA-95). Because NCA-95 and NCA-90 have previously been found to be phosphatidylinositol (PI)-linked, paroxysmal nocturnal hemoglobinuria (PNH) neutrophils (which lack PI-linked surface proteins) were tested as well. PNH neutrophils showed a diminished binding to activated EC. CD66 (on PNH cells still recognizing the transmembrane NCA-160 form) still inhibited the adherence of PNH cells to IL-1-beta-activated EC, but to a limited extent. Soluble CEA(-related) antigens inhibited normal neutrophil adherence as well, whereas neutrophil transmigration was unaffected. Sialidase-treatment as well as CD66 preclearing abolished the inhibitory capacity of the CEA(-related) antigens. The binding of soluble CEA antigens to IL-1-beta-pretreated EC was blocked by anti-ELAM-1. These soluble antigens, as well as the neutrophil NCA-160 and NCA-90, both recognized by CD66 antibodies, presented the SLe(x) determinant. Together, these findings indicate that the CD66 antigens (i.e., NCA-160/NCA-90) function as presenter molecules of the SLe(x) oligosaccharide structures on neutrophils that bind to ELAM-1 on EC.     

Downregulation of GMP-140 (CD62 or PADGEM) expression on platelets by N,N-dimethyl and N,N,N-trimethyl derivatives of sphingosine.

GMP-140 (CD62 or PADGEM), a member of the selectin family, is a membrane glycoprotein in secretory granules of platelets and endothelial cells. When these cells are activated by agonists such as thrombin or AMP, GMP-140 is rapidly redistributed to the cell surface. The carbohydrate epitope defined by GMP-140 was identified as sialosyl-Le(x) (as for ELAM-1), which may play an essential role in adhesion of leukocytes or tumor cells on endothelial cells, through aggregation with platelets. Redistribution of GMP-140 from alpha-granules of platelets to the cell surface, induced by thrombin and PMA, was strongly inhibited by preincubation of platelets with N,N-dimethylsphingosine (DMS) or N,N,N-trimethylsphingosine (TMS) at 10-20 microM concentration for a brief period (5 min). Inhibition of GMP-140 redistribution to the cell surface by DMS or TMS was also detected by a cell adhesion assay using HL60 cells, which highly express sialosyl-Le(x); i.e., HL60 cells adhered on platelets activated by thrombin or PMA but not on platelets which were briefly preincubated with DMS or TMS followed by activation. The inhibitory effect of DMS or TMS on GMP-140 redistribution is not due to cytotoxicity, since the TMS-treated platelets were fully capable of aggregating in the presence of ristocetin. Sphingosine (SPN) and protein kinase C inhibitors such as H-7 and calphostin C showed weaker inhibitory activity than DMS and TMS. Our results indicate that both DMS and TMS could be useful reagents to inhibit cell surface expression of crucial selectins which promote adhesion of Le(x-) or sialosyl-Le(x)-expressing cells with platelets and endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Insulin/protein kinase B signalling pathway upregulates metastasis-related phenotypes and molecules in H7721 human hepatocarcinoma cell line.

The effect of insulin on cancer metastatic potential was studied in a human hepatocarcinoma cell line, H7721. Cell adhesion to human umbilical vein endothelial cells (HUVECs) and laminin as well as chemotactic cell migration and invasion were selected as the indices of metastasis-related phenotypes for assessment of metastatic potential ex vivo. The results indicated that insulin enhanced all of these metastasis-related phenotypes. After the cells were treated with specific inhibitor of PI3K (LY294002) or transfected with antisense cDNA of PKB (AS-PKB), all of the above phenotypes were attenuated, and they could not be significantly stimulated by insulin, indicating that the insulin effect on metastatic potential was mediated by PI3K and PKB. Only the monoclonal antibody to the sialyl Lewis X (SLe(x)), but not antibodies to other Lewis antigens, significantly blocked the cell adhesion to HUVECs, cell migration and invasion, suggesting that SLe(x) played a crucial role in the metastatic potential of H7721 cells. The upregulation of cell surface SLe(x) and alpha-1,3-fucosyltransferase-VII (alpha-1,3 Fuc T-VII, enzyme for SLe(x) synthesis) was also mediated by PI3K and PKB, since LY294002 and AS-PKB also reduced the expressions of SLe(x) and alpha-1,3 FucT-VII, and attenuated the response to insulin. Furthermore, the alterations in the expressions of PKB protein and activity were correlated to the changes of metastatic phenotypes and SLe(x) expression. Taken together, the insulin/PKB signalling pathway participated in the enhancement of metastatic potential of H7721 cells, which was mediated by the upregulation of the expression of SLe(x) and alpha-1,3 FucT-VII.     

Transfection of the c-erbB2/neu gene upregulates the expression of sialyl Lewis X, alpha1,3-fucosyltransferase VII, and metastatic potential in a human hepatocarcinoma cell line.

The pCMV4 plasmid containing the cancer-promoting gene, c-erbB2/neu, was cotransfected into the human hepatocarcinoma cell line 7721 with the pcDNA3 vector, which contains the 'neo' selectable marker. Several clones showing stable expression of c-erbB2/neu were established and characterized by determination of c-erbB2/neu mRNA and its encoded protein p185. Expression of Lewis antigens and alpha1,3-fucosyltransferases and the biological behavior of 7721 cells after c-erbB2/neu transfection were studied using mock cells transfected with the vectors pCMV4 and pcDNA3 as controls. SLe(x) expression on the surface of mock cells was high, whereas expression of SDLe(x), Lex and SLe(a) was absent or negligible. This is compatible with the abundant expression of alpha1,3-fucosyltransferase VII, very low expression of alphafucosyltransferase III/VI, and almost absent expression of alpha1,3-fucosyltransferase IV in the mock cells. After transfection of c-erbB2/neu, expression of SLe(x) and alpha1,3-fucosyltransferase VII were simultaneously elevated, but that of alphafucosyltransferase III/VI was not altered. The expression of both SLe(x) and alpha1,3-fucosyltransferase VII correlated positively with the expression of c-erbB2/neu in different clones, being highest in clone 13, medium in clone 6, and lowest in clone 7. In addition, the adhesion of 7721 cells to human umbilical vein endothelial cells (HUVECs) or P-selectin, as well as cell migration and invasion, were increased in c-erbB2/neu-transfected cells. These increases also correlated positively with the expression intensities of c-erbB2/neu, SLe(x) and alpha1,3-fucosyltransferase VII in the different clones, whereas cell adhesion to fibronectin correlated negatively with these variables. mAbs to SLe(x) (KM93) and SDLe(x) (FH6) significantly and slightly, respectively, abolished cell adhesion to HUVECs or P-selectin and cell migration and invasion. mAbs to SDLe(x) and SLe(a) did not suppress cell adhesion to HUVECs nor inhibit cell migration and invasion. Transfection of alpha1,3-fucosyltransferase VII cDNA into 7721 cells showed similar results to transfection of c-erbB2/neu, and the increased adhesion to HUVECs, cell migration, and invasion were also inhibited significantly by KM93 and slightly by FH6. These results indicate that expression of alpha1,3-fucosyltransferase VII and its specific product, SLe(x), and their capacity for cell adhesion, migration and invasion are closely related. Therefore, the c-erbB2/neu gene is proposed to be a metastasis-promoting gene, and its effects are at least partially mediated by the increased expression of alpha1,3-fucosyltransferase VII and SLe(x).     

Conformational dynamics of sialyl Lewisx in aqueous solution and its interaction with selectinE. A study by molecular dynamics

Three dimensional structures of sialyl Lewis(x) (SLe(x)) in aqueous solution and bound to selectinE are described based on an exhaustive conformational analysis and several long molecular dynamics simulations using different glycosidic regions as starting conformations. It appears from this study that when the oligosaccharide is free in solution the NeuNAcalpha(2-3)Gal segment favors glycosidic conformation in three different regions in the (Phi,Psi) plane with propensity of populations in the ratio 1:8:1. Each one of these structures is characteristically stabilized by specific hydrogen bonding interaction between NeuNAc and Gal. On the other hand, the Gal-GlcNAc-Fuc segment can exist in four different conformational states. Based on the topology of SLe(x) we are able to predict that out of all the allowed conformations in solution only two of these structures possess a geometry that would fit without steric clashes into the binding location of selectinE. In both of these binding modes, segment Gal-GlcNAc-Fuc adopts a unique conformation. The only difference between the two SLe(x) conformers that can successfully bind to selectinE is given by two possible regions in glycosidic space in the fragment NeuNAcalpha(2-3)Gal. A large conformational departure from the crystallographic data is observed for two lysine residues at the binding site of selectinE. These two residues play an important role when SLe(x) binds selectinE in aqueous solution. These findings help reconcile the X-ray data, in which these residues appear to be 1 nm away from SLe(x), with recent liquid NMR data reporting couplings between these protein residues and the sugar.     

Cloning and characterization of a viral α2-3-sialyltransferase (vST3Gal-I) for the synthesis of sialyl Lewisx

Sialyl Lewis(x) (SLe(x), Siaα2-3Galβ1-4(Fucα1-3)GlcNAcβOR) is an important sialic acid-containing carbohydrate epitope involved in many biological processes such as inflammation and cancer metastasis. In the biosynthetic process of SLe(x), α2-3-sialyltransferase-catalyzed sialylation generally proceeds prior to α1-3-fucosyltransferase-catalyzed fucosylation. For the chemoenzymatic synthesis of SLe(x) containing different sialic acid forms, however, it would be more efficient if diverse sialic acid forms are transferred in the last step to the fucosylated substrate Lewis(x) (Le(x)). An α2-3-sialyltransferase obtained from myxoma virus-infected European rabbit kidney RK13 cells (viral α2-3-sialyltransferase (vST3Gal-I)) was reported to be able to tolerate fucosylated substrate Le(x). Nevertheless, the substrate specificity of the enzyme was only determined using partially purified protein from extracts of cells infected with myxoma virus. Herein we demonstrate that a previously reported multifunctional bacterial enzyme Pasteurella multocida sialyltransferase 1 (PmST1) can also use Le(x) as an acceptor substrate, although at a much lower efficiency compared to nonfucosylated acceptor. In addition, N-terminal 30-amino-acid truncated vST3Gal-I has been successfully cloned and expressed in Escherichia coli Origami? B(DE3) cells as a fusion protein with an N-terminal maltose binding protein (MBP) and a C-terminal His(6)-tag (MBP-Δ30vST3Gal-I-His(6)). The viral protein has been purified to homogeneity and characterized biochemically. The enzyme is active in a broad pH range varying from 5.0 to 9.0. It does not require a divalent metal for its α2-3-sialyltransferase activity. It has been used in one-pot multienzyme sialylation of Le(x) for the synthesis of SLe(x) containing different sialic acid forms with good yields.     

Effect of the sialyl Lewis X (SLe(x)) moiety on splenic accumulation of SLe(x)-carboxymethylpullulan conjugate

Sialyl Lewis X (SLe(x)), an E-selectin ligand, was conjugated with carboxymethylpullulan (CMPul) and the disposition characteristics of this conjugate after intravenous administration were investigated using mice with ear edema. The concentration of 3H-labeled SLe(x)-CMPul in the spleen was significantly high. When CMPul was modified with a saccharide unable to bind to E-selectin, this splenic accumulation was not observed. The uptake of radiolabeled SLe(x)-CMPul by the spleen was completely inhibited by a 100-fold molar of cold SLe(x)-CMPul but not by a sialyl N-acetyllactosamine-CMPul conjugate (SLN-CMPul). Microautoradiography analyses revealed that SLe(x)-CMPul accumulated in the marginal zone of the spleen.     

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.     

alpha1,3 fucosyltransferase-VII up-regulates the mRNA of alpha5 integrin and its biological function

After transfection of alpha1,3fucosyltransferase (FucT)-VII cDNA into H7721 human hepatocarcinoma cells, the expression of alpha5, but not beta1 integrin was significantly up-regulated. This was evidenced by the increase of alpha5 integrin on cell surface as well as the increase of alpha5 mRNA and protein in the cells. However, the expressions of sialyl Lewis X (SLe(x), the product of alpha1,3FucT-VII) on both alpha5 and beta1 integrin subunits were unchanged. Concomitantly, the tyrosine autophosphorylated FAK and dephosphorylated Src (FAK and Src involve in the signal transduction of integrin alpha5beta1) were up-regulated, while the Tyr-527 phosphorylated Src was down-regulated. The above-mentioned alterations were correlated to the expressions of alpha1,3FucT-VII in different alpha1,3FucT-VII transfected H7721 cell lines. In addition, after alpha1,3FucT-VII transfection, cell adhesion to fibronectin (Fn) and chemotaxic cell migration were obviously promoted. The cell adhesion could be blocked by alpha5 integrin antibody, and cell migration was obviously attenuated by the antibodies to both alpha5 integrin and SLe(x). These findings suggest that the increased surface alpha5 integrin caused by the up-regulation of alpha5 mRNA promotes the cell adhesion to Fn, cell migratiom, and Fn-induced signaling of alpha5beta1 integrin. The up-regulation of surface SLe(x) originated from the over expression of alpha1,3FucT-VII also led to the stimulation of cell migration. This is the first time to report that alpha1,3FucT-VII can regulate the mRNA expression of integrin.     

Selectin GMP-140 (CD62; PADGEM) binds to sialosyl-Le(a) and sialosyl-Le(x), and sulfated glycans modulate this binding.

GMP-140 (CD62; PADGEM) is a member of the selectin family expressed highly at the surface of platelets and endothelial cells by agonists such as thrombin or phorbol esters. Previous studies indicate that the lectin domain of GMP-140 recognizes sialosyl-Le(x) (SLex) and to a lesser extent Le(x) (Polley MJ, et al., Proc Natl Acad Sci USA 88:6224, 1991). We now report that GMP-140 binds to sialosyl Lea (SLea) and to SLex, and that degree of binding to SLea is greater than that to SLex under our experimental conditions. Binding of activated platelets to SLea or SLex was inhibited to various degrees in the presence of sulfated glycans, suggesting that sulfated glycans induce conformational change in the lectin domain of GMP-140 and modulates its binding affinity to SLea and SLex.     

Design, synthesis and biological evaluation of aryl-substituted sialyl Lewis X mimetics prepared via cross-metathesis of C-fucopeptides.

Several aryl substituted C-fucopeptides have been developed as sialyl Lewis X mimetics. Although the compounds have a much simpler structure compared to SLe(x), up to 3-times higher binding affinity toward E-selectin and > 1000 times toward P-selectin was observed. Furthermore, a convenient strategy for generating a number of analogues from a SLe(x) mimetic template at a very late stage of the synthesis was introduced, using a ruthenium catalyzed cross olefin metathesis under benchtop conditions.     

Epistatic interactions between modifier genes confer strain-specific redundancy for Tgfb1 in developmental angiogenesis

Tgfbm1 (chromosome 5, P = 8 x 10(-5)) and Tgfbm3 (chromosome 12, P = 6 x 10(-11)) were identified as loci that modify developmental angiogenesis of Tgfb1 -/- mice. Congenic mice validated these loci and demonstrated epistatic interaction between them. The novel locus, Tgfbm3, encompasses approximately 22 genes, colocalizes with both tumor susceptibility and atherosclerosis susceptibility loci, and is enriched in genes regulating cell growth and morphogenesis. The use of gene knockout and/or transgenic mice that predispose to a complex trait, such as vascular development/angiogenesis, facilitates the identification of modifiers by simplifying genetic analysis. Identification of genes that modify response to lack of transforming growth factor beta1 (TGFbeta1) will enhance the understanding of TGFbeta1 action in vivo and may help predict which patients would respond well to anti-TGFbeta therapy. Identification of angiogenesis-modifying genes may provide new targets for angiogenesis therapies and analysis of polymorphisms therein may contribute to assessment of risk for diseases involving angiogenesis.