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In this research article, we report on the strengthening of a non‐classical hydrogen bond (C−H⋅⋅⋅O) by introducing electron withdrawing groups at the carbon atom. The approach is demonstrated on the example of derivatives of the physiological E‐selectin ligand sialyl Lewisx (1, sLex). Its affinity is mainly due to a beneficial entropy term, which is predominantly caused by the pre‐organization of sLex in its binding conformation. We have shown, that among the elements responsible for the pre‐organization, the stabilization by a non‐classical hydrogen bond between the H−C5 of l‐fucose and the ring oxygen O5 of the neighboring d‐galactose moiety is essential and yields 7.4 kJ mol−1. This effect could be further strengthened by replacing l‐fucose by 6,6,6‐trifluoro‐l‐fucose leading to an improved non‐classical H‐bond of 14.9 kJ mol−1, i.e. an improved pre‐organization in the bioactive conformation. For a series of glycomimetics of sLex (1), this outcome could be confirmed by high field NMR‐shifts of the H−C5Fuc, by X‐ray diffraction analysis of glycomimetics co‐crystallized with E‐selectin as well as by isothermal titration calorimetry. Furthermore, the electron‐withdrawing character of the CF3‐group beneficially influences the pharmacokinetic properties of sLex mimetics. Thus, acid‐stability, a prerequisite for gastrointestinal stability, could be substantially improved. [ABSTRACT FROM AUTHOR]

[Display omitted] Neutrophil binding to vascular P- and E-selectin is the rate-limiting step in the recruitment of immune cells to sites of inflammation. Many diseases, including sickle cell anemia, post-myocardial infarction reperfusion injury, and acute respiratory distress syndrome are characterized by dysregulated inflammation. We have recently reported sialyl Lewisx analogues as potent antagonists of P- and E-selectin and demonstrated their in vivo immunosuppressive activity. A key component of these molecules is a tartrate diester that serves as an acyclic tether to orient the fucoside and the galactoside moiety in the required gauche conformation for optimal binding. The next stage of our study involved attaching an extended carbon chain onto one of the esters. This chain could be utilized to tether other pharmacophores, lipids, and contrast agents in the context of enhancing pharmacological applications through the sialyl Lewisx / receptor-mediated mechanism. Herein, we report our preliminary studies to generate a small library of tartrate based sialyl Lewisx analogues bearing extended carbon chains. Anionic charged chemical entities are attached to take advantage of proximal charged amino acids in the carbohydrate recognition domain of the selectin receptors. Starting with a common azido intermediate, synthesized using copper-catalyzed Huisgen 1,3-dipolar cycloadditions, these molecules demonstrate E- and P-selectin binding properties. [ABSTRACT FROM AUTHOR]

E-selectin is a cell-adhesion molecule of the vascular endothelium that promotes essential leukocyte rolling in the early inflammatory response by binding to glycoproteins containing the tetrasaccharide sialyl Lewisx (sLex). Efficient leukocyte recruitment under vascular flow conditions depends on an increased lifetime of E-selectin/ligand complexes under tensile force in a so-called catch-bond binding mode. Co-crystal structures of a representative fragment of the extracellular E-selectin region with sLex and a glycomimetic antagonist thereof reveal an extended E-selectin conformation, which is identified as a high-affinity binding state of E-selectin by molecular dynamics simulations. Small-angle X-ray scattering experiments demonstrate a direct link between ligand binding and E-selectin conformational transition under static conditions in solution. This permits tracing a series of concerted structural changes connecting ligand binding to conformational stretching as the structural basis of E-selectin catch-bond-mediated leukocyte recruitment. The detailed molecular view of the binding site paves the way for the design of a new generation of selectin antagonists. This is of special interest, since their therapeutic potentialwas recently demonstrated with the pan-selectin antagonists GMI-1070 (Rivipansel). [ABSTRACT FROM AUTHOR]

Selectins play a key role in leukocyte trafficking during the inflammatory response of the organism, i.e. the recruitment and extravasation of leukocytes from the blood stream into inflamed tissue. Antagonizing the interaction of selectins with their physiological ligands was shown to be a validated approach for the treatment of inflammatory disorders like rheumatoid arthritis, stroke or reperfusion injuries. Although numerous research efforts to identify small molecule selectin antagonists have been reported, no successful drug has been identified so far. This mini-review describes selectin antagonists, where the N-acetylneuraminic acid moiety of the natural ligand sialyl Lewisx is replaced by mimetics containing the essential carboxylic acid function. The prerequisite of a pre-organization of the carboxylate in the bioactive conformation is discussed.

Highlights: [•] Total synthesis of a new biomolecule. [•] The glycosylations are highly regio- and stereoselective. [•] The route of total synthesis is concise. [•] The overall yield of aminoethyl glycoside of sialyl Lewisx is high. [ABSTRACT FROM AUTHOR]

Among influenza A viruses, subtype H3N2 is the major cause of human influenza morbidity and is associated with seasonal epidemics causing annually half million deaths worldwide. Influenza A virus infection is initiated via hemagglutinin that binds to terminally sialylated glycoconjugates exposed on the surface of target cells. Gangliosides from human granulocytes were probed using thin-layer chromatography overlay assays for their binding potential to H3N2 virus strains A/Victoria/3/75 and A/Hiroshima/52/2005. Highly polar gangliosides with poly-N-acetyllactosaminyl chains showing low chromatographic mobility exhibited strong virus adhesion which was entirely abolished by sialidase treatment. Auxiliary overlay assays using anti-sialyl Lewisx (sLex) monoclonal antibodies showed identical binding patterns compared with those performed with the viruses. A comprehensive structural analysis of fractionated gangliosides by electrospray ionization quadrupole time-of-flight mass spectrometry revealed sLex gangliosides with terminal Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc epitope and extended neolacto (nLc)-series core structures as the preferential virus binding gangliosides. More precisely, sLex gangliosides with nLc8, nLc10 and nLc12Cer cores, carrying sphingosine (d18:1) and a fatty acid with variable chain length (mostly C24:0, C24:1 or C16:0) in the ceramide moiety and one or two additional internal fucose residues in the oligosaccharide portion, were identified as the preferred receptors recognized by H3N2 virus strains A/Victoria/3/75 and A/Hiroshima/52/2005. This study describes glycan-binding requirements of hemagglutinin beyond binding to glycans with a specific sialic acid linkage of as yet undefined neutrophil receptors acting as ligands for H3N2 viruses. In addition, our results pose new questions on the biological and clinical relevance of this unexpected specificity of a subtype of influenza A viruses. [ABSTRACT FROM AUTHOR]

Abstract: Cancer cell adhesion to vascular endothelium is a critical process in hematogenous metastasis. We hypothesized that breast cancer cells express ligands that bind under blood flow conditions to E-selectin expressed by endothelial cells. At a hemodynamic wall shear rate, BT-20 and MDA-MB-468 breast cancer cells adhered to cytokine-activated human umbilical cord vein endothelial cells (HUVECs) but not to anti-E-selectin monoclonal antibody treated HUVECs, demonstrating that adhesion was specifically mediated by E-selectin. Characterization of glycans expressed on breast cancer cells by a panel of antibodies revealed that BT-20 cells expressed sialyl Lewis X (sLex) and sialyl Lewis A (sLea) but MDA-MB-468 cells did not, suggesting that the former possess classical glycans involved in E-selectin mediated adhesion while the latter have novel binding epitopes. Protease treatment of the breast cancer cells failed to significantly alter the carbohydrate expression profiles, binding to soluble E-selectin–Ig chimera, or the ability of the cells to tether and roll on E-selectin expressed by HUVECs, indicating that glycosphingolipids are functional E-selectin ligands on these cells. Furthermore, extracted breast cancer cell gangliosides supported binding of E-selectin–Ig chimera and adhesion of E-selectin transfected cells under physiological flow conditions. In summary, our results demonstrate that breast cancer cells express sialylated glycosphingolipids (gangliosides) as E-selectin ligands that may be targeted for prevention of metastasis. [Copyright &y& Elsevier]

Sialyl Lewisx (SLex, 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 SLex, α2–3-sialyltransferase-catalyzed sialylation generally proceeds prior to α1–3-fucosyltransferase-catalyzed fucosylation. For the chemoenzymatic synthesis of SLex 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 Lewisx (Lex). 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 Lex. 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 Lex 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 His6-tag (MBP-Δ30vST3Gal-I-His6). 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 Lex for the synthesis of SLex containing different sialic acid forms with good yields. [ABSTRACT FROM AUTHOR]

Abstract: Although intravenous administration of high levels of cisplatin (CDDP) are limited due to its severe side effects, efficient delivery of CDDP directly to the tumor should improve the therapeutic response while potentially by-passing significant side effects. High loading of CDDP into liposomes is one technique that could be used as a potential drug delivery system. Since cis-diamminedinitratoplatinum (CDDP3) is highly soluble in water and converts to CDDP in the presence of chloride ions, we encapsulated CDDP3 into liposomes in the absence of chloride ions and supplemented chloride ions to prepare CDDP-encapsulated liposomes (CDDP-Lip) resulting in a significantly improved loading efficiency of CDDP. We further conjugated the CDDP-Lip with Sialyl LewisX (CDDP-SLX-Lip) because we previously demonstrated Sialyl LewisX enhanced efficient accumulation of liposomes into tumors in vivo. CDDP-SLX-Lip treated mice showed a survival rate of 75% at 14 days even if a lethal level of CDDP was injected into mice. Loss of body weight was negligible and no histological abnormality was found in a variety of normal tissues. Accumulation of CDDP-SLX-Lip was about 6 times more than that of CDDP-Lip or CDDP. As the result, there was better antitumor activity of CDDP-SLX-Lip than that of CDDP-Lip with significantly less toxic effects in normal tissues. [Copyright &y& Elsevier]

Abstract: The selectin–leukocyte interaction is the initial event in the early inflammatory cascade. This interplay proceeds via the terminal tetrasaccharide sialyl Lewisx (sLex), present on physiological selectin ligands and E- and P-selectins located on the endothelial surface. Blocking this process is regarded as a promising therapeutic approach for inflammatory diseases where excessive leukocyte efflux is responsible for tissue damage. Selectin antagonists are generally based on sLex as lead structure, containing the essential pharmacophores pre-oriented in the bioactive conformation. In this work, we describe a set of competitive sLex mimetics possessing the carboxylic acid pharmacophore equipped with additional hydrophobic substituents as neuraminic acid (Neu5Ac) replacements. This small library of antagonists derived from Huisgen-1,3-dipolar cycloadditions allows to further probe the carbohydrate recognition domain of E-selectin. [Copyright &y& Elsevier]

Summary: This study aimed to establish the clinical significance of preoperative serum cytokeratin 19 fragment (CYFRA21-1) and Sialyl Lewisx (SLX) in patients with stage I non-small cell lung cancer (NSCLC). The study involved 137 patients (87 male, 50 female; median age 69 years) with completely resected stage I NSCLC. SLX, carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), and CYFRA21-1 were examined. Receiver operator characteristic (ROC) curves were constructed to determine prognostic cut-off values. Among the 137 patients, we identified 30 with recurrence within 3 years. The 5-year survival rates in patients with (n =30) and without (n =107) recurrence were 14% and 81%, respectively. The serum concentrations of SLX, CEA, and CYFRA21-1 in the recurrence group were significantly higher than those in the non-recurrence group. The areas under the ROC curve (AUC) were 0.72, 0.65, 0.53, and 0.64 for SLX, CEA, SCC, and CYFRA21-1, respectively. The prognostic cut-off values were 36U/ml, 7.8ng/ml, 1.5ng/ml, and 3.2ng/ml for SLX, CEA, SCC, and CYFRA21-1, respectively. A log-rank test revealed that age, performance status, T factor, lymphatic invasion, vascular invasion, SLX, CEA, SCC, and CYFRA21-1 were all significantly associated with survival. By multivariate analysis, age, performance status, lymphatic invasion, SLX (risk ratio, 4.11) and CYFRA21-1 (risk ratio, 3.47) were independent prognostic factors. For patients positive for both CYFRA21-1 and SLX, the relative risk was 5.32 compared with patients who were negative for both markers. The 5-year survival rates were 80% in the group negative for both markers (n =86); 52% in the group positive for one of the markers (n =43); and 13% for the group positive for both markers (n =8) (p <0.001). We concluded that serum SLX and CYFRA21-1 were prognostic markers for stage I NSCLC. Their combination should contribute to the classification of stage I NSCLC patients. There is a need to consider adjuvant and neoadjuvant therapies to improve prognosis in patients positive for both tumor markers. [Copyright &y& Elsevier]

Abstract: Completely protected sialyl LewisX azide was synthesized from a neolactosamine azide precursor carrying a 3-O-allyloxycarbonyl group as the temporary protecting group. After its Pd(0)-catalyzed deprotection and stereoselective α-fucosylation, the obtained LewisX azide was subjected to O-deacetylation in the galactose unit and subsequent regio- and stereoselective sialylation. Reduction of the anomeric azido group afforded the sialyl LewisX amine building block. Two molecules of this tetrasaccharide ligand were conjugated to a preformed cyclooctapeptide containing two equidistant l-asparagine units equipped with carboxy-terminated tetraethyleneglycol side chains to give, after deprotection, the target glycopeptide conjugate. Preliminary biological evaluation of the synthesized bivalent sialyl LewisX cyclopeptide conjugate showed only slightly enhanced inhibition of E-selectin binding in spite of the given flexibility of the two linked saccharide determinants. [Copyright &y& Elsevier]

The purpose of this study was to analyze the clinical significance of serum Sialyl Lewisx (SLX) concentrations as a predictor of N2 disease in patients with non–small-cell lung cancer. The study included 272 patients with non–small-cell lung cancer who underwent pulmonary resection in our institution between January 1998 and December 2003. Of 272 patients, the serum concentrations of SLX were measured by using a commercially available radioimmunoassay kit. The 5-year survival rates of patients with concentrations of SLX > 38 U/mL and those with lower concentrations were 32% and 69%, respectively ( P < .0001). The median serum concentration of SLX in patients with multilevel N2 or N3, single-level N2, and N0/1 disease were 44, 30, and 27 U/mL, respectively. The concentrations of serum SLX in patients with multilevel N2 disease were significantly higher than those in patients with single-level N2 or those with N0/1 disease (Mann-Whitney U-test; P < .0001). Although the sensitivity of SLX for identifying patients with non–small-cell lung cancer was only 24% in all patients, the sensitivity of SLX increased as the N-factor increased; the sensitivity of N0/1 disease was 15%, that of single-level N2 disease was 22%, and that of multilevel N2 or N3 disease was 71%. High serum concentrations of SLX predicted multilevel N2 disease and the associated poor outcome. Although the sensitivity of serum SLX is not acceptable for use as a screening tumor marker, we suggest that the serum concentration of SLX is useful as a staging marker to determine the strategy of treatment. [ABSTRACT FROM AUTHOR]

Abstract: (−)-Quinic acid was used as a starting material for the preparation of sialyl Lewisx mimetics in order to target E-selectin. Spatial orientation of the hydroxyl groups of quinic acid could mimic the l-fucose ones. Introduction of a side chain ending with a carboxylic acid was effected to replace the sialic acid interaction at the carbohydrate recognition domain. A first series of derivatives, incorporating amino acids linked to quinic acid, were tested for their affinity and found to interact with E-selectin with IC50 within the millimolar range. [Copyright &y& Elsevier]

Uromodulin is the pregnancy-associated Tamm–Horsfall glycoprotein, with the enhanced ability to inhibit T-cell proliferation. Pregnancy-associated structural changes mainly occur in the O-glycosylation of this glycoprotein. These include up to 12 glycan structures, made up of an unusual core type 2 sequence terminated with one, two, or three sialyl Lewisx sequences; this type of O-glycans could serve as E- and P-selectin ligands. The present work focuses on the most complex one; a tetradecamer made up of a type 2 core carrying three sialyl Lewisx branches. Five different monosaccharides are assembled by 14 glycosidic linkages. The conformational behavior of the constituting disaccharide segments was evaluated using the flexible residue procedure of the MM3 molecular mechanics procedure. For each disaccharide, the adiabatic energy surface, along with the local energy minima were established. All these results were used for the generation, prior to complete optimization of the tetradecamer. This was followed by a complete exploration of conformational hyperspace throughout the use of the single coordinate method as implemented in the CICADA program. Despite the potential flexibility of the tetradecasaccharide, only four conformational families occur, accounting for more than 95% of the total low energy conformations. For each family, the molecular properties (electrostatic, lipophilicity, and hydrogen potential) were studied. The shape of the tetradecasaccharide is best described as a flat ribbon, flanked by three branches having terminal sialyl residues. Two of the branches interact through nonbonded interactions, bringing further energy stabilization, and limiting the conformational flexibility of the sialyl residues. Only one branch maintains the original conformational features of sialyl Lewisx. This O-glycan can be seen as a fascinating example of ‘dendrimeric’ structure, where the spatial arrangement of three S-Lex epitopes may favor its complementary ‘presentations’ for the interactions with E- and P-selectins. [Copyright &y& Elsevier]

The aim of this study was to evaluate the potential of different types of sialyl Lewis X-conjugated liposomes as competitive inhibitors for tumour cell adhesion to endothelial E-selectin.Sterically stabilised liposomes with the sLeX ligand at the terminal end of the polyethyleneglycol (PEG) chain, as well as vesicles that had the ligand embedded within the PEG-layer, were compared to ligand-bearing liposomes without sterical stabilisation.First, 14 different tumour cell lines were characterised for their expression of sialyl Lewis X and/or A. Tumour cell adhesion was characterised in three static assays in vitro using: (i) immobilised E-selectin, (ii) CHO cells, transfected to express E-selectin and (iii) human umbilical vein endothelial cells (HUVEC).Sterically stabilised liposomes with the ligand at the terminal end of the polyethylene chain were the most effective inhibitors in all three assays and inhibited the adhesion of HT29 colon- and Lewis lung (LL) carcinoma cells by about 60–80%. The binding was not affected by a PEG-coating of the liposomes. Sterical stabilisation, on the other hand, completely prevented macrophage uptake (J774 cell line) independently of the presence of the ligand, while plain liposomes were taken up in an amount of 5.4 nmol liposomal lipids/106 macrophages. [Copyright &y& Elsevier]

Macrosphelide B (MSB), a 16-membered macrolide from Microsphaeropsis sp. FO-5050, inhibits adhesion of sialyl Lewisx (sLex)-expressing HL-60 cells to LPS-activated (E-selectin-expressing) human umbilical vein endothelial cells (HUVECs) in vitro. This study examines MSB effects on metastasis of B16/BL6 mouse melanoma cells (B16/BL6 cells) and L5178Y-ML mouse lymphoma cells in vivo and analyzes the MSB antimetastatic activity mechanism. When administered MSB at 20 mg/kg/day, lung metastatic nodules of B16/BL6 cells were significantly decreased (T/C = 45%). However, no inhibition of metastasis of L5178Y-ML cells to the spleen and liver was observed. Flow cytometry analysis showed that B16/BL6 cells expressed high levels of sLex antigen, whereas expression on L5178Y-ML cells was low. Under in vitro conditions, B16/BL6 cells demonstrated a greater degree of adhesion to LPS-activated HUVECs than L5178Y-ML cells, but adhesion was significantly inhibited by MSB and sLex antibody. Combined therapy of MSB and cisplatin (CDDP) induced remarkable lung metastasis inhibition without adverse effects of CDDP to the host. All these findings suggest that MSB suppresses lung metastasis of B16/BL6 cells by inhibiting cell adhesion to endothelial cells through the sLex molecule. [Copyright &y& Elsevier]

Although a large body of structural data exists for bronchial mucins from cystic fibrosis (CF) and chronic bronchitis (CB) patients, little is known about terminal structures carried on poly- N-acetyllactosamine antennae. Such structures are of interest because they are potential ligands for bacterial adhesins and other lectins. In this study, we have used fast atom bombardment mass spectrometry (FAB-MS) to examine terminal sequences released by endo-β-galactosidase from O-glycans obtained by reductive elimination of bronchial mucins purified from the sputum of 8 CF and 8 CB patients. Our data show that, although the polylactosamine antennae of CF and CB mucins have several terminal sequences in common, they differ significantly in their sialyl Lewisx (NeuAcα2-3Galβ1-4[Fucα1-3]GlcNAcβ1-) content. Thus all examined mucins from CF patients carry sialyl Lewisx on their polylactosamine antennae, whereas this type of epitope is present on only three out of the eight CB mucins examined, notably in the airways of one CB patient which were heavily infected by Pseudomonas aeruginosa as are the airways of all the CF patients. This suggests that, in airway mucins, the expression of sialyl Lewisx on polylactosamine antennae is probably more related to inflammation and infection than to a direct effect of the CF defect. [ABSTRACT FROM AUTHOR]

The serum levels of sialyl Lewisa (CA19-9), sialyl Lewisx (SLX), sialyl Tn (STN) and carcinoembryonic antigen (CEA) in peripheral venous blood and tumor drainage venous blood of colorectal cancer patients were examined to determine their value as prognostic factors after surgery. Blood samples were obtained from the peripheral vein and from the tumor drainage vein from 124 colorectal cancer patients during surgical excision of the tumor. The patients were divided into high-antigen and low-antigen groups for each antigen in each location. Serum levels of SLX, STN and CEA in tumor drainage venous blood (d-SLX, d-STN and d-CEA, respectively) were significantly higher than in peripheral venous blood (p-SLX, p-STN and p-CEA, respectively). The survival time after surgery for patients with high d-SLX or d-CEA levels and low levels of the same antigen in peripheral venous blood was significantly shorter than the survival time for those patients with low levels of antigen at both sites (p = 0.0003 or p = 0.0406, respectively). Cox's regression analysis revealed that a high d-SLX or high d-CEA level was an independent prognostic variable for patient survival. In conclusion, determining d-SLX or d-CEA is more useful than p-SLX or p-CEA in predicting the outcome for colorectal cancer patients. Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

A soluble sulfotransferase from porcine serum which catalyzes the transfer of sulfate from adenosine 3′-phosphate 5′-phosphosulphate (PAPS) to 2′-fucosyllactose (2′-FL) was purified 36,333-fold using a combination of conventional and affinity chromatographic steps. The purified enzyme preparation after non-denaturing discontinuous-PAGE exhibited a molecular mass of about 80 kDa by reducing SDS-PAGE. However, when a partially purified enzyme preparation was subjected to gel filtration on Sephacryl S-300, the enzyme activity eluted in the void volume, which indicated that the native enzyme existed as an oligomer. The purified enzyme showed Km values of 9.15 µM for PAPS and 15.38 mM for 2′-FL at the optimum pH value of 7.4. The substrate specificity of the purified enzyme was evaluated with various sugars that are structurally similar to sialyl Lewisx (sLex). Results indicated that 3′-sialyllactose and lactose were efficient acceptors of sulfation, whereas 6′-sialyllactose and 6′-sialyllactosamine were poor substrates for this sulfotransferase. Further, the reaction product analysis revealed that the sulfate substitution, when using 2′-FL as the substrate, was at the C-6 position of the galactose residue. Coincidentally, a similar enzyme activity was also found in porcine lymphoid tissues such as, lymph nodes (peripheral and mesenteric) and spleen. Collectively, these findings suggest that this enzyme might be involved in the synthesis of the ligand for L-selectin. [ABSTRACT FROM AUTHOR]

Both infiltrating leukocytes and soluble immunoglobulin form aggregates in synovial fluid during the inflammatory process in rheumatoid arthritis (RA). Some of these changes are probably mediated by the adhesion molecule, E-selectin, which increases its expression with disease activity. As glycosylation changes in IgG in RA are well established, the current study was undertaken to measure the expression of the carbohydrate antigen sialyl Lewis x (sLex), on IgG in RA. sLex is a major ligand for E-selectin. Using a recently developed ELISA, sLex expression was determined in IgG isolated from 8 healthy individuals, 20 RA sufferers (10 early and 10 with more long-standing disease) and 20 patients with other rheumatic conditions (osteoarthritis, ankylosing spondylitis, systemic lupus erythematosus). S Lexexpression on IgG was elevated above the reference range in all but one of the RA patients and this change was highly significant (P < 0.0006). Expression of this antigen on IgG was also significantly different from normal in the other arthritic groups (P < 0.02), but the changes were much less than that observed for RA. In early RA, sLex was inversely correlated with parameters used to measure disease activity. This was not observed with the established RA, where there was weak positive association. These preliminary results indicate that a change in sLex expression on IgG is an early finding in the development of RA, which may be important in the development of the disease or for predicting its outcome. [ABSTRACT FROM AUTHOR]

Mice are frequently used in models for the study of immunological processes related to inflammation. Since it is known that the degree of fucosylation of human acute phase proteins (APPs) is altered as a consequence of an inflammatory response, we have undertaken this study to gain more insight into the fucosylation of acute phase proteins as it occurs in mouse liver. Mice carrying the cluster of the three genes encoding human α1-acid glycoprotein (AGP), one of the well known APPs, were used and the fucosylation of AGP was assessed. A complete absence of fucosylation on the transgenic human AGP was found, which is in sharp contrast to AGP in human serum, of which a major proportion is normally α3-fucosylated. Remarkably, a large proportion of mouse AGP did contain fucose residues. Fucosylation was also detected on another APP, mouse protease inhibitor (PI). α3-Fucosylation of the transgenic human AGP can be achieved in vitro, using an α3/4-fucosyltransferase (α3/4-FucT) isolated from human milk, showing that the glycoprotein is not intrinsically resistant to fucosylation. Upon subsequent measurement of the activities of the possible fucosyltransferases present in liver membranes of parent and transgenic mice, only an N-linked-core α6-FucT and no α2-, α3- or α4-FucT activity was detected. This indicates that fucose residues found on the mouse serum proteins AGP and PI, which are synthesized in the liver, are most probably in α6-linkage to the core chitobiosyl unit. Interestingly, both α6- and α3-FucT activity was detectable in human liver membranes. None of the above mentioned findings were influenced by the induction of an acute phase response by administration of bacterial lipopolysaccharide. This study shows that: (a) α6-FucT is probably a protein specific-glycosyltransferase, since mouse AGP, but not human AGP, may be used as an acceptor; (b) in contrast to human liver, mouse liver does not express any α3-FucT-activity, thereby making the mouse incapable of producing the Sialyl Lewisx epitope on APPs, which is an important part of the inflammatory reaction in humans. This last finding indicates that the mouse is not suitable as a model for the study of those phenomena related to inflammation in humans, in which glycosylation of acute phase proteins could play a significant role. © 1998 Rapid Science Ltd [ABSTRACT FROM AUTHOR]

Acute and chronic inflammation-induced expression of sialyl Lewisx has already been shown to occur on α1-acid glycoprotein. We now demonstrate that this phenomenon is not restricted to α1-acid glycoprotein but also occurs on two other acute-phase proteins. ie on α-antichymotrypsin and on haptoglobin. The level of expression of sialyl Lewisx on these proteins was lower than on α1-acid glycoprotein, in all likelihood because α1-acid glycoprotein is the only acute-phase protein containing tetraantennary glycans. No expression of sialyl Lewisx was detectable on α1-protease inhibitor, a protein with a high diantennary glycan content. Non-sialylated Lewisx was not detectable on these major acute-phase proteins in any of the conditions studied. This indicates that the majority of the α3-linked fucose residues are present as sialyl Lewisx on α1-acid glycoprotein, α1-antichymotrypsin and haptoglobin. The absolute contribution to the total phenotype in plasma of protein containing this determinant in a multivalent form was highest for α1-acid glycoprotein. This leads us to propose that α1-acid glycoprotein is, among the acute-phase proteins studied, the one with the highest potential for interference with the extravasation of leukocytes by binding to the selectins. [ABSTRACT FROM AUTHOR]

This study was performed in order to gain insight into the occurrence, glycosylation and the possible origin of the acute-phase proteins α1-acid glycoprotein (AGP) and α1-protease inhibitor (PI) in sera and synovial fluid from patients with rheumatoid arthritis (RA). Therefore paired sera and synovial fluid samples from patients with RA, and paired synovial fluid samples from right and left knees of patients with varying degrees of arthritis were studied. Crossed affinity immunoelectrophoresis (CAIE) was used with concanavalin A and Aleuria aurantia lectin for the detection of the degree of branching and fucosylation, respectively, and the monoclonal CSLEX-1 for the detection of Sialyl Lewisx (SLex) groups on AGP. For PI, not only CAIE, but also high-pressure-anion-exchange chromatography with pulsed amperometric detection was used to study the glycosylation. It was established that the concentrations of AGP and PI were increased in the serum of RA patients compared to normal healthy controls, but that the concentration of both proteins, as well as albumin, was significantly lower in synovial fluid than in serum. Furthermore, the type of glycosylation of both AGP and PI found in RA was significantly different from that found in normals, with increased fucosylation, but there were no major differences in the degree of branching of AGP- or PI-glycans in RA, compared to normals. No differences in glycosylation could be established between serum and synovial fluid in RA. For PI an increased fucosylation was found, both in serum and synovial fluid, using both methods of detection, and it could be established that only the α1→3- and not the α1→6-fucosylation of PI was affected by RA. The increased fucosylation of AGP resulted in an increased expression of SLex on AGP-glycans. Since the α1→3- fucosylation of AGP was significantly increased in both serum and synovial fluid from RA patients, and this correlated with systemic but not with local disease parameters, it can be suggested that acute phase proteins in synovial fluid are most probably of hepatic origin. Abbreviations: AGP, α1-acid glycoprotein; AAL, Aleuria Aurantia Lectin; Con A, concanavalin A; PI, α1-protease inhibitor; CAIE, crossed affino-immunoelectrophoresis; SLex, sialyl Lewis X; IL-6, interleukin-6; RA, rheumatoid arthritis; PMN, polymorphonuclear cells; HPAEC, high pressure anion exchange chromatography [ABSTRACT FROM AUTHOR]

The conformation of the sialyl Lewis x tetrasaccharidebound to E-selectin was previously determined from transfer NOE (trNOE)experiments in conjunction with a distance-geometry analysis. However, theorientation of the tetrasaccharide ligand in the binding site of E-selectinis still unknown. It can be predicted that the accurate quantitativeanalysis of all trNOEs, including those originating from spin diffusion, isone key to analyze the orientation of sialyl Lewisx in thebinding pocket of E-selectin. Therefore, we applied homonuclear 3D NMRexperiments and 1D analogs to obtain trNOEs that could not unambiguously beassigned from previous 2D trNOESY spectra, due to severe resonance-signaloverlap. A 3D TOCSY-trNOESY experiment, a 1D TOCSY-trNOESY experiment, and a1D trNOESY-TOCSY experiment of the sialyl Lewisx/E-selectincomplex furnished new interglycosidic trNOEs and provided additionalinformation for the interpretation of trNOEs that have been describedbefore. A 2D trROESY spectrum of the sialyl Lewisx/E-selectincomplex allowed one to identify the amount of spin-diffusion contributionsto trNOEs. Finally, an unambiguous assignment of all trNOEs, and an analysisof spin-diffusion pathways, was obtained, creating a basis for aquantitative analysis of trNOEs in the sialylLewisx/E-selectin complex. [ABSTRACT FROM AUTHOR]