Sialyl Lewis(a): a tumor-associated carbohydrate antigen involved in adhesion and metastatic potential of cancer cells

Neoplastic transformation is often associated with characteristic changes in the expression of the sialyl Lewis(a) and sialyl Lewis(x) antigens, representing typical tumor-associated carbohydrate antigens. High amounts of sialyl Lewis(a) are present in human adenocarcinomas of the colon, pancreas and stomach. A growing amount of data suggests that this carbohydrate structure is the ligand for E-selectin. Sialylated Lewis structures present on the surface of tumor cells are carried by the carbohydrate chains of glycoproteins and glycolipids. There are several lines of evidence showing that sialyl Lewis(a) is responsible for the adhesion of human cancer cells to endothelium. E-selectin present on endothelial cells mediates these interactions. Selectins and their carbohydrate ligands can thus play an important role in the selective homing of tumor cells during metastasis. However, the presence of sialyl Lewis(a) antigen on the surface of tumor cells and their adhesion to E-selectin-expressing cells in in vitro adhesion assay by itself can not be directly related to metastatic properties of all cancer cells.     

Sialosignaling: Sialyltransferases as engines of self-fueling loops in cancer progression

Background

Glycosylation is increasingly recognized as one of the most relevant postranslational modifications. Sialic acids are negatively charged sugars which frequently terminate the carbohydrate chains of glycoproteins and glycolipids. The addition of sialic acids is mediated by sialyltransferases, a family of glycosyltransferases with a crucial role in cancer progression.

Scope of the review

To describe the phenotypic and clinical implications of altered expression of sialyltransferases and of their cognate sialylated structures in cancer. To propose a unifying model of the role of sialyltransferases and sialylated structures on cancer progression.

Major conclusions

We first discuss the biosynthesis and the role played by the major cancer-associated sialylated structures, including Thomsen–Friedenreich-associated antigens, sialyl Lewis antigens, α2,6-sialylated lactosamine, polysialic acid and gangliosides. Then, we show that altered sialyltransferase expression in cancer, consequence of genetic and epigenetic alterations, generates a flow of information toward the membrane through the biosynthesis of aberrantly sialylated molecules (inside-out signaling). In turn, the presence of aberrantly sialylated structures on cell membrane receptors generates a flow of information toward the nucleus, which can exacerbate the neoplastic phenotype (outside-in signaling). We provide examples of self-fueling loops generated by these flows of information.

General significance

Sialyltransferases have a wide impact on the biology of cancer and can be the target of innovative therapies. Our unified view provides a conceptual framework to understand the impact of altered glycosylation in cancer.     

唾液酸转移酶对肿瘤中唾液酸化结构的影响

随着侵袭能力的增强,肿瘤细胞表面经常会出现唾液酸化糖链结构的增加。现在已经证实许多特殊的唾液酸化结构,如TF类抗原、sLew类抗原、α2-6唾液酸化的乳糖胺结构、PSA结构、神经节苷脂结构都参与细胞间的相互作用。本文综述了与肿瘤相关的特殊的唾液酸化结构的改变与相应的唾液酸转移酶在其中发挥的作用。     

The discovery, biology, and drug development of sialyl Lea and sialyl Lex

The discoveries of sialylated, fucosylated lacto-, and neolacto-type carbohydrate structures were accomplished with the aid of analytical methods and monoclonal antibodies such as the immunostaining of thin layer chromatograms. Based on the use of such antibodies, these structures, notably sialyl Le(a) and sialyl Le(x), were demonstrated to be highly expressed in many malignant cancers. A diagnostic assay using one of these antibodies (CA19-9) is now established as one of the more commonly used assays for pancreatic and gastrointestinal cancers worldwide. Upon further study, several laboratories have demonstrated that the level of expression of these carbohydrate tumor markers is also positively correlated with patient survival and is a prognostic indicator of metastatic disease. Concurrent with this finding, both sialyl Le(a) and sialyl Le(x) were shown to bind to a family of carbohydrate-binding proteins involved in the extravasation of cells from the bloodstream, called the selectins. Thus, sialyl Le(a) and sialyl Le(x) expressed on cell surfaces play functional roles in medical conditions that require extravasation of cells from the bloodstream which include a wide range of inflammatory diseases and cancer metastasis. Many studies have confirmed the function of sialyl Le(a) and sialyl Le(x) in animal models of these diseases and the inhibition of binding of sialyl Le(a) and sialyl Le(x) to the selectins is a validated drug target in the pharmaceutical industry. Thus, a new class of drugs, arising from the field of glycobiology, is based on the rational design of small molecule drugs that mimic the structures sialyl Le(a) and sialyl Le(x) and can potently inhibit their functional binding to the selectins.     

sLea and sLex expression in colorectal cancer: implications for tumourigenesis and disease prognosis

The glycoconjugates expressed by cancer cells frequently contain sialylated oligosaccharide chains. Among these oligosaccharides the sialyl Lewis a (sLe(a)) and sialyl Lewis x (sLe(x)) antigens are found to be overexpressed in tumours of different origin. The current study assesses sLe(a) and sLe(x) expression in different colorectal specimens in order to establish the correlation of these biomarkers with both malignant transformation of colorectal mucosa and the progression of colorectal cancer (CRC). Healthy disease-free and inflammatory mucosa specimens showed no presence of the antigens. sLe(a) was expressed in 6.7% of the healthy tissue from CRC patients, in 20.8% of the adenomas, and in 33.3% and 42.6% of the transitional tissue and tumour tissue, respectively. sLe(x) expression was observed in 6.7% of the healthy tissue from CRC patients, in 27.0% of the adenomas, and in 25.6% and 74.8% of the transitional and the tumour tissue, respectively. The expression of the sLe(a) and sLe(x) antigens was correlated in adenomas, as well as in healthy and tumour tissue from CRC. Moreover, the high expression of sLe(x) in adenomas was correlated with a high degree of dysplasia (p=0.042). Finally, the survival analysis suggested that sLe(a) expression may be a prognostic factor for predicting disease-free survival in colorectal cancer (p=0.012).     

Significant decrease in alpha1,3-linked fucose in association with increase in 6-sulfated N-acetylglucosamine in peripheral lymph node addressin of FucT-VII-deficient mice exhibiting diminished lymphocyte homing

Lymphocyte homing is mediated by binding of L-selectin on lymphocytes with L-selectin ligands present on high-endothelial venules (HEV) of peripheral and mesenteric lymph nodes. L-selectin ligands are specific O-linked carbohydrates, 6-sulfo sialyl Lewis X, composed of sialylated, fucosylated, and sulfated glycans. Abrogation of fucosyltransferase-VII (FucT-VII) results in almost complete loss of lymphocyte homing, but structural analysis of carbohydrates has not been carried out on FucT-VII null mice. To determine whether functional losses seen in FucT-VII null mice are caused by structural changes in carbohydrates, we elucidated the carbohydrate structure of GlyCAM-1, a major L-selectin counter-receptor. Our results show that most alpha1,3-fucosylated structures in 6-sulfo sialyl Lewis X are absent and 6-sulfo N-acetyllactosamine is increased in the mutant mice. Surprisingly, the amount of 6'-sulfated galactose (Gal) that bound to Sumbucus nigra agglutinin column was also increased. We found that structures of those oligosaccharides containing 6'-sulfated Gal are almost identical to those synthesized by keratan sulfate sulfotransferase (KSST). We then showed that overexpression of KSST suppresses the expression of sialyl Lewis X on Chinese hamster ovary (CHO) cells engineered to express sialyl Lewis X. Moreover, KSST expression in those cells suppressed lymphocyte rolling compared with mock-transfected CHO cells expressing 6-sulfo sialyl Lewis X. 6'-Sulfo sialyl Lewis X can neither be found in GlyCAM-1 from CHO cells expressing both KSST and FucT-VII nor be found in GlyCAM-1 from HEV of mice. These results combined together suggest that KSST competes with FucT-VII for the same acceptor substrate and downregulates the synthesis of L-selectin ligand by inhibiting alpha1,3-fucosylation.     

Characterization of cancer associated mucin type O-glycans using the exchange sialylation properties of mammalian sialyltransferase ST3Gal-II

Our previous studies suggest that the α2,3sialylated T-antigen (NeuAcα2,3Galβ1,3GalNac-) and associated glycan structures are likely to be elevated during cancer. An easy and reliable strategy to label mucinous glycans that contain such carbohydrates can enable the identification of novel glycoproteins that are cancer associated. To this end, the present study demonstrates that the exchange sialylation property of mammalian ST3Gal-II can facilitate the labeling of mucin glycoproteins in cancer cells, tumor specimens, and glycoproteins in cancer sera. Results show that (i) the radiolabeled mucin glycoproteins of each of the cancer cell lines studied (T47D, MCF7, LS180, LNCaP, SKOV3, HL60, DU4475, and HepG2) is distinct either in terms of the specific glycans presented or their relative distribution. While some cell lines like T47D had only one single sialylated O-glycan, others like LS180 and DU4475 contained a complex mixture of mucinous carbohydrates. (ii) [14C]sialyl labeling of primary tumor cells identified a 25-35 kDa mucin glycoprotein unique to pancreatic tumor. Labeled glycoproteins for other cancers had higher molecular weight. (iii) Studies of [14C] sialylated human sera showed larger mucin glycopeptides and >2-fold larger mucin-type chains in human serum compared to [14C]sialyl labeled glycans of fetuin. Overall, the exchange sialylation property of ST3Gal-II provides an efficient avenue to identify mucinous proteins for applications in glycoproteomics and cancer research.     

Regulation of sialyl Lewis antigen expression in colon cancer cells by sialidase NEU4

Sialyl Lewis antigens, sialyl Lewis a and sialyl Lewis x, are utilized as tumor markers, and their increase in cancer is associated with tumor progression by enhancement of cancer cell adhesion to endothelial E-selectin. However, regulation mechanisms are not fully understood. We previously demonstrated that NEU4 is the only sialidase efficiently acting on mucins and it is down-regulated in colon cancer. To elucidate the significance of NEU4 down-regulation, we investigated sialyl Lewis antigens as endogenous substrates for the sialidase. NEU4 was found to hydrolyze the antigens in vitro and decrease cell surface levels much more effectively than other sialidases. Western blot, thin layer chromatography, and metabolic inhibition studies of desialylation products revealed NEU4 to preferentially catalyze sialyl Lewis antigens expressed on O-glycans. Cell adhesion to and motility and growth on E-selectin were significantly reduced by NEU4. E-selectin stimulation of colon cancer cells enhanced cell motility through activation of the p38/Hsp27/actin reorganization pathway, whereas NEU4 attenuated the signaling. On immunocytochemical analysis, some NEU4 molecules were localized at cell surfaces. Under hypoxia conditions whereby the antigens were increased concomitantly with several sialyl- and fucosyltransferases, NEU4 expression was markedly decreased. These results suggest that NEU4 plays an important role in control of sialyl Lewis antigen expression and its impairment in colon cancer.     

Structures of sialylated fucosyl polylactosaminoglycans isolated from chronic myelogenous leukemia cells

Polylactosaminoglycans were isolated from human chronic myelogenous leukemia cells and their structures were elucidated. The lactosaminoglycan saccharides were isolated by hydrazinolysis and fractionated by QAE-Sephadex. The structures of fractionated oligosaccharides were analyzed by fast atom bombardment-mass spectrometry and methylation before and after treatment with specific exoglycosidases, such as alpha 2----3 specific neuraminidase. Based on these experiments, the structures of sialyl polylactosaminoglycans of chronic myelogenous leukemia cells were found to contain the following unique structure which is absent in normal mature granulocytes: (formula; see text) In addition to this, chronic myelogenous leukemia polylactosaminoglycans can be distinguished from normal granulocyte polylactosaminoglycans by the following characteristics. Leukemic polylactosaminoglycans are (a) shorter, (b) more highly sialylated and contain fully sialylated, tetrasialosyl polylactosaminoglycans, (c) are less fucosylated at C-3 of N-acetylglucosamine of polylactosaminyl side chains, and (d) contain a significant amount of sialyl Lex, NeuNAc alpha 2----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----3, structure. These results indicate that chronic myelogenous leukemia cells express unique polylactosaminoglycan structures which are distinct from normal mature granulocytes.     

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

Mammalian brains contain relatively high amounts of common and uncommon sialylated N-glycan structures. Sialic acid linkages were identified for voltage-gated potassium channels, Kv3.1, 3.3, 3.4, 1.1, 1.2 and 1.4, by evaluating their electrophoretic migration patterns in adult rat brain membranes digested with various glycosidases. Additionally, their electrophoretic migration patterns were compared with those of NCAM (neural cell adhesion molecule), transferrin and the Kv3.1 protein heterologously expressed in B35 neuroblastoma cells. Metabolic labelling of the carbohydrates combined with glycosidase digestion reactions were utilized to show that the N-glycan of recombinant Kv3.1 protein was capped with an oligo/poly-sialyl unit. All three brain Kv3 glycoproteins, like NCAM, were terminated with alpha2,3-linked sialyl residues, as well as atypical alpha2,8-linked sialyl residues. Additionally, at least one of their antennae was terminated with an oligo/poly-sialyl unit, similar to recombinant Kv3.1 and NCAM. In contrast, brain Kv1 glycoproteins consisted of sialyl residues with alpha2,8-linkage, as well as sialyl residues linked to internal carbohydrate residues of the carbohydrate chains of the N-glycans. This type of linkage was also supported for Kv3 glycoproteins. To date, such a sialyl linkage has only been identified in gangliosides, not N-linked glycoproteins. We conclude that all six Kv channels (voltage-gated K+ channels) contribute to the alpha2,8-linked sialylated N-glycan pool in mammalian brain and furthermore that their N-glycan structures contain branched sialyl residues. Identification of these novel and unique sialylated N-glycan structures implicate a connection between potassium channel activity and atypical sialylated N-glycans in modulating and fine-tuning the excitable properties of neurons in the nervous system.     

Role of sialyltransferases involved in the biosynthesis of Lewis antigens in human pancreatic tumour cells

The sialylated carbohydrate antigens, sialyl-Lewis^x and sialyl-Lewis^a, are expressed in pancreatic tumour cells and are related to their metastatic potential. While the action of the fucosyltransferases involved in the synthesis of these antigens has already been investigated, no studies have been carried out on the activity and expression of the α 2,3-sialyltransferases in pancreatic tumour cells. We describe the sialyltransferase (ST) activity, mRNA expression, and analysis of the cell carbohydrate structures in four human pancreatic adenocarcinoma cell lines of a wide range of neoplastic differentiation stages and in normal human pancreatic tissues. Total ST activity measured on asialofetuin, employing a CMP fluorescent sialic acid, varied among the pancreatic cell lines and could be correlated to the expression of their cell surface antigens. However, in some of the pancreatic cell lines, no relationship could be established with their ST3Gal III and IV mRNA expression. Human pancreatic tissues also showed ST expression and activity. However, it presented a much higher expression of neutral fucosylated structures than sialylated structures. In conclusion, ST activity levels in pancreatic cells could be correlated to their expression of sialylated epitopes, which indicates their involvement in the formation of the sialyl-Lewis antigens, in addition to fucosyltransferase activities. Published in 2005.     

Qualitative difference of subcellular localization of tumor-associated carbohydrate (Le(x)) antigens in renal cell carcinoma and normal kidney.

Renal cell carcinomas are immunohistochemically positive for oligosaccharides with the Le(x) determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc) and its derivatives, as oncofetal antigens, and their expression is closely related to a better prognosis of the patients. This study was designed to clarify the difference in antigen localization at the ultrastructural level between renal cell carcinoma and normal tissues. In normal kidneys, Le(x) detected by monoclonal antibody (MAb) FH 2 and sialylated extended Le(x) (sialyl Le(x)-i) by MAb FH 6 were identified along the plasma membrane of microvilli of proximal tubule epithelial cells, with occasional immunoreactivity along the basolateral plasma membranes. Intracellular localization was very sparse. Renal cell carcinoma showed localization of Le(x) and sialyl Le(x)-i antigens along the cell membrane and in the cytosol as aggregates or filaments. Immunoreactive materials were also observed in the lumen formed among carcinoma cells. The cytosolic immunoreactivity, not observed in the normal kidney, was regarded as "abnormal cytosolic accumulation" of the antigens. This pattern was more pronounced in clear-cell carcinoma. Pretreatment of specimens with chloroform-methanol, which extracts glycolipids, decreased immunoreactivity in carcinoma tissues, particularly that in the cytosol. The extracts contained substances immunoreactive for MAb FH6. Our study has demonstrated that (a) remarkable changes occur in the ultrastructural localization patterns of sialyl Le(x)-i and Le(x) in renal cell carcinoma and (b) considerable amounts of glycolipids are contained in the substances with sialyl Le(x)-i deposited in the cytosol of clear-cell carcinoma.     

Consequences of the expression of sialylated antigens in breast cancer

Changes in cell surface glycosylation are common modifications that occur during oncogenesis, leading to the over-expression of tumour-associated carbohydrate antigens (TACA). Most of these antigens are sialylated and the increase of sialylation is a well-known feature of transformed cells. In breast cancer, expression of TACA such as sialyl-Lewis^x or sialyl-Tn is usually associated with a poor prognosis and a decreased overall survival of patients. However, the specific role of these sialylated antigens in breast tumour development and aggressiveness is not clearly understood. These glycosylation changes result from the modification of the expression of genes encoding specific glycosyltransferases involved in glycan biosynthesis and the level of expression of sialyltransferase genes has been proposed to be a prognostic marker for the follow-up of breast cancer patients. Several human cellular models have been developed in order to explain the mechanisms by which carbohydrate antigens can reinforce breast cancer progression and aggressiveness. TACA expression is associated with changes in cell adhesion, migration, proliferation and tumour growth. In addition, recent data on glycolipid biosynthesis indicate an important role of G_D3 synthase expression in breast cancer progression. The aim of this review is to summarize our current knowledge of sialylation changes that occur in breast cancer and to describe the cellular models developed to analyze the consequences of these changes on disease progression and aggressiveness.     

Altered expression of sialylated carbohydrate antigens in HT29 colonic carcinoma cells

To determine whether cell growth conditions impacted carbohydrate expression, HT29 cells were gradually transferred from a conventional glucose-containing media to a glucose-free galactose containing media. Indirect immunofluorescence on acetone fixed cells showed increased expression of sialyl Lewis A antigen (CA19-9), sialyl Lewis C (DUPAN2) and Tn/sialyl-Tn on the surface of HT29 cells grown in the glucose-free galactose containing media compared to those grown in the glucose containing media. Sialyltransferases responsible for the synthesis for these sialylated epitopes were increased in the galactose-fed HT29 cells. Media overlying the cells was subjected to isopycnic ultracentrifugation in cesium chloride and the fractions derived from both glucose and galactose media with equivalent buoyant densities of 1.56 g/L, which are predicted to contain mucin glycoforms, were further separated by HPLC using a Mono-Q anion exchange column. The chromatograph of eluent from the sample derived from the cells growing in the galactose containing media showed an increased peak that reacted with the anti-sialyl Lewis A antibody, CA19-9. These results show that alteration of in vitro culture conditions may cause HT29 colonic carcinoma cells to alter the expression of sialylated carbohydrates.     

Gangliosides expressed on breast cancer cells are E-selectin ligands

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 (sLe^x) and sialyl Lewis A (sLe^a) 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.     

Modulation of MUC1 and blood group antigen expression in gastric adenocarcinoma cells by cytokines

Immunohistological studies demonstrated that MUC1 expression in gastric cancer is associated with a poor prognosis. As a mediator of cell-cell interactions, MUC1 may also be involved in metastasis. However, these aspects are of relevance since cytokine levels are locally increased as a consequence of peritumorous inflammatory response and coexisting chronic gastritis. Therefore we analyzed the potential influence of several cytokines on the expression of tumor-associated MUC1 and Lewis blood group antigens in gastric carcinoma cells. Gastric cancer cell lines AGS and KATOIII were incubated with the cytokines interleukin-1beta, interferon-gamma, tumor necrosis factor-alpha (TNF-alpha), and hepatocyte growth factor over a period of 72 h. Expressions of mucin antigens and cytokine secretion were measured by immunocytochemistry and/or enzyme-linked immunosorbent assay (ELISA). Analysis by fluorescence-activated cell sorter (FACS) demonstrated that MUC1 and sialyl Lewis A reactivities of AGS cells were increased significantly following TNF-alpha stimulation but not by other cytokines. Expression of mucin-associated antigens by cell line KATOIII was not affected by any of the employed cytokines. These data provide evidence that TNF-alpha can raise the expression of important mucin peptide as well as mucin-associated carbohydrate antigens and thereby potentially influence the progression of gastric carcinomas.     

Heparan sulfate-like proteoglycans mediate adhesion of human malignant melanoma A375 cells to P-selectin under flow

Selectins, a family of cell adhesion molecules, bind to sialylated and fucosylated carbohydrates, such as sialyl Lewisx (SLex) and its derivatives, as their minimal recognition motif. Here we report that P-selectin bound to human malignant melanoma A375 cells and mediated their adhesion under flow. However, probing with a specific Ab failed to detect any apparent expression of SLex. This finding was bolstered by reduced expression of alpha-1,3-fucosyltransferase VII mRNA and by absence of the cell surface expression of P-selectin glycoprotein ligand-1. Instead, they expressed heparan sulfate-like proteoglycans on their cell surfaces. Treatment with beta-d -xyloside (a proteoglycan biosynthesis inhibitor) or heparinases could reduce the binding of these cells to P-selectin. In the competition assays, heparin, but not other proteoglycans, could abolish the P-selectin recognition. Further, we found that P-selectin could bind specifically to human tongue squamous cancer Tca-8113 cells, which had negative staining of SLex but positive staining of heparan sulfates. Both beta-d -xyloside and heparinases could reduce the binding of P-selectin to Tca-8113 cells. Our results thus indicate that heparan sulfate-like proteoglycans can mediate adhesion of certain types of non-blood borne, "epithelial-like" human cancer cells to P-selectin.     

Stable expression of a cDNA encoding a human beta 1 --> 3galactosyltransferase responsible for lacto-series type 1 core chain synthesis in non-expressing cells: variation in the nature of cell surface antigens expressed.

Transient expression of a human colonic adenocarcinoma Colo 205 cell derived cDNA in cell lines which ordinarily express only neolacto-series glycolipids has resulted in the expression of a beta 1 --> 3galactosyltransferase gene responsible for synthesis of glycolipids based upon the lacto-series type 1 core chain. Calcium phosphate transfected cells were panned on anti-IgM coated plates after initial treatment with a combination of monoclonal antibodies specific for type 1 chain terminal structures (TE-3) and a very broadly specific antibody reactive with multiple type 1 chain derivatives (TE-2). Adherent cells after panning were capable of efficiently transferring Gal in beta 1 --> 3-linkage to the acceptor glycolipid Lc3. Using these reagents, clones of stably transfected human colonic adenocarcinoma HCT-15 cells were produced and isolated. Parental HCT-15 cells do not express type 1 chain based antigens. The nature of the type 1 chain based antigens produced in each of these clones was analyzed by solid phase antibody binding assays. Three types of behavior were observed. Formation of type 1 terminal structures that were either exclusively sialylated or fucosylated, or a mixture of sialylated and fucosylated determinants occurred. In contrast, no difference in type 2 antigen expression between any clone and the parental cells was observed. These data suggest that coordination of subsequent reactions capable of modifying type 1 chain structures is not the same in all clones. The relationship of these results to aspects of cellular regulation of carbohydrate biosynthesis is discussed.     

Enzymatic synthesis of alpha3'sialylated and multiply alpha3fucosylated biantennary polylactosamines. A bivalent [sialyl diLex]-saccharide inhibited lymphocyte-endothelium adhesion organ-selectively.

Multifucosylated sialo-polylactosamines are known to be high affinity ligands for E-selectin. PSGL-1, the physiological ligand of P-selectin, is decorated in HL-60 cells by a sialylated and triply fucosylated polylactosamine that is believed to be of functional importance. Mimicking some of these saccharide structures, we have synthesized enzymatically a bivalent [sialyl diLex]-glycan, Neu5Acalpha2-3'Lexbeta1-3'Lexbeta1-3'(Neu5Acalpha2-3'Lexbeta1-3Lexbe ta1-6')LN [where Neu5Ac is N-acetylneuraminic acid, Lex is the trisaccharide Galbeta1-4(Fucalpha1-3)GlcNAc and LN is the disaccharide Galbeta1-4GlcNAc]. Several structurally related, novel polylactosamine glycans were also constructed. The inhibitory effects of these glycans on two L-selectin-dependent, lymphocyte-to-endothelium adhesion processes of rats were analysed in ex-vivo Stamper-Woodruff binding assays. The IC50 value of the bivalent [sialyl diLex]-glycan at lymph node high endothelium was 50 nm, but at the capillaries of rejecting cardiac allografts it was only 5 nm. At both adhesion sites, the inhibition was completely dependent on the presence of fucose units on the sialylated LN units of the inhibitor saccharide. These data show that the bivalent [sialyl diLex]-glycan is a high affinity ligand for L-selectin, and may reduce extravasation of lymphocytes at sites of inflammation in vivo without severely endangering the normal recirculation of lymphocytes via lymph nodes.