Enzymatic basis for sialyl-Tn expression in human colon cancer cells

Sialyl-Tn antigen (SA2-6 GalNAc-Ser/Thr) is expressed as a cancer-associated antigen on the surface of cancer cells and its expression correlates with a poor prognosis in patients with colorectal and other adenocarcinomas. To understand the enzymatic basis of sialyl-Tn (STn) antigen expression, we used two clonal cell lines, LSB and LSC, derived from LS174T human colonic cancer cells. LSC cells express only the truncated carbohydrate antigen Tn (GalNAc-Ser/Thr) and sialyl-Tn on their mucin molecules, whereas LSB cells express elongated oligosaccharide chains. Both cell lines demonstrated similar activities of glycosyltransferases involved in the biosynthesis of elongated and terminal structures of complex O-glycans. However, LSC cells were unable to synthesize core 1 (Gal1-3GalNAc-) because the ubiquitous enzyme activity of UDP-Gal:GalNAc-R 3-Gal-transferase (core 1 3-Gal-transferase) was lacking. Core 1 3-Gal-transferase could not be reactivated in LSC cells by treatment with sodium butyrate or by in vivo growth of LSC cells in nude mice. In contrast, LSB cells were able to synthesize and process core 1 and core 2 (GlcNAc1-6 (Gal1-3) GalNAc-). LSC cells represent the first example of a non-hematopoietic cell line which lacks core 1 3-Gal-transferase activity. The lack of core 1 3-Gal-transferase in LSC cells explains why they are incapable of forming the common mucin O-glycan core structures and are committed to synthesizing the short Tn and STn oligosaccharides. These findings suggest that the activity of core 1 3-Gal-transferase is an important determinant of the STn phenotype of colon cancer cells.     

Glycoprotein biosynthesis in porcine aortic endothelial cells and changes in the apoptotic cell population

Porcine aortic endothelial cells (PAECs) produce glycoproteins with important biological functions, such as the control of cell adhesion, blood clotting, blood pressure, the immune system, and apoptosis. Cell surface glycoproteins play important roles in these biological activities. To understand the control of cell surface glycosylation, we elucidated biosynthetic pathways leading to N- and O-glycans in PAECs. Based on the enzyme activities, PAECs should be rich in complex biantennary N-glycans. In addition, the enzymes synthesizing complex O-glycans with core 1 and 2 structures are present in PAECs. The first enzyme of the O-glycosylation pathway, polypeptide GalNAc-transferase, was particularly active. Its specificity toward synthetic peptide substrates was found to be similar to that of purified bovine colostrum enzyme T1. A significant fraction of PAECs treated with tumour necrosis factor alpha or human serum detached from the culture plate, and most of these cells were apoptotic. The apoptotic cell population exhibited decreased core 2 beta 6-GlcNAc-transferase activity. In contrast, the activities of core 1 beta 3-Gal-transferase, which synthesizes O-glycan core 1, and of alpha 3-sialyltransferase (O), which sialylates core 1, were increased in apoptotic PAECs. Thus, apoptotic PAECs are predicted to have fewer complex O-glycans and a higher proportion of short, sialylated core 1 chains.     

The effect of TNF-alpha on glycosylation pathways in bovine synoviocytes.

Synoviocytes are fibroblastic cells that line joint cavities. These cells synthesize numerous cell-surface and extracellular-matrix glycoproteins that are required for maintenance of the joint. Joint inflammation, such as occurs in arthritis, has been shown to have major effects on synoviocyte proliferation and on the biosynthesis of glycoproteins. The structures of the carbohydrate moieties of glycoproteins, however, and the enzymes involved in their synthesis have not yet been described for synoviocytes. Therefore, to characterize the cell-surface glycoconjugates, synoviocytes were isolated from bovine ankles, and the cells were grown in primary cultures. Lectin-binding assays were used to identify exposed N- and O-glycan carbohydrate determinants on synoviocytes, and specific enzyme assays were used to identify some of the glycosyltransferases involved in the synthesis of the glycan chains. A number of the enzymes that synthesize N- and O-linked oligosaccharides were found to be active in cell-free extracts of synoviocytes, including those that synthesize core-1-based O-glycans and the more complex bi-antennary N-glycans. To understand the molecular events underlying the inflammatory response in the synovium of arthritis patients, we examined the effect of the inflammatory cytokine tumour necrosis factor alpha (TNF-alpha) on synoviocytes and on glycosylation profiles. TNF-alpha treatment, which induces apoptosis in synoviocytes, was accompanied by changes in lectin-binding patterns, indicating alterations in the expression of cell-surface oligosaccharides. Concurrently, changes in specific enzyme activities were observed in treated cells. Two enzymes potentially important to the inflammatory process, core 2 beta6-GlcNAc-transferase and beta4-Gal-transferase, increased after TNF-alpha treatment. This is the first study of glycoprotein biosynthesis in synoviocytes, and it shows that synoviocytes have a characteristic glycosylation phenotype that is altered in the presence of inflammatory cytokines.     

Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2

Background

The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.

Methods

We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.

Results

Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.

Conclusions

The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.

General significance

Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.     

Control of mucin synthesis: the peptide portion of synthetic O-glycopeptide substrates influences the activity of O-glycan core 1 UDPgalactose:N-acetyl-alpha-galactosaminyl-R beta 3-galactosyltransferase

Synthetic O-glycopeptides containing one or two GalNAc residues attached to Ser or Thr were used as substrates to investigate the effect of peptide structure on the activity of crude preparations of UDP-Gal:GalNAc alpha-R beta 3-Gal-transferase from pig stomach and pig and rat colonic mucosa and of a partially purified enzyme preparation from rat liver. High-performance liquid chromatography used to separate enzyme products revealed that uncharged glycopeptides with an acetyl group at the amino-terminal end and a tertiary butyl or an amide group at the carboxy-terminal end were resistant to proteolysis in crude preparations. The activity of beta 3-Gal-transferase varied with the sequence and length of the peptide portion of the substrate, the presence of protecting groups, the attachment site of GalNAc, and the number of GalNAc residues in the substrate. The presence and position of Pro had little effect on enzyme activity; ionizing groups near the GalNAc unit interfered with enzyme activity. Since the GalNAc-Thr moieties in many of these O-glycopeptides have been shown to assume similar rigid conformations, the variation in enzyme activity indicates that the beta 3-Gal-transferase recognizes both the peptide and carbohydrate moieties of the substrate. Rat and pig colonic mucosal homogenates contain beta 3- and beta 6-GlcNAc-transferases that synthesize respectively O-glycan core 3 (GlcNAc beta 3GalNAc alpha-R) and core 4 [GlcNAc beta 6(GlcNAc beta 3)GalNAc alpha-R]. These enzymes also showed variations in activity with different peptide structures; these effects did not parallel those observed with beta 3-Gal-transferase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of glycosyltransferases in the golden hamster (Mesocricetus auratus) oviduct and evidence for the regulation of O-glycan biosynthesis during the estrous cycle

Recently, we provided evidence that the glycosylation of hamster oviductin, a member of the mucin family of glycoproteins, is regulated during the estrous cycle. In order to further elucidate the glycosylation process of oviductal glycoproteins, we identified biosynthetic pathways involved in the assembly of mucin-type O-linked oligosaccharide (O-glycan) chains in the hamster oviduct. Our results demonstrated that the hamster oviduct has high activities of glycosyltransferases that synthesize O-glycans with core 1, 2, 3 and 4 structures as well as elongated structures. Oviduct therefore represents a typical mucin-secreting tissue. Our results also showed that specific glycosyltransferase activities are regulated during the estrous cycle. Mucin-type core 2 beta6-GlcNAc-transferase (C2GnT2) is responsible for synthesizing core 2 and core 4 structures in the oviduct. Specific assays for C2GnT2 revealed a cyclical pattern throughout the estrous cycle with high activity at the stages of proestrus and estrus and low activity at diestrus 1. Using semiquantitative RT-PCR, the mRNA levels for C2GnT2 in the estrous cycle stages could be correlated with the enzyme activities. An increase in glycosyltransferase activity in the hamster oviduct at the time of ovulation suggests that glycosylation of oviductal glycoproteins may be necessary for these proteins to exert their functions during the process of fertilization.     

Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions

The glycoproteins of tumour cells are often abnormal, both in structure and in quantity. In particular, the mucin-type O-glycans have several cancer-associated structures, including the T and Tn antigens, and certain Lewis antigens. These structural changes can alter the function of the cell, and its antigenic and adhesive properties, as well as its potential to invade and metastasize. Cancer-associated mucin antigens can be exploited in diagnosis and prognosis, and in the development of cancer vaccines. The activities and Golgi localization of glycosyltransferases are the basis for the glycodynamics of cancer cells, and determine the ranges and amounts of specific O-glycans produced. This review focuses on the glycosyltransferases of colon and breast cancer cells that determine the pathways of mucin-type O-glycosylation, and the proposed functional and pathological consequences of altered O-glycans.     

The action of TNFalpha and TGFbeta include specific alterations of the glycosylation of bovine and human chondrocytes

Joint destruction in arthritis is often associated with high levels of inflammatory cytokines. Previous work has shown that inflammatory conditions can alter the activities of glycosyltransferases that synthesize the glycan chains of glycoproteins, and that these changes in turn can influence the functions of glycoproteins. We therefore examined glycosyltransferases involved in glycoprotein biosynthesis in primary cultures of bovine articular chondrocytes and human chondrocytes isolated from knee cartilage of osteoarthritis patients. Bovine chondrocytes exhibited enzyme activities involved in the synthesis of bi-antennary complex Asn-linked N-glycans, as well as the enzymes involved in the synthesis of GalNAc-Ser/Thr-linked O-glycans with the core 1 structure. Human chondrocytes, in addition, were able to synthesize more complex O-glycans with core 2 structures. TNFalpha was found to induce apoptosis in chondrocytes, and this process was associated with significant changes in lectin binding to chondrocyte cell surface glycans. TGFbeta increased cell proliferation, and had significant effects on cell surface glycosylation in bovine but not in human cells. These cytokine-specific effects were partially correlated with changes in glycosyltransferase activities. Thus, chondrocytes have many of the enzymes necessary for the synthesis of N- and O-glycan chains of glycoproteins. The O-glycosylation pathways and the effects of TNFalpha and TGFbeta on glycosylation differed between bovine and human chondrocytes. These alterations are of potential importance for the regulation of the functions of cell surface receptors on chondrocytes, and for an understanding of the pathophysiology of arthritis.     

Pathways of O-glycan biosynthesis in cancer cells

Glycoproteins with O-glycosidically linked carbohydrate chains of complex structures and functions are found in secretions and on the cell surfaces of cancer cells. The structures of O-glycans are often unusual or abnormal in cancer, and greatly contribute to the phenotype and biology of cancer cells. Some of the mechanisms of changes in O-glycosylation pathways have been determined in cancer model systems. However, O-glycan biosynthesis is a complex process that is still poorly understood. The glycosyltransferases and sulfotransferases that synthesize O-glycans appear to exist as families of related enzymes of which individual members are expressed in a tissue- and growth-specific fashion. Studies of their regulation in cancer may reveal the connection between cancerous transformation and glycosylation which may help to understand and control the abnormal biology of tumor cells. Cancer diagnosis may be based on the appearance of certain glycosylated epitopes, and therapeutic avenues have been designed to attack cancer cells via their glycans.     

Oncofetal expression of Lex carbohydrate antigens in human colonic adenocarcinomas. Regulation through type 2 core chain synthesis rather than fucosylation

The mechanism of expression of a series of glycolipid antigens carrying the Lex determinant structure, Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----, and characterized by oncofetal expression in fetal colon and colonic adenocarcinomas has been studied in human fetal and adult proximal colon tissue. Results presented from TLC immunostain analysis of neutral glycolipids isolated from normal adult colonic mucosa have indicated the presence of only barely detectable quantities of both an Lex-active glycolipid that co-migrated with III3V3Fuc2nLc6 and its precursor nLc6. These structures were found in large quantities in glycolipid fractions from human adenocarcinoma tumors and human small cell lung carcinoma NCI-H69 cells. In contrast, type 1 chain-based Lea antigen structures were found in both normal mucosa and adenocarcinomas. Analysis of gangliosides of normal colonic mucosa by TLC immunostain indicated the presence of a series of type 2 chain-based gangliosides; however, sialyl-Lex was not detected. The ability of normal colonic mucosa to synthesize type 2 chain core structures was demonstrated by the presence of a beta 1----4 galactosyltransferase activity with Lc3 as an acceptor in an amount equivalent to 60-65% of the total galactosyltransferase activity. An alpha 1----3 fucosyltransferase was also found to be expressed in significant quantity in adult colonic mucosa. Kinetic studies indicated that this is most probably the alpha 1----3/4 fucosyltransferase suggested to be a product of the Lewis gene (Le). Thus, although normal adult colonic mucosa contained the enzymes to synthesize Lex and sialyl-Lex structures, these antigens were not found. Tissue immunofluorescence studies indicated that type 2 chain precursors and the alpha 1----3/4 fucosyltransferase were found in different cell populations in adult proximal colonic mucosa. However, both type 2 chain core structures and their fucosylated derivatives were found to be associated with epithelial cells of fetal colon. These results indicate that oncofetal expression of Lex antigens in fetal colonic epithelium and in adenocarcinomas but not in normal adult mucosa is due to the retrogenetic expression of type 2 chain precursors which are not found in normal adult colonic epithelial cells.     

Comparative study of glycosyltransferase activities in Caco-2 cells before and after enterocytic differentiation using lectin-affinity high-performance liquid chromatography.

Human colonic adenocarcinoma Caco-2 cells differentiate into enterocytes by induction with sodium butyrate after confluence. Our previous studies have shown that there are high levels of H type 1 blood group antigen and core 2 structure present in O-glycans of the glycoproteins from these differentiated cells and these O-glycans appear to be indispensable for the process of differentiation of the cells (J. Amano and M. Oshima, 1999, J. Biol. Chem. 274, 21209-21216). Here, we have determined the glycosyltransferase activities using lectin-affinity HPLC because the method enabled easy separation and identification of mixtures of isomeric oligosaccharide structures due to the high resolution and reproducibility. The activities of beta 3-galactosyltransferase, alpha 2-fucosyltransferase, which are responsible for H type 1 antigen biosynthesis, and core 2 beta 6-N-acetylglucosaminyltransferase in differentiated Caco-2 cells were higher than those in undifferentiated cells. These results demonstrate that an increase in specific glycosyltransferase activities brought on a change of the O-glycan structures during differentiation.     

ST6GalNAc I expression in MDA-MB-231 breast cancer cells greatly modifies their O-glycosylation pattern and enhances their tumourigenicity

Sialyl-Tn is a carbohydrate antigen overexpressed in several epithelial cancers, including breast cancer, and usually associated with poor prognosis. Sialyl-Tn is synthesized by a CMP-Neu5Ac:GalNAcalpha2,6-sialyltransferase: CMP-Neu5Ac: R-GalNAcalpha1-O-Ser/Thr alpha2,6-sialyltransferase (EC 2.4.99.3) (ST6GalNAc I), which transfers a sialic acid residue in alpha2,6-linkage to the GalNAcalpha1-O-Ser/Thr structure. However, established breast cancer cell lines express neither ST6GalNAc I nor sialyl-Tn. We have previously shown that stable transfection of MDA-MB-231, a human breast cancer cell line, with ST6GalNAc I cDNA induces sialyl-Tn antigen (STn) expression. We report here the modifications of the O-glycosylation pattern of a MUC1-related recombinant protein secreted by MDA-MB-231 sialyl-Tn positive cells. We also show that sialyl-Tn expression and concomitant changes in the overall O-glycan profiles induce a decrease of adhesion and an increase of migration of MDA-MB-231. Moreover, STn positive clones exhibit an increased tumour growth in severe combined immunodeficiency (SCID) mice. These observations suggest that modification of the O-glycosylation pattern induced by ST6GalNAc I expression are sufficient to enhance the tumourigenicity of MDA-MB-231 breast cancer cells.     

The pattern of glycosyl- and sulfotransferase activities in cancer cell lines: a predictor of individual cancer-associated distinct carbohydrate structures for the structural identification of signature glycans

Carbohydrate chains of cancer glycoprotein antigens contain major outer changes dictated by tissue-specific regulation of glycosyltransferase genes, the availability of sugar nucleotides, and competition between enzymes for acceptor intermediates during glycan elongation. However, it is evident from recent studies with recombinant mucin probes that the final glycosylation profiles of mucin glycoproteins are mainly determined by the cellular repertoire of glycosyltransferases. Hence, we examined various cancer cell lines for the levels of fucosyl-, beta-galactosyl, beta-N-acetylgalactosaminyl-, sialyl-, and sulfotransferase activities that generate the outer ends of the oligosaccharide chains. We have identified glycosyltransferases activities at the levels that would give rise to O-glycan chains as reported by others in breast cancer cell lines, T47D, ZR75-1, MCF-7, and MDA-MB-231. Most breast cancer cells express Gal-3-O-sulfotransferase specific for T-hapten Gal beta1-->3GalNAc alpha-, whereas the enzyme from colon cancer cells exhibits a vast preference for the Gal beta1,4GlcNAc terminal unit in O-glycans. We also studied ovarian cancer cells SW626 and PA-1 and hepatic cancer cells HepG2. Our studies show that alpha1,2-L-fucosyl-T, alpha(2,3) sialyl-T, and 3-O-Sulfo-T capable of acting on the mucin core 2 tetrasaccharide, Gal beta1,4GlcNAc beta1,6(Gal beta1,3)GalNAc alpha-, can also act on the Globo H antigen backbone, Gal beta1,3GalNAc beta1,3Gal alpha-, suggesting the existence of unique carbohydrate moieties in certain cancer-associated glycolipids. Briefly, our study indicates the following: (i) 3'-Sulfo-T-hapten has an apparent relationship to the tumorigenic potential of breast cancer cells; (ii) the 3'-sulfo Lewis(x), the 3-O-sulfo-Globo unit, and the 3-fucosylchitobiose core could be uniquely associated with colon cancer cells; (iii) synthesis of a polylactosamine chain and T-hapten are favorable in ovarian cancer cells due to negligible sialyltransferase activities; and (iv) a 6'-sialyl LacNAc unit and 3'-sialyl T-hapten appear to be prevalent structures in hepatic cancer cell glycans. Thus, it is apparent that different cancer cells are expressing unique glycan epitopes, which could be novel targets for cancer diagnosis and treatment.     

Aberrant O-glycosylation inhibits stable expression of dysadherin,a carcinoma-associated antigen,and facilitates cell-cell adhesion

Recently, we identified dysadherin, a novel carcinoma-associated glycoprotein, and showed that overexpression of dysadherin in human hepatocarcinoma PLC/PRF/5 cells could suppress E-cadherin-mediated cell-cell adhesion and promote tumor metastasis. The present study shows evidence that dysadherin is actually O-glycosylated. This was based on a direct carbohydrate composition analysis of a chimera protein of an extracellular domain of dysadherin fused to an Fc fragment of immunoglobulin. To assess the importance of O-glycosylation in dysadherin function, dysadherin-transfected hepatocarcinoma cells were cultured in a medium containing benzyl-alpha-GalNAc, a modulator of O-glycosylation. This treatment facilitated homotypic cell adhesion among dysadherin transfectants accompanied with morphological changes, indicating that the anti-adhesive effect of dysadherin was weakened. Modification of O-glycan synthesis also resulted in down-regulation of dysadherin expression and up-regulation of E-cadherin expression in dysadherin transfectants but did not affect E-cadherin expression in mock transfectants. Structural analysis of O-glycans released from the dysadherin chimera proteins indicated that a series of O-glycans with core 1 and 2 structures are attached to dysadherin, and their sialylation is remarkably inhibited by benzyl-alpha-GalNAc treatment. However, sialidase treatment of the cells did not affect calcium-dependent cell aggregation, which excluded the possibility that sialic acid itself is directly involved in cell-cell adhesion. We suggest that aberrant O-glycosylation in carcinoma cells inhibits stable expression of dysadherin and leads to the up-regulation of E-cadherin expression by an unknown mechanism, resulting in increased cell-cell adhesion. The carbohydrate-directed approach to the regulation of dysadherin expression might be a new strategy for cancer therapy.     

Acceptor specificities and selective inhibition of recombinant human Gal- and GlcNAc-transferases that synthesize core structures 1, 2, 3 and 4 of O-glycans

Background

Modifications of proteins by O-glycosylation determine many of the properties and functions of proteins. We wish to understand the mechanisms of O-glycosylation and develop inhibitors that could affect glycoprotein functions and alter cellular behavior.

Methods

We expressed recombinant soluble human Gal- and GlcNAc-transferases that synthesize the O-glycan cores 1 to 4 and are critical for the overall structures of O-glycans. We determined the properties and substrate specificities of these enzymes using synthetic acceptor substrate analogs. Compounds that were inactive as substrates were tested as inhibitors.

Results

Enzymes significantly differed in their recognition of the sugar moieties and aglycone groups of substrates. Core 1 synthase was active with glycopeptide substrates but GlcNAc-transferases preferred substrates with hydrophobic aglycone groups. Chemical modifications of the acceptors shed light on enzyme–substrate interactions. Core 1 synthase was weakly inhibited by its substrate analog benzyl 2-butanamido-2-deoxy-α-d-galactoside while two of the three GlcNAc-transferases were selectively and potently inhibited by bis-imidazolium salts which are not substrate analogs.

Conclusions

This work delineates the distinct specificities and properties of the enzymes that synthesize the common O-glycan core structures 1 to 4. New inhibitors were found that could selectively inhibit the synthesis of cores 1, 2 and 3 but not core 4.

General significance

These studies help our understanding of the mechanisms of action of enzymes critical for O-glycosylation. The results may be useful for the re-engineering of O-glycosylation to determine the roles of O-glycans and the enzymes critical for O-glycosylation, and for biotechnology with potential therapeutic applications.     

Sialyl-Lewis(x) on P-selectin glycoprotein ligand-1 is regulated during differentiation and maturation of dendritic cells: a mechanism involving the glycosyltransferases C2GnT1 and ST3Gal I

To fulfil their function as APCs, dendritic cells (DC) and their precursors need to travel from blood to the peripheral tissues and, upon activation, migrate from tissues to draining lymph nodes. Because O-glycans play a role in T cell trafficking, we investigated the O-glycosylation profile of human monocyte-derived DC. Sialyl-Lewis(x) (sLe(x)), a glycan involved in extravasation via selectin binding, was found to be expressed exclusively on P-selectin glycoprotein ligand-1 in monocytes and immature DC. However, sLe(x) was lost from mature DC even though these cells retained expression of P-selectin glycoprotein ligand-1. Maturation of DC led to a rapid change in the expression of glycosyltransferases involved in O-linked glycosylation. A down-regulation of C2GnT1 mRNA and enzymatic activity was observed with a concurrent up-regulation of ST3Gal I and ST6GalNAc II mRNA resulting in a loss of the core 2 structures required for sLe(x) expression as a P-selectin ligand. Interestingly, the early regulation of these glycosyltransferases was mediated by PGE(2), which is known to be required for human DC migration. The pattern of O-glycosylation seen in mature cells was very similar to that expressed by naive T cells, which home to lymph nodes. Our data show that the regulation of O-glycosylation controls sLe(x) expression, and also suggest that O-glycans may have a function in DC migration.     

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.     

X-ray crystal structure of leukocyte type core 2 beta1,6-N-acetylglucosaminyltransferase. Evidence for a convergence of metal ion-independent glycosyltransferase mechanism

Leukocyte type core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT-L) is a key enzyme in the biosynthesis of branched O-glycans. It is an inverting, metal ion-independent family 14 glycosyltransferase that catalyzes the formation of the core 2 O-glycan (Galbeta1-3[GlcNAcbeta1-6]GalNAc-O-Ser/Thr) from its donor and acceptor substrates, UDP-GlcNAc and the core 1 O-glycan (Galbeta1-3GalNAc-O-Ser/Thr), respectively. Reported here are the x-ray crystal structures of murine C2GnT-L in the absence and presence of the acceptor substrate Galbeta1-3GalNAc at 2.0 and 2.7A resolution, respectively. C2GnT-L was found to possess the GT-A fold; however, it lacks the characteristic metal ion binding DXD motif. The Galbeta1-3GalNAc complex defines the determinants of acceptor substrate binding and shows that Glu-320 corresponds to the structurally conserved catalytic base found in other inverting GT-A fold glycosyltransferases. Comparison of the C2GnT-L structure with that of other GT-A fold glycosyltransferases further suggests that Arg-378 and Lys-401 serve to electrostatically stabilize the nucleoside diphosphate leaving group, a role normally played by metal ion in GT-A structures. The use of basic amino acid side chains in this way is strikingly similar to that seen in a number of metal ion-independent GT-B fold glycosyltransferases and suggests a convergence of catalytic mechanism shared by both GT-A and GT-B fold glycosyltransferases.     

Recombinant MUC1 probe authentically reflects cell-specific O-glycosylation profiles of endogenous breast cancer mucin. High density and prevalent core 2-based glycosylation

Knowledge about the O-linked glycan chains of tumor-associated MUC1 is primarily based on enzymatic and immunochemical evidence. To obtain structural information and to overcome limitations by the scarcity of endogenous mucin, we expressed a recombinant glycosylation probe corresponding to six MUC1 tandem repeats in four breast cancer cell lines. Comparative analyses of the O-glycan profiles were performed after hydrazinolysis and normal phase chromatography of 2-aminobenzamide-labeled glycans. Except for a general reduction in the O-glycan chain lengths and a high density glycosylation, no common structural pattern was revealed. T47D fusion protein exhibits an almost complete shift from core 2 to core 1 expression with a preponderance of sialylated glycans. By contrast, MCF-7, MDA-MB231, and ZR75-1 cells glycosylate the MUC1 repeat peptide preferentially with core 2-based glycans terminating mostly with alpha 3-linked sialic acid (MDA-MB231, ZR75-1) or alpha 2/3-linked fucose (MCF-7). Endogenous MUC1 from T47D and MCF-7 cell supernatants revealed almost identical O-glycosylation profiles compared with the respective recombinant probes, indicating that the fusion proteins reflected the authentic O-glycan profiles of the cells. The structural patterns in the majority of cells under study are in conflict with biosynthetic models of MUC1 O-glycosylation in breast cancer, which claim that the truncation of normal core 2-based polylactosamine structures to short sialylated core 1-based glycans is due to the reduced activity of core 2-forming beta 6-N-acetylglucosaminyltransferases and/or to overexpression of competitive alpha 3- sialyltransferase.