Cellular Glycans Research Articles

Glycomics Profiling of Chinese Hamster Ovary Cell Glycosylation Mutants Reveals N-Glycans of a Novel Size and Complexity

Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z ∼13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Galβ1–4GlcNAc)n units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewisx and sialyl-Lewisx determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.

Exploring genomes for glycosyltransferases

Glycosyltransferases are one of the largest and most diverse enzyme groups in Nature. They catalyse the synthesis of glycosidic linkages by the transfer of a sugar residue from a donor to an acceptor substrate. These enzymes have been classified into families on the basis of amino acid sequence similarity that are kept updated in the Carbohydrate Active enZyme database (CAZy, ). The repertoire of glycosyltransferases in genomes is believed to determine the diversity of cellular glycan structures, and current estimates suggest that for most genomes about 1% of the coding regions are glycosyltransferases. However, plants tend to have far more glycosyltransferase genes than any other organism sequenced to date, and this can be explained by the highly complex polysaccharide network that form the cell wall and also by the numerous glycosylated secondary metabolites. In recent years, various bioinformatics strategies have been used to search bacterial and plant genomes for new glycosyltransferase genes. These are based on the use of remote homology detection methods that act at the 1D, 2D, and 3D level. The combined use of methods such as profile Hidden Markov Model (HMM) and fold recognition appears to be appropriate for this class of enzyme. Chemometric tools are also particularly well suited for obtaining an overview of multivariate data and revealing hidden latent information when dealing with large and highly complex datasets.

Centralized Modularity of N-Linked Glycosylation Pathways in Mammalian Cells

Glycosylation is a highly complex process to produce a diverse repertoire of cellular glycans that are attached to proteins and lipids. Glycans are involved in fundamental biological processes, including protein folding and clearance, cell proliferation and apoptosis, development, immune responses, and pathogenesis. One of the major types of glycans, N-linked glycans, is formed by sequential attachments of monosaccharides to proteins by a limited number of enzymes. Many of these enzymes can accept multiple N-linked glycans as substrates, thereby generating a large number of glycan intermediates and their intermingled pathways. Motivated by the quantitative methods developed in complex network research, we investigated the large-scale organization of such N-linked glycosylation pathways in mammalian cells. The N-linked glycosylation pathways are extremely modular, and are composed of cohesive topological modules that directly branch from a common upstream pathway of glycan synthesis. This unique structural property allows the glycan production between modules to be controlled by the upstream region. Although the enzymes act on multiple glycan substrates, indicating cross-talk between modules, the impact of the cross-talk on the module-specific enhancement of glycan synthesis may be confined within a moderate range by transcription-level control. The findings of the present study provide experimentally-testable predictions for glycosylation processes, and may be applicable to therapeutic glycoprotein engineering.

Carbamate-Linked Lactose: Design of Clusters and Evidence for Selectivity to Block Binding of Human Lectins to (Neo)Glycoproteins with Increasing Degree of Branching and to Tumor Cells

Various pathogenic processes are driven by protein(lectin)-glycan interactions, especially involving beta-galactosides at branch ends of cellular glycans. These emerging insights fuel the interest to design potent inhibitors to block lectins. As a step toward this aim, we prepared a series of ten mono- to tetravalent glycocompounds with lactose as a common headgroup. To obtain activated carbonate for ensuing carbamate formation, conditions for the facile synthesis of pure isomers from anomerically unprotected lactose were identified. To probe for the often encountered intrafamily diversity of human lectins, we selected representative members from the three subgroups of adhesion/growth-regulatory galectins as receptors. Diversity of the glycan display was accounted for by using four (neo)glycoproteins with different degrees of glycan branching as matrices in solid-phase assays. Cases of increased inhibitory potency of lactose clusters compared to free lactose were revealed. Extent of relative inhibition was not directly associated with valency in the glycocompound and depended on the lectin type. Of note for screening protocols, efficacy of blocking appeared to decrease with increased degree of glycan branching in matrix glycoproteins. Binding to tumor cells was impaired with selectivity for galectins-3 and -4. Representative compounds did not impair growth of carcinoma cells up to a concentration of 5 mM of lactose moieties (valence-corrected value) per assay. The reported bioactivity and the delineation of its modulation by structural parameters of lectins and glycans set instructive examples for the further design of selective inhibitors and assay procedures.

Metabolic glycoengineering: Sialic acid and beyond

This report provides a perspective on metabolic glycoengineering methodology developed over the past two decades that allows natural sialic acids to be replaced with chemical variants in living cells and animals. Examples are given demonstrating how this technology provides the glycoscientist with chemical tools that are beginning to reproduce Mother Nature's control over complex biological systems - such as the human brain - through subtle modifications in sialic acid chemistry. Several metabolic substrates (e.g., ManNAc, Neu5Ac, and CMP-Neu5Ac analogs) can be used to feed flux into the sialic acid biosynthetic pathway resulting in numerous - and sometime quite unexpected - biological repercussions upon nonnatural sialoside display in cellular glycans. Once on the cell surface, ketone-, azide-, thiol-, or alkyne-modified glycans can be transformed with numerous ligands via bioorthogonal chemoselective ligation reactions, greatly increasing the versatility and potential application of this technology. Recently, sialic acid glycoengineering methodology has been extended to other pathways with analog incorporation now possible in surface-displayed GalNAc and fucose residues as well as nucleocytoplasmic O-GlcNAc-modified proteins. Finally, recent efforts to increase the "druggability" of sugar analogs used in metabolic glycoengineering, which have resulted in unanticipated "scaffold-dependent" activities, are summarized.

Chemical Approaches To Perturb, Profile, and Perceive Glycans

Glycosylation is an essential form of post-translational modification that regulates intracellular and extracellular processes. Regrettably, conventional biochemical and genetic methods often fall short for the study of glycans, because their structures are often not precisely defined at the genetic level. To address this deficiency, chemists have developed technologies to perturb glycan biosynthesis, profile their presentation at the systems level, and perceive their spatial distribution. These tools have identified potential disease biomarkers and ways to monitor dynamic changes to the glycome in living organisms. Still, glycosylation remains the underexplored frontier of many biological systems. In this Account, we focus on research in our laboratory that seeks to transform the study of glycan function from a challenge to routine practice.In studies of proteins and nucleic acids, functional studies have often relied on genetic manipulations to perturb structure. Though not directly subject to mutation, we can determine glycan structure−function relationships by synthesizing defined glycoconjugates or by altering natural glycosylation pathways. Chemical syntheses of uniform glycoproteins and polymeric glycoprotein mimics have facilitated the study of individual glycoconjugates in the absence of glycan microheterogeneity. Alternatively, selective inhibition or activation of glycosyltransferases or glycosidases can define the biological roles of the corresponding glycans. Investigators have developed tools including small molecule inhibitors, decoy substrates, and engineered proteins to modify cellular glycans. Current approaches offer a precision approaching that of genetic control.Genomic and proteomic profiling form a basis for biological discovery. Glycans also present a rich matrix of information that adapts rapidly to changing environs. Glycomic and glycoproteomic analyses via microarrays and mass spectrometry are beginning to characterize alterations in glycans that correlate with disease. These approaches have already identified several cancer biomarkers. Metabolic labeling can identify recently synthesized glycans and thus directly track glycan dynamics. This approach can highlight changes in physiology or environment and may be more informative than steady-state analyses. Together, glycomic and metabolic labeling techniques provide a comprehensive description of glycosylation as a foundation for hypothesis generation.Direct visualization of proteins via the green fluorescent protein (GFP) and its congeners has revolutionized the field of protein dynamics. Similarly, the ability to perceive the spatial organization of glycans could transform our understanding of their role in development, infection, and disease progression. Fluorescent tagging in cultured cells and developing organisms has revealed important insights into the dynamics of these structures during growth and development. These results have highlighted the need for additional imaging probes.

Structural basis of Clostridium perfringens toxin complex formation

The virulent properties of the common human and livestock pathogen Clostridium perfringens are attributable to a formidable battery of toxins. Among these are a number of large and highly modular carbohydrate-active enzymes, including the mu-toxin and sialidases, whose catalytic properties are consistent with degradation of the mucosal layer of the human gut, glycosaminoglycans, and other cellular glycans found throughout the body. The conservation of noncatalytic ancillary modules among these enzymes suggests they make significant contributions to the overall functionality of the toxins. Here, we describe the structural basis of an ultra-tight interaction (K(a) = 1.44 x 10(11) M(-1)) between the X82 and dockerin modules, which are found throughout numerous C. perfringens carbohydrate-active enzymes. Extensive hydrogen-bonding and van der Waals contacts between the X82 and dockerin modules give rise to the observed high affinity. The mu-toxin dockerin module in this complex is positioned approximately 180 degrees relative to the orientation of the dockerin modules on the cohesin module surface within cellulolytic complexes. These observations represent a unique property of these clostridial toxins whereby they can associate into large, noncovalent multitoxin complexes that allow potentiation of the activities of the individual toxins by combining complementary toxin specificities.

Probing Glycans With the Copper(I)‐Catalyzed [3+2] Azide–Alkyne Cycloaddition

AbstractThe imposing structural complexity and heterogeneity of glycans has made the study of their precise biological roles challenging. However, recently developed synthetic and screening techniques have enabled the study of many aspects of glycan activity that were previously inaccessible. Among these new methods, the Copper(I)‐catalyzed Azide–Alkyne [3+2] Cycloaddition (CuAAC) has provided a means to mimic multivalent glycan display, manipulate cellular glycans in vivo for visualization and isolation, and generate useful inhibitors of glycoprotein processing.

Metabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligation

Metabolic labeling of glycans with a bioorthogonal chemical reporter such as the azide enables their visualization in cells and organisms as well as the enrichment of specific glycoprotein types for proteomic analysis. This process involves two steps. Azido sugars are fed to cells or organisms and integrated by the glycan biosynthetic machinery into various glycoconjugates. The azido sugars are then covalently tagged with imaging probes or epitope tags, either ex vivo or in vivo, using an azide-specific reaction. This protocol details the syntheses of the azido sugars N-azidoacetylmannosamine (ManNAz), N-azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine (GlcNAz) and 6-azidofucose (6AzFuc), and the detection reagents phosphine-FLAG and phosphine-FLAG-His6. Applications to the visualization of cellular glycans and enrichment of glycoproteins for proteomic analysis are described. The synthesis of the azido sugars (ManNAz, GalNAz, GlcNAz or 6AzFuc) or detection reagents (phosphine-FLAG or phosphine-FLAG-His6) can be completed in approximately 1 week. A cell metabolic labeling experiment can be completed in approximately 4 d.

Three-dimensional Structure of a Putative Non-cellulosomal Cohesin Module from a Clostridium perfringens Family 84 Glycoside Hydrolase

The genomes of myonecrotic strains of Clostridium perfringens encode a large number of secreted glycoside hydrolases. The activities of these enzymes are consistent with degradation of the mucosal layer of the human gastrointestinal tract, glycosaminoglycans and other cellular glycans found throughout the body. In many cases this is thought to aid in the propagation of the major toxins produced by C. perfringens. One such example is the family 84 glycoside hydrolases, which contains five C. perfringens members ( CpGH84A–E), each displaying a unique modular architecture. The smallest and most extensively studied member, CpGH84C, comprises an N-terminal catalytic domain with β- N-acetylglucosaminidase activity, a family 32 carbohydrate-binding module, a family 82 X-module (X82) of unknown function, and a fibronectin type-III-like module. Here we present the structure of the X82 module from CpGH84C, determined by both NMR spectroscopy and X-ray crystallography. CpGH84C X82 adopts a jell-roll fold comprising two β-sheets formed by nine β-strands. CpGH84C X82 displays distant amino acid sequence identity yet close structural similarity to the cohesin modules of cellulolytic anaerobic bacteria. Cohesin modules are responsible for the assembly of numerous hydrolytic enzymes in a cellulose-degrading multi-enzyme complex, termed the cellulosome, through a high-affinity interaction with the calcium-binding dockerin module. A planar surface is located on the face of the CpGH84 X82 structure that corresponds to the dockerin-binding region of cellulolytic cohesin modules and has the approximate dimensions to accommodate a dockerin module. The presence of cohesin-like X82 modules in glycoside hydrolases of C. perfringens is an indication that the formation of novel X82-dockerin mediated multi-enzyme complexes, with potential roles in pathogenesis, is possible.

A novel function of VCP (valosin-containing protein; p97) in the control of N-glycosylation of proteins in the endoplasmic reticulum

α-Chain of T-cell receptor (TCR) is a typical ERAD (ER-associated degradation) substrate degraded in the absence of other TCR subunits. Depletion of derlin 1 fails to induce accumulation of αTCR despite inducing accumulation of α1-antitrypsin, another ERAD substrate. Furthermore, while depletion of VCP does not affect levels of α1-antitrypsin, it induces an increase in levels of αTCR. RNAi of VCP induces preferential accumulation of αTCR with less mannose residues, suggesting its retention within the ER. Mass spectrometric analysis of cellular N-linked glycans revealed that depletion of VCP decreases the level of high-mannose glycoproteins, increases the levels of truncated low-mannose glycoproteins and induces changes in the abundance of complex glycans assembled in post-ER compartments. Since proteasome inhibition was unable to mimic those changes, they cannot be regarded as a simple consequence of inhibited ERAD but represent a complex effect of VCP on the function of the ER.

Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells

Developing tools for investigating the cellular activity of glycans will help to delineate the molecular basis for aberrant glycosylation in pathological processes such as cancer. Metabolic oligosaccharide engineering, which inserts sugar-reporting groups into cellular glycoconjugates, represents a powerful method for imaging the localization, trafficking, and dynamics of glycans and isolating them for glyco-proteomic analysis. Herein, we show that the alkyne-reporting group can be incorporated into cellular glycans. The alkyne group is a small, inert, bio-orthogonal handle that can be chemoselectively labeled by using the Cu(I) catalyzed [3 + 2] azide-alkyne cycloaddition, or click chemistry. Alkynyl sugar monomers, based on fucose (Fuc) and N-acetylmannosamine (ManNAc), were incorporated into fucosylated and sialylated glycans in several cancer cell lines, allowing for cell surface and intracellular visualization of glycoconjugates, as well as, observation of alkyne-bearing glycoproteins. Similarly to our previous results with an azido Fuc/alkynyl probe system, we demonstrated that click-activated fluorogenic probes are practical tools for efficiently and selectively labeling alkynyl-modified glycans. Because Fuc and sialic acid are terminal glycan residues with a notably increased presence in many tumors, we hope that our method will provide useful information about their roles in cancer and ultimately can be used for diagnostic and therapeutic purposes.

Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo.

Glycomics is emerging as a new field for the biology of complex glycoproteins and glycoconjugates. The lack of versatile glycan-labeling methods has presented a major obstacle to visualizing at the cellular level and studying glycoconjugates. To address this issue, we developed a fluorescent labeling technique based on the Cu(I)-catalyzed [3 + 2] cycloaddition, or click chemistry, which allows rapid, versatile, and specific covalent labeling of cellular glycans bearing azide groups. The method entails generating a fluorescent probe from a nonfluorescent precursor, 4-ethynyl-N-ethyl-1,8-naphthalimide, by clicking the fluorescent trigger, the alkyne at the 4 position, with an azido-modified sugar. Using this click-activated fluorescent probe, we demonstrate incorporation of an azido-containing fucose analog into glycoproteins via the fucose salvage pathway. Distinct fluorescent signals were observed by flow cytometry when cells treated with 6-azidofucose were labeled with the click-activated fluorogenic probe or biotinylated alkyne. The intracellular localization of fucosylated glycoconjugates was visualized by using fluorescence microscopy. This technique will allow dynamic imaging of cellular fucosylation and facilitate studies of fucosylated glycoproteins and glycolipids.

Chemical remodelling of cell surfaces in living animals.

Cell surfaces are endowed with biological functionality designed to mediate extracellular communication. The cell-surface repertoire can be expanded to include abiotic functionality through the biosynthetic introduction of unnatural sugars into cellular glycans, a process termed metabolic oligosaccharide engineering. This technique has been exploited in fundamental studies of glycan-dependent cell-cell and virus-cell interactions and also provides an avenue for the chemical remodelling of living cells. Unique chemical functional groups can be delivered to cell-surface glycans by metabolism of the corresponding unnatural precursor sugars. These functional groups can then undergo covalent reaction with exogenous agents bearing complementary functionality. The exquisite chemical selectivity required of this process is supplied by the Staudinger ligation of azides and phosphines, a reaction that has been performed on cultured cells without detriment to their physiology. Here we demonstrate that the Staudinger ligation can be executed in living animals, enabling the chemical modification of cells within their native environment. The ability to tag cell-surface glycans in vivo may enable therapeutic targeting and non-invasive imaging of changes in glycosylation during disease progression.

Expanding the diversity of unnatural cell-surface sialic acids.

Novel chemical reactivity can be introduced onto cell surfaces through metabolic oligosaccharide engineering. This technique exploits the substrate promiscuity of cellular biosynthetic enzymes to deliver unnatural monosaccharides bearing bioorthogonal functional groups into cellular glycans. For example, derivatives of N-acetylmannosamine (ManNAc) are converted by the cellular biosynthetic machinery into the corresponding sialic acids and subsequently delivered to the cell surface in the form of sialoglycoconjugates. Analogs of N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) are also metabolized and incorporated into cell surface glycans, likely through the sialic acid and GalNAc salvage pathways, respectively. Furthermore, GlcNAc analogs can be incorporated into nucleocytoplasmic proteins in place of {beta}-O-GlcNAc residues. These pathways have been exploited to integrate unique electrophiles such as ketones and azides into the target glycoconjugate class. These functional groups can be further elaborated in a chemoselective fashion by condensation with hydrazides and by Staudinger ligation, respectively, thereby introducing detectable probes onto the cell. In conclusion, sialic acid derivatives are efficient vehicles for delivery of bulky functional groups to cell surfaces and masking of their hydroxyl groups improves their cellular uptake and utilization. Furthermore, the successful introduction of photoactivatable aryl azides into cell surface glycans opens up new avenues for studying sialic acid-binding proteins and elucidating the role of sialic acid in essential processes such as signaling and cell adhesion.

Two distinct size classes of immature and mature subviral particles from tick-borne encephalitis virus.

Flaviviruses assemble in the endoplasmic reticulum by a mechanism that appears to be driven by lateral interactions between heterodimers of the envelope glycoproteins E and prM. Immature intracellular virus particles are then transported through the secretory pathway and converted to their mature form by cleavage of the prM protein by the cellular protease furin. Earlier studies showed that when the prM and E proteins of tick-borne encephalitis virus are expressed together in mammalian cells, they assemble into membrane-containing, icosahedrally symmetrical recombinant subviral particles (RSPs), which are smaller than whole virions but retain functional properties and undergo cleavage maturation, yielding a mature form in which the E proteins are arranged in a regular T = 1 icosahedral lattice. In this study, we generated immature subviral particles by mutation of the furin recognition site in prM. The mutation resulted in the secretion of two distinct size classes of particles that could be separated by sucrose gradient centrifugation. Electron microscopy showed that the smaller particles were approximately the same size as the previously described mature RSPs, whereas the larger particles were approximately the same size as the virus. Particles of the larger size class were also detected with a wild-type construct that allowed prM cleavage, although in this case the smaller size class was far more prevalent. Subtle differences in endoglycosidase sensitivity patterns suggested that, in contrast to the small particles, the E glycoproteins in the large subviral particles and whole virions might be in nonequivalent structural environments during intracellular transport, with a portion of them inaccessible to cellular glycan processing enzymes. These proteins thus appear to have the intrinsic ability to form alternative assembly products that could provide important clues about the role of lateral envelope protein interactions in flavivirus assembly.

Expression of galectin-3-reactive ligands in squamous cancer and normal epithelial cells as a marker of differentiation

The definition of biological markers for oropharynx and larynx cancer is essential to predict their clinical behavior. Since cellular glycans play an important role in biological information transfer, we have employed an endogenous lectin, galectin-3, to examine in primary squamous carcinomas, lymph node metastases, and physiological squamous epithelia whether glycans recognized by this lectin are altered in relation to the state of differentiation. The expression of galectin-3 was concomitantly evaluated by immunohistochemistry using the A1D6 monoclonal antibody. In addition, other antibodies were used for the detection of cytokeratins and desmosomal proteins (desmoplakin-1 and desmoglein). The results show the expression of galectin-3-reactive ligands in moderately/highly differentiated carcinomas only in areas exhibiting a high level of keratinization. Except for one patient out of 14, metastatic cells in lymph nodes expressed no accessible binding sites for galectin-3. No galectin-3-reactivity was detected in the basal cell layer of all studied normal epithelia (which contains the proliferating cells). The suprabasal layers were positive in epidermis and epithelium of tongue and cornea and negative in epithelium of palatine tonsil. The tumor cells expressed galectin-3 with an intensity positively correlated with tumor differentiation. The position of galectin-3-reactive sites colocalized with the two tested desmosomal proteins. However, presence of these proteins was also detected in areas of tumor and suprabasal layers of tonsil epithelium where no binding reactivity for galectin-3 was found. The present study showed that expression of galectin-3-reactive glycoligands is differentiation-dependent in normal as well as malignant squamous cells. Colocalization of galectin-3-reactive sites with desmosomal proteins (desmoplakin-1 and desmoglein) suggests an association of the galectin-3 ligand(s) with the cell surface, pointing to a potential participation of galectin-3 in mediation of intercellular contacts in these tumor types.

Phorbol ester treatment of HL 60 leukemia cells results in increase of β-(1→4)-galactosyltransferase

We previously showed that HL 60 leukemia cells exhibit various changes in their cellular glycans after phorbol 12-myristate 13-acetate (PMA) treatment. These changes could originate largely from changes in one or several glycosyltransferases. In this report, we show using enzymatic measures, fluorescence microscopy, immunoblotting and Northern blot that β-(1→4)-galactosyltransferase I (GalT I) activity was higher (>×2) in PMA-treated compared with untreated HL 60 cells. Immunoblotting showed an increased intensity of the GalT I band at 49 kDa and Northern blot a weak increase of the GalT I transcript band, after PMA treatment. Moreover, Northern blot performed after actinomycin-D treatment of the cells, which inhibits transcription, suggests that the observed increase of GalT I expression could originate, in part, from increase of the stability of GalT I transcripts.

Tumor cell surface beta 1-4-linked galactose binds to lectin(s) on microvascular endothelial cells and contributes to organ colonization.

Cell surface carbohydrate structures acting as ligands for tissue specific mammalian lectins have been implicated in cell-cell interactions during embryogenesis, lymphocyte homing, and tumor cell metastasis. In this report, we provide evidence that beta 1-4 linked galactose (Gal) residues in N-linked oligosaccharides on the surface of blood born tumor cells serve as a ligand for binding to microvascular endothelial cells. D36W25, a class 1 glycosylation mutant of the MDAY-D2 lymphoreticular tumor cell line, lacks sialic acid and Gal in cellular glycans due to a defect in the Golgi UDP-Gal transporter. Using UDP-Gal and bovine galactosyltransferase in vitro, beta 1-4 Gal was restored to the surface of the cells and 70% of the galactosylated glycans persisted for 8 h in vitro at 37 degrees C. Compared to mock-treated D36W25 cells, galactosylated D36W25 cells showed an 80% increase in binding to microvascular endothelial cell monolayers in vitro. The enhanced binding of galactosylated D36W25 cells to endothelial cell was inhibited by the addition of lactosamine-conjugated albumin to the assay. Consistent with these observations, swainsonine and castinospermine, two inhibitors of N-linked processing that result in loss of lactosamine antennae inhibited the binding of wild-type MDAY-D2 cells to endothelial cells in vitro. Injection of radiolabeled tumor cells into the circulation of syngeneic mice, showed that galactosylation of D36W25 cells resulted in 2-3 more tumor cells retained in the lungs and livers. In addition, galactosylation of D36W25 cells increased by 30-fold the number of visible liver metastases on inspection 4 wk after tumor cell injection. These results suggest that beta 1-4Gal-binding lectins on microvascular endothelial cells can contribute to retention and secondary tumor formation of blood born tumor cells. With the increasing availability of purified glycosyltransferases, reconstruction of a variety of carbohydrate sequences on the surface of class 1 mutants provides a controlled means of studying carbohydrate-lectin interactions on viable cells.

Cellular glycans alterations accompanying malignant phenotype in human uroepithelial cells

Cellular glycans alterations accompanying malignant phenotype in human uroepithelial cells