Chemistry-driven glycoscience

Carbohydrates are the most prominent features of the cell’s exterior—they are the cell’s “face” and serve as the cell’s identification card. The features of cell surface glycans (e.g. glycoproteins, glycolipids, polysaccharides) can be read by proteins, other cells, or organisms. In all of these contexts, glycan-binding proteins typically recognize (“read”) glycan identity. This recognition mediates important host-microbe interactions, as well as critical physiological functions, including fertilization, development, and immune system function. This article focuses on how proteins recognize glycans with an emphasis on three objectives: 1) to understand the molecular basis for carbohydrate recognition, 2) to implement that understanding to develop functional probes of protein-carbohydrate interactions, and 3) to apply those probes to elucidate and exploit the physiological consequences of protein–carbohydrate interactions. In this context, our group has focused on two key aspects of carbohydrate recognition: CH-π and multivalent interactions. We are applying the foundational knowledge gained from our studies for purposes ranging from illuminating host-microbe interactions to probing immune system function.     

A novel approach for the topographical localization of glycolipids on the cell surface

In this study we have developed a prototype system for distinguishing between the topographical distribution of glycolipids versus glycoproteins on the ultrastructural level. Direct modification of membrane-based sialic acids with biotin groups labels both glycolipids and glycoproteins. In this case, subsequent ultrastructural localization of biotinylated sites would not discern between these two classes of glycoconjugate in an unambiguous manner. When biotinylated cells are fixed prior to interaction with ferritin-conjugated avidin, the mean distance of marker molecules from the membrane bilayer is 8.0 nm. In contrast, if the cells are allowed to cap through the action of ferritin-avidin conjugates on unfixed cells, the average distance (13.0 nm) of the marker molecules appears even more distant from the membrane on the capped portion of the cell (uropod), whereas those on the head region are positioned in close proximity to the bilayer (3.7 nm). In order to exclusively label cell surface glycolipids on the ultrastructural level, bovine brain gangliosides were biotinylated in vitro and the haptenized gangliosides were incorporated into intact cells. In this case, marker molecules denoting the incorporated gangliosides were found in relatively close juxtaposition to the membrane surface, in a manner strikingly similar to the labeling pattern of the head region on capped cells. These results support the concept that, in the native state, the carbohydrate portion of glycolipids is positioned closer to the membrane bilayer than that of glycoproteins.     

Sialic acids in T cell development and function

Virtually all cell surface proteins and many cell membrane lipids are glycosylated, creating a cell surface glycocalyx. The glycan chains attached to cell surface glycoproteins and glycolipids are complex structures with specific additions that determine functions of the glycans in cell–cell communication and cell sensing of the environment. One type of specific modification of cell surface glycans is decoration of glycan termini by sialic acids. On T cells, these terminal sialic acid residues are involved in almost every aspect of T cell fate and function, from cell maturation, differentiation, and migration to cell survival and cell death. The roles that sialylated glycans play in T cell development and function, including binding to specific sialic acid-binding lectins, are reviewed here.     

Glycan antagonists and inhibitors: a fount for drug discovery

Glycans, the carbohydrate chains of glycoproteins, proteoglycans, and glycolipids, represent a relatively unexploited area for drug development compared with other macromolecules. This review describes the major classes of glycans synthesized by animal cells, their mode of assembly, and available inhibitors for blocking their biosynthesis and function. Many of these agents have proven useful for studying the biological activities of glycans in isolated cells, during embryological development, and in physiology. Some are being used to develop drugs for treating metabolic disorders, cancer, and infection, suggesting that glycans are excellent targets for future drug development.     

The expression and functions of glycoconjugates in neural stem cells

The mammalian central nervous system is organized by a variety of cells such as neurons and glial cells. These cells are generated from a common progenitor, the neural stem cell (NSC). NSCs are defined as undifferentiated neural cells that are characterized by their high proliferative potential while retaining the capacity for self-renewal and multipotency. Glycoconjugates carrying carbohydrate antigens, including glycoproteins, glycolipids, and proteoglycans, are primarily localized on the plasma-membrane surface of cells and serve as excellent biomarkers at various stages of cellular differentiation. Moreover, they also play important functional roles in determining cell fate such as self-renewal, proliferation, and differentiation. In the present review, we discuss the expression pattern and possible functions of glycoconjugates and carbohydrate antigens in NSCs, with an emphasis on stage-specific embryonic antigen-1, human natural killer antigen-1, polysialic acid-neural cell-adhesion molecule, prominin-1, gp130, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, cystatin C, galectin-1, glycolipids, and Notch.     

High-molecular-weight glycoproteins are the major carriers of the carbohydrate differentiation antigens I, i and SSEA-1 of mouse teratocarcinoma cells.

The carriers of the carbohydrate differentiation antigens I, i and SSEA-1 were investigated in embryonal carcinoma cell lines of mouse and differentiated cell lines derived from them. Glycoproteins were studied by immunostaining ('Western blotting') of total cell lysates and immunoprecipitation from lysates of galactose oxidase/NaB3H4-labelled cells; glycolipids were investigated by immunostaining of thin layer chromatograms. The antigenic activities detected by immunofluorescence of cell smears were reflected in the antigenicities of high-molecular-weight glycoproteins. These were polydisperse and markedly susceptible to digestion with endo-beta-galactosidase. Only the I antigen was detected on minor glycolipids. These observations indicate that glycoproteins rather than glycolipids are the major carriers of carbohydrate differentiation antigens I, i and SSEA-1 in the teratocarcinoma cell lines.     

Antibodies against tumor cell glycolipids and proteins, but not mucins, mediate complement-dependent cytotoxicity

One of several effector mechanisms thought to contribute to Ab efficacy against cancer is complement-dependent cytotoxicity (CDC). Serological analysis of a series of clinical trials conducted over a 10-year period suggested that six vaccines containing different glycolipids induced Abs mediating CDC whereas four vaccines containing carbohydrate or peptide epitopes carried almost exclusively by mucin molecules induced Abs that did not mediate CDC. To explore this further, we have now compared cell surface reactivity using flow cytometry assays (FACS), complement-fixing ability, and CDC activity of a panel of mAbs and immune sera from these trials on the same two tumor cell lines. Abs against glycolipids GM2, globo H and Lewis Y, protein KSA (epithelial cell adhesion molecule, also known as EpCAM) and mucin Ags Tn, sialylated Tn, Thomsen Friedenreich (TF), and MUC1 all reacted comparably by FACS with tumor cells expressing these Ags. Compared with the strong complement binding and CDC with Abs against glycolipids and KSA, complement binding was diminished with Abs against mucin Ags and no CDC was detected. A major difference between these two groups of Ags is proximity to the cell membrane. Glycolipids and globular glycoproteins extend less than 100 A from the cell membrane while mucins extend up to 5000 A. Although complement activation at sites remote from the cell membrane has long been known as a mechanism for resistance from complement lysis in bacteria, it is identified here for the first time as a factor which may contribute to resistance from CDC against cancer cells.     

Glycan Antagonists and Inhibitors: A Fount for Drug Discovery

ABSTRACT

Glycans, the carbohydrate chains of glycoproteins, proteoglycans, and glycolipids, represent a relatively unexploited area for drug development compared with other macromolecules. This review describes the major classes of glycans synthesized by animal cells, their mode of assembly, and available inhibitors for blocking their biosynthesis and function. Many of these agents have proven useful for studying the biological activities of glycans in isolated cells, during embryological development, and in physiology. Some are being used to develop drugs for treating metabolic disorders, cancer, and infection, suggesting that glycans are excellent targets for future drug development.     

Mucin-carbohydrate directed monoclonal antibody

To raise monoclonal antibodies recognizing cancer-associated alterations of the carbohydrate structure of glycoproteins, Balb/c mice were immunized with human colonic cancer cells (LS 180 from ATCC). One of the generated hybridomas produced a monoclonal antibody that bound to the carbohydrate moiety of mucin-type glycoproteins from LS 180. The antibody did not bind to glycoproteins from another colonic cancer cell line, SW 1116, or to glycolipids from any of the colonic cancer cell lines. The antibody bound to ovine and bovine submaxillary mucins (OSM and BSM). NeuAc alpha 2----6Ga1NAc seemed to be involved in the epitope.     

Natural glycan microarrays

Glycan microarrays are emerging as increasingly used screening tools with a high potential for unraveling protein–carbohydrate interactions: probing hundreds or even thousands of glycans in parallel, they provide the researcher with a vast amount of data in a short time-frame, while using relatively small amounts of analytes. Natural glycan microarrays focus on the glycans’ repertoire of natural sources, including both well-defined structures as well as still-unknown ones. This article compares different natural glycan microarray strategies. Glycan probes may comprise oligosaccharides from glycoproteins as well as glycolipids and polysaccharides. Oligosaccharides may be purified from scarce biological samples that are of particular relevance for the carbohydrate-binding protein to be studied. We give an overview of strategies for glycan isolation, derivatization, fractionation, immobilization and structural characterization. Detection methods such as fluorescence analysis and surface plasmon resonance are summarized. The importance of glycan density and multivalency is discussed. Furthermore, some applications of natural glycan microarrays for studying lectin and antibody binding are presented.     

Membrane biogenesis in embryonal carcinomas: glycoproteins destined for the cell surface are delayed in a pre-Golgi compartment

Embryonal carcinoma and early embryonic cells assemble a family of unusually large and complex carbohydrates. These glycans are highly branched, repeating copolymers of the sugars galactose and N-acetylglucosamine, referred to as polylactosamines, and are frequently decorated with fucose, sulfate, and sialic acid. We have previously shown that in teratocarcinoma cells these glycans are part of a large spectrum of glycans assembled on mannose cores derived from a common precursor glycan. Metabolic studies revealed a large excess of high-mannose glycans at a time when complex-type glycans cease to accumulate. The present studies demonstrate that these high-Man glycans are not degraded internally or secreted directly but are on glycoproteins destined for the cell surface. These unprocessed glycoproteins replace material lost during the extensive membrane turnover that occurs in these cells. Their export to the cell surface is delayed in a pre-Golgi compartment.     

Detection of the L2/HNK-1 Carbohydrate Epitope on Glycoproteins and Acidic Glycolipids of the Insect Calliphora vicina

The same or a very similar carbohydrate determinant, as represented by some sulfated, glucuronic acid-containing glycosphingolipids of human peripheral nerve, occurs on several adhesion molecules in the mammalian nervous system. In the present study, the occurrence of this epitope on glycoproteins and glycolipids of the fly, Calliphora vicina, was investigated by Western blot analysis and thin-layer chromatogram immunostaining. Several monoclonal antibodies recognizing an epitope on various neural cell adhesion molecules, designated L2 (334, 336, 349, and 412); the monoclonal antibody HNK-1 (recognizing an epitope on human natural killer cells); and a human IgM M-protein were found to react by Western blot analysis with various glycoproteins from larval and adult brains, although the intensity of staining of bands recognized by each antibody varied. Acidic glycolipids from pupae were also recognized, but only by the L2 antibody 334 and IgM M-protein. After desulfation of the acidic glycolipid fraction, the immunostaining pattern remained the same, an observation suggesting that the L2/HNK-1 epitope on insect acidic glycolipids contains a nonsulfated, glucuronic acid moiety. These observations indicate that the L2/HNK-1 carbohydrate structure occurs not only in vertebrates but also in insects on both glycoproteins and glycolipids, a finding suggesting a high degree of phylogenetic stability of this functionally important carbohydrate.     

Analysis by lectin affinity chromatography of N-linked glycans of BHK cells and ricin-resistant mutants.

Normal baby hamster kidney (BHK) fibroblasts and ricin-resistant (RicR) mutants of BHK cells derived from them were labelled metabolically with [3H]mannose or [3H]fucose. Glycopeptides obtained by digestion of disrupted cells with Pronase were separated by affinity chromatography on concanavalin A-Sepharose. In the normal BHK cells major glycopeptide fractions were obtained consisting of tetra- and tri-antennary sialylated complex glycans, bi-antennary sialylated glycans, and neutral oligomannosidic chains. The majority of bi-antennary chains were shown to contain a fucosyl-(alpha 1-6)-N-acetylglucosaminyl sequence in the core region by their ability to bind to a lentil lectin affinity column. All of the mutant cell lines examined were found to accumulate oligomannosidic glycans in cellular glycoproteins: complex sialylated glycans were either absent or greatly reduced in amount. Analysis of fractions isolated from concanavalin A-Sepharose by Bio-Gel P-4 chromatography and glycosidase degradation indicated that the glycans accumulating in RicR14 cells have the general structure: (formula; see text) and derivatives having fewer alpha-mannosyl units. We have also analysed the glycopeptides released by trypsin treatment from the surface of the normal and mutant cells, as well as those obtained by proteolysis of fibronectin isolated from the medium. The glycopeptide profiles of the cell-surface-derived material and of fibronectin showed for the mutant cells a marked accumulation of oligomannosidic chains at the expense of complex oligosaccharide chains. Hence, the alterations in glycan structure detected in bulk cellular glycoproteins of RicR cells are expressed also in cell surface glycoproteins and in fibronectin, a secreted glycoprotein.     

Antibody responses to glycolipid-borne carbohydrates require CD4+ T cells but not CD1 or NKT cells

Naturally occurring anti-carbohydrate antibodies play a major role in both the innate and adaptive immune responses. To elicit an anti-carbohydrate immune response, glycoproteins can be processed to glycopeptides and presented by the classical antigen-presenting molecules, major histocompatibility complex (MHC) Class I and II. In contrast, much less is known about the mechanism(s) for anti-carbohydrate responses to glycolipids, although it is generally considered that the CD1 family of cell surface proteins presents glycolipids to T cells or natural killer T (NKT) cells. Using model carbohydrate systems (isogloboside 3 and B blood group antigen), we examined the anti-carbohydrate response on glycolipids using both antibody neutralisation and knockout mouse-based experiments. These studies showed that CD4(+) T cells were required to generate antibodies to the carbohydrates expressed on glycolipids, and unexpectedly, these antibody responses were CD1d and NKT cell independent. They also did not require peptide help. These data provide new insight into glycolipid antigen recognition by the immune system and indicate the existence of a previously unrecognised population of glycolipid antigen-specific, CD1-independent, CD4(+) T cells.     

Immobilized glycoconjugates for cell recognition studies

Specific cell-cell recognition and adhesion may involve cell surface glycoconjugates on one cell binding the complementary carbohydrate receptors on an apposing cell surface. Such interactions have been modeled by immobilizing simple synthetic glycosides, glycoproteins, glycosaminoglycans, and glycolipids on otherwise inert plastic surfaces and incubating them with intact cells. Using this approach, the ability of several cell types to recognize specific carbohydrates has been demonstrated. This carbohydrate-directed cell adhesion may depend on cell surface carbohydrate receptors which mediate both the initial specific adhesion and complex postrecognition cellular responses. While the relationship of the cell adhesion demonstrated here to cell-cell recognition in vivo has yet to be determined, this well-controlled biochemical approach may reveal new information on the way in which cells analyze and respond to their immediate external environment.     

Evaluation of analogues of GalNAc as substrates for enzymes of the mammalian GalNAc salvage pathway

Changes in glycosylation are correlated to disease and associated with differentiation processes. Experimental tools are needed to investigate the physiological implications of these changes either by labeling of the modified glycans or by blocking their biosynthesis. N-Acetylgalactosamine (GalNAc) is a monosaccharide widely encountered in glycolipids, proteoglycans, and glycoproteins; once taken up by cells it can be converted through a salvage pathway to UDP-GalNAc, which is further used by glycosyltransferases to build glycans. In order to find new reporter molecules able to integrate into cellular glycans, synthetic analogues of GalNAc were prepared and tested as substrates of both enzymes acting sequentially in the GalNAc salvage pathway, galactokinase 2 (GK2) and uridylpyrophosphorylase AGX1. Detailed in vitro assays identified the GalNAc analogues that can be transformed into sugar nucleotides and revealed several bottlenecks in the pathway: a modification on C6 is not tolerated by GK2; AGX1 can use all products of GK2 although with various efficiencies; and all analogues transformed into UDP-GalNAc analogues except those with alterations on C4 are substrates for the polypeptide GalNAc transferase T1. Besides, all analogues that could be incorporated in vitro into O-glycans were also integrated into cellular O-glycans as attested by their detection on the cell surface of CHO-ldlD cells. Altogether our results show that GalNAc analogues can help to better define structural requirements of the donor substrates for the enzymes involved in GalNAc metabolism, and those that are incorporated into cells will prove valuable for the development of novel diagnostic and therapeutic tools.     

Inhibition of fusion of embryonic muscle cells in culture by tunicamycin is prevented by leupeptin

The carbohydrate requirement for alignment and fusion of embryonic quail muscle cells has been examined in tissue culture by use of tunicamycin (TM). The mononucleated, spindle-shaped proliferating myoblasts were treated with TM at various times before fusion and differentiation into multinucleated muscle fibers capable of spontaneous contraction. Tm blocked protein glycosylation and expression of glycoproteins on the cell surface, and strongly inhibited fusion when added to cultures of differentiating muscle cells before the fusion "burst," but had no apparent effect on cell alignment. The inhibition of fusion was partially prevented when TM was administered in the presence of protease inhibitors such as leupeptin and pepstatin, but the inhibition of glycosylation was not prevented. Both glycosylation and fusion were completely restored to normal by the removal of the antibiotic from the medium. These studies provide strong support for the idea that myoblast fusion is partially mediated by glycoproteins with asparagine-linked oligosaccharides. However, the requirement for the carbohydrate portion of the glycoprotein appears to be indirect in that it acts to stabilize the protein moiety against proteolytic degradation. Our findings do not rule out the possibility that oligosaccharide units of surface glycolipids have some role in myoblast fusion.     

Biomolecular Recognition of the Glycan Neoantigen CA19-9 by Distinct Antibodies

Glycans decorate the cell surface, secreted glycoproteins and glycolipids, and altered glycans are often found in cancers. Despite their high diagnostic and therapeutic potential, however, glycans are polar and flexible molecules that are quite challenging for the development and design of high-affinity binding antibodies. To understand the mechanisms by which glycan neoantigens are specifically recognized by antibodies, we analyze the biomolecular recognition of the tumor-associated carbohydrate antigen CA19-9 by two distinct antibodies using X-ray crystallography. Despite the potential plasticity of glycans and the very different antigen-binding surfaces presented by the antibodies, both structures reveal an essentially identical extended CA19-9 conformer, suggesting that this conformer’s stability selects the antibodies. Starting from the bound structure of one of the antibodies, we use the AbLIFT computational algorithm to design a variant with seven core mutations in the variable domain’s light-heavy chain interface that exhibits tenfold improved affinity for CA19-9. The results reveal strategies used by antibodies to specifically recognize glycan antigens and show how automated antibody-optimization methods may be used to enhance the clinical potential of existing antibodies.     

Sulfoglycolipids bind to adhesive protein amphoterin (P30) in the nervous system.

HNK-1 antibody reactive carbohydrate epitope carried by glycolipids and glycoproteins has been shown to be involved in cell to cell interactions. It has been proposed that the HNK-1 reactive 3-sulfoglucuronyl carbohydrate epitope in glycolipids may interact with a cell surface receptor to promote the biological response in the developing nervous system. The possible occurrence of such a receptor was examined in rat nervous system. A specific binding of sulfoglycolipids to a 30 kD protein from adult rat cerebellum is described. Little binding was found with neutral glycolipids and gangliosides. The 30 kD protein from cerebellum was partially purified on a sulfatide-octyl-Sepharose affinity column. Binding of sulfoglucuronyl glycolipids to a similar 30 kD protein from forebrain previously identified as amphoterin is also shown. Amphoterin is developmentally regulated and is involved in neural cell adhesion and neurite extension.     

Structural characterization of free glycolipids which are potential precursors for glycophosphatidylinositol anchors in mouse thymoma cell lines.

Biosynthesis of glycophosphatidylinositol-anchored membrane glycoproteins proceeds through the attachment of a preformed glycolipid onto a C-terminal amino acid rapidly after translation. Here we describe the structural analysis of two very polar glycolipids which can be observed after metabolic labeling of lymphoma cell lines S1A and EL-4 with either tritiated myo-inositol, mannose, or ethanolamine. These lipids are not made by mutant cells deficient in the biosynthesis of glycophosphatidylinositol anchors. The lipids were isolated, and their carbohydrate moiety was characterized using hydrofluoric acid dephosphorylation, nitrous acid deamination, acetolysis, exoglycosidase treatments, and combinations thereof to produce labeled fragments which could be analyzed by paper chromatography. Results are compatible with the structure (X-->)Man alpha 1,2 Man alpha 1,6(Y-->)Man alpha-GlcN-acylinositol, X and Y being hydrofluoric acid-sensitive substituents (most likely phosphoethanolamine). The anchor oligosaccharide of the glycophosphatidylinositol protein anchors of S1A cells was isolated, similarly characterized, and found to contain the identical carbohydrate structure. Pulse-chase experiments indicate that the very polar glycolipids have half-lives which are much longer than the one of phosphatidylinositol. The results suggest that these very polar glycolipids represent supernumerary precursor glycolipids which did not get transferred onto proteins or represent processed forms of such precursors.