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.     

Characterizing the glycome of the mammalian immune system

The outermost layer of all immune cells, the glycocalyx, is composed of a complex mixture of glycoproteins, glycolipids and lectins, which specifically recognize particular glycan epitopes. As the glycocalyx is the cell's primary interface with the external environment many biologically significant events can be attributed to glycan recognition. For this reason the rapidly expanding glycomics field is being increasingly recognized as an important component in our quest to better understand the functioning of the immune system. In this review, we highlight the current status of immune cell glycomics, with particular attention being paid to T- and B-lymphocytes and dendritic cells. We also describe the strategies and methodologies used to define immune cell glycomes.     

Ultracytochemical study of lytic complex insertion in the glycocalyx of red cells during immune hemolysis mediated by complement

Ruthenium red (RR), a cationic dye and an ultrastructural tracer of cell membrane permeability, was used on sheep red blood cells after lysis produced by a specific antibody and guinea pig complement. In addition to the opacification of the glycocalyx, RR stained structures related to lytic complexes, which appeared as rod-like structures with variable dimensions (generally 45 nm in width, 75 nm in height) inserted in the glycocalyx of red cells. They extended across the external layer of the trilaminar plasma membrane without reaching the internal layer or the cytoplasm. RR staining visualized the internal configuration of the lytic complexes and revealed small channels measuring 10 nm in diameter localized within the complexes. These lytic complexes are thought to correspond to membrane attack complex of complement. To the best of our knowledge, this is the first report of ultrastructural positive staining of lytic complexes in thin sections, allowing visualization of their internal configuration and their insertion in the plasma membrane glycocalyx.     

Localization of Galectins within Glycocalyx

Galectins are involved in various biological processes, e.g. cell–cell and cell–matrix adhesion and the transmission of cellular signals. Despite the diversity of functions, little is known about the nature of their physiological cognate ligands on the cell surface and the localization of galectins in the glycocalyx, although this information is important for understanding the functional activity of galectins. In this work, localization of endogenous and exogenously loaded galectins in the glycocalyx was studied. The following main conclusions are drawn: 1) galectins are not evenly distributed within the glycocalyx, they are accumulated in patches. Patching is not the result of a cross-linking of cellular glycans by galectins. Instead, patch-wise localization is the consequence of irregular distribution of glycans forming the glycocalyx; 2) galectins are accumulated in the inner zone of the glycocalyx rather than at its outer face or directly in vicinity of the cell membrane; 3) patches are not associated with cell rafts.     

Surface determinants of Leishmania parasites and their role in infectivity in the mammalian host

Leishmania are intracellular protozoan parasites that reside primarily in host mononuclear phagocytes. Infection of host macrophages is initiated by infective promastigote stages and perpetuated by an obligate intracellular amastigote stage. Studies undertaken over the last decade have shown that the composition of the complex surface glycocalyx of these stages (comprising lipophosphoglycan, GPI-anchored glycoproteins, proteophosphoglycans and free GPI glycolipids) changes dramatically as promastigotes differentiate into amastigotes. Marked stage-specific changes also occur in the expression of other plasma membrane components, including type-1, polytopic and peripheral membrane proteins, reflecting the distinct microbicidal responses and nutritional environments encountered by these stages. More recently, a number of Leishmania mutants lacking single or multiple surface components have been generated. While some of these mutants are less virulent than wild type parasites, many of these mutants exhibit only mild or no loss of virulence. These studies suggest that, 1) the major surface glycocalyx components of the promastigote stage (i.e. LPG, GPI-anchored proteins) only have a transient or minor role in macrophage invasion, 2) that there is considerable functional redundancy in the surface glycocalyx and/or loss of some components can be compensated for by the acquisition of equivalent host glycolipids, 3) the expression of specific nutrient transporters is essential for life in the macrophage and 4) the role(s) of some surface components differ markedly in different Leishmania species. These mutants will be useful for identifying other surface or intracellular components that are required for virulence in macrophages.     

Intermittent contact mode AFM investigation of native plasma membrane of <Emphasis Type="Italic">Xenopus laevis</Emphasis> oocyte

Intermittent contact mode atomic force microscopy (AFM) was used to visualize the native plasma membrane of Xenopus laevis oocytes. Oocyte membranes were purified via ultracentrifugation on a sucrose gradient and adsorbed on mica leaves. AFM topographs and the corresponding phase images allowed for visualization and identification of both oocyte plasma membrane patches and pure lipid bilayer regions with a height of about 5 nm within membrane patches. The quantitative analysis showed a normal distribution for the lateral dimension and height of the protein complexes centered on 16.7 ± 0.2 nm (mean ± SE, n = 263) and 5.4 ± 0.1 nm (n = 262), respectively. The phase signal, providing material-dependent information, allowed for the recognition of structural features observed in AFM topographs.     

Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components

Lateral assemblies of glycolipids and cholesterol, “rafts,” have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T–lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.     

Similar Endothelial Glycocalyx Structures in Microvessels from a Range of Mammalian Tissues: Evidence for a Common Filtering Mechanism?

The glycocalyx or endocapillary layer on the luminal surface of microvessels has a major role in the exclusion of macromolecules from the underlying endothelial cells. Current structural evidence in the capillaries of frog mesentery indicates a regularity in the structure of the glycocalyx, with a center-to-center fiber spacing of 20 nm and a fiber width of 12 nm, which might explain the observed macromolecular filtering properties. In this study, we used electron micrographs of tissues prepared using perfusion fixation and tannic acid treatment. The digitized images were analyzed using autocorrelation to find common spacings and to establish whether similar structures, hence mechanisms, are present in the microvessel glycocalyces of a variety of mammalian tissues. Continuous glycocalyx layers in mammalian microvessels of choroid, renal tubules, glomerulus, and psoas muscle all showed similar lateral spacings at ∼19.5 nm (possibly in a quasitetragonal lattice) and longer spacings above 100 nm. Individual glycocalyx tufts above fenestrations in the first three of these tissues and also in stomach fundus and jejunum showed evidence for similar short-range structural regularity, but with more disorder. The fiber diameter was estimated as 18.8 (± 0.2) nm, but we believe this is an overestimate because of the staining method used. The implications of these findings are discussed.     

Where catabolism meets signalling: neuraminidase 1 as a modulator of cell receptors

Terminal sialic acid residues are found in abundance in glycan chains of glycoproteins and glycolipids on the surface of all live cells forming an outer layer of the cell originally known as glycocalyx. Their presence affects the molecular properties and structure of glycoconjugates, modifying their function and interactions with other molecules. Consequently, the sialylation state of glycoproteins and glycolipids has been recognized as a critical factor modulating molecular recognitions inside the cell, between the cells, between the cells and the extracellular matrix, and between the cells and certain exogenous pathogens. Sialyltransferases that attach sialic acid residues to the glycan chains in the process of their initial synthesis were thought to be mainly responsible for the creation and maintenance of a temporal and spatial diversity of sialylated moieties. However, the growing evidence also suggests that in mammalian cells, at least equally important roles belong to sialidases/neuraminidases, which are located on the cell surface and in intracellular compartments, and may either initiate the catabolism of sialoglycoconjugates or just cleave their sialic acid residues, and thereby contribute to temporal changes in their structure and functions. The current review summarizes emerging data demonstrating that neuraminidase 1 (NEU1), well known for its lysosomal catabolic function, can be also targeted to the cell surface and assume the previously unrecognized role as a structural and functional modulator of cellular receptors.     

Pulsed Low-Frequency Magnetic Fields Induce Tumor Membrane Disruption and Altered Cell Viability

Tumor cells express a unique cell surface glycocalyx with upregulation of sulfated glycosaminoglycans and charged glycoproteins. Little is known about how electromagnetic fields interact with this layer, particularly with regard to harnessing unique properties for therapeutic benefit. We applied a pulsed 20-millitesla (mT) magnetic field with rate of rise (dB/dt) in the msec range to cultured tumor cells to assess whether this affects membrane integrity as measured using cytolytic assays. A 10-min exposure of A549 human lung cancer cells to sequential 50- and 385-Hz oscillating magnetic fields was sufficient to induce intracellular protease release, suggesting altered membrane integrity after the field exposure. Heparinase treatment, which digests anionic sulfated glycan polymers, before exposure rendered cells insensitive to this effect. We further examined a non-neoplastic human primary cell line (lung lymphatic endothelial cells) as a typical normal host cell from the lung cancer microenvironment and found no effect of field exposure on membrane integrity. The field exposure was also sufficient to alter proliferation of tumor cells in culture, but not that of normal lymphatic cells. Pulsed magnetic field exposure of human breast cancer cells that express a sialic-acid rich glycocalyx also induced protease release, and this was partially abrogated by sialidase pretreatment, which removes cell surface anionic sialic acid. Scanning electron microscopy showed that field exposure may induce unique membrane “rippling” along with nanoscale pores on A549 cells. These effects were caused by a short exposure to pulsed 20-mT magnetic fields, and future work may examine greater magnitude effects. The proof of concept herein points to a mechanistic basis for possible applications of pulsed magnetic fields in novel anticancer strategies.     

Desialylation of surface receptors as a new dimension in cell signaling

Terminal sialic acid residues are found in abundance in glycan chains of glycoproteins and glycolipids on the surface of all live cells forming an outer layer of the cell originally known as glycocalyx. Their presence affects the molecular properties and structure of glycoconjugates, modifying their function and interactions with other molecules. Consequently, the sialylation state of glycoproteins and glycolipids has been recognized as a critical factor modulating molecular recognitions inside the cell, between the cells, between the cells and the extracellular matrix, and between the cells and certain exogenous pathogens. Until recently sialyltransferases that catalyze transfer of sialic acid residues to the glycan chains in the process of their biosynthesis were thought to be mainly responsible for the creation and maintenance of a temporal and spatial diversity of sialylated moieties. However, the growing evidence suggests that in mammalian cells, at least equally important roles belong to sialidases/neuraminidases, which are located on the cell surface and in intracellular compartments, and may either initiate the catabolism of sialoglycoconjugates or just cleave their sialic acid residues, and thereby contribute to temporal changes in their structure and functions. The current review summarizes emerging data demonstrating that mammalian neuraminidase 1, well known for its lysosomal catabolic function, is also targeted to the cell surface and assumes the previously unrecognized role as a structural and functional modulator of cellular receptors.     

Quantification of the effect of glycocalyx condition on membrane receptor interactions using an acoustic wave sensor

The effect of the cell glycocalyx on the binding of a membrane receptor, class I major histocompatibility complex (MHC) human leukocyte antigen (HLA)-A2, to an immobilized anti-HLA antibody was investigated using an acoustic sensor based on a Love wave geometry. The enzyme neuraminidase was used to remove sialic acid residues from the cell glycocalyx. Real-time measurements of the amplitude of the acoustic wave showed that treatment with neuraminidase facilitates HLA/anti-HLA-mediated cell attachment via a 3.6-fold increase of the two-dimensional (2D) binding constant of the interaction. This could be attributed to better approach of binding partners due to favorable condition of the desialylated glycocalyx. The results underline the importance of microtopological factors in membrane receptor binding and reveal the potential of the Love wave sensor and 2D binding parameters for studying cell–substrate binding events.     

‘Porosome’ discovered nearly 20 years ago provides molecular insights into the kiss‐and‐run mechanism of cell secretion

Secretion is a fundamental cellular process in living organisms, from yeast to cells in humans. Since the 1950s, it was believed that secretory vesicles completely merged with the cell plasma membrane during secretion. While this may occur, the observation of partially empty vesicles in cells following secretion suggests the presence of an additional mechanism that allows partial discharge of intra‐vesicular contents during secretion. This proposed mechanism requires the involvement of a plasma membrane structure called ‘porosome’, which serves to prevent the collapse of secretory vesicles, and to transiently fuse with the plasma membrane (Kiss‐and‐run), expel a portion of its contents and disengage. Porosomes are cup‐shaped supramolecular lipoprotein structures at the cell plasma membrane ranging in size from 15 nm in neurons and astrocytes to 100–180 nm in endocrine and exocrine cells. Neuronal porosomes are composed of nearly 40 proteins. In comparison, the 120 nm nuclear pore complex is composed of >500 protein molecules. Elucidation of the porosome structure, its chemical composition and functional reconstitution into artificial lipid membrane, and the molecular assembly of membrane‐associated t‐SNARE and v‐SNARE proteins in a ring or rosette complex resulting in the establishment of membrane continuity to form a fusion pore at the porosome base, has been demonstrated. Additionally, the molecular mechanism of secretory vesicle swelling, and its requirement for intra‐vesicular content release during cell secretion has also been elucidated. Collectively, these observations provide a molecular understanding of cell secretion, resulting in a paradigm shift in our understanding of the secretory process.     

Scolopendin 2, a cationic antimicrobial peptide from centipede,and its membrane-active mechanism

Scolopendin 2 is a 16-mer peptide (AGLQFPVGRIGRLLRK) derived from the centipede Scolopendra subspinipes mutilans. We observed that this peptide exhibited antimicrobial activity in a salt-dependent manner against various fungal and bacterial pathogens and showed no hemolytic effect in the range of 1.6 μM to 100 μM. Circular dichroism analysis showed that the peptide has an α-helical properties. Furthermore, we determined the mechanism(s) of action using flow cytometry and by investigating the release of intracellular potassium. The results showed that the peptide permeabilized the membranes of Escherichia coli O157 and Candida albicans, resulting in loss of intracellular potassium ions. Additionally, bis-(1,3-dibutylbarbituric acid) trimethine oxonol and 3,3′-dipropylthiacarbocyanine iodide assays showed that the peptide caused membrane depolarization. Using giant unilamellar vesicles encapsulating calcein and large unilamellar vesicles containing fluorescein isothiocyanate-dextran, which were similar in composition to typical E. coli O157 and C. albicans membranes, we demonstrated that scolopendin 2 disrupts membranes, resulting in a pore size between 4.8 nm and 5.0 nm. Thus, we have demonstrated that a cationic antimicrobial peptide, scolopendin 2, exerts its broad-spectrum antimicrobial effects by forming pores in the cell membrane.     

The ultrastructure of the integument and proventriculus in the raptorial rotifer Cupelopagis vorax (Monogononta: Collothecaceae: Atrochidae)

Members of the sessile rotifer species Cupelopagis vorax are unusual ambush predators that live permanently attached to submerged freshwater plants. Previous light microscopical research has revealed several uncommon features in this species including a stellate‐patterned integument and an expansive foregut region called the proventriculus. In this study, we apply transmission electron microscopy to explore the ultrastructure of both the integument and foregut to determine how they differ from other rotifers. Our results reveal that the integument is covered by a thick glycocalyx and is patterned with tubercles that originate from the intracytoplasmic lamina (ICL) within the syncytial epidermis. The ICL forms an apical layer within the syncytium, is electron dense and mostly amorphous, and forms tubercles up to 2.3 μm; these tubercles probably account for the patterned appearance of the integument and are similar to what has been found in other gnesiotrochan rotifers. The basal cytoplasm is highly granular and contains two types of membrane‐bound vesicles: large ovoid vesicles (320–411 nm) with amorphous, opaque contents, and secretory bulbs (110–264 nm) with electron‐lucent cores and occasionally electron‐dense contents. Only the secretory bulbs were observed to form connections to the apical plasmalemma, and so are probably exocytotic. Internally, the proventriculus is a large distensible sack that connects the anterior pharyngeal tube to the posterior mastax. The proventricular epithelium is a thin syncytium mostly covered with a dense glycocalyx and a strong brush border of microvilli underlain by a thin terminal web. The cytoplasm contains few organelles and there is no evidence that it is either secretory or has features (e.g., ICL) that might aid in maceration. We hypothesize that the thick glycocalyx might serve a protective function against the movements of live prey and/or against enzymes released from the rotifer's gastric glands that become regurgitated during feeding.     

Biosynthesis of glycolipid precursors for glycosylphosphatidylinositol membrane anchors in a Toxoplasma gondii cell-free system.

Toxoplasmosis, a disease that affects humans and a wide variety of mammals is caused by Toxoplasma gondii, the obligate intracellular coccidian protozoan parasite. Most T. gondii research has focused on the rapidly growing invasive form, the tachyzoite, which expresses five major surface proteins attached to the parasite membrane by glycosylphosphatidylinositol (GPI) anchors. We have recently reported the purification and partial characterization of candidate precursor glycolipids (GPIs) from metabolically labeled parasites and have presented evidence that these GPIs have a linear glycan backbone sequence indistinguishable from the GPI core glycan of the major tachyzoite surface protein, P30. In this report, we describe a cell-free system derived from tachyzoite membranes which is capable of catalyzing GPI biosynthesis. Incubation of the membrane preparations with radioactive sugar nucleotides (GDP-[3H]mannose or UDP-[3H]GlcNAc) resulted in incorporation of radiolabeled into numerous glycolipids. By using a combination of chemical/enzymatic tests and chromatographic analysis, a series of incompletely glycosylated lipid species and mature GPIs have been identified. We have also established the involvement of Dol-P-mannose in the synthesis of T. gondii GPIs by demonstrating that the incorporation of [3H]mannose into the mannosylated GPIs is stimulated by dolichylphosphate and inhibited by amphomycin. In addition, increasing the concentration of nonradioactive GDP mannose resulted in a loss of radiolabel from the first easily detectable GPI precursor, GlcN-PI, and a concomittant appearance of the radio-activity into mannosylated glycolipids. Altogether, our data suggest that the GPI core glycan in T. gondii is assembled via sequential glycosylation of phosphatidylinositol, as proposed for the biosynthesis of GPIs in Trypanosoma brucei. In contrast to T. brucei, preliminary experiments indicate that the core glycan of some GPIs synthesized by the T. gondii cell-free system is modified by N-acetylgalactosamine similar to the situation for mammalian Thy-1.     

Nanomapping of CD1d–glycolipid complexes on THP1 cells by using simultaneous topography and recognition imaging

CD1d molecule, a monomorphic major histocompatibility complex class I‐like molecule, presents different types of glycolipids to invariant natural killer T (iNKT) cells that play an important role in immunity to infection and tumors, as well as in regulating autoimmunity. Here, we present simultaneous topography and recognition imaging (TREC) analysis to detect density, distribution and localization of single CD1d molecules on THP1 cells that were loaded with different glycolipids. TREC was conducted using magnetically coated atomic force microscopy tips functionalized with a biotinylated iNKT cell receptor (TCR). The recognition map revealed binding sites visible as dark spots, resulting from oscillation amplitude reduction during specific binding between iNKT TCR and the CD1d–glycolipid complex. THP1 cells were pulsed with three different glycolipids (α‐GalCer, C20 and OCH12) for 4 and 16 hr. Whereas CD1d–α‐GalCer and CD1d–C20:2 complexes on cellular membrane formed smaller microdomains up to ~10 000 nm² (dimension area), OCH12 loaded CD1d complexes presented larger clusters with a dimension up to ~30 000 nm². Moreover, the smallest size of recognition spots was about 25 nm, corresponding to a single CD1d binding site. TREC successfully revealed the distribution and localization of CD1d–glycolipid complexes on THP1 cell with single molecule resolution under physiological conditions. Copyright © 2013 John Wiley & Sons, Ltd.     

The Apical Membrane Glycocalyx of MDCK Cells

The microenvironment near the apical membrane of MDCK cells was studied by quantitation of the fluorescence of wheat germ agglutin attached to fluorescein (WGA). WGA was shown to bind to sialic acid residues attached to galactose at the α-2,3 position in the glycocalyx on the apical membrane. Young MDCK cells (5–8 days after splitting) showed a patchy distribution of WGA at stable sites that returned to the same locations after removal of sialic acid residues by neuraminidase treatment. Other lectins also showed stable binding to patches on the apical membrane of young cells. The ratio of WGA fluorescence emission at two excitation wavelengths was used to measure near-membrane pH. The near-membrane pH was markedly acidic to the pH 7.4 bathing solution in both young and older cells (13–21 days after splitting). Patches on the apical membrane of young cells exhibited a range of near-membrane pH values with a mean ±sem of 6.86 ± 0.04 (n= 121) while the near-membrane pH of older cells was 6.61 ± 0.04 (n= 120) with a uniform WGA distribution. We conclude that the distribution of lectin binding sites in young cells reflects the underlying nonrandom location of membrane proteins in the apical membrane and that nonuniformities in the pH of patches may indicate regional differences in membrane acid-base transport as well as in the location of charged sugars in the glycocalyx. Received: 15 December 1999/Revised: 16 March 2000     

Tubulohelical membrane arrays: Novel association of helical structures with intracellular membranes

A novel organelle-like membrane specialization has been found in an epithelial cell line. Characteristically, the helical membrane arrays (referred to hereafter as TUHMAs) are organized around tubular, proteinaceous electron-dense cores of 80 nm in diameter. Depending on the cell status, up to 8 of these cores provide the basis for an intermingled membrane scaffold of an overall length of 3-5 μm. TUHMAs exist as single organelles in transient association with the nucleus, the rough endoplasmic reticulum, patches of annulate lamellae, and the Golgi complex. While most of the constituents are still unknown, evidence for an involvement of nucleoporins in TUHMA organization is presented, as shown by fluorescence immunochemistry. This should make TUHMAs more easily accessible for future studies on their structure and function.     

Profiling of Glycan Receptors for Minute Virus of Mice in Permissive Cell Lines Towards Understanding the Mechanism of Cell Recognition

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-Le^X identified in a previous glycan microarray screen.