Glycans as cancer biomarkers

Background

Non-invasive biomarkers, such as those from serum, are ideal for disease prognosis, staging and monitoring. In the past decade, our understanding of the importance of glycosylation changes with disease has evolved.

Scope of review

We describe potential biomarkers derived from serum glycoproteins for liver, pancreatic, prostate, ovarian, breast, lung and stomach cancers. Methods for glycan analysis have progressed and newly developed high-throughput platform technologies have enabled the analysis of large cohorts of samples in an efficient manner. We also describe this evolution and trends to follow in the future.

Major conclusions

Many convincing examples of aberrant glycans associated with cancer have come about from glycosylation analyses. Most studies have been carried out to identify changes in serum glycan profiles or through the isolation and identification of glycoproteins that contain these irregular glycan structures. In a majority of cancers the fucosylation and sialylation expression are found to be significantly modified. Therefore, these aberrations in glycan structures can be utilized as targets to improve existing cancer biomarkers.

General significance

The ability to distinguish differences in the glycosylation of proteins between cancer and control patients emphasizes glycobiology as a promising field for potential biomarker identification. Furthermore, the high-throughput and reproducible nature of the chromatography platform have highlighted extensive applications in biomarker discovery and allowed integration of glycomics with other -omics fields, such as proteomics and genomics, making systems glycobiology a reality. This article is part of a Special Issue entitled Glycoproteomics.     

Glycans as endocytosis signals: the cases of the asialoglycoprotein and hyaluronan/chondroitin sulfate receptors

Animal cells internalize specific extracellular macromolecules (ligands) by using specialized cell surface receptors that operate through a complex and highly regulated process known as receptor-mediated endocytosis, which involves the binding, internalization, and transfer of ligands through a series of distinct intracellular compartments. For the uptake of a variety of carbohydrate-containing macromolecules, such as glycoproteins, animal cells use specialized membrane-bound lectins as endocytic receptors that recognize different sugar residues or carbohydrate structures present on various ligands. The hepatic asialoglycoprotein receptor, which recognizes glycoconjugates containing terminal galactose or N-acetylgalactosamine residues, was the first membrane lectin discovered and has been a classical system for studying receptor-mediated endocytosis. Studies of how the asialoglycoprotein receptor functions have led to the discovery of two functionally distinct, parallel pathways of clathrin-mediated endocytosis (called the State 1 and State 2 pathways), which may also be utilized by all the other endocytic recycling receptor systems. Another endocytic membrane lectin, the hyaluronan/chondroitin sulfate receptor, which has recently been purified and cloned, is responsible for the turnover in mammals of these glycosaminoglycans, which are important components of extracellular matrices. We discuss the characteristics and physiological importance of these two proteins as examples of how lectins can function as endocytic receptors.     

Glycans as targets for monoclonal antibodies that protect rats against Trichinella spiralis

We have investigated the role of glycans on Trichinella spiralisantigens in recognition by rat monoclonal antibodies (mAbs)which protect rat pups against challenge with the parasite.In pups born to infected dams or pups passively immunized withmAbs, antibodies eliminate a challenge dose from the intestinewithin hours (‘rapid expulsion’). Because such dramaticprotection can be afforded by mAbs, we have sought to characterizethe parasite antigens they target In this report we show thatprotective antibodies were unable to bind excretory/secretory(ES) antigens de-glycosylated with trifluoromethanesulphonicacid (TFMS). In addition, oligosaccharides isolated from glycoproteinsby alkaline hydrolysis or peptlde: Nglycosidase F (PNGase F)digestion were bound by protective, but not non-protective,mAbs. Glycans affinity purified with protective mAb 9D boundto all but one protective mAb. These antibodies have been shownpreviously to bind to the surfaces of intact larvae, indicatingthat the glycan is exposed on the parasite surface. Candidateglycans that may be involved in binding protective mAbs haveunusual tri- and tetra-antennary structures with terminal tyvelosemoieties (Reason et al., Glycobiology, 4, 000-000, 1994). Coatingof the larval surface with such glycans may serve to protectthe parasite and its secreted products from enzymatic attackas the parasite travels to and resides in its epithelial niche. glycans protective antibodies Trichinella spiralis     

Glycans as biochemical markers of human endometrial secretory differentiation

Uterine fluid contains a mixture of glycoprotein components the quantity and composition of which vary during the menstrual cycle. Their analysis may give clues to the involvement of maternally derived components in modulating the state of the peri-implantation blastocyst and aid in assessing the differentiation of the endometrium in preparation for implantation. Endometrial epithelium also exhibits an apical glycocalyx, the composition of which varies with the state of tissue differentiation. Evidence from animal systems suggests that glycans may be involved in molecular recognition between the embryo and maternal surface at implantation. Lectins and monoclonal antibodies to glycan epitopes have been used as sensitive and specific probes to examine the carbohydrates associated with endometrial epithelium as a function of the ovarian cycle. Numerous glycan structures are detected specifically in epithelial cells. Hitherto unsuspected biosynthetic heterogeneity is present in the glands. A secretory sialokeratan sulphate epitope is described, the occurrence of which coincides with the implantation phase of the cycle.     

Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment

Exosomes, also known as microvesicles (EMVs), are nano-sized membranous particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation, and suppression and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein composition, which can dictate pathogenicity. Although some mechanisms have been proposed for the regulation of EMV protein trafficking, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from that of the parent cell membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we further explore the role of glycosylation in EMV protein trafficking. We identify EMV glycoproteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Furthermore, we show that genetic manipulation of the glycosylation levels of a specific EMV glycoprotein, EWI-2, directly impacts its recruitment as a function of N-linked glycan sites. Taken together, our data provide strong evidence that N-linked glycosylation directs glycoprotein sorting into EMVs.     

Galectin-1 binds to influenza virus and ameliorates influenza virus pathogenesis

Innate immune response is important for viral clearance during influenza virus infection. Galectin-1, which belongs to S-type lectins, contains a conserved carbohydrate recognition domain that recognizes galactose-containing oligosaccharides. Since the envelope proteins of influenza virus are highly glycosylated, we studied the role of galectin-1 in influenza virus infection in vitro and in mice. We found that galectin-1 was upregulated in the lungs of mice during influenza virus infection. There was a positive correlation between galectin-1 levels and viral loads during the acute phase of viral infection. Cells treated with recombinant human galectin-1 generated lower viral yields after influenza virus infection. Galectin-1 could directly bind to the envelope glycoproteins of influenza A/WSN/33 virus and inhibit its hemagglutination activity and infectivity. It also bound to different subtypes of influenza A virus with micromolar dissociation constant (K(d)) values and protected cells against influenza virus-induced cell death. We used nanoparticle, surface plasmon resonance analysis and transmission electron microscopy to further demonstrate the direct binding of galectin-1 to influenza virus. More importantly, we show for the first time that intranasal treatment of galectin-1 could enhance survival of mice against lethal challenge with influenza virus by reducing viral load, inflammation, and apoptosis in the lung. Furthermore, galectin-1 knockout mice were more susceptible to influenza virus infection than wild-type mice. Collectively, our results indicate that galectin-1 has anti-influenza virus activity by binding to viral surface and inhibiting its infectivity. Thus, galectin-1 may be further explored as a novel therapeutic agent for influenza.     

Dictyostelium as host model for pathogenesis

The haploid social soil amoeba Dictyostelium discoideum has been established as a host model for several pathogens including Pseudomonas aeruginosa, Cryptococcus neoformans, Mycobacterium spp. and Legionella pneumophila. The research areas presently pursued include (i) the use of Dictyostelium wild-type cells as screening system for virulence of extracellular and intracellular pathogens and their corresponding mutants, (ii) the use of Dictyostelium mutant cells to identify genetic host determinants of susceptibility and resistance to infection and (iii) the use of reporter systems in Dictyostelium cells which allow the dissection of the complex host-pathogen cross-talk. The body of information presented in this review demonstrates that the availability of host cell markers, the knowledge of cell signalling pathways, the completion of the genome sequencing project and the tractability for genetic studies qualifies Dictyostelium for the study of fundamental cellular processes of pathogenesis.     

Properties of the erythrocyte receptors for influenza C virus.

Properties of the receptor for influenza C virus were studied. Although the receptor for influenza C virus on chicken erythrocytes was destroyed by the homologous virion, neuraminidase activity could not be detected in any of the influenza C virus strains tested. The receptor activity of chicken erythrocytes for influenza C virus was diminished by formaldehyde treatment but not by periodate oxidation. There was a considerable variation in the pattern and the titer of hemagglutination of influenza C virus when human erythrocytes of different blood types were used; the virus agglutinated most type B erythrocytes but not type A erythrocytes. By using human type B erythrocytes, differences among strains of influenza C virus in the hemagglutinating activity were also demonstrated. These results showed that both the receptor for and the receptor-destroying activity of influenza C virus were completely different from those of influenza A or B virus and also that carbohydrates were not involved in the receptor for influenza C virus.     

Environmental predators as models for bacterial pathogenesis

Environmental bacteria are constantly threatened by bacterivorous predators such as free-living protozoa and nematodes. In the course of their coevolution with environmental predators, some bacteria developed sophisticated defence mechanisms, including the secretion of toxins, or the capacity to avoid lysosomal killing and to replicate intracellularly within protozoa. To analyse the interactions with bacterial pathogens on a molecular, cellular or organismic level, protozoa and other non-mammalian hosts are increasingly used. These include amoebae, as well as genetically tractable hosts, such as the social amoeba Dictyostelium discoideum, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Using these hosts, the virulence mechanisms of opportunistic pathogenic bacteria such as Legionella, Mycobacterium, Pseudomonas or Vibrio were found to be not only relevant for the interactions of the bacteria with protozoa, nematodes and insect phagocytes, but also with mammalian hosts including humans. Thus, non-mammalian model hosts provide valuable insight into the pathogenesis of environmental bacteria.     

Macrophages as a battleground for toxoplasma pathogenesis

In this issue of Cell Host & Microbe, Jensen et?al. (2011) show that clonal lineages of Toxoplasma gondii have evolved distinct ways of subverting their favored host cell, the macrophage. The results suggest that T.?gondii and the ROP kinases can be used to probe immune signaling pathways.     

Glycans and neural cell interactions

Carbohydrate-carrying molecules in the nervous system have important roles during development, regeneration and synaptic plasticity. Carbohydrates mediate interactions between recognition molecules, thereby contributing to the formation of a complex molecular meshwork at the cell surface and in the extracellular matrix. The tremendous structural diversity of glycan chains allows for immense combinatorial possibilities that might underlie the fine-tuning of cell-cell and cell-matrix interactions.