The biological recognition of human milk glycans (HMGs) is poorly understood. Because HMGs are rich in galactose we explored whether they might interact with human galectins, which bind galactose-containing glycans and are highly expressed in epithelial cells and other cell types. We screened a number of human galectins for their binding to HMGs on a shotgun glycan microarray consisting of 247 HMGs derived from human milk, as well as to a defined HMG microarray. Recombinant human galectins (hGal)-1, -3, -4, -7, -8 and -9 bound selectively to glycans, with each galectin recognizing a relatively unique binding motif; by contrast hGal-2 did not recognize HMGs, but did bind to the human blood group A Type 2 determinants on other microarrays. Unlike other galectins, hGal-7 preferentially bound to glycans expressing a terminal Type 1 (Galβ1-3GlcNAc) sequence, a motif that had eluded detection on non-HMG glycan microarrays. Interactions with HMGs were confirmed in a solution setting by isothermal titration microcalorimetry and hapten inhibition experiments. These results demonstrate that galectins selectively bind to HMGs and suggest the possibility that galectin-HMG interactions may play a role in infant immunity.
Glycans are known as the third major class of biopolymers, next to DNA and proteins. They cover the surfaces of many cells, serving as the 'face' of cells, whereby other biomolecules and viruses interact. The structure of glycans, however, differs greatly from DNA and proteins in that they are branched, as opposed to linear sequences of amino acids or nucleotides. Therefore, the storage of glycan information in databases, let alone their curation, has been a difficult problem. This has caused many duplicated efforts when integration is attempted between different databases, making an international repository for glycan structures, where unique accession numbers are assigned to every identified glycan structure, necessary. As such, an international team of developers and glycobiologists have collaborated to develop this repository, called GlyTouCan and is available at http://glytoucan.org/, to provide a centralized resource for depositing glycan structures, compositions and topologies, and to retrieve accession numbers for each of these registered entries. This will thus enable researchers to reference glycan structures simply by accession number, as opposed to by chemical structure, which has been a burden to integrate glycomics databases in the past.
BACKGROUND: Primary deficiencies in mannosylation of N-glycans are seen in a majority of patients with congenital disorders of glycosylation (CDG). We report the discovery of a series of novel N-glycans in sera, plasma, and cultured skin fibroblasts from patients with CDG having deficient mannosylation.
METHOD: We used LC-MS/MS and MALDI-TOF-MS analysis to identify and quantify a novel N-linked tetrasaccharide linked to the protein core, an N-tetrasaccharide (Neu5Acα2,6Galβ1,4-GlcNAcβ1,4GlcNAc) in plasma, serum glycoproteins, and a fibroblast lysate from patients with CDG caused by ALG1 [ALG1 (asparagine-linked glycosylation protein 1), chitobiosyldiphosphodolichol β-mannosyltransferase], PMM2 (phosphomannomutase 2), and MPI (mannose phosphate isomerase).
RESULTS: Glycoproteins in sera, plasma, or cell lysate from ALG1-CDG, PMM2-CDG, and MPI-CDG patients had substantially more N-tetrasaccharide than unaffected controls. We observed a >80% decline in relative concentrations of the N-tetrasaccharide in MPI-CDG plasma after mannose therapy in 1 patient and in ALG1-CDG fibroblasts in vitro supplemented with mannose.
CONCLUSIONS: This novel N-tetrasaccharide could serve as a diagnostic marker of ALG1-, PMM2-, or MPI-CDG for screening of these 3 common CDG subtypes that comprise >70% of CDG type I patients. Its quantification by LC-MS/MS may be useful for monitoring therapeutic efficacy of mannose. The discovery of these small N-glycans also indicates the presence of an alternative pathway in N-glycosylation not recognized previously, but its biological significance remains to be studied.
Genetic evidence suggests that the Schistosoma mansoni genome contains six genes that encode α1,3-fucosyltransferases (smFuTs). To date, the activities and specificities of these putative fucosyltransferases are unknown. As Schistosoma express a variety of fucosylated glycans, including the Lewis X antigen Galβ1-4(Fucα1-3)GlcNAcβ-R, it is likely that this family of genes encode enzymes that are partly responsible for the generation of those structures. Here, we report the molecular cloning of fucosyltransferase-F (smFuT-F) from S. mansoni, as a soluble, green fluorescent protein fusion protein and its acceptor specificity. The gene smFuT-F was expressed in HEK freestyle cells, purified by affinity chromatography, and analyzed toward a broad panel of glycan acceptors. The enzyme product of smFuT-F effectively utilizes a type II chain acceptor Galβ1-4GlcNAc-R, but notably not the LDN sequence GalNAcβ1-4GlcNAc-R, to generate Lewis X type-glycans, and smFuT-F transcripts are present in all intramammalian life stages.
The acquisition of mannose 6-phosphate (Man6P) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6P monosaccharide that, through an array-screening approach against a number of phosphorylated N-glycans, is shown to bind mono- and diphosphorylated Man6 and Man7 glycans that contain terminal αMan6P(1 → 2)αMan(1 → 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man8 or mono- or diphosphorylated Man9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6P reveals that specificity and affinity is achieved via multiple hydrogen bonds to the mannose ring and two salt bridges to the phosphate moiety. In common with both MPRs, loss of binding was observed for scFv M6P-1 at pH values below the second pKa of Man6P (pKa = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6P is the main driving force for the loss of binding at acidic lysosomal pH (e.g. lysosome pH ∼ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6P recognition.
Polymorphonuclear leukocytes (PMNs) are innate immune cells whose principal function is to migrate from the blood to sites of inflammation, where they exert crucial anti-infectious and immunomodulatory effects. However, dysregulated migration of PMNs into mucosal epithelial tissues is characteristic of chronic inflammatory disorders, including inflammatory bowel disease. Carbohydrate-mediated binding interactions between PMN Lewis glycans and endothelial glycan-binding proteins are critical for initial migration of PMN out of the vasculature. However, the role of Lewis glycans during transepithelial migration (TEM) has not been well characterized. Herein, we show that antibody blockade of Lewis X (Le(x)) displayed as terminal glycan residues on the PMN surface blocks chemotaxis and TEM while enhancing PMN-adhesive interactions with intestinal epithelia. Unexpectedly, targeting of subterminal Le(x) residues within glycan chains had no effect on PMN migration or adhesive interactions. There was increased surface expression of Le(x) on PMN after TEM, and blockade of terminal Le(x) regulated post-migratory PMN functions, increasing PMN phagocytosis and the surface mobilization of azurophilic (CD63, myeloperoxidase, and neutrophil elastase) and specific (CD66b and lactoferrin) granule markers. These findings suggest that terminal Le(x) represents a potential target for regulating PMN trafficking and function in inflamed mucosa. Furthermore, given its abundant expression on migrating PMN, Le(x) may be a rational target for modulating inflammation in diseases where dysregulated PMN influx is associated with host tissue damage.
Glycan microarrays have become a powerful platform to investigate the interactions of carbohydrates with a variety of biomolecules. However, the number and diversity of glycans available for use in such arrays represent a key bottleneck in glycan array fabrication. To address this challenge, we describe a novel glycan array platform based on surface patterning of engineered glycophages that display unique carbohydrate epitopes. Specifically, we show that glycophages are compatible with surface immobilization procedures and that phage-displayed oligosaccharides retain the ability to be recognized by different glycan-binding proteins (e.g. antibodies and lectins) after immobilization. A key advantage of glycophage arrays is that large quantities of glycophages can be produced biosynthetically from recombinant bacteria and isolated directly from bacterial supernatants without laborious purification steps. Taken together, the glycophage array technology described here should help to expand the diversity of glycan libraries and provide a complement to the existing toolkit for high-throughput analysis of glycan-protein interactions.
Galectins are an evolutionarily ancient family of glycan-binding proteins (GBPs) and are found in all animals. Although they were discovered over 30 years ago, ideas about their biological functions continue to evolve. Current evidence indicates that galectins, which are the only known GBPs that occur free in the cytoplasm and extracellularly, are involved in a variety of intracellular and extracellular pathways contributing to homeostasis, cellular turnover, cell adhesion, and immunity. Here we review evolving insights into galectin biology from a historical perspective and explore current evidence regarding biological roles of galectins.
Galectins can display unique sensitivity to oxidative changes that result in significant conformational alterations that prevent carbohydrate recognition. While a variety of approaches can be utilized to prevent galectin oxidation, several of these require inclusion of reducing agents that not only prevent galectins from undergoing oxidative inactivation, but can also interfere with normal redox potentials required for fundamental cellular processes. To overcome limitations associated with placing cells in an artificial reducing environment, cysteine residues on galectins can be directly alkylated with iodoacetamide to form a stable thioether adduct that is resistant to further modification. Iodoacetamide alkylated galectin remains stable over prolonged periods of time and retains the carbohydrate binding and biological activities of the native protein. As a result, this approach allows examination of the biological roles of a stabilized form of galectin-1 without introducing the confounding variables that can occur when typical soluble reducing agents are employed.
Cellular turnover represents a fundamental aspect of immunological homeostasis. While many factors appear to regulate leukocyte removal during inflammatory resolution, recent studies suggest that members of the galectin family play a unique role in orchestrating this process. Unlike cellular removal through apoptotic cell death, several members of the galectin family induce surface expression of phosphatidylserine (PS), a phagocytic marker on cells undergoing apoptosis, in the absence of cell death. However, similar to PS on cells undergoing apoptosis, galectin-induced PS exposure sensitizes cells to phagocytic removal. As galectins appear to prepare cells for phagocytic removal without actually inducing apoptotic cell death, this process has recently been coined preaparesis. Given the unique characteristics of galectin-induced PS exposure in the context of preaparesis, we will examine important considerations when evaluating the potential impact of different galectin family members on PS exposure and cell viability.
Over a century ago, Karl Landsteiner discovered that blood group antigens could predict the immunological outcome of red blood cell transfusion. While the discovery of ABO(H) blood group antigens revolutionized transfusion medicine, many questions remain regarding the development and regulation of naturally occurring anti-blood group antibody formation. Early studies suggested that blood group antibodies develop following stimulation by bacteria that express blood group antigens. While this may explain the development of anti-blood group antibodies in blood group negative individuals, how blood group positive individuals, who cannot generate anti-blood group antibodies, protect themselves against blood group positive microbes remained unknown. Recent studies suggest that several members of the galectin family specifically target blood group positive microbes, thereby providing innate immune protection against blood group antigen positive microbes regardless of the blood group status of an individual. Importantly, subsequent studies suggest that this unique form of immunity may not be limited to blood group expressing microbes, but may reflect a more generalized form of innate immunity against molecular mimicry. As this form of antimicrobial activity represents a unique and unprecedented form of immunity, we will examine important considerations and methodological approaches that can be used when seeking to ascertain the potential antimicrobial activity of various members of the galectin family.
We have utilized simple flow cytometric and fluorescence-based solid phase assays to study the interaction of glycan-binding proteins (GBP) to cell surface glycoconjugates. These methods utilize commonly employed flow cytometry techniques and commercially available streptavidin-coated microplates to immobilize various biotinylated ligands, such as glycopeptides, oligosaccharides, and whole cells. Using this approach, fluorescently labeled GBPs, in particular, members of the galectin family, can be interrogated for potential interactions with cell surface carbohydrates, including elucidation of the potential impact of alterations in glycosylation on carbohydrate recognition. Using these approaches, we present examples of flow cytometric and fluorescence-based solid phase assays to study galectin-carbohydrate interactions.
Glycan binding proteins (GBPs) possess the unique ability to regulate a wide variety of biological processes through interactions with highly modifiable cell surface glycans. While many studies demonstrate the impact of glycan modification on GBP recognition and activity, the relative contribution of subtle changes in glycan structure on GBP binding can be difficult to define. To overcome limitations in the analysis of GBP-glycan interactions, recent studies utilized glycan microarray platforms containing hundreds of structurally defined glycans. These studies not only provided important information regarding GBP-glycan interactions, but have also resulted in significant insight into the binding specificity and biological activity of the galectin family. We will describe the methods used when employing glycan microarray platforms to examine galectin-glycan binding specificity and function.
Despite the paradigm that carbohydrates are T cell-independent antigens, isotype-switched glycan-specific immunoglobulin G (IgG) antibodies and polysaccharide-specific T cells are found in humans. We used a systems-level approach combined with glycan array technology to decipher the repertoire of carbohydrate-specific IgG antibodies in intravenous and subcutaneous immunoglobulin preparations. A strikingly universal architecture of this repertoire with modular organization among different donor populations revealed an association between immunogenicity or tolerance and particular structural features of glycans. Antibodies were identified with specificity not only for microbial antigens but also for a broad spectrum of host glycans that serve as attachment sites for viral and bacterial pathogens and/or exotoxins. Tumor-associated carbohydrate antigens were differentially detected by IgG antibodies, whereas non-IgG2 reactivity was predominantly absent. Our study highlights the power of systems biology approaches to analyze immune responses and reveals potential glycan antigen determinants that are relevant to vaccine design, diagnostic assays, and antibody-based therapies.
The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays an essential role in lysosome biogenesis by targeting ∼ 60 different phosphomannosyl-containing acid hydrolases to the lysosome. This type I membrane glycoprotein has a large extracellular region comprised of 15 homologous domains. Two mannose 6-phosphate (M6P) binding sites have been mapped to domains 3 and 9, whereas domain 5 binds preferentially to the phosphodiester, M6P-N-acetylglucosamine (GlcNAc). A structure-based sequence alignment predicts that the C-terminal domain 15 contains three out of the four conserved residues identified as essential for carbohydrate recognition by domains 3, 5 and 9 of the CI-MPR, but lacks two cysteine residues that are predicted to form a disulfide bond. To determine whether domain 15 of the CI-MPR has lectin activity and to probe its carbohydrate-binding specificity, truncated forms of the CI-MPR were tested for binding to acid hydrolases with defined N-glycans in surface plasmon resonance analyses, and used to interrogate a phosphorylated glycan microarray. The results show that a construct encoding domains 14-15 binds both M6P and M6P-GlcNAc with similar affinity (Kd = 13 and 17 μM, respectively). Site-directed mutagenesis studies demonstrate the essential role of the conserved Tyr residue in domain 15 for phosphomannosyl binding. A structural model of domain 15 was generated that predicted an Arg residue to be in the binding pocket and mutagenesis studies confirmed its important role in carbohydrate binding. Together, these results show that the CI-MPR contains a fourth carbohydrate-recognition site capable of binding both phosphomonoesters and phosphodiesters.
Neoplastic transformation results in a wide variety of cellular alterations that impact the growth, survival, and general behavior of affected tissue. Although genetic alterations underpin the development of neoplastic disease, epigenetic changes can exert an equally significant effect on neoplastic transformation. Among neoplasia-associated epigenetic alterations, changes in cellular glycosylation have recently received attention as a key component of neoplastic progression. Alterations in glycosylation appear to not only directly impact cell growth and survival but also facilitate tumor-induced immunomodulation and eventual metastasis. Many of these changes may support neoplastic progression, and unique alterations in tumor-associated glycosylation may also serve as a distinct feature of cancer cells and therefore provide novel diagnostic and even therapeutic targets.
Bacteriophage receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.
This review discusses the challenges facing research in 'functional glycomics' and the novel technologies that are being developed to advance the field. The structural complexity of glycans and glycoconjugates makes studies of both their structures and recognition difficult. However, these intricate structures can be captured from their natural sources, isolated and fluorescently-tagged for detailed structural analysis and for presentation on glycan microarrays for functional recognition by glycan-binding proteins. These advances in glycan preparation and manipulation enable the streamlining of functional glycomics studies and will help to propel the field forward in studying natural, biologically relevant glycans.
Blockade of P-selectin (P-sel)/PSGL-1 interactions holds significant potential for treatment of disorders of innate immunity, thrombosis and cancer. Current inhibitors remain limited due to low binding affinity or by the recognized disadvantages inherent to chronic administration of antibody therapeutics. Here we report an efficient approach for generating glycosulfopeptide mimics of N-terminal PSGL-1 through development of a stereoselective route for multi-gram scale synthesis of the C2 O-glycan building block and replacement of hydrolytically labile tyrosine sulfates with isosteric sulfonate analogues. Library screening afforded a compound of exceptional stability, GSnP-6, that binds to human P-sel with nanomolar affinity (Kd~22 nM). Molecular dynamics simulation defines the origin of this affinity in terms of a number of critical structural contributions. GSnP-6 potently blocks P-sel/PSGL-1 interactions in vitro and in vivo and represents a promising candidate for the treatment of diseases driven by acute and chronic inflammation.
The mammalian immune system responds to eukaryotic glycan antigens during infections, cancer, and autoimmune disorders, but the immunological bases for such responses are unclear. Conjugate vaccines containing bacterial polysaccharides linked to carrier proteins (neoglycoconjugates) have proven successful, but these often contain repeating epitopes and the reducing end of the glycan is less important, unlike typical glycan determinants in eukaryotes, which are shorter in length and may include the reducing end. Here, we have compared the effects of two linkage methods, one that opens the ring at the reducing end of the glycan, and one that leaves the reducing end closed, on the glycan specificity of the vaccine response in rabbits and mice. We immunized rabbits and mice with bovine serum albumin (BSA) conjugates of synthetic open- and closed-ring forms (OR versus CR) of a simple tetrasaccharide lacto-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and tested reactivity to the immunogens and several related glycans in both OR and CR versions on glycan microarrays. We found that in rabbits the immune response to the CR conjugate was directed toward the glycan, whereas the OR conjugate elicited antibodies to the reducing end of the glycan and linker region but not specifically to the glycan itself. Unexpectedly, mice did not generate a glycan-specific response to the CR conjugate. Our findings indicate that the reducing end of the sugar is crucial for generation of a glycan-specific response to some eukaryotic vaccine epitopes, and that there are species-specific differences in the ability to make a glycan-specific response to some glycoconjugates. These findings warrant further investigation with regard to rational design of glycoconjugate vaccines.