Rotaviruses (RVs) cause life-threatening diarrhea in infants and children worldwide. Recent biochemical and epidemiological studies underscore the importance of histo-blood group antigens (HBGA) as both cell attachment and susceptibility factors for the globally dominant P, P, and P genotypes of human RVs. How these genotypes interact with HBGA is not known. Here, our crystal structures of P and a neonate-specific P VP8*s alone and in complex with H-type I HBGA reveal a unique glycan binding site that is conserved in the globally dominant genotypes and allows for the binding of ABH HBGAs, consistent with their prevalence. Remarkably, the VP8* of P RVs isolated from neonates displays subtle structural changes in this binding site that may restrict its ability to bind branched glycans. This provides a structural basis for the age-restricted tropism of some P RVs as developmentally regulated unbranched glycans are more abundant in the neonatal gut.
Filariases are diseases caused by infection with filarial nematodes and transmitted by insect vectors. The filarial roundworm Dirofilaria immitis causes heartworm disease in dogs and other carnivores. D. immitis is closely related to Onchocerca volvulus, Wuchereria bancrofti and Brugia malayi, which cause onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis) in humans and are neglected tropical diseases. Serum N-glycosylation is very sensitive to both pathological infections and changes in mammalian biology due to normal aging or lifestyle choices. Here, we report significant changes in the serum N-glycosylation profiles of dogs infected with D. immitis. Our data derive from analysis of serum from dogs with established patent infections and from a longitudinal infection study. Overall, galactosylation and core fucosylation increase, while sialylation decreases in infected dog sera. We also identify individual glycan structures that change significantly in their relative abundance during infection. Notably, the abundance of the most dominant N-glycan in canine serum (biantennary, disialylated A2G2S2) decreases by over 10 percentage points during the first 6 months of infection in each dog analyzed. This is the first longitudinal study linking changes in mammalian serum N-glycome to progression of a parasitic infection
Glycosylation is the most common post-translational modification of serum proteins, and changes in the type and abundance of glycans in human serum have been correlated with a growing number of human diseases. While the glycosylation pattern of human serum is well studied, little is known about the profiles of other mammalian species. Here, we report detailed glycosylation profiling of canine serum by hydrophilic interaction chromatography-ultraperformance liquid chromatography (HILIC-UPLC) and mass spectrometry. The domestic dog (Canis familiaris) is a widely used model organism and of considerable interest for a large veterinary community. We found significant differences in the serum N-glycosylation profile of dogs compared to that of humans, such as a lower abundance of galactosylated and sialylated glycans. We also compare the N-glycan profile of canine serum to that of canine IgG - the most abundant serum glycoprotein. Our data will serve as a baseline reference for future studies when performing serum analyses of various health and disease states in dogs.
To better understand the role of B cells, potential mechanisms for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjögren's Syndrome (SS), we explored selection pressures and N-glycosylation acquired by somatic mutation (acN-glyc) in the immunoglobulin (Ig) variable regions (V-regions) of antibody secreting cells (ASCs) isolated from the minor salivary glands of SS patients and non-SS controls with sicca symptoms.
We report a novel method to produce and characterize recombinant monoclonal antibodies (mAbs) from SS patient and control labial salivary gland single-cell sorted ASC infiltrates that can be utilized to concurrently probe any other expressed genes. V-regions were amplified by RT-PCR, sequenced, and analyzed for incidence of N-glycosylation and selection pressure, then expressed as the native mAbs, or mutant mAbs lacking the acN-glyc for specificity testing. Protein modeling was used to demonstrate how even acN-glycs outside of the complementarity-determining region (CDR) could participate in, or inhibit, antigen binding.
V-region sequence analyses revealed clonal expansions and evidence for secondary light chain editing and allelic inclusion not previously reported in SS. We found increased acN-glycs in the sequences from SS patients and that acN-glycs were associated with increased replacement mutations and lowered selection pressure. We also identified a clonal set of polyreactive mAbs with differential FWR1 acN-glycs and demonstrated that removal of the acN-glyc could nearly abolish binding to the autoantigens.
Our findings support an alternative mechanism involving V-region N-glycosylation for the selection and proliferation of some autoreactive B cells in SS patients.
Mucin-type O-glycans decorate >80% of secretory and cell surface proteins and contribute to health and disease. However, dynamic alterations in the O-glycome are poorly understood because current O-glycomic methodologies are not sufficiently sensitive nor quantitative. Here we describe a novel isotope labeling approach termed Isotope-Cellular O-glycome Reporter Amplification (ICORA) to amplify and analyze the O-glycome from cells. In this approach, cells are incubated with Ac3GalNAc-Bn (Ac3GalNAc-[1H7]Bn) or a heavy labeled Ac3GalNAc-BnD7 (Ac3GalNAc-[2D7]Bn) O-glycan precursor (7 Da mass difference), which enters cells and upon de-esterification is modified by Golgi enzymes to generate Bn-O-glycans secreted into the culture media. After recovery, heavy and light Bn-O-glycans from two separate conditions are mixed, analyzed by MS, and statistically interrogated for changes in O-glycan abundance using a semi-automated approach. ICORA enables ~100–1000 fold enhanced sensitivity and increased throughput compared to traditional O-glycomics. We validated ICORA with model cell lines and used it to define alterations in the O-glycome in colorectal cancer. ICORA is a useful tool to explore the dynamic regulation of the O-glycome in health and disease.
Glycan-binding proteins (GBPs) play critical roles in diverse cellular functions such as cell adhesion, signal transduction and immune response. Studies of the interaction between GBPs and glycans have been hampered by the availability of high throughput and high-content technologies. Here we report multiplex glycan bead array (MGBA) that allows simultaneous analyses of 384 samples and up to 500 glycans in a single assay. The specificity, sensitivity and reproducibility of MGBA are evaluated using 39 plant lectins, 13 recombinant anti-glycan antibodies, and mammalian GBPs. We demonstrate the utility of this platform by the analyses of natural anti-glycan IgM and IgG antibodies in 961 human serum samples and the discovery of anti-glycan antibody biomarkers for ovarian cancer. Our data indicate that the MGBA platform is particularly suited for large population-based studies that require the analyses of large numbers of samples and glycans.
Here we present a Caenorhabditis elegans N-glycan shotgun array. This nematode serves as a model organism for many areas of biology including but not limited to tissue development, host–pathogen interactions, innate immunity, and genetics. Caenorhabditis elegans N-glycans contain structural motifs that are also found in other nematodes as well as trematodes and lepidopteran species. Glycan binding toxins that interact with C. elegansglycoconjugates also do so with some agriculturally relevant species, such as Haemonchus contortus, Ascaris suum, Oesophagostomum dentatum and Trichoplusia ni. This situation implies that protein–carbohydrate interactions seen with C. elegans glycans may also occur in other species with related glycan structures. Therefore, this array may be useful to study these relationships in other nematodes as well as trematode and insect species. The array contains 134 distinct glycomers spanning a wide range of C. elegans N-glycans including the subclasses high mannose, pauci mannose, high fucose, mammalian-like complex and phosphorylcholine substituted forms. The glycans presented on the array have been characterized by two-dimensional separation, ion trap mass spectrometry, and lectin affinity. High fucose glycans were well represented and contain many novel core structures found in C. elegans as well as other species. This array should serve as an investigative platform for carbohydrate binding proteins that interact with N-glycans of C. elegans and over a range of organisms that contain glycan motifs conserved with this nematode.
Oligomannose-type glycans are among the major targets on the gp120 component of the HIV envelope protein (Env) for broadly neutralizing antibodies (bnAbs). However, attempts to elicit oligomannose-specific nAbs by immunizing with natural or synthetic oligomannose have so far not been successful, possibly due to B cell tolerance checkpoints. Here we design and synthesize oligomannose mimetics, based on the unique chemical structure of a recently identified bacterial lipooligosaccharide, to appear foreign to the immune system. One of these mimetics is bound avidly by members of a family of oligomannose-specific bnAbs and their putative common germline precursor when presented as a glycoconjugate. The crystal structure of one of the mimetics bound to a member of this bnAb family confirms the antigenic resemblance. Lastly, immunization of human-antibody transgenic animals with a lead mimetic evokes nAbs with specificities approaching those of existing bnAbs. These results provide evidence for utilizing antigenic mimicry to elicit oligomannose-specific bnAbs to HIV-1.
Variable lymphocyte receptors (VLRs), the leucine-rich repeat (LRR)-based antigen receptors of jawless fish, have great utility in a wide variety of biochemical and biological applications, similar to classical Ig-based antibodies. VLR-based reagents may be particularly useful when traditional antibodies are not available. An anti-idiotype lamprey VLR, VLR39, has previously been identified that recognizes the heavy-chain CDR3 of the B-cell receptor (BCR) of a leukemic clone from a patient with chronic lymphocytic leukemia (CLL). VLR39 was used successfully to track the re-emergence of this clone in the patient following chemotherapy. Here, the crystal structure of VLR39 is presented at 1.5 Å resolution and compared with those of other protein-specific VLRs. VLR39 adopts a curved solenoid fold and exhibits substantial structural similarity to other protein-binding VLRs. VLR39 has a short LRRCT loop that protrudes outwards away from the concave face and is similar to those of its protein-specific VLR counterparts. Analysis of the VLR39-BCR interaction by size-exclusion chromatography and biolayer interferometry using the scFv version of the BCR confirms that VLR39 recognizes the BCR Fv region. Such VLR-based reagents may be useful for identifying and monitoring leukemia in CLL patients and in other clinical diagnostic assays.
Glycans possess significant chemical diversity; glycan binding proteins (GBPs) recognize specific glycans to translate their structures to functions in various physiological and pathological processes. Therefore, the discovery and characterization of novel GBPs and characterization of glycan-GBP interactions are significant to provide potential targets for therapeutic intervention of many diseases. Here, we report the biochemical, functional, and structural characterization of a 130-amino-acid protein, Y3, from the mushroom Biochemical studies of recombinant Y3 from a yeast expression system demonstrated the protein is a unique GBP. Additionally, we show that Y3 exhibits selective and potent cytotoxicity toward human T-cell leukemia Jurkat cells compared with a panel of cancer cell lines via inducing caspase-dependent apoptosis. Screening of a glycan array demonstrated GalNAcβ1-4(Fucα1-3)GlcNAc (LDNF) as a specific Y3-binding ligand. To provide a structural basis for function, the crystal structure was solved to a resolution of 1.2 Å, revealing a single-domain αβα-sandwich motif. Two monomers were dimerized to form a large 10-stranded, antiparallel β-sheet flanked by α-helices on each side, representing a unique oligomerization mode among GBPs. A large glycan binding pocket extends into the dimeric interface, and docking of LDNF identified key residues for glycan interactions. Disruption of residues predicted to be involved in LDNF/Y3 interactions resulted in the significant loss of binding to Jurkat T-cells and severely impaired their cytotoxicity. Collectively, these results demonstrate Y3 to be a GBP with selective cytotoxicity toward human T-cell leukemia cells and indicate its potential use in cancer diagnosis and treatment.
Mannose binding lectin (MBL) is a serum collagenous C-type lectin that plays an important role in the innate immune protection against pathogens. Previously, human and mouse studies have demonstrated that MBL binds a broad range of pathogens that results in their neutralization through agglutination, enhanced phagocytosis, and/or complement activation via the lectin pathway. The role of MBL in chicken is not well understood although the MBL concentration in serum seems to correlate with protection against infections. To investigate the role of MBL in chicken further, recombinant chicken MBL (RcMBL) was produced in HeLa R19 cells and purified using mannan affinity chromatography followed by gel filtration. RcMBL was shown to be structurally and functionally similar to native chicken MBL (NcMBL) isolated from serum. RcMBL is expressed as an oligomeric protein (mixture of trimers and oligomerized trimers) with a monomeric mass of 26kDa as determined by mass spectrometry, corresponding to the predicted mass. Glycan array analysis indicated that RcMBL bound most strongly to high-mannose glycans but also glycans with terminal fucose and GlcNac residues. The biological activity of RcMBL was demonstrated via its capacity to agglutinate Salmonella Typhimurium and to inhibit the hemagglutination activity of influenza A virus. The production of a structurally well-characterized and functionally active RcMBL will facilitate detailed studies into the protective role of MBL in innate defense against pathogens in chicken and other avian species.
JAA-F11 is a highly specific mouse monoclonal to the Thomsen-Friedenreich Antigen (TF-Ag) which is an alpha-O-linked disaccharide antigen on the surface of ~80% of human carcinomas, including breast, lung, colon, bladder, ovarian, and prostate cancers, and is cryptic on normal cells. JAA-F11 has potential, when humanized, for cancer immunotherapy for multiple cancer types. Humanization of JAA-F11, was performed utilizing complementarity determining regions grafting on a homology framework. The objective herein is to test the specificity, affinity and biology efficacy of the humanized JAA-F11 (hJAA-F11). Using a 609 target glycan array, 2 hJAA-F11 constructs were shown to have excellent chemical specificity, binding only to TF-Ag alpha-linked structures and not to TF-Ag beta-linked structures. The relative affinity of these hJAA-F11 constructs for TF-Ag was improved over the mouse antibody, while T20 scoring predicted low clinical immunogenicity. The hJAA-F11 constructs produced antibody-dependent cellular cytotoxicity in breast and lung tumor lines shown to express TF-Ag by flow cytometry. Internalization of hJAA-F11 into cancer cells was also shown using a surface binding ELISA and confirmed by immunofluorescence microscopy. Both the naked hJAA-F11 and a maytansine-conjugated antibody (hJAA-F11-DM1) suppressed in vivo tumor progression in a human breast cancer xenograft model in SCID mice. Together, our results support the conclusion that the humanized antibody to the TF-Ag has potential as an adjunct therapy, either directly or as part of an antibody drug conjugate, to treat breast cancer, including triple negative breast cancer which currently has no targeted therapy, as well as lung cancer.
F-type lectins are fucose binding lectins with characteristic fucose binding and calcium binding motifs. Although they occur with a selective distribution in viruses, prokaryotes and eukaryotes, most biochemical studies have focused on vertebrate F-type lectins. Recently, using sensitive bioinformatics search techniques on the non-redundant database, we had identified many microbial F-type lectin domains with diverse domain organizations. We report here the biochemical characterization of F-type lectin domains from Cyanobium sp. PCC 7001, Myxococcus hansupus and Leucothrix mucor. We demonstrate that while all these three microbial F-type lectin domains bind to the blood group H antigen epitope on fucosylated glycans, there are fine differences in their glycan binding specificity. Cyanobium sp. PCC 7001 F-type lectin domain binds exclusively to extended H type-2 motif, Myxococcus hansupus F-type lectin domain binds to B, H type-1 and Lewis motifs, and Leucothrix mucor F-type lectin domain binds to a wide range of fucosylated glycans, including A, B, H and Lewis antigens. We believe that these microbial lectins will be useful additions to the glycobiologist's toolbox for labeling, isolating and visualizing glycans.
Large quantities of immunoglobulin A (IgA) are constitutively secreted by intestinal plasma cells to coat and contain the commensal microbiota, yet the specificity of these antibodies remains elusive. Here we profiled the reactivities of single murine IgA plasma cells by cloning and characterizing large numbers of monoclonal antibodies. IgAs were not specific to individual bacterial taxa but rather polyreactive, with broad reactivity to a diverse, but defined, subset of microbiota. These antibodies arose at low frequencies among naïve B cells and were selected into the IgA repertoire upon recirculation in Peyer's patches. This selection process occurred independent of microbiota or dietary antigens. Furthermore, although some IgAs acquired somatic mutations, these did not substantially influence their reactivity. These findings reveal an endogenous mechanism driving homeostatic production of polyreactive IgAs with innate specificity to microbiota.
Infection with parasitic helminths affects humanity and animal welfare. Parasitic helminths have the capacity to modulate host immune responses to promote their survival in infected hosts, often for a long time leading to chronic infections. In contrast to many infectious microbes, however, the helminths are able to induce immune responses that show positive bystander effects such as the protection to several immune disorders, including multiple sclerosis, inflammatory bowel disease, and allergies. They generally promote the generation of a tolerogenic immune microenvironment including the induction of type 2 (Th2) responses and a sub-population of alternatively activated macrophages. It is proposed that this anti-inflammatory response enables helminths to survive in their hosts and protects the host from excessive pathology arising from infection with these large pathogens. In any case, there is an urgent need to enhance understanding of how helminths beneficially modulate inflammatory reactions, to identify the molecules involved and to promote approaches to exploit this knowledge for future therapeutic interventions. Evidence is increasing that C-type lectins play an important role in driving helminth-mediated immune responses. C-type lectins belong to a large family of calcium-dependent receptors with broad glycan specificity. They are abundantly present on immune cells, such as dendritic cells and macrophages, which are essential in shaping host immune responses. Here, we will focus on the role of the C-type lectin macrophage mannose receptor (MR) in helminth-host interactions, which is a critically understudied area in the field of helminth immunobiology. We give an overview of the structural aspects of the MR including its glycan specificity, and the functional implications of the MR in helminth-host interactions focusing on a few selected helminth species.
Secretory granules released by cytotoxic T lymphocytes (CTLs) are powerful weapons against intracellular microbes and tumor cells. Despite significant progress, there is still limited information on the molecular mechanisms implicated in target-driven degranulation, effector cell survival and composition and structure of the lytic granules. Here, using a proteomic approach we identified a panel of putative cytotoxic granule proteins, including some already known granule constituents and novel proteins that contribute to regulate the CTL lytic machinery. Particularly, we identified galectin-1 (Gal1), an endogenous immune regulatory lectin, as an integral component of the secretory granule machinery and unveil the unexpected function of this lectin in regulating CTL killing activity. Mechanistic studies revealed the ability of Gal1 to control the non-secretory lytic pathway by influencing Fas-Fas ligand interactions. This study offers new insights on the composition of the cytotoxic granule machinery, highlighting the dynamic cross talk between secretory and non-secretory pathways in controlling CTL lytic function.
Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, our approach involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplified purification scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases including key N-acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated and subsequently used to develop a glycan microarray for high-throughput, fluorescence-based screening of glycan-binding proteins. Taken together, these results confirm our combined synthesis strategy as a new, user-friendly route for supplying chemically defined human glycans simply by combining biosynthetically-derived precursors with enzymatic remodeling.
LewisX (LeX) is a branched trisaccharide Galβ1→4(Fucα1→3)GlcNAc that is expressed on many cell surface glycoproteins and plays critical roles in innate and adaptive immune responses. However, efficient synthesis of glycopeptides bearing LeX remains a major limitation for structure-function studies of the LeX determinant. Here we report a total synthesis of a LeX pentasaccharide 1 using a regioselective 1-benzenesulfinyl piperidine/triflic anhydride promoted [3 + 2] glycosylation. The presence of an Fmoc-threonine amino acid facilitates incorporation of the pentasaccharide in solid phase peptide synthesis, providing a route to diverse O-linked LeX glycopeptides. The described approach is broadly applicable to the synthesis of a variety of complex glycopeptides containing O-linked LeX or sialyl LewisX (sLeX).
High-quality reagents to study and detect glycans with high specificity for research and clinical applications are severely lacking. Here, we structurally and functionally characterize several variable lymphocyte receptor (VLR)-based antibodies from lampreys immunized with O erythrocytes that specifically recognize the blood group H-trisaccharide type II antigen. Glycan microarray analysis and biophysical data reveal that these VLRs exhibit greater specificity for H-trisaccharide compared with the plant lectin UEA-1, which is widely used in blood typing. Among these antibodies, O13 exhibits superior specificity for H-trisaccharide, the basis for which is revealed by comparative analysis of high-resolution VLR:glycan crystal structures. Using a structure-guided approach, we designed an O13 mutant with further enhanced specificity for H-trisaccharide. These insights into glycan recognition by VLRs suggest that lampreys can produce highly specific glycan antibodies, and are a valuable resource for the production of next-generation glycan reagents for biological and biomedical research and as diagnostics and therapeutics.
PMN-expressed fucosylated glycans from the Lewis glycan family, including Lewis-x (Lex) and sialyl Lewis-x (sLex), have previously been implicated in the regulation of important PMN functions, including selectin-mediated trafficking across vascular endothelium. Although glycans, such as Lex and sLex, which are based on the type 2 sequence (Galβ1-4GlcNAc-R), are abundant on PMNs, the presence of type 1 Galβ1-3GlcNAc-R glycans required for PMN expression of the closely related stereoisomer of Lex, termed Lewis-A (Lea), has not, to our knowledge, been reported. Here, we show that Lea is abundantly expressed by human PMNs and functionally regulates PMN migration. Using mAbs whose precise epitopes were determined using glycan array technology, Lea function was probed using Lea-selective mAbs and lectins, revealing increased PMN transmigration across model intestinal epithelia, which was independent of epithelial-expressed LeaAnalyses of glycan synthetic machinery in PMNs revealed expression of β1-3 galactosyltransferase and α1-4 fucosyltransferase, which are required for Lea synthesis. Specificity of functional effects observed after ligation of Lea was confirmed by failure of anti-Lea mAbs to enhance migration using PMNs from individuals deficient in α1-4 fucosylation. These results demonstrate that Lea is expressed on human PMNs, and its specific engagement enhances PMN migration responses. We propose that PMN Lea represents a new target for modulating inflammation and regulating intestinal, innate immunity.