Publications

2019
Byrd-Leotis L, Jia N, Dutta S, Trost JF, Gao C, Cummings SF, Braulke T, Müller-Loennies S, Heimburg-Molinaro J, Steinhauer DA, et al. Influenza binds phosphorylated glycans from human lung. Sci Adv. [Internet]. 2019;5 (2). Publisher's VersionAbstract
Influenza A viruses can bind sialic acid-terminating glycan receptors, and species specificity is often correlated with sialic acid linkage with avian strains recognizing α2,3-linked sialylated glycans and mammalian strains preferring α2,6-linked sialylated glycans. These paradigms derive primarily from studies involving erythrocyte agglutination, binding to synthetic receptor analogs or binding to undefined surface markers on cells or tissues. Here, we present the first examination of the N-glycome of the human lung for identifying natural receptors for a range of avian and mammalian influenza viruses. We found that the human lung contains many α2,3- and α2,6-linked sialylated glycan determinants bound by virus, but all viruses also bound to phosphorylated, nonsialylated glycans.
Gao C, Hanes MS, Byrd-Leotis L, Wei M, Jia N, Kardish RJ, McKitrick TR, Steinhauer DA, Cummings RD. Unique Binding Specificities of Proteins toward Isomeric Asparagine-Linked Glycans. Cell Chem Biol. [Internet]. 2019;26 (4) :535-347. Publisher's VersionAbstract
The glycan ligands recognized by Siglecs, influenza viruses, and galectins, as well as many plant lectins, are not well defined. To explore their binding to asparagine (Asn)-linked N-glycans, we synthesized a library of isomeric multiantennary N-glycans that vary in terminal non-reducing sialic acid, galactose, and N-acetylglucosamine residues, as well as core fucose. We identified specific recognition of N-glycans by several plant lectins, human galectins, influenza viruses, and Siglecs, and explored the influence of sialic acid linkages and branching of the N-glycans. These results show the unique recognition of complex-type N-glycans by a wide variety of glycan-binding proteins and their abilities to distinguish isomeric structures, which provides new insights into the biological roles of these proteins and the uses of lectins in biological applications to identify glycans.
Smith DF, Cummings RD, Song X. History and future of shotgun glycomics. Biochem Soc Trans. [Internet]. 2019;47 (1) :1-11. Publisher's VersionAbstract
Glycans in polysaccharides and glycoconjugates of the hydrophilic exterior of all animal cells participate in signal transduction, cellular adhesion, intercellular signaling, and sites for binding of pathogens largely through protein-glycan interactions. Microarrays of defined glycans have been used to study the binding specificities of biologically relevant glycan-binding proteins (GBP), but such arrays are limited by their lack of diversity or relevance to the GBP being investigated. Shotgun glycan microarrays are made up of structurally undefined glycans that were released from natural sources, labeled with bifunctional reagents so that they can be monitored during their purification using multidimensional chromatographic procedures, stored as a tagged glycan library (TGL) and subsequently printed onto microarrays at equal molar concentrations. The shotgun glycan microarray is then interrogated with a biologically relevant GBP and the corresponding glycan ligands can be retrieved from the TGL for detailed structural analysis and further functional analysis. Shotgun glycomics extended the defined glycan microarray to a discovery platform that supports functional glycomic analyses and may provide a useful process for ultimately defining the human glycome.
Mehta A, Cummings RD. GLAD: GLycan Array Dashboard, a Visual Analytics Tool for Glycan Microarrays. Bioinformatics [Internet]. 2019. Publisher's VersionAbstract

MOTIVATION:

Traditional glycan microarray data is typically presented as excel files with limited visualization and interactivity. Thus, comparisons and analysis of glycan array data have been difficult, and there is need for a tool to facilitate data mining of glycan array data.

RESULTS:

GLAD (GLycan Array Dashboard) is a web-based tool to visualize, analyze, present, and mine glycan microarray data. GLAD allows users to input multiple data files to create comparisons. GLAD extends the capability of the microarray data to produce more comparative visualizations in the form of grouped bar charts, heatmaps, calendar heatmaps, force graphs and correlation maps in order to analyze broad sets of samples. Additionally, it allows users to filter, sort and normalize the data and view glycan structures in an interactive manner, to facilitate faster visual data mining.

AVAILABILITY:

GLAD is freely available for use on the Web at https://glycotoolkit.com/Tools/GLAD/ with all major modern browsers (Edge, Firefox, Chrome, Safari).

SUPPLEMENTARY INFORMATION:

Full documentation and video tutorials for GLAD can be found on https://glycotoolkit.com/GLAD.

Lu LL, Smith MT, Yu KKQ, Luedemann C, Suscovich TJ, Grace PS, Cain A, Yu WH, McKitrick TR, Lauffenburger D, et al. IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure. Nat Med. [Internet]. 2019;9 (6) :977-987. Publisher's VersionAbstract
Exposure to Mycobacterium tuberculosis (Mtb) results in heterogeneous clinical outcomes including primary progressive tuberculosis and latent Mtb infection (LTBI). Mtb infection is identified using the tuberculin skin test and interferon-γ (IFN-γ) release assay IGRA, and a positive result may prompt chemoprophylaxis to prevent progression to tuberculosis. In the present study, we report on a cohort of Ugandan individuals who were household contacts of patients with TB. These individuals were highly exposed to Mtb but tested negative disease by IFN-γ release assay and tuberculin skin test, 'resisting' development of classic LTBI. We show that 'resisters' possess IgM, class-switched IgG antibody responses and non-IFN-γ T cell responses to the Mtb-specific proteins ESAT6 and CFP10, immunologic evidence of exposure to Mtb. Compared to subjects with classic LTBI, 'resisters' display enhanced antibody avidity and distinct Mtb-specific IgG Fc profiles. These data reveal a distinctive adaptive immune profile among Mtb-exposed subjects, supporting an expanded definition of the host response to Mtb exposure, with implications for public health and the design of clinical trials.
Pettinato G, Lehoux S, Ramanathan R, Salem MM, He LX, Muse O, Flaumenhaft R, Thompson MT, Rouse EA, Cummings RD, et al. Generation of fully functional hepatocyte-like organoids from human induced pluripotent stem cells mixed with Endothelial Cells. Sci Rep. [Internet]. 2019;9 (1). Publisher's VersionAbstract
Despite advances in stem cell research, cell transplantation therapy for liver failure is impeded by a shortage of human primary hepatocytes (HPH), along with current differentiation protocol limitations. Several studies have examined the concept of co-culture of human induced pluripotent cells (hiPSCs) with various types of supporting non-parenchymal cells to attain a higher differentiation yield and to improve hepatocyte-like cell functions both in vitro and in vivo. Co-culturing hiPSCs with human endothelial cells (hECs) is a relatively new technique that requires more detailed studies. Using our 3D human embryoid bodies (hEBs) formation technology, we interlaced Human Adipose Microvascular Endothelial Cells (HAMEC) with hiPSCs, leading to a higher differentiation yield and notable improvements across a wide range of hepatic functions. We conducted a comprehensive gene and protein secretion analysis of our HLCs coagulation factors profile, showing promising results in comparison with HPH. Furthermore, a stage-specific glycomic analysis revealed that the differentiated hepatocyte-like clusters (HLCs) resemble the glycan features of a mature tissue rather than cells in culture. We tested our HLCs in animal models, where the presence of HAMEC in the clusters showed a consistently better performance compared to the hiPSCs only group in regard to persistent albumin secretion post-transplantation.
Tuccinardi D, Farr OM, Upadhyay J, Oussaada SM, Klapa MI, Candela M, Rampelli S, Lehoux S, Lázaro I, Sala-Vila A, et al. Mechanisms Underlying the Cardiometabolic Protective Effect of Walnut Consumption in Obese Subjects: A Cross-Over, Randomized, Double-Blinded, Controlled Inpatient Physiology Study. Diabetes Obes Metab. [Internet]. 2019. Publisher's VersionAbstract

AIMS:

To assess the effects of walnuts on cardiometabolic outcomes in obese subjects and to explore underlying mechanisms using novel methods including metabolomic, lipidomic, glycomic, and microbiome analysis integrated with lipid particle fractionation, appetite-regulating hormones and hemodynamic measurements.

MATERIALS AND METHODS:

10 obese subjects were enrolled in this cross-over, randomized, double-blind, placebo-controlled clinical trial. Patients participated in two 5-day inpatient stays during which they consumed a smoothie containing 48g walnuts or a macronutrient-matched placebo smoothie without nuts, with a one-month washout period between the two visits.

RESULTS:

Walnut consumption improved aspects of the lipid profile, i.e. reduced fasting small and dense LDL particles (p<.02) and increased postprandial large HDL particles (p<.01). Lipoprotein Insulin Resistance Score, glucose and insulin AUC decreased significantly after walnut consumption (p<.01, p<.02, p<.04, respectively). Consuming walnuts significantly increased 10 N-glycans, with 8 of them carrying a fucose core. Lipidomic analysis showed a robust reduction in harmful ceramides, hexosylceramides and sphingomyelins, which have been shown to mediate effects on cardiometabolic risk. Peptide YY AUC significantly increased after walnut consumption (p<.03). No major significant changes in hemodynamic, metabolomic analysis or in host health-promoting bacteria such as Faecalibacterium were found.

CONCLUSIONS:

These data provide a more comprehensive mechanistic perspective of the effect of dietary walnut consumption on cardiometabolic parameters. Lipidomic and lipid nuclear magnetic resonance spectroscopy analysis showed an early but significant reduction in ceramides and other atherogenic lipids with walnut consumption that may explain the longer-term benefits of walnuts on insulin resistance, cardiovascular risk and mortality. This article is protected by copyright. All rights reserved.

Woodward AM, Lehoux S, Mantelli F, Di Zazzo A, Brockhausen I, Bonini S, Argüeso P. Inflammatory Stress Causes N-Glycan Processing Deficiency in Ocular Autoimmune Disease. Am J Pathol. [Internet]. 2019;189 (2) :283-294. Publisher's VersionAbstract
High levels of proinflammatory cytokines have been associated with a loss of tissue function in ocular autoimmune diseases, but the basis for this relationship remains poorly understood. Here we investigate a new role for tumor necrosis factor α in promoting N-glycan-processing deficiency at the surface of the eye through inhibition of N-acetylglucosaminyltransferase expression in the Golgi. Using mass spectrometry, complex-type biantennary oligosaccharides were identified as major N-glycan structures in differentiated human corneal epithelial cells. Remarkably, significant differences were detected between the efficacies of cytokines in regulating the expression of glycogenes involved in the biosynthesis of N-glycans. Tumor necrosis factor α but not IL-1β had a profound effect in suppressing the expression of enzymes involved in the Golgi branching pathway, including N-acetylglucosaminyltransferases 1 and 2, which are required for the formation of biantennary structures. This decrease in gene expression was correlated with a reduction in enzymatic activity and impaired N-glycan branching. Moreover, patients with ocular mucous membrane pemphigoid were characterized by marginal N-acetylglucosaminyltransferase expression and decreased N-glycan branching in the conjunctiva. Together, these data indicate that proinflammatory cytokines differentially influence the expression of N-glycan-processing enzymes in the Golgi and set the stage for future studies to explore the pathophysiology of ocular autoimmune diseases.
2018
Sun X, Ju T, Cummings RD. Differential expression of Cosmc, T-synthase and mucins in Tn-positive colorectal cancers. BMC Cancer. [Internet]. 2018;18 (1) :827. Publisher's VersionAbstract

BACKGROUND:

The Tn neoantigen (GalNAcα1-O-Ser/Thr) is an O-glycan expressed in various types of human cancers. Studies in several Tn-expressing cancer cell lines and pancreatic tumors have identified loss of Cosmc expression caused by either mutations or promoter hypermethylation. In this study, we explored the mechanism(s) for Tn expression in human colorectal cancers (CRC).

METHODS:

Tn-expressing cell populations were isolated from CRC cell lines by Fluorescence-associated cell sorting (FACS). The expression of the Tn and sialylated Tn (STn) antigens, Cosmc, T-synthase, and mucins was characterized in paired specimens with CRC and in CRC cell lines by immunostaining, western blot, and qPCR.

RESULTS:

Using well-defined monoclonal antibodies, we confirmed prevalent Tn/STn expression in CRC samples. However, a majority of these tumors had elevated T-synthase activity and expression of both Cosmc and T-synthase proteins. Meanwhile, Tn antigen expression was not caused by mucin overproduction. In addition, we found that Tn-expressing CRC cell lines had either loss-of-function mutations in Cosmc or reversible Tn antigen expression, which was not caused by the deficiency of T-synthase activity.

CONCLUSIONS:

Our results demonstrate multiple mechanisms for Tn expression in CRCs.

KEYWORDS:

Colorectal carcinoma; Cosmc; Mutation; STn antigen; T-synthase; Tn antigen

Ferreira RG, Rodrigues LC, Nascimento DC, Kanashiro A, Melo PH, Borges VF, Gozzi A, da Silva Prado D, Borges MC, Ramalho FS, et al. Galectin-3 aggravates experimental polymicrobial sepsis by impairing neutrophil recruitment to the infectious focus. J Infect. [Internet]. 2018;77 (5) :391-397. Publisher's VersionAbstract
Sepsis is an overwhelming systemic inflammation resulting from an uncontrolled infection that causes extensive tissue damage, organ dysfunction and eventually death. A growing body of evidence indicates that impaired neutrophil migration to the site of infection is associated with poor outcome in sepsis. Here we show that galectin-3 (Gal-3), an endogenous glycan-binding protein, plays a critical role in sepsis outcome. We found that serum Gal-3 concentration increased in patients with septic shock and mice undergoing sepsis induced by cecal ligation and puncture (CLP). Mice deficient in Gal-3 (Gal-3 KO) are more resistant to sepsis induced by CLP, showing lower levels of biochemical markers and neutrophil infiltration for organ injury/dysfunction than those observed in wild-type mice (WT). Furthermore, Gal-3 KO mice show an increased number of neutrophils in the primary focus of infection and reduced bacterial loads in the peritoneal cavity, blood, and lungs. Mechanistically, blood neutrophils from septic mice show higher levels of surface-bound Gal-3 than neutrophils from naive mice. The deficiency of Gal-3 was associated with increased rolling and adhesion of these cells in mesenteric venules. Our results indicate that Gal-3, secreted during sepsis, inhibits neutrophil migration into the infectious focus, which promotes the bacterial spread and worsens the outcome of sepsis.
Giovannone N, Antonopoulos A, Liang J, Geddes Sweeney J, Kudelka MR, King SL, Lee GS, Cummings RD, Dell A, Barthel SL, et al. Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans. Front Immunol. [Internet]. 2018;9 (2857). Publisher's VersionAbstract
Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-β1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase, ST3GAL1 (ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells in vitro induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation.
Purohit S, Li T, Guan W, Song X, Song J, Tian Y, Li L, Sharma A, Dun B, Mysona D, et al. Multiplex glycan bead array for high throughput and high content analyses of glycan binding proteins. Nat Commun. [Internet]. 2018;9 (1) :258. Publisher's VersionAbstract
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.
DeCicco RePass MA, Bhat N, Heimburg-Molinaro J, Bunnell S, Cummings RD, Ward HD. Molecular cloning, expression, and characterization of UDP N-acetyl-α-d-galactosamine: Polypeptide N-acetylgalactosaminyltransferase 4 from Cryptosporidium parvum. Mol Biochem Parasitol. [Internet]. 2018;221 :56-65. Publisher's VersionAbstract
Cryptosporidium spp. are the causative agents of diarrheal disease worldwide, but effective treatments are lacking. Cryptosporidium employs mucin-like glycoproteins with O-glycans to attach to and infect host intestinal epithelial cells. The Tn antigen (GalNAcα1-Ser/Thr) is an O-glycan essential for these processes, as Tn-specific lectins and a Tn-specific monoclonal antibody block attachment to and infection of host cells in vitro. The enzymes in Cryptosporidium catalyzing their synthesis, however, have not been studied. Previously, we identified four genes encoding putative UDP N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) in the genomes of three Cryptosporidium spp. Here we report the in silico analysis, cloning, expression, purification, and characterization of one of the four enzymes Cryptosporidium parvum (Cp)-ppGalNAc-T4. This enzyme contains the characteristic domains and motifs conserved in ppGalNAc-Ts and is expressed at multiple time points during in vitro infection. Recombinant soluble Cp-ppGalNAc-T4 was enzymatically active against an unmodified EA2 peptide suggesting that it may function as an "initiating" ppGalNAc-T. Cp-ppGalNAc-T4 also exhibited a strong preference for UDP-GalNAc over other nucleotide sugar donors and was active against unmodified and O-glycosylated versions of the C. parvum gp40-derived peptide, with a preference for the former, suggesting it may play a role in modifying this glycoprotein in vivo. Given the importance of mucin-type O-glycosylation in Cryptosporidium spp., the enzymes that catalyze their synthesis may serve as potential therapeutic targets.
Sardar MYR, Mandhapati AR, Park S, Wever WJ, Cummings RD, Chaikof EL. Convergent Synthesis of Sialyl LewisX- O-Core-1 Threonine. J Org Chem. [Internet]. 2018;83 (9) :4963-4972. Publisher's VersionAbstract
Selectins are a class of cell adhesion molecules that play a critical role during the initial steps of inflammation. The N-terminal domain of P-selectin glycoprotein ligand-1 (PSGL-1) binds to all selectins, but with the highest affinity to P-selectin. Recent evidence suggests that the blockade of P-selectin/PSGL-1 interactions provides a viable therapeutic option for the treatment of many inflammatory diseases. Herein, we report the total synthesis of threonine bearing sialyl LewisX (sLeX) linked to a Core-1- O-hexasaccharide 1, as a key glycan of the N-terminal domain of PSGL-1. A convergent synthesis using α-selective sialylation and a regioselective [4+2] glycosylation are the key features of this synthesis.
Hu L, Sankaran B, Laucirica DR, Patil K, Salmen W, Ferreon ACM, Tsoi PS, Lasanajak Y, Smith DF, Ramani S, et al. Glycan recognition in globally dominant human rotaviruses. Nat Commun. [Internet]. 2018;9 (1) :2631. Publisher's VersionAbstract
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[4], P[6], and P[8] genotypes of human RVs. How these genotypes interact with HBGA is not known. Here, our crystal structures of P[4] and a neonate-specific P[6] 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[6] 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[6] RVs as developmentally regulated unbranched glycans are more abundant in the neonatal gut.
Behrens AJ, Duke RM, Petralia LMC, Lehoux S, Carlow CKS, Taron CH, Foster JM. Changes in canine serum N-glycosylation as a result of infection with the heartworm parasite Dirofilaria immitis. Sci Rep. [Internet]. 2018;8 (1) :16625. Publisher's VersionAbstract
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
Behrens AJ, Duke RM, Petralia LM, Harvey DJ, Lehoux S, Magnelli PE, Taron CH, Foster JM. Glycosylation profiling of dog serum reveals differences compared to human serum. Glycobiology [Internet]. 2018;28 (11) :825-831. Publisher's VersionAbstract
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.
Koelsch KA, Cavett J, Smith K, Moor JS, Lehoux SD, Jia N, Mather T, Quadri SMS, Rasmussen A, Kaufman EC, et al. Evidence for Alternate Modes of B cell Activation Involving Fab Acquired-N-Glycosylations in Antibody Secreting Cells Infiltrating the Labial Salivary Glands of Sjögren's Syndrome Patients. Arthritis Rheumatol. [Internet]. 2018. Publisher's VersionAbstract

OBJECTIVE:

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.

METHODS:

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.

RESULTS:

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.

CONCLUSION:

Our findings support an alternative mechanism involving V-region N-glycosylation for the selection and proliferation of some autoreactive B cells in SS patients.

Kudelka MR, Nairn AV, Sardar MY, Sun X, Chaikof EL, Ju T, Moremen KW, Cummings RD. Isotopic Labeling with Cellular O-glycome Reporter/Amplification (ICORA) for Comparative O-glycomics of Cultured Cells. Glycobiology. 2018.Abstract
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.
Purohit S, Li T, Guan W, Song X, Song J, Yanna T, Li L, Sharma A, Dun B, Mysona D, et al. Multiplex glycan bead array for high throughput and high content analyses of glycan binding proteins. Nature Commun [Internet]. 2018;9 (1) :258. Publisher's VersionAbstract
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.

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