Publications

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.

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.

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.

Individual lectin-carbohydrate interactions are usually of low affinity. However, high avidity is frequently attained by the multivalent presentation of glycans on biological surfaces coupled with the occurrence of high order lectin oligomers or tandem repeats of lectin domains in the polypeptide. F-type lectins are l-fucose binding lectins with a typical sequence motif, HX(26)RXDX(4)R/K, whose residues participate in l-fucose binding. We previously reported the presence of a few eukaryotic F-type lectin domains with partial sequence duplication that results in the presence of two l-fucose-binding sequence motifs. We hypothesized that such partial sequence duplication would result in greater avidity of lectin-ligand interactions. Inspired by this example from Nature, we attempted to engineer a bacterial F-type lectin domain from Streptosporangium roseum to attain avid binding by mimicking partial duplication. The engineered lectin demonstrated 12-fold greater binding strength than the wild-type lectin to multivalent fucosylated glycoconjugates. However, the affinity to the monosaccharide l-fucose in solution was similar and partial sequence duplication did not result in an additional functional l-fucose binding site. We also cloned, expressed and purified a Branchiostoma floridae F-type lectin domain with naturally occurring partial sequence duplication and confirmed that the duplicated region with the F-type lectin sequence motif did not participate in l-fucose binding. We found that the greater binding strength of the engineered lectin from S. roseum was instead due to increased oligomerization. We believe that this Nature-inspired strategy might be useful for engineering lectins to improve binding strength in various applications.

Plant lectins are gaining interest because of their interesting biological properties. Several Adenia species, that are being used in traditional medicine to treat many health ailments have shown presence of lectins or carbohydrate binding proteins. Here, we report the purification, characterization and biological significance of N-Acetyl galactosamine specific lectin from Adenia hondala (AHL) from Passifloraceae family. AHL was purified in a single step by affinity chromatography on asialofetuin Sepharose 4B column, characterized and its fine sugar specificity determined by glycan array analysis. AHL is human blood group non specific and also agglutinates rabbit erythrocytes. AHL is a glycoprotein with 12.5% of the carbohydrate, SDS-PAGE, MALDI-TOF-MS and ESI-MS analysis showed that AHL is a monomer of 31.6 kDa. AHL is devoid of DNase activity unlike other Ribosome inactivating proteins (RIPs). Glycan array analysis of AHL revealed its highest affinity for terminal lactosamine or polylactosamine of N- glycans, known to be over expressed in hepatocellular carcinoma and colon cancer. AHL showed strong binding to human hepatocellular carcinoma HepG2 cells with MFI of 59.1 expressing these glycans which was effectively blocked by 93.1% by asialofetuin. AHL showed dose and time dependent growth inhibitory effects on HepG2 cells with IC of 4.8 μg/ml. AHL can be explored for its clinical potential.

OBJECTIVE: To better understand the role of B cells, the potential mechanisms responsible for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjögren's syndrome (SS), this study explored patterns of selection pressure and sites of N-glycosylation acquired by somatic mutation (acN-glyc) in the IgG variable (V) regions of antibody-secreting cells (ASCs) isolated from the minor salivary glands of patients with SS and non-SS control patients with sicca symptoms. METHODS: A novel method to produce and characterize recombinant monoclonal antibodies (mAb) from single cell-sorted ASC infiltrates was applied to concurrently probe expressed genes (all heavy- and light-chain isotypes as well as any other gene of interest not related to immunoglobulin) in the labial salivary glands of patients with SS and non-SS controls. V regions were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed for the incidence of N-glycosylation and selection pressure. For specificity testing, the amplified regions were expressed as either the native mAb or mutant mAb lacking the acN-glyc motif. Protein modeling was used to demonstrate how even an acN-glyc site outside of the complementarity-determining region could participate in, or inhibit, antigen binding. RESULTS: V-region sequence analyses revealed clonal expansions and evidence of secondary light-chain editing and allelic inclusion, of which neither of the latter two have previously been reported in patients with SS. Increased frequencies of acN-glyc were found in the sequences from patients with SS, and these acN-glyc regions were associated with an increased number of replacement mutations and lowered selection pressure. A clonal set of polyreactive mAb with differential framework region 1 acN-glyc motifs was also identified, and removal of the acN-glyc could nearly abolish binding to autoantigens. CONCLUSION: These findings support the notion of an alternative mechanism for the selection and proliferation of some autoreactive B cells, involving V-region N-glycosylation, in patients with SS.

Group/species C rotaviruses (RVCs) have been identified as important pathogens of acute gastroenteritis (AGE) in children, family-based outbreaks, as well as animal infections. However, little is known regarding their host-specific interaction, infection, and pathogenesis. In this study, we performed serial studies to characterize the function and structural features of a human G4P[2] RVC VP8* that is responsible for the host receptor interaction. Glycan microarrays demonstrated that the human RVC VP8* recognizes type A histo-blood group antigens (HBGAs), which was confirmed by synthetic glycan-/saliva-based binding assays and hemagglutination of red blood cells, establishing a paradigm of RVC VP8*-glycan interactions. Furthermore, the high-resolution crystal structure of the human RVC VP8* was solved, showing a typical galectin-like structure consisting of two β-sheets but with significant differences from cogent proteins of group A rotaviruses (RVAs). The VP8* in complex with a type A trisaccharide displays a novel ligand binding site that consists of a particular set of amino acid residues of the C-D, G-H, and K-L loops. RVC VP8* interacts with type A HBGAs through a unique mechanism compared with that used by RVAs. Our findings shed light on the host-virus interaction and the coevolution of RVCs and will facilitate the development of specific antivirals and vaccines. Group/species C rotaviruses (RVCs), members of family, infect both humans and animals, but our knowledge about the host factors that control host susceptibility and specificity is rudimentary. In this work, we characterized the glycan binding specificity and structural basis of a human RVC that recognizes type A HBGAs. We found that human RVC VP8*, the rotavirus host ligand binding domain that shares only ∼15% homology with the VP8* domains of RVAs, recognizes type A HBGA at an as-yet-unknown glycan binding site through a mechanism distinct from that used by RVAs. Our new advancements provide insights into RVC-cell attachment, the critical step of virus infection, which will in turn help the development of control and prevention strategies against RVs.

Rotaviruses (RVs), which cause severe gastroenteritis in infants and children, recognize glycan ligands in a genotype-dependent manner via the distal VP8* head of the spike protein VP4. However, the glycan binding mechanisms remain elusive for the P[II] genogroup RVs, including the widely prevalent human RVs (P[8], P[4], and P[6]) and a rare P[19] RV. In this study, we characterized the glycan binding specificities of human and porcine P[6]/P[19] RV VP8*s and found that the P[II] genogroup RV VP8*s could commonly interact with mucin core 2, which may play an important role in RV evolution and cross-species transmission. We determined the first P[6] VP8* structure, as well as the complex structures of human P[19] VP8*, with core 2 and lacto--tetraose (LNT). A glycan binding site was identified in human P[19] VP8*. Structural superimposition and sequence alignment revealed the conservation of the glycan binding site in the P[II] genogroup RV VP8*s. Our data provide significant insight into the glycan binding specificity and glycan binding mechanism of the P[II] genogroup RV VP8*s, which could help in understanding RV evolution, transmission, and epidemiology and in vaccine development. Rotaviruses (RVs), belonging to the family , are double-stranded RNA viruses that cause acute gastroenteritis in children and animals worldwide. Depending on the phylogeny of the VP8* sequences, P[6] and P[19] RVs are grouped into genogroup II, together with P[4] and P[8], which are widely prevalent in humans. In this study, we characterized the glycan binding specificities of human and porcine P[6]/P[19] RV VP8*s, determined the crystal structure of P[6] VP8*, and uncovered the glycan binding pattern in P[19] VP8*, revealing a conserved glycan binding site in the VP8*s of P[II] genogroup RVs by structural superimposition and sequence alignment. Our data suggested that mucin core 2 may play an important role in P[II] RV evolution and cross-species transmission. These data provide insight into the cell attachment, infection, epidemiology, and evolution of P[II] genogroup RVs, which could help in developing control and prevention strategies against RVs.

Variable lymphocyte receptors (VLRs) are unconventional adaptive immune receptors relatively recently discovered in the phylogenetically ancient jawless vertebrates, lamprey and hagfish. VLRs bind antigens using a leucine-rich repeat fold and are the only known adaptive immune receptors that do not utilize an immunoglobulin fold for antigen recognition. While immunoglobulin antibodies have been studied extensively, there are comparatively few studies on antigen recognition by VLRs, particularly for protein antigens. Here we report isolation, functional and structural characterization of three VLRs that bind the protein toll-like receptor 5 (TLR5) from zebrafish. Two of the VLRs block binding of TLR5 to its cognate ligand flagellin in functional assays using reporter cells. Co-crystal structures revealed that these VLRs bind to two different epitopes on TLR5, both of which include regions involved in flagellin binding. Our work here demonstrates that the lamprey adaptive immune system can be used to generate high-affinity VLR clones that recognize different epitopes and differentially impact natural ligand binding to a protein antigen.