The Zika virus outbreak has captivated the attention of the global audience and information has spread rapidly and wildly through the internet and other media channels. This virus was first identified in 1947, when it was isolated from a sentinel rhesus monkey placed by British scientists working at the Yellow Fever Research Laboratory located in the Zika forest area of Uganda, hence its name, and is transmitted primarily by the mosquito vector, Aedes aegypti. The fact that the rhesus macaque is an Asian species being placed in an African forest brings to mind the possibility of rapid adaptation of the virus from an African to Asian species, an issue that has not been considered. Whether such adaptation has played any role in acquiring pathogenicity due to cross species transmission remains to be identified. The first human infection was described in Nigeria in 1954, with only scattered reports of about a dozen human infections identified over a 50-year period.
It was not until 2007 that Zika virus raised its ugly head with infections noted in three-quarters of the population on the tiny island of Yap located between the Philippines and Papua New Guinea in the western Pacific Ocean, followed by a major outbreak in French Polynesia in 2013. The virus remained confined to a narrow equatorial band in Africa and Asia until 2014 when it began to spread eastward, first toward Oceania and then to South America. Since then, millions of infected individuals have been identified in Brazil, Colombia, Venezuela, including 25 additional countries in the Americas. While the symptoms associated with Zika virus infection are generally mild, consisting of fever, maculopapular rash, arthralgia and conjunctivitis, there have been reports of more severe reactions that are associated with neurological complications. In pregnant women, fetal neurological complications include brain damage and microcephaly, while in adults there have been several cases of virus-associated Guillain-Barre syndrome. The virus was until recently believed to only be transmitted via mosquitoes. But when the Zika virus was isolated from the semen specimens from a patient in Texas, this provided the basis for the recent report of possible sexual transmission of the Zika virus. Due to the neurological complications, various vectors for infection as well as the rapid spread throughout the globe, it has prompted the World Health Organization to issue a global health emergency. Various governmental organizations have recommended that pregnant women do not travel to countries where the virus is epidemic, and within the countries affected by the virus, recommendations were provided for women of childbearing age to delay pregnancy.
The overall public health impact of these above findings highlights the need for a rapid but specific diagnostic test for blood banks worldwide to identify those infected and for the counseling of women who are pregnant or contemplating pregnancy. As of this date, there are neither commercially licensed diagnostic tests nor a vaccine. Because cross-reactivity of the Zika virus with dengue and Chikungunya virus is common, it may pose difficulty in being able to quickly develop such tests and vaccines. So far the most effective public health measures include controlling the mosquito populations via insecticides and preventing humans from direct exposure to mosquitoes.
Glycosylation of Env defines pathogenic properties of simian immunodeficiency virus (SIV). We previously demonstrated that pathogenic SIVmac239 and a live-attenuated, quintuple deglycosylated Env mutant (Δ5G) virus target CD4 + T cells residing in different tissues during acute infection. SIVmac239 and Δ5G preferentially infected distinct CD4 + T cells in secondary lymphoid organs (SLOs) and within the lamina propria of the small intestine, respectively (C. Sugimoto et al., J Virol 86: 9323-9336, 2012, https://doi.org/10.1128/JVI.00948-12). Here, we studied the host responses relevant to SIV targeting of CXCR3 + CCR5 + CD4 + T cells in SLOs. Genomewide transcriptome analyses revealed that Th1-polarized inflammatory responses, defined by expression of CXCR3 chemokines, were distinctly induced in the SIVmac239- infected animals. Consistent with robust expression of CXCL10, CXCR3 + T cells were depleted from blood in the SIVmac239-infected animals. We also discovered that elevation of CXCL10 expression in blood and SLOs was secondary to the induction of CD14 + CD16 + monocytes and MAC387 + macrophages, respectively. Since the significantly higher levels of SIV infection in SLOs occurred with a massive accumulation of infiltrated MAC387 + macrophages, T cells, dendritic cells (DCs), and residential macrophages near high endothelial venules, the results highlight critical roles of innate/ inflammatory responses in SIVmac239 infection. Restricted infection in SLOs by Δ5G also suggests that glycosylation of Env modulates innate/inflammatory responses elicited by cells of monocyte/macrophage/DC lineages.
T lymphocytes, a key arm of adaptive immunity, are known to dynamically regulate O-glycosylation during T cell maturation and when responding to stimuli; however, the direct role of O-glycans in T cell maturation remains largely unknown. Using a conditional knockout of the gene (C1GalT1C1 or Cosmc) encoding the specific chaperone Cosmc, we generated mice whose T cells lack extended O-glycans (T cell conditional Cosmc knock out or TCKO mice) and homogeneously express the truncated Tn antigen. Loss of Cosmc is highly deleterious to T cell persistence, with near-complete elimination of Cosmc-null T cells from spleen and lymph nodes. Total T cell counts are 20% of wild type (WT), among which only 5% express the truncated glycans, with the remaining 95% consisting of escapers from Cre-mediated recombination. TCKO thymocytes were able to complete thymic maturation but failed to populate the secondary lymphoid organs both natively and upon adoptive transfer to WT recipients. Our results demonstrate that extended O-glycosylation is required for the establishment and maintenance of the peripheral T cell population.
Two urea transporters, UT-A1 and UT-A3, are expressed in the kidney terminal inner medullary collecting duct (IMCD) and are important for the production of concentrated urine. UT-A1, as the largest isoform of all UT-A urea transporters, has gained much attention and been extensively studied; however, the role and the regulation of UT-A3 are less explored. In this study, we investigated UT-A3 regulation by glycosylation modification. A site-directed mutagenesis verified a single glycosylation site in UT-A3 at Asn279. Loss of the glycosylation reduced forskolin-stimulated UT-A3 cell membrane expression and urea transport activity. UT-A3 has two glycosylation forms, 45 and 65 kDa. Using sugar-specific binding lectins, the UT-A3 glycosylation profile was examined. The 45-kDa form was pulled down by lectin concanavalin A (Con A) and Galant husnivalis lectin (GNL), indicating an immature glycan with a high amount of mannose (Man), whereas the 65-kDa form is a mature glycan composed of acetylglucosamine (GlcNAc) and poly-N-acetyllactosame (poly-LacNAc) that was pulled down by wheat germ agglutinin (WGA) and tomato lectin, respectively. Interestingly, the mature form of UT-A3 glycan contains significant amounts of sialic acid. We explored the enzymes responsible for directing UT-A3 sialylation. Sialyltransferase ST6GalI, but not ST3GalIV, catabolizes UT-A3 α2,6-sialylation. Activation of protein kinase C (PKC) by PDB treatment promoted UT-A3 glycan sialylation and membrane surface expression. The PKC inhibitor chelerythrine blocks ST6GalI-induced UT-A3 sialylation. Increased sialylation by ST6GalI increased UT-A3 protein stability and urea transport activity. Collectively, our study reveals a novel mechanism of UT-A3 regulation by ST6GalI-mediated sialylation modification that may play an important role in kidney urea reabsorption and the urinary concentrating mechanism.
Despite the prominence of carbohydrate-specific antibodies in human sera, data on their emergence and antigen specificities are limited. Whereas maternal IgG are transferred prenatally to the fetal circulation, IgM present in cord blood originate from fetal B lymphocytes. Considering the limited exposure of the fetus to foreign antigens, we assessed the repertoire of carbohydrate-specific antibodies in human cord blood and matched maternal blood samples using glycan arrays. Carbohydrate-specific IgM was absent in cord blood, whereas low cord blood IgG reactivity to glycans was detectable. Comparing IgG reactivities of matched pairs, we observed a general lack of correlation in the antigen specificity of IgG from cord blood and maternal blood due to a selective exclusion of most carbohydrate-specific IgG from maternofetal transfer.
Given the importance of intestinal bacteria in inducing carbohydrate-specific antibodies, we analyzed global antibody specificities toward commensal bacteria. Similar IgG reactivities to specific Bacteroides species were detected in matched cord and maternal blood samples, thus pointing to an efficient maternal transfer of anti-microbial IgG. Due to the observed selectivity in maternofetal IgG transfer, the lack of fetal antibodies to carbohydrate epitopes is only partially compensated by maternal IgG, thus resulting in a weak response to carbohydrate antigens in neonates.
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Beatriz Trastoy;
Jonathan J Du;
Chao Li;
Mikel García-Alija;
Erik H Klontz;
Blaine Roberts;
Thomas C Donahue;
Lai-Xi Wang;
Eric Sundberg;
Marcelo E Guerin
N-glycosylation is one of the most abundant post-translational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-β-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid-type glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for hybrid-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.
The advancement of glycoscience is critically dependent on the access to a large number of glycans for their functional study. Naturally occurring glycans are considered a viable source for diverse and biologically relevant glycan libraries. A mixture of free reducing glycans released from natural sources can be fluorescently tagged and separated by chromatography to produce a natural glycan library. Anthranilic acid (AA) has been widely used to fluorescently tag reducing glycans for HPLC or LC/MS analysis. However, AA conjugated glycans are not efficiently immobilized on microarray slides due to the lack of a primary alkylamine functional group. In this study, we have developed simple and efficient chemistry for bioconjugation and further functionalization of glycan-AA conjugates. This new approach enables quick preparation of glycan microarrays and neoglycoproteins from glycan-AA conjugates, which can be separated by weak anion exchange (WAX) and C18 reversed-phase HPLC.
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Filipa Marcelo;
Nitin Supekar;
Francisco Corzana;
Joost C. van der Horst;
Iiona Vuist;
David Live;
Geert-Jan P.H. Boons;
David Smith;
Sandra J. van Vliet
The human macrophage galactose-type lectin (MGL) is a C-type lectin characterized by a unique specificity for terminal GalNAc residues present in the tumor-associated Tn antigen (GalNAc-Ser/Thr) and its sialylated form, the sialyl-Tn antigen. However, human MGL has multiple splice variants, and whether these variants have distinct ligand-binding properties is unknown. Here, using glycan microarrays, we compared the binding properties of the short MGL 6C (MGLshort) and the long MGL 6B (MGLlong) splice variants, as well as of a histidine-to-threonine mutant (MGLshort H259T). Although the MGLshort and MGLlong variants displayed similar binding properties on the glycan array, the MGLshort H259T mutant failed to interact with the sialyl-Tn epitope. As the MGLshort H259T variant could still bind a single GalNAc monosaccharide on this array, we next investigated its binding characteristics to Tn-containing glycopeptides derived from the MGL ligands mucin 1 (MUC1), MUC2, and CD45. Strikingly, in the glycopeptide microarray, the MGLshort H259T variant lost high-affinity binding toward Tn-containing glycopeptides, especially at low probing concentrations. Moreover, MGLshort H259T was unable to recognize cancer-associated Tn epitopes on tumor cell lines. Molecular dynamics simulations indicated that in WT MGLshort, His259 mediates H bonds directly or engages the Tn-glycopeptide backbone through water molecules. These bonds were lost in MGLshort H259T, thus explaining its lower binding affinity. Together, our results suggest that MGL not only connects to the Tn carbohydrate epitope, but also engages the underlying peptide via a secondary binding pocket within the MGL carbohydrate recognition domain containing the His259 residue.
The Zika virus (ZIKV) is a newly emerging pathogen that has resulted in a worldwide epidemic. It primarily spreads either through infected Aedes aegypti or Aedes albopictus mosquitos leading to severe neurological disorders such as microcephaly and Guillain-Barré syndrome in susceptible individuals. The mode of ZIKV entry into specific cell types such as: epidermal keratinocytes, fibroblasts, immature dendritic cells (iDCs), and stem-cell-derived human neural progenitors has been determined through its major surface envelope glycoprotein. It has been known that oligosaccharides that are covalently linked to viral envelope proteins are crucial in defining host-virus interactions. However, the role of sugars/glycans in exploiting host-immune mechanisms and aiding receptor-mediated virus entry is not well defined. Therefore, this review focuses on host-pathogen interactions to better understand ZIKV pathogenesis.