Heavy glycosylation of the envelope (Env) surface subunit, gp120, is a key adaptation of HIV-1; however, the precise effects of glycosylation on the folding, conformation and dynamics of this protein are poorly understood. Here we explore the patterns of HIV-1 Env gp120 glycosylation, and particularly the enrichment in glycosylation sites proximal to the disulfide linkages at the base of the surface-exposed variable domains. To dissect the influence of glycans on the conformation these regions, we focused on an antigenic peptide fragment from a disulfide bridge-bounded region spanning the V1 and V2 hyper-variable domains of HIV-1 gp120. We used replica exchange molecular dynamics (MD) simulations to investigate how glycosylation influences its conformation and stability. Simulations were performed with and without N-linked glycosylation at two sites that are highly conserved across HIV-1 isolates (N156 and N160); both are contacts for recognition by V1V2-targeted broadly neutralizing antibodies against HIV-1. Glycosylation stabilized the pre-existing conformations of this peptide construct, reduced its propensity to adopt other secondary structures, and provided resistance against thermal unfolding. Simulations performed in the context of the Env trimer also indicated that glycosylation reduces flexibility of the V1V2 region, and provided insight into glycan-glycan interactions in this region. These stabilizing effects were influenced by a combination of factors, including the presence of a disulfide bond between the Cysteines at 131 and 157, which increased the formation of beta-strands. Together, these results provide a mechanism for conservation of disulfide linkage proximal glycosylation adjacent to the variable domains of gp120 and begin to explain how this could be exploited to enhance the immunogenicity of those regions. These studies suggest that glycopeptide immunogens can be designed to stabilize the most relevant Env conformations to focus the immune response on key neutralizing epitopes.
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.
Protein O-glycosylation has key roles in many biological processes, but the repertoire of O-glycans synthesized by cells is difficult to determine. Here we describe an approach termed Cellular O-Glycome Reporter/Amplification (CORA), a sensitive method used to amplify and profile mucin-type O-glycans synthesized by living cells. Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS). Relative to conventional O-glycan analyses, CORA resulted in an ∼100-1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus. Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.
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.
A recent study of plasma neutralization breadth in HIV-1 infected individuals at nine International AIDS Vaccine Initiative (IAVI) sites reported that viral load, HLA-A*03 genotype, and subtype C infection were strongly associated with the development of neutralization breadth. Here, we refine the findings of that study by analyzing the impact of the transmitted/founder (T/F) envelope (Env), early Env diversification, and autologous neutralization on the development of plasma neutralization breadth in 21 participants identified during recent infection at two of those sites: Kigali, Rwanda (n = 9) and Lusaka, Zambia (n = 12). Single-genome analysis of full-length T/F Env sequences revealed that all 21 individuals were infected with a highly homogeneous population of viral variants, which were categorized as subtype C (n = 12), A1 (n = 7), or recombinant AC (n = 2). An extensive amino acid sequence-based analysis of variable loop lengths and glycosylation patterns in the T/F Envs revealed that a lower ratio of NXS to NXT-encoded glycan motifs correlated with neutralization breadth. Further analysis comparing amino acid sequence changes, insertions/deletions, and glycan motif alterations between the T/F Env and autologous early Env variants revealed that extensive diversification focused in the V2, V4, and V5 regions of gp120, accompanied by contemporaneous viral escape, significantly favored the development of breadth. These results suggest that more efficient glycosylation of subtype A and C T/F Envs through fewer NXS-encoded glycan sites is more likely to elicit antibodies that can transition from autologous to heterologous neutralizing activity following exposure to gp120 diversification. This initiates an Env-antibody co-evolution cycle that increases neutralization breadth, and is further augmented over time by additional viral and host factors. These findings suggest that understanding how variation in the efficiency of site-specific glycosylation influences neutralizing antibody elicitation and targeting could advance the design of immunogens aimed at inducing antibodies that can transition from autologous to heterologous neutralizing activity.
Background: The evidence for an association between particulate air pollution and type 2 diabetes mellitus (T2DM) in developing countries was very scarce. Objective: To investigate the associations of long-term exposure to fine particulate matter (PM2.5) with T2DM prevalence and with fasting glucose and glycosylated hemoglobin (HbA1c) levels in China. Methods: This is a cross-sectional study based on a nation-wide baseline survey of 11,847 adults who participated in the China Health and Retirement Longitudinal Study from June 2011 to March 2012. The average residential exposure to PM2.5 for each participant in the same period was estimated using a satellite-based spatial statistical model. We determined the association between PM2.5 and T2DM prevalence by multivariable logistic regression models. We also evaluated the association between PM2.5 and fasting glucose and HbA1c levels using multivariable linear regression models. Stratification analyses were conducted to explore potential effect modification. Results: We identified 1760 cases of T2DM, corresponding to 14.9% of the study population. The average PM2.5 exposure for all participants was 72.6 μg/m3 during the study period. An interquartile range increase in PM2.5 (41.1 μg/m3) was significantly associated with increased T2DM prevalence (prevalence ratio, PR = 1.14), and elevated levels of fasting glucose (0.26 mmol/L) and HbA1c (0.08%). The associations of PM2.5 with T2DM prevalence and with fasting glucose and HbA1c were stronger in several subgroups. Conclusions: This nationwide cross-sectional study suggested that long-term exposure to PM2.5 might increase the risk of T2DM in China.
The HIV/AIDS epidemic, which was first reported on in 1981, progressed in just 10 years to a disease afflicting 10 million people worldwide including 1 million in the US. In 1987, AZT was approved for treating HIV/AIDS. Unfortunately, its clinical usefullness was severly limited by associated toxicities and the emergence of resistance. Three other drugs that were approved in the early 1990s suffered from similar liabilities.In 1990, the Liotta group at Emory University developed a highly diastereoselective synthesis of racemic 3′-thia-2′,3′-dideoxycytidine and 3′-thia-2′,3′-5-fluorodideoxycytidine and demonstrated that these compounds exhibited excellent anti-HIV activity with no apparent cytotoxicity. Subsequently, the enantiomers of these compounds were separated using enzyme-mediated kinetic resolutions and their (-)-enantiomers (3TC and FTC, respectively) were found to have exceptionally attractive preclinical profiles. In addition to their anti-HIV activity, 3TC and FTC potently inhibit the replication of hepatitis B virus.The development of FTC, which was being carried out by Burroughs Wellcome, had many remarkable starts and stops. For example, passage studies indicated that the compound rapidly selected for a single resistant mutant, M184V, and that this strain was 500-1000-fold less sensitive to FTC than was wild-type virus. Fortunately, it was found that combinations of AZT with either 3TC or FTC were synergistic. The effectiveness of AZT-3TC combination therapy was subsequently demonstrated in four independent clinical trials, and in 1997, the FDA approved Combivir, a fixed dose combination of AZT and 3TC.In phase 1 clinical trials, FTC was well tolerated by all subjects with no adverse events observed. However, the development of FTC was halted by the aquistition of Wellcome PLC by Glaxo PLC in January 1995. In 1996, Triangle Pharmaceuticals licensed FTC from Emory and initiated a series of phase I/II clinical studies that demonstrated the safety and efficacy of the drug. In August 1998, FTC was granted "Fast Track" status, based primarily on its potential for once daily dosing. While the outcomes of two subsequent phase III trials were positive, a third phase III clinical trial involving combinations of 3TC or FTC with stavudine and neviripine had to be terminated due to serious liver-related adverse events. Although analysis of the data suggested that the liver toxicity was due to neviripine, the FDA decided that the study could not be used for drug registration. Ultimately, in January 2003, Gilead Sciences acquired Triangle Pharmaceuticals and completed the development of FTC (emtricitabine), which was approved for once a day, oral administration in July 2003. A year later, Truvada, a once a day, oral, fixed dose combination of emtricitabine and tenofovir disoproxyl fumarate received FDA approval and quickly became the accepted first line therapy when used with a third antiretroviral agent. In July 2006, the FDA approved Atripla, a once a day, oral, fixed dose combination of emtricitabine, tenofovir disoproxyl fumarate, and efavirenz, which represented the culmination of two decades of research that had transformed AIDS from a death sentence to a manageable chronic disease.