Potential N-glycosylation Sites Research Articles

Engineering the Pattern of Protein Glycosylation Modulates the Thermostability of a GH11 Xylanase

Protein glycosylation is a common post-translational modification, the effect of which on protein conformational and stability is incompletely understood. Here we have investigated the effects of glycosylation on the thermostability of Bacillus subtilis xylanase A (XynA) expressed in Pichia pastoris. Intact mass analysis of the heterologous wild-type XynA revealed two, three, or four Hex(8-16)GlcNAc2 modifications involving asparagine residues at positions 20, 25, 141, and 181. Molecular dynamics (MD) simulations of the XynA modified with various combinations of branched Hex9GlcNAc2 at these positions indicated a significant contribution from protein-glycan interactions to the overall energy of the glycoproteins. The effect of glycan content and glycosylation position on protein stability was evaluated by combinatorial mutagenesis of all six potential N-glycosylation sites. The majority of glycosylated enzymes expressed in P. pastoris presented increased thermostability in comparison with their unglycosylated counterparts expressed in Escherichia coli. Steric effects of multiple glycosylation events were apparent, and glycosylation position rather than the number of glycosylation events determined increases in thermostability. The MD simulations also indicated that clustered glycan chains tended to favor less stabilizing glycan-glycan interactions, whereas more dispersed glycosylation patterns favored stabilizing protein-glycan interactions.

Cloning and Characterization of the Glycoside Hydrolases That Remove Xylosyl Groups from 7-β-xylosyl-10-deacetyltaxol and Its Analogues

Paclitaxel, a natural antitumor compound, is produced by yew trees at very low concentrations, causing a worldwide shortage of this important anticancer medicine. These plants also produce significant amounts of 7-β-xylosyl-10-deacetyltaxol, which can be bio-converted into 10-deacetyltaxol for the semi-synthesis of paclitaxel. Some microorganisms can convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol, but the bioconversion yield needs to be drastically improved for industrial applications. In addition, the related β-xylosidases of these organisms have not yet been defined. We set out to discover an efficient enzyme for 10-deacetyltaxol production. By combining the de novo sequencing of β-xylosidase isolated from Lentinula edodes with RT-PCR and the rapid amplification of cDNA ends, we cloned two cDNA variants, Lxyl-p1-1 and Lxyl-p1-2, which were previously unknown at the gene and protein levels. Both variants encode a specific bifunctional β-d-xylosidase/β-d-glucosidase with an identical ORF length of 2412 bp (97% identity). The enzymes were characterized, and their 3.6-kb genomic DNAs (G-Lxyl-p1-1, G-Lxyl-p1-2), each harboring 18 introns, were also obtained. Putative substrate binding motifs, the catalytic nucleophile, the catalytic acid/base, and potential N-glycosylation sites of the enzymes were predicted. Kinetic analysis of both enzymes showed kcat/Km of up to 1.07 s(-1)mm(-1) against 7-β-xylosyl-10-deacetyltaxol. Importantly, at substrate concentrations of up to 10 mg/ml (oversaturated), the engineered yeast could still robustly convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol with a conversion rate of over 85% and a highest yield of 8.42 mg/ml within 24 h, which is much higher than those reported previously. Therefore, our discovery might lead to significant progress in the development of new 7-β-xylosyl-10-deacetyltaxol-converting enzymes for more efficient use of 7-β-xylosyltaxanes to semi-synthesize paclitaxel and its analogues. This work also might lead to further studies on how these enzymes act on 7-β-xylosyltaxanes and contribute to the growing database of glycoside hydrolases.

Molecular characterization and quantification of sablefish (Anoplopoma fimbria) gonadotropins and their receptors: Reproductive dysfunction in female captive broodstock

Efforts to establish an aquaculture industry for sablefish (Anoplopoma fimbria) are constrained by reproductive dysfunction in wild-caught fish and by lack of reproduction of F1 females. Toward a better understanding of the reproductive dysfunction of captive broodstock, full-length cDNAs encoding the sablefish gonadotropin subunits (fshb, lhb and cga) and their receptors (fshr and lhcgr) were cloned, sequenced and quantitative real-time PCR assays developed. Sablefish gonadotropin subunits display some unique features, such as two additional Cys residues in the N-terminal region of Fshb and a lack of potential N-glycosylation sites in Fshb and Lhb, whereas Fshr and Lhcgr possess conserved structural characteristics described in other vertebrates. Wild females captured in fall completed gametogenesis in captivity the next spawning season, whereas females captured three months earlier, during summer, failed to mature. Interestingly, these wild non-maturing females exhibited similar reproductive features as prepubertal F1 females, including low levels of pituitary gonadotropin and ovarian receptor mRNAs and plasma sex steroids, and ovarian follicles arrested at the perinucleolus stage. In conclusion, this study described the cloning, molecular characterization and development of qPCRs for sablefish gonadotropins and their receptors. Rearing conditions may impair vitellogenic growth of ovarian follicles in sablefish, compromising the reproductive success of broodstock.

Genomic structure, characterization and expression analysis of a manganese superoxide dismutase from pearl oyster Pinctada fucata

Manganese superoxide dismutase (MnSOD) is a major component of the cellular defense mechanisms against oxidative damage. We cloned and analyzed the expression pattern and genomic structure of the MnSOD gene of pearl oyster Pinctada fucata, hereafter designated as PoMnSOD. The full-length PoMnSOD cDNA was 1080bp in length and consisted of a 5′-untranslated region (UTR) of 222bp, a 3′-UTR of 318bp with a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly (A) tail, and an open reading frame (ORF) of 540bp encoding a polypeptide of 180 amino acids with an estimated molecular mass of 20.4kDa and a predicted pI of 6.72. Sequence analysis showed that PoMnSOD contained MnSOD family signatures F44NGGGHLNH52, I97QGSGWGWLA106 and D138VWEHAYY145, four conserved residues for manganese metal binding (H4, H52, D138 and H142), and two potential N-glycosylation sites (N33 and N51). Homology analysis revealed that PoMnSOD shared 47.6–55.9% identity and 57.4–65.6% similarity to the other known PoMnSOD amino acid sequences. PoMnSOD genomic DNA was 5040bp in length and contained three exons and two introns, which was a tripartite organization and coincided with the consensus GT-AG splicing rule. PoMnSOD promoter contained the various transcription factors associated with the immune modulation and stress responses. Quantitative RT-PCR analysis demonstrated that PoMnSOD was constitutively expressed in all detected tissues, and PoMnSOD mRNA expression was significantly up-regulated in intestine, mantle, gills, digestive gland and haemocytes after Vibrio alginolyticus injection. These results suggested that PoMoSOD was an acute-response protein involved in the innate immune responses of pearl oyster, and provided general information about the mechanisms of innate immune defense against bacterial infection in pearl oyster.

Signal Peptide Cleavage from GP5 of PRRSV: A Minor Fraction of Molecules Retains the Decoy Epitope, a Presumed Molecular Cause for Viral Persistence

Porcine reproductive and respiratory syndrome virus (PRRSV) is the major pathogen in the pig industry. Variability of the antigens and persistence are the biggest challenges for successful control and elimination of the disease. GP5, the major glycoprotein of PRRSV, is considered an important target of neutralizing antibodies, which however appear only late in infection. This was attributed to the presence of a “decoy epitope” located near a hypervariable region of GP5. This region also harbors the predicted signal peptide cleavage sites and (dependent on the virus strain) a variable number of potential N-glycosylation sites. Molecular processing of GP5 has not been addressed experimentally so far: whether and where the signal peptide is cleaved and (as a consequence) whether the “decoy epitope” is present in virus particles. We show that the signal peptide of GP5 from the American type 2 reference strain VR-2332 is cleaved, both during in vitro translation in the presence of microsomes and in transfected cells. This was found to be independent of neighboring glycosylation sites and occurred in a variety of porcine cells for GP5 sequences derived from various type 2 strains. The exact signal peptide cleavage site was elucidated by mass spectrometry of virus-derived and recombinant GP5. The results revealed that the signal peptide of GP5 is cleaved at two sites. As a result, a mixture of GP5 proteins exists in virus particles, some of which still contain the “decoy epitope” sequence. Heterogeneity was also observed for the use of glycosylation sites in the hypervariable region. Lastly, GP5 mutants were engineered where one of the signal peptide cleavage sites was blocked. Wildtype GP5 exhibited exactly the same SDS-PAGE mobility as the mutant that is cleavable at site 2 only. This indicates that the overwhelming majority of all GP5 molecules does not contain the “decoy epitope”.

N-Glycosylation of Gel1 or Gel2 is vital for cell wall -glucan synthesis in Aspergillus fumigatus

Fungal cell wall is a dynamic structure that communicates with and protects the cell from outside stress. In Aspergillus fumigatus, the cell wall β-glucans are mainly elongated by β-1,3-glucanosyltransferases Gels, which consist of seven family members (Gel1-7) utilizing β-1,3-glucan chains as substrates. Previously, we have shown that the mutant deficient of N-glycan processing displays a reduction in the cell wall β-glucans, suggesting that N-glycosylation is required for the proper function of β-1,3-glucanosyltransferase. To verify this hypothesis, in this study, the gene encoding β-1,3-glucanosyltransferase Gel1 or Gel2 was deleted in the Δcwh41 mutant to construct a double-mutant Δgel1Δcwh41 or Δgel2Δcwh41. The growth phenotypes of both double mutants were similar to the single-mutant Δcwh41, suggesting that Gel1 and Gel2 are proteins that are mainly affected by deficient N-glycan processing in Δcwh41. Furthermore, the mutant Δgel1(Gel1-NM) or Δgel2(Gel2-NM), in which all potential N-glycosylation sites on Gel1 or Gel2 were removed by site-directed mutagenesis, showed phenotypes similar to the single-mutant Δgel1 or Δgel2. Biochemical analysis revealed that N-glycosylation was essential for the function of Gel1 or Gel2 and thus required for β-glucan synthesis in A. fumigatus.

Characterization of a glycoside hydrolase family-51 α-l-arabinofuranosidase gene from Aureobasidium pullulans ATCC 20524 and its encoded product

The genomic DNA and cDNA encoding α-l-arabinofuranosidase were cloned from the dimorphic fungus Aureobasidium pullulans ATCC 20524 and sequenced. The open reading frame (2097bp) of the α-l-arabinofuranosidase gene abfB was interrupted by five introns of 49, 49, 50, 65, and 49bp. The gene encoded a presumed signal peptide of 17 residues and a mature protein of 682 residues with a calculated Mr of 74,230Da and a theoretical isoelectric point of 4.95. Glu-362 and Glu-440 residues are likely involved in catalytic reactions as an acid/base and a nucleophile, respectively. The protein possessed 15 potential N-glycosylation sites. The deduced amino acid sequence of the abfB gene product was 58% identical to the Penicillium purpurogenum ABF 2, which belongs to the glycoside hydrolase family-51 α-l-arabinofuranosidase. The abfB cDNA was functionally expressed in the yeast Pichia pastoris. The recombinant enzyme, AbfB, was purified from the culture filtrate, and it appeared as a single band on SDS-PAGE with an apparent Mr of 110kDa. AbfB showed α-l-arabinofuranosidase activity of 56.6U/mg of protein toward p-nitrophenyl (pNP) α-l-arabinofuranoside at optimal pH 4.5 and 75°C. The enzyme exhibited apparent Km and Vmax values of 6.27mM and 78.1μmol/mg/min, respectively, for pNP α-l-arabinofuranoside. The enzyme was highly active on rye arabinoxylan as well as pNP α-l-arabinofuranoside, but it showed weak activity toward α-(1→5)-l-arabinobiose, α-(1→5)-l-arabinotriose, branched l-arabinan, linear α-(1→5)-l-arabinan, and arabinogalactan.

Hypo‐glycosylated hFSH binds and activates FSH receptors more rapidly than fully‐glycosylated hFSH

Follitropin (FSH) heterodimers possess two potential N‐glycosylation sites on both common α and hormone‐specific β subunits. Hypo‐glycosylated hFSH isolated from lutropin preparations possessed a partially glycosylated FSHβ subunit decorated with one glycan, while there were two in the hFSHα subunit. Mass spectrometry revealed the presence of oligomannose glycans, which are normally rare in hFSH preparations. Hypo‐glycosylated hFSH was 5‐ to 26‐fold more active than fully‐glycosylated hFSH in several FSH radioligand assays. In rat testis homogenate, 125I‐hypo‐hFSH bound receptors immediately, without the 45–60 min lag observed for 125I‐fully‐glycosylated hFSH. In porcine granulosa cells, short‐term incubations revealed an earlier onset of cAMP accumulation, protein kinase A (PKA) activity and CREB phosphorylation. Hypo‐glycosylated hFSH increased intracellular cAMP accumulation within one min and PKA‐dependent CREB phosphorylation within 5 min; whereas fully‐glycosylated hFSH induced CREB phosphorylation after a 10–15 min lag. Additionally, CREB phosphorylation was sustained for greater than 60 min in response to hypo‐glycosylated hFSH, but returned to basal within 60 min in response to fully‐glycosylated hFSH. Thus, partial glycosylation of hFSH results in faster binding to its cognate receptor and this is attended by a more rapid cellular response. Supported by NIH grant 5P01 AG‐029531.

Molecular cloning and expression of the cDNA sequence encoding a novel aspartic protease from Uncinaria stenocephala

Uncinaria stenocephala belongs to Ancylostomatidae family. Members of this family - hookworms - infect millions of people and animals worldwide. U. stenocephala is most pathogenic in dogs and other Canidae, which are the main hosts, and infection causes anemia or even death. So far no effective hookworm vaccine has been developed that is economically viable. Attempts to identify vaccine antigens have led to a group of aspartic proteases, which play a key role in parasite feeding, migration through host tissues and immune evasion. The cDNA of an aspartic protease from U. stenocephala was cloned using the RACE-PCR method. Computational analysis showed that the cDNA encodes a 447 amino acid protein with a molecular mass of 52kDa that shows high homology to aspartic proteases from related hookworms. Analysis identified 1 potential N-glycosylation site, 3 potential disulfide bonds and no O-glycosylation sites. The recombinant protein was expressed in Escherichia coli followed by purification and mouse immunization. Using raised anti-Us-APR-1(2) (Uncinaria stenocephala Aspartic protease-1) serum the presence of Us-APR-1 in the adult stage of U. stenocephala and the expression of homologous protease in L3 and adult stages of A. ceylanicum was confirmed. This analysis is the first phase of work exploring the biological role of Us-APR-1 in parasite-host interactions and raises hope for successful vaccine development against Uncinaria sp. and possibly Ancylostoma sp.

Molecular and phylogenetic analysis of influenza A H1N1 pandemic viruses in Cuba, May 2009 to August 2010

The influenza A(H1N1)pdm09 virus was detected in Cuba in May 2009. The introduction of a new virus with increased transmissibility into a population makes surveillance of the pandemic strain to the molecular level necessary. The aim of the present study was the molecular and phylogenetic analysis of pandemic influenza A(H1N1)pdm09 strains that circulated in Cuba between May 2009 and August 2010. Seventy clinical samples were included in the study. Nucleotide sequences from the hemagglutinin HA1 region segment were obtained directly from clinical samples. Genetic distances were calculated using MEGA v.5.05. A phylogenetic tree was constructed using MrBayes v.3.1.2 software. Potential N-glycosylation sites were predicted using NetNGlyc server 1.0. The 48 Cuban sequences of influenza A(H1N1)pdm09 obtained were similar to the A/California/07/2009 (H1N1) vaccine strain. Most of the Cuban strains belonged to clade 7. Cuban viruses showed amino acid changes, some of them located at three antigenic sites: Ca, Sa, and Sb. Two dominant mutations were detected: P83S (100%) and S203T (85.7%). Glycosylation site analysis revealed the gain of one site at position 162 in 13 sequences. The findings in this study contribute to our understanding of the progress of the influenza A(H1N1)pdm09 virus, since this virus is at the starting point of its evolution in humans.

Mucin-type proteins produced in the Trichoplusia ni and Spodoptera frugiperda insect cell lines carry novel O-glycans with phosphocholine and sulfate substitutions

The O-glycans of a recombinant mucin-type protein expressed in insect cell lines derived from Trichoplusia ni (Hi-5) and Spodoptera frugiperda (Sf9) were characterized. The P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b) fusion protein carrying 106 potential O-glycosylation sites and 6 potential N-glycosylation sites was expressed and purified from the Hi-5 and Sf9 cell culture medium using affinity chromatography and gel filtration. Liquid chromatography mass spectrometry (LC-MS) of O-glycans released from PSGL-1/mIgG2b revealed a large repertoire of structurally diverse glycans, which is in contrast to previous reports of only simple glycans. O-Glycans containing hexuronic acid (HexA, here glucuronic acid and galacturonic acid) were found to be prevalent. Also sulfate (Hi-5 and Sf9) and phosphocholine (PC; Sf9) O-glycan substitutions were detected. Western blotting confirmed the presence of O-linked PC on PSGL-1/mIG2b produced in Sf9 cells. To our knowledge, this is the first structural characterization of PC-substituted O-glycans in any species. The MS analyses revealed that Sf9 oligosaccharides consisted of short oligosaccharides (<6 residues) low in hexose (Hex) and with terminating N-acetylhexosamine (HexNAc) units, whereas Hi-5 produced a family of large O-glycans with (HexNAc-HexA-Hex) repeats and sulfate substitution on terminal residues. In both cell lines, the core N-acetylgalactosamine was preferentially non-branched, but small amounts of O-glycan cores with single fucose or hexose branches were found.

Molecular cloning, characterization and tissue expression of CD4 in Chinese goose

CD4 protein is a single chain transmembrane glycoprotein belonging to the immunoglobulin superfamily that recognizes the major histocompatibility complex (MHC) class II molecules. It plays an important role in cell-mediated immunity. Here, the full-length cDNA of CD4 in Chinese goose (Anser cygnoides) was cloned and identified. The goose CD4 is 1940bp in length and encodes a single open-reading frame of 480 amino acids. The putative amino acid sequence of goose CD4 consisted of a signal peptide, four potential N-glycosylation sites, a transmembrane region and a cytoplasmic tail. The multiple sequence alignment and phylogenetic analysis suggested that goose CD4 shared a higher similarity with avian than other vertebrates. Semi-quantitative RT-PCR analysis showed that the highest level of CD4 mRNA transcripts was presented in the thymus, and relatively lower in the spleen, small intestine, brain and trachea. Low expression was seen in the bursa of fabricius, cecal tonsil, cecum, skin, lung, kidney and liver. In gosling, the CD4 transcript was expressed with high abundance in the thymus, and relatively lower in the spleen, cecal tonsil and small intestine. However, in adult goose, high expression was seen in the thymus, spleen and cecum, and relatively lower in the cecal tonsil and small intestine. During the course of NGVEV infection, the obvious increase in CD4 gene expression was observed in the spleen, bursa of fabricius and harderian gland. Interestingly, a notable decrease in CD4 mRNA expression in the small intestine at 5d PI and followed by an increase of that at 19d PI were shown.

N-glycosylation deficiency enhanced heterologous production of a Bacillus licheniformis thermostable α-amylase in Saccharomyces cerevisiae

Expression of foreign enzymes in yeast is a traditional genetic engineering approach; however, useful secretory enzymes are not produced in every case. The hyperthermostable α-amylase encoded by the AmyL gene of Bacillus licheniformis was expressed in Saccharomyces cerevisiae; however, it was only weakly produced and was degraded by the proteasome. To determine the cause of low α-amylase production, AmyL was expressed in a panel of yeast mutants harboring knockouts in non-essential genes. Elevated AmyL production was observed in 44 mutants. The knockout genes were classified into six functional categories. Remarkably, all non-essential genes required for N-linked oligosaccharide synthesis and a gene encoding an oligosaccharyl transferase subunit were identified. Immunoblotting demonstrated that differently underglycosylated forms of AmyL were secreted from oligosaccharide synthesis-deficient mutants, while a fully glycosylated form was produced by wild-type yeast, suggesting that N-linked glycosylation of AmyL inhibited its secretion in yeast. Mutational analysis of six potential N-glycosylation sites in AmyL revealed that the N33Q and N309Q mutations remarkably affected AmyL production. To achieve higher AmyL production in yeast, all six N-glycosylation sites of AmyL were mutated. In wild-type yeast, production of the resulting non-glycosylated form of AmyL was threefold higher than that of the glycosylated form.

N-glycosylation enhances functional and structural stability of recombinant β-glucuronidase expressed in Pichia pastoris

Recombinant β-glucuronidase (GUS) expressed in Pichia pastoris GS115 is an important glycoprotein, encoded by a gene with four potential N-glycosylation sites. To investigate the impact of N-linked carbohydrate moieties on the stability of recombinant GUS, it was deglycosylated by peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic activities of the glycosylated and deglycosylated GUS were compared under various conditions such as temperature, pH, organic solvents, detergents and chaotropic agent. The results demonstrated that the glycosylated GUS retained greater fraction of maximum enzymatic activity against various types of denaturants compared with the deglycosylated. The conformational stabilities of both GUS were analyzed by monitoring the unfolding equilibrium by using the denaturant guanidinium chloride (dn-HCl). The glycosylated GUS displayed a significant increase in its conformational stability than the deglycosylated counterpart. These results affirmed the key role of N-glycosylation on the structural and functional stability of β-glucuronidase and could have potential applications in the functional enhancement of industrial enzymes.

Neutralization sensitivity of HIV-1 subtype B’ clinical isolates from former plasma donors in China

BackgroundHIV-1 subtype B’ isolates have been predominantly circulating in China. Their intra- and inter-subtype neutralization sensitivity to autologous and heterologous plasmas has not been well studied.ResultsTwelve HIV-1 B’ clinical isolates obtained from patients were tested for their intra- and inter-subtype neutralization sensitivity to the neutralization antibodies in the plasmas from patients infected by HIV-1 B’ and CRF07_BC subtypes, respectively. We found that the plasmas from the HIV-1 B’-infected patients could potently neutralize heterologous viruses of subtype B’ with mean ID50 titer (1/x) of about 67, but they were not effective in neutralizing autologous viruses of subtype B’ with mean ID50 titer (1/x) of about 8. The plasmas from HIV-1 CRF07_BC-infected patients exhibited weak inter-subtype neutralization activity against subtype B’ viruses with ID50 titer (1/x) is about 22. The neutralization sensitivity of HIV-1 B’ isolates was inversely correlated with the neutralizing activity of plasmas from HIV-1 B’-infected patients (Spearman’s r = −0.657, P = 0.020), and with the number of potential N-glycosylation site (PNGS) in V1-V5 region (Spearman’s r = −0.493, P = 0.034), but positively correlated with the viral load (Spearman’s r = 0.629, P = 0.028). It had no correlation with the length of V1-V5 regions or the CD4+ T cell count. Virus AH259V has low intra-subtype neutralization sensitivity, it can be neutralized by 17b (IC50: 10μg/ml) and 447-52D (IC50: 1.6μg/ml), and the neutralizing antibodies (nAbs) in plasma AH259P are effective in neutralizing infection by the primary HIV-1 isolates with different subtypes with ID50 titers (1/x) in the range of 32–396.ConclusionsThese findings suggest that the HIV-1 subtype B’ viruses may mutate under the immune pressure, thus becoming resistant to the autologous nAbs, possibly by changing the number of PNGS in the V1-V5 region of the viral gp120. Some of primary HIV-1 isolates are able to induce both intra- and inter-subtype cross-neutralizing antibody responses.

Impact of antiretroviral pressure on selection of primary human immunodeficiency virus type 1 envelope sequences in vitro

The initiation of drug therapy results in a reduction in the human immunodeficiency virus type 1 (HIV-1) population, which represents a potential genetic bottleneck. The effect of this drug-induced genetic bottleneck on the population dynamics of the envelope (Env) regions has been addressed in several in vivo studies. However, it is difficult to investigate the effect on the env gene of the genetic bottleneck induced not only by entry inhibitors but also by non-entry inhibitors, particularly in vivo. Therefore, this study used an in vitro selection system using unique bulk primary isolates established in the laboratory to observe the effects of the antiretroviral drug-induced bottleneck on the integrase and env genes. Env diversity was decreased significantly in one primary isolate [KP-1, harbouring both CXCR4 (X4)- and CCR5 (R5)-tropic variants] when passaged in the presence or absence of raltegravir (RAL) during in vitro selection. Furthermore, the RAL-selected KP-1 variant had a completely different Env sequence from that in the passage control (particularly evident in the gp120, V1/V2 and V4-loop regions), and a different number of potential N-glycosylation sites. A similar pattern was also observed in other primary isolates when using different classes of drugs. This is the first study to explore the influence of anti-HIV drugs on bottlenecks in bulk primary HIV isolates with highly diverse Env sequences using in vitro selection.

Toward Animal Cell Culture–Based Influenza Vaccine Design: Viral Hemagglutinin N-Glycosylation Markedly Impacts Immunogenicity

The glycoproteins hemagglutinin (HA) and neuraminidase are the major determinants of host range and tissue tropism of the influenza virus. HA is the most abundant protein in the virus particle membrane and represents the basis of most influenza vaccines. It has been reported that influenza virus HA N-glycosylation markedly depends on the host cell line used for virus production. However, little is known about how differential glycosylation affects immunogenicity of the viral proteins. This is of importance for virus propagation in chicken eggs as well as for innovative influenza vaccine production in mammalian cell lines. In this study, we investigated the impact of the differential N-glycosylation patterns of two influenza A virus PR/8/34 (H1N1) variants on immunogenicity. Madin-Darby canine kidney cell-derived and Vero cell-derived glycovariants were analyzed for immunogenicity in a TCR-HA transgenic mouse model. Next-generation pyrosequencing validated the congruence of the potential HA N-glycosylation sites as well as the presence of the HA peptide recognized by the TCR-HA transgenic T cells. We show that differential HA N-glycosylation markedly affected T cell activation and cytokine production in vitro and moderately influenced IL-2 production in vivo. Cocultivation assays indicated that the difference in immunogenicity was mediated by CD11c(+) dendritic cells. Native virus deglycosylation by endo- and exoglycosidases dramatically reduced cytokine production by splenocytes in vitro and markedly decreased HA-specific Ab production in vivo. In conclusion, this study indicates a crucial importance of HA N-glycosylation for immunogenicity. Our findings have implications for cell line-based influenza vaccine design.

N-glycosylation of the premembrane protein of Japanese encephalitis virus is critical for folding of the envelope protein and assembly of virus-like particles

Premembrane (prM) and envelope (E) proteins, the major structural proteins of Japanese encephalitis virus (JEV) each contain single potential N-glycosylation site. In this study, the role of N-glycosylation of these proteins on their folding and activity were investigated. Three mutant prM and/or E (prM-E) genes lacking N-glycosylation sites were generated by site-directed mutagenesis. The effects of the N-glycan on folding, secretion and cytotoxicity of mutant proteins were determined by comparison with their wild type (wt) counterparts. Removal of N-glycan from the prM protein resulted in a complete misfolding of the E protein and failure to form virus-like particles (VLPs). A similar removal of N-glycan from the E protein led to a low efficiency of its folding and VLPs formation. The secretion and cytotoxicity of the E protein was also markedly impaired in case the glycosylation sites in the prM or E or both proteins were removed. These results suggest that the N-glycosylation of the prM protein is critical to the folding of the E protein, which makes it pivotal in the cytotoxicity of JEV particles and their production.

Arctic-like Rabies Virus, Bangladesh

Arctic/Arctic-like rabies virus group 2 spread into Bangladesh ≈32 years ago. Because rabies is endemic to and a major public health problem in this country, we characterized this virus group. Its glycoprotein has 3 potential N-glycosylation sites that affect viral pathogenesis. Diversity of rabies virus might have public health implications in Bangladesh.

Heterologous expression and biochemical characterization of acetyl xylan esterase from Coprinopsis cinerea

Acetyl xylan esterase (AXE) from basidiomycete Coprinopsis cinerea Okayama 7 (#130) was functionally expressed in Pichia pastoris with a C-terminal tag under the alcohol oxidase 1 (AOX1) promoter and secreted into the medium at 1.5 mg l(-1). Its molecular mass was estimated to be 65.5 kDa based on the SDS-PAGE analysis, which is higher than the calculated molecular mass of 40 kDa based on amino acid composition. In-silico analysis of the amino acid sequence predicted two potential N-glycosylation sites. Results from PNGase F deglycosylation and mass spectrum confirmed the presence of N-glycosylation on the recombinant AXE with predominant N-glycans HexNAc2Hex9-16. The recombinant AXE showed best activity at 40 °C and pH 8. It showed not only acetyl esterase activity with a Km of 4.3 mM and a Vmax of 2.15 U mg(-1) for hydrolysis of 4-nitrophenyl acetate but also a butyl esterase activity for hydrolysis of 4-nitrophenyl butyrate with a Km of 0.11 mM and Vmax of 0.78 U mg(-1). The presence of two additional amino acid residues at its native N-terminus was found to help stabilize the enzyme against the protease cleavages without affecting its activity.