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1
Liang, Yu, Miklos Guttman, James A. Williams, Hans Verkerke, Daniel Alvarado, Shiu-Lok Hu, and Kelly K. Lee. "Changes in Structure and Antigenicity of HIV-1 Env Trimers Resulting from Removal of a Conserved CD4 Binding Site-Proximal Glycan." Journal of Virology 90, no. 20 (August 3, 2016): 9224–36. http://dx.doi.org/10.1128/jvi.01116-16.
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ABSTRACTThe envelope glycoprotein (Env) is the major target for HIV-1 broadly neutralizing antibodies (bNAbs). One of the mechanisms that HIV has evolved to escape the host's immune response is to mask conserved epitopes on Env with dense glycosylation. Previous studies have shown that the removal of a particular conserved glycan at N197 increases the neutralization sensitivity of the virus to antibodies targeting the CD4 binding site (CD4bs), making it a site of significant interest from the perspective of vaccine design. At present, the structural consequences that result from the removal of the N197 glycan have not been characterized. Using native-like SOSIP trimers, we examine the effects on antigenicity and local structural dynamics resulting from the removal of this glycan. A large increase in the binding of CD4bs and V3-targeting antibodies is observed for the N197Q mutant in trimeric Env, while no changes are observed with monomeric gp120. While the overall structure and thermostability are not altered, a subtle increase in the flexibility of the variable loops at the trimeric interface of adjacent protomers is evident in the N197Q mutant by hydrogen-deuterium exchange mass spectrometry. Structural modeling of the glycan chains suggests that the spatial occupancy of the N197 glycan leads to steric clashes with CD4bs antibodies in the Env trimer but not monomeric gp120. Our results indicate that the removal of the N197 glycan enhances the exposure of relevant bNAb epitopes on Env with a minimal impact on the overall trimeric structure. These findings present a simple modification for enhancing trimeric Env immunogens in vaccines.IMPORTANCEThe HIV-1 Env glycoprotein presents a dense patchwork of host cell-derived N-linked glycans. This so-called glycan shield is considered to be a major protective mechanism against immune recognition. While the positions of many N-linked glycans are isolate specific, some are highly conserved and are believed to play key functional roles. In this study, we examine the conserved, CD4 binding site-proximal N197 glycan and demonstrate that its removal both facilitates neutralizing antibody access to the CD4 binding site and modestly impacts the structural dynamics at the trimer crown without drastically altering global Env trimer stability. This indicates that surgical glycosylation site modification may be an effective way of sculpting epitope presentation in Env-based vaccines.
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CHITLARU, Theodor, Chanoch KRONMAN, Baruch VELAN, and Avigdor SHAFFERMAN. "Overloading and removal of N-glycosylation targets on human acetylcholinesterase: effects on glycan composition and circulatory residence time." Biochemical Journal 363, no. 3 (April 24, 2002): 619–31. http://dx.doi.org/10.1042/bj3630619.
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Optimization of post-translational modifications was shown to affect the ability of recombinant human acetylcholinesterase (rHuAChE) produced in HEK-293 cells to be retained in the circulation for prolonged periods of time [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959–967; Chitlaru, Kronman, Zeevi, Kam, Harel, Ordentlich, Velan and Shafferman (1998) Biochem. J. 336, 647–658; Chitlaru, Kronman, Velan and Shafferman (2001) Biochem. J. 354, 613–625]. To evaluate the possible contribution of the number of appended N-glycans in determining the pharmacokinetic behaviour of AChE, a series of sixteen recombinant human AChE glycoforms, differing in their number of appended N-glycans (2, 3, 4 or 5 glycans), state of assembly (dimeric or tetrameric) and terminal glycan sialylation (partially or fully sialylated) were generated. Extensive structural analysis of N-glycans demonstrated that the various glycan types associated with all the different rHuAChE glycoforms are essentially similar both in structure and abundance, and that production of the various glycoforms in the sialyltransferase-overexpressing 293ST-2D6 cell line resulted in the generation of enzyme species that carry glycans sialylated to the same extent. Pharmacokinetic profiling of the rHuAChE glycoforms in their fully tetramerized and sialylated state clearly demonstrated that circulatory longevity correlated directly with the number of attached N-glycans (mean residence times for rHuAChE glycoforms harbouring 2, 3, and 4 glycans = 200, 740, and 1055min respectively). This study provides evidence that glycan loading, together with N-glycan terminal processing and enzyme subunit oligomerization, operate in a hierarchical and concerted manner in determining the pharmacokinetic characteristics of AChE.
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Binley, James M., Yih-En Andrew Ban, Emma T. Crooks, Dirk Eggink, Keiko Osawa, William R. Schief, and Rogier W. Sanders. "Role of Complex Carbohydrates in Human Immunodeficiency Virus Type 1 Infection and Resistance to Antibody Neutralization." Journal of Virology 84, no. 11 (March 24, 2010): 5637–55. http://dx.doi.org/10.1128/jvi.00105-10.
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ABSTRACT Complex N-glycans flank the receptor binding sites of the outer domain of HIV-1 gp120, ostensibly forming a protective “fence” against antibodies. Here, we investigated the effects of rebuilding this fence with smaller glycoforms by expressing HIV-1 pseudovirions from a primary isolate in a human cell line lacking N-acetylglucosamine transferase I (GnTI), the enzyme that initiates the conversion of oligomannose N-glycans into complex N-glycans. Thus, complex glycans, including those that surround the receptor binding sites, are replaced by fully trimmed oligomannose stumps. Conversely, the untrimmed oligomannoses of the silent domain of gp120 are likely to remain unchanged. For comparison, we produced a mutant virus lacking a complex N-glycan of the V3 loop (N301Q). Both variants exhibited increased sensitivities to V3 loop-specific monoclonal antibodies (MAbs) and soluble CD4. The N301Q virus was also sensitive to “nonneutralizing” MAbs targeting the primary and secondary receptor binding sites. Endoglycosidase H treatment resulted in the removal of outer domain glycans from the GnTI- but not the parent Env trimers, and this was associated with a rapid and complete loss in infectivity. Nevertheless, the glycan-depleted trimers could still bind to soluble receptor and coreceptor analogs, suggesting a block in post-receptor binding conformational changes necessary for fusion. Collectively, our data show that the antennae of complex N-glycans serve to protect the V3 loop and CD4 binding site, while N-glycan stems regulate native trimer conformation, such that their removal can lead to global changes in neutralization sensitivity and, in extreme cases, an inability to complete the conformational rearrangements necessary for infection.
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Vyas, Heema K. N., Anuk D. Indraratna, Arun Everest-Dass, Nicolle H. Packer, David M. P. De Oliveira, Marie Ranson, Jason D. McArthur, and Martina L. Sanderson-Smith. "Assessing the Role of Pharyngeal Cell Surface Glycans in Group A Streptococcus Biofilm Formation." Antibiotics 9, no. 11 (November 4, 2020): 775. http://dx.doi.org/10.3390/antibiotics9110775.
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Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Antibiotic treatment failure rates of 20–40% have been observed. The role host cell glycans play in GAS biofilm formation in the context of GAS pharyngitis and subsequent antibiotic treatment failure has not been previously investigated. GAS serotype M12 GAS biofilms were assessed for biofilm formation on Detroit 562 pharyngeal cell monolayers following enzymatic removal of all N-linked glycans from pharyngeal cells with PNGase F. Removal of N-linked glycans resulted in an increase in biofilm biomass compared to untreated controls. Further investigation into the removal of terminal mannose and sialic acid residues with α1-6 mannosidase and the broad specificity sialidase (Sialidase A) also found that biofilm biomass increased significantly when compared to untreated controls. Increases in biofilm biomass were associated with increased production of extracellular polymeric substances (EPS). Furthermore, it was found that M12 GAS biofilms grown on untreated pharyngeal monolayers exhibited a 2500-fold increase in penicillin tolerance compared to planktonic GAS. Pre-treatment of monolayers with exoglycosidases resulted in a further doubling of penicillin tolerance in resultant biofilms. Lastly, an additional eight GAS emm-types were assessed for biofilm formation in response to terminal mannose and sialic acid residue removal. As seen for M12, biofilm biomass on monolayers increased following removal of terminal mannose and sialic acid residues. Collectively, these data demonstrate that pharyngeal cell surface glycan structures directly impact GAS biofilm formation in a strain and glycan specific fashion.
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White, K. D., R. D. Cummings, and F. J. Waxman. "Ig N-glycan orientation can influence interactions with the complement system." Journal of Immunology 158, no. 1 (January 1, 1997): 426–35. http://dx.doi.org/10.4049/jimmunol.158.1.426.
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Abstract This study was prompted by the paradoxical observation that a pair of dinitrophenyl-specific murine monoclonal IgG2a Abs had similar monosaccharide content and yet differed in their binding to lectins. The differential lectin-binding properties were lost when the Abs were denatured, suggesting that variations in lectin binding reflected the conformational accessibility of the N-glycans rather than intrinsic differences in the lectin binding capacity of the glycans themselves. This hypothesis was supported by experiments indicating that the degree to which the N-glycans on the Abs were reactive with beta-1,4-galactosyltransferase or susceptible to peptide N-glycosidase F corresponded directly to their relative accessibility to lectins. Moreover, the relative susceptibility to these enzymes and accessibility to lectins was inversely related to the capacity of the Abs to activate the classical pathway, suggesting that the orientation of the more accessible N-glycan might inhibit C1q binding. This hypothesis was supported by evidence that enzymatic cleavage of the more accessible N-glycan resulted in enhanced Clq, C4b, and C3b deposition. Conversely, removal of the less accessible N-glycan expressed by the other Ab inhibited C1q, C4b, and C3b deposition. The respective increase or decrease in C3b deposition on the two deglycosylated Abs was magnified when complement activation was performed in factor B-depleted serum, suggesting that N-glycan conformation primarily affects the classical pathway. Collectively, these data suggest that the orientation of the N-glycan expressed on Igs can profoundly influence interaction with the complement system.
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Townsley, Samantha, Yun Li, Yury Kozyrev, Brad Cleveland, and Shiu-Lok Hu. "Conserved Role of an N-Linked Glycan on the Surface Antigen of Human Immunodeficiency Virus Type 1 Modulating Virus Sensitivity to Broadly Neutralizing Antibodies against the Receptor and Coreceptor Binding Sites." Journal of Virology 90, no. 2 (October 28, 2015): 829–41. http://dx.doi.org/10.1128/jvi.02321-15.
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ABSTRACTHIV-1 establishes persistent infection in part due to its ability to evade host immune responses. Occlusion by glycans contributes to masking conserved sites that are targets for some broadly neutralizing antibodies (bNAbs). Previous work has shown that removal of a highly conserved potential N-linked glycan (PNLG) site at amino acid residue 197 (N7) on the surface antigen gp120 of HIV-1 increases neutralization sensitivity of the mutant virus to CD4 binding site (CD4bs)-directed antibodies compared to its wild-type (WT) counterpart. However, it is not clear if the role of the N7 glycan is conserved among diverse HIV-1 isolates and if other glycans in the conserved regions of HIV-1 Env display similar functions. In this work, we examined the role of PNLGs in the conserved region of HIV-1 Env, particularly the role of the N7 glycan in a panel of HIV-1 strains representing different clades, tissue origins, coreceptor usages, and neutralization sensitivities. We demonstrate that the absence of the N7 glycan increases the sensitivity of diverse HIV-1 isolates to CD4bs- and V3 loop-directed antibodies, indicating that the N7 glycan plays a conserved role masking these conserved epitopes. However, the effect of the N7 glycan on virus sensitivity to neutralizing antibodies directed against the V2 loop epitope is isolate dependent. These findings indicate that the N7 glycan plays an important and conserved role modulating the structure, stability, or accessibility of bNAb epitopes in the CD4bs and coreceptor binding region, thus representing a potential target for the design of immunogens and therapeutics.IMPORTANCEN-linked glycans on the HIV-1 envelope protein have been postulated to contribute to viral escape from host immune responses. However, the role of specific glycans in the conserved regions of HIV-1 Env in modulating epitope recognition by broadly neutralizing antibodies has not been well defined. We show here that a single N-linked glycan plays a unique and conserved role among conserved glycans on HIV-1 gp120 in modulating the exposure or the stability of the receptor and coreceptor binding site without affecting the integrity of the Env in mediating viral infection or the ability of the mutant gp120 to bind to CD4. The observation that the antigenicity of the receptor and coreceptor binding sites can be modulated by a single glycan indicates that select glycan modification offers a potential strategy for the design of HIV-1 vaccine candidates.
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Aguilar, Hector C., Kenneth A. Matreyek, Claire Marie Filone, Sara T. Hashimi, Ernest L. Levroney, Oscar A. Negrete, Andrea Bertolotti-Ciarlet, et al. "N-Glycans on Nipah Virus Fusion Protein Protect against Neutralization but Reduce Membrane Fusion and Viral Entry." Journal of Virology 80, no. 10 (May 15, 2006): 4878–89. http://dx.doi.org/10.1128/jvi.80.10.4878-4889.2006.
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ABSTRACT Nipah virus (NiV) is a deadly emerging paramyxovirus. The NiV attachment (NiV-G) and fusion (NiV-F) envelope glycoproteins mediate both syncytium formation and viral entry. Specific N-glycans on paramyxovirus fusion proteins are generally required for proper conformational integrity and biological function. However, removal of individual N-glycans on NiV-F had little negative effect on processing or fusogenicity and has even resulted in slightly increased fusogenicity. Here, we report that in both syncytium formation and viral entry assays, removal of multiple N-glycans on NiV-F resulted in marked increases in fusogenicity (>5-fold) but also resulted in increased sensitivity to neutralization by NiV-F-specific antisera. The mechanism underlying the hyperfusogenicity of these NiV-F N-glycan mutants is likely due to more-robust six-helix bundle formation, as these mutants showed increased fusion kinetics and were more resistant to neutralization by a fusion-inhibitory reagent based on the C-terminal heptad repeat region of NiV-F. Finally, we demonstrate that the fusogenicities of the NiV-F N-glycan mutants were inversely correlated with the relative avidities of NiV-F's interactions with NiV-G, providing support for the attachment protein “displacement” model of paramyxovirus fusion. Our results indicate that N-glycans on NiV-F protect NiV from antibody neutralization, suggest that this “shielding” role comes together with limiting cell-cell fusion and viral entry efficiencies, and point to the mechanisms underlying the hyperfusogenicity of these N-glycan mutants. These features underscore the varied roles that N-glycans on NiV-F play in the pathobiology of NiV entry but also shed light on the general mechanisms of paramyxovirus fusion with host cells.
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Zhang, Nanyan, Brian R. Curtis, and Peter J. Newman. "Genetic Removal of Terminal Sialic Acid Residues from the O-Linked Glycans Adjacent to the HPA-9b Polymorphism of Platelet Membrane Glycoprotein IIb Improves the Binding and Detection of HPA-9b Patient Alloantibodies." Blood 138, Supplement 1 (November 5, 2021): 352. http://dx.doi.org/10.1182/blood-2021-144718.
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Abstract Sialic acids occupy the terminal position of glycan chains, and have the potential to influence the antigenicity of glycoproteins. Antibody binding sites on a glycoprotein can be solely protein in nature, or can include or be affected by nearby glycan chains, which may either mask the epitope, or conversely comprise part of the antibody binding site. The polymorphisms responsible for formation of the Human Platelet Alloantigens (HPA)-3 (Ile843Ser) and HPA-9 (Val837Met) are next to each other near the C-terminus of the extracellular domain of platelet membrane glycoprotein (GP)IIb, and are adjacent to sialyl-Core 1 O-glycans that emanate from serines 845 and 847. Previous studies have shown that these O-linked glycans are required to support the binding of some of HPA-3a alloantibodies. Loss of these glycans, especially terminal sialic acid residues, during platelet storage or preparation, can present major difficulties in detecting clinically important anti-HPA-3a alloantibodies in suspected cases of fetal/neonatal alloimmune thrombocytopenia (FNAIT). Similarly, detection and identification of anti-HPA-9b alloantibodies from FNAIT patient sera can also be extremely challenging, resulting in the inability to resolve clinical cases of this bleeding disorder. Whether the nearby O-glycans on serines 845 and 847 of GPIIb affect the antigenicity of HPA-9b, and/or influence the binding of anti-HPA-9b alloantibodies in clinically significant cases of FNAIT is unknown. We previously reported the generation of bioengineered, HLA class I-negative, HPA-9a or -9b allele-specific megakaryocytes (MKs) from human induced pluripotent stem cells (iPSCs) that are suitable for whole-cell flow cytometric detection of anti-HPA-9b alloantibodies (Blood 2019;134(22):e1-e8). Unexpectedly, treatment of these allele-specific MKs with neuraminidase actually enhanced the binding of anti-HPA-9b alloantibodies, suggesting that terminal sialic acids on GPIIb partially mask the HPA-9b epitope. To test the hypothesis that removal of terminal sialic acids on nearby O-glycans, or removal of the entire O-glycan chains emanating from Ser845/847 of GPIIb, might enhance the detection of anti-HPA-9b patient alloantibodies, we created a series of deletion mutants in two major sialidases, ST3GAL1 and ST3GAL2, known to be responsible for transferring terminal sialic acid residues to Core 1 O-glycans, in our HPA-9a and -9b allele-specific iPSCs. Immunoprecipitation/western blot analysis confirmed the complete removal of terminal sialic acids on the O-glycan chains of GPIIb in ST3GAL1/2 knockout (KO) iPSC-derived MKs, as reported by the binding of the lectin PNA to the exposed Core 1 structure on GPIIb. These sialylation-deficient ST3GAL1/2 KO HPA-9b MKs exhibited dramatically increased anti-HPA-9b alloantibody binding, further confirming the notion that HPA-9b epitopes are partially masked by terminal sialic acids on nearby GPIIb O-glycan chains. Finally, allele-specific iPSCs lacking the complete O-glycan chains attached to serines 845 and 847 of GPIIb were generated by mutating those residues to alanines using a similar CRISPR/Cas9 gene editing approach. Interestingly, O-glycan chain-deficient Ala845/847 mutant MKs carrying the HPA-9b polymorphism exhibited slightly to moderately reduced binding of anti-HPA-9b alloantibodies, indicating that the presence of the Core 1 O-glycan chains attached to GPIIb serine residues 845 and 847 contribute to the presentation of the HPA-9b epitope - perhaps by stabilizing the conformation of the glycoprotein in this region. Taken together, these data suggest that detection of anti-HPA-9b alloantibodies may be enhanced through the use of iPSC-derived HPA-9b-specific MKs that have been genetically altered to lack nearby terminal sialic acid residues, but retain the glycan chains to which they are attached. Disclosures Curtis: Rallybio: Consultancy. Newman: Rallybio: Consultancy, Research Funding.
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Chen, Ping, Ran Liu, Mengmeng Huang, Jinlu Zhu, Dong Wei, Francis J. Castellino, Guanghui Dang, et al. "A unique combination of glycoside hydrolases in Streptococcus suis specifically and sequentially acts on host-derived αGal-epitope glycans." Journal of Biological Chemistry 295, no. 31 (June 9, 2020): 10638–52. http://dx.doi.org/10.1074/jbc.ra119.011977.
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Infections by many bacterial pathogens rely on their ability to degrade host glycans by producing glycoside hydrolases (GHs). Here, we discovered a conserved multifunctional GH, SsGalNagA, containing a unique combination of two family 32 carbohydrate-binding modules (CBM), a GH16 domain and a GH20 domain, in the zoonotic pathogen Streptococcus suis 05ZYH33. Enzymatic assays revealed that the SsCBM-GH16 domain displays endo-(β1,4)-galactosidase activity specifically toward the host-derived αGal epitope Gal(α1,3)Gal(β1,4)Glc(NAc)-R, whereas the SsGH20 domain has a wide spectrum of exo-β-N-acetylhexosaminidase activities, including exo-(β1,3)-N-acetylglucosaminidase activity, and employs this activity to act in tandem with SsCBM-GH16 on the αGal-epitope glycan. Further, we found that the CBM32 domain adjacent to the SsGH16 domain is indispensable for SsGH16 catalytic activity. Surface plasmon resonance experiments uncovered that both CBM32 domains specifically bind to αGal-epitope glycan, and together they had a KD of 3.5 mm toward a pentasaccharide αGal-epitope glycan. Cell-binding and αGal epitope removal assays revealed that SsGalNagA efficiently binds to both swine erythrocytes and tracheal epithelial cells and removes the αGal epitope from these cells, suggesting that SsGalNagA functions in nutrient acquisition or alters host signaling in S. suis. Both binding and removal activities were blocked by an αGal-epitope glycan. SsGalNagA is the first enzyme reported to sequentially act on a glycan containing the αGal epitope. These findings shed detailed light on the evolution of GHs and an important host-pathogen interaction.
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Kong, Leopold, Alba Torrents de la Peña, Marc C. Deller, Fernando Garces, Kwinten Sliepen, Yuanzi Hua, Robyn L. Stanfield, Rogier W. Sanders, and Ian A. Wilson. "Complete epitopes for vaccine design derived from a crystal structure of the broadly neutralizing antibodies PGT128 and 8ANC195 in complex with an HIV-1 Env trimer." Acta Crystallographica Section D Biological Crystallography 71, no. 10 (September 26, 2015): 2099–108. http://dx.doi.org/10.1107/s1399004715013917.
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The HIV-1 envelope gp160 glycoprotein (Env) is a trimer of gp120 and gp41 heterodimers that mediates cell entry and is the primary target of the humoral immune response. Broadly neutralizing antibodies (bNAbs) to HIV-1 have revealed multiple epitopes or sites of vulnerability, but mapping of most of these sites is incomplete owing to a paucity of structural information on the full epitope in the context of the Env trimer. Here, a crystal structure of the soluble BG505 SOSIP gp140 trimer at 4.6 Å resolution with the bNAbs 8ANC195 and PGT128 reveals additional interactions in comparison to previous antibody–gp120 structures. For 8ANC195, in addition to previously documented interactions with gp120, a substantial interface with gp41 is now elucidated that includes extensive interactions with the N637 glycan. Surprisingly, removal of the N637 glycan did not impact 8ANC195 affinity, suggesting that the antibody has evolved to accommodate this glycan without loss of binding energy. PGT128 indirectly affects the N262 glycan by a domino effect, in which PGT128 binds to the N301 glycan, which in turn interacts with and repositions the N262 glycan, thereby illustrating the important role of neighboring glycans on epitope conformation and stability. Comparisons with other Env trimer and gp120 structures support an induced conformation for glycan N262, suggesting that the glycan shield is allosterically modified upon PGT128 binding. These complete epitopes of two broadly neutralizing antibodies on the Env trimer can now be exploited for HIV-1 vaccine design.
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Fan, Rui Xia, Qing Wei Ping, Jian Zhang, and Hai Qiang Shi. "Degradation Rules of Glycan in Reed Ethanol/Water Cooking Process." Advanced Materials Research 781-784 (September 2013): 2618–22. http://dx.doi.org/10.4028/www.scientific.net/amr.781-784.2618.
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The degradation rules of glycan in reed ethanol/water cooking process were studied. The degradation rules of polydextrose and polyxylose were obtained in the cooking process by means of ion chromatography determination of glucose and xylose in the reed ethanol pulp content under different holding time. Results showed that cellulose (polydextrose) removal rate obviously was divided into two stages, rapid removal stage at the holding time of 0-90min and slow removal stage at the holding time of 90-210min; hemicellulose (xylan) removal rate was also divided into two stages, slow removal stage at the holding time of 0-60 min and rapid removal stage at the holding time from 60min until the end; meanwhile the change rules of lignin and ash content were achieved.
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Luallen, Robert J., Jianqiao Lin, Hu Fu, Karen K. Cai, Caroline Agrawal, Innocent Mboudjeka, Fang-Hua Lee, et al. "An Engineered Saccharomyces cerevisiae Strain Binds the Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody 2G12 and Elicits Mannose-Specific gp120-Binding Antibodies." Journal of Virology 82, no. 13 (April 23, 2008): 6447–57. http://dx.doi.org/10.1128/jvi.00412-08.
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ABSTRACT The glycan shield of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein serves as a barrier to antibody-mediated neutralization and plays a critical role in transmission and infection. One of the few broadly neutralizing HIV-1 antibodies, 2G12, binds to a carbohydrate epitope consisting of an array of high-mannose glycans exposed on the surface of the gp120 subunit of the Env protein. To produce proteins with exclusively high-mannose carbohydrates, we generated a mutant strain of Saccharomyces cerevisiae by deleting three genes in the N-glycosylation pathway, Och1, Mnn1, and Mnn4. Glycan profiling revealed that N-glycans produced by this mutant were almost exclusively Man8GlcNAc2, and four endogenous glycoproteins that were efficiently recognized by the 2G12 antibody were identified. These yeast proteins, like HIV-1 gp120, contain a large number and high density of N-linked glycans, with glycosidase digestion abrogating 2G12 cross-reactivity. Immunization of rabbits with whole Δoch1 Δmnn1 Δmnn4 yeast cells produced sera that recognized a broad range of HIV-1 and simian immunodeficiency virus (SIV) Env glycoproteins, despite no HIV/SIV-related proteins being used in the immunization procedure. Analyses of one of these sera on a glycan array showed strong binding to glycans with terminal Manα1,2Man residues, and binding to gp120 was abrogated by glycosidase removal of high-mannose glycans and terminal Manα1,2Man residues, similar to 2G12. Since S. cerevisiae is genetically pliable and can be grown easily and inexpensively, it will be possible to produce new immunogens that recapitulate the 2G12 epitope and may make the glycan shield of HIV Env a practical target for vaccine development.
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Yahashiri, Atsushi, Matthew A. Jorgenson, and David S. Weiss. "Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides." Proceedings of the National Academy of Sciences 112, no. 36 (August 24, 2015): 11347–52. http://dx.doi.org/10.1073/pnas.1508536112.
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Bacterial SPOR domains bind peptidoglycan (PG) and are thought to target proteins to the cell division site by binding to “denuded” glycan strands that lack stem peptides, but uncertainties remain, in part because septal-specific binding has yet to be studied in a purified system. Here we show that fusions of GFP to SPOR domains from theEscherichia colicell-division proteins DamX, DedD, FtsN, and RlpA all localize to septal regions of purified PG sacculi obtained fromE.coliandBacillus subtilis. Treatment of sacculi with an amidase that removes stem peptides enhanced SPOR domain binding, whereas treatment with a lytic transglycosylase that removes denuded glycans reduced SPOR domain binding. These findings demonstrate unequivocally that SPOR domains localize by binding to septal PG, that the physiologically relevant binding site is indeed a denuded glycan, and that denuded glycans are enriched in septal PG rather than distributed uniformly around the sacculus. Accumulation of denuded glycans in the septal PG of bothE.coliandB.subtilis, organisms separated by 1 billion years of evolution, suggests that sequential removal of stem peptides followed by degradation of the glycan backbone is an ancient feature of PG turnover during bacterial cell division. Linking SPOR domain localization to the abundance of a structure (denuded glycans) present only transiently during biogenesis of septal PG provides a mechanism for coordinating the function of SPOR domain proteins with the progress of cell division.
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Burock, Robert, Samanta Cajic, René Hennig, Falk F. R. Buettner, Udo Reichl, and Erdmann Rapp. "Reliable N-Glycan Analysis–Removal of Frequently Occurring Oligosaccharide Impurities by Enzymatic Degradation." Molecules 28, no. 4 (February 15, 2023): 1843. http://dx.doi.org/10.3390/molecules28041843.
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Glycosylation, especially N-glycosylation, is one of the most common protein modifications, with immense importance at the molecular, cellular, and organismal level. Thus, accurate and reliable N-glycan analysis is essential in many areas of pharmaceutical and food industry, medicine, and science. However, due to the complexity of the cellular glycosylation process, in-depth glycoanalysis is still a highly challenging endeavor. Contamination of samples with oligosaccharide impurities (OSIs), typically linear glucose homo-oligomers, can cause further complications. Due to their physicochemical similarity to N-glycans, OSIs produce potentially overlapping signals, which can remain unnoticed. If recognized, suspected OSI signals are usually excluded in data evaluation. However, in both cases, interpretation of results can be impaired. Alternatively, sample preparation can be repeated to include an OSI removal step from samples. However, this significantly increases sample amount, time, and effort necessary. To overcome these issues, we investigated the option to enzymatically degrade and thereby remove interfering OSIs as a final sample preparation step. Therefore, we screened ten commercially available enzymes concerning their potential to efficiently degrade maltodextrins and dextrans as most frequently found OSIs. Of these enzymes, only dextranase from Chaetomium erraticum and glucoamylase P from Hormoconis resinae enabled a degradation of OSIs within only 30 min that is free of side reactions with N-glycans. Finally, we applied the straightforward enzymatic degradation of OSIs to N-glycan samples derived from different standard glycoproteins and various stem cell lysates.
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Jones, D., A. Mehlert, and M. A. J. Ferguson. "The N-glycan glucosidase system in Trypanosoma brucei." Biochemical Society Transactions 32, no. 5 (October 26, 2004): 766–68. http://dx.doi.org/10.1042/bst0320766.
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Reactions involving removal and addition of glucose to N-glycans in the ER (endoplasmic reticulum) are performed in higher eukaryotes by glucosidases I and II and the UDP-glucose:glycoprotein glucosyltransferase respectively. Monoglucosylated N-glycan structures have been implicated in glycoprotein folding or ER quality control. Components of the system appear across a range of organisms; however, the precise combination differs between organisms. We have identified putative components of the system in the protozoal organism Trypanosoma brucei by local alignment searching. The function of one of these components, a glucosidase II α-subunit homologue, has been confirmed by phenotyping a null mutant, and an ectopic expression cell line. A combination of MS, methylation linkage analysis, exoglycosidase digestion and partial acetolysis have been used to characterize three novel N-glycan structures on the variant surface glycoprotein of the null mutant. On the basis of our results, we propose that two N-glycan precursors are available for transfer to variant surface glycoprotein (variant 221) in the ER of T. brucei; only one of these precursors is glucosylated after transfer.
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Brockhausen, Inka, Gabriele Möller, Annette Pollex-Krüger, Volker Rutz, Hans Paulsen, and Khushi L. Matta. "Control of O-glycan synthesis: specificity and inhibition of O-glycan core 1 UDP-galactose:N-acetylgalactosamine-α-R β3-galactosyltransferase from rat liver." Biochemistry and Cell Biology 70, no. 2 (February 1, 1992): 99–108. http://dx.doi.org/10.1139/o92-015.
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The specificity of glycosyltransferases is a major control factor in the biosynthesis of O-glycans. The enzyme that synthesizes O-glycan core 1, i.e., UDP-galactose:N-acetylgalactosamine-α-R β3-galactosyltransferase (β3-Gal-T; EC 2.4.1.122), was partially purified from rat liver. The enzyme preparation, free of pyrophosphatases, β4-galactosyltransferase, β-galactosidase, and N-acetylglucosaminyltransferase I, was used to study the specificity and inhibition of the β3-Gal-T. β3-Gal-T activity is sensitive to changes in the R-group of the GalNAcα-R acceptor substrate and is stimulated when the R-group is a peptide or an aromatic group. Derivatives of GalNAcα-benzyl were synthesized and tested as potential substrates and inhibitors. Removal or substitution of the 3-hydroxyl or removal of the 4-hydroxyl of GalNAc abolished β3-Gal-T activity. Compounds with modifications of the 3- or 4-hydroxyl of GalNAcα-benzyl did not show significant inhibition. Removal or substitution of the 6-hydroxyl of GalNAc reduced activity slightly and these derivatives acted as competitive substrates. Derivatives with epoxide groups attached to the 6-position of GalNAc acted as substrates and not as inhibitors, with the exception of the photosensitive 6-O-(4,4-azo)pentyl-GalNAcα-benzyl, which inhibited Gal incorporation into GalNAcα-benzyl. The results indicate that the enzyme does not require the 6-hydroxyl of GalNAc, but needs the 3- and the axial 4-hydroxyl as essential requirements for binding and activity. In the usual biochemical O-glycan pathway, core 2 (GlcNAcβ6[Galβ3]GalNAcα-) is formed from core 1 (Galβ3GalNAc-R). We have now demonstrated an alternate pathway that may be of importance in human tissues.Key words: β3-Gal-transferase, mucin synthesis, O-glycan core 1, enzyme specificity, enzyme inhibition.
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Pikora, Cheryl, Christine Wittish, and Ronald C. Desrosiers. "Identification of Two N-Linked Glycosylation Sites within the Core of the Simian Immunodeficiency Virus Glycoprotein Whose Removal Enhances Sensitivity to Soluble CD4." Journal of Virology 79, no. 19 (October 1, 2005): 12575–83. http://dx.doi.org/10.1128/jvi.79.19.12575-12583.2005.
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ABSTRACT Using PCR mutagenesis to disrupt the NXT/S N-linked glycosylation motif of the Env protein, we created 27 mutants lacking 1 to 5 of 14 N-linked glycosylation sites within regions of gp120 lying outside of variable loops 1 to 4 within simian immunodeficiency virus strain 239 (SIV239). Of 18 mutants missing N-linked glycosylation sites predicted to lie within 10 Å of CD4 contact sites, the infectivity of 12 was sufficient to measure sensitivity to neutralization by soluble CD4 (sCD4), pooled immune sera from SIV239-infected rhesus macaques, and monoclonal antibodies known to neutralize certain derivatives of SIV239. Three of these 12 mutants (g3, lacking the 3rd glycan at position 79; g11, lacking the 11th glycan at position 212; and g3,11, lacking both the 3rd and 11th glycans) were approximately five times more sensitive to neutralization by sCD4 than wild-type (WT) SIV239. However, these same mutants were no more sensitive to neutralization than WT by pooled immune sera. The other 9 of 12 replication-competent mutants in this group were no more sensitive to neutralization than the WT by any of the neutralizing reagents. Six of the nine mutants that did not replicate appreciably had three or more glycosylation sites eliminated; the other three replication-deficient strains involved mutation of site 15. Our results suggest that elimination of glycan attachment sites 3 and 11 enhanced the exposure of contact residues for CD4. Thus, glycans at positions 3 and 11 of SIV239 gp120 may be particularly important for shielding the CD4-binding site from antibody recognition.
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Campbell, Lee Ann, Amy Lee, and Cho-chou Kuo. "Cleavage of the N-Linked Oligosaccharide from the Surfaces of Chlamydia Species Affects Infectivity in the Mouse Model of Lung Infection." Infection and Immunity 74, no. 5 (May 2006): 3027–29. http://dx.doi.org/10.1128/iai.74.5.3027-3029.2006.
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ABSTRACT Previous studies determined that the Chlamydia glycan contains a high-mannose oligosaccharide, which is involved in attachment and infectivity of the organism, and that removal of the glycan decreases infectivity in vitro. The present study demonstrates that treatment of the organism with N-glycanase decreases or ablates infectivity in vivo.
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Prentoe, Jannick, Rodrigo Velázquez-Moctezuma, Elias H. Augestad, Andrea Galli, Richard Wang, Mansun Law, Harvey Alter, and Jens Bukh. "Hypervariable region 1 and N-linked glycans of hepatitis C regulate virion neutralization by modulating envelope conformations." Proceedings of the National Academy of Sciences 116, no. 20 (April 30, 2019): 10039–47. http://dx.doi.org/10.1073/pnas.1822002116.
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About two million new cases of hepatitis C virus (HCV) infections annually underscore the urgent need for a vaccine. However, this effort has proven challenging because HCV evades neutralizing antibodies (NAbs) through molecular features of viral envelope glycoprotein E2, including hypervariable region 1 (HVR1) and N-linked glycans. Here, we observe large variation in the effects of removing individual E2 glycans across HCV strains H77(genotype 1a), J6(2a), and S52(3a) in Huh7.5 cell infections. Also, glycan-mediated effects on neutralization sensitivity were completely HVR1-dependent, and neutralization data were consistent with indirect protection of epitopes, as opposed to direct steric shielding. Indeed, the effect of removing each glycan was similar both in type (protective or sensitizing) and relative strength across four nonoverlapping neutralization epitopes. Temperature-dependent neutralization (e.g., virus breathing) assays indicated that both HVR1 and protective glycans stabilized a closed, difficult to neutralize, envelope conformation. This stabilizing effect was hierarchical as removal of HVR1 fully destabilized closed conformations, irrespective of glycan status, consistent with increased instability at acidic pH and high temperatures. Finally, we observed a strong correlation between neutralization sensitivity and scavenger receptor BI dependency during viral entry. In conclusion, our study indicates that HVR1 and glycans regulate HCV neutralization by shifting the equilibrium between open and closed envelope conformations. This regulation appears tightly linked with scavenger receptor BI dependency, suggesting a role of this receptor in transitions from closed to open conformations during entry. This importance of structural dynamics of HCV envelope glycoproteins has critical implications for vaccine development and suggests that similar phenomena could contribute to immune evasion of other viruses.
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Mishin, Vasiliy P., Dmitri Novikov, Frederick G. Hayden, and Larisa V. Gubareva. "Effect of Hemagglutinin Glycosylation on Influenza Virus Susceptibility to Neuraminidase Inhibitors." Journal of Virology 79, no. 19 (October 1, 2005): 12416–24. http://dx.doi.org/10.1128/jvi.79.19.12416-12424.2005.
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ABSTRACT Inhibition of neuraminidase (NA) activity prevents release of progeny virions from influenza-infected cells and removal of neuraminic (sialic) acid moieties from glycans attached to hemagglutinin (HA). Neuraminic acid moieties situated near the HA receptor-binding site can reduce the efficiency of virus binding and decrease viral dependence on NA activity for replication. With the use of reverse genetics technique, we investigated the effect of glycans attached at Asn 94a, 129, and 163 on the virus susceptibility to NA inhibitors in MDCK cells and demonstrated that the glycan attached at Asn 163 plays a dominant role in compensation for the loss of NA activity.
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Kamikubo, Yuichi, Claudia Dellas, David J. Loskutoff, James P. Quigley, and Zaverio M. Ruggeri. "Contribution of leptin receptor N-linked glycans to leptin binding." Biochemical Journal 410, no. 3 (February 27, 2008): 595–604. http://dx.doi.org/10.1042/bj20071137.
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The extracellular domain of the human leptin receptor (Ob-R) contains 20 potential N-glycosylation sites whose role in leptin binding remains to be elucidated. We found that a mammalian cell-expressed sOb-R (soluble Ob-R) fragment (residues 22–839 of the extracellular domain) bound leptin with a dissociation constant of 1.8 nM. This binding was inhibited by Con A (concanavalin A) or wheatgerm agglutinin. Treatment of sOb-R with peptide N-glycosidase F reduced leptin binding by ∼80% concurrently with N-linked glycan removal. The human megakaryoblastic cell line, MEG-01, expresses two forms of the Ob-R, of approx. 170 and 130 kDa molecular mass. Endo H (endoglycosidase H) treatment and cell culture with α-glucosidase inhibitors demonstrated that N-linked glycans are of the complex mature type in the 170 kDa form and of the high-mannose type in the 130 kDa form. Both isoforms bound leptin, but not after peptide N-glycosidase F treatment. An insect-cell-expressed sOb-R fragment, consisting of the Ig (immunoglobulin), CRH2 (second cytokine receptor homology) and FNIII (fibronectin type III) domains, bound leptin with affinity similar to that of the entire extracellular domain, but this function was abolished after N-linked glycan removal. The same treatment had no effect on the leptin-binding activity of the isolated CRH2 domain. Our findings show that N-linked glycans within Ig and/or FNIII domains regulate Ob-R function, but are not involved in essential interactions with the ligand.
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McKinnon, Thomas A. J., Alain C. K. Chion, and Mike A. Laffan. "The Effect of Von Willebrand Factor Glycans on the Interaction with ADAMTS13." Blood 108, no. 11 (November 16, 2006): 1701. http://dx.doi.org/10.1182/blood.v108.11.1701.1701.
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Abstract Von Willebrand factor (VWF) is a large plasma glycoprotein that mediates platelet tethering at sites of vascular injury. This function is dependent upon its multimeric size, which is modulated in plasma through proteolysis by ADAMTS13. Terminal ABO blood group sugars presented on the N-linked glycans of VWF alter the susceptibility of VWF to cleavage by ADAMTS13. Two predicted N-linked glycan sites (N1515 & N1574) are in close proximity to the ADAMTS13 cleavage site (Y1605-M1606). However, is it not known whether these predicted sites are occupied, or indeed, present the ABO sugars. In this study, purified plasma VWF was digested with trypsin, and the resulting fragments separated by ion-exchange chromatography. A 55kDa fragment observed under reducing SDS-PAGE was identified as VWF residues 1493–1849 by mass spectrocosopy and N-terminal sequencing. This fragment encompassed the predicted N-linked glycan sites at N1515 & N1574. Incomplete PNGase F digestion produced three protein bands, indicating that both glycosylation sites were occupied. Furthermore, analysis with blood group specific lectins demonstrated presentation of the ABO sugars on these glycan chains. This data suggests that the N1515 and N1574 glycosylation sites and subsequent ABO(H) sugar modification may influence VWF proteolysis. To further elucidate the role of other VWF glycan structures on susceptibility to ADAMTS13 cleavage, purified VWF was treated with neuraminidase to remove terminal sialic acid residues, producing asialo VWF (As-VWF), and PNGase F to remove whole N-linked glycan chains. (Nless-VWF). Lectin analysis demonstrated removal of >95% of terminal sialic residues and >75% whole N-linked glycan chains. Both AsVWF and Nless-VWF bound to type III collagen with similar affinity to wild type VWF (KD 1.9nM, 1.6nM, 1.4nM respectively) and demonstrated similar multimeric composition. Interestingly, As-VWF had decreased susceptibility to ADAMTS13 cleavage, but bound ADAMTS13 with comparable affinity to wild type VWF (KD 11nM & 12nM respectively). Nless-VWF was cleaved faster by ADAMTS13 and bound ADAMTS13 with ~ 4-fold increased affinity (KD 3.4nM). This data demonstrates that the glycan moiety of VWF plays an important role in mediating the interaction with ADAMTS13.
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Hoffmeister, Karin M. "Novel Glycan Dependent Platelet Clearance Mechanisms." Blood 130, Suppl_1 (December 7, 2017): SCI—31—SCI—31. http://dx.doi.org/10.1182/blood.v130.suppl_1.sci-31.sci-31.
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Platelet numbers are intricately regulated to avoid spontaneous bleeding or arterial occlusion and organ damage. The growth factor thrombopoietin (TPO) promotes platelet biogenesis by controlling megakaryocyte maturation and differentiation. Recently, we identified a feedback mechanism by which clearance of aged, desialylated platelets stimulates TPO synthesis by hepatocytes in mice. The Ashwell-Morell receptor (AMR) of hepatocytes, originally termed the hepatic asialoglycoprotein receptor, was the first cellular receptor to be identified and isolated and the first lectin (carbohydrate-binding) to be detected in mammals. Our work showed that the AMR recognizes and removes senescent, sialic acid-depleted platelets (desialylated) under steady state conditions. Desialylated platelets and the AMR are the physiological ligand-receptor pair regulating hepatic TPO mRNA production. The AMR-mediated removal of desialylated platelets regulates hepatic TPO synthesis by recruiting JAK2 and STAT3 to increase thrombopoiesis. Recent genetics studies have revealed that AMR haplodeficiency provides protection from atherosclerosis by regulating plasma glycolipids and platelets. The potential interactions of AMR with LDL receptor may regulate the rate of LDL uptake and as a result may lower plasma non-HDL cholesterol. Taken together, the AMR appears to be a multifaceted receptor, specializing in the clearance of desialylated platelets and plasma glycolipids. Thus, platelet clearance, its biogenesis via TPO, and vascular integrity appear to be regulated by intricately interwoven mechanisms dependent on the AMR. Disclosures Hoffmeister: Amgen: Consultancy.
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Lowe, M. E. "Site-specific mutations in the COOH-terminus of placental alkaline phosphatase: a single amino acid change converts a phosphatidylinositol-glycan-anchored protein to a secreted protein." Journal of Cell Biology 116, no. 3 (February 1, 1992): 799–807. http://dx.doi.org/10.1083/jcb.116.3.799.
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Placental alkaline phosphatase (PLAP) is anchored in the plasma membrane by a phosphatidylinositol-glycan moiety (PI-glycan). PI-glycan is added posttranslationally to the nascent peptide chain after the removal of 29 amino acids from the COOH-terminus. The contribution of selected COOH-terminal amino acids to the signal for PI-glycan addition was tested by creating a fusion protein with the COOH-terminus of PLAP and a secreted protein and by mutagenesis of specific PLAP COOH-terminal amino acids. The cDNA encoding the COOH-terminus of PLAP was fused in frame to the cDNA for human clotting Factor X and expressed in transfected COS-1 cells. Fusion proteins containing 32 amino acids of the PLAP COOH-terminus were modified by PI-glycan addition. Thus, the signal for PI-glycan modification must reside in these amino acids. Next, the region between the hydrophobic domain and the cleavage site was examined for additional determinants. Mutations of the hydrophilic residues in the spacer region demonstrated that these amino acids do not contribute to the signal for PI-glycan addition. Deletion of amino acids in the spacer region prevented the addition of PI-glycan suggesting that the length of the spacer domain or the amino acids around the cleavage site are important determinants. Finally, we demonstrated that interruption of the hydrophobic domain by a charged residue prevents PI-glycan addition and results in a protein that is secreted into the medium. The finding that a single Leu to Arg substitution in the hydrophobic domain converts a PI-glycan anchored, membrane protein to a secreted protein suggests that an essential signal for the correct sorting of PI-glycan anchored proteins versus secreted proteins resides in the hydrophobic domain. Substitution of a charged amino acid for a hydrophobic amino acid may be a mechanism for producing membrane bound and secreted forms of the same protein.
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Otsubo, N., H. Ishida, and M. Kiso. "Synthetic Studies on Sialoglycoconjugates. CXXIV. Total Synthesis of O-Glycan on the L-Selectin Ligand GlyCAM-1." Australian Journal of Chemistry 55, no. 2 (2002): 105. http://dx.doi.org/10.1071/ch02006.
Full textAbstract:
The total synthesis of O-glycans containing the sulfated sialyl Lex (sLex) determinant on GlyCAM-1 (1), the counter-receptor glycoprotein for L-selectin, was accomplished by a simultaneous glycosylation procedure. The glycosylation of the lactoside acceptor (4) with the 2-azidogalactopyranosyl trichloroacetimidate donor (3) resulted in the desired trisaccharide (5). This trisaccharide, after transformation to the glycosyl acceptor, was successfully coupled with the phenylthioglycoside of glucosamine (13) to give the core 6 structure (14). Following proper manipulation of the protecting groups for the hydroxyl, as well as the amino function, the resulting free hydroxyl of the tetrasaccharide at C3 of the GlcNAc was then fucosylated to give the desired pentasaccharide (19). This pentasaccharide was converted into the key glycosyl acceptor (21), which was then simultaneously glycosylated with the sialyl α (2→3)-galactosyl trichloroacetimidate (22), forming the desired nonasaccharide (23). Finally, sulfation of the 6-OH of the GlcNAc residue and removal of all the protecting groups furnished the spacer-armed O-glycans on GlyCAM-1 (1).
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Mochizuki, Kaori, Tatehiro Kagawa, Asano Numari, Matthew J. Harris, Johbu Itoh, Norihito Watanabe, Tetsuya Mine, and Irwin M. Arias. "Two N-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 292, no. 3 (March 2007): G818—G828. http://dx.doi.org/10.1152/ajpgi.00415.2006.
Full textAbstract:
The aim of this study was to determine the role of N-linked glycosylation in protein stability, intracellular trafficking, and bile acid transport activity of the bile salt export pump [Bsep (ATP-binding cassette B11)]. Rat Bsep was fused with yellow fluorescent protein, and the following mutants, in which Asn residues of putative glycosylation sites (Asn109, Asn116, Asn122, and Asn125) were sequentially replaced with Gln, were constructed by site-directed mutagenesis: single N109Q, double N109Q + N116Q, triple N109Q + N116Q + N122Q, and quadruple N109Q + N116Q + N122Q + N125Q. Immunoblot and glycosidase cleavage analysis demonstrated that each site was glycosylated. Removal of glycans decreased taurocholate transport activity as determined in polarized MDCK II cells. This decrease resulted from rapid decay of the mutant Bsep protein; biochemical half-lives were 3.76, 3.65, 3.24, 1.35, and 0.52 h in wild-type, single-mutant, double-mutant, triple-mutant, and quadruple-mutant cells, respectively. Wild-type and single- and double-mutant proteins were distributed exclusively along the apical membranes, whereas triple- and quadruple-mutant proteins remained intracellular. MG-132 but not bafilomycin A1 extended the half-life, suggesting a role for the proteasome in Bsep degradation. To determine whether a specific glycosylation site or the number of glycans was critical for protein stability, we studied the protein expression of combinations of N-glycan-deficient mutants and observed that Bsep with one glycan was considerably unstable compared with Bsep harboring two or more glycans. In conclusion, at least two N-linked glycans are required for Bsep protein stability, intracellular trafficking, and function in the apical membrane.
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Mittermayr, Stefan, Jonathan Bones, Giao N. Le, and Peter O'Gorman. "N-Glycan Analysis of Polyclonal IgG from Patients with Multiple Myeloma Enables Classification of Stage Specific Pathologies." Blood 126, no. 23 (December 3, 2015): 1761. http://dx.doi.org/10.1182/blood.v126.23.1761.1761.
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Abstract Background Multiple myeloma (MM) is an incurable plasma cell malignancy, with eventual disease refractory and relapse. Its benign precursor, monoclonal gammopathy of undetermined significance (MGUS), has an annual transformation rate of 1%, while that of the asymptomatic smouldering myeloma (SMM) is 10%. The pathognomonic feature is the presence abnormal monoclonal immunoglobulin, of which immunoglobulin G (IgG) paraprotein is the most common. All subclasses of Igs are post-translationally modified by the addition of N-glycans, reportedly influencing structure, stability, and biological function. Previous studies in MM IgG had suggested an increase in the glycosylation of the antigen-binding fragment (Fab), and an overall elevation of sialylation. Using glycomic platforms, we aimed to investigate and characterise the IgG N-glycosylation profiles across the myeloma disease spectrum. Methods Polyclonal IgG was extracted from sera of patients with MM, SMM, MGUS, and age-matched control, using Protein G affinity chromatography. The quantity of extracted IgG was determined using the Bradford assay. N-glycans were enzymatically liberated from a normalised quantity of purified IgG, fluorescently labelled and profiled using hydrophilic interaction ultra-performance liquid chromatography. A combination of exoglycosidase digestions and mass spectrometry were used to elucidate the glycan structures with full linkage and positional specificity. Localisation analyses were performed to determine the distribution of N-glycans at asparagine 297 in the Fc region of the antibody and those present at any additional glycosylation sites present in the Fab region using a combination of enzymatic digestion using the commercially available IdeS enzyme and chemical reduction followed by SDS-PAGE separation of the resulting protein fragments and subsequent in-gel digestion of the N-glycans. Non-supervised principal component analysis (PCA) was employed to detect distinguishable chromatographic features among the studied groups. Longitudinal analyses of samples from individual patients collected during multiple clinical assessments were also performed. Results N-glycan analysis of polyclonal IgG showed unique N-glycan peaks with statistically significant chromatogram variation across the 4 studied groups. PCA identified specific patterns of glycosylation present in the glycan profiles, thus demonstrating the ability to distinguish between MGUS, SMM, MM and control. Sialylated biantennary N-glycans and N-glycans containing bisecting GlcNAc residues contributed most to the PCA separation. Further characterisation of the glycans using sialic acid linkage specific derivatisation and LC-MS analysis confirmed that sialic acids were present in an α2-6 linked configuration. Localisation analysis revealed N-glycans present in the Fc region of the extracted polyclonal antibodies consisted of the standard biantennary type glycans with core fucosylation, variable degrees of galactosylation and low levels of sialylation. Such oligosaccharide structures suggest that the Fc regions of polyclonal IgG present in patients with varying stages of plasma cell disorder maintain a correct orientation to facilitate interaction with Fcγ receptors. Sialylated biantennary N-glycans, identified during global polyclonal IgG glycosylation profiling, were found to be located predominantly in the Fab region of the antibody. The formation of these larger highly sialylated N-glycan structures is likely due to the removal of steric hindrance resulting in more facilitated access by the associated glycosyltransferases required for oligosaccharide biosynthesis. The presence of these charged oligosaccharide structures, with their inherent structural dynamics, are likely to affect the ability of the antibody to recognise antigen and form an immune complex. Conclusions Glycan analysis of polyclonal IgG extracted from the sera of patients with varying stages of myeloma progression is reported. Differential glycosylation on polyclonal IgG was observed between the patients with MGUS, SMM, and MM, with alterations in the levels of sialylated biantennary structures and glycans bearing bisecting GlcNAc residues capable of distinguishing between the patient groups in the spectrum of plasma cell disorders. Disclosures No relevant conflicts of interest to declare.
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Dester, Emma, and Evangelyn Alocilja. "Current Methods for Extraction and Concentration of Foodborne Bacteria with Glycan-Coated Magnetic Nanoparticles: A Review." Biosensors 12, no. 2 (February 11, 2022): 112. http://dx.doi.org/10.3390/bios12020112.
Full textAbstract:
Rapid and accurate food pathogen detection is an essential step to preventing foodborne illnesses. Before detection, removal of bacteria from the food matrix and concentration to detectable levels are often essential steps. Although many reviews discuss rapid concentration methods for foodborne pathogens, the use of glycan-coated magnetic nanoparticles (MNPs) is often omitted. This review seeks to analyze the potential of this technique as a rapid and cost-effective solution for concentration of bacteria directly from foods. The primary focus is the mechanism of glycan-coated MNP binding, as well as its current applications in concentration of foodborne pathogens. First, a background on the synthesis, properties, and applications of MNPs is provided. Second, synthesis of glycan-coated particles and their theorized mechanism for bacterial adhesion is described. Existing research into extraction of bacteria directly from food matrices is also analyzed. Finally, glycan-coated MNPs are compared to the magnetic separation technique of immunomagnetic separation (IMS) in terms of cost, time, and other factors. At its current state, glycan-coated MNPs require more research to fully identify the mechanism, potential for optimization, and extraction capabilities directly in food matrices. However, current research indicates glycan-coated MNPs are an incredibly cost-effective method for rapid food pathogen extraction and concentration.
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Naegeli, Andreas, Eleni Bratanis, Christofer Karlsson, Oonagh Shannon, Raja Kalluru, Adam Linder, Johan Malmström, and Mattias Collin. "Streptococcus pyogenes evades adaptive immunity through specific IgG glycan hydrolysis." Journal of Experimental Medicine 216, no. 7 (May 15, 2019): 1615–29. http://dx.doi.org/10.1084/jem.20190293.
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Streptococcus pyogenes (Group A streptococcus; GAS) is a human pathogen causing diseases from uncomplicated tonsillitis to life-threatening invasive infections. GAS secretes EndoS, an endoglycosidase that specifically cleaves the conserved N-glycan on IgG antibodies. In vitro, removal of this glycan impairs IgG effector functions, but its relevance to GAS infection in vivo is unclear. Using targeted mass spectrometry, we characterized the effects of EndoS on host IgG glycosylation during the course of infections in humans. Substantial IgG glycan hydrolysis occurred at the site of infection and systemically in the severe cases. We demonstrated decreased resistance to phagocytic killing of GAS lacking EndoS in vitro and decreased virulence in a mouse model of invasive infection. This is the first described example of specific bacterial IgG glycan hydrolysis during infection and thereby verifies the hypothesis that EndoS modifies antibodies in vivo. This mechanisms of immune evasion could have implications for treatment of severe GAS infections and for future efforts at vaccine development.
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Hurevich, Mattan, Mamidi Samarasimhareddy, Israel Alshanski, and Evgeniy Mervinetsky. "Photodeprotection of up to Eight Photolabile Protecting Groups from a Single Glycan." Synlett 29, no. 07 (February 6, 2018): 880–84. http://dx.doi.org/10.1055/s-0036-1591915.
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Permanent protecting groups are essential for oligosaccharide synthesis. However, the removal of the traditionally used protecting groups is not trivial and demands considerable expertise. Using photolabile protecting groups as permanent protection for glycan can overcome many limitations associated with the traditional oligosaccharide synthesis approach. It is demonstrated here that up to eight photolabile protecting groups can be readily removed from a single glycan using a benchtop LED setup that is very easy to operate. This report suggests that further development of the strategy will offer an attractive alternative for oligosaccharide synthesis.
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ZHANG, Wenli, Pinjiang CAO, Shihao CHEN, Andrew M. SPENCE, Shaoxian ZHU, Erika STAUDACHER, and Harry SCHACHTER. "Synthesis of paucimannose N-glycans by Caenorhabditis elegans requires prior actions of UDP-N-acetyl-d-glucosamine:alpha-3-d-mannoside beta1,2-N-acetylglucosaminyltransferase I, alpha3,6-mannosidase II and a specific membrane-bound beta-N-acetylglucosaminidase." Biochemical Journal 372, no. 1 (May 15, 2003): 53–64. http://dx.doi.org/10.1042/bj20021931.
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We have previously reported three Caenorhabditis elegans genes (gly-12, gly-13 and gly-14) encoding UDP-N-acetyl-d-glucosamine:α-3-d-mannoside β1,2-N-acetylglucosaminyltransferase I (GnT I), an enzyme essential for hybrid and complex N-glycan synthesis. GLY-13 was shown to be the major GnT I in worms and to be the only GnT I cloned to date which can act on [Manα1,6(Manα1,3)Manα1,6](Manα1,3)Manβ1, 4GlcNAcβ1,4GlcNAc-R, but not on Manα1,6(Manα1,3)Manβ1-O-R substrates. We now report the kinetic constants, bivalent-metal-ion requirements, and optimal pH, temperature and Mn2+ concentration for this unusual enzyme. C. elegans glycoproteins are rich in oligomannose (Man6–9GlcNAc2) and ‘paucimannose’ Man3–5GlcNAc2(±Fuc) N-glycans, but contain only small amounts of complex and hybrid N-glycans. We show that the synthesis of paucimannose Man3GlcNAc2 requires the prior actions of GnT I, α3,6-mannosidase II and a membrane-bound β-N-acetylglucosaminidase similar to an enzyme previously reported in insects. The β-N-acetylglucosaminidase removes terminal N-acetyl-d-glucosamine from the GlcNAcβ1, 2Manα1,3Manβ- arm of Manα1,6(GlcNAcβ1,2Manα1,3) Manβ1,4GlcNAcβ1,4GlcNAc-R to produce paucimannose Man3GlcNAc2 N-glycan. N-acetyl-d-glucosamine removal was inhibited by two N-acetylglucosaminidase inhibitors. Terminal GlcNAc was not released from [Manα1,6(Manα1,3)Manα1,6] (GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-R nor from the GlcNAcβ1,2Manα1,6Manβ- arm. These findings indicate that GLY-13 plays an important role in the synthesis of N-glycans by C. elegans and that therefore the worm should prove to be a suitable model for the study of the role of GnT I in nematode development.
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Valbuena, Galder, Juan Francisco Madrid, María Martínez de Ubago, Laura Gómez-Santos, Edurne Alonso, Lucio Díaz-Flores, and Francisco J. Sáez. "N-Glycans in Xenopus laevis testis characterised by lectin histochemistry." Reproduction, Fertility and Development 28, no. 3 (2016): 337. http://dx.doi.org/10.1071/rd14077.
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Analysis of glycan chains of glycoconjugates is difficult because of their considerable variety. Despite this, several functional roles for these glycans have been reported. N-Glycans are oligosaccharides linked to asparagine residues of proteins. They are synthesised in the endoplasmic reticulum (ER) in a unique way, and later modified in both the ER and Golgi apparatus, developing different oligosaccharide chains. An essential role for complex N-glycans in mammalian spermatogenesis has been reported. The aim of the present study was to analyse the N-glycans of the Xenopus laevis testis by means of lectin histochemistry. Five lectins were used that specifically recognise mannose-containing and complex glycans, namely Galanthus nivalis agglutinin (GNA) from snowdrops, concanavalin A (Con A) from the Jack bean, Lens culinaris agglutinin (LCA) from lentils and Phaseolus vulgaris erythroagglutinin (PHA-E) and P. vulgaris leukoagglutinin (PHA-L) from the common bean. GNA and Con A labelled the interstitium and most of the germ cell types, whereas LCA and PHA-E showed affinity only for the interstitium. A granular cytoplasmic region was labelled in spermatogonia and spermatocytes by GNA and PHA-L, whereas GNA and LCA labelled a spermatid region that is probably associated with the centriolar basal body of the nascent flagellum. There was no specific labelling in the acrosome. Some unexpected results were found when deglycosylative pretreatments were used: pre-incubation of tissue sections with peptide N glycosidase F, which removes N-linked glycans, reduced or removed labelling with most lectins, as expected. However, after this pretreatment, the intensity of labelling remained or increased for Con A in the follicle (Sertoli) and post-meiotic germ cells. The β-elimination procedure, which removes O-linked glycans, revealed new labelling patterns with GNA, LCA and PHA-L, suggesting that some N-glycans were masked by O-glycans, and thus they became accessible to these lectins only after removal of the O-linked oligosaccharides. The functional role of the glycan chains identified could be related to the role of N-glycans involved in mammalian spermatogenesis reported previously.
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LONG, Yannick, Jean-Louis FRANC, Jacques KANIEWSKI, Jeanne LANET, and Annie GIRAUD. "Effect of N-glycan removal on the enzymatic activity of porcine thyroid peroxidase." European Journal of Biochemistry 202, no. 2 (December 1991): 501–5. http://dx.doi.org/10.1111/j.1432-1033.1991.tb16401.x.
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Li, Rasia C., Madeline Y. Wong, Andrew S. DiChiara, Azade S. Hosseini, and Matthew D. Shoulders. "Collagen’s enigmatic, highly conserved N-glycan has an essential proteostatic function." Proceedings of the National Academy of Sciences 118, no. 10 (March 5, 2021): e2026608118. http://dx.doi.org/10.1073/pnas.2026608118.
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Intracellular procollagen folding begins at the protein’s C-terminal propeptide (C-Pro) domain, which initiates triple-helix assembly and defines the composition and chain register of fibrillar collagen trimers. The C-Pro domain is later proteolytically cleaved and excreted from the body, while the mature triple helix is incorporated into the extracellular matrix. The procollagen C-Pro domain possesses a single N-glycosylation site that is widely conserved in all the fibrillar procollagens across humans and diverse other species. Given that the C-Pro domain is removed once procollagen folding is complete, the N-glycan might be presumed to be important for folding. Surprisingly, however, there is no difference in the folding and secretion of N-glycosylated versus non-N-glycosylated collagen type-I, leaving the function of the N-glycan unclear. We hypothesized that the collagen N-glycan might have a context-dependent function, specifically, that it could be required to promote procollagen folding only when proteostasis is challenged. We show that removal of the N-glycan from misfolding-prone C-Pro domain variants does indeed cause serious procollagen and ER proteostasis defects. The N-glycan promotes folding and secretion of destabilized C-Pro variants by providing access to the ER’s lectin-based chaperone machinery. Finally, we show that the C-Pro N-glycan is actually critical for the folding and secretion of even wild-type procollagen under ER stress conditions. Such stress is commonly incurred during development, wound healing, and other processes in which collagen production plays a key role. Collectively, these results establish an essential, context-dependent function for procollagen’s previously enigmatic N-glycan, wherein the carbohydrate moiety buffers procollagen folding against proteostatic challenge.
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Sim, Gyuri, Moonkyung Jeong, Hyunseok Seo, Jangrae Kim, and Soojin Lee. "The Role of N-Glycosylation in the Intracellular Trafficking and Functionality of Neuronal Growth Regulator 1." Cells 11, no. 7 (April 6, 2022): 1242. http://dx.doi.org/10.3390/cells11071242.
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Neuronal growth regulator 1 (NEGR1) is a brain-enriched membrane protein that is involved in neural cell communication and synapse formation. Accumulating evidence indicates that NEGR1 is a generic risk factor for various psychiatric diseases including autism and depression. Endoglycosidase digestion of single NEGR1 mutants revealed that the wild type NEGR1 has six putative N-glycosylation sites partly organized in a Golgi-dependent manner. To understand the role of each putative N-glycan residue, we generated a series of multi-site mutants (2MT–6MT) with additive mutations. Cell surface staining and biotinylation revealed that NEGR1 mutants 1MT to 4MT were localized on the cell surface at different levels, whereas 5MT and 6MT were retained in the endoplasmic reticulum to form highly stable multimer complexes. This indicated 5MT and 6MT are less likely to fold correctly. Furthermore, the removal of two N-terminal sites N75 and N155 was sufficient to completely abrogate membrane targeting. An in vivo binding assay using the soluble NEGR1 protein demonstrated that glycans N286, N294 and N307 on the C-terminal immunoglobulin-like domain play important roles in homophilic interactions. Taken together, these results suggest that the N-glycan moieties of NEGR1 are closely involved in the folding, trafficking, and homodimer formation of NEGR1 protein in a site-specific manner.
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Stigliano, Ivan D., Julio J. Caramelo, Carlos A. Labriola, Armando J. Parodi, and Cecilia D'Alessio. "Glucosidase II β Subunit Modulates N-Glycan Trimming in Fission Yeasts and Mammals." Molecular Biology of the Cell 20, no. 17 (September 2009): 3974–84. http://dx.doi.org/10.1091/mbc.e09-04-0316.
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Glucosidase II (GII) plays a key role in glycoprotein biogenesis in the endoplasmic reticulum (ER). It is responsible for the sequential removal of the two innermost glucose residues from the glycan (Glc3Man9GlcNAc2) transferred to Asn residues in proteins. GII participates in the calnexin/calreticulin cycle; it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. GII is a heterodimer whose α subunit (GIIα) bears the glycosyl hydrolase active site, whereas its β subunit (GIIβ) role is controversial and has been reported to be involved in GIIα ER retention and folding. Here, we report that in the absence of GIIβ, the catalytic subunit GIIα of the fission yeast Schizosaccharomyces pombe (an organism displaying a glycoprotein folding quality control mechanism similar to that occurring in mammalian cells) folds to an active conformation able to hydrolyze p-nitrophenyl α-d-glucopyranoside. However, the heterodimer is required to efficiently deglucosylate the physiological substrates Glc2Man9GlcNAc2 (G2M9) and Glc1Man9GlcNAc2 (G1M9). The interaction of the mannose 6-phosphate receptor homologous domain present in GIIβ and mannoses in the B and/or C arms of the glycans mediates glycan hydrolysis enhancement. We present evidence that also in mammalian cells GIIβ modulates G2M9 and G1M9 trimming.
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Kołaczkowski, Bartłomiej M., Christian I. Jørgensen, Nikolaj Spodsberg, Mary A. Stringer, Nitin T. Supekar, Parastoo Azadi, Peter Westh, Kristian B. R. M. Krogh, and Kenneth Jensen. "Analysis of fungal high-mannose structures using CAZymes." Glycobiology 32, no. 4 (December 22, 2021): 304–13. http://dx.doi.org/10.1093/glycob/cwab127.
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Abstract Glycoengineering ultimately allows control over glycosylation patterns to generate new glycoprotein variants with desired properties. A common challenge is glycan heterogeneity, which may affect protein function and limit the use of key techniques such as mass spectrometry. Moreover, heterologous protein expression can introduce nonnative glycan chains that may not fulfill the requirement for therapeutic proteins. One strategy to address these challenges is partial trimming or complete removal of glycan chains, which can be obtained through selective application of exoglycosidases. Here, we demonstrate an enzymatic O-deglycosylation toolbox of a GH92 α-1,2-mannosidase from Neobacillus novalis, a GH2 β-galactofuranosidase from Amesia atrobrunnea and the jack bean α-mannosidase. The extent of enzymatic O-deglycosylation was mapped against a full glycosyl linkage analysis of the O-glycosylated linker of cellobiohydrolase I from Trichoderma reesei (TrCel7A). Furthermore, the influence of deglycosylation on TrCel7A functionality was evaluated by kinetic characterization of native and O-deglycosylated forms of TrCel7A. This study expands structural knowledge on fungal O-glycosylation and presents a ready-to-use enzymatic approach for controlled O-glycan engineering in glycoproteins expressed in filamentous fungi.
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Wang, Junyao, Wenjing Peng, Mojibola Fowowe, Oluwatosin Daramola, and Yehia Mechref. "An Efficient and Economical N-Glycome Sample Preparation Using Acetone Precipitation." Metabolites 12, no. 12 (December 17, 2022): 1285. http://dx.doi.org/10.3390/metabo12121285.
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Due to the critical role of the glycome in organisms and its close connections with various diseases, much time and effort have been dedicated to glycomics-related studies in the past decade. To achieve accurate and reliable identification and quantification of glycans extracted from biological samples, several analysis methods have been well-developed. One commonly used methodology for the sample preparation of N-glycomics usually involves enzymatic cleavage by PNGase F, followed by sample purification using C18 cartridges to remove proteins. PNGase F and C18 cartridges are very efficient both for cleaving N-glycans and for protein removal. However, this method is most suitable for a limited quantity of samples. In this study, we developed a sample preparation method focusing on N-glycome extraction and purification from large-scale biological samples using acetone precipitation. The N-glycan yield was first tested on standard glycoprotein samples, bovine fetuin and complex biological samples, and human serum. Compared to C18 cartridges, most of the sialylated N-glycans from human serum were detected with higher abundance after acetone precipitation. However, C18 showed a slightly higher efficiency for protein removal. Using the unfiltered human serum as the baseline, around 97.7% of the proteins were removed by acetone precipitation, while more than 99.9% of the proteins were removed by C18 cartridges. Lastly, the acetone precipitation was applied to N-glycome extraction from egg yolks to demonstrate large-scale glycomics sample preparation.
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Wang, Chao. "Synthesis of a Disaccharide with a Thiol Spacer Used in Gold Nanoparticles." Advanced Materials Research 643 (January 2013): 153–56. http://dx.doi.org/10.4028/www.scientific.net/amr.643.153.
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The synthesis of a mannose-bearing disaccharide containing a thiol spacer at the reducing end was carried out to provide a tethered sugar suitable for attaching to gold nanoparticles. Such sugar is designed to mimic carbohydrates involved in cell-surface interactions. The molecule was constructed via Schmidt glycosylation of an appropriately protected glycosyl donor and an acceptor, followed by removal of protective groups and reductive amination to introduce a protected thiol spacer at the reducing end of the glycan. Subsequent removal of the thiol protective group gave the target disaccharide in a satisfactory yield.
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Wei, Zuzhang, Tao Lin, Lichang Sun, Yanhua Li, Xiaoming Wang, Fei Gao, Runxia Liu, Chunyan Chen, Guangzhi Tong, and Shishan Yuan. "N-Linked Glycosylation of GP5 of Porcine Reproductive and Respiratory Syndrome Virus Is Critically Important for Virus ReplicationIn Vivo." Journal of Virology 86, no. 18 (July 3, 2012): 9941–51. http://dx.doi.org/10.1128/jvi.07067-11.
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It has been proposed that the N-linked glycan addition at certain sites in GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) is important for production of infectious viruses and viral infectivity. However, such specific N-linked glycosylation sites do not exist in some field PRRSV isolates. This implies that the existence of GP5-associated glycanper seis not vital to the virus life cycle. In this study, we found that mutation of individual glycosylation sites at N30, N35, N44, and N51 in GP5 did not affect virus infectivity in cultured cells. However, the mutants carrying multiple mutations at N-linked glycosylation sites in GP5 had significantly reduced virus yields compared with the wild-type (wt) virus. As a result, no viremia and antibody response were detected in piglets that were injected with a mutant without all N-linked glycans in GP5. These results suggest that the N-linked glycosylation of GP5 is critically important for virus replicationin vivo. The study also showed that removal of N44-linked glycan from GP5 increased the sensitivity of mutant virus to convalescent-phase serum samples but did not elicit a high-level neutralizing antibody response to wt PRRSV. The results obtained from the present study have made significant contributions to better understanding the importance of glycosylation of GP5 in the biology of PRRSV.
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Kumar, Rajnish, Michael Tuen, Hualin Li, Doris B. Tse, and Catarina E. Hioe. "Improving immunogenicity of HIV-1 envelope gp120 by glycan removal and immune complex formation." Vaccine 29, no. 48 (November 2011): 9064–74. http://dx.doi.org/10.1016/j.vaccine.2011.09.057.
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Jarrell, Ken F., Gareth M. Jones, Lina Kandiba, Divya B. Nair, and Jerry Eichler. "S-Layer Glycoproteins and Flagellins: Reporters of Archaeal Posttranslational Modifications." Archaea 2010 (2010): 1–13. http://dx.doi.org/10.1155/2010/612948.
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Many archaeal proteins undergo posttranslational modifications. S-layer proteins and flagellins have been used successfully to study a variety of these modifications, including N-linked glycosylation, signal peptide removal and lipid modification. Use of these well-characterized reporter proteins in the genetically tractable model organisms,Haloferax volcanii, Methanococcus voltaeandMethanococcus maripaludis,has allowed dissection of the pathways and characterization of many of the enzymes responsible for these modifications. Such studies have identified archaeal-specific variations in signal peptidase activity not found in the other domains of life, as well as the enzymes responsible for assembly and biosynthesis of novel N-linked glycans. In vitro assays for some of these enzymes have already been developed. N-linked glycosylation is not essential for eitherHfx. volcaniior theMethanococcusspecies, an observation that allowed researchers to analyze the role played by glycosylation in the function of both S-layers and flagellins, by generating mutants possessing these reporters with only partial attached glycans or lacking glycan altogether. In future studies, it will be possible to consider questions related to the heterogeneity associated with given modifications, such as differential or modulated glycosylation.
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Sachs, Ulrich J., Kathrin Walek, Annika Krautwurst, Mathias J. Rummel, Gregor Bein, Sentot Santoso, and Tamam Bakchoul. "N-Glycosylation Of Human ITP Autoantibodies Affects Complement Activation, Platelet Phagocytosis, and Platelet Survival In a Murine Model." Blood 122, no. 21 (November 15, 2013): 569. http://dx.doi.org/10.1182/blood.v122.21.569.569.
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Abstract Introduction Immune thrombocytopenia (ITP) is a bleeding disorder caused by IgG autoantibodies (AAbs) directed against platelets. The IgG effector functions of autoantibodies depend on their Fc-constant region which undergoes posttranslational glycosylation. We investigated the role of Asn279-linked N-glycan of AAbs in vitro and in vivo. Material and Methods AAbs were purified from ITP patients (n=15) and controls (n=10) and N-glycans were enzymatically cleaved by endoglycosidase F. The effects of native AAbs and deglycosylated AAbs (deAAbs) were compared in vitro on enhancement of phagocytosis of platelets by monocytes and complement fixation and activation applying flow cytometry, laser scanning microscopy, and a complement consumption assay. The capability of AAbs and deAAbs to eliminate human platelets in vivo was studied in a NOD/SCID mouse model in presence and absence of a complement source. Results AAb-induced platelet phagocytosis was inhibited by N-glycan cleavage (median phagocytic activity: 8% vs. 0.8%, p=0.004). Seven out of 15 native AAbs bound C1q and induced complement consumption. N-glycan cleavage significantly reduced C1q binding (MFI 16.4 vs. 4.9, p=0.017) and complement consumption. In vivo survival of human PLTs was assessed after cotransfusion with native or deAAbs in NOD/SCID mice. Injection of AAbs resulted in rapid clearance of human platelets compared to control (platelet clearance after 5h (CL5h) 75% vs. 30%, p<0.001). AAbs that were able to activate complement induced more pronounced platelet clearance in the presence of complement compared to the clearance in the absence of complement (CL5h 82% vs. 62%, p=0.003). AAbs lost their ability to destroy platelets in vivo after deglycosylation (CL5h42%, p<0.001). Conclusion Removal of N-glycan from AAbs interferes with Fc-mediated phagocytosis and complement activation and thereby prolongs platelet survival in vivo. Our study provides tools for better characterizing ITP AAbs and sheds light on the heterogeneity of AAbs in ITP. Clinical studies should aim to assess such additional characteristics, since this could lead to the identification of ITP patient subgroups with increased responses to specific or new interventions such as, targetting complement factors. Disclosures: No relevant conflicts of interest to declare.
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Hargett, Audra A., Aaron M. Marcella, Huifeng Yu, Chao Li, Jared Orwenyo, Marcos D. Battistel, Lai-Xi Wang, and Darón I. Freedberg. "Glycosylation States on Intact Proteins Determined by NMR Spectroscopy." Molecules 26, no. 14 (July 16, 2021): 4308. http://dx.doi.org/10.3390/molecules26144308.
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Protein glycosylation is important in many organisms for proper protein folding, signaling, cell adhesion, protein-protein interactions, and immune responses. Thus, effectively determining the extent of glycosylation in glycoprotein therapeutics is crucial. Up to now, characterizing protein glycosylation has been carried out mostly by liquid chromatography mass spectrometry (LC-MS), which requires careful sample processing, e.g., glycan removal or protein digestion and glycopeptide enrichment. Herein, we introduce an NMR-based method to better characterize intact glycoproteins in natural abundance. This non-destructive method relies on exploiting differences in nuclear relaxation to suppress the NMR signals of the protein while maintaining glycan signals. Using RNase B Man5 and RNase B Man9, we establish reference spectra that can be used to determine the different glycoforms present in heterogeneously glycosylated commercial RNase B.
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Shade, Kai-Ting C., Barbara Platzer, Nathaniel Washburn, Vinidhra Mani, Yannic C. Bartsch, Michelle Conroy, Jose D. Pagan, et al. "A single glycan on IgE is indispensable for initiation of anaphylaxis." Journal of Experimental Medicine 212, no. 4 (March 30, 2015): 457–67. http://dx.doi.org/10.1084/jem.20142182.
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Immunoglobulin ε (IgE) antibodies are the primary mediators of allergic diseases, which affect more than 1 in 10 individuals worldwide. IgE specific for innocuous environmental antigens, or allergens, binds and sensitizes tissue-resident mast cells expressing the high-affinity IgE receptor, FcεRI. Subsequent allergen exposure cross-links mast cell–bound IgE, resulting in the release of inflammatory mediators and initiation of the allergic cascade. It is well established that precise glycosylation patterns exert profound effects on the biological activity of IgG. However, the contribution of glycosylation to IgE biology is less clear. Here, we demonstrate an absolute requirement for IgE glycosylation in allergic reactions. The obligatory glycan was mapped to a single N-linked oligomannose structure in the constant domain 3 (Cε3) of IgE, at asparagine-394 (N394) in human IgE and N384 in mouse. Genetic disruption of the site or enzymatic removal of the oligomannose glycan altered IgE secondary structure and abrogated IgE binding to FcεRI, rendering IgE incapable of eliciting mast cell degranulation, thereby preventing anaphylaxis. These results underscore an unappreciated and essential requirement of glycosylation in IgE biology.
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Ward, Soracha E., Jamie M. O'Sullivan, Sonia Aguila Martinez, Clive Drakeford, Thomas Anthony Jude McKinnon, Alain Chion, and James S. O'Donnell. "Defining the Molecular Mechanisms through Which the Macrophage Galactose Lectin (MGL) Receptor Regulates Von Willebrand Factor Clearance." Blood 132, Supplement 1 (November 29, 2018): 980. http://dx.doi.org/10.1182/blood-2018-99-114775.
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Abstract Introduction Although the biological mechanisms underpinning VWF clearance remain poorly understood, accumulating data has shown that increased clearance is a common pathophysiology in type 1 VWD patients. Moreover, enhanced clearance has been implicated in the etiology of types 2 and 3 VWD. Previous studies have shown that loss of terminal sialic acid from VWF glycans results in enhanced clearance. Furthermore, ST3Gal-IV knockout mice exhibit reduced plasma VWF levels due to rapid in vivo clearance. Initial studies suggested that clearance of hyposialylated VWF was mediated via the asialoglycoprotein receptor (ASGPR). However, we recently reported a novel role for the Macrophage Galactose Lectin (MGL) in regulating clearance of VWF (Ward et al, Blood 2018). In this study, we sought to elucidate the molecular mechanisms through which MGL interacts with human VWF. Methods Recombinant VWF variants, truncated A1A2A3 and isolated A1, A2 and A3 domains were expressed in HEK293T cells. Human plasma derived (pdVWF) was purified from commercial VWF concentrates as before. VWF glycoforms were generated by digestion with exoglycosidases. VWF-MGL interaction was assessed using plate binding ELISA assays. In vivo clearance of VWF variants was assessed in both Asgr1+/+VWF-/-and Asgr1-/-VWF-/-mice in the presence/absence of anti-MGL blocking antibodies. Results Previous studies have shown 80% of total sialic acid on VWF is expressed on N glycans in α2-6 linkage. In contrast, the other 20% of VWF sialylation is α2-3 linked on O linked glycans. Removal O linked sialylation (with α2-3 neuraminidase) was sufficient to increase VWF clearance in VWF-/- and Asgr1-/-VWF-/-mice (half life = 9.0 ± 1 mins and 8.3 ± 2mins). Interestingly, in vivo clearance of α2-3 Neu VWF in VWF-/-andAsgr1-/-VWF-/-mice was almost as rapid as that of asialo-VWF (digested with α2-3,6,8,9 neuraminidase). Critically, the increased clearance of α2-3 Neu VWF in VWF-/- is attenuated in the presence of MGL blocking antibodies (120mins residual α2-3 Neu VWF is 10.6% ±2% , compared to 10.2% ±1% for that of pdVWF). Plate-binding studies confirmed that α2-3 Neu VWF and α2-3,6,8,9 Neu VWF demonstrated enhanced binding to MGL compared to pdVWF (155% and 134% versus 100%; p =0.017 and 0.006). In keeping with in vivo clearance data, removal of α2-6 linked sialic acid from N glycans did not further promote binding to MGL. Finally, although PNGase removal of VWF N glycans did not affect MGL binding, treatment with O glycosidase significantly attenuated binding (p<0.0001). All together, these data demonstrate that O-linked sialylation plays a critical role in protecting VWF from MGL mediated clearance. The mature VWF monomer contains 10 O linked glycans, with eight clustered around the A1 domain. To further investigate the role of these glycans in regulating MGL interaction, site directed mutagenesis was used to generate VWF molecules lacking either O-linked cluster 1 (T1248, T1255, T1256, T1263) or O-linked cluster 2 (T1468, T1477, S1486, T1487) respectively. Binding of Δcluster 1 VWF to MGL was similar to that of wild-type VWF. In contrast, binding of the Δcluster 2 VWF variant to MGL was markedly reduced (84 % vs 40%; p=0.0027). O linked glycan structures are known to play important roles in maintaining glycoprotein conformation. To further define the mechanism through which these O glycan structures influence MGL mediated clearance, binding of a truncated A1A2A3 fragment and isolated recombinant A1, A2 and A3 domains were examined. Interestingly, MGL-binding to full length rVWF and A1A2A3 were both significantly increased in the presence of ristocetin. Interestingly, no binding of isolated A2 or A3 domains to MGL was observed, while A1 domain binding to MGL was seen both in the presence and absence of ristocetin. Conclusions In conclusion, these findings define a novel role for O linked sialylation in protecting VWF from MGL mediated clearance. Our data further demonstrate that the cluster of O linked glycans located at the C-terminal end of the A1 domain play a specific role in regulating VWF clearance via MGL, and that conformation of A1A2A3 may be important in determining accessibility of these glycans for the clearance receptor. Further studies will be required to determine whether abnormalities in these O-glycan determinants may be important in the pathophysiology of VWD, particularly in patients with type 1C VWD who lack VWF gene coding mutations. Disclosures O'Donnell: Bayer: Research Funding, Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Leo Pharma: Speakers Bureau; Octapharma: Speakers Bureau; CSL Behring: Consultancy; Daiichi Sankyo: Consultancy; Pfizer: Consultancy, Research Funding; Baxter: Research Funding, Speakers Bureau; Shire: Research Funding, Speakers Bureau.
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Tracey, W. R., R. A. Johns, G. Romero, and M. J. Peach. "Mechanism of phospholipase C-induced release of EDRF from pulmonary artery endothelium." American Journal of Physiology-Cell Physiology 261, no. 5 (November 1, 1991): C927—C935. http://dx.doi.org/10.1152/ajpcell.1991.261.5.c927.
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The mechanism of phospholipase (PL) C-induced release of endothelium-derived relaxing factor (EDRF) was investigated. Bovine pulmonary artery endothelial cells (BPAEC) were treated with phosphatidylinositol (PI)-selective PLCs, nonselective PLCs, or a nonselective PLD. PI-PLCs elicited PI-glycan anchor hydrolysis but did not alter either intracellular Ca2+ ([Ca2+]i) in fura-2-loaded BPAEC or EDRF production in BPAEC-vascular smooth muscle cocultures. In contrast, non-selective PLCs increased [Ca2+]i, an effect prevented by prior exposure to the PLCs, and EDRF production in a time- and concentration-dependent manner. Antibodies raised against PI-glycan anchors did not alter, while heat denaturation abolished, the PLC-dependent effects. Removal of extracellular Ca2+ with [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid both prevented and reversed PLC-stimulated increases in [Ca2+]i and inhibited EDRF production. Although Mn2+ quenched PLC-induced increases in fura-2 fluorescence, high PLC concentrations elicited significant dye loss from fura-2-loaded BPAEC. We conclude that the effects of exogenous PLC on EDRF production are not dependent on release of a membrane PI-glycan-linked moiety. Rather, the PLC actions are mediated by a graded increase in cell membrane permeability, probably related to pore formation by the hemolytic activity of the enzyme, followed by an influx of extracellular Ca2+.
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Li, Yun, Bradley Cleveland, Igor Klots, Bruce Travis, Barbra A. Richardson, David Anderson, David Montefiori, Patricia Polacino, and Shiu-Lok Hu. "Removal of a Single N-Linked Glycan in Human Immunodeficiency Virus Type 1 gp120 Results in an Enhanced Ability To Induce Neutralizing Antibody Responses." Journal of Virology 82, no. 2 (October 24, 2007): 638–51. http://dx.doi.org/10.1128/jvi.01691-07.
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ABSTRACT Glycans on human immunodeficiency virus (HIV) envelope protein play an important role in infection and evasion from host immune responses. To examine the role of specific glycans, we introduced single or multiple mutations into potential N-linked glycosylation sites in hypervariable regions (V1 to V3) of the env gene of HIV type 1 (HIV-1) 89.6. Three mutants tested showed enhanced sensitivity to soluble CD4. Mutant N7 (N197Q) in the carboxy-terminal stem of the V2 loop showed the most pronounced increase in sensitivity to broadly neutralizing antibodies (NtAbs), including those targeting the CD4-binding site (IgG1b12) and the V3 loop (447-52D). This mutant is also sensitive to CD4-induced NtAb 17b in the absence of CD4. Unlike the wild-type (WT) Env, mutant N7 mediates CD4-independent infection in U87-CXCR4 cells. To study the immunogenicity of mutant Env, we immunized pig-tailed macaques with recombinant vaccinia viruses, one expressing SIVmac239 Gag-Pol and the other expressing HIV-1 89.6 Env gp160 in WT or mutant forms. Animals were boosted 14 to 16 months later with simian immunodeficiency virus gag DNA and the cognate gp140 protein before intrarectal challenge with SHIV89.6P-MN. Day-of-challenge sera from animals immunized with mutant N7 Env had significantly higher and broader neutralizing activities than sera from WT Env-immunized animals. Neutralizing activity was observed against SHIV89.6, SHIV89.6P-MN, HIV-1 SF162, and a panel of subtype B primary isolates. Compared to control animals, immunized animals showed significant reduction of plasma viral load and increased survival after challenge, which correlated with prechallenge NtAb titers. These results indicate the potential advantages for glycan modification in vaccine design, although the role of specific glycans requires further examination.
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Shamsi Kazem Abadi, Saeideh, Matthew C. Deen, Jacqueline N. Watson, Fahimeh S. Shidmoossavee, and Andrew J. Bennet. "Directed evolution of a remarkably efficient Kdnase from a bacterial neuraminidase." Glycobiology 30, no. 5 (December 4, 2019): 325–33. http://dx.doi.org/10.1093/glycob/cwz099.
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Abstract N-acetylneuraminic acid (5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid), which is the principal sialic acid family member of the non-2-ulosonic acids and their various derivatives, is often found at the terminal position on the glycan chains that adorn all vertebrate cells. This terminal position combined with subtle variations in structure and linkage to the underlying glycan chains between humans and other mammals points to the importance of this diverse group of nine-carbon sugars as indicators of the unique aspects of human evolution and is relevant to understanding an array of human conditions. Enzymes that catalyze the removal N-acetylneuraminic acid from glycoconjugates are called neuraminidases. However, despite their documented role in numerous diseases, due to the promiscuous activity of many neuraminidases, our knowledge of the functions and metabolism of many sialic acids and the effect of the attachment to cellular glycans is limited. To this end, through a concerted effort of generation of random and site-directed mutagenesis libraries, subsequent screens and positive and negative evolutionary selection protocols, we succeeded in identifying three enzyme variants of the neuraminidase from the soil bacterium Micromonospora viridifaciens with markedly altered specificity for the hydrolysis of natural Kdn (3-deoxy-d-glycero-d-galacto-non-2-ulosonic acid) glycosidic linkages compared to those of N-acetylneuraminic acid. These variants catalyze the hydrolysis of Kdn-containing disaccharides with catalytic efficiencies (second-order rate constants: kcat/Km) of greater than 105 M−1 s−1; the best variant displayed an efficiency of >106 M−1 s−1 at its optimal pH.
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Jejcic, Alenka, Robert Daniels, Laura Goobar-Larsson, Daniel N. Hebert, and Anders Vahlne. "Small Molecule Targets Env for Endoplasmic Reticulum-Associated Protein Degradation and Inhibits Human Immunodeficiency Virus Type 1 Propagation." Journal of Virology 83, no. 19 (July 29, 2009): 10075–84. http://dx.doi.org/10.1128/jvi.01700-08.
Full textAbstract:
ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is dependent on its envelope glycoprotein (Env) to bind, fuse, and subsequently infect a cell. We show here that treatment of HIV-1-infected cells with glycyl-prolyl-glycine amide (GPG-NH2), dramatically reduced the infectivity of the released viral particles by decreasing their Env incorporation. The mechanism of GPG-NH2 was uncovered by examining Env expression and maturation in treated cells. GPG-NH2 treatment was found to affect Env by significantly decreasing its steady-state levels, its processing into gp120/gp41, and its mass by inducing glycan removal in a manner dependent on its native signal sequence and the proteasome. Therefore, GPG-NH2 negatively impacts Env maturation, facilitating its targeting for endoplasmic reticulum-associated protein degradation, where Env is deglycosylated en route to its degradation. These findings illustrate that nontoxic drugs such as GPG-NH2, which can selectively target glycoproteins to existing cellular degradation pathways, may be useful for pathogen therapy.