scholarly journals Involvement of oligomerization, N-glycosylation and sialylation in the clearance of cholinesterases from the circulation

1995 ◽  
Vol 311 (3) ◽  
pp. 959-967 ◽  
Author(s):  
C Kronman ◽  
B Velan ◽  
D Marcus ◽  
A Ordentlich ◽  
S Reuveny ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Half Life ◽  
Sharp Decrease ◽  
Life Time ◽  
Absolute Number ◽  
Glycosylation Site ◽  
Protein Glycosylation ◽  
Wild Type ◽  
Attachment Sites ◽  
Glycosylation Sites

The possible role of post-translational modifications such as subunit oligomerization, protein glycosylation and oligosaccharide processing on the circulatory life-time of proteins was studied using recombinant human acetylcholinesterase (rHuAChE). Different preparations of rHuAChE containing various amounts of tetramers, dimers and monomers are cleared at similar rates from the circulation, suggesting that oligomerization does not play an important role in determining the rate of clearance. An engineered rHuAChE mutant containing only one N-glycosylation site was cleared from the circulation more rapidly than the wild-type triglycosylated enzyme. On the other hand, hyperglycosylated mutants containing either four or five occupied N-glycosylation sites, analagous to those present on the slowly cleared fetal bovine serum acetylcholinesterase (FBS-AChE), were also cleared more rapidly from the bloodstream than the wild-type species. Furthermore, the two different tetraglycosylated mutants were cleared at different rates while the pentaglycosylated mutant exhibited the most rapid clearance profile. These results imply that though the number of N-glycosylation sites plays a role in the circulatory life-time of the enzyme, the number of N-glycan units in itself does not determine the rate of clearance. When saturating amounts of asialofetuin were administered together with rHuAChE, the circulatory half-life of the enzyme was dramatically increased (from 80 min to 19 h) and was found to be similar to that displayed by plasma-derived cholinesterases while desialylation of these enzymes caused a sharp decrease in the circulatory half-life to approximately 3-5 min. Determination of the average number of sialic acid residues per enzyme subunit of the five different N-glycosylation species generated, revealed that the rate of clearance is not a function of the absolute number of appended sialic acid moieties but rather of the number of unoccupied sialic acid attachment sites per enzyme molecule. Specifically, we demonstrate an inverse-linear relationship between the number of vacant sialic acid attachment sites and the values of the enzyme residence time within the bloodstream.

Plasma membrane delivery of the gastric H,K-ATPase: the role of β-subunit glycosylation

2003 ◽  
Vol 285 (4) ◽  
pp. C968-C976 ◽  
Author(s):  
O. Vagin ◽  
S. Denevich ◽  
G. Sachs
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Glycosylation Site ◽  
Β Subunit ◽  
Wild Type ◽  
Α Subunit ◽  
Hek 293 ◽  
Glycosylation Sites ◽  
293 Cells

The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive. To identify such determinants in the gastric H,K-ATPase, fusion proteins of yellow fluorescent protein (YFP) and the gastric H,K-ATPase β-subunit (YFP-β) and cyan fluorescent protein (CFP) and the gastric H,K-ATPase α-subunit (CFP-α) were expressed in HEK-293 cells. Then plasma membrane delivery of wild-type CFP-α, wild-type YFP-β, and YFP-β mutants lacking one or two of the seven β-subunit glycosylation sites was determined using confocal microscopy and surface biotinylation. Expression of the wild-type YFP-β resulted in the plasma membrane localization of the protein, whereas the expressed CFP-α was retained intracellularly. When coexpressed, both CFP-α and YFP-β were delivered to the plasma membrane. Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP-β prevented plasma membrane delivery of the protein. Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane. A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein. Therefore, six of the seven glycosylation sites in the β-subunit are essential for the plasma membrane delivery of the β-subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asn103), which is not conserved among the β-subunits from different species, is not critical for plasma delivery of the protein.

scholarly journals DeepNGlyPred: A Deep Neural Network-Based Approach for Human N-Linked Glycosylation Site Prediction

Molecules ◽  
2021 ◽  
Vol 26 (23) ◽  
pp. 7314
Author(s):  
Subash C. Pakhrin ◽  
Kiyoko F. Aoki-Kinoshita ◽  
Doina Caragea ◽  
Dukka B. KC
Keyword(s):  
Computational Prediction ◽  
Structural Features ◽  
Glycosylation Site ◽  
Protein Glycosylation ◽  
Evolutionary Information ◽  
Computational Technique ◽  
Post Translational Modification ◽  
Glycosylation Sites ◽  
Better Than

Protein N-linked glycosylation is a post-translational modification that plays an important role in a myriad of biological processes. Computational prediction approaches serve as complementary methods for the characterization of glycosylation sites. Most of the existing predictors for N-linked glycosylation utilize the information that the glycosylation site occurs at the N-X-[S/T] sequon, where X is any amino acid except proline. Not all N-X-[S/T] sequons are glycosylated, thus the N-X-[S/T] sequon is a necessary but not sufficient determinant for protein glycosylation. In that regard, computational prediction of N-linked glycosylation sites confined to N-X-[S/T] sequons is an important problem. Here, we report DeepNGlyPred a deep learning-based approach that encodes the positive and negative sequences in the human proteome dataset (extracted from N-GlycositeAtlas) using sequence-based features (gapped-dipeptide), predicted structural features, and evolutionary information. DeepNGlyPred produces SN, SP, MCC, and ACC of 88.62%, 73.92%, 0.60, and 79.41%, respectively on N-GlyDE independent test set, which is better than the compared approaches. These results demonstrate that DeepNGlyPred is a robust computational technique to predict N-Linked glycosylation sites confined to N-X-[S/T] sequon. DeepNGlyPred will be a useful resource for the glycobiology community.

scholarly journals Quantitative glycoproteomics reveals new classes of STT3A- and STT3B-dependent N-glycosylation sites

2019 ◽  
Vol 218 (8) ◽  
pp. 2782-2796 ◽  
Author(s):  
Natalia A. Cherepanova ◽  
Sergey V. Venev ◽  
John D. Leszyk ◽  
Scott A. Shaffer ◽  
Reid Gilmore
Keyword(s):  
Protein Translocation ◽  
Site Occupancy ◽  
Glycosylation Site ◽  
Wild Type ◽  
New Class ◽  
Cysteine Rich Protein ◽  
Glycosylation Sites ◽  
Flanking Sequences ◽  
Dependent Site ◽  
Mutant Cells

Human cells express two oligosaccharyltransferase complexes (STT3A and STT3B) with partially overlapping functions. The STT3A complex interacts directly with the protein translocation channel to mediate cotranslational glycosylation, while the STT3B complex can catalyze posttranslocational glycosylation. We used a quantitative glycoproteomics procedure to compare glycosylation of roughly 1,000 acceptor sites in wild type and mutant cells. Analysis of site occupancy data disclosed several new classes of STT3A-dependent acceptor sites including those with suboptimal flanking sequences and sites located within cysteine-rich protein domains. Acceptor sites located in short loops of multi-spanning membrane proteins represent a new class of STT3B-dependent site. Remarkably, the lumenal ER chaperone GRP94 was hyperglycosylated in STT3A-deficient cells, bearing glycans on five silent sites in addition to the normal glycosylation site. GRP94 was also hyperglycosylated in wild-type cells treated with ER stress inducers including thapsigargin, dithiothreitol, and NGI-1.

Effects of N224 glycosylation in Saccharomycopsis fibuligera R64 α-amylase on enzyme activity and stability

2021 ◽  
Vol 17 ◽  
Author(s):  
Yovin Sugijo ◽  
Tina Dewi Rosahdi ◽  
Fernita Puspasari ◽  
Wangsa Tirta Ismaya ◽  
Khomaini Hasan ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Enzyme Activity ◽  
Evolutionary Relationship ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Saccharomycopsis Fibuligera ◽  
Glycosylation Sites ◽  
Starch Substrate ◽  
Relationship Of

Background: The amino acid sequence of an α-amylase of the yeast Saccharomycopsis fibuligera R64 (SfamyR64) contains the two putative N-linked glycosylation sites N153 and N224. N224 is hypothetically responsible for the binding of starch substrate because it is highly conserved among SfamyR64 homologs. Objective: To test whether N224 plays a key role in enzyme activity and stability. Methods: N224Q substitution was introduced by site-directed mutagenesis. The wild type and the mutant were independently over-produced in Pichia pastoris KM71. Activity of the wild type and of the mutant were compared, and their thermal-stability was assessed using heat treatments. The evolutionary relationship of SfamyR64 with its structural homologs with different glycosylation patterns was examined. Results: Activity of the N224Q mutant was approximately 80% lower than that of the wild type. The mutant showed no activity after 10 min of pre-incubation at 50 °C, whereas the wild type SfamyR64 showed activity until 30 min of treatment. Sfamy appeared to have evolved earlier than its structural homolog. Conclusion: SfamyR64 N224 is crucial for enzyme activity and thermal stability. This glycosylation site is unique for fungal and bacterial α-amylases.

scholarly journals Mutation Profiles, Glycosylation Site Distribution and Codon Usage Bias of Human Papillomavirus Type 16

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1281
Author(s):  
Wei Liu ◽  
Junhua Li ◽  
Hongli Du ◽  
Zhihua Ou
Keyword(s):  
Human Papillomavirus ◽  
Codon Usage ◽  
Codon Usage Bias ◽  
Vaccine Development ◽  
Surface Protein ◽  
Glycosylation Site ◽  
Protein Glycosylation ◽  
Adaptation Mechanism ◽  
Human Papillomavirus Type 16 ◽  
Glycosylation Sites

Human papillomavirus type 16 (HPV16) is the most prevalent HPV type causing cervical cancers. Herein, using 1597 full genomes, we systemically investigated the mutation profiles, surface protein glycosylation sites and the codon usage bias (CUB) of HPV16 from different lineages and sublineages. Multiple lineage- or sublineage-conserved mutation sites were identified. Glycosylation analysis showed that HPV16 lineage D contained the highest number of different glycosylation sites from lineage A in both L1 and L2 capsid proteins, which might lead to their antigenic distances between the two lineages. CUB analysis showed that the HPV16 open reading frames (ORFs) preferred codons ending with A/T. The CUB of HPV16 ORFs was mainly affected by natural selection except for E1, E5 and L2. HPV16 only shared some of the preferred codons with humans, which might help reduce competition in translational resources. These findings increase our understanding of the heterogeneity between HPV16 lineages and sublineages, and the adaptation mechanism of HPV in human cells. In summary, this study might facilitate HPV classification and improve vaccine development and application.

Glycosylation decreases aggregation and immunogenicity of adalimumab fab secreted from Pichia pastoris

2020 ◽  
Author(s):  
Hitomi Nakamura ◽  
Masato Kiyoshi ◽  
Makoto Anraku ◽  
Noritaka Hashii ◽  
Naoko Oda-Ueda ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Half Life ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Induced Stress ◽  
Glycan Chain ◽  
Elimination Half ◽  
Pharmacokinetic Behavior

Abstract Glycoengineering of therapeutic proteins has been applied to improve the clinical efficacy of several therapeutics. Here, we examined the effect of glycosylation on the properties of the Fab of the therapeutic antibody, adalimumab. An N-glycosylation site was introduced at position 178 of the H-chain constant region of adalimumab Fab through site-directed mutagenesis (H: L178N Fab), and the H: L178N Fab was produced in Pichia pastoris. Expressed mutant Fab contained long and short glycan chains (L-glyco Fab and S-glyco Fab, respectively). Under the condition of aggregation of Fab upon pH shift-induced stress, both of L-glyco Fab and S-glyco Fab were less prone to aggregation, with L-glyco Fab suppressing aggregation more effectively than the S-glyco Fab. Moreover, the comparison of the antigenicity of glycosylated and wild-type Fabs in mice revealed that glycosylation resulted in the suppression of antigenicity. Analysis of the pharmacokinetic behavior of the Fab, L-glyco Fab, and S-glyco Fab indicated that the half-lives of glycosylated Fabs in the rats were shorter than that of wild-type Fab, with L-glyco Fab having a shorter half-life than S-glyco Fab. Thus, we demonstrated that the glycan chain influences Fab aggregation and immunogenicity, and glycosylation reduces the elimination half-life in vivo.

scholarly journals N-glycosylation Site Analysis Reveals Sex-related Differences in Protein N-glycosylation in the Rice Brown Planthopper (Nilaparvata lugens)

2020 ◽  
Vol 19 (3) ◽  
pp. 529-539 ◽  
Author(s):  
Freja Scheys ◽  
Els J. M. Van Damme ◽  
Jarne Pauwels ◽  
An Staes ◽  
Kris Gevaert ◽  
...  
Keyword(s):  
Pest Control ◽  
Control Strategies ◽  
Brown Planthopper ◽  
Original Data ◽  
Nilaparvata Lugens ◽  
Glycosylation Site ◽  
Protein Glycosylation ◽  
Biological Pest Control ◽  
Glycosylation Sites ◽  
Wide Range

Glycosylation is a common modification of proteins and critical for a wide range of biological processes. Differences in protein glycosylation between sexes have already been observed in humans, nematodes and trematodes, and have recently also been reported in the rice pest insect Nilaparvata lugens. Although protein N-glycosylation in insects is nowadays of high interest because of its potential for exploitation in pest control strategies, the functionality of differential N-glycosylation between sexes is yet unknown. In this study, therefore, the occurrence and role of sex-related protein N-glycosylation in insects were examined. A comprehensive investigation of the N-glycosylation sites from the adult stages of N. lugens was conducted, allowing a qualitative and quantitative comparison between sexes at the glycopeptide level. N-glycopeptide enrichment via lectin capturing using the high mannose/paucimannose-binding lectin Concanavalin A, or the Rhizoctonia solani agglutinin which interacts with complex N-glycans, resulted in the identification of over 1300 N-glycosylation sites derived from over 600 glycoproteins. Comparison of these N-glycopeptides revealed striking differences in protein N-glycosylation between sexes. Male- and female-specific N-glycosylation sites were identified, and some of these sex-specific N-glycosylation sites were shown to be derived from proteins with a putative role in insect reproduction. In addition, differential glycan composition between males and females was observed for proteins shared across sexes. Both lectin blotting experiments as well as transcript expression analyses with complete insects and insect tissues confirmed the observed differences in N-glycosylation of proteins between sexes. In conclusion, this study provides further evidence for protein N-glycosylation to be sex-related in insects. Furthermore, original data on N-glycosylation sites of N. lugens adults are presented, providing novel insights into planthopper's biology and information for future biological pest control strategies.

scholarly journals Restoration of virulence of escape mutants of H5 and H9 influenza viruses by their readaptation to mice

2005 ◽  
Vol 86 (10) ◽  
pp. 2831-2838 ◽  
Author(s):  
Irina A. Rudneva ◽  
Natalia A. Ilyushina ◽  
Tatiana A. Timofeeva ◽  
Robert G. Webster ◽  
Nikolai V. Kaverin
Keyword(s):  
Amino Acid ◽  
Amino Acid Change ◽  
Glycosylation Site ◽  
Influenza Viruses ◽  
Phenotypic Characterization ◽  
Escape Mutant ◽  
Wild Type ◽  
Glycosylation Sites ◽  
Escape Mutants ◽  
The One

Antigenic mapping of the haemagglutinin (HA) molecule of H5 and H9 influenza viruses by selecting escape mutants with monoclonal anti-HA antibodies and subjecting the selected viruses to immunological analysis and sequencing has previously been performed. The viruses used as wild-type strains were mouse-adapted variants of the original H5 and H9 isolates. Phenotypic characterization of the escape mutants revealed that the amino acid change in HA that conferred resistance to a monoclonal antibody was sometimes associated with additional effects, including decreased virulence for mice. In the present study, the low-virulence H5 and H9 escape mutants were readapted to mice. Analysis of the readapted variants revealed that the reacquisition of virulence was not necessarily achieved by reacquisition of the wild-type HA gene sequence, but was also associated either with the removal of a glycosylation site (the one acquired previously by the escape mutant) without the exact restoration of the initial wild-type amino acid sequence, or, for an H5 escape mutant that had no newly acquired glycosylation sites, with an additional amino acid change in a remote part of the HA molecule. The data suggest that such ‘compensating’ mutations, removing the damaging effects of antibody-selected amino acid changes, may be important in the course of influenza virus evolution.

scholarly journals Prediction of O-glycosylation Sites Using Random Forest and GA-Tuned PSO Technique

2015 ◽  
Vol 9 ◽  
pp. BBI.S26864 ◽  
Author(s):  
Hebatallah Hassan ◽  
Amr Badr ◽  
M. B. Abdelhalim
Keyword(s):  
Genetic Algorithm ◽  
Random Forest ◽  
Classification Accuracy ◽  
Characteristic Curve ◽  
Pso Algorithm ◽  
Glycosylation Site ◽  
Protein Glycosylation ◽  
Parameters Optimization ◽  
Minority Class ◽  
Glycosylation Sites

O-glycosylation is one of the main types of the mammalian protein glycosylation; it occurs on the particular site of serine (S) or threonine (T). Several O-glycosylation site predictors have been developed. However, a need to get even better prediction tools remains. One challenge in training the classifiers is that the available datasets are highly imbalanced, which makes the classification accuracy for the minority class to become unsatisfactory. In our previous work, we have proposed a new classification approach, which is based on particle swarm optimization (PSO) and random forest (RF); this approach has considered the imbalanced dataset problem. The PSO parameters setting in the training process impacts the classification accuracy. Thus, in this paper, we perform parameters optimization for the PSO algorithm, based on genetic algorithm, in order to increase the classification accuracy. Our proposed genetic algorithm-based approach has shown better performance in terms of area under the receiver operating characteristic curve against existing predictors. In addition, we implemented a glycosylation predictor tool based on that approach, and we demonstrated that this tool could successfully identify candidate glycosylation sites in case study protein.

scholarly journals Determinants of attenuation in the envelope protein of the flavivirus Alfuy

2011 ◽  
Vol 92 (10) ◽  
pp. 2286-2296 ◽  
Author(s):  
Natalie A. Prow ◽  
Fiona J. May ◽  
Daniel J. Westlake ◽  
Robert J. Hurrelbrink ◽  
Rebecca M. Biron ◽  
...  
Keyword(s):  
Infectious Clone ◽  
Glycosylation Site ◽  
Protein Glycosylation ◽  
Hinge Region ◽  
Wild Type ◽  
E Proteins ◽  
Time To Death ◽  
Naturally Occurring ◽  
High Doses ◽  
Delayed Time

Murray Valley encephalitis virus (MVEV) is a mosquito-borne flavivirus endemic to Australia and Papua New Guinea. Most strains of MVEV cause potentially fatal cases of encephalitis in humans and horses, and have been shown to be highly neuroinvasive in weanling mice. In contrast, the naturally occurring subtype Alfuy virus (ALFV) has never been associated with human disease, nor is it neuroinvasive in weanling mice, even at high doses. To identify viral factors associated with ALFV attenuation, a chimeric infectious clone was constructed containing the structural genes premembrane (prM) and envelope (E) of ALFV swapped into the MVEV genome. The resulting virus (vMVEV/ALFVstr) was no longer neuroinvasive in mice, suggesting that motifs within prM–E of ALFV confer attenuation. To define these motifs further, mutants were constructed by targeting divergent sequences between the MVEV and ALFV E proteins that are known markers of virulence in other encephalitic flaviviruses. MVEV mutants containing a unique ALFV sequence in the flexible hinge region (residues 273–277) or lacking the conserved glycosylation site at position 154 were significantly less neuroinvasive in mice than wild-type MVEV, as determined by delayed time to death or increased LD50. Conversely, when the corresponding MVEV sequences were inserted into the vMVEV/ALFVstr chimera, the mutant containing the MVEV hinge sequence was more neuroinvasive than the parental chimera, though not to the same level as wild-type MVEV. These results identify the hinge region and E protein glycosylation as motifs that contribute to the attenuation of ALFV.

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