In vitro mutagenesis of HLA-B27: Single and multiple amino acid substitutions at consensus B27 sites identify distinct monoclonal antibody-defined epitopes

1992 ◽  
Vol 33 (4) ◽  
pp. 243-248 ◽  
Author(s):  
Fouad A.K. El-Zaatari ◽  
Joel D. Taurog
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Amino Acid Substitutions ◽  
In Vitro Mutagenesis ◽  
Hla B27 ◽  
Multiple Amino Acid

scholarly journals Specific Substitutions in Region V2 of gp120 env confer SHIV Neutralisation Resistance

Pathogens ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 181
Author(s):  
Yalcin Pisil ◽  
Zafer Yazici ◽  
Hisatoshi Shida ◽  
Shuzo Matsushita ◽  
Tomoyuki Miura
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitutions ◽  
In Vitro Mutagenesis ◽  
Tier 2 ◽  
Infected Monkey ◽  
Tier 1 ◽  
V3 Region ◽  
Monkey Plasma

A tier 2 SHIV-MK38 strain was obtained after two in vivo passages of tier 1 SHIV-MK1. SHIV-MK38#818, cloned from the MK38 strain, was neutralisation-resistant, like the parental MK38 strain, to SHIV-infected monkey plasma (MP), HIV-1-infected human pooled plasma (HPP), and KD247 monoclonal antibody (mAb) (anti-V3 gp120 env). We investigated the mechanisms underlying the resistance of #818, specifically the amino acid substitutions that confer resistance to MK1. We introduced amino acid substitutions in the MK1 envelope by in vitro mutagenesis and then compared the neutralisation resistance to MP, HPP, and KD247 mAb with #818 in a neutralisation assay using TZM-bl cells. We selected 11 substitutions in the V1, V2, C2, V4, C4, and V5 regions based on the alignment of env of MK1 and #818. The neutralisation resistance of the mutant MK1s with 7 of 11 substitutions in the V1, C2, C4, and V5 regions did not change significantly. These substitutions did not alter any negative charges or N-glycans. The substitutions N169D and K187E, which added negative charges, and S190N in the V2 region of gp120 and A389T in V4, which created sites for N-glycan, conferred high neutralisation resistance. The combinations N169D+K187E, N169D+S190N, and N169D+A389T resulted in MK1 neutralisation resistance close to that of #818. The combinations without 169D were neutralisation-sensitive. Therefore, N169D is the most important substitution for neutralisation resistance. This study demonstrated that although the V3 region sequences of #818 and MK1 are the same, V3 binding antibodies cannot neutralise #818 pseudovirus. Instead, mutations in the V2 and V4 regions inhibit the neutralisation of anti-V3 antibodies. We hypothesised that 169D and 190N altered the MK1 Env conformation so that the V3 region is buried. Therefore, the V2 region may block KD247 from binding to the tip of the V3 region.

scholarly journals Genotypic and Phenotypic Analyses of Hepatitis C Virus from Patients Treated with JTK-853 in a Three-Day Monotherapy

2012 ◽  
Vol 57 (1) ◽  
pp. 436-444 ◽  
Author(s):  
Naoki Ogura ◽  
Yukiyo Toyonaga ◽  
Izuru Ando ◽  
Kunihiro Hirahara ◽  
Tsutomu Shibata ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Viral Resistance ◽  
Hcv Rna ◽  
Amino Acid Substitutions ◽  
Sequencing Analysis ◽  
Resistance Mutations ◽  
In The Wild

ABSTRACTJTK-853, a palm site-binding NS5B nonnucleoside polymerase inhibitor, shows antiviral activityin vitroand in hepatitis C virus (HCV)-infected patients. Here, we report the results of genotypic and phenotypic analyses of resistant variants in 24 HCV genotype 1-infected patients who received JTK-853 (800, 1,200, or 1,600 mg twice daily or 1,200 mg three times daily) in a 3-day monotherapy. Viral resistance in NS5B was investigated using HCV RNA isolated from serum specimens from the patients. At the end of treatment (EOT) with JTK-853, the amino acid substitutions M414T (methionine [M] in position 414 at baseline was replaced with threonine [T] at EOT), C445R (cysteine [C] in position 445 at baseline was replaced with arginine [R] at EOT), Y448C/H (tyrosine [Y] in position 448 at baseline was replaced with cysteine [C] or histidine [H] at EOT), and L466F (leucine [L] in position 466 at baseline was replaced with phenylalanine [F] at EOT), which are known to be typical resistant variants of nonnucleoside polymerase inhibitors, were observed in a clonal sequencing analysis. These substitutions were also selected by a treatment with JTK-853in vitro, and the 50% effective concentration of JTK-853 in the M414T-, C445F-, Y448H-, and L466V-harboring replicons attenuated the susceptibility by 44-, 5-, 6-, and 21-fold, respectively, compared with that in the wild-type replicon (Con1). These findings suggest that amino acid substitutions of M414T, C445R, Y448C/H, and L466F are thought to be viral resistance mutations in HCV-infected patients receiving JTK-853 in a 3-day monotherapy.

scholarly journals TheEscherichia coliβ-Barrel Assembly Machinery Is Sensitized to Perturbations under High Membrane Fluidity

2018 ◽  
Vol 201 (1) ◽  
Author(s):  
Kelly M. Storek ◽  
Rajesh Vij ◽  
Dawei Sun ◽  
Peter A. Smith ◽  
James T. Koerber ◽  
...  
Keyword(s):  
Amino Acid ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Membrane Fluidity ◽  
Amino Acid Substitutions ◽  
Inhibitory Antibody ◽  
Critical Determinant ◽  
Gram Negative ◽  
Growth Environment

ABSTRACTIntegral β-barrel membrane proteins are folded and inserted into the Gram-negative bacterial outer membrane by the β-barrel assembly machine (BAM). This essential complex, composed of a β-barrel protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE, resides in the outer membrane, a unique asymmetrical lipid bilayer that is difficult to recapitulatein vitro. Thus, the probing of BAM function in living cells is critical to fully understand the mechanism of β-barrel folding. We recently identified an anti-BamA monoclonal antibody, MAB1, that is a specific and potent inhibitor of BamA function. Here, we show that the inhibitory effect of MAB1 is enhanced when BAM function is perturbed by either lowering the level of BamA or removing the nonessential BAM lipoproteins, BamB, BamC, or BamE. The disruption of BAM reduces BamA activity, increases outer membrane (OM) fluidity, and activates the σEstress response, suggesting the OM environment and BAM function are interconnected. Consistent with this idea, an increase in the membrane fluidity through changes in the growth environment or alterations to the lipopolysaccharide in the outer membrane is sufficient to provide resistance to MAB1 and enable the BAM to tolerate these perturbations. Amino acid substitutions in BamA at positions in the outer membrane spanning region or the periplasmic space remote from the extracellular MAB1 binding site also provide resistance to the inhibitory antibody. Our data highlight that the outer membrane environment is a critical determinant in the efficient and productive folding of β-barrel membrane proteins by BamA.IMPORTANCEBamA is an essential component of the β-barrel assembly machine (BAM) in the outer membranes of Gram-negative bacteria. We have used a recently described inhibitory anti-BamA antibody, MAB1, to identify the molecular requirements for BAM function. Resistance to this antibody can be achieved through changes to the outer membrane or by amino acid substitutions in BamA that allosterically affect the response to MAB1. Sensitivity to MAB1 is achieved by perturbing BAM function. By using MAB1 activity and functional assays as proxies for BAM function, we link outer membrane fluidity to BamA activity, demonstrating that an increase in membrane fluidity sensitizes the cells to BAM perturbations. Thus, the search for potential inhibitors of BamA function must consider the membrane environment in which β-barrel folding occurs.

scholarly journals Comparing mutagenesis and simulations as tools for identifying functionally important sequence changes for protein thermal adaptation

2018 ◽  
Vol 116 (2) ◽  
pp. 679-688 ◽  
Author(s):  
Ming-ling Liao ◽  
George N. Somero ◽  
Yun-wei Dong
Keyword(s):  
Amino Acid ◽  
Thermal Adaptation ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
Amino Acid Substitutions ◽  
Thermal Stabilities ◽  
Specific Amino Acid ◽  
Enzyme Thermal Stability ◽  
And Function

Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from −1.9 °C (Antarctica) to ∼55 °C (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability, we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme’s thermal responses and foster evolutionary adaptation of function.

Multiple amino acid substitutions are involved in the adaptation of H9N2 avian influenza virus to mice

2009 ◽  
Vol 138 (1-2) ◽  
pp. 85-91 ◽  
Author(s):  
Rui Wu ◽  
Hongbo Zhang ◽  
Keli Yang ◽  
Wangwang Liang ◽  
Zhongliang Xiong ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Amino Acid Substitutions ◽  
H9n2 Avian Influenza Virus ◽  
Multiple Amino Acid

scholarly journals Roles of Disulfide Linkage and Calcium Ion-Mediated Interactions in Assembly and Disassembly of Virus-Like Particles Composed of Simian Virus 40 VP1 Capsid Protein

2001 ◽  
Vol 75 (1) ◽  
pp. 61-72 ◽  
Author(s):  
Ken-Ichiro Ishizu ◽  
Hajime Watanabe ◽  
Song-Iee Han ◽  
Shin-Nosuke Kanesashi ◽  
Mainul Hoque ◽  
...  
Keyword(s):  
Amino Acid ◽  
Calcium Ion ◽  
Insect Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Ion Binding ◽  
Amino Acid Substitutions ◽  
Disulfide Linkage ◽  
Calcium Ion Binding

ABSTRACT The simian virus 40 capsid is composed of 72 pentamers of VP1 protein. Although the capsid is known to dissociate to pentamers in vitro following simultaneous treatment with reducing and chelating agents, the functional roles of disulfide linkage and calcium ion-mediated interactions are not clear. To elucidate the roles of these interactions, we introduced amino acid substitutions in VP1 at cysteine residues and at residues involved in calcium binding. We expressed the mutant proteins in a baculovirus system and analyzed both their assembly into virus-like particles (VLPs) in insect cells and the disassembly of those VLPs in vitro. We found that disulfide linkages at both Cys-9 and Cys-104 conferred resistance to proteinase K digestion on VLPs, although neither linkage was essential for the formation of VLPs in insect cells. In particular, reduction of the disulfide linkage at Cys-9 was found to be critical for VLP dissociation to VP1 pentamers in the absence of calcium ions, indicating that disulfide linkage at Cys-9 prevents VLP dissociation, probably by increasing the stability of calcium ion binding. We found that amino acid substitutions at carboxy-terminal calcium ion binding sites (Glu-329, Glu-330, and Asp-345) resulted in the frequent formation of unusual tubular particles as well as VLPs in insect cells, indicating that these residues affect the accuracy of capsid assembly. In addition, unexpectedly, amino acid substitutions at any of the calcium ion binding sites tested, especially at Glu-157, resulted in increased stability of VLPs in the absence of calcium ions in vitro. These results suggest that appropriate affinities of calcium ion binding are responsible for both assembly and disassembly of the capsid.

scholarly journals Identification of a Secreted Cholesterol-Dependent Cytolysin (Mitilysin) from Streptococcus mitis

2006 ◽  
Vol 189 (2) ◽  
pp. 627-632 ◽  
Author(s):  
Johanna Jefferies ◽  
Leena Nieminen ◽  
Lea-Ann Kirkham ◽  
Calum Johnston ◽  
Andrew Smith ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Immunosorbent Assay ◽  
Pneumococcal Disease ◽  
Neutralization Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Substitutions ◽  
Streptococcus Mitis ◽  
Hemolytic Toxin

ABSTRACT We have detected a cholesterol-dependent cytolysin, which we have named mitilysin, in a small number of Streptococcus mitis isolates. We have sequenced the mitilysin gene from seven isolates of S. mitis. Comparisons with the pneumococcal pneumolysin gene show 15 amino acid substitutions. S. mitis appear to release mitilysin extracellularly. Certain alleles of mitilysin are not recognized by a monoclonal antibody raised to the related toxin pneumolysin. Based on enzyme-linked immunosorbent assay and neutralization assay results, one isolate of S. mitis may produce a further hemolytic toxin in addition to mitilysin. As genetic exchange is known to occur between S. mitis and Streptococcus pneumoniae, this finding may have implications for the development of vaccines or therapies for pneumococcal disease that are based on pneumolysin.

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