scholarly journals High-resolution epitope mapping of anti-Hu and anti-Yo autoimmunity by programmable phage display

2020 ◽  
Vol 2 (2) ◽  
Author(s):  
Brian O’Donovan ◽  
Caleigh Mandel-Brehm ◽  
Sara E Vazquez ◽  
Jamin Liu ◽  
Audrey V Parent ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Resolution ◽  
Neurological Disorders ◽  
Epitope Mapping ◽  
Single Point ◽  
Single Amino Acid ◽  
Phage Library ◽  
Sequencing Library ◽  
Restricted Region ◽  
Paraneoplastic Neurological Disorders

Abstract Paraneoplastic neurological disorders are immune-mediated diseases understood to manifest as part of a misdirected anti-tumor immune response. Paraneoplastic neurological disorder-associated autoantibodies can assist with diagnosis and enhance our understanding of tumor-associated immune processes. We designed a comprehensive library of 49-amino-acid overlapping peptides spanning the entire human proteome, including all splicing isoforms and computationally predicted coding regions. Using this library, we optimized a phage immunoprecipitation and sequencing protocol with multiple rounds of enrichment to create high-resolution epitope profiles in serum and cerebrospinal fluid (CSF) samples from patients suffering from two common paraneoplastic neurological disorders, the anti-Yo (n = 36 patients) and anti-Hu (n = 44 patients) syndromes. All (100%) anti-Yo patient samples yielded enrichment of peptides from the canonical anti-Yo (CDR2 and CDR2L) antigens, while 38% of anti-Hu patients enriched peptides deriving from the nELAVL (neuronal embryonic lethal abnormal vision like) family of proteins, the anti-Hu autoantigenic target. Among the anti-Hu patient samples that were positive for nELAVL, we noted a restricted region of immunoreactivity. To achieve single amino acid resolution, we designed a novel deep mutational scanning phage library encoding all possible single-point mutants targeting the reactive nELAVL region. This analysis revealed a distinct preference for the degenerate motif, RLDxLL, shared by ELAVL2, 3 and 4. Lastly, phage immunoprecipitation sequencing identified several known autoantigens in these same patient samples, including peptides deriving from the cancer-associated antigens ZIC and SOX families of transcription factors. Overall, this optimized phage immunoprecipitation sequencing library and protocol yielded the high-resolution epitope mapping of the autoantigens targeted in anti-Yo and anti-Hu encephalitis patients to date. The results presented here further demonstrate the utility and high-resolution capability of phage immunoprecipitation sequencing for both basic science and clinical applications and for better understanding the antigenic targets and triggers of paraneoplastic neurological disorders.

scholarly journals A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 289
Author(s):  
Kathleen K. M. Glover ◽  
Danica M. Sutherland ◽  
Terence S. Dermody ◽  
Kevin M. Coombs
Keyword(s):  
Amino Acid ◽  
Temperature Sensitivity ◽  
Reverse Genetics ◽  
Core Protein ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

scholarly journals Intrinsic resistance to terbinafine among human and animal isolates of Trichophyton mentagrophytes related to amino acid substitution in the squalene epoxidase

Infection ◽  
2020 ◽  
Vol 48 (6) ◽  
pp. 889-897 ◽  
Author(s):  
Dominik Łagowski ◽  
Sebastian Gnat ◽  
Aneta Nowakiewicz ◽  
Marcelina Osińska ◽  
Mariusz Dyląg
Keyword(s):  
Amino Acid ◽  
Fungal Infections ◽  
Single Point ◽  
Antifungal Susceptibility ◽  
Point Mutations ◽  
Trichophyton Mentagrophytes ◽  
Single Amino Acid ◽  
Squalene Epoxidase ◽  
Intrinsic Resistance ◽  
Resistant Strains

Abstract Background Dermatomycoses are the most common fungal infections in the world affecting a significant part of the human and animal population. The majority of zoophilic infections in humans are caused by Trichophyton mentagrophytes. Currently, the first-line drug for both oral and topical therapy is terbinafine. However, an increasing number of cases that are difficult to be cured with this drug have been noted in Europe and Asia. Resistance to terbinafine and other allylamines is very rare and usually correlated with point mutations in the squalene epoxidase gene resulting in single amino acid substitutions in the enzyme, which is crucial in the ergosterol synthesis pathway. Purpose Here, we report terbinafine-resistant T. mentagrophytes isolates among which one was an etiological factor of tinea capitis in a man and three were obtained from asymptomatic foxes in Poland. Methods We used the CLSI protocol to determine antifungal susceptibility profiles of naftifine, amphotericin B, griseofulvin, ketoconazole, miconazole, itraconazole, voriconazole, and ciclopirox. Moreover, the squalene epoxidase gene of the terbinafine-resistant strains was sequenced and analysed. Results In the genomes of all four resistant strains exhibiting elevated MICs to terbinafine (16 to 32 µg/ml), single-point mutations leading to Leu393Phe substitution in the squalene epoxidase enzyme were revealed. Among the other tested substances, a MIC50 value of 1 µg/ml was shown only for griseofulvin. Conclusion Finally, our study revealed that the terbinafine resistance phenomenon might not be acquired by exposure to the drug but can be intrinsic. This is evidenced by the description of the terbinafine-resistant strains isolated from the asymptomatic animals.

A Single Point Mutation Gln716His on the α2 Integrin: The New Platelet Alloantigen Casa.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3400-3400
Author(s):  
Gerald Bertrand ◽  
Vincent Jallu ◽  
Dominique Kervran ◽  
Corinne Martageix ◽  
Cecile Kaplan-Gouet
Keyword(s):  
Amino Acid ◽  
Cho Cells ◽  
Single Point ◽  
Low Frequency ◽  
Caucasian Population ◽  
Single Amino Acid ◽  
Nucleotide Sequence Analysis ◽  
Single Point Mutation ◽  
Severe Thrombocytopenia ◽  
Transfusion Therapy

Abstract We report here the identification and characterization of a new low-frequency platelet alloantigen Casa involved in a case of neonatal alloimmune thrombocytopenia (NAIT). A 29-year-old mother gave birth to a full-term male infant who exhibited petechiae at birth. Nine hours post-delivery the platelet counts revealed a severe thrombocytopenia (16.109platelets/L) leading to platelet transfusion therapy associated with IVIG. The outcome was uneventful. Blood samples from the parents and infant were referred to our laboratory for investigation because of suspected NAIT. Maternal serum showed a specific positive reaction with the antigen-capture assay (MAIPA) only when it was tested with the paternal platelets and the monoclonal antibodies Gi9 (Immunotech, Marseille, France), P16 (NIBSC, Bristol, UK), and AK7 (Abcys, Paris, France) directed against the GPIa-IIa (a2b1 integrin). Nucleotide sequence analysis of GPIa cDNA of the father and newborn showed a nucleotide substitution at position 2235 (2235G>T according to the International Nomenclature). This substitution induces a Q716H amino acid change in the GPIa mature protein, located outside the I domain involved in cell-adhesion for collagen. In vitro analysis of recombinant CHO cells expressing wild-type or mutant (Q716H) human GPIa allowed us to demonstrate that this single amino-acid substitution is responsible and sufficient for inducing Casa antigen expression. Adhesion of CHO cells to collagen coated on microtiter plates was not modified by the Cas polymorphism, nor by the maternal anti Casa alloantibodies, indicating that the mutation does not affect the function of the integrin a2b1. PCR-SSP was developed for Casa genotyping. In a Caucasian population study none of the 100 unrelated blood donors was found to be Casa carrier. In conclusion, the Casa antigen described here, implicated in a case of severe neonatal thrombocytopenia is a low-frequency platelet antigen in the Caucasian population. This study highlights the high polymorphism of the GPIa gene.

scholarly journals Tuning Structure and Dynamics of Blue Copper Azurin Junctions via Single Amino-Acid Mutations

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 611 ◽  
Author(s):  
Ortega ◽  
Vilhena ◽  
Zotti ◽  
Díez-Pérez ◽  
Cuevas ◽  
...  
Keyword(s):  
Amino Acid ◽  
Single Molecule ◽  
Single Point ◽  
Point Mutations ◽  
Point Of View ◽  
Single Amino Acid ◽  
Theoretical Point ◽  
Blue Copper ◽  
Structure And Dynamics ◽  
Amino Acid Mutations

In the growing field of biomolecular electronics, blue-copper Azurin stands out as one of the most widely studied protein in single-molecule contacts. Interestingly, despite the paramount importance of the structure/dynamics of molecular contacts in their transport properties, these factors remain largely unexplored from the theoretical point of view in the context of single Azurin junctions. Here we address this issue using all-atom Molecular Dynamics (MD) of Pseudomonas Aeruginosa Azurin adsorbed to a Au(111) substrate. In particular, we focus on the structure and dynamics of the free/adsorbed protein and how these properties are altered upon single-point mutations. The results revealed that wild-type Azurin adsorbs on Au(111) along two well defined configurations: one tethered via cysteine groups and the other via the hydrophobic pocket surrounding the Cu 2 + . Surprisingly, our simulations revealed that single amino-acid mutations gave rise to a quenching of protein vibrations ultimately resulting in its overall stiffening. Given the role of amino-acid vibrations and reorientation in the dehydration process at the protein-water-substrate interface, we suggest that this might have an effect on the adsorption process of the mutant, giving rise to new adsorption configurations.

scholarly journals High-resolution mapping and characterization of epitopes in COVID-19 patients

2020 ◽  
Author(s):  
Winston A. Haynes ◽  
Kathy Kamath ◽  
Joel Bozekowski ◽  
Elisabeth Baum-Jones ◽  
Melissa Campbell ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Resolution ◽  
Antibody Response ◽  
Severe Disease ◽  
Host Protein ◽  
Single Amino Acid ◽  
Furin Cleavage ◽  
Disease Heterogeneity ◽  
Furin Cleavage Site ◽  
Wide Range

AbstractFine scale delineation of epitopes recognized by the antibody response to SARS-CoV-2 infection will be critical to understanding disease heterogeneity and informing development of safe and effective vaccines and therapeutics. The Serum Epitope Repertoire Analysis (SERA) platform leverages a high diversity random bacterial display library to identify epitope binding specificities with single amino acid resolution. We applied SERA broadly, across human, viral and viral strain proteomes in multiple cohorts with a wide range of outcomes from SARS-CoV-2 infection. We identify dominant epitope motifs and profiles which effectively classify COVID-19, distinguish mild from severe disease, and relate to neutralization activity. We identify a repertoire of epitopes shared by SARS-CoV-2 and endemic human coronaviruses and determine that a region of amino acid sequence identity shared by the SARS-CoV-2 furin cleavage site and the host protein ENaC-alpha is a potential cross-reactive epitope. Finally, we observe decreased epitope signal for mutant strains which points to reduced antibody response to mutant SARS-CoV-2. Together, these findings indicate that SERA enables high resolution of antibody epitopes that can inform data-driven design and target selection for COVID-19 diagnostics, therapeutics and vaccines.

scholarly journals A single lysine in the N-terminal region of store-operated channels is critical for STIM1-mediated gating

2010 ◽  
Vol 136 (6) ◽  
pp. 673-686 ◽  
Author(s):  
Annette Lis ◽  
Susanna Zierler ◽  
Christine Peinelt ◽  
Andrea Fleig ◽  
Reinhold Penner
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Calcium Release ◽  
Transmembrane Domain ◽  
Single Point ◽  
Point Mutations ◽  
Terminal Region ◽  
Single Amino Acid ◽  
Critical Element ◽  
Crac Channels

Store-operated Ca2+ entry is controlled by the interaction of stromal interaction molecules (STIMs) acting as endoplasmic reticulum ER Ca2+ sensors with calcium release–activated calcium (CRAC) channels (CRACM1/2/3 or Orai1/2/3) in the plasma membrane. Here, we report structural requirements of STIM1-mediated activation of CRACM1 and CRACM3 using truncations, point mutations, and CRACM1/CRACM3 chimeras. In accordance with previous studies, truncating the N-terminal region of CRACM1 or CRACM3 revealed a 20–amino acid stretch close to the plasma membrane important for channel gating. Exchanging the N-terminal region of CRACM3 with that of CRACM1 (CRACM3-N(M1)) results in accelerated kinetics and enhanced current amplitudes. Conversely, transplanting the N-terminal region of CRACM3 into CRACM1 (CRACM1-N(M3)) leads to severely reduced store-operated currents. Highly conserved amino acids (K85 in CRACM1 and K60 in CRACM3) in the N-terminal region close to the first transmembrane domain are crucial for STIM1-dependent gating of CRAC channels. Single-point mutations of this residue (K85E and K60E) eliminate store-operated currents induced by inositol 1,4,5-trisphosphate and reduce store-independent gating by 2-aminoethoxydiphenyl borate. However, short fragments of these mutant channels are still able to communicate with the CRAC-activating domain of STIM1. Collectively, these findings identify a single amino acid in the N terminus of CRAC channels as a critical element for store-operated gating of CRAC channels.

scholarly journals Terbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Tsuyoshi Yamada ◽  
Mari Maeda ◽  
Mohamed Mahdi Alshahni ◽  
Reiko Tanaka ◽  
Takashi Yaguchi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Single Point ◽  
Point Mutations ◽  
Trichophyton Mentagrophytes ◽  
Antifungal Drugs ◽  
Clinical Isolates ◽  
Single Amino Acid ◽  
Squalene Epoxidase ◽  
Amino Acid Substitutions ◽  
Content Type

ABSTRACT Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu393, Phe397, Phe415, and His440) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.

Single amino acid substitutions determine mouse CD8 allotypes: epitope mapping of mouse CD8

1991 ◽  
Vol 33 (3) ◽  
pp. 206-209 ◽  
Author(s):  
Kei-ichi Nakayama ◽  
Akinori Sarai ◽  
Hiromitsu Nakauchi
Keyword(s):  
Amino Acid ◽  
Epitope Mapping ◽  
Single Amino Acid ◽  
Amino Acid Substitutions

scholarly journals Systematic profiling of SARS-CoV-2 specific IgG epitopes at single amino acid resolution

2020 ◽  
Author(s):  
Huan Qi ◽  
Ming-liang Ma ◽  
Jeremy Jiang ◽  
Jian-ya Ling ◽  
Ling-yun Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nucleocapsid Protein ◽  
Epitope Mapping ◽  
Spike Protein ◽  
Single Amino Acid ◽  
Therapeutic Antibodies ◽  
Igg Binding ◽  
Mapping Technology ◽  
Specific Proteins ◽  
Specific Igg

SARS-CoV-2 specific IgG responses play critical roles for patients to recover from COVID-19, in-depth dissecting of the IgG responses on systems level is of great interest. Herein, we adopted a newly developed high-throughput epitope mapping technology (AbMap), analyzed 55 COVID-19 convalescent sera and 226 antibody samples enriched by specific proteins or peptides from these sera. We revealed three areas that are rich of IgG epitopes, two are on Spike protein but outside of RBD, and one is on Nucleocapsid protein. We identified 29 significant epitopes on Spike protein, from two of these significant epitopes, two critical epitope residues were found, i. e., D936 and P1263, which are highly related to the infectivity of SARS-CoV-2. In summary, we provided the first global map of IgG binding epitopes for SARS-CoV-2 at single amino acid resolution. This map will facilitate the precise development of therapeutic antibodies and vaccines.

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