scholarly journals Antibody Mapping of the Linear Epitopes of CMY-2 and SHV-1 β-Lactamases

2004 ◽  
Vol 48 (10) ◽  
pp. 3980-3988 ◽  
Author(s):  
Andrea M. Hujer ◽  
Christopher R. Bethel ◽  
Robert A. Bonomo
Keyword(s):  
Amino Acids ◽  
Polyclonal Antibodies ◽  
Synthesis Method ◽  
Hydrolytic Activity ◽  
Spot Synthesis ◽  
Antibody Recognition ◽  
Molar Excess ◽  
Omega Loop ◽  
Direct Binding ◽  
Linear Epitopes

ABSTRACT Knowledge of the amino acids that define recognition of anti-β-lactamase antibodies is critical to the interpretation of sensitivity and specificity of these antibodies when they are used in a clinical or research setting. To this end, we mapped the epitopes of the CMY-2 and SHV-1 β-lactamases by using the SPOT synthesis method. Eight linear epitopes in SHV-1 and seven linear epitopes in CMY-2 were identified by using anti-SHV-1 and anti-CMY-2 polyclonal antibodies, respectively. The epitopes of SHV-1 were mapped to amino acids at the Ambler positions ABL 28 to 38, 42 to 54, 88 to 100, 102 to 114, 170 to 182, 186 to 194, 202 to 210, and 276 to 288. In the epitope spanning amino acids 102 to 114, alanine and X-Scan analysis demonstrated that D104, Y105, P107, and S109 are essential residues for antibody recognition. In the epitope containing amino acids 170 to 182, N170, L173, P174, G175, and D176 were immunodominant. In CMY-2 β-lactamase, amino acids 4 to 16, 70 to 79, 211 to 223, 274 to 286, 289 to 298, 322 to 334, and 343 to 358 of the mature enzyme defined the major linear epitopes. A detailed analysis of the recognition sites that are located in an area analogous to the omega loop of class A β-lactamases (V211 to V223) showed that the amino acids Q215 to E219 are important in antibody binding. Incubation of CMY-2 β-lactamase with a 10-fold molar excess of anti-CMY-2 antibody for 60 min resulted in greater than 80% inhibition of nitrocefin hydrolysis. A 10-fold molar excess of anti-SHV-1 antibody reduced the activity of SHV-1 by 69%. Analysis of the CMY-2 and SHV-1 structures suggest that this reduction of hydrolytic activity may be due in part to the direct binding of antibodies to the omega loop, thereby hindering access of substrate to the active site.

scholarly journals Synthetic Peptide Immunogens Elicit Polyclonal and Monoclonal Antibodies Specific for Linear Epitopes in the D Motifs ofStaphylococcus aureus Fibronectin-Binding Protein, Which Are Composed of Amino Acids That Are Essential for Fibronectin Binding

2000 ◽  
Vol 68 (3) ◽  
pp. 1156-1163 ◽  
Author(s):  
Mario Huesca ◽  
Qing Sun ◽  
Robert Peralta ◽  
Gulnar M. Shivji ◽  
Daniel N. Sauder ◽  
...  
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Synthetic Peptide ◽  
Polyclonal Antibodies ◽  
Ofstaphylococcus Aureus ◽  
Antibody Preparation ◽  
Linear Epitopes ◽  
Fibronectin Binding Protein ◽  
Fibronectin Binding ◽  
Repeated Motifs

ABSTRACT A fibronectin (Fn)-binding adhesin of Staphylococcus aureus contains three tandem 37- or 38-amino-acid motifs (D1, D2, and D3), which function to bind Fn. Plasma from patients with S. aureus infections contain antibodies that preferentially recognize ligand induced binding sites in the D motifs and do not inhibit Fn binding (F. Casolini, L. Visai, D. Joh, P. G. Conaldi, A. Toniolo, M. Höök, and P. Speziale, Infect. Immun. 66:5433–5442, 1998). To eliminate the influence of Fn binding on antibody development, we used synthetic peptide immunogens D121–34 and D320–33, which each contain a conserved pattern of amino acids that is essential for Fn binding but which cannot bind Fn without N- or C-terminal extensions. The D320–33 immunogen promoted the production of polyclonal antibodies that were 10-fold more effective as inhibitors of Fn-binding to the D3 motif than antibodies obtained by immunizing with an extended peptide D316–36, which exhibits functional Fn binding. The D320–33 immunogen also facilitated the production of a monoclonal antibody, 9C3, which was highly specific for the epitope SVDFEED, and abolished Fn binding by the D3 motif. When mixed with polyclonal anti-D121–34 immunoglobulin G, 70% inhibition of Fn binding to the three tandem D motifs was achieved compared to no more than 30% inhibition with either antibody preparation alone. Therefore, by immunizing with short synthetic peptides that are unable to bind Fn, we have effectively stimulated the production of antibodies specific for epitopes comprised of amino acids that are essential for Fn binding. Although these epitopes occur within a conserved pattern of amino acids that is required for Fn binding, the antibodies recognized specific linear epitope sequences and not a conserved structure common to all repeated motifs.

scholarly journals Enzymatic Preparation of Bioactive Peptides Exhibiting ACE Inhibitory Activity from Soybean and Velvet Bean: A Systematic Review

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3822
Author(s):  
Azis Boing Sitanggang ◽  
Jessica Eka Putri ◽  
Nurheni Palupi ◽  
Emmanuel Hatzakis ◽  
Elvira Syamsir ◽  
...  
Keyword(s):  
Systematic Review ◽  
Amino Acids ◽  
Reaction Time ◽  
Hydrolytic Activity ◽  
Converting Enzyme ◽  
Enzymatic Preparation ◽  
Angiotensin I ◽  
Velvet Bean ◽  
Angiotensin I Converting Enzyme ◽  
Aspergillus Sp

The Angiotensin-I-converting enzyme (ACE) is a peptidase with a significant role in the regulation of blood pressure. Within this work, a systematic review on the enzymatic preparation of Angiotensin-I-Converting Enzyme inhibitory (ACEi) peptides is presented. The systematic review is conducted by following PRISMA guidelines. Soybeans and velvet beans are known to have high protein contents that make them suitable as sources of parent proteins for the production of ACEi peptides. Endopeptidase is commonly used in the preparation of soybean-based ACEi peptides, whereas for velvet bean, a combination of both endo- and exopeptidase is frequently used. Soybean glycinin is the preferred substrate for the preparation of ACEi peptides. It contains proline as one of its major amino acids, which exhibits a potent significance in inhibiting ACE. The best enzymatic treatments for producing ACEi peptides from soybean are as follows: proteolytic activity by Protease P (Amano-P from Aspergillus sp.), a temperature of 37 °C, a reaction time of 18 h, pH 8.2, and an E/S ratio of 2%. On the other hand, the best enzymatic conditions for producing peptide hydrolysates with high ACEi activity are through sequential hydrolytic activity by the combination of pepsin-pancreatic, an E/S ratio for each enzyme is 10%, the temperature and reaction time for each proteolysis are 37 °C and 0.74 h, respectively, pH for pepsin is 2.0, whereas for pancreatin it is 7.0. As an underutilized pulse, the studies on the enzymatic hydrolysis of velvet bean proteins in producing ACEi peptides are limited. Conclusively, the activity of soybean-based ACEi peptides is found to depend on their molecular sizes, the amino acid residues, and positions. Hydrophobic amino acids with nonpolar side chains, positively charged, branched, and cyclic or aromatic residues are generally preferred for ACEi peptides.

scholarly journals Alanine scans of IgE-binding to linear epitopes of Ara h 2 reveal critical amino acids

2021 ◽  
Vol 147 (2) ◽  
pp. AB89
Author(s):  
Nicole Canon ◽  
Catherine Schein ◽  
Xueni Chen ◽  
Marina Pozzoli ◽  
Vidhya Pathy ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ara H 2 ◽  
Ige Binding ◽  
Linear Epitopes

scholarly journals Identification of Two Cross-Neutralizing Linear Epitopes within the L1 Major Capsid Protein of Human Papillomaviruses

2002 ◽  
Vol 76 (13) ◽  
pp. 6480-6486 ◽  
Author(s):  
Alba-Lucia Combita ◽  
Antoine Touzé ◽  
Latifa Bousarghin ◽  
Neil D. Christensen ◽  
Pierre Coursaget
Keyword(s):  
Neutralizing Antibodies ◽  
Polyclonal Antibodies ◽  
Human Papillomaviruses ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
Linear Epitopes ◽  
Cross Neutralization ◽  
Definition Of ◽  
L1 Protein

ABSTRACT The neutralizing activities of polyclonal antibodies and monoclonal antibodies (MAbs) obtained by immunization of mice with L1 virus-like particles (VLPs) were investigated by using pseudovirion infectivity assays for human papillomavirus type 16 (HPV-16), HPV-31, HPV-33, HPV-45, HPV-58, and HPV-59 to obtain a better definition of cross-neutralization between high-risk HPVs. In this study, we confirmed and extended previous studies indicating that most genital HPV genotypes represent separate serotypes, and the results suggest that the classification of serotypes is similar to that of genotypes. In addition, three cross-neutralizing MAbs were identified (HPV-16.J4, HPV-16.I23, and HPV-33.E12). MAb HPV-16.J4 recognized a conserved linear epitope located within the FG loop of the L1 protein, and HPV-16.I23 recognized another located within the DE loop. The results suggested that reactivity of MAb HPV-16.I23 to L1 protein is lost when leucine 152 of the HPV-16 L1 protein is replaced by phenylalanine. This confirmed the existence of linear epitopes within the L1 protein that induce neutralizing antibodies, and this is the first evidence that such linear epitopes induce cross-neutralization. However, the cross-neutralization induced by L1 VLPs represents less than 1% of the neutralizing activity induced by the dominant conformational epitopes, and it is questionable whether this is sufficient to offer cross-protection in vivo.

Evaluating the coupling efficiency of phosphorylated amino acids for SPOT synthesis

2008 ◽  
Vol 14 (12) ◽  
pp. 1309-1314 ◽  
Author(s):  
Victor Tapia ◽  
Bernhard Ay ◽  
Julia Triebus ◽  
Eike Wolter ◽  
Prisca Boisguerin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Coupling Efficiency ◽  
Spot Synthesis

Identification of amino acids involved in the hydrolytic activity of lipase LipBL from Marinobacter lipolyticus

Microbiology ◽  
2012 ◽  
Vol 158 (8) ◽  
pp. 2192-2203 ◽  
Author(s):  
Dolores Pérez ◽  
Filip Kovačić ◽  
Susanne Wilhelm ◽  
Karl-Erich Jaeger ◽  
María Teresa García ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hydrolytic Activity

DIRECTED MUTAGENESIS OF HUMAN FIBRINOGEN: Aα CHAIN SUBSTITUTIONS THAT ALTER THROMBIN CLEAVAGE AND ANTIBODY RECOGNITION

1987 ◽  
Author(s):  
S T Lord
Keyword(s):  
Amino Acids ◽  
Blot Analysis ◽  
Fibrin Clot ◽  
Fibrinopeptide A ◽  
Human Fibrinogen ◽  
Antibody Recognition ◽  
Amino Terminal ◽  
Thrombin Cleavage ◽  
Initial Event ◽  
Bacterial Lysates

The initial event in fibrin clot formation is the thrombin catalized cleavage of the Aa chain of fibrinogen between Argl6 and Glyl7, releasing fibrinopeptide A. Previous data indicate that most of the information required for thrombin recognition and cleavage of the Aa chain lies within the amino terminal 51 residue CNBr fragment. In order to use protein engineering techniques to study the interaction of thrombin with the Aa chain, we have constructed a plasmid expression vector which encodes a tripartite protein consisting of amino acids 1-50 of the Aa chain of human fibrinogen followed by 60 amino acids of chicken collagen, and the beta-galactosidase protein from Escherichia coli. The codons for an initiator methionine and amino acids 1-50 were assembled from 7 oligonucleotides. Protein blot analysis of bacterial lysates of cells induced to synthesize this tribrid protein show a single band (MW = 125,000) crossreactive with a monoclonal antibody, Y-18, which recognizes the Aa chain of fibrinogen but not the products of thrombin cleavage. When these lysates are incubated with thrombin, fibrinopeptide A is released as demonstrated both by protein blot analysis and radioimmunoassay. By including one heterogeneous oligonucleotide in the assembly process, we have constructed plasmids which encode specific amino acid substitutions within residues 1-23. One of these substitutions, Glyl4 to val, significantly alters both cleavage by thrombin and recognition by Y-18. Substitution of ilu for Arg23 alters neither thrombin cleavage nor monoclonal recognition while substitution of leu for Argl6 alters thrombin cleavage, but not recognition by Y-18.

scholarly journals Rational design of peptides for identification of linear epitopes and generation of neutralizing monoclonal antibodies against DKK2 for cancer therapy

2020 ◽  
Vol 3 (2) ◽  
pp. 63-70 ◽  
Author(s):  
Rongqing Zhao ◽  
Qian Xiao ◽  
Maohua Li ◽  
Wenlin Ren ◽  
Chenxi Xia ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Growth ◽  
Wnt Signaling ◽  
Rational Design ◽  
Polyclonal Antibodies ◽  
Wnt Signaling Pathway ◽  
Keyhole Limpet Hemocyanin ◽  
Linear Epitopes

Abstract Dickkopf-related protein 2 (DKK2)is a member of the Dickkopf family in Wnt signaling pathway. Recently, we found that antibodies against DKK2 could activate natural killer (NK) and CD8+ T cells in tumors and inhibit tumor growth. In this paper, we report the rational design of peptides for identification of linear epitopes and generation of neutralizing monoclonal anti-DKK2 antibodies. To break the immune tolerance, we designed and chemically synthesized six peptides corresponding to different regions of DKK2 as immunogens and found five of them could generate mouse polyclonal antibodies that can bind to the active recombinant human DKK2 protein. Neutralizing mouse monoclonal antibodies (5F8 and 1A10) against human DKK2 were successfully developed by immunizing the mice with two different peptides (34KLNSIKSSL42 and 240KVWKDATYS248) conjugated to Keyhole limpet hemocyanin (KLH). The monoclonal antibodies not only abolish DKK2’s suppression of Wnt signaling in vitro but also inhibits tumor growth in vivo. Currently, those two mAbs are undergoing humanization as immunotherapy candidates and may offer a new drug for treatment of human cancers.

Identification of B-cell linear epitopes in domains 1–3 of pyolysin of Trueperella pyogenes using polyclonal antibodies

2017 ◽  
Vol 210 ◽  
pp. 24-31 ◽  
Author(s):  
Lingxiao Yang ◽  
Yue Zhang ◽  
Haili Wang ◽  
Bo Ma ◽  
Li Xu ◽  
...  
Keyword(s):  
B Cell ◽  
Polyclonal Antibodies ◽  
Trueperella Pyogenes ◽  
Linear Epitopes

scholarly journals Identification of specific B cell linear epitopes of mycoplasma hyorhinis P37 protein using monoclonal antibodies against baculovirus-expressed P37 protein

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Hongzhen Zhu ◽  
Yanwu Wei ◽  
Liping Huang ◽  
Dan Liu ◽  
Yongxing Xie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
B Cell ◽  
Etiologic Agent ◽  
Mycoplasma Hyorhinis ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Precise Analysis ◽  
Linear Epitopes ◽  
Specific Antigens

Abstract Background Mycoplasma hyorhinis (Mhr) is the etiologic agent of lameness and polyserositis in swine. P37 is a membrane protein of Mhr that may be an important immunogen and is a potential target for diagnostic development. However, there is little information concerning Mhr P37 protein epitopes. A precise analysis of the P37 protein epitopes should extend our understanding of the antigenic composition of the P37 protein and the humoral immune responses to Mhr infection. Investigating the epitopes of Mhr P37 will help to establish a detection method for Mhr in tissue and provide an effective tool for detecting Mhr infection. Results Western blot and indirect immunofluorescence assays (IFA) confirmed that the expressed P37 protein was recognized by Mhr-positive porcine and mouse sera. Furthermore, the P37 protein was purified using affinity chromatography and used to immunize mice for hybridoma cell fusion. Four monoclonal antibodies (mAbs) found to be positive for Mhr were detected in infected lung tissue. A panel of truncated P37 proteins was used to identify the minimal B cell linear epitopes of the protein based on these mAbs. The core epitope was determined to be 206KIKKAWNDKDWNTFRNF222. Conclusions In this study, we identified 17 critical amino acids that determine the epitope of the P37 protein of Mhr. This study identified mAbs that could provide useful tools for investigating the Mhr P37 antigenic core epitope (amino acids 206–222) and detecting Mhr-specific antigens in infected tissue.

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