Mutational Analysis of Two Stefin A Epitopes

1999 ◽  
Vol 380 (6) ◽  
Author(s):  
N. Kopitar-Jerala ◽  
R. Jerala ◽  
B. Turk ◽  
F. Gubensek ◽  
V. Turk
Keyword(s):  
Monoclonal Antibodies ◽  
Tertiary Structure ◽  
Mutational Analysis ◽  
Sequence Similarity ◽  
Mammalian Species ◽  
Cross Reactivity ◽  
Binding Affinities ◽  
Amino Acid Residues ◽  
Stefin A ◽  
Carboxy Terminal

AbstractStefin A, an intracellular inhibitor of cysteine proteinases, is expressed most abundantly in epithelial cells and in cells of lymphatic origin. In order to study its role in normal and pathological conditions we have prepared and characterized monoclonal antibodies against recombinant stefin A. Two high affinity monoclonal antibodies (mAbs) (A22 and C52) were tested for binding to free and papain-complexed stefin A and to a chimeric inhibitor, consisting of 61 amino acid residues of stefin A and 37 carboxy-terminal residues of stefin B. mAb A22 recognized not only free stefin A but also stefin A in complex with papain. The mAbs were further tested for their cross-reactivity against stefin A and B isolated from different mammalian species. On the basis of sequence similarity and tertiary structure of human stefin A we have prepared three mutants–Glu33Lys, Asp61Gly and Asn62Tyr–and their reactivity with the mAbs was tested. The binding affinities of mAb A22 for the Asp61Gly and Asn62Tyr mutants were significantly lower, indicating that the two amino acids are part of the stefin A epitope recognized by A22. The binding of both mAbs to the mutants Gly4Arg and Gly4Glu was comparable to wild-type stefin A.

scholarly journals Investigation of major amino acid residues of anti-norfloxacin monoclonal antibodies responsible for binding with fluoroquinolones

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Patamalai Boonserm ◽  
Songchan Puthong ◽  
Thanaporn Wichai ◽  
Sajee Noitang ◽  
Pongsak Khunrae ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
3D Structure ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Residues ◽  
Variable Regions ◽  
Complementarity Determining Regions ◽  
Crucial Part ◽  
Major Amino Acid

AbstractIt is important to understand the amino acid residues that govern the properties of the binding between antibodies and ligands. We studied the binding of two anti-norfloxacins, anti-nor 132 and anti-nor 155, and the fluoroquinolones norfloxacin, enrofloxacin, ciprofloxacin, and ofloxacin. Binding cross-reactivities tested by an indirect competitive enzyme-linked immunosorbent assay indicated that anti-nor 132 (22–100%) had a broader range of cross-reactivity than anti-nor 155 (62–100%). These cross-reactivities correlated with variations in the numbers of interacting amino acid residues and their positions. Molecular docking was employed to investigate the molecular interactions between the fluoroquinolones and the monoclonal antibodies. Homology models of the heavy chain and light chain variable regions of each mAb 3D structure were docked with the fluoroquinolones targeting the crucial part of the complementarity-determining regions. The fluoroquinolone binding site of anti-nor 155 was a region of the HCDR3 and LCDR3 loops in which hydrogen bonds were formed with TYR (H:35), ASN (H:101), LYS (H:106), ASN (L:92), and ASN (L:93). These regions were further away in anti-nor 132 and could not contact the fluoroquinolones. Another binding region consisting of HIS (L:38) and ASP (H:100) was found for norfloxacin, enrofloxacin, and ciprofloxacin, whereas only ASP (H:100) was found for ofloxacin.

scholarly journals Characterization of Chimpanzee/Human Monoclonal Antibodies to Vaccinia Virus A33 Glycoprotein and Its Variola Virus Homolog In Vitro and in a Vaccinia Virus Mouse Protection Model

2007 ◽  
Vol 81 (17) ◽  
pp. 8989-8995 ◽  
Author(s):  
Zhaochun Chen ◽  
Patricia Earl ◽  
Jeffrey Americo ◽  
Inger Damon ◽  
Scott K. Smith ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Vaccinia Virus ◽  
Protective Efficacy ◽  
Variola Virus ◽  
Binding Affinities ◽  
Amino Acid Residues ◽  
C Terminus ◽  
20 Nm

ABSTRACT Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human γ1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 (Kd of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.

scholarly journals Mutational Analysis of RsbT, an Activator of the Bacillus subtilis Stress Response Transcription Factor, σB

2004 ◽  
Vol 186 (9) ◽  
pp. 2789-2797 ◽  
Author(s):  
Robyn L. Woodbury ◽  
Tingqiu Luo ◽  
Lindsay Grant ◽  
W. G. Haldenwang
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Tertiary Structure ◽  
Mutational Analysis ◽  
Physical Stress ◽  
Negative Regulator ◽  
Alanine Scanning Mutagenesis ◽  
Charged Amino Acids ◽  
Carboxy Terminal ◽  
Stress Pathway

ABSTRACT σB, the stress-activated σ factor of Bacillus subtilis, requires the RsbT protein as an essential positive regulator of its physical stress pathway. Stress triggers RsbT to both inactivate the principal negative regulator of the physical stress pathway (RsbS) by phosphorylation and activate a phosphatase (RsbU) required for σB induction. Neither the regions of RsbT that are involved in responding to stress signaling nor those required for downstream events have been established. We used alanine scanning mutagenesis to examine the contributions of RsbT's charged amino acids to the protein's stability and activities. Eleven of eighteen rsbT mutations blocked σB induction by stress. The carboxy terminus of RsbT proved to be particularly important for accumulation in Bacillus subtilis. Four of the five most carboxy-terminal mutations yielded rsbT alleles whose products were undetectable in B. subtilis extracts. Charged amino acids in the central region of RsbT were less critical, with four of the five substitutions in this region having no measurable effect on RsbT accumulation or activity. Only when the substitutions extended into a region of kinase homology was σB induction affected. Six other RsbT variants, although present at levels adequate for activity, failed to activate σB and displayed significant changes in their ability to interact with RsbT's normal binding partners in a yeast dihybrid assay. These changes either dramatically altered the proteins' tertiary structure without affecting their stability or defined regions of RsbT that are involved in multiple interactions.

An ancient prevertebrate Na+-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti)

2002 ◽  
Vol 283 (1) ◽  
pp. C155-C168 ◽  
Author(s):  
Sylvia Y. Yao ◽  
Amy M. Ng ◽  
Shaun K. Loewen ◽  
Carol E. Cass ◽  
Stephen A. Baldwin ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Mammalian Species ◽  
Functional Characterization ◽  
Structural Determinants ◽  
Pathogenic Yeast ◽  
Uridine Uptake ◽  
The Pacific ◽  
Selective System ◽  
Nucleoside Drugs ◽  
Carboxy Terminal

The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1, hCNT2, and hCNT3 are pyrimidine nucleoside-selective (system cit), purine nucleoside-selective (system cif), or broadly selective for both pyrimidine and purine nucleosides (system cib), respectively. All have orthologs in other mammalian species and belong to a gene family (CNT) that has members in insects, nematodes, pathogenic yeast, and bacteria. Here, we report the cDNA cloning and functional characterization of a CNT family member from an ancient marine prevertebrate, the Pacific hagfish ( Eptatretus stouti). This Na+-nucleoside symporter, designated hfCNT, is the first transport protein to be characterized in detail in hagfish and is a 683-amino acid residue protein with 13 predicted transmembrane helical segments (TMs). hfCNT was 52, 50, and 57% identical in sequence to hCNT1, hCNT2, and hCNT3, respectively. Similarity to hCNT3 was particularly marked in the TM 4–13 region. When produced in Xenopus oocytes, hfCNT exhibited the transport properties of system cib, with uridine, thymidine, and inosine apparent K m values of 10–45 μM. The antiviral nucleoside drugs 3′-azido-3′-deoxythymidine, 2′,3′-dideoxycytidine, and 2′,3′-dideoxyinosine were also transported. Simultaneous measurement of uridine-evoked currents and radiolabeled uridine uptake under voltage-clamp conditions gave a Na+-to-uridine coupling ratio of 2:1 (cf. 2:1 for hCNT3 and 1:1 for hCNT1/2). The apparent K 50 value for Na+ activation was >100 mM. A 50:50 chimera between hfCNT and hCNT1 (TMs 7–13 of hfCNT replaced by those of hCNT1) exhibited hCNT1-like cation interactions, establishing that the structural determinants of cation stoichiometry and binding affinity were located within the carboxy-terminal half of the protein. The high degree of sequence similarity between hfCNT and hCNT3 may indicate functional constraints on the primary structure of the transporter and suggests that cib-type CNTs fulfill important physiological functions.

scholarly journals Cross-Reactivity, Epitope Mapping, and Potency of Monoclonal Antibodies to Class 5 Fimbrial Tip Adhesins of Enterotoxigenic Escherichia coli

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Yang Liu ◽  
Sakina Shahabudin ◽  
Sami Farid ◽  
Lanfong H. Lee ◽  
Annette L. McVeigh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Sequence Similarity ◽  
Bacterial Pathogen ◽  
Cross Reactivity ◽  
Enterotoxigenic Escherichia Coli ◽  
Content Type ◽  
Functional Activities ◽  
Colonization Factor ◽  
Protective Antibodies

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is a leading diarrheagenic bacterial pathogen among travelers and children in resource-limited regions. Adherence to host intestinal cells mediated by ETEC fimbriae is believed to be a critical first step in ETEC pathogenesis. These fimbriae are categorized into related classes based on sequence similarity, with members of the class 5 fimbrial family being the best characterized. The eight related members of the ETEC class 5 fimbrial family are subdivided into three subclasses (5a, 5b, and 5c) that share similar structural arrangements, including a fimbrial tip adhesin. However, sequence variability among the class 5 adhesins may hinder the generation of cross-protective antibodies. To better understand functional epitopes of the class 5 adhesins and their ability to induce intraclass antibody responses, we produced 28 antiadhesin monoclonal antibodies (MAbs) to representative adhesins CfaE, CsbD, and CotD, respectively. We determined the MAb cross-reactivities, localized the epitopes, and measured functional activities as potency in inhibition of hemagglutination induced by class 5 fimbria-bearing ETEC. The MAbs’ reactivities to a panel of class 5 adhesins in enzyme-linked immunosorbent assays (ELISAs) revealed several reactivity patterns, including individual adhesin specificity, intrasubclass specificity, intersubclass specificity, and class-wide cross-reactivity, suggesting that some conserved epitopes, including two conserved arginines, are shared by the class 5 adhesins. However, the cross-reactive MAbs had functional activities limited to strains expressing colonization factor antigen I (CFA/I), coli surface antigen 17 (CS17), or CS1, suggesting that the breadth of functional activities of the MAbs was more restricted than the repertoire of cross-reactivities measured by ELISA. The results imply that multivalent adhesin-based ETEC vaccines or prophylactics need more than one active component to reach broad protection.

scholarly journals Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis.

1992 ◽  
Vol 12 (10) ◽  
pp. 4634-4642 ◽  
Author(s):  
B Rubinfeld ◽  
W J Crosier ◽  
I Albert ◽  
L Conroy ◽  
R Clark ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Amino Terminus ◽  
Amino Acid Residues ◽  
Gtpase Activating Protein ◽  
Mutant Proteins ◽  
Carboxy Terminal

rap1GAP is a GTPase-activating protein that specifically stimulates the GTP hydrolytic rate of p21rap1. We have defined the catalytic domain of rap1GAP by constructing a series of cDNAs coding for mutant proteins progressively deleted at the amino- and carboxy-terminal ends. Analysis of the purified mutant proteins shows that of 663 amino acid residues, only amino acids 75 to 416 are necessary for full GAP activity. Further truncation at the amino terminus resulted in complete loss of catalytic activity, whereas removal of additional carboxy-terminal residues dramatically accelerated the degradation of the protein in vivo. The catalytic domain we have defined excludes the region of rap1GAP which undergoes phosphorylation on serine residues. We have further defined this phosphoacceptor region of rap1GAP by introducing point mutations at specific serine residues and comparing the phosphopeptide maps of the mutant proteins. Two of the sites of phosphorylation by cyclic AMP (cAMP)-dependent kinase were localized to serine residues 490 and 499, and one site of phosphorylation by p34cdc2 was localized to serine 484. In vivo, rap1GAP undergoes phosphorylation at four distinct sites, two of which appear to be identical to the sites phosphorylated by cAMP-dependent kinase in vitro.

Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis

1992 ◽  
Vol 12 (10) ◽  
pp. 4634-4642
Author(s):  
B Rubinfeld ◽  
W J Crosier ◽  
I Albert ◽  
L Conroy ◽  
R Clark ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Amino Terminus ◽  
Amino Acid Residues ◽  
Gtpase Activating Protein ◽  
Mutant Proteins ◽  
Carboxy Terminal

rap1GAP is a GTPase-activating protein that specifically stimulates the GTP hydrolytic rate of p21rap1. We have defined the catalytic domain of rap1GAP by constructing a series of cDNAs coding for mutant proteins progressively deleted at the amino- and carboxy-terminal ends. Analysis of the purified mutant proteins shows that of 663 amino acid residues, only amino acids 75 to 416 are necessary for full GAP activity. Further truncation at the amino terminus resulted in complete loss of catalytic activity, whereas removal of additional carboxy-terminal residues dramatically accelerated the degradation of the protein in vivo. The catalytic domain we have defined excludes the region of rap1GAP which undergoes phosphorylation on serine residues. We have further defined this phosphoacceptor region of rap1GAP by introducing point mutations at specific serine residues and comparing the phosphopeptide maps of the mutant proteins. Two of the sites of phosphorylation by cyclic AMP (cAMP)-dependent kinase were localized to serine residues 490 and 499, and one site of phosphorylation by p34cdc2 was localized to serine 484. In vivo, rap1GAP undergoes phosphorylation at four distinct sites, two of which appear to be identical to the sites phosphorylated by cAMP-dependent kinase in vitro.

scholarly journals The role of plant antimicrobial peptides (AMPs) in response to biotic and abiotic environmental factors

2020 ◽  
Vol 65 (2) ◽  
Author(s):  
Olga Kulaeva ◽  
Marina Kliukova ◽  
Alexey Afonin ◽  
Anton Sulima ◽  
Vladimir Zhukov ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Tertiary Structure ◽  
Plant Protection ◽  
Sequence Similarity ◽  
Abiotic Factors ◽  
Cascade Reactions ◽  
Fine Tuning ◽  
Amino Acid Residues ◽  
Biotic And Abiotic Factors ◽  
High Or Low Temperature

Plants are continuously exposed to various biotic and abiotic factors that may trigger cascade reactions aimed at maintaining homeostasis. One of the most important components of plant protection from biotic factors is the synthesis of antimicrobial peptides (AMPs). AMPs are a large group of peptides present in insects, animals and plants. Plant innate immunity is provided by AMPs from different families that are categorized according to sequence similarity, the number and order of amino acid residues, and the tertiary structure of the mature peptide. AMPs may also participate in plant response to abiotic stresses such as high salinity, drought, high or low temperature, and heavy metals. In nitrogen-fixing nodules of some members of the Fabaceae family, AMP-like molecules named NCR peptides promote the differentiation of the symbiotic bacteria into bacteroids. Thus, AMPs are used by plants for fine tuning their responses to biotic and abiotic factors alike.

Bovine Temporal Bones as a Source of Inner Ear Antigen

1992 ◽  
Vol 101 (8) ◽  
pp. 688-690 ◽  
Author(s):  
Jose SanMartin ◽  
Steven D. Rauch ◽  
Richard A. Moscicki
Keyword(s):  
Monoclonal Antibodies ◽  
Inner Ear ◽  
Human Tissue ◽  
Mammalian Species ◽  
Temporal Bones ◽  
Auditory Research

Modern immunologic techniques of immunostaining, immunoblotting, and creation of monoclonal antibodies are gaining wide application in studies of development, function, and pathology of the ear. These techniques require a source of inner ear tissue for production of antigen extract. Human tissue is not readily available, and other mammalian species common in auditory research are small in size. Bovine temporal bones are readily available, and the membranous portions of the inner ear are abundant and easily accessible. Herein we report our technique for acquisition and dissection of bovine temporal bones and preparation and preservation of inner ear antigen.

scholarly journals Development of a Colloidal Gold Immunochromatographic Assay for Duck Enteritis Virus Detection Using Monoclonal Antibodies

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 365
Author(s):  
Fengli Liu ◽  
Yanxin Cao ◽  
Maokai Yan ◽  
Mengxu Sun ◽  
Qingshui Zhang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Colloidal Gold ◽  
Preventive Intervention ◽  
Virus Detection ◽  
Cross Reactivity ◽  
Duck Enteritis Virus ◽  
Detection Sensitivity ◽  
Immunochromatographic Assay ◽  
Efficient Detection ◽  
Enteritis Virus

Duck viral enteritis is a highly contagious and fatal disease of commercial waterfowl flocks. The disease occurs sporadically or epizootically in mainland China due to insufficient vaccinations. Early and rapid diagnosis is important for preventive intervention and the control of epizootic events in clinical settings. In this study, we generated two monoclonal antibodies (MAbs) that specifically recognized the duck enteritis virus (DEV) envelope glycoprotein B and tegument protein UL47, respectively. Using these MAbs, a colloidal gold-based immunochromatographic assay (ICA) was developed for the efficient detection of DEV antigens within 15 min. Our results showed that the detection limit of the developed ICA strip was 2.52 × 103 TCID50/mL for the virus infected cell culture suspension with no cross-reactivity with other pathogenic viruses commonly encountered in commercially raised waterfowl. Using samples from experimentally infected ducks, we demonstrated that the ICA detected the virus in cloacal swab samples on day three post-infection, demonstrating an 80% concordance with the PCR. For tissue homogenates from ducks succumbing to infection, the detection sensitivity was 100%. The efficient and specific detection by this ICA test provides a valuable, convenient, easy to use and rapid diagnostic tool for DVE under both laboratory and field conditions.

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