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.

Expression of Na+-d-glucose cotransporter in brush-border membrane of the chicken intestine

1999 ◽  
Vol 276 (2) ◽  
pp. R627-R631 ◽  
Author(s):  
Carles Garriga ◽  
Nativitat Rovira ◽  
Miquel Moretó ◽  
Joana M. Planas
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Western Blot ◽  
Brush Border ◽  
Synthetic Peptide ◽  
Brush Border Membrane ◽  
Polyclonal Antibodies ◽  
Membrane Vesicles ◽  
Dependent Transport

We have studied the expression of Na+-d-glucose cotransporter in brush-border membrane vesicles (BBMVs) of chicken enterocytes to correlate the changes in the apical Na+-dependent transport with the changes in the amounts of transporter determined by Western blot analysis. Two different rabbit polyclonal antibodies were used simultaneously. The antibody raised against amino acids 564–575 of the deduced amino acid sequence of rabbit intestinal SGLT-1 ( antibody 1) specifically detects a single 75-kDa band in the three segments, and this band disappeared when the antibody was preabsorbed with the antigenic peptide. The antibody raised against the synthetic peptide corresponding to amino acids 402–420 of the same protein ( antibody 2) only reacts with jejunal and ileal samples, but no signal is found in BBMVs of rectum. Only when antibody 1 was used was there a linear correlation between the maximal transport rates of hexoses in BBMVs and the relative protein amounts determined by Western blot. These results indicate that the Na+-d-glucose cotransport in the jejunum, the ileum, and the rectum of chickens is due to an SGLT-1 type protein.

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 Fibronectin binding to complement subcomponent C1q. Localization of their respective binding sites

1985 ◽  
Vol 226 (1) ◽  
pp. 207-215 ◽  
Author(s):  
J Sorvillo ◽  
I Gigli ◽  
E Pearlstein
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Binding Sites ◽  
Solid Phase ◽  
Fluid Phase ◽  
Globular Domain ◽  
Plasma Fibronectin ◽  
Fibronectin Binding ◽  
Inhibition Studies ◽  
Radiobinding Assay

The interaction of purified human plasma fibronectin with the C1q subcomponent of complement was investigated by using a solid-phase radiobinding assay. 125I-fibronectin binding to native C1q, purified collagen domain (C1q-c) or globular domain (C1q-g) was compared. When the purified domains were insolubilized by binding to plastic, the C1q-c exhibited 59% of the binding demonstrated with intact C1q, whereas the C1q-g exhibited 35% of the binding. N-Terminal sequencing of the globular domain showed that a sequence of seven collagen-like amino acids was retained on each chain of the C1q-g fragment. 125I-fibronectin binding to C1q could be inhibited equally well by fluid-phase C1q and C1q-c, but not by fluid-phase C1q-g, implying that the collagen-like region retained on the C1q-g is masked in the fluid phase. In addition, studies were performed to determine which subunit(s) of C1q bind(s) fibronectin. The percentages of fibronectin bound by the A, B, and C chain of C1q were found to be 38, 21 and 41% respectively. Inhibition studies with purified 200-180 kDa, 50 kDa or 29 kDa fragments of fibronectin show that the binding site on fibronectin for C1q is the 50 kDa gelatin-binding domain.

scholarly journals Thrombospondin interaction with plasminogen. Evidence for binding to a specific region of the kringle structure of plasminogen

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 976-982 ◽  
Author(s):  
P DePoli ◽  
T Bacon-Baguley ◽  
S Kendra-Franczak ◽  
MT Cederholm ◽  
DA Walz
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Binding Sites ◽  
Synthetic Peptide ◽  
Specific Region ◽  
The Other ◽  
Direct Result ◽  
Amino Terminal ◽  
Binding Domains ◽  
Kringle 5

Abstract Platelet thrombospondin interacts with plasminogen in a specific and saturable manner. Thrombospondin was found to specifically bind to plasminogen and the nonenzyme chain of plasmin. Preincubation of 125I- labeled thrombospondin with 30 mmol/L lysine was without effect in the binding of thrombospondin to immobilized plasminogen; preincubation of 125I-labeled plasminogen with 30 mmol/L lysine, on the other hand, significantly reduced the binding of plasminogen to immobilized thrombospondin, suggesting that the interaction of thrombospondin with plasminogen is not the direct result of the lysine binding sites of plasminogen. Arginine and benzamidine, ligands known to specifically bind to the kringle 5 domain of plasminogen, blocked the binding of thrombospondin to plasminogen. Limited elastase proteolysis of plasminogen and plasmin resulted in the generation of two distinct thrombospondin binding domains, one of which was retained on lysine- agarose. The isolation and amino-terminal analysis of these domains following elastase proteolysis of plasminogen identified them, respectively, as a domain containing kringle structures 4 and 5 and plasmin and the other domain consisting of kringle 5-plasmin. A 16- residue synthetic peptide, which represents the amino acids linking kringle 4 to kringle 5 (residues 435–450 of native plasminogen), was without effect in either binding to thrombospondin or blocking the binding of thrombospondin to plasminogen. Plasminogen, therefore, possesses a single thrombospondin interactive site that is independent of, but influenced by, the lysine binding site containing kringle structures and most likely is located within the kringle 5 domain.

scholarly journals Thrombospondin interaction with plasminogen. Evidence for binding to a specific region of the kringle structure of plasminogen

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 976-982
Author(s):  
P DePoli ◽  
T Bacon-Baguley ◽  
S Kendra-Franczak ◽  
MT Cederholm ◽  
DA Walz
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Binding Sites ◽  
Synthetic Peptide ◽  
Specific Region ◽  
The Other ◽  
Direct Result ◽  
Amino Terminal ◽  
Binding Domains ◽  
Kringle 5

Platelet thrombospondin interacts with plasminogen in a specific and saturable manner. Thrombospondin was found to specifically bind to plasminogen and the nonenzyme chain of plasmin. Preincubation of 125I- labeled thrombospondin with 30 mmol/L lysine was without effect in the binding of thrombospondin to immobilized plasminogen; preincubation of 125I-labeled plasminogen with 30 mmol/L lysine, on the other hand, significantly reduced the binding of plasminogen to immobilized thrombospondin, suggesting that the interaction of thrombospondin with plasminogen is not the direct result of the lysine binding sites of plasminogen. Arginine and benzamidine, ligands known to specifically bind to the kringle 5 domain of plasminogen, blocked the binding of thrombospondin to plasminogen. Limited elastase proteolysis of plasminogen and plasmin resulted in the generation of two distinct thrombospondin binding domains, one of which was retained on lysine- agarose. The isolation and amino-terminal analysis of these domains following elastase proteolysis of plasminogen identified them, respectively, as a domain containing kringle structures 4 and 5 and plasmin and the other domain consisting of kringle 5-plasmin. A 16- residue synthetic peptide, which represents the amino acids linking kringle 4 to kringle 5 (residues 435–450 of native plasminogen), was without effect in either binding to thrombospondin or blocking the binding of thrombospondin to plasminogen. Plasminogen, therefore, possesses a single thrombospondin interactive site that is independent of, but influenced by, the lysine binding site containing kringle structures and most likely is located within the kringle 5 domain.

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.

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