Bacterial lactoferrin receptors: insights from characterizing theMoraxella bovisreceptors

2002 ◽  
Vol 80 (1) ◽  
pp. 81-90 ◽  
Author(s):  
Rong-Hua Yu ◽  
Anthony B Schryvers
Keyword(s):  
Amino Acid ◽  
Binding Protein ◽  
Protein A ◽  
Bovine Lactoferrin ◽  
Predicted Amino Acid Sequence ◽  
Amino Acid Homology ◽  
Affinity Ligand ◽  
Affinity Isolation ◽  
Moraxella Bovis ◽  
Isogenic Mutants

Moraxella bovis is the causative agent of infectious conjunctivitis in cattle. Moraxella bovis isolates were shown to specifically bind bovine lactoferrin (bLf) and bovine transferrin (bTf) and to use these proteins as a source of iron to support the growth of iron-limited cells. Affinity isolation experiments with immobilized bTf yielded two proteins readily resolved by SDS-PAGE analysis, whereas only a single band of approximately 100 kDa was detected when immobilized bLf was used as the affinity ligand. Using a novel cloning strategy, regions containing the genes encoding the lactoferrin (Lf) and transferrin (Tf) receptor proteins were isolated and sequenced, demonstrating that they both consisted of two genes, with the tbpB or lbpB gene preceding the tbpA or lbpA gene. The cloned lbp genes were used to generate isogenic mutants deficient in lactoferrin binding protein A and (or) B, and the resulting strains were tested in growth and binding assays. The isogenic mutants were deficient in their use of bLf for growth and had substantially diminished bLf binding capability. The predicted amino acid sequence from the segment encoding Lf binding protein B revealed an internal amino acid homology suggesting it is a bi-lobed protein, with a C-lobe enriched in acidic amino acids, but without the evident clustering observed in Lf-binding proteins from other species.Key words: outer membrane protein, iron-binding protein, lactoferrin, receptor, iron, transport, specificity.

Structural and functional studies on C4b-binding protein, a regulatory component of the human complement system

1985 ◽  
Vol 5 (10-11) ◽  
pp. 855-865 ◽  
Author(s):  
L. Ping Chung ◽  
Kenneth B. M. Reid
Keyword(s):  
Amino Acid ◽  
Binding Protein ◽  
Protein A ◽  
Limited Proteolysis ◽  
Binding Activity ◽  
Amino Acid Sequencing ◽  
Functional Studies ◽  
Before And After ◽  
Cofactor Activity ◽  
Long Chain

The binding and cofactor activities of C4b-binding protein were examined before and after limited proteolysis by pepsin, trypsin and chymotrypsin. The major fragments generated were characterized by amino acid sequencing, thus establishing the precise points of limited proteolysis. These studies allow a tentative assignment of the cofactor activity site to the residues 177–322 of the 549 amino acid long chain of C4b-binding protein but indicated that residues in the region 332–395 are important in the binding activity.

scholarly journals Characterization of a glutathione S-transferase and a related glutathione-binding protein from gill of the blue mussel, Mytilus edulis

1995 ◽  
Vol 305 (1) ◽  
pp. 145-150 ◽  
Author(s):  
P J Fitzpatrick ◽  
T O B Krag ◽  
P Højrup ◽  
D Sheehan
Keyword(s):  
Amino Acid ◽  
Mytilus Edulis ◽  
Blue Mussel ◽  
Binding Protein ◽  
Protein A ◽  
Gel Filtration ◽  
Cumene Hydroperoxide ◽  
Gill Tissue ◽  
Glutathione S Transferase ◽  
Sequencing Studies

The major isoenzyme of glutathione S-transferase (GST 1) was purified to homogeneity from cytosolic extracts of Mytilus edulis gill tissue by GSH-agarose affinity chromatography followed by Mono Q ion-exchange f.p.l.c. This enzyme was particularly active with 1-chloro-2,4-dinitrobenzene, ethacrynic acid and cumene hydroperoxide as substrates. Immunoblotting and amino acid sequencing studies indicate that the enzyme belongs to the Pi class of GSTs. A related protein which binds to GSH-agarose was also purified. This GSH-binding protein did not immunoblot with GST antisera and showed no detectable catalytic activity with GST substrates although its N-terminal sequence was similar to Mu-class GSTs. Gel-filtration chromatography indicated that GST 1 is a dimer and the GSH-binding protein a monomer. Mass spectrometry and SDS/PAGE indicate subunit molecular masses of 24 kDa (GST 1) and 25 kDa (GSH-binding protein), respectively. Both proteins have amino acid compositions typical of GSTs.

scholarly journals Identification of Discrete Domains within Gonococcal Transferrin-Binding Protein A That Are Necessary for Ligand Binding and Iron Uptake Functions

2000 ◽  
Vol 68 (12) ◽  
pp. 6988-6996 ◽  
Author(s):  
Ian C. Boulton ◽  
Mary Kate Yost ◽  
James E. Anderson ◽  
Cynthia Nau Cornelissen
Keyword(s):  
Ligand Binding ◽  
Iron Uptake ◽  
Binding Protein ◽  
Protein A ◽  
Ligand Interaction ◽  
Whole Cells ◽  
Affinity Ligand ◽  
Discrete Domains ◽  
Transferrin Binding Proteins

ABSTRACT The availability of free iron in vivo is strictly limited, in part by the iron-binding protein transferrin. The pathogenicNeisseria spp. can sequester iron from this protein, dependent upon two iron-repressible, transferrin-binding proteins (TbpA and TbpB). TbpA is a TonB-dependent, integral, outer membrane protein that may form a β-barrel exposing multiple surface loops, some of which are likely to contain ligand-binding motifs. In this study we propose a topological model of gonococcal TbpA and then test some of the hypotheses set forth by the model by individually deleting three putative loops (designated loops 4, 5, and 8). Each mutant TbpA could be expressed without toxicity and was surface exposed as assessed by immunoblotting, transferrin binding, and protease accessibility. Deletion of loop 4 or loop 5 abolished transferrin binding to whole cells in solid- and liquid-phase assays, while deletion of loop 8 decreased the affinity of the receptor for transferrin without affecting the copy number. Strains expressing any of the three mutated TbpAs were incapable of growth on transferrin as a sole iron source. These data implicate putative loops 4 and 5 as critical determinants for receptor function and transferrin-iron uptake by gonococcal TbpA. The phenotype of the ΔL8TbpA mutant suggests that high-affinity ligand interaction is required for transferrin-iron internalization.

scholarly journals Molecular cloning and characterization of the cDNA coding for C4b-binding protein, a regulatory protein of the classical pathway of the human complement system

1985 ◽  
Vol 230 (1) ◽  
pp. 133-141 ◽  
Author(s):  
L P Chung ◽  
D R Bentley ◽  
K B Reid
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Sequence ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Protein A ◽  
Classical Pathway ◽  
Amino Acid Residues ◽  
Cloned Cdna

By using synthetic oligonucleotides as probes, plasmid clones containing portions of cDNA coding for human C4b-binding protein were isolated from a liver cDNA library. The entire amino acid sequence of the C4b-binding protein can be predicted from this study of the cloned cDNA when allied to a previous sequence study at the protein level [Chung, Gagnon & Reid (1985) Mol. Immunol. 22, 427-435], in which over 55% of the amino acid sequence, including the N-terminal 62 residues, was obtained. The plasmid clones isolated allowed the unambiguous determination of 1717 nucleotides of cDNA sequence between the codon for the 32nd amino acid in the sequence of C4b-binding protein and the 164th nucleotide in the 3′ non-translated region. The sequence studies show that the secreted form of C4b-binding protein, found in plasma, is composed of chains of apparent Mr 70 000 that contains 549 amino acid residues. Examination of the protein and cDNA sequence results show that there are at least two polymorphic sites in the molecule. One is at position 44, which can be glutamine or threonine, and the other is at position 309, which can be tyrosine or histidine. Northern-blot analysis indicated that the mRNA for C4b-binding protein is approx. 2.5 kilobases long. The N-terminal 491 amino acids of C4b-binding protein can be divided into eight internal homologous regions, each approx. 60 amino acids long, which can be aligned by the presence in each region of four half-cystine, one tryptophan and several other conserved residues. These regions in C4b-binding protein are homologous with the three internal-homology regions that have been reported to be present within the Ba region of the complement enzyme factor B and also to the internal-homology regions found in the non-complement beta 2-glycoprotein I.

Crystal structure of the N-lobe of lactoferrin binding protein B from Moraxella bovis1 1This paper is an invited article as a result of a presentation at the International Lactoferrin Conference held in Mazatlan, Mexico (May 2011), and has undergone the Journal’s usual peer review process.

2012 ◽  
Vol 90 (3) ◽  
pp. 351-361 ◽  
Author(s):  
Elena Arutyunova ◽  
Cory L. Brooks ◽  
Amanda Beddek ◽  
Michelle W. Mak ◽  
Anthony B. Schryvers ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structural Information ◽  
Peer Review Process ◽  
Binding Protein ◽  
Iron Acquisition ◽  
Protein A ◽  
Bacterial Surface ◽  
Moraxella Bovis ◽  
Surface Receptor ◽  
Protein B

Lactoferrin (Lf) is a bi-lobed, iron-binding protein found on mucosal surfaces and at sites of inflammation. Gram-negative pathogens from the Neisseriaceae and Moraxellaceae families are capable of using Lf as a source of iron for growth through a process mediated by a bacterial surface receptor that directly binds host Lf. This receptor consists of an integral outer membrane protein, lactoferrin binding protein A (LbpA), and a surface lipoprotein, lactoferrin binding protein B (LbpB). The N-lobe of the homologous transferrin binding protein B, TbpB, has been shown to facilitate transferrin binding in the process of iron acquisition. Currently there is little known about the role of LbpB in iron acquisition or how Lf interacts with the bacterial receptor proteins. No structural information on any LbpB or domain is available. In this study, we express and purify from Escherichia coli the full-length LbpB and the N-lobe of LbpB from the bovine pathogen Moraxella bovis for crystallization trials. We demonstrate that M. bovis LbpB binds to bovine but not human Lf. We also report the crystal structure of the N-terminal lobe of LbpB from M. bovis and compare it with the published structures of TbpB to speculate on the process of Lf mediated iron acquisition.

scholarly journals Molecular cloning and in vitro expression of a cDNA clone for human cellular tumor antigen p53.

1985 ◽  
Vol 5 (7) ◽  
pp. 1601-1610 ◽  
Author(s):  
E Harlow ◽  
N M Williamson ◽  
R Ralston ◽  
D M Helfman ◽  
T E Adams
Keyword(s):  
Amino Acid ◽  
Tumor Antigen ◽  
Human Tumor ◽  
Northern Analysis ◽  
Predicted Amino Acid Sequence ◽  
Coding Region ◽  
A431 Cells ◽  
Amino Acid Homology ◽  
P53 Mrna

Three clones for the human tumor antigen p53 were isolated from a cDNA library prepared from A431 cells. One of these clones, pR4-2, contains the entire coding region for human p53. This clone directs the synthesis of a polypeptide with the correct molecular weight and immunological epitopes of an authentic p53 molecule in an in vitro transcription-translation reaction. Although the pR4-2 clone contains the coding region for p53, it is not a full-length copy of the human p53 mRNA. Northern analysis showed that the p53 mRNA is approximately 2,500 nucleotides long, whereas the pR4-2 insert is only 1,760 base pairs in length. Analysis of the DNA sequence of this clone suggests that the human p53 polypeptide has 393 amino acids. We compared the predicted amino acid sequence of the pR4-2 clone with similar clones for the mouse p53 and found long regions of amino acid homology between these two molecules.

scholarly journals Specificity of human tissue kallikrein towards substrates containing Phe–Phe pair of amino acids

1999 ◽  
Vol 339 (2) ◽  
pp. 473-479 ◽  
Author(s):  
Daniel C. PIMENTA ◽  
Julie CHAO ◽  
Lee CHAO ◽  
Maria A. JULIANO ◽  
Luiz JULIANO
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Human Tissue ◽  
Binding Protein ◽  
General Structure ◽  
Protein A ◽  
Tissue Kallikrein ◽  
Hybrid Peptides ◽  
Loop Sequence ◽  
Reactive Centre Loop

We have explored in detail the determinants of specificity for the hydrolysis by human tissue kallikrein (hK1) of substrates containing the Phe–Phe amino acid pair, after which hK1 cleaves kallistatin (human kallikrein-binding protein), a specific serpin for this protease, as well as somatostatin 1–14. Internally quenched fluorogenic peptides were synthesized with the general structure Abz-peptidyl-EDDnp [Abz, o-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine], based on the natural reactive-centre loop sequence of kallistatin from P9 to P´13, and the kinetic parameters of their hydrolysis by hK1 were determined. All these peptides were cleaved after the Phe–Phe pair. For comparison, we have also examined peptides containing the reactive-centre loop sequences of human protein-C inhibitor (PCI) and rat kallikrein-binding protein, which were hydrolysed after Phe–Arg and Leu–Lys bonds, respectively. Hybrid peptides containing kallistatin–PCI sequences showed that the efficiency of hK1 activity on the peptides containing kallistatin and PCI sequences depended on both the nature of the P1 amino acid as well as on residues at the P- and P´-sides. Moreover, we have made systematic modifications on the hydrophobic pair Phe–Phe, and on Lys and Ile at the P3 and P4 positions according to the peptide substrate, Abz-AIKFFSRQ-EDDnp. All together, we concluded that tissue kallikrein was very effective on short substrates that are cleaved after the Phe–Arg pair; however, hydrolysis after Phe–Phe or other hydrophobic pairs of amino acids was more restrictive, requiring additional enzyme–substrate interaction and/or particular substrate conformations.

scholarly journals FL-160 proteins of Trypanosoma cruzi are expressed from a multigene family and contain two distinct epitopes that mimic nervous tissues.

1993 ◽  
Vol 178 (2) ◽  
pp. 681-694 ◽  
Author(s):  
W C Van Voorhis ◽  
L Barrett ◽  
R Koelling ◽  
A G Farr
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Sciatic Nerve ◽  
Trypanosoma Cruzi ◽  
Gene Family ◽  
Molecular Mimicry ◽  
Surface Membrane ◽  
Apparent Molecular Weight ◽  
Predicted Amino Acid Sequence ◽  
Amino Acid Homology

The partial sequence of a gene encoding the COOH terminus of a protein of apparent molecular weight of 160 kD associated with the flagellum of trypomastigotes of Trypanosoma cruzi (FL-160 now renamed to FL-160-1) has been previously reported. The COOH terminus of FL-160-1 has an epitope, defined by 12 amino acids, which molecularly miMics a nervous tissue antigen of 48 kD found in myenteric plexus, sciatic nerve, and a subset of cells in the central nervous system. We now report that FL-160 is a family of highly related genes. The sequence has been determined for the entire open reading frame (ORF) of one of the members of the FL-160 gene family (FL-160-2) and three other partial ORFs. Sequence analysis reveals the various members of the FL-160 gene family to be approximately 80% homologous in the predicted amino acid sequence, but all retain the 12-amino acid molecular mimicry epitope on the COOH terminus. Comparison of the sequence of FL-160-2 to other sequences demonstrates amino acid homology to bacterial sialidase (27%), members of the SA85 gene family (25-30%) and the shed acute-phase antigen/neuraminidase/trans-sialidase gene family (25-30%). Quantitative hybridization at high stringency suggests 750 copies of FL-160 are present in the DNA of each parasite. Reverse transcription and sequence analysis demonstrates that at least five of the members of the FL-160 gene family are transcribed. The NH2 terminus of one of the FL-160 gene products was expressed and antibodies prepared. Antibodies directed to either the COOH or the NH2 terminus of FL-160 bind a 160-kD T. cruzi protein. Both antibodies bind the surface membrane in the flagellar pocket of the trypomastigote. Antibodies to the NH2 terminus bind epineurium and scattered linear densities in sciatic nerve in a pattern distinct from the pattern with antibodies to the COOH terminus. Thus, there are at least two distinct molecular mimicry epitopes on the FL-160 molecule and both mimic epitopes found in nervous tissues. FL-160 may be involved in the generation of autoimmunity to nervous tissues by molecular mimicry, observed in chronic Chagas' disease.

scholarly journals Molecular Characteristics of VP1 Region of Enterovirus 71 Strains in China

2020 ◽  
Author(s):  
Haiyan Sun ◽  
Min Gao ◽  
Dawei Cui
Keyword(s):  
Amino Acid ◽  
Vaccine Strain ◽  
Repetitive Sequences ◽  
Protein A ◽  
Enterovirus 71 ◽  
Molecular Characteristics ◽  
Continuous Change ◽  
Amino Acid Homology ◽  
Genetic Characteristics ◽  
Vp1 Protein

Abstract Background Enterovirus 71 (EV71) is the most common causative agent of severe and fetal hand, foot, and mouth disease (HFMD) outbreaks worldwide, and the VP1 protein, a capsid protein of EV71, is responsible for the genotype of EV71, which is important for vaccine selection and effectiveness. We performed an observational study of the genetic characteristics and genotype of EV71 isolates in China. Methods The VP1 gene sequences of 3712 EV71 virus strains from China, excluding repetitive sequences, and 30 known EV71 genotypes, as reference strains, between 1986 and 2019 were obtained from GenBank. A phylogenetic tree, amino acid homology, genetic variation and genotype analysis of the EV71-VP1 protein were performed with MEGA 6.0 software. Results The amino acid identity of all Chinese EV71 strains was 88.33%-100%, 93.47%-100% with the vaccine strain H07, and 93.04%-100% with the vaccine strains FY7VP5 and FY-23K-B. Since 2000, the prevalent strains of EV71 were mainly the C4 genotype; the C4a subgenotype was predominant, the C4b subgenotype was the second, and other subgenotypes appeared sporadically between 2005 and 2018 in mainland China. The B4 genotype was the main genotype in Taiwan, and the epidemic strains were constantly changing. Some amino acid variations in VP1 of EV71 occurred with high frequencies: A289T (20.99%), H22Q (16.49%), A293S (15.95%), S283T (15.11%), V249I (7.76%), N31D (7.25%), and E98K (6.65%). Conclusion The C4 genotype of EV71 in China can match the vaccine, which can effectively control EV71 epidemiology. However, the efficacy of the vaccine strain is partially affected by the continuous change in epidemic strains in Taiwan, China. These studies suggested that the genetic characteristics of the EV71-VP1 region should be continuously monitored, which is critical for epidemic control and vaccine design against EV71 infection in children.

scholarly journals Antigenic and Molecular Conservation of the Gonococcal NspA Protein

1999 ◽  
Vol 67 (6) ◽  
pp. 2855-2861 ◽  
Author(s):  
Martin Plante ◽  
Nathalie Cadieux ◽  
Clément R. Rioux ◽  
Josée Hamel ◽  
Bernard R. Brodeur ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Protein A ◽  
Surface Protein ◽  
Meningococcal Infection ◽  
Predicted Amino Acid Sequence ◽  
Amino Acid Residues ◽  
Intact Cells ◽  
Reading Frame ◽  
Calculated Molecular Weight

ABSTRACT A low-molecular-weight protein named NspA (neisserial surface protein A) was recently identified in the outer membrane of allNeisseria meningitidis strains tested. Antibodies directed against this protein were shown to protect mice against an experimental meningococcal infection. Hybridization experiments clearly demonstrated that the nspA gene was also present in the genomes of the 15 Neisseria gonorrhoeae strains tested. Cloning and sequencing of the nspA gene of N. gonorrhoeaeB2 revealed an open reading frame of 525 nucleotides coding for a polypeptide of 174 amino acid residues, with a calculated molecular weight of 18,316 and a pI of 10.21. Comparison of the predicted amino acid sequence of the NspA polypeptides from the gonococcal strains B2 and FA1090, together with that of the meningococcal strain 608B, revealed an identity of 93%, suggesting that the NspA protein is highly conserved among pathogenic Neisseria strains. The level of identity rose to 98% when only the two gonococcal predicted NspA polypeptides were compared. To evaluate the level of antigenic conservation of the gonococcal NspA protein, monoclonal antibodies (MAbs) were generated. Four of the seven NspA-specific MAbs described in this report recognized their corresponding epitope in 100% of the 51 N. gonorrhoeae strains tested. Radioimmunobinding assays clearly indicated that the gonococcal NspA protein is exposed at the surface of intact cells.

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