scholarly journals Allelic Diversity of the Two Transferrin Binding Protein B Gene Isotypes among a Collection of Neisseria meningitidisStrains Representative of Serogroup B Disease: Implication for the Composition of a Recombinant TbpB-Based Vaccine

2000 ◽  
Vol 68 (9) ◽  
pp. 4938-4947 ◽  
Author(s):  
Bachra Rokbi ◽  
Geneviève Renauld-Mongenie ◽  
Michèle Mignon ◽  
B. Danve ◽  
David Poncet ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bactericidal Activity ◽  
Immunoglobulin G ◽  
Binding Protein ◽  
Restriction Analysis ◽  
Allelic Diversity ◽  
Gene Variants ◽  
C Terminus ◽  
Specific Antisera

ABSTRACT The distribution of the two isotypes of tbpB in a collection of 108 serogroup B meningococcal strains belonging to the four major clonal groups associated with epidemic and hyperendemic disease (the ET-37 complex, the ET-5 complex, lineage III, and cluster A4) was determined. Isotype I strains (with a 1.8-kbtbpB gene) was less represented than isotype II strains (19.4 versus 80.6%). Isotype I was restricted to the ET-37 complex strains, while isotype II was found in all four clonal complexes. The extent of the allelic diversity of tbpB in these two groups was studied by PCR restriction analysis and sequencing of 10 newtbpB genes. Four major tbpB gene variants were characterized: B16B6 (representative of isotype I) and M982, BZ83, and 8680 (representative of isotype II). The relevance of these variants was assessed at the antigenic level by the determination of cross-bactericidal activity of purified immunoglobulin G preparations raised to the corresponding recombinant TbpB (rTbpB) protein against a panel of 27 strains (5 of isotype I and 22 of isotype II). The results indicated that rTbpB corresponding to each variant was able to induce cross-bactericidal antibodies. However, the number of strains killed with an anti-rTbpB serum was slightly lower than that obtained with an anti-TbpA+B complex. None of the sera tested raised against an isotype I strain was able to kill an isotype II strain and vice versa. None of the specific antisera tested (anti-rTbpB or anti-TbpA+B complex) was able to kill all of the 22 isotype II strains tested. Moreover, using sera raised against the C-terminus domain of TbpB M982 (amino acids 352 to 691) or BZ83 (amino acids 329 to 669) fused to the maltose-binding protein, cross-bactericidal activity was detected against 12 and 7 isotype II strains, respectively, of the 22 tested. These results suggest surface accessibility of the C-terminal end of TbpB. Altogether, these results show that although more than one rTbpB will be required in the composition of a TbpB-based vaccine to achieve a fully cross-bactericidal activity, rTbpB and its C terminus were able by themselves to induce cross-bactericidal antibodies.

scholarly journals Stem-Loop Binding Protein Facilitates 3′-End Formation by Stabilizing U7 snRNP Binding to Histone Pre-mRNA

1999 ◽  
Vol 19 (5) ◽  
pp. 3561-3570 ◽  
Author(s):  
Zbigniew Dominski ◽  
Lian-Xing Zheng ◽  
Ricardo Sanchez ◽  
William F. Marzluff
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Binding Protein ◽  
Nuclear Extract ◽  
Stable Complex ◽  
Stem Loop ◽  
Terminal Domain ◽  
C Terminus ◽  
Loop Sequence ◽  
U7 Snrnp

ABSTRACT The 3′ end of histone mRNA is formed by an endonucleolytic cleavage of the primary transcript after a conserved stem-loop sequence. The cleavage reaction requires at least two trans-acting factors: the stem-loop binding protein (SLBP), which binds the stem-loop sequence, and the U7 snRNP that interacts with a sequence downstream from the cleavage site. Removal of SLBP from a nuclear extract abolishes 3′-end processing, and the addition of recombinant SLBP restores processing activity of the depleted extract. To determine the regions of human SLBP necessary for 3′ processing, various deletion mutants of the protein were tested for their ability to complement the SLBP-depleted extract. The entire N-terminal domain and the majority of the C-terminal domain of human SLBP are dispensable for processing. The minimal protein that efficiently supports cleavage of histone pre-mRNA consists of 93 amino acids containing the 73-amino-acid RNA-binding domain and 20 amino acids located immediately next to its C terminus. Replacement of these 20 residues with an unrelated sequence in the context of the full-length SLBP reduces processing >90%. Coimmunoprecipitation experiments with the anti-SLBP antibody demonstrated that SLBP and U7 snRNP form a stable complex only in the presence of pre-mRNA substrates containing a properly positioned U7 snRNP binding site. One role of SLBP is to stabilize the interaction of the histone pre-mRNA with U7 snRNP.

scholarly journals Fine Antigenic Specificity and Cooperative Bactericidal Activity of Monoclonal Antibodies Directed at the Meningococcal Vaccine Candidate Factor H-Binding Protein

2008 ◽  
Vol 76 (9) ◽  
pp. 4232-4240 ◽  
Author(s):  
Peter T. Beernink ◽  
Jo Anne Welsch ◽  
Michal Bar-Lev ◽  
Oliver Koeberling ◽  
Maurizio Comanducci ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Bactericidal Activity ◽  
Binding Protein ◽  
Factor H ◽  
Antigenic Specificity ◽  
Complement Protein ◽  
Antigenic Variant ◽  
Classical Complement Pathway ◽  
Group B

ABSTRACT No broadly protective vaccine is available for the prevention of group B meningococcal disease. One promising candidate is factor H-binding protein (fHbp), which is present in all strains but often sparsely expressed. We prepared seven murine immunoglobulin G monoclonal antibodies (MAbs) against fHbp from antigenic variant group 2 (v.2) or v.3 (∼40% of group B strains). Although none of the MAbs individually elicited bactericidal activity with human complement, all had activity in different combinations. We used MAb reactivity with strains expressing fHbp polymorphisms and site-specific mutagenesis to identify residues that are important for epitopes recognized by six of the v.2 or v.3 MAbs and by two v.1 MAbs that were previously characterized. Residues affecting v.2 or v.3 epitopes resided between amino acids 174 and 216, which formed an eight-stranded beta-barrel in the C domain, while residues affecting the v.1 epitopes included amino acids 121 and 122 of the B domain. Pairs of MAbs were bactericidal when their respective epitopes involved residues separated by 16 to 20 Å and when at least one of the MAbs inhibited the binding of fH, a downregulatory complement protein. In contrast, there was no cooperative bactericidal activity when the distance between residues was ≥27 Å or ≤14 Å, which correlated with the inhibition of the binding of one MAb by the other MAb. Thus, a model for anti-fH MAb bactericidal activity against strains expressing low levels of fHbp requires the binding of two MAbs directed at nonoverlapping epitopes, which activates the classical complement pathway as well as inhibits fH binding. The latter increases the susceptibility of the organism to complement-mediated bacteriolysis.

scholarly journals Purification of human urinary prokallikrein. Identification of the site of activation by the metalloproteinase thermolysin

1985 ◽  
Vol 232 (3) ◽  
pp. 851-858 ◽  
Author(s):  
Y Takada ◽  
R A Skidgel ◽  
E G Erdös
Keyword(s):  
Amino Acids ◽  
Human Plasma ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Plasma Kallikrein ◽  
Double Immunodiffusion ◽  
C Terminus ◽  
Hydrophobic Amino Acids

Human urinary active kallikrein and prokallikrein were separated on DEAE-cellulose and octyl-Sepharose columns and both purified to homogeneity by affinity chromatography, gel filtration and hydrophobic h.p.l.c. Prokallikrein was monitored during purification by trypsin activation followed by determination of both amidase and kininogenase activity. After trypsin activation, purified prokallikrein had a specific kininogenase activity of 39.4 micrograms of bradykinin equivalent/min per mg and amidase activity of 16.5 mumol/min per mg with D-Val-Leu-Arg-7-amino-4-trifluoromethylcoumarin. Purified active kallikrein had a specific activity of 47 micrograms of bradykinin/min per mg. The molecular mass of prokallikrein was 48 kDa on electrophoresis and 53 kDa on gel filtration whereas active kallikrein gave values of 46 kDa and 53 kDa respectively. Antisera to active and prokallikrein were obtained. In double immunodiffusion and immunoelectrophoresis, antiserum to active kallikrein reacted with active and pro-kallikrein. Antiserum to prokallikrein contained antibodies to determinants not found in active kallikrein, presumably due to the presence of the activation peptide in the proenzyme. Human prokallikrein can be activated by thermolysin, trypsin and human plasma kallikrein. Activation of 50% of the prokallikrein (1.35 microM) was achieved in 30 min with 25 nM-thermolysin, 78 nM-trypsin or 180 nM-human plasma kallikrein. Thus thermolysin was the most effective activator. Thermolysin activated prokallikrein by releasing active kallikrein with N-terminal Ile1-Val2. Thus human tissue (glandular) prokallikrein can be activated by two types of enzymes: serine proteinases, which cleave at the C-terminus of basic amino acids, and by a metalloproteinase that cleaves at the N-terminus of hydrophobic amino acids.

scholarly journals The Enigmatic N-Terminal Domain of Proglucagon; A Historical Perspective

2021 ◽  
Vol 12 ◽  
Author(s):  
J. Michael Conlon
Keyword(s):  
Amino Acids ◽  
Erroneous Conclusion ◽  
Subsequent Work ◽  
Terminal Domain ◽  
C Terminus ◽  
Specific Antisera ◽  
Pancreatic Peptide ◽  
Insulin Radioimmunoassay ◽  
Gut Gli

Enteroglucagon refers to the predominant peptide with glucagon-like immunoreactivity (GLI) that is released by the intestine into the circulation in response to nutrients. Development of a radioimmunoassay for glucagon revealed issues that were not apparent in applications of the insulin radioimmunoassay. The fact that some antisera raised against glucagon recognized glucagon-related peptides in extracts of both pancreas and gut whereas others recognized only components in the pancreas remained a mystery until it was realized that the “gut GLI cross-reactive” antisera were directed against an epitope in the N-terminal to central region of glucagon whereas the “pancreatic glucagon specific” antisera were directed against an epitope in the C-terminal region. Unlike the cross-reactive antisera, the glucagon specific antisera did not recognize components in which glucagon was extended from its C-terminus by additional amino acids. Initial attempts to purify enteroglucagon from porcine ileum led to the erroneous conclusion that enteroglucagon comprised 100 amino acids with an apparent molecular mass of 12,000 Da and was consequently given the name glicentin. Subsequent work established that the peptide constituted residues (1-69) of proglucagon (Mr 8128). In the 40 years since the structural characterization of glicentin, attempts to establish an unambiguous physiological function for enteroglucagon have not been successful. Unlike the oxyntomodulin domain at the C-terminus of enteroglucagon, the primary structure of the N-terminal domain (glicentin-related pancreatic peptide) has been poorly conserved among mammals. Consequently, most investigations of the bioactivity of porcine glicentin may have been carried out in inappropriate animal models. Enteroglucagon may simply represent an inactive peptide that ensures that the intestine does not release equimolar amounts of a hyperglycemic agent (glucagon) and a hypoglycemic agent (GLP-1) after ingestion of nutrients.

scholarly journals New genotypes of Helicobacter Pylori VacA d-region identified from global strains

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Djaleel Muhammad Soyfoo ◽  
Yussriya Hanaa Doomah ◽  
Dong Xu ◽  
Chao Zhang ◽  
Huai-Ming Sang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Helicobacter Pylori ◽  
Allelic Diversity ◽  
Loop Region ◽  
C Terminus ◽  
D Region ◽  
Nucleotide Database ◽  
Vaca Gene ◽  
Loop Conformation ◽  
Type Strains

Abstract Background Pathogenesis of Helicobacter Pylori (HP) vacuolating toxin A (vacA) depends on polymorphic diversity within the signal (s), middle (m), intermediate (i), deletion (d) and c-regions. These regions show distinct allelic diversity. The s-region, m-region and the c-region (a 15 bp deletion at the 3′-end region of the p55 domain of the vacA gene) exist as 2 types (s1, s2, m1, m2, c1 and c2), while the i–region has 3 allelic types (i1, i2 and i3). The locus of d-region of the vacA gene has also been classified into 2 genotypes, namely d1 and d2. We investigated the “d-region”/“loop region” through bioinformatics, to predict its properties and relation to disease. One thousand two hundred fifty-nine strains from the NCBI nucleotide database and the dryad database with complete vacA sequences were included in the study. The sequences were aligned using BioEdit and analyzed using Lasergene and BLAST. The secondary structure and physicochemical properties of the region were predicted using PredictProtein. Results We identified 31 highly polymorphic genotypes in the “d-region”, with a mean length of 34 amino acids (9 ~ 55 amino acids). We further classified the 31 genotypes into 3 main types, namely K-type (strains starting with the KDKP motif in the “d-region”), Q-type (strains starting with the KNQT motif), and E-type (strains starting with the ESKT motif) respectively. The most common type, K-type, is more prevalent in cancer patients (80.87%) and is associated with the s1i1m1c1 genotypes (P < .01). Incidentally, a new region expressing sequence diversity (2 aa deletion) at the C-terminus of the p55 domain of vacA was identified during bioinformatics analysis. Conclusions Prediction of secondary structures shows that the “d-region” adopts a loop conformation and is a disordered region.

Human Platelet Antigen 3 (HPA-3): Localization of the Determinant of the Alloantibody Leka (HPA-3a) to the C-Terminus of Platelet Glycoprotein IIb Heavy Chain and Contribution of O-Linked Carbohydrates

1993 ◽  
Vol 69 (05) ◽  
pp. 485-489 ◽  
Author(s):  
Isabelle Djaffar ◽  
Didier Vilette ◽  
Dominique Pidard ◽  
Jean-Luc Wautier ◽  
Jean-Philippe Rosa
Keyword(s):  
Amino Acids ◽  
Heavy Chain ◽  
Human Platelet ◽  
Platelet Glycoprotein ◽  
Fibrinogen Receptor ◽  
C Terminus ◽  
Platelet Antigen ◽  
Human Platelet Antigen ◽  
Determinant Structure ◽  
Polymerase Chain

SummaryThe human platelet antigen (HPA) 3 system is expressed on GPIIb, one subunit of GPIIb-IIIa, the platelet fibrinogen receptor. It was recently shown that HPA-3 was associated with an Ile843/Ser polymorphism. To investigate further HPA-3 determinant structure, we localized an HPA-3a determinant, recognized by the alloantiserum Leka, within the last 29 amino acids of GPIIbα. This region encompasses the polymorphic Ile843, which, as expected, is substituted into Ser in Leka-negative individuals, as shown by DNA sequence after polymerase chain reaction on platelet RNA. In addition, contribution of glycosylation to the determinant structure was demonstrated since the Leka antigenicity was strongly decreased after specifically removing nonterminal O-linked sugars, but not terminal sialic acids. We have thus refined the localization of an HPA-3a determinant within the last 29 amino acids, including Ile843, of GPIIb heavy chain, and shown that the Leka HPA-3a determinant is dependent, in part, upon the serine-linked carbohydrates adjacent to Ile/Ser843.

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