scholarly journals Determination of the immunogenic region in the LipL32 protein of Leptospira

2018 ◽  
pp. 27-33
Author(s):  
Mohammad Iskandar Jumat ◽  
Kenneth Francis Rodrigues ◽  
Azlyna Laribe ◽  
Rashidah Mohammad ◽  
Timothy William ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
High Specificity ◽  
Full Length ◽  
Immunogenic Region ◽  
C Terminus ◽  
Initial Symptoms ◽  
N Terminus ◽  
Low Sensitivity

Leptospirosis is a zoonotic disease caused by the pathogenic species of Leptospira. The initial symptoms include fever, myalgia, nausea, skin rash, chills, and headache, which can be misdiagnosed. LipL32 is the highly conserved and abundant outer membrane protein (OMP) of Leptospira, which is used as an antigen in serodiagnostic assays. We used three in silico methods to predict the immunodominant regions in the full-length LipL32 protein. We identified three regions, namely the N-terminus (NrLipL32, amino acid sequence 20th-120th), intermediate (amino acid sequence 120th-150th), and C-terminus (CrLipL32, amino acid sequence 160th-260th) regions. The full-length protein and two larger fragments were cloned into the pET22b plasmid and expressed in Escherichia coli BL21 (DE3). The purified proteins were used as antigens in an ELISA to detect Leptospira-specific antibodies. The CrLipL32 ELISA showed the highest sensitivity for IgM (73.3%) and IgG (65%), followed by the full-length rLipL32 ELISA (IgM 68% and IgG 60%). The full-length rLipL32 ELISA showed high specificity (IgM 85% and IgG 75%), followed by the NrLipL32 ELISA (IgM 75% and IgG 60%). The intermediate fragment showed very low sensitivity (IgM 17% and IgG 2%). The sensitivity of the rLipL32 ELISA could be enhanced by adding other OMPs of Leptospira.

The amino acid sequence of yeast phosphoglycerate mutase

1982 ◽  
Vol 215 (1198) ◽  
pp. 19-44 ◽  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Proteolytic Enzymes ◽  
Phosphoglycerate Mutase ◽  
Crystallographic Structure ◽  
X Ray ◽  
C Terminus ◽  
Detailed Interpretation ◽  
High Degree

The complete amino acid sequence of yeast phosphoglycerate mutase comprising 241 residues has been determined. The sequence was deduced from the two cyanogen bromide fragments, and from the peptides derived from these fragments after digestion by a number of proteolytic enzymes. Determination of this sequence now allows a detailed interpretation of the existing high-resolution X-ray crystallographic structure. A comparison of the sequence reported here with the sequences of peptides from phosphoglycerate mutases from other species, and with the sequence of erythrocyte diphosphoglycerate mutase, indicates that these enzymes have a high degree of structural homology. Autolysis of phosphoglycerate mutase by yeast extracts leads to the complete loss of mutase activity, and the formation of electrophoretically distinguishable forms (R. Sasaki, E. Sugimoto & H. Chiba, Archs Biochem. Biophys. 115, 53-61 (1966)). It is apparent from the amino acid sequence that these changes are due to the loss of an 8─12 residue peptide from the C-terminus.

Amino acid sequence and d/l-configuration determination of peptides utilizing liberated N-terminus phenylthiohydantoin amino acids

1998 ◽  
Vol 813 (2) ◽  
pp. 267-275 ◽  
Author(s):  
Takayuki Iida ◽  
Hirokazu Matsunaga ◽  
Tomofumi Santa ◽  
Takeshi Fukushima ◽  
Hiroshi Homma ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
N Terminus

scholarly journals The amino acid sequence of protein SCMK-B2C from the high-sulphur fraction of wool keratin

1972 ◽  
Vol 128 (5) ◽  
pp. 1229-1239 ◽  
Author(s):  
T. C. Elleman
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Wool Fibre ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Unusual Distribution ◽  
Mechanical And Physical Properties ◽  
N Terminus ◽  
Fibre Matrix ◽  
Wool Keratin

1. The amino acid sequence of a protein from the reduced and carboxymethylated high-sulphur fraction of wool has been determined. 2. The sequence of this S-carboxymethylkerateine (SCMK-B2C) of 151 amino acid residues displays much internal homology and an unusual residue distribution. Thus a ten-residue sequence occurs four times near the N-terminus and five times near the C-terminus with few changes. These regions contain much of the molecule's half-cystine, whereas between them there is a region of 19 residues that are mainly small and devoid of cystine and proline. 3. Certain models of the wool fibre based on its mechanical and physical properties propose a matrix of small compact globular units linked together to form beaded chains. The unusual distribution of the component residues of protein SCMK-B2C suggests structures in the wool-fibre matrix compatible with certain features of the proposed models.

scholarly journals Localization of the Intracellular Activity Domain of Pasteurella multocida Toxin to the N Terminus

1999 ◽  
Vol 67 (1) ◽  
pp. 80-87 ◽  
Author(s):  
Brenda A. Wilson ◽  
Virgilio G. Ponferrada ◽  
Jefferson E. Vallance ◽  
Mengfei Ho
Keyword(s):  
Amino Acid ◽  
Pasteurella Multocida ◽  
Mutational Analysis ◽  
Vero Cells ◽  
Full Length ◽  
Induced Response ◽  
Intracellular Activity ◽  
C Terminus ◽  
N Terminus ◽  
Activity Domain

ABSTRACT We have shown that Pasteurella multocida toxin (PMT) directly causes transient activation of Gqα protein that is coupled to phosphatidylinositol-specific phospholipase Cβ1 inXenopus oocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268–1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response in Xenopus oocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using theXenopus oocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain of P. multocida the entiretoxA gene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein inEscherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments in E. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in the Xenopus oocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells.

Molecular Identification and Functional Characterization of a Novel Protein That Mediates the Attachment of Erythroblasts to Macrophages

Blood ◽  
1998 ◽  
Vol 92 (8) ◽  
pp. 2940-2950 ◽  
Author(s):  
Manjit Hanspal ◽  
Yva Smockova ◽  
Quang Uong
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transmembrane Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Functional Characterization ◽  
Sodium Dodecyl ◽  
Full Length ◽  
Human Macrophage ◽  
N Terminus ◽  
Novel Protein

We have previously identified a novel protein that mediates the attachment of erythroblasts to macrophages in vitro. This attachment promotes terminal maturation and enucleation of erythroblasts (Hanspal and Hanspal, Blood 84:3494, 1994). This protein is referred to here as Emp for erythroblast macrophageprotein. Two immunologically related isoforms of Emp with apparent molecular weights of 33 kD and 36 kD were detected in macrophage membranes. The complete amino acid sequence of the larger isoform of Emp was deduced from the nucleotide sequence of a full-length 2.0-kb cDNA that was isolated from a human macrophage cDNA library using affinity-purified anti-Emp antibodies. Of the 2,005 bp, 1,185 bp encode for 395 amino acids representing 43 kD (the sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] molecular mass is 36 kD). Northern blot analysis of human macrophage poly(A) RNA detected a message for Emp of 2.1 kb. The deduced amino acid sequence contains a putative transmembrane domain near the N-terminus. To investigate the structure/function relationships of Emp, recombinant fusion proteins of full-length and truncated Emp were produced in bacteria, COS-7, and HeLa cells. Cell binding assays showed that the N-terminus is exposed on the cell surface. The recombinant Emp functions as a cell attachment molecule when expressed in heterologous cells. Furthermore, we showed that the demise of erythroblasts in the absence of Emp-mediated erythroblast-macrophage association is accompanied by apoptosis. We postulate that Emp-mediated contact between erythroblasts and macrophages promotes terminal maturation of erythroid cells by suppressing apoptosis. © 1998 by The American Society of Hematology.

Molecular Identification and Functional Characterization of a Novel Protein That Mediates the Attachment of Erythroblasts to Macrophages

Blood ◽  
1998 ◽  
Vol 92 (8) ◽  
pp. 2940-2950 ◽  
Author(s):  
Manjit Hanspal ◽  
Yva Smockova ◽  
Quang Uong
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transmembrane Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Functional Characterization ◽  
Sodium Dodecyl ◽  
Full Length ◽  
Human Macrophage ◽  
N Terminus ◽  
Novel Protein

Abstract We have previously identified a novel protein that mediates the attachment of erythroblasts to macrophages in vitro. This attachment promotes terminal maturation and enucleation of erythroblasts (Hanspal and Hanspal, Blood 84:3494, 1994). This protein is referred to here as Emp for erythroblast macrophageprotein. Two immunologically related isoforms of Emp with apparent molecular weights of 33 kD and 36 kD were detected in macrophage membranes. The complete amino acid sequence of the larger isoform of Emp was deduced from the nucleotide sequence of a full-length 2.0-kb cDNA that was isolated from a human macrophage cDNA library using affinity-purified anti-Emp antibodies. Of the 2,005 bp, 1,185 bp encode for 395 amino acids representing 43 kD (the sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] molecular mass is 36 kD). Northern blot analysis of human macrophage poly(A) RNA detected a message for Emp of 2.1 kb. The deduced amino acid sequence contains a putative transmembrane domain near the N-terminus. To investigate the structure/function relationships of Emp, recombinant fusion proteins of full-length and truncated Emp were produced in bacteria, COS-7, and HeLa cells. Cell binding assays showed that the N-terminus is exposed on the cell surface. The recombinant Emp functions as a cell attachment molecule when expressed in heterologous cells. Furthermore, we showed that the demise of erythroblasts in the absence of Emp-mediated erythroblast-macrophage association is accompanied by apoptosis. We postulate that Emp-mediated contact between erythroblasts and macrophages promotes terminal maturation of erythroid cells by suppressing apoptosis. © 1998 by The American Society of Hematology.

Polypeptides Folding: Rules for the Calculation of the Backbone Dihedral Angles φ starting from the Amino Acid Sequence

2020 ◽  
Author(s):  
Michele Larocca
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Theoretical Approach ◽  
Polypeptide Chain ◽  
Hydrophobic Effect ◽  
Side Chain ◽  
Dihedral Angles ◽  
Mechanical Forces ◽  
Specific Chemical

<p>Protein folding is strictly related to the determination of the backbone dihedral angles and depends on the information contained in the amino acid sequence as well as on the hydrophobic effect. To date, the type of information embedded in the amino acid sequence has not yet been revealed. The present study deals with these problematics and aims to furnish a possible explanation of the information contained in the amino acid sequence, showing and reporting rules to calculate the backbone dihedral angles φ. The study is based on the development of mechanical forces once specific chemical interactions are established among the side chain of the residues in a polypeptide chain. It aims to furnish a theoretical approach to predict backbone dihedral angles which, in the future, may be applied to computational developments focused on the prediction of polypeptide structures.</p>

Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

1995 ◽  
Vol 69 (11) ◽  
pp. 7274-7277 ◽  
Author(s):  
J I Casal ◽  
J P Langeveld ◽  
E Cortés ◽  
W W Schaaper ◽  
E van Dijk ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Peptide Vaccine ◽  
Canine Parvovirus ◽  
N Terminus

scholarly journals Terminal deoxynucleotidyltransferase. Alignment of α- and β-subunits of the core enzyme along the primary translation product

1985 ◽  
Vol 227 (3) ◽  
pp. 1003-1007 ◽  
Author(s):  
C M Beach ◽  
S K Chan ◽  
T C Vanaman ◽  
M S Coleman
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Beta Subunits ◽  
Isolation And Purification ◽  
C Terminus ◽  
Human Lymphoblast ◽  
Catalytically Active ◽  
Single Polypeptide ◽  
Dna Nucleotide Sequence

Terminal deoxynucleotidyltransferase exists in multiple Mr forms, all apparently generated from a single polypeptide of 62kDa. On isolation and purification, the smallest catalytically active protein of this enzyme consists of two subunits, alpha (12kDa) and beta (30kDa). Recently a complementary-DNA nucleotide sequence has been reported for a portion of the enzyme from human lymphoblast. We have pinpointed the locations of the alpha- and beta-subunits within the elucidated nucleotide sequence. From these data, the portions of the nucleotide sequence coding for the catalytically important area of the transferase can be estimated. Here the amino acid sequence of a number of tryptic peptides from calf alpha- and beta-subunits is presented. Because of the striking homology between the amino acid sequence of the calf enzyme and that predicted for human lymphoblast enzyme, it is possible for us to conclude that the alpha-subunit was generated from the C-terminus of the precursor protein and the beta-subunit was non-overlapping and proximal.

scholarly journals Prospective Characterization of Full-Length Hepatitis C Virus NS5A Quasispecies during Induction and Combination Antiviral Therapy

2000 ◽  
Vol 74 (19) ◽  
pp. 9028-9038 ◽  
Author(s):  
J.-B. Nousbaum ◽  
S. J. Polyak ◽  
S. C. Ray ◽  
D. G. Sullivan ◽  
A. M. Larson ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Antiviral Therapy ◽  
Variable Region ◽  
Response To Therapy ◽  
Full Length ◽  
Variable Regions ◽  
C Terminus

ABSTRACT The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR237–276) sequence associated with IFN resistance was not found, although the presence of Ala245 within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P < 0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P < 0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P < 0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.

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