scholarly journals Detection of four genetic subgroup-specific antibodies to human metapneumovirus attachment (G) protein in human serum

2008 ◽  
Vol 89 (8) ◽  
pp. 1970-1977 ◽  
Author(s):  
Rika Endo ◽  
Takashi Ebihara ◽  
Nobuhisa Ishiguro ◽  
Shinobu Teramoto ◽  
Tadashi Ariga ◽  
...  
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
G Proteins ◽  
Trichoplusia Ni ◽  
Recombinant Baculovirus ◽  
Human Metapneumovirus ◽  
Amino Acid Sequences ◽  
Serum Samples ◽  
Paired Samples ◽  
Genetic Subgroup

Human metapneumovirus (hMPV) strains are classified into two genetic groups, A and B, each of which is further divided in two genetic subgroups, A1, A2, B1 and B2. hMPV encodes two major surface glycoproteins, the fusion (F) and attachment (G) proteins, which may be immunogenic and protective antigens. Although the amino acid sequences of hMPV F protein are highly conserved, those of the G protein are highly variable with low amino acid identity between the two groups. To address the antigenic variation between the genetic subgroups, we developed an immunofluorescence assay (IFA) method using Trichoplusia ni (Tn5) insect cells infected with each recombinant baculovirus-expressed hMPV G (Bac-G) protein of the four genetic subgroups. The titre of each antibody to the four Bac-G proteins was measured by the IFA in 12 paired serum samples obtained from children infected with hMPV of each genetic subgroup. Although 11 of the 12 acute-phase serum samples in paired samples were negative for the antibody to any Bac-G proteins, all of the convalescent-phase serum samples in those paired samples were positive for the antibody to only one of the four Bac-G proteins of the infecting genotype of hMPV. Since the antibody response to hMPV G protein was transient and genetic subgroup-specific without cross-reactivity, four genetic subgroups on the basis of hMPV G protein could be identified as different serotypes. This assay may be useful for the study of immune responses of humans to different hMPV strains, especially for clarifying the risk of reinfection with hMPV.

scholarly journals Immunofluorescence Assay for Detection of Human Metapneumovirus-Specific Antibodies by Use of Baculovirus-Expressed Fusion Protein

2005 ◽  
Vol 12 (1) ◽  
pp. 202-205 ◽  
Author(s):  
Nobuhisa Ishiguro ◽  
Takashi Ebihara ◽  
Rika Endo ◽  
Xiaoming Ma ◽  
Ryo Shirotsuki ◽  
...  
Keyword(s):  
Trichoplusia Ni ◽  
Respiratory Infections ◽  
Recombinant Baculovirus ◽  
Immunofluorescence Assay ◽  
Human Metapneumovirus ◽  
Specific Method ◽  
Serum Samples ◽  
F Protein ◽  
Hmpv Infection ◽  
Well Correlation

ABSTRACT Human metapneumovirus (hMPV) has recently been identified as an etiological agent of acute respiratory infections. The hMPV fusion (F) protein has been indicated to be a major antigenic determinant that mediates effective neutralization and protection against hMPV infection. We developed a new immunofluorescence assay (IFA) using Trichoplusia ni (Tn5) insect cells infected with a recombinant baculovirus-expressing hMPV F protein (Bac-F IFA). A total of 200 serum samples from Japanese people 1 month to 41 years old were tested for immunoglobulin G antibodies to hMPV F protein by Bac-F IFA. The results were compared with those of the conventional IFA based on hMPV-infected LLC-MK2 cells (hMPV IFA). The titers obtained by the two IFAs correlated well (correlation coefficient of 0.88), and the concordance of seroreactivities between the two IFAs was 91% (κ = 0.76). For 192 of the 200 serum samples, the titers obtained by the Bac-F IFA were equal to or higher than those obtained by the hMPV IFA. These results indicated that the Bac-F IFA was more sensitive than the hMPV IFA and that the majority of the antibodies detected by the hMPV IFA reacted with the hMPV F protein. The Bac-F IFA is a more reliable, sensitive, and specific method for the detection of hMPV antibodies than is the hMPV IFA.

scholarly journals Molecular cloning, expression and regulatory activity of Gα11- and βγ-subunit-stimulated phospholipase C-β from avian erythrocytes

1996 ◽  
Vol 316 (2) ◽  
pp. 559-568 ◽  
Author(s):  
Gary L. WALDO ◽  
Andrew PATERSON ◽  
José L. BOYER ◽  
Robert A. NICHOLAS ◽  
T. Kendall HARDEN
Keyword(s):  
Amino Acid ◽  
Phospholipase C ◽  
G Protein ◽  
Recombinant Baculovirus ◽  
Recombinant Enzyme ◽  
Amino Acid Sequences ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Terminal Domain ◽  
Α Subunits

A turkey erythrocyte phospholipase C (PLC) has been instrumental in delineating the role of G-proteins in receptor-regulated inositol lipid signalling. This isoenzyme is uniquely regulated both by α-subunits of the Gq family and by G-protein βγ-subunits. A 4819 bp cDNA encoding this PLC has been cloned from a turkey erythrocyte cDNA library. The open reading frame of this cDNA encodes a 1211-amino-acid protein (calculated molecular mass 139050 Da) that contains amino acid sequences of 16 peptides sequenced from the turkey erythrocyte PLC. The predicted sequence of the turkey PLC shows considerable similarity with the sequences of previously cloned members of the PLC-β family, with the highest identity (71%) shared with PLC-β2 and lesser identities observed with PLC-β1 (49%), PLC-β3 (46%) and PLC-β4 (37%). The largest differences in sequence between the turkey PLC-β and other PLC-β isoenzymes occur in the C-terminal domain and in the region between the X- and Y-domains. The turkey isoenzyme and PLC-β2, which differ in their regulation by G-protein α-subunits, are only 44% similar across the approx. 400 amino acid residues of the C-terminal domain that has been implicated in αq activation of these proteins. Recombinant turkey PLC-β was purified to homogeneity following expression from a recombinant baculovirus in Sf9 insect cells. The immunoreactivity and mobility on SDS/PAGE of the recombinant enzyme were the same as observed with native turkey erythrocyte PLC-β. Moreover, the catalytic activities of the recombinant enzyme were indistinguishable from those of native turkey erythrocyte PLC-β in assays carried out in the presence of cholate and Ca2+, or in assays of activity after reconstitution with Gα11 or G-protein βγ-subunits. The turkey PLC-β was more sensitive to activation by Gα11 than was PLC-β2, and was more sensitive to activation by βγ-subunits than either PLC-β2 or PLC-β1.

scholarly journals Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

1991 ◽  
Vol 2 (2) ◽  
pp. 135-154 ◽  
Author(s):  
M A Lochrie ◽  
J E Mendel ◽  
P W Sternberg ◽  
M I Simon
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
G Proteins ◽  
Amino Acid Sequences ◽  
Alpha Subunit ◽  
Predicted Amino Acid Sequence ◽  
C Elegans ◽  
Alpha Subunits ◽  
G Protein Alpha Subunit ◽  
Protein Alpha Subunit

A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology.

scholarly journals Physicochemical properties of amino acid sequences of G-proteins for understanding GPCR-G-protein coupling

2006 ◽  
Vol 6 (1) ◽  
pp. 1-16 ◽  
Author(s):  
Ganga D. Ghimire ◽  
Kenichiro Imai ◽  
Fumitsugu Akazawa ◽  
Toshiyuki Tsuji ◽  
Masashi Sonoyama ◽  
...  
Keyword(s):  
Amino Acid ◽  
Physicochemical Properties ◽  
G Protein ◽  
G Proteins ◽  
Amino Acid Sequences ◽  
G Protein Coupling ◽  
Protein Coupling

scholarly journals Single Passage of Human Metapneumovirus in LLC-MK2 Cells Does Not Affect Viral Protein-Coding Capacity

2018 ◽  
Vol 6 (21) ◽  
Author(s):  
Simon Loevenich ◽  
Aleksandr Ianevski ◽  
Eneli Oitmaa ◽  
Denis E. Kainov ◽  
Marit W. Anthonsen
Keyword(s):  
Amino Acid ◽  
Viral Protein ◽  
Complete Genome ◽  
Human Metapneumovirus ◽  
Amino Acid Sequences ◽  
Paired Comparisons ◽  
Genome Sequences ◽  
Protein Coding ◽  
Single Passage ◽  
Coding Capacity

ABSTRACT Here, we report the complete genome sequences of human metapneumovirus (HMPV) prior to and after passaging in LLC-MK2 cells. Paired comparisons of the 13,335-nucleotide genomes revealed that the virus acquired the T10736C transition in its genome, which did not affect the amino acid sequences of HMPV proteins.

scholarly journals Phage Display Detection of Mimotopes that Are Shared Epitopes of Clinically and Epidemiologically Relevant Enterobacteria

2020 ◽  
Vol 8 (5) ◽  
pp. 780
Author(s):  
Armando Navarro ◽  
Delia Licona-Moreno ◽  
Alejandro Monsalvo-Reyes ◽  
Ulises Hernández-Chiñas ◽  
Carlos A. Eslava-Campos
Keyword(s):  
Amino Acid ◽  
Western Blot ◽  
Phage Display ◽  
Synthetic Peptides ◽  
Amino Acid Sequences ◽  
Serum Samples ◽  
E Coli ◽  
Peptide Mimotopes ◽  
Human Sera ◽  
Shared Epitopes

Background: Escherichia coli and Salmonella are etiologic agents of intestinal infections. A previous study showed the presence of shared epitopes between lipopolysaccharides (LPSs) of E. coli O157 and Salmonella. Aim: Using phage display, the aim of this study is to identify mimotopes of shared epitopes in different enterobacterial LPSs. Methods: We use anti-LPS IgG from E. coli O157 and Salmonella to select peptide mimotopes of the M13 phage. The amino acid sequence of the mimotopes is used to synthesize peptides, which are in turn used to immunize rabbits. The antibody response of the resulting sera against the LPSs and synthetic peptides (SPs) is analyzed by ELISA and by Western blot assays, indicating that LPS sites are recognized by the same antibody. In a complementary test, the reactions of human serum samples obtained from the general population against the SPs and LPSs are also analyzed. Results: From the last biopanning phase, sixty phagotopes are selected. The analysis of the peptide mimotope amino acid sequences shows that in 4 of them the S/N/A/PF motif is a common sequence. Antibodies from the sera of immunized rabbits with SP287/3, SP459/1, SP308/3, and SP073/14 react against both their own peptide and the different LPSs. The Western blot test shows a sera reaction against both the lateral chains and the cores of the LPSs. The analysis of the human sera shows a response against the SPs and LPSs. Conclusion: The designed synthetic peptides are mimotopes of LPS epitopes of Salmonella and E. coli that possess immunogenic capacity. These mimotopes could be considered for use in the design of vaccines against both enterobacteria.

scholarly journals IMMUNOLOGICAL INVESTIGATIONS OF HHV-6 U12 AND U51 ENCODED PROTEINS INVOLVEMENT IN AUTOIMMUNE THYROIDITIS DEVELOPMENT

2020 ◽  
Author(s):  
Alina Sultanova ◽  
Maksims Cistjakovs ◽  
Liba Sokolovska ◽  
Egils Cunskis ◽  
Modra Murovska
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
Autoimmune Thyroiditis ◽  
Synthetic Peptides ◽  
Human Herpesvirus ◽  
Amino Acid Sequences ◽  
G Protein Coupled Receptors ◽  
Igm Antibodies ◽  
Specific Igg ◽  
G Protein Coupled

AbstractHuman herpesvirus 6 (HHV-6) is a human pathogen with a wide cell tropism and many immunomodulating properties. HHV-6 has been linked to the development of multiple diseases, among them – autoimmune. Conflicting evidence implicates HHV-6 in autoimmune thyroiditis (AIT). HHV-6 contains two genes (U12 and U51) that encode putative homologues of human G-protein-coupled receptors (GPCR) like CCR1, CCR3 and CCR5. It has been shown that proteins encoded by HHV-6 U12 and U51 genes can be expressed on the surface of epithelial and some peripheral blood mononuclear cells populations, which makes them a potential cause for evoking autoimmunity.The aim of this study was to identify potentially immunogenic synthetic peptides derived from HHV-6 U12 and U51 amino acid sequences and to find evidences of the possible involvement of these proteins in AIT development. 62 AIT patients positive for HHV-6 infection were enrolled in this study. 30 different synthetic peptides designed from HHV-6 U12 and U51 proteins’ amino acid sequences, as well as, recombinant human CCR1, CCR3 and CCR5 proteins were used for suspension multiplex immunological assay (SMIA) to detect specific IgG, and IgM antibodies.HHV-6 peptide specific IgG and IgM antibodies were found in patient’s samples, with higher signals for IgM antibodies, which is indicative of reactivation and active HHV-6 infection. As well recombinant CCR1 and CCR5 showed high signals on IgM antibodies which is indicating on the presence of potential auto-antibodies against human G protein-coupled receptors. No cross reactivity between HHV-6 peptide specific antibodies and human recombinant CCR1, CCR3 and CCR5 was found, however, the possibility of cross-reactive autoantibodies specific for structural epitopes cannot be excluded.

scholarly journals Hemagglutination Inhibition (HAI) Antibody Landscapes after Vaccination with diverse H7 hemagglutinin (HA) proteins

2021 ◽  
Author(s):  
Hyesun Jang ◽  
Ted M Ross
Keyword(s):  
Amino Acid ◽  
Hemagglutination Inhibition ◽  
Lethal Dose ◽  
Antigenic Site ◽  
Glycosylation Site ◽  
Amino Acid Sequences ◽  
Serum Samples ◽  
Influenza Hemagglutinin ◽  
Amino Acid Mutations ◽  
Antigenic Profile

AbstractBackgroundA systemic evaluation of the antigenic differences of the H7 influenza hemagglutinin (HA) proteins, especially for the viruses isolated after 2016, are limited. The purpose of this study was to investigate the antigenic differences of major H7 strains with an ultimate aim to discover H7 HA proteins that can elicit protective receptor-blocking antibodies against co-circulating H7 influenza strains.MethodA panel of nine H7 influenza strains were selected from 3,633 H7 HA amino acid sequences identified over the past two decades (2000-2018). The sequences were expressed on the surface of virus like particles (VLPs) and used to vaccinate C57BL/6 mice. Serum samples were collected and tested for hemagglutination-inhibition (HAI) activity. The vaccinated mice were challenged with lethal dose of H7N9 virus, A/Anhui/1/2013.ResultsVLPs expressing the H7 HA antigens elicited broadly reactive antibodies each of the selected H7 HAs, except the A/Turkey/Italy/589/2000 (Italy/00) H7 HA. A putative glycosylation due to an A169T substitution in antigenic site B was identified as a unique antigenic profile of Italy/00. Introduction of the putative glycosylation site (H7 HA-A169T) significantly altered the antigenic profile of HA of the A/Anhui/1/2013 (H7N9) strain.ConclusionThis study identified key amino acid mutations that result in severe vaccine mismatches for future H7 epidemics. Future universal influenza vaccine candidates will need to focus on viral variants with these key mutations.

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