STRUCTURE-FUNCTION RELATIONSHIPS IN ABNORMAL FIBRINOGEN WITH Bβ14 ARG→CYS SUBSTITUTION: FIBRINOGENS SEATTLE I AND CHRISTCHURCH II

1987 ◽  
Author(s):  
H Kaudewitz ◽  
A Henschen ◽  
H Pirkle ◽  
D Heaton ◽  
J Soria ◽  
...  
Keyword(s):  
Amino Acid ◽  
Structure Function ◽  
Amino Acid Substitution ◽  
Cleavage Site ◽  
Disulfide Bridge ◽  
Thrombin Cleavage ◽  
Parallel Arrangement ◽  
Fibrinogen Molecule

Genetically abnormal, dysfunctional fibrinogen variants may be used as unique models for studies of structure-function relationships both in vitro and in vivo. Out of the over kO so far structurally elucidated abnormal fibrinogens only 4 have an amino acid substitution in the Bg-chain. These variants are named Fibrinogen Pontoise, Nev York I, Christchurch II and Seattle I.Fibrinogens Seattle I and Christchurch II are slow-clotting fibrinogens which on thrombin-treatment release only half the normal amount of fibrinopeptide B and therefore were expected to contain an amino acid substitution close to the thrombin cleavage site in the Bβ-chain. In order to sequence the abnormal Bg-chains the fibrinogens were cleaved with thrombin and cyanogen bromide. The abnormal Bβ-chain components were isolated from the mercapto-lysed-pyridylethylated N-terminal disulfide knots. After pyroglu-tamyl-peptidase digestion the Bβ 14 Arg→Cys substitutions could be demonstrated for both variants by direct N-terminal sequence analysis. The form of the cyst(e)ine residue was determined by amino acid analysis of the alkylated native fibrinogen. As no alkylated cysteine was detected it was concluded that Bβ 14 Cys participates in a disulfide bridge.Fibrinogens Seattle I and Christchurch II are the first two elucidated fibrinogens with substitutions at the Bβ-chain thrombin cleavage site. Surprisingly, both the thrombin and Reptilase times are prolonged. It may be assumed that the half-cystine residues in position 14 of the two Bβ-chains within one fibrinogen molecule are disulfide-linked to each other, in an analogous way to that already established for the half-cystine residues in position 16 of the Aα-chains of several abnormal fibrinogens.This additional disulfide bridge might change the conformation and charge in the N-terminal region sufficiently to explain the prolonged clotting times. Furthermore, this bridge would provide the evidence for the parallel arrangement of the Bβ-chains at the fibrinogen N-terminus in a similar way as previously shown for the Aα-chains.

A novel polymorphism of human gene has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo

2004 ◽  
Vol 76 (6) ◽  
pp. 519-527 ◽  
Author(s):  
T FUKAMI ◽  
M NAKAJIMA ◽  
R YOSHIDA ◽  
Y TSUCHIYA ◽  
Y FUJIKI ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Amino Acid Substitution ◽  
Human Gene

DIRECTED MUTAGENESIS IN THE STUDY OF THE REQUIREMENTS FOR FACTOR VIII ACTIVITY IN VITRO AND IN VIVO

1987 ◽  
Author(s):  
Randal J Kaufman ◽  
Debra D Pittman ◽  
Louise C Wasley ◽  
W Barry Foster ◽  
Godfrey W Amphlett ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Cleavage Site ◽  
Factor Xa ◽  
Cleavage Sites ◽  
Thrombin Cleavage ◽  
Terminal Fragment ◽  
Cofactor Activity

Factor VIII is a high molecular weight plasma glycoprotein that functions in the blood clotting cascade as the cofactor for factor DCa proteolytic activation of factor X. Factor VIII does not function proteolytically in this reaction hut itself can be proteolytically activated by other coagulation enzymes such as factor Xa and thrombin. In the plasma, factor VIII exists as a 200 kDa amino-terminal fragment in a metal ion stabilized complex with a 76 kDa carboxy-terminal fragment. The isolation of the cENA for human factor VIII provided the deduced primary amino acid sequence of factor VIIT and revealed three distinct structural domains: 1) a triplicated A domain of 330 amino acids which has homology to ceruloplasmin, a plasma copper binding protein, 2) a duplicated C domain of 150 amino acids, and 3) a unique B domain of 980 amino acids. These domains are arranged as shown below. We have previously reported the B domain is dispensible far cofactor activity in vitro (Toole et al. 1986 Proc. Natl. Acad 5939). The in vivo efficacy of factor VIII molecules harboring the B domain deletion was tested by purification of the wildtype and modified forms and infusion into factor VIII deficient, hemophilic, dogs. The wildtype and the deleted forms of recombinant derived factor VIII exhibited very similar survival curves (Tl/2 = 13 hrs) and the cuticle bleeding times suggested that both preparations appeared functionally equivalent. Sepharose 4B chromatography indicated that both factor VIII molecules were capable of binding canine plasma vWF.Further studies have addressed what cleavages are necessary for activation of factor VIII. The position of the thrombin, factor Xa, and activated protein C (AFC) cleavage sites within factor VIII are presented below, site-directed ENA medicated mutagenesis has been performed to modify the arginine at the amino side of each cleavagesite to an soleucine. In all cases this modification resulted in molecules that were resistant to cleavage by thrombin at the modified site. Modification of the thrombin cleavage sites at 336 and 740 and modification of the factor Xa cleavage site at 1721 resulted in no loss of cofactor activity. Modification of the thrombin cleavage site at either 372 or 1689 destroyed oofactor activity. Modification of the thrombin cleavage site at 336 resulted in a factor VIII having an increased activity, possibly due to resistance to inactivation. These results suggest the requirement of cleavage at residues 372 and 1689 for cofactor activity.

scholarly journals Virus Susceptibility Analyses from a Phase IV Clinical Trial of Inhaled Zanamivir Treatment in Children Infected with Influenza

2013 ◽  
Vol 57 (4) ◽  
pp. 1677-1684 ◽  
Author(s):  
Phillip J. Yates ◽  
Nalini Mehta ◽  
Joseph Horton ◽  
Margaret Tisdale
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Drug Pressure ◽  
Receptor Binding Site ◽  
Virus Susceptibility ◽  
Susceptibility Data

ABSTRACTA zanamivir postapproval efficacy study was conducted in children (n= 279) in Japan during three influenza seasons. Pharyngeal swab specimens (n= 714) were obtained for detailed resistance analysis. From 371 cultured viruses, 3 viruses (A/H1N1) from two subjects showed reduced susceptibility to zanamivir at day 1 (before treatment), 1 had an N74S amino acid substitution (fold shift, 46), and 2 (day 1 and day 2) had a Q136K amino acid substitution (fold shifts, 292 and 301). Q136K was detected only in cultured virus and not in the swab. From the remaining 118 cultured viruses obtained during or after treatment with zanamivir, no shifts in virus susceptibility were detected. Neuraminidase (NA) population sequencing showed that viruses from 12 subjects had emergent amino acid substitutions, but 3 with susceptibility data were not zanamivir resistant. The remainder may be natural variants. Further analysis is planned. Hemagglutinin (HA) sequencing showed that viruses from 20 subjects had 9 HA amino acid substitutions that were previously implicated in resistance to neuraminidase inhibitors inin vitroassays or that were close to the receptor binding site. Their role inin vivoresistance appears to be less important but is not well understood. NA clonal sequence analysis was undertaken to determine if minority species of resistant viruses were present. A total of 1,682 clones from 90 subjects were analyzed. Single clones from 12 subjects contained amino acid substitutions close to the NA active site. It is unclear whether these single amino acid substitutions could have been amplified after drug pressure or are just chance mutations introduced during PCR.

scholarly journals An amino acid substitution (V3I) in the Japanese encephalitis virus NS4A protein increases its virulence in mice, but not its growth rate in vitro

2011 ◽  
Vol 92 (7) ◽  
pp. 1601-1606 ◽  
Author(s):  
Yukie Yamaguchi ◽  
Yoko Nukui ◽  
Shigeru Tajima ◽  
Reiko Nerome ◽  
Fumihiro Kato ◽  
...  
Keyword(s):  
Amino Acid ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Amino Acid Substitution ◽  
Encephalitis Virus ◽  
Virus Growth ◽  
High Virulence ◽  
Isoleucine Residue

Our previous studies have shown that the Japanese encephalitis virus (JEV) strain Mie/40/2004 is the most virulent of the strains isolated by us in Japan from 2002 to 2004. Comparison of the amino acid sequence of Mie/40/2004 with those of low-virulence strains revealed that an isoleucine residue at position 3 of the Mie/40/2004 NS4A protein may increase viral pathogenicity. A recombinant virus with a single valine-to-isoleucine substitution (V3I) at position 3 in the low-virulence Mie/41/2002 background (rJEV-Mie41-NS4AV3I) exhibited increased virulence in mice compared with the Mie/41/2002 parent strain. The V3I mutation did not affect virus growth in several cell lines. These results demonstrate that the isoleucine at position 3 in the NS4A protein of Mie/40/2004 is responsible for its high virulence in vivo. This is the first report to show that an amino acid substitution in a flavivirus NS4A protein alters viral pathogenicity in mice.

scholarly journals Murine Coronavirus Evolution In Vivo: Functional Compensation of a Detrimental Amino Acid Substitution in the Receptor Binding Domain of the Spike Glycoprotein

2005 ◽  
Vol 79 (12) ◽  
pp. 7629-7640 ◽  
Author(s):  
Sonia Navas-Martin ◽  
Susan T. Hingley ◽  
Susan R. Weiss
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Receptor Binding ◽  
Amino Acid Substitution ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Spike Glycoprotein ◽  
Functional Compensation

ABSTRACT Murine coronavirus A59 strain causes mild to moderate hepatitis in mice. We have previously shown that mutants of A59, unable to induce hepatitis, may be selected by persistent infection of primary glial cells in vitro. These in vitro isolated mutants encoded two amino acids substitutions in the spike (S) gene: Q159L lies in the putative receptor binding domain of S, and H716D, within the cleavage signal of S. Here, we show that hepatotropic revertant variants may be selected from these in vitro isolated mutants (Q159L-H716D) by multiple passages in the mouse liver. One of these mutants, hr2, was chosen for more in-depth study based on a more hepatovirulent phenotype. The S gene of hr2 (Q159L- R654H -H716D- E1035D ) differed from the in vitro isolates (Q159L-H716D) in only 2 amino acids (R654H and E1035D). Using targeted RNA recombination, we have constructed isogenic recombinant MHV-A59 viruses differing only in these specific amino acids in S (Q159L-R654H-H716D-E1035D). We demonstrate that specific amino acid substitutions within the spike gene of the hr2 isolate determine the ability of the virus to cause lethal hepatitis and replicate to significantly higher titers in the liver compared to wild-type A59. Our results provide compelling evidence of the ability of coronaviruses to rapidly evolve in vivo to highly virulent phenotypes by functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein.

scholarly journals Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair

2017 ◽  
Vol 114 (19) ◽  
pp. 4930-4935 ◽  
Author(s):  
Jochen Genschel ◽  
Lyudmila Y. Kadyrova ◽  
Ravi R. Iyer ◽  
Basanta K. Dahal ◽  
Farid A. Kadyrov ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mismatch Repair ◽  
Amino Acid Substitution ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Endonuclease Domain ◽  
Substitution Mutations ◽  
Proliferating Cell

Eukaryotic MutLα (mammalian MLH1–PMS2 heterodimer; MLH1–PMS1 in yeast) functions in early steps of mismatch repair as a latent endonuclease that requires a mismatch, MutSα/β, and DNA-loaded proliferating cell nuclear antigen (PCNA) for activation. We show here that human PCNA and MutLα interact specifically but weakly in solution to form a complex of approximately 1:1 stoichiometry that depends on PCNA interaction with the C-terminal endonuclease domain of the MutLα PMS2 subunit. Amino acid substitution mutations within a PMS2 C-terminal 721QRLIAP motif attenuate or abolish human MutLα interaction with PCNA, as well as PCNA-dependent activation of MutLα endonuclease, PCNA- and DNA-dependent activation of MutLα ATPase, and MutLα function in in vitro mismatch repair. Amino acid substitution mutations within the corresponding yeast PMS1 motif (723QKLIIP) reduce or abolish mismatch repair in vivo. Coupling of a weak allele within this motif (723AKLIIP) with an exo1Δ null mutation, which individually confer only weak mutator phenotypes, inactivates mismatch repair in the yeast cell.

scholarly journals Biochemical and Structural Characterization of Quizalofop-Resistant Wheat Acetyl-Coa Carboxylase

2021 ◽  
Author(s):  
Raven Bough ◽  
Franck Dayan
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Specific Activity ◽  
Reference Sequence ◽  
Cross Resistance ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Nucleotide Mutation

Abstract A novel nucleotide mutation in ACC1 resulting in an alanine to valine amino acid substitution in acetyl-CoA carboxylase (ACCase) at position 2004 of the Alopecurus myosuroides reference sequence (A2004V) imparts quizalofop resistance in wheat. Genotypes endowed with one or two homozygous mutant ACC1 homoelogs are 7- and 68-fold more resistant to quizalofop than a wildtype variety in greenhouse experiments, respectively. In vitro assays of ACCase activities in protein extracts from these varieties reveal a 3.8- and 39.4-fold increase in resistance to quizalofop in the single and double-mutants relative to the wildtype. The A2004V mutation does not alter the specific activity of wheat ACCase, suggesting that ACCase mutants retain their normal catalytic functions. Modeling of wildtype and quizalofop-resistant wheat ACCase demonstrates that the A2004V amino acid substitution causes a reduction in the volume of the binding pocket that hinders quizalofop’s interaction with ACCase. Docking studies confirm that the mutation reduces the binding affinity of quizalofop. Interestingly, the models suggest that the A2004V mutation does not affect haloxyfop binding. Follow up in vivo and in vitro experiments reveal that the mutation, in fact, imparts negative cross-resistance to haloxyfop, with quizalofop-resistant varieties exhibiting more sensitivity to haloxyfop than the wildtype variety.

scholarly journals A Region in the Bacillus subtilisTranscription Factor Spo0A That Is Important for spoIIGPromoter Activation

1998 ◽  
Vol 180 (14) ◽  
pp. 3578-3583 ◽  
Author(s):  
Cindy M. Buckner ◽  
Ghislain Schyns ◽  
Charles P. Moran
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Amino Acid Substitution ◽  
Dna Binding Protein ◽  
Single Amino Acid ◽  
Promoter Activation ◽  
Additional Amino Acid

ABSTRACT Spo0A is a DNA binding protein in Bacillus subtilisrequired for the activation of spoIIG and other promoters at the onset of endospore formation. Activation of some of these promoters may involve interaction of Spo0A and the ςAsubunit of RNA polymerase. Previous studies identified two single-amino-acid substitutions in ςA, K356E and H359R, that specifically impaired Spo0A-dependent transcription in vivo. Here we report the identification of an amino acid substitution in Spo0A (S231F) that suppressed the sporulation deficiency due to the H359R substitution in ςA. We also found that the S231F substitution partially restored use of the spoIIG promoter by the ςA H359R RNA polymerase in vitro. Alanine substitutions in the 231 region of Spo0A revealed an additional amino acid residue important for spoIIG promoter activation, I229. This amino acid substitution in Spo0A did not affect repression of abrB transcription, indicating that the alanine-substituted Spo0A was not defective in DNA binding. Moreover, the alanine-substituted Spo0A protein activated the spoIIApromoter; therefore, this region of Spo0A is probably not required for Spo0A-dependent, ςH-directed transcription. These and other results suggest that the region of Spo0A near position 229 is involved in ςA-dependent promoter activation.

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