scholarly journals Analysis of Integrin αIIb Subunit Dynamics Reveals Long-Range Effects of Missense Mutations on Calf Domains

2022 ◽  
Vol 23 (2) ◽  
pp. 858
Author(s):  
Sali Anies ◽  
Vincent Jallu ◽  
Julien Diharce ◽  
Tarun J. Narwani ◽  
Alexandre G. de Brevern
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
3D Structure ◽  
Single Amino Acid ◽  
Structural Basis ◽  
Missense Mutations ◽  
Structural Alphabet ◽  
Platelet Surface ◽  
Integrin Αiibβ3 ◽  
The Impact

Integrin αIIbβ3, a glycoprotein complex expressed at the platelet surface, is involved in platelet aggregation and contributes to primary haemostasis. Several integrin αIIbβ3 polymorphisms prevent the aggregation that causes haemorrhagic syndromes, such as Glanzmann thrombasthenia (GT). Access to 3D structure allows understanding the structural effects of polymorphisms related to GT. In a previous analysis using Molecular Dynamics (MD) simulations of αIIb Calf-1 domain structure, it was observed that GT associated with single amino acid variation affects distant loops, but not the mutated position. In this study, experiments are extended to Calf-1, Thigh, and Calf-2 domains. Two loops in Calf-2 are unstructured and therefore are modelled expertly using biophysical restraints. Surprisingly, MD revealed the presence of rigid zones in these loops. Detailed analysis with structural alphabet, the Proteins Blocks (PBs), allowed observing local changes in highly flexible regions. The variant P741R located at C-terminal of Calf-1 revealed that the Calf-2 presence did not affect the results obtained with isolated Calf-1 domain. Simulations for Calf- 1+ Calf-2, and Thigh + Calf-1 variant systems are designed to comprehend the impact of five single amino acid variations in these domains. Distant conformational changes are observed, thus highlighting the potential role of allostery in the structural basis of GT.

Impaired Platelet Function in Sept8-Deficient Mice In Vitro

2020 ◽  
Author(s):  
Kerstin Jurk ◽  
Katharina Neubauer ◽  
Victoria Petermann ◽  
Elena Kumm ◽  
Barbara Zieger
Keyword(s):  
Platelet Function ◽  
Thrombin Generation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Integrin Αiibβ3 ◽  
Glycoprotein Vi ◽  
Fibrinogen Receptor ◽  
Static Conditions ◽  
The Impact

AbstractSeptins (Septs) are a widely expressed protein family of 13 mammalian members, recognized as a unique component of the cytoskeleton. In human platelets, we previously described that SEPT4 and SEPT8 are localized surrounding α-granules and move to the platelet surface after activation, indicating a possible role in platelet physiology. In this study, we investigated the impact of Sept8 on platelet function in vitro using Sept8-deficient mouse platelets. Deletion of Sept8 in mouse platelets caused a pronounced defect in activation of the fibrinogen receptor integrin αIIbβ3, α-granule exocytosis, and aggregation, especially in response to the glycoprotein VI agonist convulxin. In contrast, δ-granule and lysosome exocytosis of Sept8-deficient platelets was comparable to wild-type platelets. Sept8-deficient platelet binding to immobilized fibrinogen under static conditions was diminished and spreading delayed. The procoagulant activity of Sept8-deficient platelets was reduced in response to convulxin as determined by lactadherin binding. Also thrombin generation was decreased relative to controls. Thus, Sept8 is required for efficient integrin αIIbβ3 activation, α-granule release, platelet aggregation, and contributes to platelet-dependent thrombin generation. These results revealed Sept8 as a modulator of distinct platelet functions involved in primary and secondary hemostatic processes.

P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency

2020 ◽  
Vol 168 (5) ◽  
pp. 557-567
Author(s):  
Wanitcha Rachadech ◽  
Yusuke Kato ◽  
Rabab M Abou El-Magd ◽  
Yuji Shishido ◽  
Soo Hyeon Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Active Site ◽  
Structural Changes ◽  
Catalytic Efficiency ◽  
Acid Oxidase ◽  
Wild Type ◽  
Amino Acid Oxidase ◽  
Adenine Ring ◽  
The Impact

Abstract Human D-amino acid oxidase (DAO) is a flavoenzyme that is implicated in neurodegenerative diseases. We investigated the impact of replacement of proline with leucine at Position 219 (P219L) in the active site lid of human DAO on the structural and enzymatic properties, because porcine DAO contains leucine at the corresponding position. The turnover numbers (kcat) of P219L were unchanged, but its Km values decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lower Ki value (0.7–0.9 µM) compared with wild-type (1.2–2.0 µM). Crystal structure of P219L in complex with flavin adenine dinucleotide (FAD) and benzoate at 2.25 Å resolution displayed conformational changes of the active site and lid. The distances between the H-bond-forming atoms of arginine 283 and benzoate and the relative position between the aromatic rings of tyrosine 224 and benzoate were changed in the P219L complex. Taken together, the P219L substitution leads to an increase in the catalytic efficiency and binding affinity for substrates/inhibitors due to these structural changes. Furthermore, an acetic acid was located near the adenine ring of FAD in the P219L complex. This study provides new insights into the structure–function relationship of human DAO.

Adhesive and Signaling Properties of a Naturally Occurring Allele of Glycoprotein IIIa With an Amino Acid Substitution Within the Ligand Binding Domain—The Pena/PenbPlatelet Alloantigenic Epitopes

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3260-3267 ◽  
Author(s):  
Ronggang Wang ◽  
Peter J. Newman
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Amino Acid Substitution ◽  
Polyclonal Antibodies ◽  
Chinese Hamster ◽  
Binding Domain ◽  
Integrin Subunit ◽  
Adhesive Properties ◽  
Platelet Surface ◽  
Glycoprotein Iiia

Platelet membrane glycoprotein IIIa (GPIIIa) is the most polymorphic integrin subunit in man, with at least seven recognized allelic isoforms present in the human gene pool. Whether these allelic variants of the GPIIb-IIIa complex differ in the ability to interact with the adhesive ligand fibrinogen (Fg) is still unknown. Since the Pena and Penb allelic forms of GPIIIa are distinguished by a single Arg143Gln amino acid substitution within the RGD binding domain of GPIIIa and anti-Pena human alloantibodies have been shown to bind GPIIb-IIIa on the platelet surface and inhibit ADP-induced platelet aggregation, we expressed both forms of this integrin in Chinese hamster ovary (CHO) cells and examined the relative adhesive properties. Both allelic forms of GPIIb-IIIa were expressed on the cell surface and were recognized by a well-characterized panel of murine and human monoclonal and polyclonal antibodies. Like Pena, the Penb form of GPIIb-IIIa could undergo conformational changes in response to RGD peptide binding, and could be induced by activating antibodies to bind Fg and the Fg mimetic antibody P1-55. The binding affinity for Fg of the Pena form of the GPIIb-IIIa complex was not significantly different from that of the Penb form, nor was its ability to signal to focal adhesion kinase, suggesting that Arg143Gln polymorphism has little or no effect on integrin function. Examination of the functional consequences of other integrin polymorphisms may be necessary to determine whether they constitute a risk factor for thrombosis or hemorrhage. © 1998 by The American Society of Hematology.

scholarly journals Epistatic interactions can moderate the antigenic effect of substitutions in hemagglutinin of influenza H3N2 virus

2018 ◽  
Author(s):  
Björn F. Koel ◽  
David F. Burke ◽  
Stefan van der Vliet ◽  
Theo M. Bestebroer ◽  
Guus F. Rimmelzwaan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Context Dependence ◽  
Single Amino Acid ◽  
H3n2 Virus ◽  
Amino Acid Substitutions ◽  
Epistatic Interactions ◽  
Context Dependent ◽  
The Impact ◽  
Influenza H3n2

AbstractWe previously showed that single amino acid substitutions at seven positions in hemagglutinin determined major antigenic change of influenza H3N2 virus. Here, the impact of two such substitutions was tested in eleven representative H3 hemagglutinins to investigate context-dependence effects. The antigenic effect of substitutions introduced at hemagglutinin position 145 was fully independent of the amino acid context of the representative hemagglutinins. Antigenic change caused by substitutions introduced at hemagglutinin position 155 was variable and context-dependent. Our results suggest that epistatic interactions with contextual amino acids in the hemagglutinin can moderate the magnitude of antigenic change.

scholarly journals The Impacts of G4S Mutation on N-glycosylation and conformation of the Human Coagulation Factor IX GLA domain: In silico and In vitro Analysis

2021 ◽  
Author(s):  
Fahimeh Ghasemi ◽  
Mina Maddah ◽  
Hourieh Kalhor ◽  
Mohsen Khorashadizadeh ◽  
Alireza Zomorodipour
Keyword(s):  
Amino Acid ◽  
Coagulation Factor ◽  
Glycosylation Site ◽  
Factor Ix ◽  
Hemophilia B ◽  
Phospholipid Bilayer ◽  
Single Amino Acid ◽  
Missense Mutations ◽  
Coagulation Factor Ix ◽  
Gla Domain

Abstract Missense mutations are the most prevalent form of mutation in hemophilia B patients. These alterations may result in the creation of novel and non-native N-glycosylation sites (Asn-X-Ser/Thr) through single amino acid substitutions. The pathogenic mechanisms of N-glycosylation mutations in hemophilia B patients have not been extensively studied yet. By survey among known missense mutations, we found only one N-glycosylation mutation in the γ-carboxyglutamic-rich (GLA) domain of the human coagulation factor IX (hFIX). This mutation that was reported in patients with mild and moderate hemophilia B, is caused by G4S amino acid substitution. To investigate the possibility of glycan attachment to the novel N-glycosylation site in G4S-mutant hFIX and the occurrence of hyperglycosylation, site-directed mutagenesis was applied to introduce the selected mutation into the coding sequence of the hFIX. The nucleotide sequences of the both native and G4S-mutant hFIX were separately cloned into the pcDNA3.1 expression plasmid and transiently expressed in HEK293T cells. Our results from gradient SDS-PAGE and western blotting analysis of the both recombinant native and mutant hFIX demonstrated no glycan attachment to the new N-glycosylation site in the G4S-mutant hFIX. Molecular dynamics (MD) simulation was also conducted to provide atomistic insights into structure and behavior of the native and G4S-mutant GLA domains in the both free and membrane-bound states. The results revealed that the mutation slightly affected the dynamic behavior of the mutant GLA domain. The conformational analysis proved that the native GLA domain had less fluctuation and more stability than the mutant GLA domain. The slight conformational changes may influence the binding capacity and interaction of the mutant GLA domain to phospholipid bilayer which is necessary for coagulation activity of the hFIX. These findings were in accordance with the nature of the G4S mutation which causes mild hemophilia B.

Single amino acid substitution in human platelet glycoprotein Ibβ is responsible for the formation of the platelet-specific alloantigen Iya

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1849-1855 ◽  
Author(s):  
Ulrich J. H. Sachs ◽  
Volker Kiefel ◽  
Micaela Böhringer ◽  
Vahid Afshar-Kharghan ◽  
Hartmut Kroll ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Human Platelet ◽  
Population Analysis ◽  
Severe Case ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Platelet Glycoprotein ◽  
Nucleotide Sequence Analysis ◽  
Platelet Surface

We recently described a new low-frequency platelet alloantigen on the human platelet glycoprotein (GP) Ib-IX complex, termed Iya, which was implicated in a severe case of neonatal alloimmune thrombocytopenia. Immunoprecipitation studies with trypsin-treated platelets indicated that the Iyaalloantigenic determinants are formed by the membrane-associated remnant moiety of GP Ib (GP Ibr) together with GP Ibβ and GP IX. To elucidate the molecular basis underlying the Iya alloantigen, we amplifiedGPIbr, GPIbβ, andGPIX genes by polymerase chain reaction (PCR). Nucleotide-sequence analysis of these 3 genes showed a G to A transition at position 141 on GPIbβ gene in a subject positive for Iya. This transition resulted in a Gly15Glu dimorphism on the N-terminal domain ofGPIbβ. This finding was confirmed by genotyping analysis of 6 Iya-positive subjects by restriction fragment length polymorphism (RFLP) studies using NarI endonuclease. In 300 randomly selected healthy blood donors, one Iya-positive individual was found. Phenotypes determined by monoclonal antibody-specific immobilization of platelet antigens assay and genotypes determined by RFLP were identical in this population. Analysis of Iya-positive platelets showed that the point mutation affected neither the degree of surface expression nor the function of the GP Ib-GP Ibβ-IX complex on the platelet surface. Transient expression of the GP Ib-IX complex in CHO cells using wild-type GP Ibβ (Gly15) or mutant GP Ibβ (Glu15) allowed us to demonstrate that this single amino acid substitution is sufficient to induce Iya epitope(s).

scholarly journals Analysis of Large-Scale Mutagenesis Data To Assess the Impact of Single Amino Acid Substitutions

Genetics ◽  
2017 ◽  
Vol 207 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Vanessa E. Gray ◽  
Ronald J. Hause ◽  
Douglas M. Fowler
Keyword(s):  
Amino Acid ◽  
Large Scale ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
The Impact

scholarly journals A Single Amino Acid in the Reverse Transcriptase Domain of Hepatitis B Virus Affects Virus Replication Efficiency

2001 ◽  
Vol 75 (23) ◽  
pp. 11827-11833 ◽  
Author(s):  
Xu Lin ◽  
Zheng-Hong Yuan ◽  
Li Wu ◽  
Jian-Ping Ding ◽  
Yu-Mei Wen
Keyword(s):  
Amino Acid ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Single Amino Acid ◽  
Structural Basis ◽  
Replication Efficiency ◽  
P Gene ◽  
B Virus ◽  
The Difference

ABSTRACT To explore functional domains in the hepatitis B virus (HBV) polymerase, two naturally occurring HBV isolates (56 and 2-18) with 98.7% nucleic acid sequence homology but different replication efficiencies were studied. After transfection into HepG2 cells, HBV DNA isolated from intracellular virus core particles was much higher in 56-transfected cells than in cells transfected with 2-18. The structural basis for the difference in replication efficiency between these two isolates was studied by functional domain gene substitution. The complete polymerase (P) gene and its gene segments coding for the terminal protein (TP), spacer (SP), reverse transcriptase (RT), and RNase H in 2-18 were separately replaced with their counterparts from 56 to construct full-length chimeric genomes. Cell transfection analysis revealed that substitution of the complete P gene of 2-18 with the P gene from 56 slightly enhanced viral replication. The only chimeric genome that regained the high replication efficiency of the original 56 isolate was the one with substitution of the RT gene of 2-18 with that from 56. Within the RT region, amino acid differences between isolates 2-18 and 56 were located at positions 617 (methionine versus leucine), 652 (serine versus proline), and 682 (valine versus leucine). Point mutation identified amino acid 652 as being responsible for the difference in replication efficiency. Homologous modeling studies of the HBV RT domain suggest that the mutation of residue 652 from proline to serine might affect the conformation of HBV RT which interacts with the template-primer, leading to impaired polymerase activity.

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