Missense mutations in the β3 subunit have a different impact on the expression and function between αIIbβ3 and αvβ3

Abstract αIIbβ3 and αvβ3 belong to the β3integrin subfamily. Although the β3 subunit is a key regulator for the biosynthesis of β3 integrins, it remains obscure whether missense mutations in β3 may induce the same defects in both αIIbβ3 and αvβ3. In this study, it is revealed that thrombasthenic platelets with a His280Pro mutation in β3, which is prevalent in Japanese patients with Glanzmann thrombasthenia, did contain significant amounts of αvβ3 (about 50% of control) using sensitive enzyme-linked immunosorbent assay. Expression studies showed that the His280Proβ3 mutation impaired αIIbβ3 expression but not αvβ3 expression in 293 cells. To extend these findings, the effects of several β3 missense mutations leading to an impaired αIIbβ3expression on αvβ3 function as well as expression was examined: Leu117Trp, Ser162Leu, Arg216Gln, Cys374Tyr, and a newly created Arg216Gln/Leu292Ser mutation. Leu117Trp and Cys374Tyr β3 mutations did impair αvβ3 expression, while Ser162Leu, Arg216Gln, and Arg216Gln/Leu292Ser mutations did not. With regard to ligand binding function, Ser162Leu mutation induced especially distinct effects between 2 β3 integrins: it markedly impaired ligand binding to αIIbβ3 but not to αvβ3 at all. These data clearly demonstrate that the biosynthesis and the ligand binding function of αIIbβ3 and those of αvβ3 are regulated in part by different mechanisms. Present data would be a clue to elucidate the regulatory mechanism of expression and function of β3 integrins.

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A naturally occurring Tyr143HisαIIb mutation abolishes αIIbβ3 function for soluble ligands but retains its ability for mediating cell adhesion and clot retraction: comparison with other mutations causing ligand-binding defects

Blood ◽

10.1182/blood-2002-07-2144 ◽

2003 ◽

Vol 101 (9) ◽

pp. 3485-3491 ◽

Cited By ~ 17

Author(s):

Teruo Kiyoi ◽

Yoshiaki Tomiyama ◽

Shigenori Honda ◽

Seiji Tadokoro ◽

Morio Arai ◽

...

Keyword(s):

Cell Adhesion ◽

Ligand Binding ◽

Binding Sites ◽

Clot Retraction ◽

Naturally Occurring ◽

Binding Function ◽

293 Cells ◽

Ligand Binding Sites ◽

Functional Defect ◽

And Function

The molecular basis for the interaction between a prototypic non–I-domain integrin, αIIbβ3, and its ligands remains to be determined. In this study, we have characterized a novel missense mutation (Tyr143His) in αIIb associated with a variant of Glanzmann thrombasthenia. Osaka-12 platelets expressed a substantial amount of αIIbβ3(36%-41% of control) but failed to bind soluble ligands, including a high-affinity αIIbβ3-specific peptidomimetic antagonist. Sequence analysis revealed that Osaka-12 is a compound heterozygote for a single 521T>C substitution leading to a Tyr143His substitution in αIIb and for the null expression of αIIb mRNA from the maternal allele. Given that Tyr143 is located in the W3 4-1 loop of the β-propeller domain of αIIb, we examined the effects of Tyr143His or Tyr143Ala substitution on the expression and function of αIIbβ3 and compared them with KO (Arg-Thr insertion between 160 and 161 residues of αIIb) and with the Asp163Ala mutation located in the same loop by using 293 cells. Each of them abolished the binding function of αIIbβ3 for soluble ligands without disturbing αIIbβ3 expression. Because immobilized fibrinogen and fibrin are higher affinity/avidity ligands for αIIbβ3, we performed cell adhesion and clot retraction assays. In sharp contrast to KO mutation and Asp163AlaαIIbβ3, Tyr143HisαIIbβ3-expressing cells still had some ability for cell adhesion and clot retraction. Thus, the functional defect induced by Tyr143HisαIIb is likely caused by its allosteric effect rather than by a defect in the ligand-binding site itself. These detailed structure–function analyses provide better understanding of the ligand-binding sites in integrins.

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Three Separate Glanzmann Thrombasthenia Mutants Affecting the αIIb β-Propeller Result in Production of Normally Stable Pro-αIIb, but Impaired Progression from Endoplasmic Reticulum to Golgi.

Blood ◽

10.1182/blood.v104.11.741.741 ◽

2004 ◽

Vol 104 (11) ◽

pp. 741-741 ◽

Cited By ~ 4

Author(s):

Everette J.R. Nelson ◽

Jihong Li ◽

Mammen Chandy ◽

Alok Srivastava ◽

Barry S. Coller

Keyword(s):

Cell Line ◽

Cell Lines ◽

Mutant Cell ◽

Immunoblot Analysis ◽

Missense Mutations ◽

Glanzmann Thrombasthenia ◽

Normal Value ◽

Hek 293 ◽

Expression Studies ◽

Surface Staining

Abstract The platelet αIIbβ3 (GPIIb/IIIa) integrin plays an important role in platelet function. Mutations in either αIIb or β3 can cause Glanzmann thrombasthenia by interfering with its biogenesis and/or function. We have previously reported three missense mutations in the β-propeller domain of αIIb, namely G128S, S287L and G357S, among three unrelated patients with Glanzmann thrombasthenia in southern India. Immunoblot analysis of platelet lysates showed no detectable αIIb in any of the patients and trace amounts of β3 in the latter two patients. Here, we describe further characterization of these mutations carried out by expression studies. Transfections were performed using HEK 293 cell lines with each of the following constructs: mock (vector only), αIIb only, normal αIIbβ3, and mutant αIIbβ3 (mutant αIIb G128S, S287L, or G357S co-transfected with normal β3). Stable cell lines were generated by G418 selection. While 52% of the normal αIIbβ3 cell lines bound antibody 10E5 (background = 5% positive), and the mean fluorescence intensity (MFI) of this subpopulation was 43 units, only 0, 2 and 8% of the mutant cell lines bound 10E5 above background, and the extent of binding among the positive cells was low (MFIs 14, 14 and 35). Immunoprecipitation of the cell lysates and immunoblotting showed no detectable mature αIIb in the G128S mutant. In contrast, when compared with results of normal αIIbβ3, the S287L mutant showed 7% of the normal value of mature αIIb, and the G357S mutant showed 24% of the normal value. Pulse-chase analysis showed maturation of pro-αIIb to mature, cleaved αIIb in the normal αIIbβ3 cell line over 0–24 h. All three mutants demonstrated pro-αIIb bands comparable to the normal pro-αIIb band, but there was no conversion to mature αIIb in the G128S mutant, and trace conversion to mature αIIb in the S287L and G357S mutants. The disappearance of pro-αIIb in the three mutants was very similar to that of the normal αIIbβ3 and the αIIb only cell lines, with about 50% lost after 4 h. Thus, the failure of the mutant pro-αIIb to progress to mature αIIb was not a result of rapid degradation of pro-αIIb. Immunofluorescence of the normal αIIbβ3 cell line demonstrated strong αIIb staining on the surface, with αIIb also co-localizing with an ER marker (calnexin) and a Golgi marker (mannosidase II). In contrast, while all three mutants showed αIIb co-localizing in the ER, the G128S mutant showed no co-localization in the Golgi apparatus, and the other two mutants showed only slight co-localization in the Golgi. Surface staining was undetectable in the G128S and S287L mutants and decreased in the G357S mutant. These experiments show that the three β-propeller mutations do not affect the production of pro-αIIb or its stability. Instead, they affect transport from ER to the Golgi, most likely as a result of reducing αIIbβ3 complex formation. Hence, the mutant pro-αIIb appears to be retained in the ER and then degraded.

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A Novel Mutation Glyl672→Arg in Type 2A and a Homozygous Mutation in Type 2B von Willebrand Disease

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650564 ◽

1996 ◽

Vol 76 (02) ◽

pp. 253-257 ◽

Cited By ~ 14

Author(s):

Takeshi Hagiwara ◽

Hiroshi Inaba ◽

Shinichi Yoshida ◽

Keiko Nagaizumi ◽

Morio Arai ◽

...

Keyword(s):

Missense Mutation ◽

Novel Mutation ◽

Point Mutations ◽

Japanese Patients ◽

Von Willebrand Disease ◽

Homozygous Mutation ◽

Missense Mutations ◽

Reaction Products ◽

Type 3 ◽

Von Willebrand

SummaryGenetic materials from 16 unrelated Japanese patients with von Willebrand disease (vWD) were analyzed for mutations. Exon 28 of the von Willebrand factor (vWF) gene, where point mutations have been found most frequent, was screened by various restriction-enzyme analyses. Six patients were observed to have abnormal restriction patterns. By sequence analyses of the polymerase chain-reaction products, we identified a homozygous R1308C missense mutation in a patient with type 2B vWD; R1597W, R1597Q, G1609R and G1672R missense mutations in five patients with type 2A; and a G1659ter nonsense mutation in a patient with type 3 vWD. The G1672R was a novel missense mutation of the carboxyl-terminal end of the A2 domain. In addition, we detected an A/C polymorphism at nucleotide 4915 with HaeIII. There was no particular linkage disequilibrium of the A/C polymorphism, either with the G/A polymorphism at nucleotide 4391 detected with Hphl or with the C/T at 4891 detected with BstEll.

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Matrix Metalloproteinases in Invertebrates

Protein and Peptide Letters ◽

10.2174/0929866527666200429110945 ◽

2020 ◽

Vol 27 (11) ◽

pp. 1068-1081

Author(s):

Xi Liu ◽

Dongwu Liu ◽

Yangyang Shen ◽

Mujie Huang ◽

Lili Gao ◽

...

Keyword(s):

Matrix Metalloproteinases ◽

Immune Responses ◽

Theoretical Basis ◽

Regulatory Mechanism ◽

Structure And Function ◽

Catalytic Domains ◽

Physiological Processes ◽

Disease Occurrence ◽

Complex Functions ◽

And Function

Matrix Metalloproteinases (MMPs) belong to a family of metal-dependent endopeptidases which contain a series of conserved pro-peptide domains and catalytic domains. MMPs have been widely found in plants, animals, and microorganisms. MMPs are involved in regulating numerous physiological processes, pathological processes, and immune responses. In addition, MMPs play a key role in disease occurrence, including tumors, cardiovascular diseases, and other diseases. Compared with invertebrate MMPs, vertebrate MMPs have diverse subtypes and complex functions. Therefore, it is difficult to study the function of MMPs in vertebrates. However, it is relatively easy to study invertebrate MMPs because there are fewer subtypes of MMPs in invertebrates. In the present review, the structure and function of MMPs in invertebrates were summarized, which will provide a theoretical basis for investigating the regulatory mechanism of MMPs in invertebrates.

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Alterations in ZnT1 expression and function lead to impaired intracellular zinc homeostasis in cancer

Cell Death Discovery ◽

10.1038/s41420-019-0224-0 ◽

2019 ◽

Vol 5 (1) ◽

Cited By ~ 2

Author(s):

Adrian Israel Lehvy ◽

Guy Horev ◽

Yarden Golan ◽

Fabian Glaser ◽

Yael Shammai ◽

...

Keyword(s):

Malignant Tumors ◽

Zinc Homeostasis ◽

Healthy Population ◽

Missense Mutations ◽

Protein Alignment ◽

Loss Of Function ◽

Intracellular Zinc ◽

Dismal Prognosis ◽

Zinc Levels ◽

And Function

Abstract Zinc is vital for the structure and function of ~3000 human proteins and hence plays key physiological roles. Consequently, impaired zinc homeostasis is associated with various human diseases including cancer. Intracellular zinc levels are tightly regulated by two families of zinc transporters: ZIPs and ZnTs; ZIPs import zinc into the cytosol from the extracellular milieu, or from the lumen of organelles into the cytoplasm. In contrast, the vast majority of ZnTs compartmentalize zinc within organelles, whereas the ubiquitously expressed ZnT1 is the sole zinc exporter. Herein, we explored the hypothesis that qualitative and quantitative alterations in ZnT1 activity impair cellular zinc homeostasis in cancer. Towards this end, we first used bioinformatics to analyze inactivating mutations in ZIPs and ZNTs, catalogued in the COSMIC and gnomAD databases, representing tumor specimens and healthy population controls, respectively. ZnT1, ZnT10, ZIP8, and ZIP10 showed extremely high rates of loss of function mutations in cancer as compared to healthy controls. Analysis of the putative functional impact of missense mutations in ZnT1-ZnT10 and ZIP1-ZIP14, using homologous protein alignment and structural predictions, revealed that ZnT1 displays a markedly increased frequency of predicted functionally deleterious mutations in malignant tumors, as compared to a healthy population. Furthermore, examination of ZnT1 expression in 30 cancer types in the TCGA database revealed five tumor types with significant ZnT1 overexpression, which predicted dismal prognosis for cancer patient survival. Novel functional zinc transport assays, which allowed for the indirect measurement of cytosolic zinc levels, established that wild type ZnT1 overexpression results in low intracellular zinc levels. In contrast, overexpression of predicted deleterious ZnT1 missense mutations did not reduce intracellular zinc levels, validating eight missense mutations as loss of function (LoF) mutations. Thus, alterations in ZnT1 expression and LoF mutations in ZnT1 provide a molecular mechanism for impaired zinc homeostasis in cancer formation and/or progression.

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Mice with cleavage-resistant N-cadherin exhibit synapse anomaly in the hippocampus and outperformance in spatial learning tasks

Molecular Brain ◽

10.1186/s13041-021-00738-1 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

M. Asada-Utsugi ◽

K. Uemura ◽

M. Kubota ◽

Y. Noda ◽

Y. Tashiro ◽

...

Keyword(s):

Memory Function ◽

Three Dimensional ◽

Excitatory Synapses ◽

Missense Mutations ◽

Glutamatergic Synapses ◽

Learning Tasks ◽

Transmission Electron ◽

Nmdar Activation ◽

And Function

AbstractN-cadherin is a homophilic cell adhesion molecule that stabilizes excitatory synapses, by connecting pre- and post-synaptic termini. Upon NMDA receptor (NMDAR) activation by glutamate, membrane-proximal domains of N-cadherin are cleaved serially by a-disintegrin-and-metalloprotease 10 (ADAM10) and then presenilin 1(PS1, catalytic subunit of the γ-secretase complex). To assess the physiological significance of the initial N-cadherin cleavage, we engineer the mouse genome to create a knock-in allele with tandem missense mutations in the mouse N-cadherin/Cadherin-2 gene (Cdh2R714G, I715D, or GD) that confers resistance on proteolysis by ADAM10 (GD mice). GD mice showed a better performance in the radial maze test, with significantly less revisiting errors after intervals of 30 and 300 s than WT, and a tendency for enhanced freezing in fear conditioning. Interestingly, GD mice reveal higher complexity in the tufts of thorny excrescence in the CA3 region of the hippocampus. Fine morphometry with serial section transmission electron microscopy (ssTEM) and three-dimensional (3D) reconstruction reveals significantly higher synaptic density, significantly smaller PSD area, and normal dendritic spine volume in GD mice. This knock-in mouse has provided in vivo evidence that ADAM10-mediated cleavage is a critical step in N-cadherin shedding and degradation and involved in the structure and function of glutamatergic synapses, which affect the memory function.

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Prediction of Functional Consequences of Missense Mutations in ANO4 Gene

International Journal of Molecular Sciences ◽

10.3390/ijms22052732 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2732

Author(s):

Nadine Reichhart ◽

Vladimir M. Milenkovic ◽

Christian H. Wetzel ◽

Olaf Strauß

Keyword(s):

Transmembrane Protein ◽

Functional Characterization ◽

Missense Mutations ◽

Mutant Proteins ◽

Functional Consequences ◽

New Perspective ◽

The Stability ◽

Dynamics Simulations ◽

And Function

The anoctamin (TMEM16) family of transmembrane protein consists of ten members in vertebrates, which act as Ca2+-dependent ion channels and/or Ca2+-dependent scramblases. ANO4 which is primarily expressed in the CNS and certain endocrine glands, has been associated with various neuronal disorders. Therefore, we focused our study on prioritizing missense mutations that are assumed to alter the structure and stability of ANO4 protein. We employed a wide array of evolution and structure based in silico prediction methods to identify potentially deleterious missense mutations in the ANO4 gene. Identified pathogenic mutations were then mapped to the modeled human ANO4 structure and the effects of missense mutations were studied on the atomic level using molecular dynamics simulations. Our data show that the G80A and A500T mutations significantly alter the stability of the mutant proteins, thus providing new perspective on the role of missense mutations in ANO4 gene. Results obtained in this study may help to identify disease associated mutations which affect ANO4 protein structure and function and might facilitate future functional characterization of ANO4.

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Consequences of two naturally occurring missense mutations in the structure and function of Bruton agammaglobulinemia tyrosine kinase

IUBMB Life ◽

10.1002/iub.1009 ◽

2012 ◽

Vol 64 (4) ◽

pp. 346-353 ◽

Cited By ~ 2

Author(s):

Alexander Vargas-Hernández ◽

Gabriela López-Herrera ◽

José L. Maravillas-Montero ◽

Felipe Vences-Catalán ◽

Dolores Mogica-Martínez ◽

...

Keyword(s):

Tyrosine Kinase ◽

Structure And Function ◽

Missense Mutations ◽

Naturally Occurring ◽

And Function

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Does Hepatic Hepcidin Play an Important Role in Exercise-Associated Anemia in Rats?

International Journal of Sport Nutrition and Exercise Metabolism ◽

10.1123/ijsnem.21.1.19 ◽

2011 ◽

Vol 21 (1) ◽

pp. 19-26 ◽

Cited By ~ 14

Author(s):

Yu-Qian Liu ◽

Yan-Zhong Chang ◽

Bin Zhao ◽

Hai-Tao Wang ◽

Xiang-Lin Duan

Keyword(s):

Regulatory Mechanism ◽

Iron Absorption ◽

White Blood Cells ◽

Strenuous Exercise ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Transcriptional Level ◽

Divalent Metal Transporter 1 ◽

Divalent Metal Transporter ◽

Sports Anemia

Some athletes are diagnosed as suffering from sports anemia because of iron deficiency, but the regulatory mechanism remains poorly understood. It is reported that hepcidin may provide a way to illuminate the regulatory mechanism of exercise-associated anemia. Here the authors investigate the hepcidin-involved iron absorption in exercise-associated anemia. Twelve male Wistar rats (300 ± 10 g) were randomly divided into 2 groups, 6 in a control group (CG) and 6 in an exercise group (EG, 5 wk treadmill exercise of different intensities with progressive loading). Serum samples were analyzed for circulating levels of IL-6 by means of enzyme-linked immunosorbent assay (ELISA). The expression of hepatic hepcidin mRNA was examined by real-time polymerase chain reaction analysis. The protein levels of divalent metal transporter 1 (DMT1), ferroportin1 (FPN1), and heme-carrier protein 1 (HCP1) of duodenum epithelium were examined by Western blot. The results showed that the amount of iron and ferritin in serum were lower in EG than in CG (p < .05). The levels of IL-6 and white blood cells were greater in EG than in CG (p < .01). The expression of DMT1, HCP1, and FPN1 was significantly lower in EG than in CG (p < .01). The mRNA expressions of hepatic hepcidin and hemojuvelin in skeletal muscle were remarkably higher in EG than in CG. The data indicated that inflammation was induced by strenuous exercise, and as a result, the transcriptional level of the hepatic hepcidin gene was increased, which further inhibited the expression of iron-absorption proteins and led to exercise-associated anemia.

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