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

To study the structure and function of the pH-regulated receptor tyrosine kinase insulin receptor-related receptor (IRR), а member of the insulin receptor family, we obtained six mouse monoclonal antibodies against the recombinant IRR ectodomain. These antibodies were characterized in experiments with exogenously expressed full-length IRR by Western blotting, immunoprecipitation, and immunocytochemistry analyses. Utilizing a previously obtained set of IRR/IR chimeras with swapped small structural domains and point amino acid substitutions, we mapped the binding sites of the obtained antibodies in IRR. Five of them showed specific binding to different IRR domains in the extracellular region, while one failed to react with the full-length receptor. Unexpectedly, we found that 4D5 antibody can activate IRR at neutral pH, and 4C2 antibody can inhibit activation of IRR by alkali. Our study is the first description of the instruments of protein nature that can regulate activity of the orphan receptor IRR and confirms that alkali-induced activation is an intrinsic property of this receptor tyrosine kinase.

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Alteration of structure and function of ATP synthase and cytochrome c oxidase by lack of Fo-a and Cox3 subunits caused by mitochondrial DNA 9205delTA mutation

Biochemical Journal ◽

10.1042/bj20141462 ◽

2015 ◽

Vol 466 (3) ◽

pp. 601-611 ◽

Cited By ~ 12

Author(s):

Kateřina Hejzlarová ◽

Vilma Kaplanová ◽

Hana Nůsková ◽

Nikola Kovářová ◽

Pavel Ješina ◽

...

Keyword(s):

Cytochrome C ◽

Cytochrome C Oxidase ◽

Atp Synthase ◽

Stop Codon ◽

Threshold Effect ◽

Structure And Function ◽

Missense Mutations ◽

Atp Production ◽

Atp6 Gene ◽

And Function

Mutations in the MT-ATP6 gene are frequent causes of severe mitochondrial disorders. Typically, these are missense mutations, but another type is represented by the 9205delTA microdeletion, which removes the stop codon of the MT-ATP6 gene and affects the cleavage site in the MT-ATP8/MT-ATP6/MT-CO3 polycistronic transcript. This interferes with the processing of mRNAs for the Atp6 (Fo-a) subunit of ATP synthase and the Cox3 subunit of cytochrome c oxidase (COX). Two cases described so far presented with strikingly different clinical phenotypes–mild transient lactic acidosis or fatal encephalopathy. To gain more insight into the pathogenic mechanism, we prepared 9205delTA cybrids with mutation load ranging between 52 and 99% and investigated changes in the structure and function of ATP synthase and the COX. We found that 9205delTA mutation strongly reduces the levels of both Fo-a and Cox3 proteins. Lack of Fo-a alters the structure but not the content of ATP synthase, which assembles into a labile, ∼60 kDa smaller, complex retaining ATP hydrolytic activity but which is unable to synthesize ATP. In contrast, lack of Cox3 limits the biosynthesis of COX but does not alter the structure of the enzyme. Consequently, the diminished mitochondrial content of COX and non-functional ATP synthase prevent most mitochondrial ATP production. The biochemical effects caused by the 9205delTA microdeletion displayed a pronounced threshold effect above ∼90% mutation heteroplasmy. We observed a linear relationship between the decrease in subunit Fo-a or Cox3 content and the functional presentation of the defect. Therefore we conclude that the threshold effect originated from a gene–protein level.

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Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function

Blood ◽

10.1182/blood.v99.10.3646 ◽

2002 ◽

Vol 99 (10) ◽

pp. 3646-3653 ◽

Cited By ~ 28

Author(s):

Gabriella Kunz ◽

Ann-Kristin Öhlin ◽

Antonella Adami ◽

Bengt Zöller ◽

Peter Svensson ◽

...

Keyword(s):

Cell Surface ◽

Protein C ◽

Thromboembolic Disease ◽

Missense Mutations ◽

Wild Type ◽

Michaelis Constant ◽

Complementary Dna ◽

Naturally Occurring ◽

Cofactor Activity ◽

And Function

Sporadic mutations in the thrombomodulin (TM) gene occur in patients with both arterial and venous thrombosis, but the effects of these mutations on expression and function are largely unexplored. Full-length wild-type TM complementary DNA (cDNA) was incorporated into vector pcDNA6 for transfection into COS-7 cells for transient expression. Mutagenesis was performed to create 7 TM mutants with natural mutations either previously identified (Ala25Thr, Gly61Ala, Asp468Tyr, Pro477Ser, Pro483Leu) or reported here (an 11-base pair [bp] deletion, del791-801, leading to STOP306, and a missense mutation, Arg385Ser). Four mutations were found to detrimentally affect the level of expression of the TM protein. Of the missense mutations, 3 had reduced expression compared to wild-type TM (100%), Arg385Ser (50.2% ± 5%, P < .001), Pro477Ser (76.8% ± 1%, P < .001), Pro483Leu (82.1% ± 8%, P < .007). No TM protein expression could be detected on the cell surface for mutation del791-801. The cofactor activity of TM in protein C activation was also evaluated. The Michaelis constant (Km) for wild-type thrombin-TM complex was 634 ± 6 nmol/L. Two mutants, with Arg385Ser and Pro477Ser, had increased (P < .0001) Km, 2967 ± 283 nM, and 2342 ± 219 nM, respectively, demonstrating impaired function of the thrombin-TM complex. This work presents biochemical evidence that certain (but not all) natural mutations in the TM gene reduce expression and impair function of the protein on the cell surface, and helps clarify the suggested contribution that these mutations might make to the risk of thromboembolic disease.

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Heterogeneity of autoantibodies to factor VIII: differences in specificity for apparently distinct antigenic determinants of factor VIII coagulant protein

Blood ◽

10.1182/blood.v62.1.133.bloodjournal621133 ◽

1983 ◽

Vol 62 (1) ◽

pp. 133-140 ◽

Cited By ~ 7

Author(s):

MP Croissant ◽

M Zuzel ◽

JP Allain

Keyword(s):

Factor Viii ◽

Protein A ◽

Structure And Function ◽

Selective Recognition ◽

Antigenic Determinants ◽

Immunoradiometric Assay ◽

Naturally Occurring ◽

Response Slope ◽

And Function ◽

Naturally Occurring Antibodies

The interference of antibodies to factor VIII coagulant protein (VIII:C) of 9 nonhemophilic patients with the binding to factor VIII coagulant antigen (VIII:CAg) of a reference hemophilic 125I-Fab' reagent, used in a liquid phase VIII:CAg assay, was studied. The binding competition was estimated from immunoradiometric assay (IRMA) dose-response slope of VIII:CAg present in patient plasma, interference of antibodies with the 125I-Fab' binding to VIII:CAg in normal plasma, and the displacement of antibody from the complexes with VIII:CAg by the 125I Fab'. Antibody populations from three patients were studied in detail; in the VIII:CAg assay, two of them interfered with the 125I- Fab' binding, and one did not (patient 1). The formation of stable complexes between antibodies of each patient and VIII:CAg was demonstrated by protein-A-Sepharose adsorption. The 125I-Fab' binding to VIII:CAg-anti-VIII:CAg IgG complexes indicated that patient 1 antibodies and the 125I-Fab' recognized different antigenic determinants, whereas the other two patient antibodies and 125I-Fab' recognized closely related or identical VIII:CAg determinants. These results demonstrate an apparently selective recognition of at least two distinct VIII:CAg determinants by naturally occurring antibodies, suggesting a possibility of a wider use of these antibodies in studies of the structure and function of factor VIII.

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