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

Blood ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3646-3653 ◽  
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.

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

IUBMB Life ◽  
2012 ◽  
Vol 64 (4) ◽  
pp. 346-353 ◽  
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

The Structure and Function of Murine Factor V and Its Inactivation by Protein C

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4593-4599 ◽  
Author(s):  
Tony L. Yang ◽  
Jisong Cui ◽  
Alnawaz Rehumtulla ◽  
Angela Yang ◽  
Micheline Moussalli ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein C ◽  
Mammalian Species ◽  
Structure And Function ◽  
Procoagulant Activity ◽  
Factor V ◽  
Cleavage Sites ◽  
Wild Type ◽  
Coding Region ◽  
And Function

Factor V (FV) is a central regulator of hemostasis, serving both as a critical cofactor for the prothrombinase activity of factor Xa and the target for proteolytic inactivation by the anticoagulant, activated protein C (APC). To examine the evolutionary conservation of FV procoagulant activity and functional inactivation by APC, we cloned and sequenced the coding region of murine FV cDNA and generated recombinant wild-type and mutant murine FV proteins. The murine FV cDNA encodes a 2,183-amino acid protein. Sequence comparison shows that the A1-A3 and C1-C2 domains of FV are highly conserved, demonstrating greater than 84% sequence identity between murine and human, and 60% overall amino acid identity among human, bovine, and murine FV sequences. In contrast, only 35% identity among all three species is observed for the poorly conserved B domain. The arginines at all thrombin cleavage sites and the R305 and R504 APC cleavage sites (corresponding to amino acid residues R306 and R506 in human FV) are invariant in all three species. Point mutants were generated to substitute glutamine at R305, R504, or both (R305/R504). Wild-type and all three mutant FV recombinant proteins show equivalent FV procoagulant activity. Single mutations at R305 or R504 result in partial resistance of FV to APC inactivation, whereas recombinant murine FV carrying both mutations (R305Q/R504Q) is nearly completely APC resistant. Thus, the structure and function of FV and its interaction with APC are highly conserved across mammalian species.

Cleavage of Factor V at Arg 506 by Activated Protein C and the Expression of Anticoagulant Activity of Factor V

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2552-2558 ◽  
Author(s):  
Elisabeth Thorelli ◽  
Randal J. Kaufman ◽  
Björn Dahlbäck
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Factor V ◽  
Wild Type ◽  
Apc Resistance ◽  
S 100 ◽  
Negative Effect ◽  
Cofactor Activity

Activated protein C (APC) inhibits coagulation by cleaving and inactivating procoagulant factor Va (FVa) and factor VIIIa (FVIIIa). FV, in addition to being the precursor of FVa, has anticoagulant properties; functioning in synergy with protein S as a cofactor of APC in the inhibition of the FVIIIa-factor IXa (FIXa) complex. FV:Q506 isolated from an individual homozygous for APC-resistance is less efficient as an APC-cofactor than normal FV (FV:R506). To investigate the importance of the three APC cleavage sites in FV (Arg-306, Arg-506, and Arg-679) for expression of its APC-cofactor activity, four recombinant FV mutants (FV:Q306, FV:Q306/Q506, FV:Q506, and FV:Q679) were tested. FV mutants with Gln (Q) at position 506 instead of Arg (R) were found to be poor APC-cofactors, whereas Arg to Gln mutations at positions 306 or 679 had no negative effect on the APC-cofactor activity of FV. The loss of APC-cofactor activity as a result of the Arg-506 to Gln mutation suggested that APC-cleavage at Arg-506 in FV is important for the ability of FV to function as an APC-cofactor. Using Western blotting, it was shown that both wild-type FV and mutant FV was cleaved by APC during the FVIIIa inhibition. At optimum concentrations of wild-type FV (11 nmol/L) and protein S (100 nmol/L), FVIIIa was found to be highly sensitive to APC with maximum inhibition occurring at less than 1 nmol/L APC. FV:Q506 was inactive as an APC-cofactor at APC-concentrations ≤ 1 nmol/L and only partially active at higher APC concentrations. Our results show that increased expression of FV anticoagulant activity correlates with APC-mediated cleavage at Arg-506 in FV, but not with cleavage at Arg-306 nor at Arg-679.

Thrombomodulin with the Asp468Tyr mutation is expressed on the cell surface with normal cofactor activity for protein C activation

1999 ◽  
Vol 106 (2) ◽  
pp. 416-420 ◽  
Author(s):  
Fumie Nakazawa ◽  
Takatoshi Koyama ◽  
Takako Saito ◽  
Misako Shibakura ◽  
Haruhiko Yoshinaga ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein C ◽  
Cofactor Activity

scholarly journals The flatiron mutation in mouse ferroportin acts as a dominant negative to cause ferroportin disease

Blood ◽  
2007 ◽  
Vol 109 (10) ◽  
pp. 4174-4180 ◽  
Author(s):  
Irene E. Zohn ◽  
Ivana De Domenico ◽  
Andrew Pollock ◽  
Diane McVey Ward ◽  
Jessica F. Goodman ◽  
...  
Keyword(s):  
Mouse Model ◽  
Cell Surface ◽  
Kupffer Cells ◽  
Transferrin Saturation ◽  
High Serum ◽  
Dominant Negative ◽  
Iron Loading ◽  
Missense Mutations ◽  
Wild Type ◽  
Export Activity

Abstract Ferroportin disease is caused by mutation of one allele of the iron exporter ferroportin (Fpn/IREG1/Slc40a1/MTP1). All reported human mutations are missense mutations and heterozygous null mutations in mouse Fpn do not recapitulate the human disease. Here we describe the flatiron (ffe) mouse with a missense mutation (H32R) in Fpn that affects its localization and iron export activity. Similar to human patients with classic ferroportin disease, heterozygous ffe/+ mice present with iron loading of Kupffer cells, high serum ferritin, and low transferrin saturation. In macrophages isolated from ffe/+ heterozygous mice and through the use of Fpn plasmids with the ffe mutation, we show that Fpnffe acts as a dominant negative, preventing wild-type Fpn from localizing on the cell surface and transporting iron. These results demonstrate that mutations in Fpn resulting in protein mislocalization act in a dominant-negative fashion to cause disease, and the Fpnffe mouse represents the first mouse model of ferroportin disease.

A functional analysis of the putative polymorphisms A91V and N252S and 22 missense perforin mutations associated with familial hemophagocytic lymphohistiocytosis

Blood ◽  
2005 ◽  
Vol 105 (12) ◽  
pp. 4700-4706 ◽  
Author(s):  
Ilia Voskoboinik ◽  
Marie-Claude Thia ◽  
Joseph A. Trapani
Keyword(s):  
Functional Analysis ◽  
Hemophagocytic Lymphohistiocytosis ◽  
Autosomal Recessive ◽  
Missense Mutations ◽  
Wild Type ◽  
Lytic Function ◽  
Functional Defect ◽  
Target Cell Membrane ◽  
And Function ◽  
Prf1 Gene

Abstract Up to 60% of cases of the autosomal recessive immunodeficiency hemophagocytic lymphohistiocytosis (HLH) are associated with mutations in the perforin (PRF1) gene. In this study, we expressed wild-type and mutated perforin in rat basophil leukemia cells to study the effect on lytic function of the substitutions A91V and N252S (commonly considered to be neutral polymorphisms) and 22 perforin missense substitutions first identified in HLH patients. Surprisingly, we found that A91V perforin was expressed at reduced levels compared with wild-type perforin, resulting in partial loss of lytic capacity. In contrast, expression and function of N252S-substituted perforin were normal. Most HLH-associated mutations resulted in protein degradation (probably due to misfolding) and complete loss of perforin activity, the exception being R232H, which retained approximately 30% wild-type activity. Several other mutated proteins (H222Q, C73R, F157V, and D313V) had no detectable lytic activity but were expressed at normal levels, suggesting that their functional defect might map downstream at the level of the target cell membrane. One further perforin substitution identified in an HLH patient (V183G) was normally expressed and displayed normal lysis. This report represents the first systematic functional analysis of HLH-associated missense mutations and the 2 most common perforin polymorphisms. (Blood. 2005;105:4700-4706)

scholarly journals Exacerbated venous thromboembolism in mice carrying a protein S K196E mutation

Blood ◽  
2015 ◽  
Vol 126 (19) ◽  
pp. 2247-2253 ◽  
Author(s):  
Fumiaki Banno ◽  
Toshiyuki Kita ◽  
José A. Fernández ◽  
Hiroji Yanamoto ◽  
Yuko Tashima ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Venous Thrombosis ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Protein S Deficiency ◽  
Wild Type ◽  
S Deficiency ◽  
Key Points ◽  
Cofactor Activity

Key Points A protein S-K196E mutation reduced its activated protein C cofactor activity in recombinant murine protein S-K196E and in K196E mutant mice. Mice carrying a protein S-K196E mutation or heterozygous protein S deficiency were more vulnerable to venous thrombosis than wild-type mice.

Vision in mice with neuronal redundancy due to inhibition of developmental cell death

1999 ◽  
Vol 16 (4) ◽  
pp. 721-726 ◽  
Author(s):  
VITTORIO PORCIATTI ◽  
TOMMASO PIZZORUSSO ◽  
LAMBERTO MAFFEI
Keyword(s):  
Cell Death ◽  
Neural Circuitry ◽  
Contrast Threshold ◽  
Wild Type ◽  
Compensatory Mechanisms ◽  
Vertical Meridian ◽  
Naturally Occurring ◽  
Brain Expansion ◽  
And Function ◽  
Developmental Cell Death

Transgenic mice overexpressing bcl-2, due to inhibition of naturally occurring cell death, have much larger brains and optic nerves as compared to wild-type mice. Since developmental cell death is believed to exert a crucial role in establishing the mature neural circuitry and function, we asked the question of whether basic aspects of vision were altered in bcl-2 mice. Local visually evoked potentials (VEPs) in response to patterned stimuli were recorded from the primary visual cortex. The representation of the vertical meridian was displaced by about 15% in the bcl-2 mouse, accounting for brain expansion. However, visual acuity, contrast threshold, and response latency were normal, indicating that compensatory mechanisms can ensure normal basic properties of vision in spite of marked neuronal redundancy.

scholarly journals Functional Characterization of Naturally Occurring Pathogenic Mutations in the Human Leptin Receptor

Endocrinology ◽  
2008 ◽  
Vol 149 (12) ◽  
pp. 6043-6052 ◽  
Author(s):  
Wendy Kimber ◽  
Frank Peelman ◽  
Xavier Prieur ◽  
Teresia Wangensteen ◽  
Stephen O'Rahilly ◽  
...  
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Binding Site ◽  
Molecular Mechanisms ◽  
Leptin Receptor ◽  
Receptor Activation ◽  
Cell Surface Expression ◽  
Missense Mutations ◽  
Naturally Occurring

We have recently reported the first naturally occurring missense mutations in the leptin receptor (LR) in patients with severe obesity. We have examined the molecular mechanisms by which these extracellular domain mutations disrupt LR signaling. The Ala409Glu mutant receptor is expressed at the cell surface, binds leptin normally but fails to signal to downstream pathways. A409 is present on the surface-exposed region of the Ig-like domain that forms the binding site III for interaction with leptin. This binding site does not appear to contribute to the binding affinity of leptin to its receptor but is critical for receptor activation in response to ligand binding. The Trp664Arg and His684Pro mutations are predicted to impair receptor folding. Both mutants result in a complete inability to signal to downstream pathways despite evidence for some residual cell surface expression and ligand binding. The Arg612His mutant falls in the second subdomain of the high-affinity binding site for leptin, and results in a receptor that shows evidence for intracellular retention but retains some residual signaling. These studies, which represent the first detailed characterization of the functional properties of naturally occurring missense mutations in the human LR, indicate that most such mutations affect receptor folding and expression at the cell surface rather than primarily impairing ligand binding. The exception is Ala409Glu, which interferes with the coupling of ligand binding to receptor activation. Naturally occurring mutations associated with human obesity are valuable tools with which to explore structure/function relationships within the LR.

scholarly journals Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning

2017 ◽  
Vol 114 (9) ◽  
pp. 2265-2270 ◽  
Author(s):  
Justin R. Klesmith ◽  
John-Paul Bacik ◽  
Emily E. Wrenbeck ◽  
Ryszard Michalczyk ◽  
Timothy A. Whitehead
Keyword(s):  
Surface Display ◽  
Protein Solubility ◽  
Fitness Landscapes ◽  
Yeast Surface Display ◽  
Crystallographic Structure ◽  
Missense Mutations ◽  
Classification Models ◽  
Wild Type ◽  
Trade Offs ◽  
And Function

Proteins are marginally stable, and an understanding of the sequence determinants for improved protein solubility is highly desired. For enzymes, it is well known that many mutations that increase protein solubility decrease catalytic activity. These competing effects frustrate efforts to design and engineer stable, active enzymes without laborious high-throughput activity screens. To address the trade-off between enzyme solubility and activity, we performed deep mutational scanning using two different screens/selections that purport to gauge protein solubility for two full-length enzymes. We assayed a TEM-1 beta-lactamase variant and levoglucosan kinase (LGK) using yeast surface display (YSD) screening and a twin-arginine translocation pathway selection. We then compared these scans with published experimental fitness landscapes. Results from the YSD screen could explain 37% of the variance in the fitness landscapes for one enzyme. Five percent to 10% of all single missense mutations improve solubility, matching theoretical predictions of global protein stability. For a given solubility-enhancing mutation, the probability that it would retain wild-type fitness was correlated with evolutionary conservation and distance to active site, and anticorrelated with contact number. Hybrid classification models were developed that could predict solubility-enhancing mutations that maintain wild-type fitness with an accuracy of 90%. The downside of using such classification models is the removal of rare mutations that improve both fitness and solubility. To reveal the biophysical basis of enhanced protein solubility and function, we determined the crystallographic structure of one such LGK mutant. Beyond fundamental insights into trade-offs between stability and activity, these results have potential biotechnological applications.

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