Recombinant Mutant Proteins Research Articles

Identification of Serpin Determinants of Specificity and Selectivity for Furin Inhibition through Studies of α1PDX (α1-Protease Inhibitor Portland)-Serpin B8 and Furin Active-site Loop Chimeras

α1-Protease inhibitor Portland (α1PDX) is an engineered serpin family inhibitor of the proprotein convertase (PC), furin, that exhibits high specificity but limited selectivity for inhibiting furin over other PC family proteases. Here, we characterize serpin B8, a natural inhibitor of furin, together with α1PDX-serpin B8 and furin-PC chimeras to identify determinants of serpin specificity and selectivity for furin inhibition. Replacing reactive center loop (RCL) sequences of α1PDX with those of serpin B8 demonstrated that both the P4-P1 RXXR recognition sequence as well as the P1'-P5' sequence are critical determinants of serpin specificity for furin. Alignments of PC catalytic domains revealed four variable active-site loops whose role in furin reactivity with serpin B8 was tested by engineering furin-PC loop chimeras. The furin(298-300) loop but not the other loops differentially affected furin reactivity with serpin B8 and α1PDX in a manner that depended on the serpin RCL-primed sequence. Modeling of the serpin B8-furin Michaelis complex identified serpin exosites in strand 3C close to the 298-300 loop whose substitution in α1PDX differentially affected furin reactivity depending on the furin loop and serpin RCL-primed sequences. These studies demonstrate that RCL-primed residues, strand 3C exosites, and the furin(298-300) loop are critical determinants of serpin reactivity with furin, which may be exploited in the design of specific and selective α1PDX inhibitors of PCs.

Novel compound VHL heterozygosity (VHL T124A/L188V) associated with congenital polycythaemia

Novel compound <scp>VHL</scp> heterozygosity (<i><scp>VHL</scp></i> T124A/L188V) associated with congenital polycythaemia

Functional deficit associated with a missense Werner syndrome mutation

Werner syndrome (WS) is a rare autosomal recessive disorder caused by mutations in the WRN gene. WRN helicase, a member of the RecQ helicase family, is involved in various DNA metabolic pathways including DNA replication, recombination, DNA repair and telomere maintenance. In this study, we have characterized the G574R missense mutation, which was recently identified in a WS patient. Our biochemical experiments with purified mutant recombinant WRN protein showed that the G574R mutation inhibits ATP binding, and thereby leads to significant decrease in helicase activity. Exonuclease activity of the mutant protein was not significantly affected, whereas its single strand DNA annealing activity was higher than that of wild type. Deficiency in the helicase activity of the mutant may cause defects in replication and other DNA metabolic processes, which in turn could be responsible for the Werner syndrome phenotype in the patient. In contrast to the usual appearance of WS, the G574R patient has normal stature. Thus the short stature normally associated with WS may not be due to helicase deficiency.

Mutations Found in Patients Leber's Congenital Amaurosis Disrupt Retinal Guanylate Cyclase Interaction with Rhodopsin In Vitro; Involvement In Phototransduction

We use bovine rod outer membranes membrane and recombinant intradiscal domain of retinal guanylate cyclase (retGC) expressed in HEK293 in pull down assay to demonstrate interaction between rhodopsin (Rho) and retGC.It has been previously shown that retGC activity in light‐exposed rod outer segment membranes was much higher than activity of the same membranes isolated under dark conditions suggesting that communication of retGC with activated Rho is required for its full activation by GCAP. To study the effect of the mutations found in Leber's congenital amaurosis (LCA) in intradiscal domain of retGC on its interaction with Rho we used pull down assay and colocalization experiments. Fluorescent protein‐tagged intradiscal domain of bovine retGC was expressed in HEK293 cells and soluble fraction containing recombinant wild type or mutant proteins were prepared. We used photobleached and hypotonically washed rod outer segment (ROS) membrane as a source of photoactivated Rho. Soluble fractions containing recombinant proteins were incubated with ROS membranes. After washing several times with isotonic buffer presence of recombinant proteins in membranes and soluble fraction were analyzed by western blot using retGC‐specific antibodies. Only wild type intradiscal domain of retGC was detected in ROS membrane fraction suggesting that mutation causing LCA disrupt the interaction between retGC and Rho.

E6AP/UBE3A Ubiquitin Ligase Harbors Two E2∼ubiquitin Binding Sites

By exploiting (125)I-polyubiquitin chain formation as a functional readout of enzyme activity, we have quantitatively examined the mechanism of human E6AP/UBE3A for the first time. Initial rate studies identify UbcH7 as the cognate E2 carrier protein for E6AP, although related Ubc5 isoforms and the ISG15-specific UbcH8 paralog also support E6AP with reduced efficacy due to impaired binding and catalytic competence. Initial rates of polyubiquitin chain formation displayed hyperbolic kinetics with respect to UbcH7 concentration (K(m) = 57.6 ± 5.7 nM and kcat = 0.032 ± 0.001 s(-1)) and substrate inhibition above 2 μM. Competitive inhibition by an isosteric UbcH7C86S-ubiquitin oxyester substrate analog (K(i) = 64 ± 18 nM) demonstrates that Km reflects intrinsic substrate affinity. In contrast, noncompetitive inhibition by a UbcH7C86A product analog (K(i) = 7 ± 0.7 μM) and substrate inhibition at high concentrations require two functionally distinct E2∼ubiquitin substrate binding sites. The kinetics of polyubiquitin chain formation reflect binding at a cryptic Site 1 not previously recognized that catalyzes E6AP∼ubiquitin thioester formation. Subsequent binding of E2∼ubiquitin at the canonical Site 2 present in the extant crystal structure is responsible for polyubiquitin chain elongation. Other rate studies show that the conserved -4 Phe(849) residue is required for polyubiquitin chain formation rather than target protein conjugation as originally suggested. The present studies unambiguously preclude earlier models for the mechanism of Hect domain-catalyzed conjugation through the canonical binding site suggested by the crystal structure and define a novel two-step mechanism for formation of the polyubiquitin degradation signal.

Familial dilated cardiomyopathy mutations uncouple troponin I phosphorylation from changes in myofibrillar Ca2+ sensitivity

The pure form of familial dilated cardiomyopathy (DCM) is mainly caused by mutations in genes encoding sarcomeric proteins. Previous measurements using recombinant proteins suggested that DCM mutations in thin filament proteins decreased myofibrillar Ca(2+) sensitivity, but exceptions were reported. We re-investigated the molecular mechanism of familial DCM using native proteins. We used the quantitative in vitro motility assay and native troponin and tropomyosin to study DCM mutations in troponin I, troponin T, and α-tropomyosin. Four mutations reduced myofilament Ca(2+) sensitivity, but one mutation (TPM1 E54K) did not alter Ca(2+) sensitivity and another (TPM1 D230N) increased Ca(2+) sensitivity. In thin filaments from normal human and mouse heart, protein kinase A (PKA) phosphorylation of troponin I caused a two- to three-fold decrease in myofibrillar Ca(2+) sensitivity. However, Ca(2+) sensitivity did not change with the level of troponin I phosphorylation in any of the DCM-mutant containing thin filaments (E40K, E54K, and D230N in α-tropomyosin; R141W and ΔK210 in cardiac troponin T; K36Q in cardiac troponin I; G159D in cardiac troponin C, and E361G in cardiac α-actin). This 'uncoupling' was observed with native mutant protein from human and mouse heart and with recombinant mutant protein expressed in baculovirus/Sf9 systems. Uncoupling was independent of the fraction of mutated protein present above 0.55. We conclude that DCM-causing mutations in thin filament proteins abolish the relationship between myofilament Ca(2+) sensitivity and troponin I phosphorylation by PKA. We propose that this blunts the response to β-adrenergic stimulation and could be the cause of DCM in the long term.

A unique IBMPFD-related P97/VCP mutation with differential binding pattern and subcellular localization

p97/VCP is a hexameric AAA type ATPase that functions in a variety of cellular processes such as endoplasmic reticulum associated degradation (ERAD), organelle biogenesis, autophagy and cell-cycle regulation. Inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder which has been attributed to mutations in p97/VCP. Several missense mutations affecting twelve different amino acids have been identified in IBMPFD patients and some of them were suggested to be involved in the observed pathology. Here, we analyzed the effect of all twelve p97/VCP variants on ERAD substrates and their cofactor binding abilities. While all mutants cause ERAD substrate accumulation, P137L mutant p97/VCP differs from other IBMPFD mutants by having a unique solubility profile and subcellular localization. Intriguingly, although almost all mutants exhibit enhanced p47 and Ufd1-Npl4 binding, the P137L mutation completely abolishes p97/VCP interactions with Ufd1, Npl4 and p47, while retaining its gp78 binding. While recombinant R155C mutant protein consistently interacts with both Ufd1 and VIM of gp78, P137L mutant protein lost binding ability to Ufd1 but not to VIM in vitro. The differential impairments in p97/VCP interactions with its functional partners and function should help our understanding of the molecular pathogenesis of IBMPFD.

Functional Characterization of the Type 1 VWD Candidate VWF Gene Variants: P.M771I, p.L881R, p.P1413L, and p.Q1475X

Abstract 97Von Willebrand Factor (VWF) is a multimeric glycoprotein that maintains hemostasis in the vascular system. VWF is secreted mainly from endothelial cells. During its biosynthesis VWF undergoes a series of post-translational modifications and some of the newly synthesized protein is released constitutively. The remainder of the protein is stored for regulated release from cytoplasmic storage granules, Weibel Palade Bodies. Abnormalities in the biosynthetic pathway or increased clearance of plasma VWF are likely to contribute to decreased plasma VWF levels. Inherited partial deficiency of VWF is classified as Type I von Willebrand disease (VWD). Clinical diagnosis of Type I VWD is complicated due to incomplete penetrance and variable expressivity of the abnormal VWF phenotype. The molecular basis of Type 1 VWD remains incompletely understood. The purpose of this study is to explore the effect of four VWF missense mutations (p.M771I, p.L881R, p.P1413L, p.Q1475X), that were reported as candidate mutations in Type I VWD patients in the ISTH SSC VWF Database. The focus of these studies is on the intracellular biosynthetic processing and localization of VWF in a heterologous cell system. For this purpose, selected VWF gene variations were generated in a VWF cDNA expression vector by in vitro site directed mutagenesis. The effect of the candidate mutations on the intracellular localization were analyzed in HEK293 cells by immunoflourescence antibody staining and confocal microscopy. In addition, the effect of the mutations on the biosynthesis of the recombinant VWF was analyzed in COS-7 cells The expression vectors WT-cDNA (wild type) and the expression vector carrying the candidate mutations were transiently transfected into COS-7 cells alone or together to generate wild-type, mutant homozygous and heterozygous genotypes, the latter situation mimicking the status in type 1 VWD patients. To determine whether the mutant recombinant protein was retained within the cell or was efficiently secreted, VWF:Ag levels were assayed in cell lysates and in the conditioned media using ELISA.Confocal analysis of the transfected HEK293 cells demonstrated impaired intracellular localization of recombinant VWF having p.M771I and p.Q1475X variations. On the other hand, a normal pattern of intracellular storage was observed for the VWF variants having p.L881R and p.P1413L changes.Expression studies for the p.P1413L and p.Q1475X mutations are currently in progress. Transient transfection of COS-7 cells with the VWF expression vector carrying p.M771I mutation revealed that secretion of the recombinant VWF protein was decreased by 73% (P=0.013, n=3) in the homozygous state, and by 47% (P=0.013, n=3) in the heterozygous state relative to the secretion of wild-type recombinant VWF protein. Intracellular levels of the mutant recombinant protein was increased by 212% in the homozygous state (P=0.005, n=3) and by 69% in the heterozygous state (P=0.255, not significant, n=3). This study demonstrated that p.M771I mutation causes increased intracellular retention of the protein and it has a dominant negative effect on the processing of the wild type protein.Transient transfection of COS-7 cells with the VWF expression vector carrying p.L881R mutation revealed that secretion of the recombinant VWF protein was decreased by 10% (P=0.61, not significant, n=3) in the homozygous state, and increased by 47% (P=0.36, not significant, n=3) in the heterozygous state relative to the secretion of wild-type recombinant VWF protein. Intracellular levels of the mutant recombinant protein were increased by 58% in the homozygous state (P=0.925, not significant, n=3,) and decreased by 35% in the heterozygous state (P=0.405, not significant, n=3). This study demonstrates that in contrast to the findings with the p.M771I mutant, the p.L881R mutation has little effect on VWF biosynthesis and secretion and does not show a dominant negative effect on the wild type VWF. The pathogenetic mechanism responsible for the type 1 VWD phenotype associated with the p.L881R mutation requires additional evaluation but may involve enhanced protein clearance. Disclosures:No relevant conflicts of interest to declare.

Slow spontaneous α-to-β structural conversion in a non-denaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein

In prion diseases, the normal prion protein is transformed by an unknown mechanism from a mainly α-helical structure to a β-sheet-rich, disease-related isomer. In this study, we surprisingly found that a slow, spontaneous α-to-coil-to-β transition could be monitored by circular dichroism spectroscopy in one full-length mouse recombinant prion mutant protein, denoted S132C/N181C, in which the endogenous cysteines C179 and C214 were replaced by Ala and S132 and N181 were replaced by Cys, during incubation in a non-denaturing neutral buffer. No denaturant was required to destabilize the native state for the conversion. The product after this structural conversion is toxic β-oligomers with high fluorescence intensity when binding with thioflavin T. Site-directed spin-labeling ESR data suggested that the structural conversion involves the unfolding of helix 2. After examining more protein mutants, it was found that the spontaneous structural conversion is due to the disulfide-deletion (C to A mutations). The recombinant wild-type mouse prion protein could also be transformed into β-oligomers and amyloid fibrils simply by dissolving and incubating the protein in 0.5 mM NaOAc (pH 7) and 1 mM DTT at 25°C with no need of adding any denaturant to destabilize the prion protein. Our findings indicate the important role of disulfide bond reduction on the structural conversion of the recombinant prion protein, and highlight the special “intrinsically disordered” conformational character of the recombinant prion protein.

Histidine 117 in the His-Gly-Ser-Asp motif is Required for the Biochemical Activities of Nucleoside Diphosphate Kinase of Mycobacterium smegmatis

Nucleoside diphosphate kinase (NDK), which is widely conserved in both prokaryotes and eukaryotes, maintains a balanced pool of nucleotide triphosphates and their deoxy derivatives. NDKs from bacterial and other systems contain the conserved HGSD motif, where the His residue is required for the biochemical activities, namely the NTPase (AT-Pase and GTPase), NTP synthesising, and autophosphorylation activities of the enzyme. Amino acid sequence homology comparison of the NDK of Mycobacterium smegmatis (MsmNDK) with the NDKs of other bacterial genera showed the presence of H117GSD motif. While the recombinant wild type MsmNDK showed the NTPase, NTP synthesising, and autophosphorylation activities, the H117Q mutation abolished the biochemical activities of the recombinant MsmNDK-H117Q mutant protein in vitro. These observations demonstrate that the H117 residue in the HGSD motif is required for the biochemical activities of MsmNDK.

Mitochondrial phenylalanyl-tRNA synthetase mutations underlie fatal infantile Alpers encephalopathy

Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding the mitochondrial phenylalanyl transfer RNA (tRNA) synthetase (mtPheRS) in two patients with fatal epileptic mitochondrial encephalopathy. The mutations affected highly conserved amino acids, p.I329T and p.D391V. Recently, a homozygous FARS2 variant p.Y144C was reported in a Saudi girl with mitochondrial encephalopathy, but the pathogenic role of the variant remained open. Clinical features, including postnatal onset, catastrophic epilepsy, lactic acidemia, early lethality and neuroimaging findings of the patients with FARS2 variants, resembled each other closely, and neuropathology was consistent with Alpers syndrome. Our structural analysis of mtPheRS predicted that p.I329T weakened ATP binding in the aminoacylation domain, and in vitro studies with recombinant mutant protein showed decreased affinity of this variant to ATP. Furthermore, p.D391V and p.Y144C were predicted to disrupt synthetase function by interrupting the rotation of the tRNA anticodon stem-binding domain from a closed to an open form. In vitro characterization indicated reduced affinity of p.D391V mutant protein to phenylalanine, whereas p.Y144C disrupted tRNA binding. The stability of p.I329T and p.D391V mutants in a refolding assay was impaired. Our results imply that the three FARS2 mutations directly impair aminoacylation function and stability of mtPheRS, leading to a decrease in overall tRNA charging capacity. This study establishes a new genetic cause of infantile mitochondrial Alpers encephalopathy and reports a new mitochondrial aminoacyl-tRNA synthetase as a cause of mitochondrial disease.

Abstract 2602: CHEK2 contribution to hereditary breast cancer in non-BRCA famillies

Abstract Mutations in the BRCA1 and BRCA2 genes are responsible for only a part of hereditary breast cancer (HBC). The origins of “non BRCA” HBC families may be attributed in part to mutations in genes giving moderate risk, such as CHEK2. We investigated the contribution of CHEK2mutations to non-BRCA HBC by direct sequencing of its entire coding sequence. Fifteen mutations were discovered among 507 non-BRCAHBC cases and four among 513 controls. The frequency of CHEK2variants was significantly higher among cases (p= 0.0076), and gave an OR for breast cancer of 4.72 for deleterious mutation carriers. We then used both in silico tools and in vitro kinase activity to evaluate recombinant mutant proteins. Tumor characteristics and tumor grade of paraffin-embedded tissue blocks from 8 CHEK2 mutated patients were evaluated by histology. To further characterize those tumors, breast cancer immunohistochemical markers such as hormone receptors, HER2 and P53 were assessed. Because the mechanisms of tumorigenesis in association with CHEK2 variants are still unclear, we performed genetic and epigenetic analysis of those tumors. Three relevant SNPs spanning the CHEK2 gene locus were used to determine loss of heterozygosity (LOH). Also, the proximal CpG islands of the CHEK2 gene were investigated for hypermethylation. Our results suggest a contribution of CHEK2 mutations to non-BRCA HBC, though the usefulness of moderate penetrance genes for genetic counseling remains controversial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2602. doi:1538-7445.AM2012-2602

Reduction of thrombus size in murine models of thrombosis following administration of recombinant α1-proteinase inhibitor mutant proteins

The variant serpin α1-PI M358R inhibits thrombin and other proteases such as activated protein C (APC) and factor XIa. We previously described recombinant proteins HAPI M358R (α1-PI M358R containing an N-terminal extension corresponding to residues 1-75 of heparin cofactor II) and HAPI RCL5 (HAPI M358R with F352-I356 and I360 substituted for the corresponding residues of antithrombin), with enhanced selectivity for thrombin over APC inhibition. We tested the hypotheses that these recombinant proteins would limit thrombosis in three mouse models, and that the HAPI chimeric proteins would be more effective than α1-PI M358R. Recombinant serpins were purified from Escherichia coli by nickel chelate and ion exchange affinity chromatography, and administered to mice intravenously. HAPI RCL5 reduced incorporation of radiolabelled fibrin(ogen) into thrombi in the ferric chloride-injured vena cava in a dose-dependent manner; HAPI M358R was less effective and α1-PI M358R was without effect. In a model of murine endotoxaemia, HAPI RCL5 was more effective than α1-PI M358R in reducing radiolabelled fibrin(ogen) deposition in heart and kidneys; immunohistochemistry of tissue sections showed lesser staining with anti-fibrin(ogen) antibodies with both treatments. In the ferric chloride-injured murine carotid artery, administration of both recombinant serpins was equally effective in lengthening the vessel's time to occlusion. Our results show that the antithrombotic efficacy of the recombinant serpins correlates with their potency as thrombin inhibitors, since HAPI RCL5 inhibits thrombin, but not factors Xa, XIa, XIIa, or neutrophil elastase, more rapidly than α1-PI M358R.

Two missense mutations identified in venous thrombosis patients impair the inhibitory function of the protein Z dependent protease inhibitor

Protein Z-dependent protease inhibitor (ZPI) is a plasma inhibitor of factor (F)Xa and FXIa. In an earlier study, five mutations were identified within the ZPI gene of venous thrombosis patients and healthy controls. Two of these were nonsense mutations and three were missense mutations in important regions of the protein. Here we report that two of these latter three mutations, F145L and Q384R, impair the inhibitory function of ZPI in vitro. Recombinant wild-type and mutant proteins were prepared; stability in response to thermal challenge was similar. Inhibition of FXa in the presence of the cofactor protein Z was reduced 68-fold by the Q384R mutant; inhibition of FXIa by the F145L mutant was reduced two- to three-fold compared to the wild-type ZPI. An analysis of all five ZPI mutations was undertaken in a cohort of venous thrombosis patients (n=550) compared to healthy controls (n=600). Overall, there was a modest increase in incidence of these mutations in the thrombosis group (odds ratio 2.0, 1.05-3.7, p=0.044). However, in contrast to W324X (nonsense mutation), the Q384R missense mutation and R88X nonsense mutation were evenly distributed in patients and controls; F145L was rare. The final mutation (S143Y) was also rare and did not significantly alter ZPI function in laboratory studies. The F145L and particularly the Q384R mutation impaired the function of the coagulation inhibitor ZPI; however, there was no convincing association between these mutations and venous thrombosis risk. The functional role for ZPI in vivo has yet to be clarified.

In Silico Studies on the Stability of Human Interferon-Gamma Mutants

ABSTRACTHuman interferon-gamma (hIFNγ) is a key cytokine in the realisation of cellular immunity. It accomplishes its biological activity upon binding to a specific cell receptor thus inducing the JAK/STAT1 signal transduction pathway. Two putative NLS sequences were pointed out to assist in the translocation of STAT1 into the nucleus. In order to employ mutational analysis for study the biological significance of the polybasic sequence Lys86-Lys87-Lys88 belonging to the upstream putative NLS, hIFNγ mutants with preserved structure and intact binding affinity to cell receptor need to be selected. To this end in silico studies of molecular stability of hIFNγ mutants was performed. The potential conformational changes in the structure of the mutant proteins were investigated employing molecular dynamics simulations. The free energy surface of Lys86 backbone torsion angles space in hIFNγ wild type and mutants was analyzed using metadynamic model. The obtained in silico results were verified by construction of selected mutant recombinant hIFNy proteins, which were analysed for biological activity. To judge for the secondary structure of the mutants the affinity to the cell receptor was investigated. High correlation between results of the molecular dynamics simulations and biological data was obtained.

Intrarectal administration of milk fat globule epidermal growth factor-8 protein ameliorates murine experimental colitis

Milk fat globule epidermal growth factor-8 (MFG-E8) promotes phagocytic clearance of apoptotic cells to maintain normal tissue homeostasis. However, its functions in intestinal inflammatory disorders are unknown. Since the pathogenesis of those disorders are due to abnormal interactions between intestinal epithelial cells (IECs) and microbial pathogens, we analyzed the effects of MFG-E8 on IECs to determine its protective role in murine experimental colitis. Expression of αvβ3-integrin in Colon-26 cells was examined by RT-PCR and immunostaining. Colon-26 cells were pretreated with recombinant wild-type and mutant MFG-E8 proteins, following stimulation with flagellin as an inducer of innate immunity, and the effects of the recombinant proteins on inhibition of nuclear factor-κB (NF-κB) and inflammatory cytokine production in flagellin-stimulated Colon-26 cells were determined using a luciferase assay and EIA, respectively. Experimental colitis was induced in mice by intrarectal administration of trinitrobenzene sulfonic acid (TNBS). Recombinant proteins were then intrarectally administered into TNBS-induced colitic mice, after which disease activity parameters (body weight, colon length, histological score), as well as interleukin (IL)-6 and MIP-2 levels were determined in inflamed tissues. Flagellin-induced inflammatory cytokine production in vitro was significantly downregulated via αvβ3-integrin following pretreatment with wild-type MFG-E8 due to inhibition of NF-κB activation. In vivo, intrarectal treatment with wild-type MFG-E8, but not its mutant counterpart, significantly inhibited body weight loss, colon shortening and histological inflammation induced by TNBS administration. Our findings suggest that MFG-E8 has anti-inflammatory effects on flagellin-induced inflamed intestinal epithelial cells and may be a useful therapeutic agent for colitis.

Severe Factor V Deficiency Caused by a Novel Compound Heterozygous Mutation: Factor V G1617V and 1-Bp Insertion

Abstract 2268 Introduction:Congenital factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. There is poor correlation between FV activity in plasma and symptoms of bleeding tendency. In the present study, we identified a novel compound heterozygous mutation in the FV gene (F5) in a Japanese patient with severe bleeding symptoms.Patient and methods: The patient was a young boy in his teens. He suffered from intracranial hemorrhage after birth and he also experienced joints and muscle hemorrhage, afterwards. His plasma FV activity was less than 1% and FV antigen was 2%. Sequence analysis of F5, FV protein analysis, and recombinant mutant protein expression experiments were performed. And to evaluate the relation between bleeding symptoms and FV phenotypes, we analyzed the FV level of platelets in the patient and his family members. Our present and previous results were verified. Results:Sequence analysis of F5 in this patient revealed a novel compound heterozygous mutation, G1617V missense mutation in exon 14 and a 1-bp insertion in exon 16 (p.G1645V and c.5255–5259 ins A). The patient’s father had a G1617V mutation and his mother had a 1-bp insertion for the heterozygote state, respectively. The patient’s two sisters each had also a 1-bp insertion for the heterozygote state. We analyzed the expression of platelet F5 mRNA by semi-quantitative RT-PCR, which showed that platelet F5 mRNA from the patient and his family members were equal to the amount in healthy subjects. Platelet FV protein from family members examined by western blotting detected equal to those of healthy subjects, although the FV bands from the patient were detected only weakly. Platelet FV antigen and activity were measured with an ELISA and a functional assay based on prothrombin time. The patient’s platelet FV activity was 1.6% and antigen was 1%, compared with 100% for those of normal subjects. Platelet FV activity and antigen of the patient’s father, mother, elder sister and younger sister were 26% and 37.1%, 39.6% and 51.0%, 23.3% and 38.0%, and 35.4% and 56.6%, respectively. In the expression study of pMT2/FV-wild-type and FV-G1617V mutant recombinant proteins in HEK293 cells, the FV antigen levels produced by the FV-G1617V mutant in cell lysates was approximately 73% of wild-type. In conditioned media of the study, FV antigen and specific activity were reduced to approximately 4% and 6% of wild-type, respectively. Discussions:Although there was similar intracellular synthesis of the FV-G1617V mutant and wild-type FV, the results in the conditioned media suggested that secretion of the FV-G1617V mutant was impaired. Platelet FV protein was detected very low by Western blot and ELISA, though platelet F5 mRNA was confirmed to be normal by RT-PCR. Our previous results raise the possibility that in the case of severe FV deficiency, such as that with a 1 % or less plasma FV level, the amount of FV in platelets is important to cope with local bleeding. This patient's platelet FV activity and antigen levels were the lowest of those of a number of FV-deficient patients whom we have analyzed so far, and this patient had severe bleeding symptoms. Conclusion:Our previous studies support the concept that the severity of bleeding is related to FV protein in platelets. Taken together, we believe the severity of this patient's bleeding tendency is caused by a reduction in both platelet and plasma FV, especially by the highly significant decrease in platelet FV. Disclosures:No relevant conflicts of interest to declare.

Thymidine kinase 2 mutations in autosomal recessive progressive external ophthalmoplegia with multiple mitochondrial DNA deletions

Autosomal-inherited progressive external ophthalmoplegia (PEO) is an adult-onset disease characterized by the accumulation of multiple mitochondrial DNA (mtDNA) deletions in post-mitotic tissues. Mutations in six different genes have been described to cause the autosomal dominant form of the disease, but only mutations in the DNA polymerase gamma gene are known to cause autosomal recessive PEO (arPEO), leaving the genetic background of arPEO mostly unknown. Here we used whole-exome sequencing and identified compound heterozygous mutations, leading to two amino acid alterations R225W and a novel T230A in thymidine kinase 2 (TK2) in arPEO patients. TK2 is an enzyme of the mitochondrial nucleotide salvage pathway and its loss-of-function mutations have previously been shown to underlie the early-infantile myopathic form of mtDNA depletion syndrome (MDS). Our TK2 activity measurements of patient fibroblasts and mutant recombinant proteins show that the combination of the identified arPEO variants, R225W and T230A, leads to a significant reduction in TK2 activity, consistent with the late-onset phenotype, whereas homozygosity for R225W, previously associated with MDS, leads to near-total loss of activity. Our finding identifies a new genetic cause of arPEO with multiple mtDNA deletions. Furthermore, MDS and multiple mtDNA deletion disorders are manifestations of the same pathogenic pathways affecting mtDNA replication and repair, indicating that MDS-associated genes should be studied when searching for genetic background of PEO disorders.

A new Caucasian case of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD): A clinical, molecular, and functional study

Citrin is the liver-specific isoform of the mitochondrial aspartate/glutamate carrier (AGC2). AGC2 deficiency is an autosomal recessive disorder with two age related phenotypes: neonatal intrahepatic cholestasis (NICCD, OMIM#605814) and adult-onset type II citrullinemia (CTLN2, OMIM#603471). NICCD arises within the first few weeks of life resulting in prolonged cholestasis and metabolic abnormalities including aminoacidemia and galactosuria. Usually symptoms disappear within the first year of life, thus making a diagnosis difficult after this time. In this study we report a new Caucasian case of NICCD, a seven week old Romanian boy with prolonged jaundice. Sequencing of the AGC2 gene showed a novel homozygous missense double-nucleotide (doublet) mutation, which produces the change of the glycine at position 437 into glutamate. Functional studies, carried out on the recombinant mutant protein, for the first time demonstrated, that NICCD is caused by a reduced transport activity of AGC2. The presence of AGC2 deficiency in other ethnic groups besides Asian population suggests further consideration for NICCD diagnosis of any neonate with an unexplained cholestasis; a prompt diagnosis is crucial to resolve the metabolic decompensation with an appropriate dietary treatment.

Expression of Bm-TFF2 mutants in Escherichia coli and their cell migration-promoting activity

Bm-TFF2, a trefoil factor from the large-webbed bell toad (Bombina maxima), can stimulate cell migration and inhibit cell apoptosis. To study the structure-function relationship of Bm-TFF2, we constructed wild-type and mutated Bm-TFF2 plasmids and expressed recombinant proteins in E. coli. The wild-type Bm-TFF2 gene encoding mature peptide was obtained by RT-PCR, while the N-terminal, C-terminal and two arginine mutated Bm-TFF2 clones were constructed, and ligated into pET-32a(+) expression vectors. The fusion proteins were induced by IPTG at 37 Degrees Celsius. The mutant Bm-TFF2 fusion proteins expressed mainly in the inclusion bodies. The mutant (TRX)/Bm-TFF2 could be purified by using Ni(2+)-chelating chromatography and reverse-phase HPLC from the inclusion body supernatant. The fusion proteins were analyzed by SDS-PAGE and Western blotting. The yield of mutant Bm-TFF2 fusion proteins of above 95% purity was about 20 mg/L. All three recombinant mutant proteins can promote the migration of AGS cells in a dose-dependent manner with no obvious activity difference.