Substitution Of Met Research Articles

Effect of Met/Leu substitutions on the stability of NAD+-dependent formate dehydrogenases from Gossypium hirsutum.

NAD+-dependent formate dehydrogenases (FDHs) are extensively used in the regeneration of NAD(P)H and the reduction of CO2 to formate. In addition to their industrial importance, FDHs also play a crucial role in the maintenance of a reducing environment to combat oxidative stress in plants. Therefore, it is important to investigate the response of NAD+-dependent FDH against both temperature and H2O2, to understand the defense mechanisms, and to increase its stability under oxidative stress conditions. In the present study, we characterized the oxidative and thermal stability of NAD+-dependent FDH isolated from cotton, Gossypium hirsutum (GhFDH), by investigating the effect of Met/Leu substitutions in the positions of 225, 234, and 243. Results showed that the single mutant, M234L (0.72 s-1 mM-1), and the triple mutant, M225L/M234L/M243L (0.55 s-1 mM-1), have higher catalytic efficiency than the native enzyme. Substitution of methionine by leucine on the position of 243 increased the free energy gain by 670 J mol-1. The most remarkable results in chemical stability were seen for double and triple mutants, cumulatively. Double and triple substitution of Met to Leu (M225L/M243L and M225L/M243L/M234L) reduce the kefin by a factor of 2 (12.3×10-5 and 12.8×10-5 s-1, respectively.Key points• The closer the residue to NAD+, in which we substituted methionine to leucine, the lower the stability against H2O2 we observed.• The significant gain in the Tm value for the M243L mutant was observed as +5°C.• Residue 234 occupies a critical position for oxidation defense mechanisms. Graphical abstract (a) Methionine amino acids on the protein surface are susceptible to oxidative stress and can be converted to methionine sulfoxide by reactive oxygen derivatives (such as hydrogen peroxide). Therefore, they are critical regions in the change of protein conformation and loss of activity. (b) Replacing the amino acid methionine, which is susceptible to oxidation due to the sulfur group, with the oxidation-resistant leucine amino acid is an important strategy in increasing oxidative stability.

Analogs of sulfakinin-related peptides demonstrate reduction in food intake in the red flour beetle, Tribolium castaneum, while putative antagonists increase consumption

The insect sulfakinins (SKs) constitute a family of neuropeptides that display both structural and functional similarities to the mammalian hormones gastrin and cholecystokinin (CCK). As a multifunctional neuropeptide, SKs are involved in muscle contractions as well as food intake regulation in many insects. In the red flour beetle Tribolium castaneum, the action on food intake by a series of synthetic SK analogs and one putative antagonist was investigated by injection in beetle adults. The most remarkable result was that both sulfated and non-sulfated SKs [FDDY(SO3H)GHMRFamide] inhibited food intake by about 70%. Strong activity observed for SK analogs featuring a residue that mimics the acidic nature of Tyr(SO3H) but lack the phenyl ring of Tyr, indicate that aromaticity is not a critical characteristic for this position of the peptide. SK demonstrated considerable tolerance to Ser and Ala substitution in position 8 (basic Arg), as analogs featuring these uncharged substitutions retained almost all of the food intake inhibitory activity. Also, the Phe in position 1 could be replaced by Ser without complete loss of activity. Conversely, substitution of Met by Nle in position 7 led to inactive compounds. Finally, the Caenorhabditis elegans sulfated neuropeptide-like protein-12 (NLP-12), that shares some sequence similarities with the SKs but features a Gln-Phe-amide rather than an Arg-Phe-amide at the C-terminus, elicited increased food intake in T. castaneum, which may indicate an antagonist activity. Co-injection of NLP-12 with nsSK blocked the food intake inhibitory effects of nsSK.

Selenomethionine Incorporation into Amyloid Sequences Regulates Fibrillogenesis and Toxicity

BackgroundThe capacity of a polypeptide chain to engage in an amyloid formation process and cause a conformational disease is contained in its sequence. Some of the sequences undergoing fibrillation contain critical methionine (Met) residues which in vivo can be synthetically substituted by selenomethionine (SeM) and alter their properties.Methodology/Principal FindingsUsing peptide synthesis, biophysical techniques and cell viability determinations we have studied the effect of the substitution of methionine (Met) by selenomethionine (SeM) on the fibrillogenesis and toxic properties of Aβ40 and HuPrP(106–140). We have found that the effects display site-specificity and vary from inhibition of fibrillation and decreased toxicity ([SeM35]Aβ40, [SeM129]HuPrP(106–140) and [SeM134]HuPrP(106–140)), retarded assembly, modulation of polymer shape and retention of toxicity ([SeM112]HuPrP(106–140) to absence of effects ([SeM109]HuPrP(106–140)).Conclusions/SignificanceThis work provides direct evidence that the substitution of Met by SeM in proamyloid sequences has a major impact on their self-assembly and toxic properties, suggesting that the SeM pool can play a major role in dictating the allowance and efficiency of a polypeptide chain to undergo toxic polymerization.

Profiling of Substrate Specificity of SARS-CoV 3CLpro

BackgroundThe 3C-like protease (3CLpro) of severe acute respiratory syndrome-coronavirus is required for autoprocessing of the polyprotein, and is a potential target for treating coronaviral infection.Methodology/Principal FindingsTo obtain a thorough understanding of substrate specificity of the protease, a substrate library of 198 variants was created by performing saturation mutagenesis on the autocleavage sequence at P5 to P3' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer. The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high β-sheet propensity; P4 prefers small hydrophobic residues; P2 prefers hydrophobic residues without β-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1' position prefers small residues, while P2' and P3' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence.Conclusions/SignificanceOur results demonstrated a strong structure-activity relationship between the 3CLpro and its substrate. The substrate specificity profiled in this study may provide insights into a rational design of peptidomimetic inhibitors.

Alternative Tasks of Drosophila Tan in Neurotransmitter Recycling Versus Cuticle Sclerotization Disclosed by Kinetic Properties

Upon a stimulus of light, histamine is released from Drosophila photoreceptor axonal endings. It is taken up into glia where Ebony converts it into beta-alanyl-histamine (carcinine). Carcinine moves into photoreceptor cells and is there cleaved into beta-alanine and histamine by Tan activity. Tan thus provides a key function in the recycling pathway of the neurotransmitter histamine. It is also involved in the process of cuticle formation. There, it cleaves beta-alanyl-dopamine, a major component in cuticle sclerotization. Active Tan enzyme is generated by a self-processing proteolytic cleavage from a pre-protein at a conserved Gly-Cys sequence motif. We confirmed the dependence on the Gly-Cys motif by in vitro mutagenesis. Processing time delays the rise to full Tan activity up to 3 h behind its putative circadian RNA expression in head. To investigate its pleiotropic functions, we have expressed Tan as a His(6) fusion protein in Escherichia coli and have purified it to homogeneity. We found wild type and mutant His(6)-Tan protein co-migrating in size exclusion chromatography with a molecular weight compatible with homodimer formation. We conclude that dimer formation is preceding pre-protein processing. Drosophila tan(1) null mutant analysis revealed that amino acid Arg(217) is absolutely required for processing. Substitution of Met(256) in tan(5), on the contrary, does not affect processing extensively but renders it prone to degradation. This also leads to a strong tan phenotype although His(6)-Tan(5) retains activity. Kinetic parameters of Tan reveal characteristic differences in K(m) and k(cat) values of carcinine and beta-alanyl-dopamine cleavage, which conclusively illustrate the divergent tasks met by Tan.

Despite its role in assembly, methionine 35 is not necessary for amyloid β‐protein toxicity

An important component of the pathologic process underlying Alzheimer's disease is oxidative stress. Met(35) in amyloid beta-protein (A beta) is prone to participating in redox reactions promoting oxidative stress, and therefore is believed to contribute significantly A beta-induced toxicity. Thus, substitution of Met(35) by residues that do not participate in redox chemistry would be expected to decrease A beta toxicity. Indeed, substitution of Met(35) by norleucine (Nle) was reported to reduce A beta toxicity. Surprisingly, however, substitution of Met(35) by Val was reported to increase toxicity. A beta toxicity is known to be strongly related to its self-assembly. However, neither substitution is predicted to affect A beta assembly substantially. Thus, the effect of these substitutions on toxicity is difficult to explain. We revisited this issue and compared A beta 40 and A beta 42 with analogs containing Met(35)-->Nle or Met(35)-->Val substitutions using multiple biophysical and toxicity assays. We found that substitution of Met(35) by Nle or Val had moderate effects on A beta assembly. Surprisingly, despite these effects, neither substitution changed A beta neurotoxicity significantly in three different assays. These results suggest that the presence of Met(35) in A beta is not important for A beta toxicity, challenging to the prevailing paradigm, which suggests that redox reactions involving Met(35) contribute substantially to A beta-induced toxicity.

ErbB-2 and Met Reciprocally Regulate Cellular Signaling via Plexin-B1

Sema4D-induced activation of plexin-B1 has been reported to evoke different and sometimes opposing cellular responses. The mechanisms underlying the versatility of plexin-B1-mediated effects are not clear. Plexin-B1 can associate with the receptor tyrosine kinases ErbB-2 and Met. Here we show that Sema4D-induced activation and inactivation of RhoA require ErbB-2 and Met, respectively. In breast carcinoma cells, Sema4D can have pro- and anti-migratory effects depending on the presence of ErbB-2 and Met, and the exchange of the two receptor tyrosine kinases is sufficient to convert the cellular response to Sema4D from pro- to anti-migratory and vice versa. This work identifies a novel mechanism by which plexin-mediated signaling can be regulated and explains how Sema4D can exert different biological activities through the differential association of its receptor with ErbB-2 and Met.

Evidence of the Importance of the Met115 for Bacillus thuringiensis subsp. israelensis Cyt1Aa Protein Cytolytic Activity in Escherichia coli

Cyt1Aa is a cytolytic toxin, found together with the delta-endotoxins in Bacillus thuringiensis subsp. israelensis parasporal insecticidal crystals. The latter are used as an environmental friendly insecticide against mosquitoes and black flies. Contrary to Cry delta-endotoxin, the mode of action of Cyt1Aa is not completely understood. In the absence of direct structural data, a novel mutated cyt1Aa gene was used to obtain indirect informations on Cyt1Aa conformation changes in the lipid membrane environment. A mutated cyt1Aa gene named cyt1A97 has been isolated from a B. thuringiensis israelensis strain named BUPM97. The nucleotide sequence predicted a protein of 249 amino acids residues with a calculated molecular mass of 27 kDa. Both nucleotide and amino acid sequences similarity analysis revealed that cyt1A97 presents one amino acid different from the native cyt1Aa gene. This mutation was located in the helix alpha C corresponding to a substitution of Met(115) by a Thr. The heterologous expression of the cyt1A97 and another cyt1Aa-type gene called cyt1A98, not affected by such mutation used as control, was performed in Escherichia coli. It revealed that the mutated Cyt1A97 protein was over produced as inclusion bodies showing a very weak toxicity to E. coli contrarily to Cyt1A98 that stopped E. coli growth. Hence, hydrophobic residue Met at position 115 of Cyt1Aa should play a very important role for the maintenance of the structure and cytolytic functions of Cyt1Aa.

The AGAAAAGA Palindrome in PrP Is Required to Generate a Productive PrPSc-PrPC Complex That Leads to Prion Propagation

The molecular hallmark of prion disease is the conversion of normal prion protein (PrPC) to an insoluble, proteinase K-resistant, pathogenic isoform (PrPSc). Once generated, PrPSc propagates by complexing with, and transferring its pathogenic conformation onto, PrPC. Defining the specific nature of this PrPSc-PrPC interaction is critical to understanding prion genesis. To begin to approach this question, we employed a prion-infected neuroblastoma cell line (ScN2a) combined with a heterologous yeast expression system to independently model PrPSc generation and propagation. We additionally applied fluorescence resonance energy transfer analysis to the latter to specifically study PrP-PrP interactions. In this report we focus on an N-terminal hydrophobic palindrome of PrP (112-AGAAAAGA-119) thought to feature intimately in prion generation via an unclear mechanism. We found that, in contrast to wild type (wt) PrP, PrP lacking the palindrome (PrPDelta112-119) neither converted to PrPSc when expressed in ScN2a cells nor generated proteinase K-resistant PrP when expressed in yeast. Furthermore, PrPDelta112-119 was a dominant-negative inhibitor of wtPrP in ScN2a cells. Both wtPrP and PrPDelta112-119 were highly insoluble when expressed in yeast and produced distinct cytosolic aggregates when expressed as fluorescent fusion proteins (PrP::YFP). Although self-aggregation was evident, fluorescence resonance energy transfer studies in live yeast co-expressing PrPSc-like protein and PrPDelta112-119 indicated altered interaction properties. These results suggest that the palindrome is required, not only for the attainment of the PrPSc conformation but also to facilitate the proper association of PrPSc with PrPC to effect prion propagation.

Role of Individual Methionines in the Fibrillation of Methionine-Oxidized α-Synuclein

The aggregation of normally soluble alpha-synuclein in the dopaminergic neurons of the substantia nigra is a crucial step in the pathogenesis of Parkinson's disease. Oxidative stress is believed to be a contributing factor in this disorder. We have previously established that oxidation of all four methionine residues in alpha-synuclein (to the sulfoxide, MetO) inhibits fibrillation of this protein in vitro and that the MetO protein also inhibits fibrillation of unmodified alpha-synuclein. Here we show that the degree of inhibition of fibrillation by MetO alpha-synuclein is proportional to the number of oxidized methionines. This was accomplished be selectively converting Met residues into Leu, prior to Met oxidation. The results showed that with one oxidized Met the kinetics of fibrillation were comparable to those for the control (nonoxidized), and with increasing numbers of methionine sulfoxides the kinetics of fibrillation became progressively slower. Electron microscope images showed that the fibril morphology was similar for all species examined, although fewer fibrils were observed with the oxidized forms. The presence of zinc was shown to overcome the Met oxidation-induced inhibition. Interestingly, substitution of Met by Leu led to increased propensity for aggregation (soluble oligomers) but slower formation of fibrils.

In Vivo Mutagenesis by Escherichia coliDNA Polymerase I: ILE709 IN MOTIF A FUNCTIONS IN BASE SELECTION

The fidelity of DNA replication by Escherichia coli DNA polymerase I (pol I) was assessed in vivo using a reporter plasmid bearing a ColE1-type origin and an ochre codon in the beta-lactamase gene. We screened 53 single mutants within the region Val(700)-Arg(712) in the polymerase active-site motif A. Only replacement of Ile(709) yielded mutator polymerases, with substitution of Met, Asn, Phe, or Ala increasing the beta-lactamase reversion frequency 5-23-fold. Steady-state kinetic analysis of the I709F polymerase revealed reductions in apparent K(m) values for both insertion of non-complementary nucleotides and extension of mispaired primer termini. Abolishment of the 3'-5' exonuclease activity of wild-type pol I increased mutation frequency 4-fold, whereas the combination of I709F and lack of the 3'-5' exonuclease yielded a 400-fold increase. We conclude that accurate discrimination of the incoming nucleotide at the polymerase domain is more critical than exonucleolytic proofreading for the fidelity of pol I in vivo. Surprisingly, the I709F polymerase enhanced mutagenesis in chromosomal DNA, although the increase was 10-fold less than in plasmid DNA. Our findings indicate the feasibility of obtaining desired mutations by replicating a target gene at a specific locus in a plasmid under continuous selection pressure.

Identification of determinants of inverse agonism in a constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor by photoaffinity cross-linking and mutational analysis.

We have investigated receptor structural components responsible for ligand-dependent inverse agonism in a constitutively active mutant of the human parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor type 1 (hP1R). This mutant receptor, hP1R-H223R (hP1R(CAM-HR)), was originally identified in Jansen's chondrodysplasia and is altered in transmembrane domain (TM) 2. We utilized the PTHrP analog, [Bpa(2),Ile(5),Trp(23),Tyr(36)]PTHrP-(1-36)-amide (Bpa(2)-PTHrP-(1-36)), which has valine 2 replaced by p-benzoyl-l-phenylalanine (Bpa); this substitution renders the peptide a photoreactive inverse agonist at hP1R(CAM-HR). This analog cross-linked to hP1R(CAM-HR) at two contiguous receptor regions as follows: the principal cross-link site (site A) was between receptor residues Pro(415)-Met(441), spanning the TM6/extracellular loop three boundary; the second cross-link site (site B) was within the TM4/TM5 region. Within the site A interval, substitution of Met(425) to Leu converted Bpa(2)-PTHrP-(1-36) from an inverse agonist to a weak partial agonist; this conversion was accompanied by a relative shift of cross-linking from site A to site B. The functional effect of the M425L mutation was specific for Bpa(2)-containing analogs, as inverse agonism of Bpa(2)-PTH-(1-34) was similarly eliminated, whereas inverse agonism of [Leu(11),d-Trp(12)]PTHrP-(5-36) was not affected. Overall, our data indicate that interactions between residue 2 of the ligand and the extracellular end of TM6 of the hP1R play an important role in modulating the conversion between active and inactive receptor states.

Nonessential role for methionines in the productive association between calmodulin and the plasma membrane Ca-ATPase.

To investigate the role of hydrophobic interactions involving methionine side chains in facilitating the productive association between calmodulin (CaM) and the plasma membrane (PM) Ca-ATPase, we have substituted the polar amino acid Gln for Met at multiple positions in both the amino- and carboxyl-terminal domains of CaM. Conformationally sensitive fluorescence signals indicate that these mutations have little effect on the backbone fold of the carboxyl-terminal domain of CaM. The insertion of multiple Gln in either globular domain results in a decrease in the apparent affinity of CaM for the PM-Ca-ATPase. However, despite the multiple substitution of Gln for four methionines at positions 36, 51, 71, and 72 in the amino-terminal domain or for three methionines at positions 124, 144, and 145 in the carboxyl-terminal domain, these mutant CaMs are able to fully activate the PM-Ca-ATPase. Thus, although these CaM mutants have a decreased affinity for the CaM-binding site on the Ca-ATPase, they retain the ability to fully activate the Ca-ATPase at saturating concentrations of CaM. The role of individual methionines in modulating the affinity between the carboxyl terminus and the PM-Ca-ATPase was further investigated through the substitution of individual Met with Gln. Upon substitution of Met(124) and Met(144) with Gln, there is a 5- and 10-fold increase in the amount of CaM necessary to obtain half-maximal activation of the PM-Ca-ATPase, indicating that these methionine side chains participate in the high-affinity association between CaM and the PM-Ca-ATPase. However, substitution of Gln for Met(145) results in no change in the apparent affinity between CaM and the PM-Ca-ATPase, indicating that in contrast to all other known CaM targets, Met(145) does not participate in the interaction between CaM and the PM-Ca-ATPase. These results emphasize differences in the binding interactions between individual methionines in CaM and different target enzymes, and suggest that hydrophobic interactions between methionines in CaM and the binding site on the PM-Ca-ATPase are not necessary for enzyme activation. Calculation of the binding affinities of individual CaM domains associated with activation of the PM-Ca-ATPase suggests that mutations of methionines located in either domain of CaM can decrease the initial high-affinity association between CaM and the PM-Ca-ATPase, but have little effect upon the subsequent binding of the opposing globular domain. These results suggest that the initial associations between CaM and the CaM-binding sequence in the PM-Ca-ATPase are guided by nonspecific hydrophobic interactions involving both domains of CaM.

General role of GDP dissociation inhibitor 2 in membrane release of Rab proteins: modulations of its functional interactions by in vitro and in vivo structural modifications.

The GDP dissociation inhibitors (GDIs) represent an important class of regulatory proteins in the functional cycle and recycling of Rab GTPases. Previous studies have demonstrated that GDI-1 can operate with multiple Rab proteins. In this study we have addressed a plausible general activity of GDI-2 in supporting Rab membrane release and have analyzed the requirements of sequence-conserved vs variable regions of GDI-2 in these functional interactions. The in vitro function of expressed recombinant GDI-2 wild-type-, point-, or deletion-mutant proteins was investigated toward several Rab family members, divergent in structure, localized and operating on different membranes, including Rab2, Rab4, Rab5, Rab8, Rab9, and Rab11. We demonstrate here a general and nearly invariant ability of GDI-2(WT) to release from membranes this subset of diverse Rabs. Deletion of an 18-residue segment from the C-terminal variable region yielded a fully functional or only slightly defective GDI-2. Conversely, substitution of Met at position 250 of the conserved region markedly abrogated the activity toward all Rabs. Surprisingly, a replacement of an adjacent conserved residue (Y249V) resulted in a selective Rab-dependent response and a profound gain of function toward specific Rabs. To further test whether the endogenous GDI-2 can adopt a gain-of-function conformation, we pharmacologically stimulated intact 3T3-L1 adipocytes to induce GDI-2 tyrosine phosphorylation. We found a pronounced increase of the Rab4 soluble form and its soluble complexes with the tyrosine-phosphorylated GDI-2. Together, these results indicate that (a) GDI-2 displays a general activity to release Rabs from membranes, (b) GDI-2-conserved residues, but not the C-terminal variable region, are essential for this activity, and (c) structural modifications in GDI-2 can enhance its functional activity, directing selective interactions with individual Rabs.

High expression and steady-state kinetic characterization of methionine site-directed mutants of Escherichia coli methionyl- and selenomethionyl-dihydrofolate reductase

A high expression system that produces Escherichia coli dihydrofolate reductase (DHFR) at 30% total cellular protein was constructed. This expression vector, named pCOCK, allowed for the purification of nearly 100 mg of homogeneous DHFR from a 1 l bacterial culture. A simple, single Q-Sepharose anion exchange column purification was developed on an FPLC instrument. Methionine site-directed mutants were constructed in DHFR to assess the role of Met within the enzymes. These mutants consisted of a Met 16leucine (Leu), Met 20Leu, Met 42Leu, Met 92Leu, Met 16,20Leu and Met 16,20,42Leu. Steady-state kinetic studies showed that the Met 16Leu, Met 42Leu and Met 92Leu mutants possessed essentially the same k cat, K m(DHF) and K m(NADPH) as that of wild-type (wt) DHFR (13.7 s −1, 0.97 μM and 2.52 μM, respectively). Mutants which contained a Leu at position 20 possessed substantially elevated specific activity and k cat values. The specific activity and k cat of wt, Met 20Leu, Met 16,20Leu and Met 16,20,42Leu were 45.9, 92.7, 90.2 and 172 μmol/min/mg and 13.7, 24.6, 25.2 and 52.7 s −1, respectively. Upon substitution of Met by selenomethionine (SeMet) in the aforementioned mutants, further information as to the effect of SeMet incorporation into proteins was ascertained. Steady-state kinetic parameters of the SeMet substituted Met 16Leu, Met 20Leu, Met 42Leu and Met 92Leu mutants were nearly identical to those of their Met containing counterparts. These data indicate that Met apparently has a limited role in the protein structure and function of DHFR and that SeMet incorporation has no effect on the steady-state kinetic constants of DHFR.

Production of selenomethionine-labeled recombinant human neutrophil collagenase in Escherichia coli

Molecular analogs of amino acids can be incorporated into proteins. The amino acid analog selenomethionine (SeMet) has been shown to be efficiently incorporated into the proteins of growing Escherichia coli. SeMet-containing proteins are known to produce sufficiently strong anomalous scatter permitting the solution of the selenomethionyl crystal structure by multiwavelength anomalous diffraction (MAD) techniques. The recombinant protein chosen for these studies is mature, truncated neutrophil collagenase (rmNC-t). The rmNC-t protein is a monomer of 163 amino acid residues featuring one active site and two Met residues. We developed a T7 polymerase expression system allowing incorporation of SeMet into rmNC-t protein produced in E. coli. Substitution of Met with SeMet was accomplished by culturing E. coli DL41(DE3), a SeMet-tolerant strain with metA lesion, in a defined medium containing SeMet as the sole source of Met. The SeMet-labeled rmNC-t was isolated from inclusion bodies by solubilizing in urea, purified by anion column chromatography, and then refolded in the presence of Ca 2+ and Zn 2+. Analysis of SeMet-labeled rmNC-t demonstrated that Met replacement was 100%. Enzymatic characterization revealed no obvious differences in activity or inhibitor binding between rmNC-t and the SeMet-labeled product. We have produced pure, active SeMet-labeled rmNC-t in sufficient quantities for macromolecular crystallography studies.

South African founder mutations in the low-density lipoprotein receptor gene causing familial hypercholesterolemia in the Dutch population.

In South African Afrikaners, three point mutations in the gene coding for the low-density lipoprotein (LDL)-receptor are responsible for more than 95% of the cases of familial hypercholesterolemia (FH). To investigate whether one or more of these mutations originated in The Netherlands, a large group of Dutch heterozygous FH patients was screened for the presence of these three mutations. Of these, a missense mutation in exon 9 of the LDL-receptor gene, resulting in a substitution of Met for Val408, responsible for 15% of FH in Afrikaners, was found in 19 (1.5%) of 1268 FH patients of Dutch descent. Nine of the patients carrying the exon 9 mutation on one allele shared the LDL-receptor DNA haplotype with an FH patient from South Africa, who was homozygous for the same mutation. This would suggest that the mutation in these patients and in the South African patient have a common ancestral background. The remaining ten FH patients all shared a common haplotype, partly identical to the Afrikaner haplotype, which could have arisen from a single recombinational event. This mutation has not been described in persons other than of Dutch ancestry and supports the hypothesis that this mutation in exon 9 originated in The Netherlands and, in all likelihood, was introduced into South Africa by early Dutch settlers in the seventeenth century.

Detection of a new mutant α-1-antichymotrypsin in patients with occlusive-cerebrovascular disease

A new mutant α-1-antichymotrypsin (variant ACT) was found by direct sequencing and PCR-single strand conformation polymorphism (PCR-SSCP). This variant ACT was a point mutation of exon V of ACT, with the substitution of Met by Val. Four out of six individuals with this variant ACT had occlusive-cerebrovascular disease, leading to one hypothesis that there might be an association between this mutation and occlusive-cerebrovascular disease.

Expressed Full-Length von Willebrand Factor Containing Missense Mutations Linked to Type IIB von Willebrand Disease Shows Enhanced Binding to Platelets

von Willebrand disease (vWD) variant type IIB is an inherited bleeding disorder resulting from the spontaneous binding of defective von Willebrand factor (vWF) to platelets in vivo. To identify the molecular basis for type IIB vWD, we used reverse transcription and the polymerase chain reaction to examine the nucleotide sequence of the platelet glycoprotein (GP) Ib-binding domain encoded by the vWF messenger RNA in an affected family, and in an unrelated affected individual. We identified two different missense mutations linked with expression of type IIB vWD. These mutations, which lead to Pro574----Leu and Val553----Met substitutions, respectively, were each introduced into the full-length vWF expression vector pvW198, and both wild-type (wt) and mutant vWF were transiently expressed in COS-7 cells. Binding assays showed that both mutant proteins showed significant non-ristocetin-dependent spontaneous binding to platelets, and that complete binding was induced by low concentrations of ristocetin that failed to induce platelet binding by wt vWF. The vWF/platelet interaction was inhibited by the anti-vWF monoclonal antibody (MoAb) AvW3, and the anti-GPIb MoAb AP1, which both block vWF binding to platelets. These results show that the identified missense mutations are the likely basis for the expression of type IIB vWD in these affected individuals.

Expressed full-length von Willebrand factor containing missense mutations linked to type IIB von Willebrand disease shows enhanced binding to platelets

von Willebrand disease (vWD) variant type IIB is an inherited bleeding disorder resulting from the spontaneous binding of defective von Willebrand factor (vWF) to platelets in vivo. To identify the molecular basis for type IIB vWD, we used reverse transcription and the polymerase chain reaction to examine the nucleotide sequence of the platelet glycoprotein (GP) Ib-binding domain encoded by the vWF messenger RNA in an affected family, and in an unrelated affected individual. We identified two different missense mutations linked with expression of type IIB vWD. These mutations, which lead to Pro574---- Leu and Val553----Met substitutions, respectively, were each introduced into the full-length vWF expression vector pvW198, and both wild-type (wt) and mutant vWF were transiently expressed in COS-7 cells. Binding assays showed that both mutant proteins showed significant non- ristocetin-dependent spontaneous binding to platelets, and that complete binding was induced by low concentrations of ristocetin that failed to induce platelet binding by wt vWF. The vWF/platelet interaction was inhibited by the anti-vWF monoclonal antibody (MoAb) AvW3, and the anti-GPIb MoAb AP1, which both block vWF binding to platelets. These results show that the identified missense mutations are the likely basis for the expression of type IIB vWD in these affected individuals.