Combined assessment of 3q26 amplification and promoter methylation in patients with high grade cervical lesions show age specific differences.

A considerable proportion of high grade cervical intraepithelial lesions (CIN2/3) are known to resolve on their own especially among young women. However, since reliable prognostic markers are still lacking, the diagnosis "CIN3" is still an indication for surgery which may result in overtreatment. It is conceivable that a combination of different, ideally independent molecular markers may provide more reliable results. In the present cross-sectional study two established triage markers, 3q26 amplification and a methylation signature, were evaluated in an age-dependent manner. The patient cohort comprised 60 patients with histologically confirmed CIN2/3 in two equally sized age groups (<30 years, ≥30 years). Cervical scrapes were analyzed by interphase fluorescence in situ hybridization for 3q26 amplification and methylation specific PCR (GynTect®) for six different genome regions. Both assays showed a significantly different pattern of test outcome independent of age (P = .001). Moreover, the combination of both assays differed significantly for double positive and double negative cases when comparing the two age groups: In patients <30 years there were clearly less cases with positive methylation signature and amplification of 3q26 as in women ≥30 years (23% vs 63%, Bonferroni adjusted P = .016). Of particular interest is the finding that double negative results were exclusive for the young age group (0% vs 27%, Bonferroni adjusted P = .020). Since regression of CIN2/3 characteristically occurs among young women it is tempting to speculate that a double negative test result could be prognostic for regression of CIN2/3. This will have to be investigated further in a prospective longitudinal intervention study.

Performance of a DNA methylation marker panel using liquid-based cervical scrapes to detect cervical cancer and its precancerous stages.

BACKGROUND: A change of cervical cancer screening algorithms to an HPV-based screening setting is discussed in many countries, due to higher sensitivity of HPV testing compared to cytology. Reliable triage methods are, however, an essential prerequisite in such a setting to avoid overtreatment and higher screening costs. RESULTS: In this study, a series of cervical scrapes collected in PreservCyt liquid-based cytology (LBC) medium from women with cervical cancer (n = 5), cervical intraepithelial neoplasia grade 1-3 (n = 74), and normal cytology (n = 201; further n = 352 collected in SureThin®) were assessed for methylation of the marker regions ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671 using the GynTect assay and compared to cobas® HPV and CINtec Plus® biomarker results. All samples from women with cervical cancer, 61.2% of CIN3, 44.4% of CIN2 and 20.0% of CIN1 cases were scored positive for the GynTect methylation assay. In contrast, all CIN, irrespective of severity grade, and carcinomas were positive by both, CINtec Plus and cobas HPV. The specificity of GynTect for CIN3+ was 94.6% compared to 69.9% for CINtec Plus and 82.6% for cobas HPV (all HPV types) and 90.6% for cobas HPV 16/18. DNA methylation analysis of this methylation marker panel (GynTect assay) in cervical scrapes consistently detects cervical cancer and the majority of CIN3 as well as a subset of CIN1/2 lesions. The detection rate among cytologically normal samples is extraordinarily low (1.5%). CONCLUSION: GynTect shows excellent performance when using cervical scrape material collected in liquid-based cytology media, a prerequisite for employing such a test as a triage in screening programs. Compared to the other test systems used in this work, GynTect showed higher specificity while still detecting all cancer cases.

Triage of hrHPV-positive women: comparison of two commercial methylation-specific PCR assays.

AIM: High-risk human papillomavirus (hrHPV)-based screening is becoming increasingly important, either by supplementing or replacing the traditional cytology-based cervical Pap smear. However, hrHPV screening lacks specificity, because it cannot differentiate between transient virus infection and clinically relevant hrHPV-induced disease. Therefore, reliable triage methods are needed for the identification of HPV-positive women with cervical intraepithelial neoplasia (CIN) in need of treatment. Promising tools discussed for the triage of these patients are molecular diagnostic tests based on epigenetic markers. Here, we compare the performance of two commercially available DNA methylation-based diagnostic assays-GynTect® and the QIAsure Methylation Test-in physician-taken cervical scrapes from 195 subjects. FINDINGS: Both GynTect® and the QIAsure Methylation Test detected all cervical carcinoma and carcinoma in situ (CIS). The differences observed in the detection rates between both assays for the different grades of cervical lesions (QIAsure Methylation Test: CIN1 26.7%, CIN2 27.8% and CIN3 74.3%; GynTect®: CIN1 13.3%, CIN2 33.3% and CIN3 60%) were not significant. Concerning the false-positive rates, significant differences were evident. For the healthy (NILM) hrHPV-positive group, the false-positive rates were 5.7% for GynTect® and 26.4% for QIAsure Methylation Test (p = 0.003) and for the NILM hrHPV-negative group 2.2% vs. 23.9% (p = 0.006), respectively. When considering hrHPV-positive samples only for comparison (n = 149), GynTect® delivered significantly higher specificity compared to the QIAsure Methylation Test for CIN2 + (87.6% vs. 67.4% (p < 0.001)) and CIN3 + (84.1% vs. 68.2% (p = 0.002)). Overall our findings suggest that DNA methylation-based tests are suitable for the triage of hrHPV-positive women. With the goal to provide a triage test that complements the limited specificity of HPV testing in HPV-based screening, GynTect® may be preferable, due to its higher specificity for CIN2+ or CIN3+ .

Methionine sulfoxide reductase A and a dietary supplement S-methyl-L-cysteine prevent Parkinson's-like symptoms.

Parkinson's disease (PD), a common neurodegenerative disease, is caused by loss of dopaminergic neurons in the substantia nigra. Although the underlying cause of the neuronal loss is unknown, oxidative stress is thought to play a major role in the pathogenesis of PD. The amino acid methionine is readily oxidized to methionine sulfoxide, and its reduction is catalyzed by a family of enzymes called methionine sulfoxide reductases (MSRs). The reversible oxidation-reduction cycle of methionine involving MSRs has been postulated to act as a catalytic antioxidant system protecting cells from oxidative damage. Here, we show that one member of the MSR family, MSRA, inhibits development of the locomotor and circadian rhythm defects caused by ectopic expression of human alpha-synuclein in the Drosophila nervous system. Furthermore, we demonstrate that one way to enhance the MSRA antioxidant system is dietary supplementation with S-methyl-L-cysteine (SMLC), found abundantly in garlic, cabbage, and turnips. SMLC, a substrate in the catalytic antioxidant system mediated by MSRA, prevents the alpha-synuclein-induced abnormalities. Therefore, interventions focusing on the enzymatic reduction of oxidized methionine catalyzed by MSRA represent a new prevention and therapeutic approach for PD and potentially for other neurodegenerative diseases involving oxidative stress.

Identification of a new functional splice variant of the enzyme methionine sulphoxide reductase A (MSRA) expressed in rat vascular smooth muscle cells.

Reactive oxygen species contribute to ageing of the vascular system and development of cardiovascular disease. Methionine-S-sulphoxide, an oxidized form of methionine, is repaired by the enzyme methionine sulphoxide reductase A (MSRA). The enzyme, targeted to mitochondria or the cytosol by alternative splicing, is vital for oxidative stress resistance. This study was designed to examine the endogenous expression and intracellular localization of MSRA in rat aortic vascular smooth muscle cells (VSMCs). We detected robust MSRA immunoreactivity exclusively in mitochondria. Sequence analysis of msrA transcripts revealed the presence of a novel mitochondrial splice variant, msrA2a, in cultured rat VSMCs as well as in aortic tissue preparations. The enzymatic activity of a recombinant MSRA2a protein was confirmed by the reduction of methionine sulphoxide in a model substrate peptide. We conclude that multiple MSRA variants participate in the repair of oxidized proteins in VSMC mitochondria, but that other protective mechanisms may exist in the cytoplasmic compartment.

Performance of a methylation specific real-time PCR assay as a triage test for HPV-positive women.

BACKGROUND: HPV DNA testing as a primary screening marker is being implemented in several countries. Due to the high HPV prevalence in the screening population, effective triage strategies for HPV-positive cases are required. The aim of this study was to evaluate the performance of a methylation-specific real-time PCR  assay (GynTect®) comprising six marker regions as a triage test. RESULTS: An analytical sensitivity of 0.1 ng genomic DNA corresponding to 15 SiHa cells was achieved. Absolute specificity was observed in the presence of 20 ng unmethylated genomic DNA. In a clinical setting, cervical scrapes of 306 women showing abnormal colposcopy were tested for cytology, HPV positivity, and the GynTect markers ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671. Of all women, histopathological data were available. The overall sensitivity for GynTect to detect CIN3+ was 67.7% (95% CI 57.3%-77.1%) whereas sensitivity was significantly higher for women of age ≥ 30 years (p = 0.04). All cancer cases (n = 5) were detected by GynTect. The overall false positive rate (= 1-specificity) for women with no CIN was 17.4% (95% CI 12.5-23.1%), with a higher proportion among HPV-positive women (24.0%, 95% CI 16.0-33.6%). In a triage screening setting, where all women underwent HPV testing and the HPV positives in addition GynTect testing, the overall sensitivity would slightly decline but specificity would reach the maximum value of 88.7% (95% CI 83.7-92.6%). CONCLUSION: The GynTect® assay is a robust easy to use assay with high analytical sensitivity and specificity. Moreover, the performance of the assay based on cervical scrapes provides further evidence for the usefulness of methylation markers to detect HPV-positive women with clinically relevant disease.

Heterogeneity and function of mammalian MSRs: enzymes for repair, protection and regulation.

Methionine sulfoxide, the physiologically relevant oxidation product of methionine, is enzymatically reduced by peptide methionine sulfoxide reductases (MSRs). Two distinct classes of these enzymes, MSRA and MSRB, which selectively reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, are found in virtually all organisms. Mammals typically possess only one gene encoding MSRA, but at least three genes encoding MSRBs. These MSRs show distinct tissue- and subcellular expression patterns and may play specific functional roles. Susceptibility of some ion channels to reversible methionine oxidation suggests that MSRs have a regulatory role in cellular excitability. Some--if not all--MSRs protect cells and organisms against a variety of oxidative stress episodes, including those by hypoxia and reperfusion, and play a modulatory role in lifespan determination. More MSR-dependent physiological phenomena await to be discovered.

The repair enzyme peptide methionine-S-sulfoxide reductase is expressed in human epidermis and upregulated by UVA radiation.

Recently, we reported that photoaging correlates well with the amount of oxidized protein accumulated in the upper dermis, while protein oxidation levels in the viable epidermis are very low. We hypothesized that this might be due to epidermal expression of the repair enzymes methionine sulfoxide reductases (MSRs). The expression of human methionine sulfoxide reductase A (MSRA) was investigated in HaCaT cells, primary human keratinocytes, and in human skin. High MSRA mRNA and protein levels as well as MSR activity were found in cultured human keratinocytes. MSRA was expressed in human epidermis, as shown by immunohistochemistry in healthy human skin. Repetitive in vivo exposure of human skin to solar-simulated light on 10 consecutive days (n=10 subjects) significantly increased epidermal MSRA expression. To further assess the functional relevance of the enzyme, its expression in response to UVB, UVA, and H(2)O(2) was investigated in HaCaT cells. While UVB lowered protein expression of MSRA, an upregulation was observed in response to low doses of UVA and H(2)O(2). In summary, MSRA represents the only enzyme so far identified in human skin that is capable of repairing oxidative protein damage. In addition to melanogenesis and DNA repair systems, a wavelength-specific activation of epidermal MSRA may be involved in epidermal photoprotection.

The muO-conotoxin MrVIA inhibits voltage-gated sodium channels by associating with domain-3.

Several families of peptide toxins from cone snails affect voltage-gated sodium (Na(V)) channels: mu-conotoxins block the pore, delta-conotoxins inhibit channel inactivation, and muO-conotoxins inhibit Na(V) channels by an unknown mechanism. The only currently known muO-conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (Na(V)1.4) and brain (Na(V)1.2) sodium channels in mammalian cells. A systematic domain-swapping strategy identified the C-terminal pore loop of domain-3 as the major determinant for Na(V)1.4 being more potently blocked than Na(V)1.2 channels. muO-conotoxins therefore show an interaction pattern with Na(V) channels that is clearly different from the related mu- and delta-conotoxins, indicative of a distinct molecular mechanism of channel inhibition.

Molecular interaction of delta-conotoxins with voltage-gated sodium channels.

Various neurotoxic peptides modulate voltage-gated sodium (Na(V)) channels and thereby affect cellular excitability. Delta-conotoxins from predatory cone snails slow down inactivation of Na(V) channels, but their interaction site and mechanism of channel modulation are unknown. Here, we show that delta-conotoxin SVIE from Conus striatus interacts with a conserved hydrophobic triad (YFV) in the domain-4 voltage sensor of Na(V) channels. This site overlaps with that of the scorpion alpha-toxin Lqh-2, but not with the alpha-like toxin Lqh-3 site. Delta-SVIE functionally competes with Lqh-2, but exhibits strong cooperativity with Lqh-3, presumably by synergistically trapping the voltage sensor in its "on" position.

Mitochondrial targeting of the human peptide methionine sulfoxide reductase (MSRA), an enzyme involved in the repair of oxidized proteins.

Peptide methionine sulfoxide reductase (MSRA) catalyzes the reduction of methionine sulfoxide to methionine. This widely expressed enzyme constitutes an important repair mechanism for oxidatively damaged proteins, which accumulate during the manifestation of certain degenerative diseases and aging processes. In addition, it is discussed to be involved in regulatory processes. Here we address the question of how the enzyme's diverse functions are reflected in its subcellular localization. Using fusions of the human version of MSRA with the enhanced green fluorescence protein expressed in various mammalian cell lines, we show a distinct localization at mitochondria. The N-terminal 23 amino acid residues contain the signal for this mitochondrial targeting. Activity tests showed that they are not required for enzyme function. Mitochondrial localization of native MSRA in mouse and rat liver slices was verified with an MSRA-specific antibody by using immunohistochemical methods. The protein was located in the mitochondrial matrix, as demonstrated by using pre-embedding immunostaining and electron microscopy. Mitochondria are the major source of reactive oxygen species (ROS). Therefore, MSRA has to be considered an important means for the general reduction of ROS release from mitochondria.

Activity, tissue distribution and site-directed mutagenesis of a human peptide methionine sulfoxide reductase of type B: hCBS1.

Human CBS1 is a methionine sulfoxide reductase of type B (MSRB) as it specifically reduced Met-R-SO in peptides with dithiothreitol or the thioredoxin system as reductants. Mutation C169S in the active site completely abolished enzymatic activity, while mutation W110A only reduced activity and C105S had no effect. Like human MSRA, hCBS1 showed in vivo reducing activity coexpressed with the Drosophila ShC/B potassium channel in oocytes, by accelerating the overall inactivation time course. hCBS1-encoding mRNA is most abundant in muscle tissues, especially in the heart and thereby shows an expression pattern different to the human MSRA.

A second human methionine sulfoxide reductase (hMSRB2) reducing methionine-R-sulfoxide displays a tissue expression pattern distinct from hMSRB1.

Peptide methionine sulfoxide reductases are important enzymes in the defense against cellular oxidative stress as they reduce methionine sulfoxide, the product of methionine oxidation by physiologically relevant reactive oxygen species. Two distinct enzyme classes, MSRA and MSRB, have evolved for selectively reducing the two epimers, methionine-S-sulfoxide and methionine-R-sulfoxide. A new human MSR enzyme (hMSRB2) specifically reducing methionine-R-sulfoxide, which showed a conversion rate for peptide-bound methionine-S-sulfoxide similar to hMSRB1, was characterized with respect to its tissue expression. As previously found for hMSRB1, expression of hMSRB2 mRNA was weak in brain, but strong in heart and skeletal muscle. In contrast to hMSRB1, its expression was high in smooth muscle-containing organs (digestive system, bladder), lung and aorta, while hMSRB1 displayed a higher expression than hMSRB2 in liver and kidney.

A promising DNA methylation signature for the triage of high-risk human papillomavirus DNA-positive women.

High-risk human papillomavirus (hrHPV)-DNA testing is frequently performed parallel to cytology for the detection of high-grade dysplasia and cervical cancer particularly in women above 30 years of age. Although highly sensitive, hrHPV testing cannot distinguish between HPV-positive women with or without clinically relevant lesions. However, in principle discrimination is possible on the basis of DNA methylation markers. In order to identify novel DNA regions which allow an effective triage of hrHPV-positive cases, hypermethylated DNA enriched from cervical cancers was compared with that from cervical scrapes of HPV16-positive cases with no evidence for disease by CpG island microarray hybridization. The most promising marker regions were validated by quantitative methylation-specific PCR (qMSP) using DNA from archived cervical tissues and cervical scrapes. The performance of these markers was then determined in an independent set of 217 hrHPV-positive cervical scrapes from outpatients with histopathological verification. A methylation signature comprising the 5' regions of the genes DLX1, ITGA4, RXFP3, SOX17 and ZNF671 specific for CIN3 and cervical cancer (termed CIN3+) was identified and validated. A high detection rate of CIN3+ was obtained if at least 2 of the 5 markers were methylated. In the subsequent cross-sectional study all cervical carcinomas (n = 19) and 56% (13/23) of CIN3 were identified by this algorithm. Only 10% (11/105) of hrHPV-positive women without histological evidence of cervical disease were scored positive by the methylation assay. Of note is that the detection rate of CIN3 differed between age groups. Eight of nine CIN3 were detected among women ≥30 years of age but only five of fourteen among <30 year old group (p = 0.03). The specificity for CIN3+ in the older age group was 76.6% (95% CI 65.6-85.5%). Clinical validation studies are required to determine the usefulness of these novel markers for triage after primary hrHPV testing in a cervical cancer screening setting.

Oxidation of multiple methionine residues impairs rapid sodium channel inactivation.

Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel Na(V)1.4 expressed in mammalian cells was studied by applying the Met-preferring oxidant chloramine-T or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile-Phe-Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of Met1470 in the putative receptor of the inactivation lid also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues in intracellular linkers connecting the membrane-spanning segments (442, 1139, 1154, 1316, 1469) were of minor importance. The results of mutagenesis, assays of other Na(V) channel isoforms (Na(V)1.2, Na(V)1.5, Na(V)1.7), and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of Na(V) channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion.

Subtype specificity of scorpion beta-toxin Tz1 interaction with voltage-gated sodium channels is determined by the pore loop of domain 3.

Voltage-gated sodium (Nav) channels are modulated by a variety of specific neurotoxins. Scorpion beta-toxins affect the voltage-dependence of channel gating: In their presence, Nav channels activate at subthreshold membrane voltages. Previous mutagenesis studies have revealed that the beta-toxin Css4 interacts with the extracellular linker between segments 3 and 4 in domain 2 of Nav channels with the effect to trap this voltage sensor in an open position (Neuron 21: 919-931, 1998 ). The voltage sensor of domain 2 was thus identified to constitute a major part of neurotoxin receptor site 4. In this work, we studied the effects of the beta-toxin Tz1 from the Venezuelan scorpion Tityus zulianus on various mammalian Nav channel types expressed in HEK 293 cells. Although skeletal muscle channels (Nav1.4) were strongly affected by Tz1, the neuronal channels Nav1.6 and Nav1.2 were less sensitive, and the cardiac Nav1.5 and the peripheral nerve channel Nav1.7 were essentially insensitive. Analysis of channel chimeras in which whole domains of Nav1.2 were inserted into a Nav1.4 background revealed that the Nav1.2 phenotype was not conferred to Nav1.4 by domain 2 but by domain 3. The interaction epitope could be narrowed down to residues Glu1251, Lys1252, and His1257 located in the C-terminal pore loop in domain 3. The receptor site for beta-toxin interaction with Nav channels thus spans domains 2 and 3, where the pore loop in domain 3 specifies the pharmacological properties of individual neuronal Nav channel types.

Heterocyst-specific excision of the Anabaena sp. strain PCC 7120 hupL element requires xisC.

In nitrogen-limiting conditions, approximately 10% of the vegetative cells in filaments of the cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 differentiate into nitrogen-fixing heterocysts. During the late stages of heterocyst differentiation, three DNA elements, each embedded within an open reading frame, are programmed to excise from the chromosome by site-specific recombination. The DNA elements are named after the genes that they interrupt: nifD, fdxN, and hupL. The nifD and fdxN elements each contain a gene, xisA or xisF, respectively, that encodes the site-specific recombinase required for programmed excision of the element. Here, we show that the xisC gene (alr0677), which is present at one end of the 9,435-bp hupL element, is required for excision of the hupL element. A strain in which the xisC gene was inactivated showed no detectable excision of the hupL element. hupL encodes the large subunit of uptake hydrogenase. The xisC mutant forms heterocysts and grows diazotrophically, but unlike the wild type, it evolved hydrogen gas under nitrogen-fixing conditions. Overexpression of xisC from a plasmid in a wild-type background caused a low level of hupL rearrangement even in nitrogen-replete conditions. Expression of xisC in Escherichia coli was sufficient to produce rearrangement of an artificial substrate plasmid bearing the hupL element recombination sites. Sequence analysis indicated that XisC is a divergent member of the phage integrase family of recombinases. Site-directed mutagenesis of xisC showed that the XisC recombinase has functional similarity to the phage integrase family.

Combinatorial interaction of scorpion toxins Lqh-2, Lqh-3, and LqhalphaIT with sodium channel receptor sites-3.

Scorpion alpha-toxins LqhalphaIT, Lqh-2, and Lqh-3 are representatives of three groups of alpha-toxins that differ in their preference for insects and mammals. These alpha-insect, antimammalian, and alpha-like toxins bind to voltage-gated sodium channels and slow down channel inactivation. Sodium channel mutagenesis studies using various alpha-toxins have shown that they interact with receptor site 3, which is composed mainly of a short stretch of amino-acid residues between S3 and S4 of domain 4. Variation in this region results in marked differences between various subtypes of sodium channels with respect to their sensitivity to the three Lqh toxins. We incorporated the S3-S4 linker of domain 4 from hNaV1.2/hNaV1.1, hNaV1.3, hNaV1.6, and hNaV1.7 channels as well as individual point mutations into the rNaV1.4 skeletal muscle sodium channel. Our data show that the affinity of Lqh-3 and LqhalphaIT to sodium channels is markedly determined by an aspartate residue (Asp1428 in rNaV1.4); when mutated to glutamate, as is present in NaV1.1-1.3 channels, Lqh-3-channel interactions are abolished. The interaction of Lqh-2 and LqhalphaIT, however, is strongly reduced when a lysine residue (Lys1432 in rNaV1.4) is replaced by threonine (as in hNaV1.7), whereas this substitution is without effect for Lqh-3. The influence of Lys1432 on Lqh-2 and LqhalphaIT strongly depends on the context of the Asp/Glu site at position 1428, giving rise to a wide variety of toxicological phenotypes by means of a combinatorial mixing and matching of only a few residues in receptor site 3.

Differential sensitivity of sodium channels from the central and peripheral nervous system to the scorpion toxins Lqh-2 and Lqh-3.

The scorpion alpha-toxins Lqh-2 and Lqh-3, isolated from the venom of the Israeli yellow scorpion Leiurus quinquestriatus hebraeus, were previously shown to be very potent in removing fast inactivation of rat skeletal muscle sodium channels (Chen et al., 2000). Here, we show that tetrodotoxin-sensitive neuronal channels NaV1.2 and NaV1.7, which are mainly expressed in mammalian central and peripheral nervous systems, respectively, are differentially sensitive to these two toxins. rNaV1.2 and hNaV1.7 channels were studied with patch-clamp methods upon expression in mammalian cells. While Lqh-3 was about 100-times more potent in removing inactivation in hNaV1.7 channels compared with rNaV1.2, Lqh-2 was about 20-times more active in the other direction. Site-directed mutagenesis showed that the differences in the putative binding sites for these toxins, the S3-4 linkers of domain 4, are of major importance for Lqh-3, but not for Lqh-2.