Patent Application
20140287433
This invention relates to a method of determining presence, amount and/or activity of a clostridial neurotoxin in a sample, the method comprising or consisting of the following steps: (a) bringing said sample into contact with a liposome, said liposome comprising (aa) at least one receptor on its outer surface, said receptor being capable of binding said neurotoxin and comprising or consisting of (i) a glycolipid and (ii) a peptide or protein; and (ab) a substrate in its interior, said substrate (i) being cleavable by the peptidase comprised in said neurotoxin and (ii) generating a detectable signal upon cleavage, said detectable signal preferably being generated by (1) the donor of a FRET pair, said donor exhibiting increased fluorescence upon cleavage by said peptidase, (2) a luminescent compound formed upon said cleavage, or (3) an enzyme formed upon said cleavage; and (b) determining whether an increase in signal occurs as compared to the absence of said sample, wherein such increase is indicative of the presence of said neurotoxin and/or the degree of such increase is indicative of the amount and/or activity of said neurotoxin in said sample.
In this specification, a number of documents including patent applications and manufacturer's manuals is cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
Clostridial neurotoxins include various serotypes of botulinum neurotoxin and tetanus neurotoxin.
Botulinum neurotoxin (BoNT) is the substance with the highest toxic activity known to man. Intoxication with BoNT causes botulism, a gradually increasing neuroparalytic disease, which can lead to death by respiratory arrest [1]. Although four of the serotypes (A, B, E, and F) are known to cause human botulism, the majority of human cases are due to serotypes A and B [2]. BoNT consists of two individual subunits, Heavy Chain (HC) and Light Chain (LC), which are connected via a disulphide bond.
In the body, BoNT specifically binds via the C-terminal part of its HC(HC) to receptors on the motoneuron's membrane, typically SV2C and the trisialoganglioside GT1b in case of BoNT/A, and Synaptotagmin-II (SytII) and GT1b in case of BoNT/B [3,4]. The toxin is internalised into the motoneuron via receptor-mediated endocytosis [5]. Following proton influx, the pH in the endosome is typically lowered from pH 7.2 to pH 5.3, and the HC N-terminal part (HN) refolds into a transmembrane channel crossing the endosomal membrane, allowing for translocation of the LC into the cytosol of the motoneuron [6]. There, depending on the toxin's serotype, the LC specifically cleaves distinct SNARE-proteins (soluble N-ethylmaleimide-sensitive fusion protein attachment receptor). For example, the LC of BoNT/A specifically cleaves SNAP-25 (synaptosome associated protein of 25 kDa), and the LC of BoNT/B specifically cleaves Synaptobrevin/VAMP-2 (vesicle associated membrane protein) [7-9]. If one or more SNARE proteins are cleaved, then the SNARE complex cannot assemble anymore, release of neurotransmitter is inhibited, and hence, the following muscle cell is paralysed. The amount of toxin needed to cause severe symptoms of botulism and even death is extremely low. From studies in mice and monkeys it was calculated that already 1 ng per kg body weight via the intravenous route might be lethal in humans [10-12].
In spite of its extreme toxicity, the neurotoxin is used in modern biomedical applications [13-15]. If applied in minute doses, the toxin exerts a locally constrained paralysing effect, which is used for the treatment of a large variety of diseases, such as strabismus, hyperhidrosis, or chronic pain. Moreover, treatment of wrinkles, frown lines, or facial asymmetries and other applications of aesthetical surgery, present a large application area [16]. However, to guarantee patients' safety, it is crucial that the toxic activity in all batches of pharmaceutical and cosmetic BoNT preparations is consistent. Accordingly, strict controls apply, where potency in each batch is measured with the mouse LD50 test. In this test, mice are injected intraperitoneally with defined volumes of different dilutions of BoNT containing preparations and observed for typical botulism symptoms for up to 96 hours. The injected amount where 50% of mice die is considered the LD50, which is defined as 1 Unit of BoNT [17]. Prior to their death by respiratory arrest this test leaves the mice with severe suffering. Due to the enormous demand for BoNT containing pharmaceutical products, it is estimated that in Europe and USA, each year, more than 600,000 mice have to die for BoNT LD50 potency testing performed by pharmaceutical companies [18,19]. Despite intensified efforts, the mouse LD50 test is still the only detection method officially validated and accepted for testing the toxic activity of BoNT in pharmaceutical products [20]. This is because the test is able to assess the complex mode of action of BoNT in the body.
If an alternative assay aims to replace the mouse LD50 test, it would have to assess the following key actions: (1) Binding of the toxin to specific receptors on the membrane of motoneurons, (2) translocation of the LC from the endosome into the cell's cytosol, and (3) LC-mediated cleavage of distinct SNARE-protein(s). Although other methods have been published, none has so far been able to present an adequate alternative to the mouse LD50 test [23]. Most in vitro assays, for instance, are only able to detect one of the key actions of BoNT's biological activity, i.e. the LC cleavage activity or HC binding to nerve cell receptors [24,25]. Although cell-based assays would in theory be ideal to test for all of the toxin's key actions, techniques for differentiation of neuronal stem cells into motoneurons are still in their infancy and primary nerve cell cultures provide only poor sensitivities, if compared with the mouse LD50 test [26-28]. Hence, detection of the key actions of BoNT toxic activity in the body requires a system that resembles that of living organisms but that offers at the same time properties such as reproducibility, easy readout and low maintenance costs.
The following Table 1 provides an overview of the currently available methods for determining botulinum neurotoxins.
1Receptor binding
2Translocation
3Cleavage
A further example of a botulinum neurotoxin assay, which fails to determine the entire mechanism of action of the neurotoxin is described in US 2011/0033866. This document describes neurotoxin substrates suitable for a FRET assay. To the extent use is made of vesicles, the vesicles merely serve as a carrier for such substrate. The vesicle is not equipped with receptors and accordingly does not provide for determining receptor binding and/or translocation. Moreover, to the extent this document refers to assays making use of vesicles, FRET is not used as detection scheme.
The technical problem underlying the present invention can therefore be seen in the provision of alternative or improved means and methods for determining clostridial neurotoxins.
This problem is solved by the subject-matter of the claims. In particular, and in a first aspect, the present invention provides a method of determining presence, amount and/or activity of a clostridial neurotoxin in a sample, the method comprising or consisting of the following steps: (a) bringing said sample into contact with a liposome, said liposome comprising (aa) at least one receptor on its outer surface, said receptor being capable of binding said neurotoxin and comprising or consisting of (i) a glycolipid and (ii) a peptide or protein; and (ab) a substrate in its interior, said substrate (i) being cleavable by the peptidase comprised in said neurotoxin and (ii) generating a detectable signal upon cleavage, said detectable signal preferably being generated by (1) the donor of a FRET pair, said donor exhibiting increased fluorescence upon cleavage by said peptidase, (2) a luminescent compound formed upon said cleavage, or (3) an enzyme formed upon said cleavage; and (b) determining whether an increase in signal occurs as compared to the absence of said sample, wherein such increase is indicative of the presence of said neurotoxin and/or the degree of such increase is indicative of the amount and/or activity of said neurotoxin in said sample.
Said determining may be a merely qualitative determining, i.e. determining presence or absence of the clostridial neurotoxin. Alternatively or in addition, said determining may be quantitatively, i.e. the determining of amount and/or activity of a clostridial neurotoxin. Amount and activity to be determined may be relative or absolute amounts and/or activities. This will depend inter alia on how the method is calibrated and/or which controls and standards are used. Such specifics of the assay design are within the abilities of the skilled person. An exemplary illustration is comprised in the examples enclosed herewith.
Generally speaking, amount and activity will be proportional to each other across a significant range of values. Having said that, it is known in the art that a defined amount of a clostridial neurotoxin may exhibit different activity depending on presence or absence of accessory non-toxin proteins, also referred to as neurotoxin associated proteins (NAPs). The neurotoxin as such consists of light and heavy chain as discussed in the introductory section herein above. In case of serotype A of botulinum toxin, said neurotoxin has an approximate molecular weight of 150 kDa. Clostridium botulinum typically produces botulinum type A toxin complexes, wherein said complexes comprise said neurotoxin on the one hand and one or more non-toxin proteins on the other hand. As a consequence, botulinum type A toxin complexes are found which have molecular weights of about 900 kDa, 500 kDa or 300 kDa. Similar findings, although different in terms of molecular weight, apply to the other botulinum toxins, which is well-known in the art. Depending on the specific conditions chosen, the presence of non-toxin proteins may have a stabilizing effect, the consequence being that a given amount of neurotoxin, when provided in complex form, may have a different, for example higher activity as compared to the same amount of neurotoxin in the absence of non-toxin proteins. The stabilizing effect of non-toxin proteins as well as conditions where non-toxin proteins are dispensable are known to the skilled person. Furthermore, formulations of clostridial neurotoxins are known or at the skilled person's disposal, which, instead of said non-toxin proteins or in addition thereto, contain further stabilizing agents such as human serum albumin, sucrose and/or gelatine.
While said neurotoxin as comprised in said sample may be associated with NAPs, it is preferred that a sample comprising neurotoxin and being free of NAPs is subjected to the methods of the invention. Within said neurotoxin, presence of heavy chain and light chain is necessary, noting that the method probes for the concomitant occurrence of receptor binding, translocation and cleavage. It is understood that, as established in the art, the term “clostridial neurotoxin” refers to an entity comprising, preferably consisting of heavy and light chain (see also the introductory section above), either fused as single chain form or as di-chain form, and providing the activities required for said receptor binding, translocation and cleavage. As noted above, in case of BoNT/A, the neurotoxin has an approximate molecular weight of 150 kD. Sometimes the term “neurotoxic component” is used in the art to designate the entity consisting of heavy and light chain.
The term “amount” includes concentration, mass, weight, and amount of substance, and preferably is expressed in terms of a concentration. To the extent molecular weights are known, mass, weight and concentration on the one hand and amount of substance on the other hand can be interconverted.
Since the third step of the mechanism of action of clostridial neurotoxins is an enzymatic activity, more specifically a proteolytic activity (herein also referred to as peptidase activity), activity may be expressed in terms of the number of cleaved substrates per unit of time. In the methods according to the invention, the measured activity results from the efficacy of all three steps of the mechanism of action of the neurotoxin, i.e. receptor binding, translocation and substrate hydrolysis, herein also referred to as cleavage of the substrate. Receptor binding is governed inter alia by the affinity of the respective neurotoxin for the receptor according to the invention. Translocation, i.e. formation of a transmembrane channel and transport of the peptidase into the lumen of the liposome according to the invention, is triggered or enhanced by a pH shift (for details see below).
Since the same amounts of a given neurotoxin may exhibit different activities, such different activities may be expressed as specific activity, with specific activity typically being the activity per unit of mass or amount of substance. Typically the methods of the invention, when used for quantitative determination of neurotoxins, yield activities in the first step. If the specific activity of the sample is known or can be determined, activity may be converted into amounts. Alternatively, the amount, in particular in case of samples, which are concentrated solutions of neurotoxin, may be determined by means such as SDS-PAGE, photometry and ELISA. Upon determination of activity by the method of the invention and of the amount by such means, the specific activity may be calculated.
A further common measure of the activity of neurotoxins, in particular of botulinum neurotoxins, are units. A Unit is defined by reference to the above-mentioned mouse LD50 assay. A Unit is the median intraperitoneal lethal dose (LD50) in mice. In preferred embodiments of the method according to the first aspect of the invention, a reference sample may be used, said reference sample comprising a defined activity of clostridial neurotoxin, said defined activity preferably being expressed in terms of units. In such a case, the activity of sample can then be specified in terms of units as well.
Clostridial neurotoxins are neurotoxins produced by bacteria of the genus Clostridium. Toxin producing species include Clostridium botulinum, Clostridium butyricum, Clostridium baratii, Clostridium argentinense as well as Clostridium tetania. Clostridial neurotoxins are further detailed below. Clostridial neurotoxins are typically characterized by a similar mechanism of action, which, as detailed above, comprises binding to receptors on the target nerve cell, translocation through the membrane and cleavage of the target protein by the peptidase component of the neurotoxin. Receptors and peptidase substrate may be different for the various toxins. Generally, the receptor is an integral or peripheral membrane protein, which consists of or comprises one or more extracellular domains, thereby being accessible to the neurotoxin heavy chain for binding. As regards the substrates, these are typically SNARE proteins. Several serotypes of botulinum neurotoxin cleave the same SNARE protein, however, at different sites. In some instances, for a substrate to be cleaved, not only the cleavage site, but also one or more recognition sites as present in the natural substrate must be present in an artificial substrate which may be used in the methods of the present invention (for details see below). Despite these subtle differences, the clostridial neurotoxins share a common mechanism of action, wherein all three steps of said common mechanism of action are assayed by the methods according to the present invention.
A sample according to the invention is known to comprise or suspected of comprising at least one clostridial neurotoxin. Depending on its origin, said sample may comprise further constituents. Preferred samples are the subject of a preferred embodiment described in detail below. The sample to be subjected to the method of determining according to the present invention may not only comprise further constituents owing to its origin, but also as a consequence of the deliberate addition of such further constituents. Such deliberately added constituent may be a test compound or a plurality of test compounds, wherein it is to be determined whether such compounds are capable of modulating the activity of clostridial neurotoxins. On the other hand, the primary sample as it might be obtained from, for example, a patient, might be known to comprise or suspected of comprising neutralizing antibodies against one or more clostridial neurotoxins, but otherwise free of neurotoxins as such. In that case, it is envisaged to deliberately add one or more neurotoxins to such primary sample, thereby obtaining the sample to be subjected to the method according to the present invention. Such preferred aspects (screen; determining of antibodies) are the subject of preferred embodiments discussed in more detail below.
Said sample is to be brought into contact with a liposome, the liposome being further characterized by items (i) and (ii) as recited in step (a) of the method according to the first aspect of the invention. It is understood that said bringing into contact is to be effected under conditions, which allow binding of said neurotoxin, if present, to said receptor. The skilled person can readily establish such conditions, such conditions including, for example, buffered solutions. Further preferred/exemplary conditions are specified in the examples enclosed herewith. Said conditions, even though disclosed in conjunction with serotype B of botulinum toxin are not confined to their use in conjunction with said serotype, but instead may be used with any clostridial neurotoxin and across all embodiments comprised by the first aspect of the present invention. Preferably, said determining according to (b) is effected at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 24, 36 or 48 hours after said bringing into contact according to (a). These preferred periods of time refer to the total incubation time.
As stated above, said liposome meets at least the requirements (i) and (ii). Such liposome may either be obtained from natural sources such as nerve tissue or nerve cell cultures, or, in the alternative, by preparation starting from defined constituents. Preference is given to the latter approach. A method of preparing a liposome is also the subject of the present invention; see the fourth aspect discussed further below. The use of defined constituents and defined procedures of manufacture of the liposomes provides for a reproducible method of determining according to the first aspect.
The liposome according to the invention, which is also subject of the third aspect described in more detail below, comprises a receptor and a substrate. A liposome is, as known in the art, a closed compartment formed by a lipid bilayer. Generally, liposomes are artificially prepared. It is understood that the term “liposome”, as used herein, extends to liposomes, which are prepared from naturally occurring cells or tissues.
The receptor is defined in both structural and functional terms. In structural terms, the receptor comprises or consists of a glycolipid on the one hand and a peptide or protein on the other hand. In functional terms, said receptor is required to be capable of binding said neurotoxin. As a consequence, the skilled person understands that said receptor is intended to mimic the neurotoxin receptor, as it is present on the surface of nerve cells. Accordingly, said glycolipid is preferably to be chosen from a glycolipid which naturally occurs in the membrane of nerve cells or a modified version thereof, said modified version being capable of acting as a constituent of said receptor being capable of binding said neurotoxin. Preferred glycolipids are described further below.
The second, proteinaceous component of said receptor is a protein naturally occurring in nerve cells and known to be involved in neurotoxin binding, or a fragment thereof (herein also referred to as peptide), or a modified version of said protein or fragment, wherein said fragment, i.e. said peptide as well as said modified version are capable of acting as a component of a receptor being capable of binding said neurotoxin. The skilled person, in view of the present knowledge on targets and mechanism of action of clostridial neurotoxins, and furthermore provided with the teaching of the present invention is in a position to devise receptors meeting both the structural and functional requirements according to item (i) of the first aspect. For example, various truncated, deleted and/or modified versions of, the respective naturally occurring protein may be prepared and tested for their capability of binding a clostridial neurotoxin or a heavy chain thereof. The testing of receptors is also described in Nishiki et al. [76], Nishiki et al. [48], Dong et al. [50] Dong et al. [77], Dong et al. [78], Rummel et al. [79] and Mahrhold et al. [32].
The substrate according to item (ii) may either comprise the cognate substrate of the respective clostridial neurotoxin to be assayed, or a subsequence thereof, such subsequence being cleavable by the peptidase comprised in the neurotoxin. Again, both substrates as well as several subsequences are known to the skilled person and further detailed herein below. Further suitable subsequences may be found by simple tests such truncation series of the natural substrate, the truncated substrates being subjected, for example, to the method according to the present invention, wherein the measured activity is compared to the activity, which is observed when the sequence of the wild type substrate is used.
The substrate further generates a detectable signal upon cleavage. In principle, any means may be used which generate a detectable signal upon cleavage. Various such means are known in the art. The signal may be emission of light, be it by fluorescence or luminescence. Light may be emitted by the substrate itself once it is cleaved, or may be generated as a result of a downstream event triggered by cleavage of the substrate. Preferred options are detailed below.
In one preferred embodiment, the substrate comprises a FRET pair, such FRET pair consisting of a fluorescence donor and a fluorescence acceptor (option (1)). The term “FRET” is well-known in the art and stands for fluorescence resonance energy transfer. Sometimes the “F” within “FRET” is also understood as an abbreviation of the name Förster, noting that a scientist named Förster described the physical laws governing the energy transfer between a fluorescence donor and a fluorescence acceptor. Said FRET pair is to be positioned such that fluorescence increases upon cleavage of the substrate by the peptidase. This implies positioning of donor and acceptor (i) on different sides of the substrate's cleavage site, and furthermore (ii) that donor and acceptor have an average spatial distance in the substrate that provides for sufficient quenching of the donor fluorescence by the acceptor in the uncleaved substrate. Distances between donor and acceptor for quenching to occur are typically in the order of up to 10 nM. Regardless thereof, suitable positions of donor and acceptor within a given substrate can be determined by the skilled person without further ado by simple tests.
In an alternative embodiment of the FRET pair of the invention, the acceptor does not (only) act as a quencher, but is capable of fluorescence emission, in particular when energy is transferred from a donor in sufficient spatial proximity. In that case, little or no donor emission, but significant acceptor emission will be measured in the uncleaved state of the substrate, while upon cleavage, the acceptor emission is decreased and the donor emission is increased. Suitable donor/acceptor pairs are known in the art and at the skilled person's disposal.
In a further preferred embodiment, a luminescent compound is formed upon cleavage (option (2)). Said luminescent compound may be, for example, oxyluciferin, which is formed from luciferin in the presence of the enzyme luciferase. Luciferase in turn may be provided in the form of two fragments of luciferase, said fragments being generated by said cleavage and, while being enzymatically inactive themselves, assemble into a functional enzyme having luciferase activity upon cleavage.
Generally speaking, it is also envisaged to use a substrate, which, upon cleavage, assembles to a yield a functional enzyme, wherein the activity of said enzyme is detected (option (3)). Such enzyme complementation system on the one hand and the generation of a luminescent compound on the other hand may characterize one and the same read-out scheme; see, for example, the luciferase system described above.
Preferred substrates are SNARE proteins comprising such FRET pair or otherwise modified to generate a signal upon cleavage, or fragments of SNARE proteins comprising FRET pairs or otherwise modified to generate a signal upon cleavage.
Step (b) provides for a comparison of signal generated by the liposome with and without sample, but otherwise preferably under the same conditions. To the extent substrates with FRET pairs are used, the following applies. For the purpose of determining any change in fluorescence, either the entire fluorescence spectrum may be recorded, or measurements may be effected at one or more specific wavelengths. A preferred wavelength is the wavelength of maximum fluorescence emission which is a characteristic known for most commercially available fluorophor or can be determined without further ado, for example by performing a spectral scan.
Said receptor is located on the outer surface of the liposome, and the substrate in the interior of the liposome, preferably only in the interior of the liposome. Preferred means of ensuring occurrence of the substrate only in the interior of the liposome include the following: (i) purification by means of size exclusion chromatography (as shown in
While it is preferred that the substrate is soluble and dissolved in the aqueous medium in the lumen of the liposome, it is also envisaged to use membrane-bound substrates. In the latter case, alternative or additional means of ensuring that the substrate is pointing only inwards, i.e. into the lumen of the liposome, may be used. For example, substrate may be incorporated into the liposomes prior to incorporation of the receptor, at least of the peptide or protein component of the receptor. To the extent substrate molecules are pointing outwards, they may be digested by a suitable enzyme such as proteinase K or the light chain (LC) of the neurotoxin/serotype to be determined by the methods of the invention. In a subsequent step, the receptors are incorporated, thereby obtaining a liposome according to the present invention with membrane-bound substrate pointing only into the interior of the liposome. Said aqueous medium in the lumen of the liposome is such that cleavage of said substrate by said neurotoxin can take place. Such aqueous medium can be chosen by the skilled person without further ado. Preferred are the aqueous media disclosed further below in relation to the method of preparing a liposome according to the fourth aspect of this invention. A particularly preferred aqueous medium comprises or consists of HEPES buffer, preferably between 10 and 100 mM such as 20 mM, with a pH between 7 and 8, preferably 7.4, preferably supplemented with ZnSO4, preferably at a concentration between 0.01 and 0.1 mM such as 0.05 mM, and furthermore preferably supplemented with TCEP, preferably at a concentration between 1 and 10 mM such as 2 mM.
In view of the above properties, said liposome mimics the characteristics of a nerve cell which are essential for the entire mechanism of action of a clostridial neurotoxin to be monitored, said mechanism of action comprising receptor binding, translocation and proteolytic cleavage of the target protein, see above. As is apparent from the review of the state of the art, the known neurotoxin assays either fail to test the entire mechanism of action or, to the extent they do, suffer from other deficiencies (see Table 1 herein above). The assay according to the first aspect of the present invention does not require mice (be it whole animals or diaphragm preparations obtained therefrom) nor are cultures of nerve cells needed. Due to the limited number of constituents and steps to be effected, the assay is robust, reproducible, amenable to standardization and capable of being effected in high throughput format. Sterile conditions are not required. Accordingly, a high throughput screen can be effected fast.
It is furthermore envisaged to use the assay according to the first aspect, wherein instead of said clostridial neurotoxins other bacterial toxins are to be determined. Such other toxins include the Bacillus anthracis toxin and diphtheria toxin. In an assay for Bacillus anthracis toxin, a commercially available substrate (MAPKKide) from List Biological Laboratories may be used. In case of diphtheria toxin, the enzymatic reaction occurring in the liposome is ADP-ribosylation of eukaryotic elongation factor 2 instead of proteolytic cleavage. This activity is described in the art and the assay according to the first aspect of the present invention may be readily adapted for the monitoring of said activity.
In a preferred embodiment, said clostridial neurotoxin is a botulinum neurotoxin, preferably type A, B, C1, D, E, F or G botulinum neurotoxin or tetanus neurotoxin. The various serotypes of botulinum toxin are known in the art and well characterized. Pharmaceutical or cosmetic compositions typically comprise botulinum neurotoxin type A (BoNT/A) or BoNT/B as active pharmaceutical ingredient.
In a further preferred embodiment, said sample is selected from a pharmaceutical composition, a diagnostic composition, a cosmetic composition, a clinical or patient sample, a food or feed sample, a beverage sample, a sample taken from a biotechnological process, a sample obtained from an animal and an environmental sample. As mentioned in the introductory section herein above, a variety of botulinum neurotoxin formulations are presently approved for medical and cosmetic uses. In the course of manufacture of these compositions, their testing is indispensable, in particular in view of the high toxicity of the active agent as well as its fragility and easy denaturation. Clinical samples, patient samples and samples obtained from an animal include samples taken from subjects or animals suspected to suffer from botulism. Alternatively, said samples may be from subjects suspected to contain neutralizing antibodies directed to one or more clostridial neurotoxins. Food, feed and beverage as well as environmental samples may be assayed for the presence of neurotoxin activity in order to determine whether any threat to the health of humans or animals is present. Samples taken from biotechnological processes include samples taken from biological processes for the manufacture of clostridial neurotoxins. For example, such a sample may be taken from a fermenter containing a bacterial culture expressing clostridial neurotoxins.
In a further preferred embodiment, said liposome comprises or consists of the following constituents: (a) (i) one or more liposome-forming lipids, preferably at least one phosphatidylcholine and cholesterol, said phosphatidylcholine preferably being selected from the group consisting of SPC, DOPC, and POPC; (ii) optionally tocopherol; (b) said at least one receptor, wherein said receptor preferably comprises or consists of (i) a glycolipid, preferably selected from the tri-sialo ganglioside GT1b, the di-sialo ganglioside GD1b and the di-sialo ganglioside GD1a; and (ii) a peptide or protein selected from (1) SV2C or a fragment thereof, wherein said fragment is capable of binding to said neurotoxin and preferably comprises or consists of the luminal domain 4 and at least one transmembrane domain; (2) synaptotagmin I or II or a fragment thereof, wherein said fragment is capable of binding to said neurotoxin and preferably comprises or consists of the N-terminal luminal domain and the transmembrane portion of synaptotagmin I or II; (3) SV2A or a fragment thereof, wherein said fragment is capable of binding to said neurotoxin and preferably comprises or consists of the luminal domain 4, preferably the C-terminal portion thereof; and (4) SV2B or a fragment thereof, wherein said fragment is capable of binding to said neurotoxin and preferably comprises or consists of the luminal domain 4, preferably the C-terminal portion thereof; (c) said substrate; and (d) an aqueous medium in the interior of said liposome.
In a further preferred embodiment, said one or more liposome formic lipids are selected from SPC, DOPC, POPC, Lecithin (egg yolk or soy bean), Asolectin (soy bean), posphatidylinositol, posphatidylserin, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, DOPG (di-oleoyl-phosphatidylglycerol), DOPE (di-oleoyl-phosphatidylethanolamine) and POPG (palmitoyl oleoyl phosphatidylglycerol). Lecithin and Asolectin are lipid mixtures from natural sources. The remainder of lipids are preferably single molecular species. Moreover, also other amphiphilic molecules, either alone or in combination with any one of the above, can be used for the purpose of generating liposomes. The skilled person is aware of such further amphiphilic molecules. The lipids, lipid mixtures and amphiphilic molecules described above can be used in conjunction with any embodiment of the present invention.
Also provided is a liposome comprising or consisting of constituents (a), (b) and (c) as defined above as well as liposomes comprising or consisting of constituents (a) and (c); or constituents (a) and (b). In either case, said liposome may further comprise or further consist of constituent (d).
These embodiments further characterize the essential constituents of the liposome according to the present invention. In particular, constituent (a) provides the lipid bilayer forming compounds, constituent (b) provides the compounds forming the receptor, and constituent (c) is the substrate. As is apparent from the definition of constituents (a) and (b), each of said constituents may—either optionally or compulsory—comprise or consist of more than one compound.
In order to mimic closely the characteristics of eukaryotic cell membranes, at least one phosphatidylcholine as well as cholesterol are preferred. Among the preferred phosphatidylcholines, there is soy phosphatidylcholine (SPC), dioleylglycerol phosphocholine (DOPC) and palmitoyl oleoyl phosphocholine (POPC). Preference is given to DOPC and POPC. Preferably, also tocopherol, in particular D,L-alpha-tocopherol is present. In a further preferred embodiment, phosphatidic acid (PA) is furthermore present. Acidic lipids such as PA may have a beneficial effect on the activity of the peptidase of said neurotoxin. Further preferred compositions forming constituent (a) are apparent from the examples enclosed herewith. The liposome forming lipids as used in the examples as well as their relative amounts are generally applicable for the purposes of the present invention.
Receptors for clostridial neurotoxins as they naturally occur in nerve cells are typically made up by two components, namely a glycolipid and a protein. Accordingly, the receptor present on the liposomes according to the present invention, said receptor being capable of binding said neurotoxin, mimics such naturally occurring neurotoxin receptors. Preferred glycolipids are gangliosides and particularly preferred are the gangliosides according to (b)(i) of the present preferred embodiment. Gangliosides are glycosphingolipids. The above used designations for preferred gangliosides according to the present invention (i.e. GT1b, GD1b and GD1a) are established in the art; see, for example, Yowler and Schengrund [75]. The structures of said gangliosides are provided in the following: GD1b, Galβ3NAcGalβ4(NAcNeuα8NAcNeuα3)Galβ4GlcβCer; GD1a, NAcNeuα3Galβ3NAcGalβ4(N AcNeuα3)Galβ4GlcβCer; GT1b, NAcNeuα3Galβ3NAcGalβ4(NAcNeuα8NAcNeuα3)Galβ4Gl cβCer; wherein Cer is Ceramide; Gal is galactose; Glc is glucose; NAcGal is N-acetylgalactosamine and NAcNeu is sialic acid.
The proteinaceous component of the receptor is defined in part (b)(ii) of this embodiment. Either the full length naturally occurring proteins may be used or fragments thereof, the fragments being capable of binding to the respective neurotoxin. SV2A, SV2B and SV2C are forms of synaptic vesicle glycoprotein 2. Synaptic vesicle proteins are membrane trafficking proteins comprising 12 transmembrane segments in case of SV2 but only one in case of Syt-II and Syt-I.
Preferred peptides and proteins comprised in said receptor are provided in SEQ ID NOs: 1 to 12. More specifically, SEQ ID NO: 1 is a fusion protein of human SV2C, including the transmembrane domain, with glutathione S transferase (GST). The SV2C component of said fusion protein are residues 454 to 603 of human SV2C. SEQ ID NO: 2 consists of residues 454 to 603 of human SV2C. SEQ ID NO: 3 is a GST fusion protein with the luminal and transmembrane domain of rat Synaptotagmin I (residues 1 to 88). SEQ ID NO: 4 consists of residues 1 to 82 of rat Synaptotagmin I and accordingly comprises the luminal domain and the transmembrane domain. SEQ ID NO: 5 consists of residues 35 to 82 of rat Synaptotagmin I which is the minimal luminal domain including the transmembrane domain. SEQ ID NO: 6 is a fusion protein of GST with the luminal domain and the transmembrane domain of rat Synaptotagmin II (residues 1 to 90). SEQ ID NO: 7 consists of luminal and transmembrane domain of rat Synaptotagmin II (residues 1 to 90). SEQ ID NO: 8 consists of the minimal luminal domain and the transmembrane domain of rat Synaptotagmin II (residues 44 to 90). SEQ ID NO: 9 is a fusion protein of GST with the luminal domain 4 and the transmembrane domain of rat SV2A. SEQ ID NO: 10 consists of luminal domain 4 and transmembrane domain of rat SV2A (residues 469 to 619). SEQ ID NO: Ills a fusion protein of GST with the luminal domain 4 and the transmembrane domain of rat SV2B. SEQ ID NO: 12 consists of luminal domain 4 and transmembrane domain of rat SV2B (residues 413 to 560).
As stated above, said substrate is located in the interior of the liposome. Preferably, it is exclusively located in the interior of the liposome. Moreover, preference is given to a soluble substrate such that the substrate is located in the lumen of the liposome. Alternatively or in addition, it is envisaged that the substrate may comprise a transmembrane segment and/or a membrane anchor, such membrane anchor being provided, for example, by covalently attached hydrophobic molecules such as fatty acids. While generally preference is given to soluble substrates, it is noted that, in conjunction with serotype C of botulinum neurotoxin, preference is given to a membrane attached substrate, said substrate still being located inside the liposome.
In a further preferred embodiment said bringing into contact is effected at a pH between 6 and 8, preferably between 7 and 7.4, more preferably at about 7.2.
In a further preferred embodiment, after step (a) and prior to step (b), the pH is changed to a value between 4 and 6, preferably between 5 and 5.4, more preferably about 5.2. Further preferred pH values are 3.8, 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.1, 5.3, 5.5, 5.6, 5.7, 5.8 and 5.9. Preferably, said change of pH is effected at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 24, 36 or 48 hours after said bringing into contact. These periods of time specify the binding incubation time.
A pH-shift may be induced by addition of an acid, preferably a membrane impermeable acid (e.g. DMG=Dimethylglutaric acid, MES=Morpholinoethanesulfonic acid or others) to the mixture of liposomes and botulinum neurotoxin in buffer (e.g. HEPES buffer at pH 7.2). Another way to adjust the pH may be performed as follows: After incubation of botulinum neurotoxin and liposomes, preferably both with as little dilution as possible, preferably at 4° C. (on ice), preferably for 5-120 minutes, the mixture is transferred, for example into the cavities of black 96-well microplates (preferably pre-heated to 37° C.) and buffer, preferably HEPES buffer, with the appropriate pH (preferably between pH 4 to 6, preferably preheated to 37° C.) is added to give a desired pH as well as a specific concentration of liposomes and botulinum neurotoxin in the final reaction volume. Desired pH values are preferably selected from 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0.
These preferred embodiments provide for a pH shift when performing the methods according to the present invention. While not strictly necessary, such shift in pH facilitates the formation of a membrane channel by a portion of the heavy chain of neurotoxin, said membrane channel providing for translocation of peptidase comprised by the light chain across the membrane.
If said bringing into contact is effected at a pH of 6, it is preferred that after step (a) and prior to step (b) the pH is changed to a value below 6.
In a further preferred embodiment, said determining according to (b) is effected at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 24, 36 or 48 hours after said change of pH. These periods of time specify the translocation and cleavage incubation time.
In a further preferred embodiment, said liposome has a mean diameter between 50 and 500 nm, preferably between 100 and 200 nm, most preferred about 150 nm. Any size within these intervals, such as 250, 300, 350, 400 and 450 nm as well as larger diameters such 600, 700, 800 and 900 nm or 1 μm are deliberately envisaged. As detailed in the examples enclosed herewith, liposomes may be prepared in such a manner that they exhibit a defined, preferably narrow size distribution. The means for ensuring such size distribution as defined in the examples are generally applicable to any liposome according to the present invention.
In a further preferred embodiment, said liposome comprises or consists of: (i) DOPC and/or POPC; (ii) cholesterol; (iii) GT1b, GD1b and/or GD1a; (iv) (1) SV2C or said fragment thereof; (2) synaptotagmin I or II or said fragment thereof; (3) SV2A or said fragment thereof; and/or (4) SV2B or said fragment thereof; (v) said substrate; (vi) an aqueous medium in the interior of said liposome; and (vii) optionally tocopherol.
The aqueous medium is preferably as defined herein above as well as further below.
In a second aspect, the present invention provides the use of a liposome as defined in any one of the preceding claims for determining presence, amount and/or activity of a clostridial neurotoxin.
The present invention furthermore relates in a third aspect to a liposome as defined in any one of the preceding claims. Such liposomes may either be obtained from nerve cells or nerve tissues via procedures known in the art (synaptosome preparations). Alternatively, the liposomes are prepared by the methods of the invention as described further below.
In a further preferred embodiment of the methods according to the first aspect, said sample is known to comprise or suspected of comprising neutralising antibodies against said neurotoxin, wherein said sample, prior to subjecting it to said method, is combined with a known amount or activity of said neurotoxin, and wherein a decreased amount or activity of said neurotoxin as determined by said method in comparison to a control sample is indicative of the presence of said neutralising antibodies, wherein said control sample comprises said known amount or activity of said neurotoxin but is free of said neutralising antibodies.
In the context of a running or intended pharmaceutical or cosmetic treatment with a botulinum neurotoxin, it may be of interest to determine whether the patient or subject has developed or is in the course of developing neutralizing antibodies against the neurotoxin. Such neutralizing antibodies may interfere with the effects triggered by the neurotoxin and may prevent any beneficial effect altogether. In such a case it might be considered, for example, to switch serotype. In order to properly decide when such measures should be considered, monitoring of presence and amount of neutralizing antibodies may be useful.
In the preferred embodiment of the method according to the invention designed for neutralizing antibody monitoring, use is made of a test sample and a control sample, wherein both samples comprise the same known amount or activity of neurotoxin.
In a further preferred embodiment, said sample comprises a test compound and a known amount or activity of said neurotoxin, wherein a decreased or increased amount or activity of said neurotoxin as determined by said method in comparison to a control sample is indicative of the test compound being an inhibitor or activator, respectively, of said neurotoxin, wherein said control sample comprises said known amount or activity of said neurotoxin but is free of said test compound.
This embodiment provides for a screening method for neurotoxin modulators, preferably inhibitors. Given the robustness and simplicity of the assay according to the present invention, it readily can be effected in high throughput format. In view of the FRET detection scheme, the readout may be effected in an automatic manner, for example by means of a CCD camera coupled to a data processing unit. Since liposomes may be kept in solution, the assay may be performed in wells of microtiter plates. Robotic systems for the handling of microtiter plates as known in the art may be employed.
In a fourth aspect, the present invention provides a method of preparing a liposome, said method comprising or consisting of the following steps: (a) dissolving (i) liposome-forming lipid(s), preferably at least one phosphatidylcholine and cholesterol, said phosphatidylcholine preferably being selected from the group consisting of SPC, DOPC, and POPC; (ii) GT1b; and optionally (iii) tocopherol in a suitable organic solvent; (b) evaporating said organic solvent; (c) resuspending the residue of step (b) in an aqueous medium, said aqueous medium comprising (ca) at least one receptor, said receptor being capable of binding a clostridial neurotoxin and comprising or consisting of a glycolipid and a peptide or protein, wherein preferably (i) said glycolipid is selected from the tri-sialo ganglioside GT1b, the di-sialo ganglioside GD1b and the di-sialo ganglioside GD1a; and (ii) said peptide or protein is selected from (1) SV2C or a fragment thereof, wherein said fragment is capable of binding to said neurotoxin and preferably comprises the luminal domain 4 and at least one transmembrane domain; (2) synaptotagmin I or II or a fragment thereof, wherein said fragment is capable of binding to said neurotoxin and preferably comprises the N-terminal extracellular domain and the transmembrane portion of synaptotagmin I or II; and (3) SV2A, SV2B or a fragment of SV2A or SV2B, wherein said fragment is capable of binding to said neurotoxin and preferably comprises or consists of the luminal domain 4; and (cb) a substrate (i) being cleavable by the peptidase comprised in said neurotoxin and (ii) generating a detectable signal upon cleavage, said detectable signal preferably being generated by (1) the donor of a FRET pair, said donor exhibiting increased fluorescence upon cleavage by said peptidase, (2) a luminescent compound formed upon said cleavage, or (3) an enzyme formed upon said cleavage; (d) extruding the suspension obtained in step (c) through a suitable membrane; and (e) optionally purifying the liposomes obtained in step (d), preferably by means of size exclusion chromatography.
Suitable organic solvents to be used in step (a) include polar solvents. Polar protic as well as polar aprotic solvents may be used, wherein preference is given to mixtures thereof. A preferred protic solvent is methanol and preferred aprotic solvents are dichloromethane and chloroform. Particularly preferred solvents are mixtures of methanol and dichloromethane and mixtures of methanol and chloroform, preferably 1:1 mixtures; see also Example 1 enclosed herewith.
The aqueous medium to be used in step (c) may comprise, in addition to the constituents recited in step (c) one or more buffers and/or salts. HEPES (4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid) buffer, preferably at a concentration between 10 and 100 mM, and preferably at a pH between 7 and 8 may be used. Particularly preferred is 20 mM HEPES buffer at a pH of 7.4 and optionally 150 mM K-Glu or NaCl. A particularly preferred aqueous medium comprises or consists of HEPES buffer, preferably between 10 and 100 mM such as 20 mM, with a pH between 7 and 8, preferably 7.4, preferably supplemented with ZnSO4, preferably at a concentration between 0.01 and 0.1 mM such as 0.05 mM, and furthermore preferably supplemented with TCEP, preferably at a concentration between 1 and 10 mM such as 2 mM. Alternatively, other known buffers with a pH between 7 and 8, preferably a pH of 7.4 may be used, for example phosphate buffered saline (for example 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4; also referred to as PBS) or Tris buffered saline (50 mM Tris.HCl and 150 mM NaCl; also referred to as TBS).
Preferably said aqueous medium is free of any detergent, in particular free of Tween-20.
Furthermore, it is preferred that said aqueous medium is free of dithiothreitol (DDT).
Suitable membranes to be used for preparing liposomes by means of extruding as recited in step (d) are known to the skilled person. Preferred membranes are polycarbonate membranes, which are available with various pore sizes.
Purification according to optional step (e) is a means of (i) removing components from the preparation obtained in step (d) which are not constituents of the formed liposomes, and (ii) obtaining a more uniform or narrow size distribution of liposomes. Other preferred means of purification are described further above.
As alternative to the method of preparing a liposome according to the fourth aspect, the present invention in addition provides the following methods.
In particular, provided is a method of preparing a liposome, said method comprising or consisting of the following steps: (a) dissolving a substrate in an aqueous medium; (b) adding liposome-forming lipids; (c) extruding the mixture obtained in (b) through a suitable membrane; (d) adding lactose or trehalose; (e) cooling the obtained mixture to a temperature between −50° C. and −100° C., preferably −80° C.; (f) subjecting the frozen mixture to freeze-drying; and (g) reconstituting the lyophilisate with aqueous medium.
Preferably, prior to said cooling according to step (e), said liposome solution is cooled in a first step to a temperature between 0 and 10° C., preferably 4° C., and in a second step to a temperature between 0 and −50° C., preferably −20° C. Said reconstituting according to (g) is preferably effected with 10 times concentrated aqueous medium, wherein after shaking, centrifuging and again shaking, distilled water is added until the desired 1-fold concentration is reached. Preferably, the receptor according to the invention is added concomitantly with said substrate.
In a further alternative, a method of preparing a liposome is provided, said method comprising or consisting of the following steps: (a) diluting a substrate in an aqueous medium; (b) adding liposome-forming lipids; (c) cooling the emulsion obtained in (b) to about 0° C.; (d) sonicating said solution. Preferably, the receptor according to the invention is added concomitantly with said substrate.
It is understood that in conjunction with these alternative methods of preparing a liposome according to the invention, the definition of terms as well as of preferred embodiments thereof is as specified herein in relation to the various aspects of the invention. This applies in particular to substrate, aqueous medium, liposome-forming lipids and membranes suitable for extruding. Further details of said alternative methods of preparing a liposome according to the invention can be taken from the examples. The methods described there are generally applicable for any substrate or receptor as defined herein. Moreover, the conditions used are applicable to all liposome-forming lipids disclosed herein.
Yet further means for preparing liposomes according to the invention are described in Estes [81] and Mertins [82] and references cited therein.
In a preferred embodiment of the methods, the use and the liposomes according to the present invention, said substrate consists of or comprises a compound of the following formula (I):
X-L-Y;
wherein L is a peptide or protein comprising or consisting of a sequence which is cleavable by said peptidase; “—” denotes a covalent bond, X-L-Y is preferably soluble in aqueous medium and/or free of any transmembrane domain or membrane anchor; and (a) X is moiety comprising or consisting of a FRET donor or acceptor; and Y is a moiety comprising or consisting of a FRET acceptor if X comprises or consists of a donor, or a FRET donor if X comprises or consists of an acceptor; or (b) X is a fragment of an enzyme, said enzyme preferably being luciferase; and Y is another fragment of said enzyme, said enzyme preferably being luciferase, wherein, upon cleavage of L by the peptidase of said neurotoxin, a functional enzyme comprising X and Y, said functional enzyme preferably having luciferase activity, is formed.
It is understood that prior to cleavage none of X and Y according to (b), be it alone or in combination, is enzymatically active.
This embodiment defines the above-mentioned substrate of the peptidase comprised in the neurotoxin more specifically in structural terms.
Noting that L is a peptide or protein, in one embodiment moieties X and Y are attached to the N- and C-terminus of said peptide or protein L, respectively. However, there is no strict necessity for such placement. With regard to option (a), it is sufficient that donor and acceptor are placed on different sides of the cleavage site. Considering that the cleavage site defines an N-terminal and a C-terminal portion of the peptide or protein, said N- and C-terminal portions being connected by the scissile bond, donor and acceptor may be placed anywhere in said N- and C-terminal portion, respectively, provided that they are in sufficient spatial proximity for fluorescence resonance transfer to occur in the uncleaved form of the substrate.
X-L-Y is preferably soluble in aqueous medium. Also, it is preferred that X-L-Y is free of any transmembrane domain or membrane anchor, such membrane anchor being, for example, a hydrophobic covalently attached moiety (for example palmitoyl side chains as present in naturally occurring SNAP-25).
In a preferred embodiment of the method, the use or the liposome of the present invention, L comprises or consists of (i) SNAP-25 or a fragment thereof, said fragment being cleavable by the peptidase comprised in said neurotoxin and preferably being selected from a sequence comprising or consisting of residues 93 to 206, 146 to 203, or 156 to 184 of SNAP-25; (ii) VAMP-2, VAMP-1, VAMP-3 or a fragment thereof, said fragment being cleavable by the peptidase comprised in said neurotoxin and preferably being selected from a sequence comprising or consisting of residues 30 to 62, 30 to 86, 38 to 62, 47 to 96, or 62 to 86 of VAMP-2; and/or (iii) Syntaxin-1, -2, -3 or a fragment thereof, said fragment being cleavable by the peptidase comprised in said neurotoxin and preferably being a sequence comprising or consisting of residues 196 to 259 of Syntaxin.
SEQ ID NOs: 13 to 21 provide sequences of preferred L moieties according to the present invention. In particular, SEQ ID NO: 13 is the sequence of residues 1 to 206 of rat SNAP-25. SEQ ID NO: 14 is a subsequence of SEQ ID NO: 13 consisting of residues 140 to 206 of SNAP-25. SEQ ID NO: 15 is the sequence of human VAMP-2/Synaptobrevin II. SEQ ID NO: 16 is the sequence of rat VAMP-2 Synaptobrevin II. SEQ ID NO: 17 is the sequence of rat VAMP1. SEQ ID NO: 18 is the sequence of rat VAMP-3. SEQ ID NO: 19 is the sequence of human Syntaxin-1. SEQ ID NO: 20 is the sequence of rat Syntaxin-1. SEQ ID NO: 21 is the sequence of mouse Syntaxin-1.
As will be discussed in more detail below, subsequences of the natural substrates are also substrates of the respective clostridial neurotoxins. The subsequence preferences may vary between serotypes or between botulinum neurotoxins and tetanus toxin.
The skilled person can determine by using simple tests whether a given fragment of any one of SNAP-25, VAMP-2, VAMP-1, VAMP-3 and Syntaxin qualifies as a substrate.
In further preferred embodiments, said substrate is selected from SNAPtide, SNAP Etide, VAMPtide, and SYNTAXide. This embodiment refers to commercially available botulinum neurotoxin substrates. In particular, SNAPtide as described in U.S. Pat. No. 6,504,006 is a SNAP-derived BoNT/A substrate, SNAP Etide is a BoNT/E substrate, VAMPtide is a VAMP-derived BoNT/B substrate and SYNTAXide is a Syntaxin-derived BoNT/C1 substrate. These substrates are available from List Biological Labs Inc.
In a further preferred embodiment of the methods, use or liposome of the present invention, (a) said neurotoxin is botulinum neurotoxin type A; said receptor comprises or consists of (i) GT1b and (ii) SV2C or said fragment thereof; and L comprises or consists of SNAP-25 or said fragment thereof, said fragment preferably consisting of residues 146 to 203 of SNAP-25; (b) said neurotoxin is botulinum neurotoxin type B; said receptor comprises or consists of (i) GT1b and (ii) synaptotagmin II or I or said fragment thereof; and L comprises or consists of VAMP-2, VAMP, VAMP-3 or said fragment of any of these, said fragment preferably consisting of residues 62 to 86 of VAMP-2; (c) said neurotoxin is botulinum neurotoxin type C1; said receptor comprises or consists of GT1b; and L comprises or consists of SNAP-25, said fragment thereof, said fragment thereof preferably consisting of residues 93 to 206 of SNAP-25, Syntaxin-1, or said fragment thereof, said fragment preferably consisting of residues 196 to 259 of Syntaxin-1; (d) said neurotoxin is botulinum neurotoxin type D; said receptor comprises or consists of (i) GT1b and (ii) SV2A, SV2B, SV2C or the fragment of any of these as defined above; and L comprises or consists of VAMP-2, VAMP, VAMP-3 or said fragment of any of these, said fragment preferably consisting of residues 38 to 62 of VAMP-2; (e) said neurotoxin is botulinum neurotoxin type E; said receptor comprises or consists of (i) GT1b and (ii) SV2A, SV2B, or the fragment of any of these as defined above; and L comprises or consists of SNAP-25 or said fragment thereof, said fragment preferably consisting of residues 156 to 184 of SNAP-25; (f) said neurotoxin is botulinum neurotoxin type F; said receptor comprises or consists of (i) GT1b and (ii) SV2A, SV2B, SV2C, or the fragment of any of these as defined above, said fragment of SV2A and SV2B preferably comprising or consisting of the three luminal domains of SV2A and SV2B, respectively; and L comprises or consists of VAMP-2, VAMP, VAMP-3 or said fragment of any of these, said fragment preferably consisting of residues 30 to 62 of VAMP-2; (g) said neurotoxin is botulinum neurotoxin type G; said receptor comprises or consists of (i) GT1b and (ii) synaptotagmin I or II or said fragment thereof; and L comprises or consists of VAMP-2, VAMP, VAMP-3 or said fragment of any of these, said fragment preferably consisting of residues 47 to 96 of VAMP-2 and/or (h) said neurotoxin is tetanus toxin; said receptor comprises or consists of (i) GT1b and (ii) SV2A, SV2B, SV2C, the fragment of any of these as defined above, or a GPI-anchored glycoprotein like Thy-1; and L comprises or consists of VAMP-2, VAMP, VAMP-3 or said fragment of any of these, said fragment preferably consisting of residues 30 to 86 of VAMP-2.
VAMP-2 is also known as synaptobrevin 2. VAMP is also known as synaptobrevin 1. VAMP-3 is also referred to as cellubrevin.
Several neurotoxins are capable of recognizing different receptors and/or cleaving different substrates. For example, both BoNT/B and BoNT/G bind to synaptotagmins I and II, wherein the affinities for binding are generally as follows: BoNT/B-synaptotagmin II>>BoNT/G-synaptotagmin I>BoNT/G-synaptotagmin II>BoNT/B-synaptotagmin I.
The recited substrates, to the extent they are fragments defined in terms of specific residue ranges, are generally susceptible to cleavage by the neurotoxin peptidase to the same extent as the protein they originate from.
This embodiment provides preferred neurotoxin/receptor/substrate combinations, wherein the substrate is defined in terms of L as recited in herein above. Accordingly, it is understood that the preferred fragments are to be modified by introduction of fluorophor A and B in order to obtain substrates according to the present invention.
In further preferred embodiments, X is o-aminobenzoyl (o-Abz) or fluorescein isothiocyanate (FITC) and/or Y is 2,4-dinitrophenyl (DNP) or DABCYL. In particular, it is envisaged to use o-Abz in conjunction with DNP and FITC in conjunction with DABCYL. In addition, a large variety of FRET pairs, available from several manufacturers, is at the skilled person's disposal.
In a fifth aspect, the present invention provides a kit comprising or consisting of (a) at least one receptor, said receptor being capable of binding a clostridial neurotoxin and comprising (1) a glycolipid and (2) a peptide or protein; (b) a substrate which (1) is cleavable by the peptidase comprised in said neurotoxin and (2) comprises a FRET pair, the donor of said FRET pair exhibiting increased fluorescence upon cleavage by said peptidase; and (c) optionally one or more liposome-forming lipids.
In a preferred embodiment, the kit according to the present invention further comprises or further consist of one or more of the following (a) a manual containing instructions for performing the method of determining according to the invention; and/or (b) a manual containing instructions for performing the method of a liposome according to the invention.
In a further preferred embodiment, the constituents (a), (b) and (c) of said kit are as defined in conjunction with any of the other aspects of the present invention as disclosed herein above.
The figures show:
The examples illustrate the invention but are not to be construed as being limiting.
The study was performed using purified 150 kDa BoNT/B and BoNT/B complex from C. botulinum strain Okra B (Metabiologics Inc., Madison, Wis., USA), as well as isolated BoNT/B (LC=residues 1-441), BoNT/B LHNB and BoNT/B scLHNB (BoNT/B truncation mutants consisting of residues 1 to 863 and lacking the HC-fragment; either as single polypeptide chain (sc) or dichain hydrolysed into LC and HC after R441; both available from A. Rummel, Hannover Medical School (MHH), Institute for Toxicology). The latter two represent toxin derivatives derived from directed mutagenesis devoid of the receptor-binding domain of BoNT/B. Also, scLHnB is still connected to the BoNT/B LC not only via a disulphide, but also via a peptide bond.
Lyophilised Trisialoganglioside GT1b from bovine brain with a total mass of 2180 Da was purchased from Merck KGaA (Darmstadt, Germany). The ganglioside was dissolved in a 2:1 mixture of Chloroform (CHCl3) and methanol (CH3OH) and stored at −20° C.
Instead of 422 amino acids found in the native SytII protein, the modified version used here presents is truncated, containing 61 amino acids of the N-terminus, with 30 amino acids representing the transmembrane domain. For purification, after production in E. coli BL21, the truncated SytII also contains GST (Glutathione S-transferase) coupled to the N-terminus, without hindering binding of BoNT/B HC. GST-SytII is bound in Triton X-100 micelles due to the purification protocol used by Andreas Rummel's group (Rummel et al. [80]).
For immunoassay experiments, mouse anti-GT1b antibodies (Millipore, Billerica, Mass., USA) or rabbit polyclonal anti-GST antibody (Bethyl Laboratories Inc., Montgomery, Tex., USA) at a concentration of 0.1 μg/mL, and peroxidase coupled goat anti-mouse, or goat anti-rabbit antibodies (Sigma-Aldrich, Buchs, Switzerland) at 1/1000 dilution were used. TMB (3, 3′,5, 5′-tetramethylbenzidine; Sigma-Aldrich, Buchs, Switzerland) or 4CN (4-chloro-1-naphthol; Bio-Rad, Laboratories Inc., Hercules, Calif., USA) were used as substrates for ELISA or dot blot experiments, respectively.
VAMPtide (List Biological Laboratories, Inc., Campbell, Calif., USA) was used as a peptide substrate for measuring proteolytic activity of BoNT/B LC. VAMPtide is an oligopeptide with o-Abz (o-Aminobenzoic acid) as donor fluorophor coupled to the N-terminus, and DNP (2,4-Dinitrophenyl) as acceptor/quencher with an absorbance spectrum overlapping the emission spectrum of the donor fluorophor coupled to the C-terminus. In between, the peptide contains a recognition sequence specific for the cleavage by BoNT/B LC. In the intact, uncleaved state, Forster resonance energy transfer (FRET) between the donor fluorophor and the quencher impedes emission of fluorescence. If VAMPtide is cleaved by BoNT/B LC, then donor fluorophor and quencher are spatially separated and, if excited, the donor fluorophor emits quantifiable fluorescence.
For immunological detection of uncleaved VAMPtide molecules, a 1/1000 dilution of polyclonal rabbit antibody generated against aa 1-81 of the cytoplasmic part of rat cellubrevin (anti-VAMP 1/2/3; Synaptic Systems GmbH, Göttingen, Germany) was used. Binding of the anti-VAMP antibody was detected via peroxidase coupled anti-rabbit antibody (Sigma-Aldrich, Buchs, Switzerland).
VAMPtide assay
Unless otherwise stated VAMPtide assay for the detection of BoNT cleavage activity was performed according to the manufacturer's instructions. In short, lyophilised VAMPtide was resuspended in DMSO resulting in a 5 mM stock solution and stored at −20° C. Prior to the experiments, stock solution was diluted in 20 mM HEPES to 250 μM. The reaction buffer for hydrolysis of VAMPtide by BoNT/B was 20 mM HEPES (pH 7.4), 0.05 mM ZnSO4, 5 mM DTT and 0.2% Tween-20. For reactions with BoNT/B LC the hydrolysis buffer contained 50 mM HEPES (pH 6.3) and 0.05% Tween-20. For the assays a final concentration of 10 μM VAMPtide was used. Assays were performed in black FluoroNunc F96 Micro-Well plates (Nunc, Langenselbold, Germany). The assay was run at 37° C. in a Spectramax Gemini XS fluorescence microplate reader (Molecular devices, Sunnyvale, Calif., USA) with an excitation wavelength of 321 nm and emission wavelength at 418 nm.
For production of receptor liposomes either a mixture of SPC (soy Phosphatidylcholine; Phospholipon 85G; Lipoid A G, Cham Switzerland), cholesterol (Sigma-Aldrich, Buchs, Switzerland), and DL-alpha-tocopherol (VW R, Dietikon, Switzerland) or a predefined mixture of DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine) or POPC (1-Palmitoyl-2-oleoyl-sn-glycero-3-phophocholine) and cholesterol (Avanti Polar Lipids Inc., Alabaster, USA) was used. DL-alpha-Tocopherol was supplemented for its antioxidant and membrane stabilising effect [36].
An alternative lipid mixture may be prepared as follows. Soybean asolectin, dioleoyl L-K-phoshatidylcholine, bovine brain phospholipids or alternatively a phosphatidic acid (PA) lipid mixture (consisting of dioleoyl L-alpha-phosphatidylcholine 70%, phosphatidic acid 20% and cholesterol 10%) are dissolved in chloroform, dried to a thin film under a gentle N2 flow and vacuum pumped for at least 2 h to remove residual traces of organic solvent.
For LUV production the required volume of the respective lipid mixture was dissolved in a 1:1 mixture of methanol (CH3OH) and dichloromethane (CH2Cl2), and dried at room temperature under vacuum at 1400 rpm in a Eppendorf concentrator 5301 with a F 45-48-11 Rotor (Eppendorf A G, Hamburg, Germany) until complete evaporation of the organic solvents (1-2 h). Upon complete evaporation of solvent, the lipids formed a thin yellow film on the walls of the vials. For longer storage, vials were sealed under N2-atmosphere and stored at −20° C. For liposome production, the lipid film was resuspended in appropriate volumes of 20 mM HEPES buffer (pH 7.4) as aqueous medium. The vial was shaken until complete solution of the lipids, giving a turbid white emulsion of multilamellar vesicles (MLV). Examination under Zeiss Dialux 20 EB light microscope using 100-fold object lens (100/1.25 oil immersion) plus 10 fold ocular (Periplan GF 10×/18) magnification allowed for visual control that the MLV had been formed. The vial with the emulsion was shaken with 1400 rpm in an Eppendorf Thermomixer comfort (Eppendorf A G, Hamburg, Germany) at 30° C. for 30 minutes until the lipid film was completely dissolved in the aqueous medium. LUVs were obtained by extrusion through polycarbonate (PC) membranes (Nucleopore track-etched polycarbonate membrane; Whatman plc, Maidstone, UK) with defined pore sizes (400, 200, and 100 nm) in a MiniExtruder (Avanti Polar Lipids Inc., Alabaster, Ala., USA). The equipment was assembled according to the manufacturer's instructions and, for production of LUVs, the MLV emulsion was passed through a 400 nm, and subsequently through 200 and 100 nm pore size PC-membrane. Homogeneity and size were controlled via DLS (Dynamic Light Scattering) with a Brookhaven Instruments BI-200SM research goniometer and particle sizer (Brookhaven Instruments, Holtsville, N.Y., USA) at an angle of 90° at 25° C. sample temperature. Data was processed with Brookhaven Instruments Particle Sizing Software (Brookhaven Instruments, Holtsville, N.Y., USA).
While LUVs are preferred, liposomes may also be prepared as follows.
VAMPtide or other any other substrate according to the invention is diluted in the reaction buffer (preferably 20 mM HEPES with ZnSO4 and TCEP at pH 7.2-7.4 as defined herein above; see “aqueous medium”) at concentrations of 100-200 μM and added to a lipid film giving an emulsion of 40 mM DOPC/Cholesterol (13/1), 100-200 μM substrate in the reaction buffer. The emulsion is extruded eleven times each through polycarbonate membranes with pore diameters of 200 nm and 100 nm (and, if preferred, 50 nm) pore diameter. Then, lactose or trehalose is added to give a final concentration of 1 to 5%, preferably 3.5%. In the following, the (clear) liposome solution is cooled to 4° C., to −20° C., and subsequently to −80° C. The frozen emulsion is then subjected to freeze-drying over night until complete evaporation of the contained liquid. Reconstitution is carried out first by adding 1/10 of the original liquid volume as 10 times concentrated buffer (preferably 10×HEPES). For resuspension, the lyophilizate plus added buffer is left for 30 minutes at room temperature, and then vigorously shaken, centrifuged, and again vigorously shaken. Subsequently, liquid (i.e. distilled H2O) is added till the original level of liquid volume is reached.
VAMPtide or other any other substrate is diluted in the reaction buffer (preferably 20 mM HEPES with ZnSO4 and TCEP at pH 7.2-7.4 as defined herein above; see “aqueous medium”) at concentrations of 100-200 μM and added to a lipid film giving an emulsion of 40 mM DOPC/Cholesterol (13/1), 100-200 μM substrate in the reaction buffer. The vessel containing the emulsion is placed into a 0° C. waterbath. The transducer tip of the sonicator (such as Branson sonifier) is immersed into the sample. Different durations and sonication times can be applied until the emulsion turns from milky to opalescent. Metallic particles from the transducer tip can be removed by centrifugation.
Unless otherwise stated, liposome emulsions were subjected to SEC (size exclusion chromatography) columns with a gel bed volume of approximately 600 μL for separation from not-integrated and/or not-encapsulated compounds. SEC columns allowed for easy and quick separation of 10-100 μl of sample via centrifugation in a tabletop centrifuge. Due to the small gel-volume the dilution effect was relatively low (1-2 times dilution). The technique works as common gravitational SEC-column, just that the gravitational force is replaced by centrifugal force. The gel, in this case a 50%-slurry of Sepharose 4B (Sigma-Aldrich, Buchs, Switzerland) in 20 mM HEPES (pH 7.4), is filled into the column, i.e. a 0.5 mL Eppendorf cup pierced on the bottom and stuffed with 1 mm glass fibres. For separation, 10-100 μL of the sample are loaded slowly onto the gel bed, assuring that none of the sample passes along the sides of the SEC column. Centrifugation of the tube at 1200 RCF for 45 s elutes the first fraction of the sample, including the components with the biggest hydrodynamic volume. Following, further application of elution buffer with the same volumes as the sample applied and further centrifugation steps elute further fractions with decreasingly smaller hydrodynamic volume. The arrangement of the system is shown in
For production of receptor liposomes, GT1b was added to the lipid mixture dissolved in organic solvent and either dried under vacuum or under constant laminar flow of gaseous N2. Due to the high transition phase of GT1b gangliosides, the lipid solution cannot be dried out completely. Accordingly, instead of a lipid film the lipid mixture is present in a highly viscous gel-like state. For complete solvation of the gel, the whole suspension is pipetted up and down in aqueous medium and subsequently shaken as described above. GST-SytII is added with the aqueous medium in the respective concentration. Likewise, for encapsulation experiments, VAMPtide is added with the aqueous medium in the respective concentration to dried lipid film. To remove Triton X-100 from liposomes with GST-SytII, the emulsion is incubated with pre-hydrated Bio-Beads SM-2 Adsorbents (Bio-Rad, Laboratories Inc., Hercules, Calif., USA). Bio-Beads plus GST-SytII liposomes are incubated under rotation at 4° C. for 2.5 h. Thereafter, another portion of SM2 beads is added and incubated with the emulsion for another 2 h. To separate Bio-Beads with bound Triton X-100 from the liposome, the mixture is kept still for 5 min to allow settling of the beads. After transferring the liposomes in the supernatant to a clean vial, the remainder is centrifuged at 5.000×g RCF for 30 seconds and the resulting supernatant pooled with the first one.
For ELISA experiments, receptors, VAMPtide, receptor liposomes, or VAMPtide liposomes are coated on MaxiSorp microtiter plates (Nunc, Langenselbold, Germany) at 4° C. overnight. After 60 min of blocking, sample cavities are washed and incubated for 60 min with the respective antibodies, followed by incubation with species-specific peroxidase coupled antibodies for 30 min.
In direct dot blot experiments, two times 1 μL of the antigen is pipetted onto nitrocellulose membranes (Bio-Rad, Laboratories Inc., Hercules, Calif., USA) and dried at ambient temperature. After 60 min of blocking, membranes are washed and incubated for 45 min with the respective antibodies, followed by incubation with species-specific peroxidase coupled antibodies for 30 min. In sandwich dot blot experiments, entire membranes are coated with the respective capture antibody for 60 min. After washing, membranes are blocked overnight at 4° C. Then, membranes are washed, dried slightly, subsequently 1 μL of the respective antigens pipetted onto the membrane and incubated in a humid chamber for 60 min. Afterwards, membranes are incubated with detection antibody complementary to the coating antibody for 45 min, followed by 30 min incubation with peroxidase coupled antibody, binding the respective detection antibody.
The aqueous liposomes lumen provides a cell-like reaction compartment. Due to the multiple and complex reactions (binding of BoNT/B, translocation of BoNT/B LC and VAMPtide cleavage) that are supposed to take place in the assay, preferably LUV with a size of approximately 100-200 nm are used to provide for a sufficiently large reaction volume. For the reproducibility and functionality of the assay it is preferable that the liposome emulsion were consistent concerning their lamellarities and diameters. Accordingly, emulsions with mainly MLVs obtained by reconstitution of lipid films were subjected to an extrusion process. We used an Avanti MiniExtruder combined with PC membranes with pore sizes between 400 and 100 nm to obtain homogenous emulsions with liposome diameters between 100 and 200 nm. Success of the extrusion process was monitored by visible control with (light microscopy; not shown) or without magnification, as shown in
Careful review of current literature suggested that artificial lipids might present an alternative to natural phosphatidylcholine [37-39,35]. Especially for reliable imitation of the characteristics of eukaryotic cell membrane it is preferred to use lipids such as DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine) or POPC (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), due to their low transition phase temperature (approximately 0° C.) and their structural properties (Steven Burgess, director R&D, Avanti Polar Lipids, personal communication). Accordingly, not only SPC, but also DOPC, both in combination with Cholesterol, were tested. DLS measurements were performed for the liposomes derived from different lipid formulations (Table 2).
The quantity of lipid molecules per liposome (Nlipid) was calculated with formula (1), using the average diameter of the lipid head groups (a), the outer liposome diameter (d), and the inner liposome diameter, which is the outer diameter reduced by 5 nm (the average thickness of a lipid double membrane).
N
lipid=4π×1/a×[(d/2)2+(d/2−5 nm)2] (1)
The total number of liposomes (Nlipid) in one millilitre of the respective emulsion was calculated using formula (2), with the concentration of the lipid mixture in Mol (clipid), the Avogadro constant of 6.022×1023 mol−1 (NA) and division by 1000 for conversion from L to mL [40].
N
lipid=(clipid×NA)/(Ntot×1000) (2)
Interestingly, average diameters were higher in DOPC liposomes in comparison to SPC liposomes, although the head groups of both lipids occupy almost the same average area. Lower cholesterol content in DOPC could not have been responsible, as SPC liposomes without any cholesterol were even smaller. An explanation for differing liposome sizes could be, that DOPC is known to have a higher bilayer bending rigidity when compared with similar lipids [41-43]. Hence, extrusion with the MiniExtruder was not able to reduce average diameters of DOPC liposomes below 170 nm. The extruded liposomes from the three formulations shown in Table 2 showed low polydispersity, i.e. high homogeneity, were easily extruded and matched the required size of 100-200 nm. Hence, both liposome types were used in the following experiments.
In order to detect the nerve cell receptors GST-SytII and GT1b, either in solution or integrated into liposome membranes, we established an immunoassay protocol for specific and sensitive detection.
A pH-shift provides for enhanced translocation of BoNT LC via the BoNT HN transmembrane channel from the cis side (i.e. endosomal lumen or medium surrounding the liposomes) to the trans side (i.e. cytosol or liposomal lumen) of a membrane [44]. Hence, it is preferred to use conditions that emulate the pH and redox gradient across endosomes, i.e. an acidic shift from pH 7.2 to 5.2, to facilitate transformation of BoNT/B HN into a transmembrane channel. Hence, to elucidate, whether a pH-shift with DMG (3,3-Dimethylglutaric acid) would influence the binding affinity of BoNT/B to the respective receptors we performed the following experiment. BoNT/B was coated on a microtiter plate and GST-SytII used as antigen, which was detected by anti-GST and a respective peroxidase coupled anti-species antibody. The pH was kept at pH 7.2 for 90 min, shifted to pH 5.2 after 60 min and incubated for another 30 min, or kept at pH 5.2 for the entire 90 min during incubation of GST-SytII with the coated toxin, respectively.
Results presented in other studies, however, confirm that binding of BoNT/B to SytII at pH 5 was unaffected or even increased [46]. Although GT1b or GD1a, which is another ganglioside, SytII, and SytI are involved in BoNT/B binding [47,48], GST-SytII has in our experiments been sufficient for binding of the toxin.
Experiments performed in hippocampal neurons cultured from ganglioside KO mice showed that toxin binding was affected by absence of gangliosides [49]. Another study, however, was able to show with PC12 cells, a neuroendocrine cell line with low ganglioside contents, that SytII present in the cell membranes was sufficient to bind and mediate entry of BoNT/B into the nerve cell [50]. Accordingly, binding of BoNT/B HCC to GST-SytII will remain active, so that even under acidic conditions, translocation of BoNT/B LC through the HCN transmembrane channel is feasible.
Unfortunately, there is little information on the exact content of Glib residing in the motoneuronal membrane. Yet, a publication on the molecular composition of synaptic vesicles (SVs) gives information on lipid and protein composition and content in SV membranes [51]. The authors suggested that ceramides present approximately 0.17-1.2% and hexylceramides 8.6% of the total lipid content. In publications, where protein and lipid composition in the synaptosomal plasma membrane (SPM) were quantified, it was found that gangliosides present approximately 8.2% of all lipids [52,53]. Accordingly, we used either 0.2 to 0.5 mM GT1b in the presented experiments, which corresponds to 0.1 to 2.5% of total lipid content. Also, similar ganglioside contents (i.e. 0.11 mM) have been used for supplementation of ganglioside deficient PC12 cells tested for BoNT/B binding [50]. As for GT1b, little is known about the exact amount of SytII receptor in the presynaptic membrane. Measurements in SV, however, indicate that Sytl, a SytII analogue, constitutes approximately 7% of all SV-Proteins [51]. Yet, as GST-SytII will be the only protein receptor in the liposome membrane, there should be sufficient protein receptor molecules present to complement GT1b receptors on the liposome membranes. Although both, N-terminal domain of SytII and ganglioside are involved in BoNT/B binding [47,48], it has been reported, that truncated SytII, with N-terminus and transmembrane domain only is sufficient to bind and mediate entry of BoNT/B into nerve cells [50]. Hence, in comparison to GT1b, we used excess GST-SytII at concentrations of 0.45 to 0.72 mg/mL, i.e. 12.5 to 20 mM for production of receptor liposomes.
As GT1b integration into liposome membranes takes place via spontaneous integration of its ceramide chain during reconstitution of the lipid film, GT1b is already added to the other lipid components at the very beginning [54,55]. In case of GST-SytII, we expected that the apolar transmembrane domain of SytII [56,57] and the associated Triton X-100 molecules facilitate integration [58]. Accordingly, the protein receptor was supplemented with the aqueous medium.
Following extrusion, liposome emulsions, containing Triton X-100 were treated with SM-2 Bio-Beads to extract detergent from the liposomes. To test for integration of the receptors in the liposome membranes, we immobilised extruded receptor liposomes either on nitrocellulose membranes or on microtiter plates and analysed them with the respective receptor antibodies.
To confirm and validate receptor integration into liposomes, SEC was used to separate the respective liposomes [40]. Accordingly, a refined and miniaturised SEC method that allowed reliable separation of minute volumes of liposome mixtures from not-encapsulated compounds or buffer components by hydrodynamic diameter was used. After separation, SEC fractions were analysed with the respective receptor antibodies in dot blot experiments on nitrocellulose membranes and in ELISA experiments (
Thus, visual control and detection in dot blot and ELISA experiments confirmed the presence of both GT1b and GST-SytII in liposomal fractions.
A sandwich immunoassay in a dot blot format was used to test whether both receptor types are present on the membrane of same liposomes (
Also, it has been suggested before, that GT1b and SytII co-localise in membranes due to binding events taking place between the ceramide portion of GT1b and the transmembrane domain of SytII [47]. Taken together, there is strong evidence that both receptors are present on the membrane of the same liposomes.
Fluorescence Characteristics of VAMPtide o-Abz Fluorophor
For measuring VAMPtide fluorescence without the necessity of the actual cleavage reaction unquenched calibration peptide of VAMPtide (uq VAMPtide; List Biological Laboratories, Inc., Campbell, Calif., USA) was used. This peptide contains the complete VAMPtide peptide sequence with a coupled o-Abz fluorophor, but without any quencher molecule.
As can be seen in
As the liposome lumen presents the reaction compartment for the cleavage reaction, the cleavage buffer has to be confined to the liposome interior. The cleavage buffer recommended by the manufacturer is comprised of HEPES, ZnSO4, DTT as reducing agent and Tween-20 to increase toxin's solubilisation. Due to its size and steric hindrance, tris(2-carboyethyl)phosphine (TCEP) cannot freely diffuse through membranes. Accordingly, it has been tested as a substitute for DTT as reducing agent [45,59,60,6,61,62]. Also the detergent Tween-20 may present a problem when used within liposomes, as it may compromise the integrity of cell membranes, possibly facilitating cells lysis [63]. Also, Tween-20 may intercalate between the membrane bilayers and form mixed micelles with isolated phospholipids and membrane proteins. Accordingly, if Tween-20 was used, there might be a certain risk, that a fraction of the liposomes could be transformed from LUV into mixed micelles with lipid and detergent molecules in the membrane. Hence, attempts were made to replace DTT in the cleavage buffer by TCEP and to elucidate how the absence of Tween-20 affects VAMPtide cleavage.
First, to find the TCEP concentration, best suited for VAMPtide cleavage, the cleavage activity of 20 nM BoNT/B was tested with different TCEP concentrations, ranging from 0.75 to 5 mM, and compared with the cleavage activity in buffer supplemented with 5 mM DTT. The cleavage reaction was performed at 37° C. and fluorescence was measured with excitation and emission wavelengths of 321 nm and 418 nm, respectively. In direct comparison of the cleavage activities, 2 mM TCEP was found to be the concentration, which yielded the highest cleavage activity (
Similar observations have been made in other studies, where 1 and 2 mM TCEP were found to be the best concentrations for VAMPtide cleavage by BoNT/B and BoNT/B LC, respectively [64,65]. In other studies that employed VAMPtide for measuring BoNT/B LC cleavage, Tween-20 has always been supplemented in the cleavage buffer. The manufacturer of VAMPtide states that in case of SNAPtide, the analogous reporter peptide for BoNT/A LC, cleavage was also measured in the absence of Tween-20, though at lower levels. The manufacturer hypothesises that Tween-20 may help to disperse non-specifically bound enzyme or substrate from the walls of the microtiter well, so that more enzyme and substrate are available for interaction [66]. So, apparently specificity of VAMPtide cleavage by BoNT/B LC should not be impaired by the absence of Tween-20 in the cleavage buffer. As our results confirm good cleavage activity even without Tween-20, the described buffer composition without Tween-20 and with 2 mM TCEP in 20 mM HEPES (pH 7.4) supplemented with 0.05 mM ZnSO4 is preferred.
Apart from BoNT/B, we tested the cleavage activity of isolated BoNT/B LC, TeNT, LHNB, and scLHNB in the modified cleavage buffer. Early experiments showed that isolated BoNT/B LC did not exhibit any cleavage activity under these conditions. Instead, supplementation of 0.2% Tween-20 restored most of the cleavage activity of isolated BoNT/B LC, while DTT supplementation restored the function only partly (data not shown). As expected, the cleavage reaction did neither work for TeNT, as VAMPtide did not contain the specific recognition site for TeNT LC. In contrast to isolated BoNT/B LC and TeNT, BoNT/B and BoNT/B complex displayed typical cleavage activity, with a limit of detection of 2 and 5 nM, respectively (
LHNBand scLHNB are BoNT/B truncation mutants devoid of the receptor-binding domain. Also, scLHNB is still connected to the BoNT/B LC not only via disulphide, but also via a peptide bond. As the LC in these toxin derivatives is still active, VAMPtide could be successfully cleaved under the modified buffer conditions, both with detection limits of 1 nM. Although the actual enzymatic cleavage activities (Vmax s−1) showed very high inter-assay variations, it can be said, that the modified cleavage buffer conditions allows cleavage of VAMPtide by BoNT/B, LHNB and scLHNB (
Other groups that used VAMPtide molecule as reporter for BoNT/B LC endopeptidase activity found similar results, e.g. detection of 0.25, 6 and 10 nM BoNT/B LC in buffer after incubation with 10, 4.2 and 20 μM VAMPtide, respectively [67,68,64,65,69]. For BoNT/B, detection was possible for 3.3 to 6.7 μg mL−1, i.e. 22 to 44 nM [64,65]. The values obtained are still above the values that could theoretically be achieved. The manufacturer described detection limits as low as 0.2 nM for BoNT/B, and even 78 μM for BoNT/B LC, using similar machinery and settings [70]. Results for BoNT/B LC, however, were only obtained when endpoint readings were obtained after 24 hours incubation at 37° C. Using magnetic bead enrichment prior to the VAMPtide cleavage assay, even allowed detection at concentrations as low as 10 ng mL−1, i.e. 0.07 nM BoNT/B [67]. Concerning the dependence of BoNT/B LC cleavage activity on Tween-20 and/or DTT, another group made similar observations [69]. They found, that nonreduced LC exists as a homodimer, which reduces the flexibility of LC molecules to move around and to coordinate with the substrate for the enzymatic reaction to occur. High cleavage activities have been found in DTT containing cleavage buffer (Table 2). Interestingly, if 2 mM TCEP is used instead of 5 mM DTT, LC displays almost no cleavage activity. In the complete neurotoxin, consisting of heavy chain (HC) and light chain (LC), however, part of the HC belt hangs above the open pocket of the LC in which the active site is located, so that latter becomes more difficult for the substrate to access, thus showing a lower level of activity than the LC in buffer supplemented with DTT [69]. In case of LHnB and scLHnB, which are devoid of the BoNT/B HC binding domain, less steric hindrance is present, thus allowing for higher cleavage activities. Here, however, TCEP seems to allow for sufficient reduction. This might, to a large part, be due to the fact that the two toxin derivatives resemble structure-wise more to the holotoxin more than the LC. BoNT/B complex showed similar, but slightly lower cleavage activities than the holotoxin if DTT was present. This exemplary assay provides sensitivity for BoNT/B cleavage activity at concentrations as low as 2-5 nM.
Incorporation of VAMPtide Reporter Peptide into the Assay Liposomes
VAMPtide molecules are incorporated into liposomes by adding them together with the aqueous medium for lipid film reconstitution. Upon forming of MLV, VAMPtide molecules become enclosed by liposomal membranes. With subsequent extrusion through PC membranes, a distinct portion of the molecules gets encapsulated, while another part stays outside the liposomes. To test for the actual encapsulation rate of VAMPtide molecules into the liposomes, without the need for any cleavage reaction, we used a VAMP antibody and corresponding peroxidase coupled anti-species antibody for detection.
In
As shown in Table 4, EE[%] values were very high, with approximately 73 and 67% of all VAMPtide molecules encapsulated into liposomes, i.e. 146.14+/−46.28 μM (original 200 μM) or 66.61+/−16.19 μM (original 100 μM) VAMPtide encapsulated. High Standard Deviations (SD) were due to the narrow range of linear detection in the ELISA immunoassay (
DNP (2,4-dinitrophenol) and o-Abz (o-Aminobenzoic acid) are coupled to the VAMPtide molecule as FRET acceptor and donor fluorophor, respectively. It is known, that DNP exhibits hydrophobic properties [71] and o-Abz may also dissolve in nonpolar solvents [72]. Hence, instead of being properly encapsulated, it might be that VAMPtide is only loosely associated to the liposomes and/or integrated only by part into the liposome membranes [73]. This could influence the calculated encapsulation efficiency, as BoNT/B could cleave the associated VAMPtide molecules without any binding or translocation needed. Accordingly, we intended to rule this out, by testing for not-encapsulated VAMPtide with a protease protection assay. In this assay, VAMPtide molecules, which are only loosely associated to the liposomes, and VAMPtide molecules that are partly integrated into the membrane, but whose peptide chains extend into the liposome exterior, would be cleaved by Proteinase K (Sigma-Aldrich, Buchs, Switzerland). Proteinase K is a broadspectrum serine protease, which predominantly cleaves the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups [74].
First, to test whether Proteinase K is able to cleave VAMPtide molecule, we subjected a solution of 200 μM VAMPtide to digestion with 100 μg/mL of Proteinase K. Digestion was performed for 1 h in a 37° C. waterbath.
This means, that Proteinase K was able to cleave VAMPtide. Subsequently, VAMPtide liposomes from the previous experiment with approximately 146.14+/−46.28 μM VAMPtide encapsulated (original 200 μM) were subjected to digestion for 60 min at 37° C. with 50 and 100 μg/mL of Proteinase K, respectively. After digestion, VAMPtide liposomes were fractioned by SEC, and measured with VAMP antibody in an ELISA assay (
In the actual assay, the respective substrate is mixed with 10 μL of the corresponding 10 times concentrated reaction buffer (RB) and the toxin or respective sample. Finally, H2O (ultrapure; 37° C.) is added to give a final volume of 100 μL (in black 96 half-well microplates pre-warmed to 37° C.). The measurement is then performed with a SpectraMax GeminiXS or any other spectro-fluorometry microplate reader at 37° C. assay temperature. For signal readout the samples are excited at the excitation and emission wavelength combination of the respective substrate (see below).
As can be seen in
To allow for later experiments to detect PL50 and PL150 e.g. in liposome fractions or for encapsulation studies, immunological detection was tested in ELISA experiments. Therefore, different concentrations of either substrate were coated over night at 4° C. on microplates (clear MaxiSorp microplates; Nunc, Langenselbold, Germany). Bound PL50 and PL150 were detected with polyclonal rabbit@SNAP25 antiserum or affinity purified rabbit@VAMP1,2,3, respectively (SynapticSystems GmbH, Goettingen, Germany). The antibodies themselves were subsequently detected with goat@rabbit-HRP (BioFX Laboratories Inc., Owings Mills Md., USA) and TMB (Pierce Biotechnology Inc., Rockford Ill., USA) as substrate. As can be seen in
SNAPtideFITC (SNAPtide (FITC/DABCYL); List Biological Laboratories Inc., Campbell, Calif., USA) is a peptide, which is intramolecularly quenched by fluorescence resonance energy transfer (FRET). The N-terminally-linked fluorophore is fluorescein-thiocarbamoyl (FITC) and the acceptor chromophore is DABCYL [89]. Lyophilised SNAPtideFITC stock is dissolved in ultrapure H2O and for experiments with SNAPtideFITC, the following reaction buffer (RBSNAPtide-FITC) is used: 20 mM HEPES (pH 7.5), 15 mM ZnCl2, 1.25 mM DTT, 0.1% Tween-20. Upon cleavage of the substrate by BoNT/A, the fluorescence can be measured with an excitation wavelength of λex=490 nm and an emission wavelength of λem=523 nm with a cutoff filter set at 495 nm.
SNAPtideo-Abz (SNAPtide (o-Abz/Dnp); List Biological Laboratories Inc., Campbell, Calif., USA) is a peptide, which is intramolecularly quenched by fluorescence resonance energy transfer (FRET). The N-terminally-linked fluorophore is o-aminobenzoic acid (o-Abz) and the acceptor chromophore is a 2,4-dinitrophenyl group (Dnp) [89,90,91]. Lyophilised SNAPtideFITC stock is dissolved in ultrapure H2O and for experiments with SNAPtideo-Abz, the following reaction buffer (RBSNAPtide-o-Abz) is used: 20 mM HEPES (pH 8.0), 0.75 mM ZnSO4, 1.25 mM DTT. Upon cleavage of the substrate by BoNT/A, the fluorescence can be measured with an excitation wavelength of λex=320 nm and an emission wavelength of λem=421 nm.
As for production of B-liposomes, the lipid receptor (ganglioside GT1b) was evaporated together with the used lipids (40 mM DOPC/Cholesterol; same as for B-liposomes) from organic solution to form a lipid film; GT1b was used at the same concentrations described for B-liposomes. The protein receptor for BoNT/A (GST-SV2c) was applied to the lipid film together with the future reaction buffer (RBPL50); GST-SV2c was used at 12.5 or 8 μg/mL (final concentration in the liposome emulsion). Except for the receptors used, the production process for A-liposomes is the same as for B-liposomes, including removal of Triton X-100 by treatment with BioBeads SM-2.
SEC separation of B-FALPL150 and B-FALVAMPtide-o-Abz, respectively, shows that the majority of PL150 and VAMPtideo-Abz molecules elute in the liposome fractions (
| Number | Date | Country | Kind |
|---|---|---|---|
| 1105915.1 | Jul 2011 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2012/064068 | 7/18/2012 | WO | 00 | 4/9/2014 |
