Journal ~fNeuroscience Methods, 42 (1992) 229-236 ~ 1992 Elsevier Science Publishers B.V. All rights reserved 0165-0270/92/$05.00
229
NSM 01366
A simplified method for the preparation of tetanus toxin binding fragment for neurobiology Paul S. Fishman ~, Dawn A. F a r r a n d ~, Jane L. Halpern 2 and William C. L a t h a m 3 / Baltimore Veterans Administration Medical Center and the Department oJ"Neurology of the Unit'ersity of Maryland School of Medicine, Baltimore, MD (USA), 2 Dil'ision of Bacterial Products, Food and Drug Administration, Bethesda, MD (USA) and ~ Massachusetts Public Health Laboratories, Boston, MA (USA) (Received 22 May 1991) (Revised version received 12 March 1992) (Accepted 12 March 1992)
Key words: Fragment C; Transneuronal transport; Axonal transport; Neuronal labeling The non-toxic binding fragment of tetanus toxin (fragment C) binds avidly to neural tissue and has a growing number of neurobiological uses. Its current utility is limited by both its high commercial cost and the complex procedure for its preparation requiring highly purified tetanus toxin. We have developed a short procedure which prepares fragments of tetanus toxin fi'om crude C. tetani extracts. The resultant proteins are atoxic with molecular sizes and immunological properties closely resembling fragment C. These proteins undergo retrograde axonal and apparent transneuronal transport in a fashion similar to fragment C.
Introduction
Tetanus toxin is well known to bind avidly to neuronal membranes and undergoes both axonal transport and transneuronal transport (Price et al., 1975; Schwab et at., 1979; Pierce et al., 1986). Fragments of the tetanus toxin molecule have been produced which are both atoxic and show neuronal binding and transport properties similar to that of native toxin (Bizzini et al., 1977; Bizzini et al., 1980). The most well studied of these peptides, fragment C, is a 47 kD fragment of the heavy chain of the toxin, produced by papain digestion of tetanus toxin (Helting and Zwisler, 1974). Fragment C contains the conformational site of tetanus toxin necessary for binding and
Correspondence to: Paul S. Fishman, M.D., Ph.D., Department of Neurology, University of Maryland School of Medicine, Baltimore, MD 21201, USA. Tel.: (410) 328-2346; Fax: (410) 328-5899.
internalization by neurons and their processes (Bizzini et al., 1977; Buttner-Ennever, 1981; Evinger and Erichsen, 1986). Increasing neurobiological interest in fragment C resulted initially in the exhaustion of commercial supplies in 1988, and fragment C has only recently again become commercially available. Published accounts of production of fragment C begin with highly purified tetanus toxin as the substrate for enzymatic degradation (Helting and Zwisler, 1974). Purification of tetanus toxin requires a significant effort in itself. Tetanus toxin has unusual solubility properties which cause it to aggregate under non-reducing conditions (Robinson and Hash, 1982). Preparations of tetanus toxin also frequently show endogenous proteolytic activity so that careful control of bacterial culture conditions, toxin extraction and handling are needed to obtain intact toxin comparable to that used in published preparations of fragment C (Weller et al., 1988). High-level purification of fragment C after digestion of tetanus toxin with
230 p a p a i n is t h e n n e e d e d to r e m o v e t h e e x t r e m e l y toxic n a t i v e t e t a n u s toxin f r o m t h e p r e p a r a t i o n ( R o b i n s o n et al., 1975). T h i s level o f p u r i f i c a t i o n is d i f f i c u l t to a c h i e v e e v e n w i t h u n i f o r m p r e p a r a t i o n s o f i n t a c t toxin. W i t h p r e p a r a t i o n s c o n t a i n ip~. e n d o g e n o u s p r o t e o l y t i c activity, t h e v a r i e t y o f conta~;~'nating p e p t i d e s m a k e s e p a r a t i o n o f fragm e n t C v i r t u a l l y i m p o s s i b l e by p u b l i s h e d t e c h n i q u e s . T h e d i f f i c u l t i e s e n c o u n t e r e d in p r o d u c -
t i o n o f f r a g m e n t C h a v e led in p a r t to successful e f f o r t s by s e v e r a l i n v e s t i g a t o r s to p r o d u c e fragm e n t C by g e n e t i c r e c o m b i n a n t m e t h o d s ( M a k o f f et al., 1989; H a l p e r n et al., 1990). O u r i n t e r e s t in t h e n e u r o b i o l o g i c a l p r o p e r t i e s o f f r a g m e n t C, r a t h e r t h a n in its p h a r m a c o l o g i c o r p h y s i c o - c h e m i c a l p r o p e r t i e s , led us to d e v e l o p a m e t h o d for its p r o d u c t i o n t h a t is s i m p l e , rapid, r e l a t i v e l y safe, i n e x p e n s i v e a n d u s e s r o u t i n e l y
Fig. 1. A: electrophoretic separation of tetanus toxin fragments, Lane MC (Massachusetts Crude) contains undigested ammonium sulfate precipitate of C. tetani cultures (180 ~zg/lane). Note the highly heterogenous mixture of peptides without a clear band corresponding to heavy chain (100 kD) or light chain (55 kD) of purified tetanus toxin. Lane D contains a trypsin digest of the MC mixture (pre-dialysis). Most of the remaining protein is contained as a doublet with molecule weight between 42 and 46 kD (75 /.,g/lane), although much of a total protein concentration has run off the gel as small peptide. Lane CF contains commercial fragment C obtained from Calbiochem (16/zg/lane) with a similar but slightly smaller doublet compared to D. Both CF and D have a small amount of protein larger than the doublet band. Samples were incubated with 5% mercaptoethanol prior to application to the 10-20% acrylamide gradient gel run in the presence of 0.19: SDS. B: immunoblot of gel electrophoresis of a similar mixture of tetanus fragment as in 1A, reacted with antibodies against fragment C (polyclonal, rabbit, 1/2000 dilution). MC lanes contain the crude precipitate, showing multiple bands that react with anti-C fragment antibodies (100 #g of protein per lane). Lane D shows that the major protein doublet of the digest mixture is also immunoreactive with the antisera (40 /zg/lane). As expected, commercial fragment C (lane CF) reacts with these antibodies, although on this occasion it did not run as a doublet (10 /xg/lane). In spite of the area of poor contact of the gel in the CF lane, it is apparent that both the digest and commercial fragment C also have a similarly immunoreactive 38 kD peptide.
231 available laboratory equipment. Previous studies of fragment C have shown it to be relatively protease resistant compared to other sites of the tetanus toxin molecule (Helting and Zwisler, 1974; Bizzini, 1977). This method produces protein fragments with molecular weight, antigenic and neural uptake properties very similar to those of fragment C produced by previously published procedures.
Methods
Preparation of the fragment Cultures of C. tetani (Harvard strain) were grown in modified Harvard medium for 4 days in 5-1 batch lots in Ehrlenmeyer flasks (Latham et al., 1962). The cultures were then filtered to remove bacteria and other particulate matter, and extracellular toxin was precipitated by the addition of solid ammonium sulfate to a final concentration of 40% at 5°C. The solution was again filtered, and the crude toxin preparation was allowed to dry onto filter paper. This solid preparation can be maintained indefinitely (over 1 year in our hands) at - 4 0 ° C . Crude toxin was resuspended in 0.1 M phosphate buffer (7.5 pH), and ammonium sulfate was removed by dialysis prior to digestion. This crude preparation would be considered highly unsuitable for the preparation of fragment C by usual methods. It is highly aggregated and will not enter conventional SDS/acrylamide gels, in spite of the addition of urea, without reduction with mercaptoethanol. Electrophoresis of this preparation under reducing conditions reveals a variety of proteolytic fragments of extracellular tetanus toxin (Fig. 1A). This preparation was digested with trypsin (type 463 from Sigma Chemical) in the presence of SDS, in an 0.1 M phosphate buffer at pH 7.5. Usual preparations consisted of 15 m g / m l of crude toxin digested with 0.1 m g / m l of trypsin in 0.1% SDS. The reaction is allowed to proceed until all high molecular weight fragments (and native toxin) had been eliminated (20 rain at 37°C). The digestion was terminated by the addition of an excess of soybean trypsin inhibitor.
Crude tetanus protein was also maintained as a concentrated solution (50 m g / m l ) at -40°C. Such solutions when assayed months later had undergone significant proteolysis. The large protein aggregates seen in freshly prepared solutions were no longer observed. This solution can yield a similar distribution of tetanus protein fragments as seen after digestion of freshly prepared solutions simply by the addition of SDS (final concentration 0.1%).
Analysis of the fragment Samples of the reaction were evaluated with SDS/polyacrylamide gel electrophoresis. Samples were prepared in a Laemmli buffer system containing TRIS, glycine, and SDS (Laemmli, 1970). Samples were run both unreduced and after reduction by heat with beta-mercaptoethanol. In general, samples were run on commercial gradient gels (Integrated Separation Systems) with polyacrylamide gradients of 10-20%. Protein concentrations were determined photometrically with a Pierce assay system prior to electrophoresis. Proteins on gels were visualized either with Coomassie blue or silver staining. The possible identity of electrophoretic bands as tetanus toxin fragment C was established after electrophoretic transfer of protein to nitrocellulose membranes previously exposed for 2 h to 5% bovine serum albumin (BSA). Immunoblots were exposed overnight to anti-tetanus toxin fragment C either as monoclonal mouse (Boehringer-Mannheim) or polyclonal rabbit (Chemicon) IgG at dilution of 1:1000 to 1:5000 in phosphate buffered saline (PBS) with 2.5% BSA. Binding was visualized with a 1 : 250 dilution of biotintylated goat antimouse or rabbit IgG, followed by incubation with the avidin-biotin-peroxidase complex (Vector Labs) and reaction with chloro-naphthol.
Assessment of neuronal uptake and transport To assess retrograde axonal transport of the fragments, intramuscular injections were made into the tongues of 12 mice (8 /xl) of solutions with protein concentrations ranging from 8 to 70 m g / m l . Preparations of digested tetanus protein as described above were concentrated by vacuum dialysis, but not further purified prior to injection
~3~
A!i !~!i!!i!~?ili!ii!ill!i!ii!¸I~~)!i i !~i ~'
Fig. 2. A: section through the level of the hypoglossal nucleus from a mouse rejected with tetanus toxin digest into the tongue (8/zl, 24 mg protein/ml) and allowed to survive for 24 h prior to perfusion. The section shows immunoreaction with anti-fragment C antibodies throughout the region of the hypoglossal nucleus (arrows). (The bar represents 500 /~m). B: sections from animals injected in the tongue with commercial fragment C (8 mg/ml). The tissue from this animal was processed together with that from the tissue from the mouse injected in A. Immunolabeling with anti-fragment C antibodies is similar to that seen in A. Arrows indicate the hypoglossal nucleus, and the bar represents 500/zm. C: higher magnification view of the hypoglossal nucleus from the digest injected mouse shown in A. Although labeled motoneuron cell bodies are visible (arrowheads), the entire nucleus is delineated by a haze of labeled neuropil (arrows at border of nucleus) characteristic of transneuronal transfer of fragment C. The bar represents 100 ~zm.
233 into animals. Injections were performed under inhalation anesthesia with methoxyflurane. Mice were then allowed to survive from 18 to 72 h after injection and were then sacrificed by intraperitoneal injection of a supralethal dose of pentobarbital (5 mg) and perfused with 10% formalin in PBS. None of these animals injected with digests electrophoretically free of high molecular weight protein fragments developed signs of clinical tetanus, with amounts of digests ranging up to 0.5 mg of protein injected (intraperitoneal). We did not attempt to establish the maximal amounts of our mixtures that could be safely administered, but limited experiments demonstrated the marked reduction in toxicity from the procedure. All 6 animals injected with 0.5 mg of this protein preparation remained healthy, while all 4 adult mice injected with crude toxin (our starting product) died of clinical tetanus within 24 h of an injection containing 0.5 ng of protein. Purified tetanus toxin has an LDs~~ in the range of 1-2 n g / k g in mice (Robinson et al., 1975).
Tissue from animals injected with tetanus digests was examined with immunohistochemical techniques, using both a polyclonal rabbit antisera raised in this laboratory to commercial fragment C and a commercial polyclonal antisera (Chemicon) raised against purified fragment C. Tissue from 8 other animals injected at the same time with the same volumes of commercial fragment C (1-8 m g / m l ) were processed along with tissue from digest-injected animals for the purpose of comparison. Tissue was sectioned on a vibrating microtome (50-75 /x) and incubated in primary antisera (1 / 1000-1/2{}{}{} dilution in 5% liquid gelatin, in PBS with 1% Triton X-l{}(}) overnight and visualized with the avidin-biotin technique with diaminobenzidine as a chromagen (Hsu et al., 1981). Results
Examination of this preparation with S D S / P A G E revealed that the largest remaining pro-
; ....
Fig. 2 (continued).
234 tein fragments had molecular weights between 40-45 kD, similar to that of commercial fragment C from Calbiochem (Fig. IA). The Calbiochem product was the sole source of commercial fragment C up until 1990, but is is no longer available. The majority of recent studies on the neurobiology of fragment C have used this product. Several smaller fragments also were detectable at lower concentration. It was evident that a large amount of small peptide had run off the gel and that the protein bands represented only a small proportion of the total protein nitrogen loaded on the gel. This suspicion was confirmed by the finding that protein concentration as assessed by protein nitrogen dropped to 20-40% of initial concentration after overnight dialysis (molecular weight cutoff 6-8 kD). We examined this digest with immunoblot analysis with both polyclonal and monoclonal antibodies against fragment C. The 40-45 kD doublet band was recognized by these antibodies with equal affinity to that of commercial fragment C (Fig. 1B), although the smaller molecular weight fragments were frequently not recognized by the monoclonal anti-fragment C antibodies. Examination of brain sections revealed immunostaining primarily at the hypoglossal nucleus. The distribution and pattern of immunostaining in tissue from digest-injected animals was similar to that previously reported by this laboratory for fragment C (Fishman and Carrigan, 1987). Tissue from all animals sacrificed showed labeling of neuronal cell bodies along with labeling of perineuronal neuropil characteristic of transneutonal transport of fragment C (Fig. 2) (Fishman and Savitt, 1989). There was a small amount of immunolabeling in all cellular groups with projections outside the blood-brain barrier, including the facial nucleus, trigeminal motor nucleus and dorsal motor nucleus of the vagus. This distribution of labeling is characteristic of systemic spread of fragment C, where leakage from the initial intramuscular injection site into the circulation results in retrograde labeling of neurons (particularly motoneurons) with peripheral terminals far from injected muscle (Fishman and Carrigan, 1988). We have not performed ultrastructurat studies with this preparation to confirm that its
transsynaptic transport properties are similar to those of fragment C. The only observable difference between tissue injected with commercial fragment C and our tetanus protein digests was the intensity of labeling on a per milligram of protein basis. Protein digests usually containing five times the protein concentration of commercial fragment C gave comparable labeling intensities of the hypoglossal nucleus following intramuscular injection. Discussion
Over the last 5 years it has become increasingly apparent that there are several neurobiological uses for fragment C not shared by tetanus toxin. Evinger and Erichsen (1986) have demonstrated that fragment C can be used to map out presynaptic connections of motoneurons through its capacity for retrograde transneuronal transport. Its strong avidity for neural membranes has allowed Robbins to use it to study the development of the neuromuscular junction in vivo (Robbins and Polak, 1988). That avidity and its relative selectivity for neural membranes also makes it an excellent vector for delivering other large proteins into the nervous system (Bizzini et al., 1977; Fishman and Savitt, 1989; Fishman et al., 1990). Unfortunately these and other neurobiological uses of fragment C require far more protein than do the pharmacological uses of tetanus toxin, and this demand quickly outstripped commercial supplies in 1988. This situation led to intensive efforts by both commercial and academic laboratories to produce fragment C by both conventional and molecular genetics techniques. In early 1991, fragment C produced by a conventional method became commercially available from List Laboratories at a cost of over 300 times that of the formerly available preparation. There are no published reports of neuronal uptake or transport of this preparation at this time. Fragment C has recently been produced by recombinant gene methods (Makoff et al., 1989; Cavanagh et al.. 1990; Halpern et al., 1990). One preparation has been shown to be non-toxic and binds to neuronal receptors in culture, while a preliminary report of another recombinant frag-
235 ment C shows axonal transport in vivo (Cavanagh et al., 1990; Halpern et al., 1990). One of these recombinant forms of fragment C has also recently become commercially available (Boehringer-Mannheim) at approximately 75 times the cost of the original digest fragment C (Cavanagh et al., 1990). The technique described here is clearly not suitable for every laboratory interested in using fragment C. Only experienced microbiologists with well controlled facilities should maintain large volume cultures of C. tetani, although the crude product of those cultures can be maintained in a small volume for an indefinite period of time. Until processed by proteolysis with SDS, this product is highly toxic and must be kept under secure conditions and handled by laboratory workers with current tetanus toxoid boosters. We recommend that all laboratory workers handling any fragment C-like product have recent tetanus toxoid boosters due to the possibility of contamination by native tetanus toxin. The procedure described above should yield a preparation free of high molecular weight (greater than 60 kD) proteins. It is our experience that preparations with detectable proteins on SDS/PAGE in the range of heavy chain of the toxin (approximately 95 kD) or native toxin (approximately 145 kD) are potentially toxic. It is advisable to assess the preparation with SDS/PAGE prior to using it in experimental animals. Any remaining larger protein can be eliminated by lengthening the duration of trypsin incubation. This product is a reasonable substitute for commercial fragment C for at least some neurobiological purposes, although we can make no claims about its pharmacological properties at this time. Our simple method takes advantage of the relative stability of the neural binding portion of the tetanus toxin molecule to proteolysis by trypsin and endogenous proteases. Extensive proteolytic digestion of tetanus toxin in the presence of SDS markedly reduces its toxicity, but is clearly an inefficient means of producing fragment C. The original procedure of Helting with more limited digests yielded 20% of the total protein as fragment C (Helting and Zwisler, 1974). Our pro-
cedure compensates for the low yield of fragment C from extensive proteolysis by using a crude tetanus precipitate rather than purified tetanus toxin. Purified tetanus toxin is regularly prepared from these cultures by one of us (WL). A typical 5-1 culture flask, such as our source of crude protein, yields usually 500 mg of purified toxin. By the limited papain digestion of Helting, this purified toxin would yield approximately 100 mg of fragment C, which would then be further purified to remove all remaining native tetanus toxin. An ammonium sulfate precipitate of such a 5-1 culture solution yields approximately 3.5 g of crude protein (our starting product). Digestion and dialysis reduced protein quantities to 20-40% of the initial amounts. Our qualitative evaluation of histological sections suggests that our digests are approximately 10-20% as efficient for neural labeling as commercial fragment C on a per milligram basis. There are several possible reasons for this reduced efficiency. It is possible that the non-fragment C proteins, although non-toxic, interfere with its uptake. It is also possible that our fragment C-like protein, although similar in size and antigenic properties, is less avidly bound and internalized than conventional fragment C. By these rough estimates the same 5-1 culture would produce a non-toxic protein digest equivalent to at least 70 mg of fragment C, a similar yield to the more demanding and time-consuming conventional procedure. Investigators interested in the neurobiological aspects of the fragment C of tetanus toxin should find this procedure useful for generating a nontoxic product capable of both retrograde and transneuronal transport in the nervous system. It is designed particularly for investigators whose experimental designs require large amounts of fragment C (mg/experiment) where commercial products would be prohibitively expensive. We will continue to explore the utility of these fragments for one such use, i.e., vectors for the delivell of macromolecule into the CNS.
Acknowledgements We would like to thank Ms. Bernadette Pasko for preparation of the typescript. This work was
236
supported by a Merit Review Grant form the Research Service of the Department of Veterans Affairs.
References Bizzini, B., Akert, K., Glicksman, M. and Grob, P. (1980) Preparation of conjugates using two tetanus toxin derived fragments: their binding to gangliosides and isolated synaptic membranes and their immunological properties, Toxicon, 18: 561-572. Bizzini, B, Stoeckel, K. and Schwab, M. (1977) An antigenic polypeptide fragment isolated from tetanus toxin: chemical characterization, binding to gangliosides and retrograde axonal transport in various neuron systems, J. Neurochem., 28: 529-542. Buttner-Ennever, J.A., Grob, P., Akert, K. and Bizzini, B. (1981) A transsynaptic autoradiographic study of the pathways controlling the extraocular eye muscles, using (125I)B-IIb tetanus toxin fragment, Ann. NY Acad. Sci., 374: 157-170. Cavanagh, T.J., Grega, D.S., Bowker, G. and Dwulet, F. (1990) Synaptic uptake and transport of recombinant tetanus toxin fragment C in the rat visual system, Neurosci. Abst., 16: 52. Evinger, C. and Erichsen, J.T. (1986) Transsynaptic retrograde transport of fragment C of tetanus toxin demonstrated by immunohistochemical localization, Brain Res., 380: 383-388. Fishman, P.S. and Carrigan, D.R. (1988) Motoneuron uptake from the circulation of the binding fragment of tetanus toxin, Arch. Neurol., 45: 558-561. Fishman, P.S. and Carrigan, D.R. (1987) Retrograde transneuronal transfer of the C-fragment of tetanus toxin. Brain Res., 406: 275-279. Fishman, P.S. and Savitt, J.M. (1989) Transsynaptic transfer of retrogradely transported tetanus protein-peroxidase conjugates, Exp. Neurol., 106: 197-203. Fishman, P.S., Savitt, J.M. and Farrand, D.A. (1990) Enhanced CNS uptake of systemically administered proteins through conjugation with tetanus C-fragment, J. Neurol. Sci., 98: 311-325.
Halpern, J.L., Habig, W.H., Neale, E.A. and Stibitz, S. (1990) Cloning and expression of functional fragment-C of tetanu~ toxin, Infect. Immun., 58: 1004-1009. Helting, T. and Zwisler, O. (1974) Enzymatic breakdown t)J tetanus toxin, Biochem. Biophys. Res. Commun., 57: 1263-127O, Hsu, S.M., Raine, L. and Fanger, H. (198t) The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures, J. Histochem. Cytochem., 29: 577-580. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage "['4, Nature, 227: 680-685. Latham, W.C., Bent, D.F. and Levine, L. (1962)Tetanus toxin production in the absence of protein, Appl. Microbiol., I(t: 146-152. Makoff, A.J.. Ballantine, S.P., Smallwood, A.E. and Fairweather. N.F. (1989) Expression of tetanus toxin fragment C in E. Coli: its purification and potential use as a vaccine, Biotechnology, 7: 1043-1046. Pierce, E.J., Davison, M.D., Parton, R.G., Habig, W.H. and Critchley, D.R. (1986) Characterization of tetanus toxin binding to rat brain membranes, Biochem. J., 236: 845-852. Price, D.L., Griffin, J., Young, A., et al. (1975) Tetanus toxin: direct evidence for retrograde intra-axonal transport, Science, 183: 945-947. Robbins, N. and Polak, J. (1988) Filopodia, lamellipodia and retractions at mouse neuromuscular junctions, J. Neurocytol., 17: 545-561. Robinson, J.P. and Hash, J.H. (1982) A review of the molecular structure of tetanus toxin. Mol. Cell. Biochem., 48: 33-44. Robinson, J.P., Picklesimer, J.B. and Puett, D. (1975) The effect of chemical modifications on toxicity, immunogenicity and conformation, J. Biol. Chem., 250: 7435-7442. Schwab, M.E., Suda, K. and Thoenen, H. (1979) Selective retrograde transsynaptic transfer of a protein tetanus toxin subsequent to its retrograde axonal transport, J. Cell. Biol., 82: 798-810. Weller, U., Mauler, F. and Habermann. E. (1988) Tetanus toxin: biochemical and pharmacological comparison between its protoxin and some isotoxins obtained by limited proteolysis, Naunyn Schmiedeberg, Arch. Pharmacol., 338: 99-106.
229
NSM 01366
A simplified method for the preparation of tetanus toxin binding fragment for neurobiology Paul S. Fishman ~, Dawn A. F a r r a n d ~, Jane L. Halpern 2 and William C. L a t h a m 3 / Baltimore Veterans Administration Medical Center and the Department oJ"Neurology of the Unit'ersity of Maryland School of Medicine, Baltimore, MD (USA), 2 Dil'ision of Bacterial Products, Food and Drug Administration, Bethesda, MD (USA) and ~ Massachusetts Public Health Laboratories, Boston, MA (USA) (Received 22 May 1991) (Revised version received 12 March 1992) (Accepted 12 March 1992)
Key words: Fragment C; Transneuronal transport; Axonal transport; Neuronal labeling The non-toxic binding fragment of tetanus toxin (fragment C) binds avidly to neural tissue and has a growing number of neurobiological uses. Its current utility is limited by both its high commercial cost and the complex procedure for its preparation requiring highly purified tetanus toxin. We have developed a short procedure which prepares fragments of tetanus toxin fi'om crude C. tetani extracts. The resultant proteins are atoxic with molecular sizes and immunological properties closely resembling fragment C. These proteins undergo retrograde axonal and apparent transneuronal transport in a fashion similar to fragment C.
Introduction
Tetanus toxin is well known to bind avidly to neuronal membranes and undergoes both axonal transport and transneuronal transport (Price et al., 1975; Schwab et at., 1979; Pierce et al., 1986). Fragments of the tetanus toxin molecule have been produced which are both atoxic and show neuronal binding and transport properties similar to that of native toxin (Bizzini et al., 1977; Bizzini et al., 1980). The most well studied of these peptides, fragment C, is a 47 kD fragment of the heavy chain of the toxin, produced by papain digestion of tetanus toxin (Helting and Zwisler, 1974). Fragment C contains the conformational site of tetanus toxin necessary for binding and
Correspondence to: Paul S. Fishman, M.D., Ph.D., Department of Neurology, University of Maryland School of Medicine, Baltimore, MD 21201, USA. Tel.: (410) 328-2346; Fax: (410) 328-5899.
internalization by neurons and their processes (Bizzini et al., 1977; Buttner-Ennever, 1981; Evinger and Erichsen, 1986). Increasing neurobiological interest in fragment C resulted initially in the exhaustion of commercial supplies in 1988, and fragment C has only recently again become commercially available. Published accounts of production of fragment C begin with highly purified tetanus toxin as the substrate for enzymatic degradation (Helting and Zwisler, 1974). Purification of tetanus toxin requires a significant effort in itself. Tetanus toxin has unusual solubility properties which cause it to aggregate under non-reducing conditions (Robinson and Hash, 1982). Preparations of tetanus toxin also frequently show endogenous proteolytic activity so that careful control of bacterial culture conditions, toxin extraction and handling are needed to obtain intact toxin comparable to that used in published preparations of fragment C (Weller et al., 1988). High-level purification of fragment C after digestion of tetanus toxin with
230 p a p a i n is t h e n n e e d e d to r e m o v e t h e e x t r e m e l y toxic n a t i v e t e t a n u s toxin f r o m t h e p r e p a r a t i o n ( R o b i n s o n et al., 1975). T h i s level o f p u r i f i c a t i o n is d i f f i c u l t to a c h i e v e e v e n w i t h u n i f o r m p r e p a r a t i o n s o f i n t a c t toxin. W i t h p r e p a r a t i o n s c o n t a i n ip~. e n d o g e n o u s p r o t e o l y t i c activity, t h e v a r i e t y o f conta~;~'nating p e p t i d e s m a k e s e p a r a t i o n o f fragm e n t C v i r t u a l l y i m p o s s i b l e by p u b l i s h e d t e c h n i q u e s . T h e d i f f i c u l t i e s e n c o u n t e r e d in p r o d u c -
t i o n o f f r a g m e n t C h a v e led in p a r t to successful e f f o r t s by s e v e r a l i n v e s t i g a t o r s to p r o d u c e fragm e n t C by g e n e t i c r e c o m b i n a n t m e t h o d s ( M a k o f f et al., 1989; H a l p e r n et al., 1990). O u r i n t e r e s t in t h e n e u r o b i o l o g i c a l p r o p e r t i e s o f f r a g m e n t C, r a t h e r t h a n in its p h a r m a c o l o g i c o r p h y s i c o - c h e m i c a l p r o p e r t i e s , led us to d e v e l o p a m e t h o d for its p r o d u c t i o n t h a t is s i m p l e , rapid, r e l a t i v e l y safe, i n e x p e n s i v e a n d u s e s r o u t i n e l y
Fig. 1. A: electrophoretic separation of tetanus toxin fragments, Lane MC (Massachusetts Crude) contains undigested ammonium sulfate precipitate of C. tetani cultures (180 ~zg/lane). Note the highly heterogenous mixture of peptides without a clear band corresponding to heavy chain (100 kD) or light chain (55 kD) of purified tetanus toxin. Lane D contains a trypsin digest of the MC mixture (pre-dialysis). Most of the remaining protein is contained as a doublet with molecule weight between 42 and 46 kD (75 /.,g/lane), although much of a total protein concentration has run off the gel as small peptide. Lane CF contains commercial fragment C obtained from Calbiochem (16/zg/lane) with a similar but slightly smaller doublet compared to D. Both CF and D have a small amount of protein larger than the doublet band. Samples were incubated with 5% mercaptoethanol prior to application to the 10-20% acrylamide gradient gel run in the presence of 0.19: SDS. B: immunoblot of gel electrophoresis of a similar mixture of tetanus fragment as in 1A, reacted with antibodies against fragment C (polyclonal, rabbit, 1/2000 dilution). MC lanes contain the crude precipitate, showing multiple bands that react with anti-C fragment antibodies (100 #g of protein per lane). Lane D shows that the major protein doublet of the digest mixture is also immunoreactive with the antisera (40 /zg/lane). As expected, commercial fragment C (lane CF) reacts with these antibodies, although on this occasion it did not run as a doublet (10 /xg/lane). In spite of the area of poor contact of the gel in the CF lane, it is apparent that both the digest and commercial fragment C also have a similarly immunoreactive 38 kD peptide.
231 available laboratory equipment. Previous studies of fragment C have shown it to be relatively protease resistant compared to other sites of the tetanus toxin molecule (Helting and Zwisler, 1974; Bizzini, 1977). This method produces protein fragments with molecular weight, antigenic and neural uptake properties very similar to those of fragment C produced by previously published procedures.
Methods
Preparation of the fragment Cultures of C. tetani (Harvard strain) were grown in modified Harvard medium for 4 days in 5-1 batch lots in Ehrlenmeyer flasks (Latham et al., 1962). The cultures were then filtered to remove bacteria and other particulate matter, and extracellular toxin was precipitated by the addition of solid ammonium sulfate to a final concentration of 40% at 5°C. The solution was again filtered, and the crude toxin preparation was allowed to dry onto filter paper. This solid preparation can be maintained indefinitely (over 1 year in our hands) at - 4 0 ° C . Crude toxin was resuspended in 0.1 M phosphate buffer (7.5 pH), and ammonium sulfate was removed by dialysis prior to digestion. This crude preparation would be considered highly unsuitable for the preparation of fragment C by usual methods. It is highly aggregated and will not enter conventional SDS/acrylamide gels, in spite of the addition of urea, without reduction with mercaptoethanol. Electrophoresis of this preparation under reducing conditions reveals a variety of proteolytic fragments of extracellular tetanus toxin (Fig. 1A). This preparation was digested with trypsin (type 463 from Sigma Chemical) in the presence of SDS, in an 0.1 M phosphate buffer at pH 7.5. Usual preparations consisted of 15 m g / m l of crude toxin digested with 0.1 m g / m l of trypsin in 0.1% SDS. The reaction is allowed to proceed until all high molecular weight fragments (and native toxin) had been eliminated (20 rain at 37°C). The digestion was terminated by the addition of an excess of soybean trypsin inhibitor.
Crude tetanus protein was also maintained as a concentrated solution (50 m g / m l ) at -40°C. Such solutions when assayed months later had undergone significant proteolysis. The large protein aggregates seen in freshly prepared solutions were no longer observed. This solution can yield a similar distribution of tetanus protein fragments as seen after digestion of freshly prepared solutions simply by the addition of SDS (final concentration 0.1%).
Analysis of the fragment Samples of the reaction were evaluated with SDS/polyacrylamide gel electrophoresis. Samples were prepared in a Laemmli buffer system containing TRIS, glycine, and SDS (Laemmli, 1970). Samples were run both unreduced and after reduction by heat with beta-mercaptoethanol. In general, samples were run on commercial gradient gels (Integrated Separation Systems) with polyacrylamide gradients of 10-20%. Protein concentrations were determined photometrically with a Pierce assay system prior to electrophoresis. Proteins on gels were visualized either with Coomassie blue or silver staining. The possible identity of electrophoretic bands as tetanus toxin fragment C was established after electrophoretic transfer of protein to nitrocellulose membranes previously exposed for 2 h to 5% bovine serum albumin (BSA). Immunoblots were exposed overnight to anti-tetanus toxin fragment C either as monoclonal mouse (Boehringer-Mannheim) or polyclonal rabbit (Chemicon) IgG at dilution of 1:1000 to 1:5000 in phosphate buffered saline (PBS) with 2.5% BSA. Binding was visualized with a 1 : 250 dilution of biotintylated goat antimouse or rabbit IgG, followed by incubation with the avidin-biotin-peroxidase complex (Vector Labs) and reaction with chloro-naphthol.
Assessment of neuronal uptake and transport To assess retrograde axonal transport of the fragments, intramuscular injections were made into the tongues of 12 mice (8 /xl) of solutions with protein concentrations ranging from 8 to 70 m g / m l . Preparations of digested tetanus protein as described above were concentrated by vacuum dialysis, but not further purified prior to injection
~3~
A!i !~!i!!i!~?ili!ii!ill!i!ii!¸I~~)!i i !~i ~'
Fig. 2. A: section through the level of the hypoglossal nucleus from a mouse rejected with tetanus toxin digest into the tongue (8/zl, 24 mg protein/ml) and allowed to survive for 24 h prior to perfusion. The section shows immunoreaction with anti-fragment C antibodies throughout the region of the hypoglossal nucleus (arrows). (The bar represents 500 /~m). B: sections from animals injected in the tongue with commercial fragment C (8 mg/ml). The tissue from this animal was processed together with that from the tissue from the mouse injected in A. Immunolabeling with anti-fragment C antibodies is similar to that seen in A. Arrows indicate the hypoglossal nucleus, and the bar represents 500/zm. C: higher magnification view of the hypoglossal nucleus from the digest injected mouse shown in A. Although labeled motoneuron cell bodies are visible (arrowheads), the entire nucleus is delineated by a haze of labeled neuropil (arrows at border of nucleus) characteristic of transneuronal transfer of fragment C. The bar represents 100 ~zm.
233 into animals. Injections were performed under inhalation anesthesia with methoxyflurane. Mice were then allowed to survive from 18 to 72 h after injection and were then sacrificed by intraperitoneal injection of a supralethal dose of pentobarbital (5 mg) and perfused with 10% formalin in PBS. None of these animals injected with digests electrophoretically free of high molecular weight protein fragments developed signs of clinical tetanus, with amounts of digests ranging up to 0.5 mg of protein injected (intraperitoneal). We did not attempt to establish the maximal amounts of our mixtures that could be safely administered, but limited experiments demonstrated the marked reduction in toxicity from the procedure. All 6 animals injected with 0.5 mg of this protein preparation remained healthy, while all 4 adult mice injected with crude toxin (our starting product) died of clinical tetanus within 24 h of an injection containing 0.5 ng of protein. Purified tetanus toxin has an LDs~~ in the range of 1-2 n g / k g in mice (Robinson et al., 1975).
Tissue from animals injected with tetanus digests was examined with immunohistochemical techniques, using both a polyclonal rabbit antisera raised in this laboratory to commercial fragment C and a commercial polyclonal antisera (Chemicon) raised against purified fragment C. Tissue from 8 other animals injected at the same time with the same volumes of commercial fragment C (1-8 m g / m l ) were processed along with tissue from digest-injected animals for the purpose of comparison. Tissue was sectioned on a vibrating microtome (50-75 /x) and incubated in primary antisera (1 / 1000-1/2{}{}{} dilution in 5% liquid gelatin, in PBS with 1% Triton X-l{}(}) overnight and visualized with the avidin-biotin technique with diaminobenzidine as a chromagen (Hsu et al., 1981). Results
Examination of this preparation with S D S / P A G E revealed that the largest remaining pro-
; ....
Fig. 2 (continued).
234 tein fragments had molecular weights between 40-45 kD, similar to that of commercial fragment C from Calbiochem (Fig. IA). The Calbiochem product was the sole source of commercial fragment C up until 1990, but is is no longer available. The majority of recent studies on the neurobiology of fragment C have used this product. Several smaller fragments also were detectable at lower concentration. It was evident that a large amount of small peptide had run off the gel and that the protein bands represented only a small proportion of the total protein nitrogen loaded on the gel. This suspicion was confirmed by the finding that protein concentration as assessed by protein nitrogen dropped to 20-40% of initial concentration after overnight dialysis (molecular weight cutoff 6-8 kD). We examined this digest with immunoblot analysis with both polyclonal and monoclonal antibodies against fragment C. The 40-45 kD doublet band was recognized by these antibodies with equal affinity to that of commercial fragment C (Fig. 1B), although the smaller molecular weight fragments were frequently not recognized by the monoclonal anti-fragment C antibodies. Examination of brain sections revealed immunostaining primarily at the hypoglossal nucleus. The distribution and pattern of immunostaining in tissue from digest-injected animals was similar to that previously reported by this laboratory for fragment C (Fishman and Carrigan, 1987). Tissue from all animals sacrificed showed labeling of neuronal cell bodies along with labeling of perineuronal neuropil characteristic of transneutonal transport of fragment C (Fig. 2) (Fishman and Savitt, 1989). There was a small amount of immunolabeling in all cellular groups with projections outside the blood-brain barrier, including the facial nucleus, trigeminal motor nucleus and dorsal motor nucleus of the vagus. This distribution of labeling is characteristic of systemic spread of fragment C, where leakage from the initial intramuscular injection site into the circulation results in retrograde labeling of neurons (particularly motoneurons) with peripheral terminals far from injected muscle (Fishman and Carrigan, 1988). We have not performed ultrastructurat studies with this preparation to confirm that its
transsynaptic transport properties are similar to those of fragment C. The only observable difference between tissue injected with commercial fragment C and our tetanus protein digests was the intensity of labeling on a per milligram of protein basis. Protein digests usually containing five times the protein concentration of commercial fragment C gave comparable labeling intensities of the hypoglossal nucleus following intramuscular injection. Discussion
Over the last 5 years it has become increasingly apparent that there are several neurobiological uses for fragment C not shared by tetanus toxin. Evinger and Erichsen (1986) have demonstrated that fragment C can be used to map out presynaptic connections of motoneurons through its capacity for retrograde transneuronal transport. Its strong avidity for neural membranes has allowed Robbins to use it to study the development of the neuromuscular junction in vivo (Robbins and Polak, 1988). That avidity and its relative selectivity for neural membranes also makes it an excellent vector for delivering other large proteins into the nervous system (Bizzini et al., 1977; Fishman and Savitt, 1989; Fishman et al., 1990). Unfortunately these and other neurobiological uses of fragment C require far more protein than do the pharmacological uses of tetanus toxin, and this demand quickly outstripped commercial supplies in 1988. This situation led to intensive efforts by both commercial and academic laboratories to produce fragment C by both conventional and molecular genetics techniques. In early 1991, fragment C produced by a conventional method became commercially available from List Laboratories at a cost of over 300 times that of the formerly available preparation. There are no published reports of neuronal uptake or transport of this preparation at this time. Fragment C has recently been produced by recombinant gene methods (Makoff et al., 1989; Cavanagh et al.. 1990; Halpern et al., 1990). One preparation has been shown to be non-toxic and binds to neuronal receptors in culture, while a preliminary report of another recombinant frag-
235 ment C shows axonal transport in vivo (Cavanagh et al., 1990; Halpern et al., 1990). One of these recombinant forms of fragment C has also recently become commercially available (Boehringer-Mannheim) at approximately 75 times the cost of the original digest fragment C (Cavanagh et al., 1990). The technique described here is clearly not suitable for every laboratory interested in using fragment C. Only experienced microbiologists with well controlled facilities should maintain large volume cultures of C. tetani, although the crude product of those cultures can be maintained in a small volume for an indefinite period of time. Until processed by proteolysis with SDS, this product is highly toxic and must be kept under secure conditions and handled by laboratory workers with current tetanus toxoid boosters. We recommend that all laboratory workers handling any fragment C-like product have recent tetanus toxoid boosters due to the possibility of contamination by native tetanus toxin. The procedure described above should yield a preparation free of high molecular weight (greater than 60 kD) proteins. It is our experience that preparations with detectable proteins on SDS/PAGE in the range of heavy chain of the toxin (approximately 95 kD) or native toxin (approximately 145 kD) are potentially toxic. It is advisable to assess the preparation with SDS/PAGE prior to using it in experimental animals. Any remaining larger protein can be eliminated by lengthening the duration of trypsin incubation. This product is a reasonable substitute for commercial fragment C for at least some neurobiological purposes, although we can make no claims about its pharmacological properties at this time. Our simple method takes advantage of the relative stability of the neural binding portion of the tetanus toxin molecule to proteolysis by trypsin and endogenous proteases. Extensive proteolytic digestion of tetanus toxin in the presence of SDS markedly reduces its toxicity, but is clearly an inefficient means of producing fragment C. The original procedure of Helting with more limited digests yielded 20% of the total protein as fragment C (Helting and Zwisler, 1974). Our pro-
cedure compensates for the low yield of fragment C from extensive proteolysis by using a crude tetanus precipitate rather than purified tetanus toxin. Purified tetanus toxin is regularly prepared from these cultures by one of us (WL). A typical 5-1 culture flask, such as our source of crude protein, yields usually 500 mg of purified toxin. By the limited papain digestion of Helting, this purified toxin would yield approximately 100 mg of fragment C, which would then be further purified to remove all remaining native tetanus toxin. An ammonium sulfate precipitate of such a 5-1 culture solution yields approximately 3.5 g of crude protein (our starting product). Digestion and dialysis reduced protein quantities to 20-40% of the initial amounts. Our qualitative evaluation of histological sections suggests that our digests are approximately 10-20% as efficient for neural labeling as commercial fragment C on a per milligram basis. There are several possible reasons for this reduced efficiency. It is possible that the non-fragment C proteins, although non-toxic, interfere with its uptake. It is also possible that our fragment C-like protein, although similar in size and antigenic properties, is less avidly bound and internalized than conventional fragment C. By these rough estimates the same 5-1 culture would produce a non-toxic protein digest equivalent to at least 70 mg of fragment C, a similar yield to the more demanding and time-consuming conventional procedure. Investigators interested in the neurobiological aspects of the fragment C of tetanus toxin should find this procedure useful for generating a nontoxic product capable of both retrograde and transneuronal transport in the nervous system. It is designed particularly for investigators whose experimental designs require large amounts of fragment C (mg/experiment) where commercial products would be prohibitively expensive. We will continue to explore the utility of these fragments for one such use, i.e., vectors for the delivell of macromolecule into the CNS.
Acknowledgements We would like to thank Ms. Bernadette Pasko for preparation of the typescript. This work was
236
supported by a Merit Review Grant form the Research Service of the Department of Veterans Affairs.
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