Table Of ContentReviews of
,ygoloisyhP
39
Biochemistry and
ygolocamrahP
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Contents
Tetanus Neurotoxin
By H.-H. ,RENOHLLEW Hannover/Federal
Republic of Germany. With 2 Figures .....
Control of Blood Volume
By R. D. MANNING, Jr. and A. C. ,NOTYUG
Jackson/Mississippi, USA. With 41 Figures 69
Proteolytic Processing of Polypeptides During
the Biosynthesis of Subcellular Structures
By P. C. HEINRICH, Freiburg/Federal Republic
ofG ermany. With 6 Figures . . . . . . . . . 511
Author Index ...... .- . . . . . . . . . 981
Subject Index ......... . . . . . . . . 209
dexednI Current in stnetnoC
Rev. Physiol. Biochem. Pharmacol., Vol. 93
© by Springer-Verlag 1982
Tetanus Neurotoxin
HANS-H. WELLH~)NER 1
Contents
1 Introduction .......................................... 2
2 Abbreviations and Definitions .............................. 2
3 Origin and Homogenicity of Toxin ........................... 4
4 Purification .......................................... 4
4.1 Tests for the Assessment of Purity ....................... 4
4.2 Purification Procedures ............................... 5
5 Structure of Tetanus Toxin ................................ 6
6 Derivatives of Tetanus Toxin ............................... 9
6.1 Iodination ....................................... 9
6.2 Modification of Tryptophanyl Residues .................... 10
6.3 Modification of Tyrosyl Residues ........................ 10
6.4 Modification of Lysyl Residues ......................... 11
6.5 Modification of Histidyl Residues ........................ 11
6.6 Formaldehyde Toxoid ............................... 11
6.7 Other Toxoids .................................... 12
7 Immunogenicity and Immunoreactivity of Tetanus Toxin and
Formaldehyde Toxoid ................................... 13
7.1 Classes, Subclasses and Allotypes of Human Antitetanus Toxoid
Immunoglobulins .................................. 13
7.2 Immunogenic Determinants ............................ 14
7.3 Immunogenicity and Immunoreactivity of Other Derivatives ...... 14
8 Fixation ............................................ 15
8.1 Fixation to Gangliosides .............................. 15
8.2 Fixation to Subcellular Structures and to Cells ............... 17
8.3 Specificity of Fixation ............................... 18
8.4 Effect of Antitoxin on Toxicity and Fixation ................ 19
8.5 Fixation of Subunits ................................ 20
8.6 Fixation of Formaldehyde Toxoid ....................... 20
9 Absorption and Distribution ............................... 21
9.1 Absorption, General Distribution ........................ 21
9.2 Neural Ascent ..................................... 22
9.3 Transsynaptic Migration .............................. 27
10 Actions of Tetanus Toxin on the Nervous System ................. 28
10.1 Action at Cholinergic Peripheral Synapses .................. 28
10.2 Action on an Inhibitory Peripheral Synapse ................. 33
I Medizinische Hochschule Hannover, Abt. Toxikologie, Postfach 610 t 80,
D-3000 Hannover 61
2 .H-.H ren6hlleW
10.3 Action on Electrically Excitable Membrane Elements ........... 33
10.4 Action on ssaM Action Potentials in Spinal Cord Efferents ....... 34
10.5 Synaptic Topology of the Action of Tetanus Toxin at Spinal Cord
Synapses ........................................ 83
10.6 Action of Tetanus Toxin After Direct Injection into Functionally
Defined NSC Structures .............................. 34
10.7 In vitro Actions of Tetanus Toxin on Transmitter Release ........ 64
10.8 Action of Tetanus Toxin on Intracellular Components of the
Neuron ......................................... 74
10.9 Species-and egA Dependent Toxicity ..................... 84
11 Nonneuronal Actions of Tetanus Toxin ........................ 84
21 Summary and Concluding Remarks ........................... 94
References .............................................. 15
1 Introduction
During the past 20 years considerable progress has been made on the biol-
ogy, biochemistry, toxicology, and immunology of tetanus toxin. These
recent developments are the main subject of this review. Epidemiological,
preventive, and therapeutic aspects have been deliberately omitted. Many
earlier observations, some of them seemingly contradictory, can now be
explained. They will not be repeated, but the reader is referred to earlier
reviews, e.g., by Fildes et al. (1929), Prevot (1955), Wright PG (1955),
Turpin and Raynaud (1959) and Laurence and Webster (1963). Of more
recent reviews, two by Zacks and Shelf ( 1970, 1971) are interesting for
their provocative theories, and one by Curtis (1971) dealing with electro-
physiological aspects. Further surveys dealing with various aspects of
tetanus toxin have been given by nav Heyningen and Mellanby (1971),
Bizzini (1976, 1979), Habermann (1978), Mellanby and Green (1981).
The extensive work of Soviet scientists on tetanus has been published in a
monograph by Kryzhanovsky (1966), these studies have also been sum-
marized in English (Kryzhanovsky 1967, 1973, 1975a, b). In the reviews of
Lamanna and rraC (1967) and of Dasgupta and Sugiyama (1977) tetanus
toxin is compared with botulinum toxin.
2 Abbreviations and Definitions
CNS Central nervous system
EPSP Excitatory postsynaptic potential
IPSP Inhibitory postsynaptic potential
Tetanus Neurotoxin 3
IU International unit for tetanus antitoxin. The IU is equiv-
alent to 0.03384 mg of the Second International Standard
for Tetanus Antitoxin (Spaun and Lyng 1970).
L+ The L+ (or L+I/10 etc.) defines an amount of toxin or
(L+I/10 etc.) toxin derivative by its toxicity. The L+ (or L+I/10 etc.) is
the lowest amount of toxin or derivative that, after incuba-
tion with 1 (or 1/10 etc.) international unit (IU) of anti-
toxin in a final volume of 0.5 ml for 1 h at 20°C and pH 7.5
and after subsequent intramuscular (i.m.) or subcutaneous
(s.c.) injection into one mouse, becomes lethal to 50% of
the injected mice within the next 4 days (lpsen 1951;van
Heyningen 1960; Barile et al. 1970; Bizzini et al. 1973b;
sensniW and nesnaitsirhC 1979).
Lf The Lf defines an amount of toxin or toxin derivative by
its immunoreactivity in a flocculation assay. If different
amounts of toxin (or of one of its immunoreactive deriv-
atives) are mixed with a constant amount of 1 IU of
tetanus antitoxin, the rapidity of flocculation runs through
a maximum for a certain amount of toxin (or derivative).
This amount of toxin (or derivative) is defined to contain
I Lf unit.
For practical purposes, the technique of Ramon (1922)is
used. In this procedure the amount and concentration of
antitoxin is held constant and the amount of toxin is
varied (see also Dean and Webb 1926; drariG et al. 1965).
mepp Miniature end-plate potential
MLD The MLD defines an amount of toxin or toxin derivative
by its toxicity. Unless special reference is made to the ani-
mal species, the MLD refers to mice. Unfortunately, three
different definitions have been used for the MLD. The
MLD is the lowest amount of toxin or toxin derivative per
mouse that, after injection i.m., s.c., intraperitoneal (i.p.):
1. becomes lethal to 100% of the mice within the next
4 days (commonly used definition, example: Bizzini et
al. 1973b)
2. becomes lethal to 50% of the mice within the next 4
days (example: Ipsen 1951)
3. becomes just not lethal to any of the mice (rarely and
no longer used definition).
The MLD according to .1 is about 1.4 times higher than
the MLD according to 2., which in turn is about 1.7 times
higher than the MLD according to 3. (van Heyningen and
4 H-.H Wellhtner
Metlanby 1971). The dose-response curve is very steep in a
toxicity assay in mice (van Heyningen 1959b).
PAGE Polyacrylamide gel electrophoresis
SDmin The SDmi n defines an amount of toxin by its toxicity. The
SDmi n is the dose of tetanus toxin per animal that can be
given such that further increase of the dose does not de-
crease survival time (minimum saturation dose). The SDmi n
has been defined and used by Zacks and Shelf (1970). Ac-
cording to them, the SDmi n in adult mice is about 4 X 401
times the MLD at 35°C.
SDS Sodium dodecylsulfate
TSH Thyroid stimulating hormone
3 Origin and Homogenicity of Toxin
Tetanus neurotoxin, termed also tetanospasmin or simply tetanus toxin,
is synthesized by some strains of Clostridium tetani. Germination of the
spores requires strictly anaerobic conditions; most of the toxin is pro-
duced at the end of the germination phase. Details of toxin production
have been given by Bizzini et al. (1969, 1974). Ten serological types of
Clostridium tetani have been described (Mclennan 1939), and some strains
of Clostridium tetani apparently do not produce tetanus toxin (Fildes
1925, 1927; Hara et al. 1977). The latter authors treated a highly toxigenic
strain with mutagenic agents and obtained stable, nontoxigenic variants
producing neither a nontoxic variant of tetanus toxin nor nontoxic sub-
units. However, some known toxigenic Clostridium tetani strains may
produce a nontoxic protein differing from tetanus toxin in only a few
amino acids. Any supposed involvement of bacteriophages in the produc-
tion of tetanus toxin has not been supported (Hara et al. 1977; Ackermann
et al. 1978). Apparently, all strains synthesize the same kind of tetanus
toxin (Largier 1956 ;Hardegree and Wannamaker 1965).
4 Purification
4.1 Tests for the Assessment of Purity
Determination of Toxicity. Already in 1893 Brieger and Cohn assessed the
efficiency of their purification procedure by demonstrating an increased
toxicity of the enriched product. Determination of toxicity is still a very
Tetanus Neurotoxin 5
sensitive test to control the quality of a purified batch of tetanus toxin.
Toxicity is commonly expressed in MLD (minimal lethal dose, see Sect. 2
for definition). Zacks and Shelf (1970) have proposed the minimum
saturation dose SDmi n (see Sect. 2 for definition). The MLD depends to
some extent on the environmental temperature. For economic reasons it
is determined in mice, in which the MLD is very low (5 ng/kg or less).
Immunoreaetivity ni the flocculation test. The immunoreactivity increases
with the purity of a preparation, but unlike the toxicity it is not affected
by toxoid formation and may even increase on degradation (Bizzini and
Raynaud 1974a, 1975b). Because of its simplicity the method of LF de-
termination (Ramon 1922; for definition see Sect. 2) is still used for pur-
poses of comparison.
Analytical chromatography and electrophoresis. Gel filtration and gel
electrophoresis are powerful methods for detecting impurities not related
to tetanus toxin, but are far less sensitive for uncoveringd egradation prod-
ucts or for separating toxin derivatives (for instance l:sI-toxin) from
toxin. At its pI (5.1 + 0.1 ; An der Lahn et al. 1980) tetanus toxin has a
low solubility. Isoelectric focusing has been used with tetanus toxin only
recently by An der Lahn et al. (1980). The resolving power for 125I deriv-
atives was disappointing.
4.2 Purification Procedures
Purification may start either from an extract of the clostridia or from the
culture filtrate. With the former approach "intracellular" toxin is obtained,
the latter procedure yields "extracellular" toxin. The extracellular toxin
differs from its intracellular precursor (Fig. 1) in that one peptide bond
in the single-chain precursor is cleaved by a clostridiaI enzyme. The
resulting two chains are still held together in the extracellular toxin by
one disulfide linkage (see Sect. 6). The purification of tetanus toxin on a
preparative scale has been reviewed by Turpin and Raynaud (1959),
Bizzini et al. (1969), Zacks and Shelf(1970) and Bizzini (1976). A puri-
fication may be considered satisfactory if it yields a toxin with 3.000-
3.200 LF/mg N and at least 10 s MLD/mg N. Affinity chromatography for
the purification of iodinated toxin was first tried by Habermann (1976)
on a synaptosome column, then by Ledley et al. (1977) on thyroid plasma
membranes. Robinson et al. (1981) used preparative gel electrophoresis.
6 .H-.H ren6hlleW
5 Structure of Tetanus Toxin
The molecular weight of tetanus toxin is about 150 000 daltons (Table 1).
An adsorbency ratio 280 nm:260 nm of 2.1 has been measured (Murphy
and Miller 1967; Bizzini et al. 1969;Matsuda and Yoneda 1974). Adsorb-
ance indices E280 %1 protein mn are between 12.4 and 12.54 for extracellular toxin
(Bizzini et al. 1969; Craven and Dawson 1973). From circular dichroic
spectra Robinson et al. (1974) calculated about 20% a-helix, 23% t3-helix,
and 57% aperiodic structure, and a stable tertiary structure. The same
authors reported a high polarity of 51%.
Table .1 Estimates of the molecular weight of tetanus toxin
Authors Method Estimate (daltons)
olagnaM et al (1968) Ultracentrif. 841 000 + 8 000
Bizzini et at. (1973a) Ultracentrif. 541 000 -+ 5 450
M6 guanidine
nosniboR et .la (1975) Ultracentrif. 051 000 + 01 000
noswaD and Nichol (1969) Ultracentrif. 671 +-000 5 000
yhpruM et .la (1968) leG filtration 041 000
semloH and Ryan ( 1791 ) legoiB P200 041 000
Bizzini et .la (1973a) Sephadex G200 051 000
+ 6 M guanidine
Bizzini et .la (1973a) EGAP-SDS a 041 000
adustaM and Yoneda (1974) EGAP-SDS a 061 000 + 5 000
a eeS Sect. 2 for definition
According to Craven and Dawson (1973) extracellular toxin consists of
two polypeptide chains (95 000 and 55 000 daltons) linked to each other
by one disulfide bond. Matsuda and Yoneda (1974, 1975) published the
same finding (mol. wt. of 53 000 and 107 000 daltons, respectively), but
reported in addition that the heavy (H) and light (L) chains can be obtain-
ed also from intracellular toxin if prior to reduction the toxin is subjected
to mild trypsination. The fragments are nontoxic, but may be reunited to
form (toxic) tetanus toxin (Matsuda and Yoneda 1976b). Without trypsin-
ation, but after reduction of the disulfide bonds, intracellular toxin (in
Tetanus Neurotoxin 7
contrast to extracellular toxin) migrates as a single component. From this
information the Craven-Dawson model can be sketched in the simple form
of Fig. .1
ralullecartnI :nixot
2HN [ ~ trypsin
/
4- stilps ereh
COOH
Fig. .1 Enzymic conversion of intraceUular into extracellular tetanus toxin
It was assumed that under "'natural" conditions intracellular toxin is
nicked by a tryptic clostridial enzyme and then becomes extracellular
toxin. Helting et al. (1979) isolated three enzymes from the culture fil-
trate. The enzyme with the lowest mol. wt. (27 000 daltons) displayed a
very high cleaving activity against tetanus toxin, the activity of the enzyme
with the intermediate mol. wt. (40 000 daltons) was much lower.
Evidence on the N terminus of intracellular toxin was provided by Neu-
bauer and Helting 1979, 1981). Dansylation of intracellular toxin revealed
dansyl-proline only, and isoleucine was next. The N-terminal proline initi-
ates the light chain. After cleavage by clostridial proteases Neubauer and
Helting (1979) found leucine as an additional N terminus, which has to be
attributed to the heavy chain. This amino acid was also identified by
Bizzini et al. (1970). In intracellular toxin, however, they identified pre-
dominantly isoleucine and only limited quantities of leucine.
Matsuda and Yoneda (1974) named the L chain derived from extra-
cellular toxin the a-fragment and the H chain the #-fragment. Later they
used the Greek letters also for the designation of components derived
from intracellular toxin. By forced trypsination of the #-fragment (ob-
tained by pepsination)Matsuda and Yoneda (1977) obtained one large
subfragment 1~ and a number of smaller fragments. By comparing the
immunological features of trypsin fragments with those of papain frag-
ments (see below) they showed that 1# was not directly attached to the
a-fragment. The region interposed between a and 1# was named 23/
(Fig. 2).
The disulfide bond has been tentatively drawn by Matsuda and Yoneda
(1977). The C-terminal amino acids of the 1# -fragment are still unknown.
Papain treatment of extracellular toxin was described by Helting and
Zwisler (1974), by Bizzini and Raynaud (1974a) and by Helting and
Zwisler (1977). Matsuda and Yoneda (1977) used intracellular toxin.
