Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-03-14
2003-04-08
Bugaisky, Gabrielle (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S370000, C514S002600, C514S012200, C424S094600
Reexamination Certificate
active
06545126
ABSTRACT:
BACKGROUND OF THE INVENTION
C. botulinum
Toxin Complex
Toxins of the different
C. botulinum
serotypes are produced in culture as aggregates of neurotoxin and other non-toxic proteins non-covalently associated into a polypeptide complex (Schantz, E., Purification and characterization of
C. botulinum
toxins, In K. Lewis and K. Cassel, Jr. (eds.), Botulism. Proceedings of a symposium. U.S. Department of Health, Education, and Welfare, Public Health Service, Cincinnati, pp. 91-104, 1964; Sugii, S. and Sakaguchi, G.,
Infect. Immun
. 12:1262-1270, 1975; Kozaki, S., et al.,
Jpn. J. Med. Sci. Biol
. 28:70-72, 1974; Miyazaki, S., et al.,
Infect. Immun
. 17:395-401, 1977; Kitamura, M., et al.,
J. Bacteriol
. 98:1173-1178, 1969; Ohishi and Sakaguchi,
Appl. Environ. Microbiol
. 28:923-928, 1974; Yang, K. and Sugiyama, H.,
Appl. Microbiol
. 29:598-603, 1975; Nukina, M., et al.,
Zbl. Bakt. Hyg
. 268:220, 1987). Toxin complexes are described as M for medium, L for large and LL for very large. These toxin complexes vary in size from ca. 900 kD for type A LL toxin complex to ca. 300 kD for the type B M complex and type E complex, to 235 kD for type F M complex (Ohishi, I. and Sakaguchi, G., supra, 1974; Kozaki, S., et al., supra, 1974; Kitamura, M., et al., supra, 1969). According to Sugii and Sakaguchi (
J. Food Safety
1:53-65, 1977), during culture the proportion of one toxin complex versus another is dependent on the growth medium and conditions. A type B culture grown in the presence of 1 mM Fe
+2
produces an equal proportion of L and M complexes while the same culture grown in the presence of 10 mM Fe
+2
produces predominantly M complex.
TABLE 1
Molecular sizes of various
C. botulinum
toxin complexes.
Toxin type
Sedimentation coefficient
ca. M, (kD)
LL
A
19S
900
L
A, B, D, G
16S
450-500
M
A, B, C
1
, D,
10-12S
235-350
E, F, G
Some of the non-toxic proteins associated with the various toxin complexes have hemagglutinating abilities (Sugiyama, H.,
Microbiol. Rev
. 44:419-448, 1980; Somers, E. and DasGupta, B.,
J. Protein Chem
. 10:415-425, 1991). In particular, non-neurotoxic fractions of the L complexes of type A, B, C, and D have been shown to have hemagglutinating activity. Hemagglutinin fractions isolated from the different serotypes show some serological cross-reactivity. Non-toxic fractions from type A and B serotypes cross-react (Goodnough, M. and Johnson, E.,
Appl. Environ. Microbiol
. 59:2339-2342, 1993) as do non-toxic fractions from types E and F. The non-toxic fractions of types C
1
and D are antigenically identical as determined by Ouchterlony diffusion (Sakaguchi, G., et al.,
Jpn. J. Med. Sci. Biol
. 27:161-170, 1974).
The non-toxic complexing proteins have been demonstrated to be essential for stabilization of the toxin during passage through the digestive tract (Ohishi and Sakaguchi, supra, 1974; Sakaguchi, G., et al., Purification and oral toxicities of
Clostridium botulinum
progenitor toxins, In Biomedical aspects of botulism, G. Lewis (ed.), Academic Press, Inc., New York, pp. 21-34, 1981). Pure neurotoxin has a peroral LD
50
about 100-10,000 times lower than that of toxin complex on a weight basis (Ohishi, I.,
Infect. Immun
. 43:487-490, 1984; Sakaguchi, G.,
Pharmacol. Therap
. 19:165-194, 1983). Presumably, the complexing proteins protect the very labile toxin molecule from proteolytic cleavage and other types of inactivation by enzymes, acids and other components present in the gut and circulatory systems since the toxin and the complexing proteins are generally stable in low pH environments.
Analysis by SDS-PAGE has shown that type A toxin complex consists of seven different nontoxic proteins ranging in size from ca. 17 kD to 118 kD in association with a neurotoxic protein of ca. 147 kD (Goodnough, M. and Johnson, E., supra, 1993; Gimenez, J. and DasGupta, B.,
J. Protein Chem
. 12:349-361, 1993; DasGupta,
Canad. J. Microbiol
. 26:992-997, 1980). Isolated type A toxin complex has a specific toxicity of 2-4×10
7
intraperitoneal LD
50
/mg in 18-22 g white mice. Specific toxicities of other
C. botulinum
toxin complexes are type B M complex—4-5×10
7
LD
50
/mg, type C
1
M complex—1-2×10
7
LD
50
/mg, type D M complex—7-8×10
7
LD
50
/mg, type E M complex—1×10
7
LD
50
/mg, type F M complex—2-3×10
7
LD50/mg (Sugiyama, H., supra, 1980), and 8-9×10
6
/mg for type G complex (Schiavo, G., et al.,
J. Biol. Chem
. 269:20213-20216, 1994).
C. Botulinum
Neurotoxin
The biologically active neurotoxin of
C. botulinum
is a dichain molecule of ca. 150 kD in molecular weight. The molecule is composed of two fragments or chains that are termed the heavy chain (Hc, ca. 100 kD) and the light chain (Lc, ca. 50 kD) that are covalently connected by one disulfide bond (FIG.
1
). The neurotoxin is synthesized by the organism as a single polypeptide called the protoxin and undergoes post-translational processing termed nicking by at least one protease to generate the two separate chains (Yokosawa, N., et al.,
J. Gen. Microbiol
. 132:1981-1988, 1986; Krysinski, E. and Sugiyama, H.,
Appl. Environ. Microbiol
. 41:675-678, 1981). The two chains are covalently bound through a disulfide bridge. The nicking event occurs in the culture fluid for proteolytic
C. botulinum
and through the activity of an added exogenous enzyme such as trypsin in non-proteolytic strains (Yokosawa, N., et al., supra, 1986; DasGupta, B.,
J. Physiol
. (Paris) 84:220-228, 1990; Kozaki, S., et al.,
FEMS Microbiol. Lett
. 27:149-154, 1985).
Functional Domains of Botulinal Neurotoxin
Binding to cell surface. The carboxyl terminus of botulinal heavy chain is responsible for receptor binding on the cell surface. Initial work done using tetanus toxin, which is very similar in structure to botulinum neurotoxin, showed binding to cell receptors involved a multiple step binding sequence. The ten C-terminal amino acids are essential for initial receptor recognition on the motor neuron via a low affinity binding site while a sequence in the middle of the heavy chain was responsible for higher affinity secondary binding through a different protein receptor (Halpern, J. and Loftus, A.,
J. Biol. Chem
. 268:11188-11192, 1993).
Evidence shows that binding by type B botulinum neurotoxin occurs in a similar fashion (Nishiki, T., et al.,
J. Biol. Chem
. 269:10498-10503, 1994). The initial binding of type B neurotoxin to synaptosomes has been shown to be related to the presence of sialic acid containing motor neuron membrane components such as gangliosides G
DIa
, and G
T1b
as well as a partially purified 58 kD protein that has been tentatively determined to be a synaptogamin isoform. There is minimal binding of the neurotoxin to the 58 kD high affinity receptor in the absence of the low affinity gangliosides. This indicates that the initial low affinity binding to gangliosides which are prevalent on the cell surface by the carboxyl-terminal amino acids is followed by a high affinity binding to the 58 kD protein by an undetermined region that is located more towards the amino terminus and possibly in the central portion of the heavy chain. Treatment of synaptosomes with proteases and or sialidase decreased binding of the neurotoxin to the synaptosomes.
Channel formation. Once the neurotoxin is bound to the motor neuron via the C-terminus end of the heavy chain, the light chain and the N-terminus of the heavy chain are endocytosed. The proteolytically active light chain is then released into the cytosol of the cell via a translocation event through the phospholipid vesicle membrane. This translocation event is driven by a sequence of amino acids contained in the N-terminal portion of the heavy chain. The predicted sequence responsible for translocation of botulinum toxin type A is from amino acids 650-681 and shows strong sequence homology to tetanus toxin amino acids 659-690 (Montal, M., et al.,
FEBS. Lett
. 313:12-18, 1992). Both of these regions contain a high number of hydrophobic amino acid residues which presumably facilitate intercalation into
Goodnough Michael C.
Johnson Eric A.
Malizio Carl J.
Scott Alan B.
Bugaisky Gabrielle
Wisconsin Alumni Research Foundation
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