Lyme Toxin Chemically Similar to Botulinum Toxin, a zinc endoproteinase
- From: Snappy <freyfaxi@xxxxxxxxxxxxx>
- Date: 4 May 2007 08:20:30 -0700
from an article on the Biochemistry of Lyme Disease, by professor
Robert Bradford
Lyme Disease Toxin
Because many of the symptoms of Lyme disease involve the nervous
system, it
was speculated that the spirochete produced a toxin that disrupted
normal nerve function. Through the use of DNA manipulations and a
database of known protein toxin DNA sequences, a match was made with a
selected Borrelia burgdorferi (Bb) gene and a specific toxin in the
database. Protein generated from this cloned Bb gene was examined
biochemically and found to have characteristics similar to that of
botulinum, the toxin of Clostridium botulinum, a zinc endoproteinase.
1
The toxin from Bb belongs to a family of toxic proteins known as "zinc
endoproteinases" or metalloproteases, and includes the toxin from the
organism causing tetanus as well as those from many other well-known
infectious diseases. The structures of this family of toxins are all
very similar, as determined by x-ray crystal analysis.2 They all
contain zinc and perform the same proteolytic function, namely,
cleaving the chemical (covalent) bond between two specific amino acids
in a particular protein found in nerve cells.3 The substrate for this
enzyme is very large, implying that any inhibitor of enzyme activity
blocking the entry of the substrate into the active site must also be
very large.
One reason for learning the structure of the toxin (including the
active site) is to determine the geometry of this site, the exact
positions of the atoms that bind other atoms in the substrate. Knowing
the arrangement of these atoms permits the development of inhibitors
of the toxin, substances that compete with the normal substrate for
active site occupancy.4
Action of Toxin
The action of botulinum (as well as the toxin from the Lyme
spirochete) is to prevent, through its action as a proteolytic enzyme,
the release of the neurotransmitter acetylcholine. Nerve endings may
be associated with other nerves or muscles (the neuromuscular
junction). To understand this mechanism in greater detail, consider
the basic principles of nerve physiology described below.
Nerve Cells
A typical nerve cell consists of a long filament or axon, the terminal
end of which
lies in close proximity to another nerve cell. The space between them
is known as the synaptic cleft (synapse). One nerve cell communicates
with another through the release of a chemical substance known as a
neurotransmitter held within small sacs (vesicles) lying near the
terminal end. An electrical pulse travels the length of the axon and,
when it reaches the nerve cell terminal, causes the vesicles to
rupture through the presynaptic membrane and discharge the
neurotransmitter into the synaptic cleft. The neurotransmitter is
bound by a protein (receptor) in the postsynaptic membrane of the
adjoining nerve cell causing, in turn, the transmission of an
electrical pulse down the axon of the second nerve cell. By this
mechanism, nerve cells communicate with one another
through the action of a neurotransmitter. One such neurotransmitter is
a simple organic substance known as acetycholine. (See Chart 1.)
The structure of acetylcholine is shown by this formula:
CH3C(O)-O-CH2-CH2-N+(CH3)3
Mechanism of Neurotransmitter Release
Only recently has the mechanism of neurotransmitter release been
understood at the molecular level. The proteins responsible for this
highly detailed process have been isolated and characterized. Some
parts of the puzzle are not as yet completely understood, for example,
the process of membrane fusion. A study of the release of
neurotransmitters from nerve endings has also revealed the mechanism
of "switching," a process by which only one nerve among several in
close proximity may be separately fired. This switching process is
analogous to a similar process occurring in computers. Our brains work
in a manner, in many ways, similar to that of computers. (See Chart
2.)
Each vesicle within a nerve ending contains only one type of
neurotransmitter. The vesicle containing a specific neurotransmitter
(NT) contains on its surface a specific protein designated VAMP
(vesicle-associated membrane protein). This protein is a member of a
family of specific proteins, differing only in the sequence of amino
acids forming a chain extending from the protein. If the NT is
designated NTA, the VAMP found in the membrane of the vesicle
containing NTA, will always be VAMPA. In other words, a specific
neurotransmitter is always associated in the vesicle with a specific
type of VAMP. Finding another type of VAMP - for example, VAMPB - on
the surface of a vesicle containing NTA will never occur. The
difference between VAMPA and VAMPB lies only in the sequence of amino
acids in the peptide (protein chain) extending from the protein.5
During the random motion of vesicles in the region of a nerve ending,
some encounter another protein embedded in the presynaptic membrane,
designated SNAP-25 (synaptosomal-associated membrane protein). All
SNAP-25 proteins belong to a family of similar proteins, differing
only in the amino acid sequences of two peptides extending from the
protein. A particular member of this family may, for example, be
designated (SNAP-25)A. If a vesicle bearing on its surface the protein
VAMPA encounters the protein (SNAP-25)A lying in the presynaptic
membrane, the three peptides (two from SNAP-25 and one from VAMP)
rapidly intertwine and automatically form a triple helix, which twists
in a manner similar to a "twist-tie" used on bread wrappers (ATP-
driven). The structure of this peptide triple helix is similar to the
triple helix found in collagen (a).5
The result of the twisting action is to draw the vesicle close to the
surface of the
presynaptic membrane. When the membrane of the vesicle contacts the
presynaptic membrane, the two membranes automatically fuse, resulting
in the vesicle contents (containing NTA) emptying into the synapse.
The membrane flattens out and the VAMP/SNAP-25 proteins (the SNARE
complex) are recycled.6 (See Chart 2.)
NSF Protein
A third protein linked to the VAMP/SNAP-25 complex is N-ethylmaleimide-
sensitive factor (NSF). N-ethylmaleimide is simply a chemical reagent
used by biochemical researchers (not a normal body metabolite),
capable of attaching acetyl groups [CH3C(O)-] to sulfhydryl groups (-
SH) as found in the amino acid cysteine, a constituent of many
proteins. The protein NSF is "sensitive" to this reagent (binds acetyl
groups when exposed to the reagent), indicating that its surface is
rich in sulfhydryl groups. This observation gives a hint about the
activity of NSF, an agent that holds together two other proteins (VAMP
and SNAP-25). Sulfhydryl groups are normally used to bind two proteins
together (cross-linking) or to bind different parts of a single
protein to each other. This is accomplished by the elimination of two
hydrogens (-H) from two sulfhydryl groups (-SH) (usually by a single
atom of oxygen, thereby forming water), resulting in a disulfide
linkage (-S-S-). For this reason, NSF is believed to function as a
link between VAMP and SNAP-25, forming a single rigid unit.5 (See
Chart 1.)
Specificity of Nerve Firing
If a vesicle having VAMPA on its surface encounters a (SNAP-25)B (or
any type other than A), no intertwining of the peptides will occur,
the vesicle will not contact the presynaptic membrane and,
consequently, no neurotransmitter will be released.
The NTA, released into the synapse, almost immediately contacts a
receptor (RA) in the postsynaptic membrane capable of binding this
neurotransmitter. If this receptor is found in nerve A (see Chart 2),
this nerve only is fired (i.e., develops an action potential that
travels down the axon). Any nerve ending in close proximity not
carrying RA in its postsynaptic membrane will not be activated. If NTB
is released into the synapse, only those nerve endings carrying RB
will be activated. By synthesizing large amounts of vesicles
containing NTA and simultaneously synthesizing an equal number of
(SNAP-
25)A, the corresponding type of nerve is activated.5
Dietary Supplements in Lyme Disease
One of the known actions of the Lyme spirochete toxin is to diminish
the release and availability of the neurotransmitter acetylcholine, a
simple organic compound (see above for chemical structure). This
substance is biosynthesized by the body as required in nerve
activation and transmission. Supplementation by the precursors of
acetylcholine synthesis would be of value to Lyme patients since they
have a deficiency of this substance. (See Listing 1.)
Listing 1: Dietary Supplements Increasing Acetylcholine
Synthesis Improving Neurologic Function
Phosphatidylcholine (Lecithin)Acetyl-L-Carnitine
Vitamin B5 (Pantothenic Acid)
Vitamin B6 (Pyridoxine)
Vitamin C (Ascorbic Acid)
Lysine (Amino Acid)
S-Adenosylmethionine (SAM) (Sulfur-bound Adenosyl Methionine)
If the inhibition of acetylcholine release were total, Lyme patients
and those suffering from food poisoning would not be able to move;
they would be completely paralyzed. Since the blockage is only
partial, any increase in the amount of available neurotransmitter
would benefit anyone experiencing neurotransmitter blockage. For this
reason, dietary supplements increasing the amount of available
acetylcholine have been shown to benefit Lyme patients.
Acetylcholine Formation
In Chart 3, we can see phopsphatidylcholine is a constituent of
lecithin, a well-
known dietary supplement. Acetylcholine is simply choline to which an
acetyl group (CH3CO-) has been attached. Lecithin is the source of
choline, and acetyl-L-carnitine (ALC) is the source of the acetyl
group. Carnitine is synthesized by the body and requires several
factors, including the amino acid lysine and vitamin C (ascorbic
acid). The supplement known as SAM (S-adenosylmethionine) supplies
methyl groups (CH3-) to lysine, forming trimethyllysine. This compound
is further processed, requiring additional vitamin C, resulting in
carnitine that supplies the necessary acetyl group.8,9
History of Lyme and Related Spirochetal Diseases
The discovery by Burgdorfer that Lyme disease was caused by a
spirochete placed it in a category of other diseases known to be
caused by spirochetes. An example of such a disease is syphilis, the
scourge of Europe for hundreds of years. Arsenic and some of its
compounds had been known for quite some time as a highly successful
and popular means of fatally poisoning someone (remember the King in
Shakespeare's Hamlet). Following the discovery of the Germ Theory of
Disease by Louis Pasteur (1822-1895), it was theorized that, if
arsenic was toxic enough to kill, it may also be effective in killing
the organisms that cause disease. In the early 1900s, the German
chemist-physician Paul Ehrlich (1854-1915) developed a chemical
treatment for syphilis. By using a "shotgun" approach of trying
hundreds of compounds in an effort to find one that worked, Ehrlich
discovered what became known as Salvarsan or "606" after 606 compounds
had been tested. Salvarsan is an organic compound of arsenic and may
be highly toxic if not properly used. For his monumental discovery,
Ehrlich was awarded the Nobel Prize in 1908. Salvarsan may be
considered the first man-made antibiotic.26 Arsenic belongs to that
column in the periodic table of chemical elements known as the "Group
V elements," which also include phosphorus, antimony and bismuth. (See
Chart 4.)
Following the success of Salvarsan as a treatment for syphilis, other
compounds of antimony and bismuth were also prepared and tried against
spirochetes. Examples of these compounds include bismuth subcitrate,
bismuth subsalicylate (Pepto-Bismol), bismuth subgallate, and many
others. An example of an antimony-containing antibiotic is Pentostam
(an antimonial, antimony sodium gluconate).27,28
A biological molecule known as ATP (adenosine triphosphate) supplies
energy to biological systems through the high energy bonds found in a
chain of three terminal phosphate groups. One of the mechanisms by
which arsenic exerts its toxic effect is the substitution of
phosphorus by arsenic in ATP, since both arsenic and phosphorus lie in
the same column of the periodic table of chemical elements and have
similar chemistry. (See Chart 5.)
When this substitution occurs, the molecule experiences immediate
hydrolysis, breaks down, and no longer functions as a source of energy
for the cell. Both antimony and bismuth are also found in this column
of the periodic table (Group V). 29,30 (See Chart 6.)
What may be the first case of Lyme disease was noted about 1974 in a
14-year old boy, taken to the hospital with extreme pains in the
muscles of his legs and unable to walk. This case, coupled with other
pertinent facts related to the boy and a highly classified US
government laboratory conducting research on contagious animal
diseases in this same area, is suggestive of a link between these two
events. The government laboratory alluded to is found on Plum Island,
just north of Long Island, NY, and south of Lyme, Connecticut. Because
of its secret nature, access to the island was only by ferry boat and
restricted to the government workers employed there. The 14-year old
boy lived near the ferry boat dock. Although not providing proof,
these considerations are highly indicative of a possible link between
this research laboratory and the subsequent outbreak in 1975 of an
unknown disease involving juveniles in the same area of Lyme,
Connecticut.32
.
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