Molecular medicine-Occurrence of Inclusion Bodies

Inclusion bodies were first identified in
the blood cells of patients with abnormal
hemoglobins, the resulting pathology being
anemia. Pathological point mutants of
hemoglobin aggregate into inclusion bodies;
this is the case for hemoglobin K¨oln
(Val98Met on the β chain) and hemoglobin
Sabine (Leu91Pro on the β chain). Similar
deposits have been described in studies on
the metabolism of abnormal proteins subjected
to covalent modification in E. coli.
The formation of aggregates also occurs
when cells are subjected to heat shock.
The in vivo folding pathway of tailspike
endorhamnosidase of Salmonella phage
22 is a well-documented system studied
by J.King’s group. Furthermore, it is one
of the few systems in which the in vivo
folding pathway has been compared with
the in vitro refolding pathway. The protein
is a trimer of 666 amino acids. The secondary
structure is predominantly β-sheet.
Newly synthesized polypeptide chains released
from the ribosome generate an
early partially folded intermediate. This intermediate
further evolves into a species
sufficiently structured for chain–chain
recognition. In the following step, an incompletely
folded trimer is formed upon
close association with the latter species.
The protrimer is then transformed into
the native tailspike. A clear difference between
the physicochemical properties of
the intermediates and the native state has
Aggregation, Protein 41
allowed their identification. Figure 3 illustrates
the folding pathway of the protein.
The native protein is highly thermostable
with a Tm of 88 ◦C; it is also resistant
to detergents and proteases. During the
in vivo folding process, the intermediates
are sensitive to these factors, allowing
their identification. At low temperature,
almost 100% of the newly synthesized
chains reach the native trimer conformation.
When the temperature increases in
the cells, the number of polypeptide chains
achieving the native state decreases. At
39 ◦C, the maturation proceeds with 30%
efficiency, while the remainder aggregates
into inclusion bodies. It has been shown
that the aggregation does not result from
an intracellular denaturation of the native
protein, but is generated from an early
thermolabile intermediate. The aggregated
chains cannot recover their proper folding
by lowering the temperature. But when
polypeptide chains that have been synthesized
at high temperatures are shifted to
low temperature early enough, they can
refold correctly.
A set of mutations that alter protein
folding without modifying the properties
and stability of native P22 tailspike
has been identified; they are referred
to as temperature-sensitive folding (tsf)
mutants. These mutations have been supposed
to destabilize the already thermolabile
intermediate and are located at more
than 30 sites in the central region of
the polypeptide chain. Starting from mutants
kinetically blocked in their folding, a
second set of mutants capable of correcting
the folding defects was selected, and
the sequences surrounding the suppressor
mutations were identified. Only two substitution
positions on the 666 amino acids
of the polypeptide chain were sufficient to
prevent inclusion body formation. Thus,
single temperature mutations that affect
the folding pathway but not the native
conformation of a protein are efficient in
preventing off-pathway and subsequent aggregation.
A similar result has been found
for heterodimeric luciferase. For recombinant
proteins such as interferon-γ and
interleukin 1β, as well as for P22 tailspike,
amino acid substitutions that can
decrease or increase the formation of inclusion
bodies without alteration of the
functional structure were found by Wetzel
and coworkers.
The formation of inclusion bodies is
frequently observed in the production of
recombinant proteins. High levels of expression
of these proteins result in the
formation of inactive amorphous aggregates,
and has been reported for proteins
expressed in E. coli and also in several
host cells, gram-negative as well as grampositive
bacteria, and eukaryotic cells such
as Saccharomyces cerevisiae, insect cells,
and even animal cells. The production
of recombinant proteins, among them
human insulin, interferon-γ, interleukin
1β, β-lactamase, prochymosin, tissue plasminogen
activator, basic fibroblast growth
hormone, and somatotropin, gives rise to
inclusion bodies.