Inclusion bodies


Inclusion bodies, sometimes called elementary bodies, are nuclear or cytoplasmic aggregates of stable substances, usually proteins. They typically represent sites of viral multiplication in a bacterium or a eukaryotic cell and usually consist of viral capsid proteins. Inclusion bodies can also be hallmarks of genetic diseases, as in the case of neuronal inclusion bodies in disorders like frontotemporal dementia and Parkinson's disease.
Inclusion bodies contain very little host protein, ribosomal components or DNA/RNA fragments. They often almost exclusively contain the over expressed protein and aggregation in inclusion bodies has been reported to be reversible. It has been suggested that inclusion bodies are dynamic structures formed by an unbalanced equilibrium between aggregated and soluble proteins of Escherichia coli. There is a growing body of information indicating that formation of inclusion bodies occurs as a result of intracellular accumulation of partially folded expressed proteins which aggregate through non-covalent hydrophobic or ionic interactions or a combination of both.
Inclusion bodies are dense electron-refractile particles of aggregated protein found in both the cytoplasmic and periplasmic spaces of E. coli during high-level expression of heterologous protein. It is generally assumed that high level expression of non-native protein and highly hydrophobic protein is more prone to lead to accumulation as inclusion bodies in E. coli. In the case of proteins having disulfide bonds, formation of protein aggregates as inclusion bodies is anticipated since the reducing environment of bacterial cytosol inhibits the formation of disulfide bonds. The diameter of spherical bacterial inclusion bodies varies from 0.5–1.3 μm and the protein aggregates have either an amorphous or paracrystalline nature depending on the localization. Inclusion bodies have higher density than many of the cellular components, and thus can be easily separated by high-speed centrifugation after cell disruption. Inclusion bodies despite being dense particles are highly hydrated and have a porous architecture.

Composition

Inclusion bodies have a non-unit lipid membrane. Protein inclusion bodies are classically thought to contain misfolded protein. However, this has recently been contested, as green fluorescent protein will sometimes fluoresce in inclusion bodies, which indicates some resemblance of the native structure and researchers have recovered folded protein from inclusion bodies.

Mechanism of formation

When genes from one organism are expressed in another organism the resulting protein sometimes forms inclusion bodies. This is often true when large evolutionary distances are crossed: a cDNA isolated from Eukarya for example, and expressed as a recombinant gene in a prokaryote risks the formation of the inactive aggregates of protein known as inclusion bodies. While the cDNA may properly code for a translatable mRNA, the protein that results will emerge in a foreign microenvironment. This often has fatal effects, especially if the intent of cloning is to produce a biologically active protein. For example, eukaryotic systems for carbohydrate modification and membrane transport are not found in prokaryotes. The internal microenvironment of a prokaryotic cell may differ from that of the original source of the gene. Mechanisms for folding a protein may also be absent, and hydrophobic residues that normally would remain buried may be exposed and available for interaction with similar exposed sites on other ectopic proteins. Processing systems for the cleavage and removal of internal peptides would also be absent in bacteria. The initial attempts to clone insulin in a bacterium suffered all of these deficits. In addition, the fine controls that may keep the concentration of a protein low will also be missing in a prokaryotic cell, and overexpression can result in filling a cell with ectopic protein that, even if it were properly folded, would precipitate by saturating its environment.

Viral inclusion bodies

Examples of viral inclusion bodies in animals are
Intracytoplasmic eosinophilic -
Intranuclear eosinophilic -
Intranuclear basophilic-
Both intranuclear and intracytoplasmic-
Examples of viral inclusion bodies in plants include aggregations of virus particles and aggregations of viral proteins. Depending on the plant and the plant virus family these inclusions can be found in epidermal cells, mesophyll cells, and stomatal cells when plant tissue is properly stained.

Inclusion bodies in Erythrocytes

Normally a red blood cell does not contain inclusions in the cytoplasm. However, it may be seen because of certain hematologic disorders.
There are three kinds of erythrocyte inclusions:
  1. Developmental Organelles
  2. #Howell-Jolly bodies: small, round fragments of the nucleus resulting from karyorrhexis or nuclear disintegration of the late reticulocyte and stain reddish-blue with Wright stain.
  3. #Basophilic stipplings - these stipplings are either fine or coarse, deep blue to purple staining inclusion that appears in erythrocytes on a dried Wright stain.
  4. #Pappenheimer bodies - are siderotic granules which are small, irregular, dark-staining granules that appear near the periphery of a young erythrocyte in a Wright stain.
  5. #Polychromatophilic red cells - young red cells that no longer have nucleus but still contain some RNA.
  6. #Cabot Rings - ring-like structure and may appear in erythrocytes in megaloblastic anemia or in severe anemias, lead poisoning, and in dyserythropoiesis, in which erythrocytes are destroyed before being released from the bone marrow.
  7. Abnormal Hemoglobin Precipitation
  8. #Heinz bodies - round bodies, refractile inclusions not visible on a Wright stain film. It is best identified by supravital staining with basic dyes.
  9. #Hemoglobin H Inclusions - alpha thalassemia, greenish-blue inclusion bodies appear in many erythrocytes after four drops of blood is incubated with 0.5mL of Brilliant cresyl blue for 20 minutes at 37 °C.
  10. Protozoan Inclusion
  11. #Malaria
  12. #Babesia

    Inclusion bodies in Bacteria

Polyhydroxyalkanoates or PHA are produced by bacteria as inclusion bodies, the size of PHA granules are limited in E. coli, due to its small bacterial size. Bacterial cell's inclusion bodies are not as abundant intracellularly, in comparison to eukaryotic cells.

Current problems with the isolation of proteins from bacterial inclusion bodies

70-80% of recombinant proteins expressed E. coli are contained in inclusion bodies. The purification of the expressed proteins from inclusion bodies usually require two main steps: extraction of inclusion bodies from the bacteria followed by the solubilisation of the purified inclusion bodies. Solubilisation of inclusions bodies often involves treatment with denaturing agents, such as urea or guanidine chloride at high concentrations, to de-aggregate the collapsed proteins. Renaturation follows the treatment with denaturing agents and often consists of dialysis and/or use of molecules that promote the refolding of denatured proteins.

Pseudo-inclusions

Pseudo-inclusions are invaginations of the cytoplasm into the cell nuclei, which may give the appearance of intranuclear inclusions. They may appear in papillary thyroid carcinoma.

Diseases involving inclusion bodies

DiseaseAffected cells
Inclusion body myositismuscle cells
Amyotrophic lateral sclerosismotor neurons
Dementia with Lewy bodiescerebral neurons

Inclusion body diseases differ from amyloid diseases in that inclusion bodies are necessarily intracellular aggregates of protein, where amyloid can be intracellular or extracellular. Amyloid also necessitates protein polymerization where inclusion bodies do not.

Preventing the formation of inclusion bodies

Inclusion bodies are often made of denatured aggregates of inactive proteins. Although, the renaturation of inclusion bodies can sometimes lead to the solubilisation and the recovery of active proteins, the process is still very empirical, uncertain and of low efficiency. Several techniques have been developed over the years to prevent the formation of inclusion bodies. These techniques include: