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Different bioactivities of human thyrotropin receptors with different signal peptides
1Department of Life Science and Biotechnology Research Center, Pohang University of Science and Technology, South Korea.
Abstract:
For investigation of the mechanism and pathogenesis of Graves' disease, availability of a large amount of functional human thyrotropin receptor (TSHR) capable of recognition by Graves' autoantibodies is essential. Many attempts have been made to produce the extracellular domain of TSH receptor (TSHRE) in a baculovirus expression system. However, the receptor is expressed as an insoluble form and the refolded protein is often not recognized by the autoantibodies. In this study, we found that the TSHRE expressed with its own signal peptide (VL3-RE) in insect cells is retained inside of the cells and found in both soluble and insoluble fractions in equal proportion. The signal peptide is not removed. The receptor in the soluble fraction is not recognized by either TSH or Graves' autoantibodies. The TSHRE with an insect-specific mellitin signal peptide (Mel-RE) is also retained inside of the cell and found in both the soluble and insoluble fractions in equal proportion. However, the signal peptide is removed and the receptor is recognized by the Graves' autoantibodies but not by TSH. Also, the amount of Mel-RE expressed was 5-10-fold higher than VL3-RE. The two receptor preparations apparently have the same degree of glycosylation as evidenced by the same increased mass (approximately 15 kDa) due to glycosylation. However, the two receptors have different affinity for an anion-exchange resin and different pI. Deglycosylated receptors have the same pI. This suggests that the composition of sugars may be different. Taken together, the results suggest that the two receptors are modified and folded differently by different pathways due to the presence of different signal peptides. Use of an insect-specific signal peptide is recommended for expression of TSHR that is recognized by Graves' autoantibodies in a baculovirus system.
Insights
Producing functional human thyrotropin receptor (TSHR) for Graves' disease research is challenging. Using an insect-specific signal peptide enhances TSHR expression and recognition by Graves' autoantibodies in a baculovirus system.
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- Graves' disease pathogenesis requires functional human thyrotropin receptor (TSHR) for autoantibody recognition.
- Previous baculovirus expression systems yielded insoluble or non-functional TSHR extracellular domain (TSHRE).
Purpose of the Study:
- To optimize the expression of functional TSHRE recognized by Graves' autoantibodies using a baculovirus system.
- To investigate the role of signal peptides in TSHRE folding, modification, and antibody binding.
Main Methods:
- Expressed TSHRE in insect cells using its native signal peptide (VL3-RE) and an insect-specific melittin signal peptide (Mel-RE).
- Analyzed TSHRE solubility, signal peptide cleavage, glycosylation, anion-exchange affinity, and isoelectric point (pI).
- Assessed recognition of expressed TSHRE by TSH and Graves' autoantibodies.
Main Results:
- Mel-RE, unlike VL3-RE, showed signal peptide cleavage and was recognized by Graves' autoantibodies but not TSH.
- Mel-RE expression levels were 5-10 fold higher than VL3-RE.
- Different signal peptides led to differential TSHRE folding and glycosylation, affecting receptor properties.
Conclusions:
- An insect-specific signal peptide is crucial for producing functional TSHRE in baculovirus systems.
- Optimized TSHRE expression facilitates Graves' disease research and autoantibody interaction studies.
- Signal peptide choice significantly impacts TSHR post-translational modifications and functional outcomes.