Splice variants of protein disulfide isomerase - identification, distribution and functional characterization in the
Thomas Chetot1, Xavier Serfaty1, Léna Carret1
1USC 1233 RS2GP, VetAgro Sup, INRAe, Université de Lyon, 69280 Marcy l'étoile, France.
Background:
Protein Disulfide Isomerase (PDI) enzyme is an emerging therapeutic target in oncology and hematology. Although PDI reductase activity has been studied with isolated fragments of the protein, natural structural variations affecting reductase activity have not been addressed.
Methods:
In this study, we discovered four coding splice variants of the Pdi pre-mRNA in rats. In vitro Michaelis constants and apparent maximum steady-state rate constants after purification and distribution in different rat tissues were determined.
Results:
The consensus sequence was found to be the most expressed splice variant while the second most expressed variant represents 15 to 35% of total Pdi mRNA. The third variant shows a quasi-null expression profile and the fourth was not quantifiable. The consensus sequence splice variant and the second splice variant are widely expressed (transcription level) in the liver and even more present in males. Measurements of the reductase activity of recombinant PDI indicate that the consensus sequence and third splice variant are fully active variants. The second most expressed variant, differing by a lack of signal peptide, was found active but less than the consensus sequence.
General Significance:
Our work emphasizes the importance of taking splice variants into account when studying PDI-like proteins to understand the full biological functionalities of PDI.
Insights
Researchers discovered four Protein Disulfide Isomerase (PDI) splice variants in rats. Two variants are fully active, while a third shows reduced activity, highlighting the importance of considering PDI variations in biological studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Protein Disulfide Isomerase (PDI) is a key enzyme in oncology and hematology.
- Previous studies focused on PDI reductase activity using isolated fragments, neglecting natural structural variations.
- The impact of PDI splice variants on its reductase activity remains largely unaddressed.
Purpose of the Study:
- To identify and characterize coding splice variants of PDI in rat pre-mRNA.
- To determine the reductase activity and tissue distribution of identified PDI splice variants.
- To understand the functional implications of PDI structural variations.
Main Methods:
- Discovery and identification of four coding splice variants of Pdi pre-mRNA in rats.
- In vitro determination of Michaelis constants and apparent maximum steady-state rate constants for purified variants.
- Analysis of PDI splice variant expression and distribution across different rat tissues.
Main Results:
- Four coding PDI splice variants were identified, with the consensus sequence being the most expressed.
- The second most expressed variant, lacking a signal peptide, showed reduced but significant reductase activity.
- Fully active PDI variants (consensus and third) and a less active variant were identified, with wide expression in liver, particularly in males.
Conclusions:
- Splice variants significantly influence the reductase activity of Protein Disulfide Isomerase.
- The characterized PDI variants exhibit differential expression and activity profiles across rat tissues.
- Accounting for PDI splice variants is crucial for a comprehensive understanding of PDI's biological functions and therapeutic potential.
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