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[Identification of the interactions between the truncated fragments of macrophage migration inhibitory factor and
Zhi-xi Shan1, Qiu-xiong Lin, Chun-yu Deng
1Research Center of Medical Sciences, Guangdong Provincial People's Hospital, Guangzhou/Guangdong Provincial Cardiovascular Disease Institute, Guangzhou 510080, China. zhixinshan@yahoo.com.cn
Objective:
To investigate the interaction domains between macrophage migration inhibitory factors (MIF) and the extracellular segment of type-II trans-membrane protein CD74 using a yeast two-hybrid system.
Methods:
By using molecular cloning techniques, the DNA fragments encoding MIF, MIF(50-65) and MIF(1-50/65-115) were introduced into the pGBKT7 vector to construct the corresponding recombinant bait plasmids, and the DNA fragments encoding CD74(73-232), CD74(73-109), CD74(1109-149) and CD74(149-232) into the pGADT7 vector to construct the recombinant activation domain (AD) plasmids. PEG/LiAC method was employed to transform the above 3 recombinant bait plasmids paired with each of the 4 recombinant AD plasmids into the chemical competent yeast AH109 cells. The transformed yeast AH109 cells were screened consecutively on SD/-Trp-Leu and SD/-Trp-Leu-Ade-His/X-alpha-gal nutritional media.
Results:
The results of restriction endonuclease digestion and DNA sequencing verified the correct construction of all the recombinant plasmids. The yeast AH109 cells transformed with each of the 3 recombinant bait plasmids could grow on SD/-trp nutritional media without autonomous activation effect on the reporter gene MEL1. The cells transformed with each of the 4 recombinant AD plasmids could also grow on SD/-leu nutritional media without activation of the reporter gene MEL1. Only the yeast AH109 cells co-transformed with MIF, MIF(50-65), or MIF(1-50/65-115) plasmid and CD74(73-232) plasmid could grow on SD/-Trp-Leu-Ade-His nutritional media with transcription activation of the reporter gene MEL1.
Conclusion:
MIF interacts with the intact extracellular segment of CD74 (CD74(73-232)) independent of the functional domain of MIF(50-65).
Insights
Macrophage migration inhibitory factor (MIF) interacts with the full extracellular segment of CD74. This interaction is independent of the MIF(50-65) functional domain, revealing key binding sites.
Area of Science:
- Molecular Biology
- Immunology
- Protein-Protein Interactions
Background:
- Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine involved in immune regulation and inflammation.
- CD74, the extracellular segment of a type-II trans-membrane protein, acts as a receptor for MIF and other ligands.
- Understanding the specific interaction domains is crucial for elucidating MIF-CD74 signaling pathways.
Purpose of the Study:
- To map the interaction domains between macrophage migration inhibitory factor (MIF) and the extracellular segment of CD74.
- To identify the specific regions of MIF and CD74 involved in their binding.
Main Methods:
- Yeast two-hybrid system was employed to investigate protein-protein interactions.
- Molecular cloning was used to generate recombinant plasmids encoding various fragments of MIF and CD74.
- Transformed yeast cells were screened on selective media to identify positive interactions.
Main Results:
- Successful construction and verification of recombinant plasmids for MIF and CD74 fragments.
- Yeast cells co-transformed with MIF or its fragments and the full-length CD74 extracellular segment (CD74(73-232)) showed growth on selective media.
- Specific interaction was confirmed between MIF and the intact extracellular segment of CD74.
Conclusions:
- Macrophage migration inhibitory factor (MIF) directly interacts with the entire extracellular segment of CD74 (CD74(73-232)).
- The interaction between MIF and CD74 is independent of the MIF(50-65) domain.
- This study defines the interaction interface between MIF and CD74, providing insights into their functional relationship.
