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Updated: Jun 2, 2026
Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens
Published on: June 6, 2020
Identification of soluble and membrane-bound isoforms of porcine tumor necrosis factor receptor 2
Mireia Uribe-Herranz1, Sandra R Casinghino, Laia Bosch-Presegué
1New Therapies of Genes and Transplants Group, Institut d'Investigació Biomèdica de Bellvitge, IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain.
Background:
TNF and its receptors TNF-Receptor 1 (TNFR1, CD120a) and TNF-Receptor 2 (TNFR2, CD120b) have been implicated in the rejection of transplanted cells and organs. Although pig TNFR1 (pTNFR1) is known to mediate the effects of human TNF in a xenogeneic setting, it is unclear whether pig TNFR2 (pTNFR2) could contribute to xenograft rejection.
Methods:
We have cloned the cDNA of various pTNFR2 variants by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. We have characterized the predicted proteins with bioinformatic tools and conducted expression, affinity, and functional studies to investigate their roles.
Results:
We have identified four isoforms of pTNFR2: one comprising the four cysteine-rich domains (CRD) conserved between species, a shorter variant (pTNFR2ΔE7-10) encoding for a soluble isoform, another with only three CRD due to the lack of exon 4 (pTNFR2ΔE4), and a fourth variant containing both modifications. Accordingly, multiple mRNA transcripts were observed by northern blotting. Quantitative RT-PCR determined high pTNFR2 expression in lung and immune cells and detected the two alternative splicings in all cells/tissues examined. The full receptor was moderately expressed on the surface of pig cells such as porcine aortic endothelial cells and PK-15 and was regulated by TNF. On the contrary, the membrane-bound pTNFR2ΔE4 was located only intracellularly. Plasmon resonance studies showed that pTNFR2 binds pig and human TNFα with high affinity, but pTNFR2ΔE4 interacts poorly with pig TNFα and does not bind to the human cytokine. Moreover, pull-down experiments with the two recombinant soluble isoforms consistently demonstrated that the two bound together and soluble pTNFR2ΔE4 was able to modulate the TNF inhibitory activity of pTNFR2-GST in a cell-based assay.
Conclusion:
The pTNFR2 may participate in the process of xenograft rejection and other related events, as well as be used in soluble form to block TNF in this setting. In addition, we have discovered other pTNFR2 isoforms that may affect the pig immune responses and have an impact on rejection of xenografts.
