Abstract: SA-PO747
THSD7A Cleavage Is Mediated by the Proprotein Convertase Furin In Vitro
Session Information
- Glomerular Diseases: Podocyte Biology - II
November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1304 Glomerular Diseases: Podocyte Biology
Authors
- Seifert, Larissa, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Dehde, Silke, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Lucas, Renke, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Zahner, Gunther, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Tomas, Nicola M., Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
Background
Thrombospondin type 1 domain-containing 7A (THSD7A) is a pathogenic autoantigen in membranous nephropathy (MN). It is prominently expressed on podocytes in humans and rodents, where it is located at the basal aspects of foot processes and it likely plays a role in podocyte adhesion. When expressed recombinantly e.g. in HEK cells, THSD7A is cleaved in the N-terminal region.
Methods
Purified, recombinantly expressed THSD7A was analyzed by mass spectrometry (MS). Following MS analysis, we applied site directed mutagenesis to further narrow down the cleavage site. We tried two additional approaches to confirm our data and identify the protease that we assumed to be responsible for the cleavage. First, we tested a specific inhibitor and second we added the recombinant commercial available protease to uncut protein, to see whether it is processed.
Results
Mass spectrometry revealed a cleavage of THSD7A in the polybasic region of the fourth TSP-1 domain. The cleavage site was located between two cysteins forming a disulfide bond, leading to adherence of the fragments even after cleavage of the peptide backbone. The identified site corresponded to the cleavage motif of proprotein convertases (PCs). Base substitution of one Arginin to Alanin within the cleavage site completely abolished the proteolytic processing. Adding an inhibitor, which dominantly inhibits the PC furin, to the cell culture medium of THSD7A expressing HEK cells achieved the same effect. Conversely, adding recombinant furin to uncut THSD7A led to cleavage of the protein.
Conclusion
The PC furin cleaves THSD7A in the fourth TSP-1 domain in vitro. Whether this processing also occurs in vivo and is related to the biological function of THSD7A needs to be investigated.
Funding
- Government Support – Non-U.S.