Protein domains of APOL1 and its risk variants

Increasing lines of evidence have demonstrated that the development of higher rates of non-diabetic glomerulosclerosis (GS) in African Americans can be attributed to two coding sequence variants (G1 and G2) in the Apolipoprotein L1 (APOL) gene. Recent studies indicate that the gene products of these APOL1 risk variants have augmented toxicity to kidney cells. However, the biological characteristics of APOL1 and its risk variants are not well elucidated. The APOL1 protein can be divided into several functional domains, including signal peptide (SP), pore forming domain (PFD), membrane address domain (MAD), and SRA-interacting domain. To investigate the relative contribution of each domain to cell injury, we constructed a serial expression vectors to delete or express each domain. These vectors were transfected into the human embryonic kidney cell line 293T, and then compared the cytotoxicity. In addition, we conducted studies in which APOL1 wild type (G0) was co-transfected in combination with G1 or G2 to see whether G0 could counteract the toxicity of the risk variants. The results showed that deleting the SP did not abolish the toxicity of APOL1, though deletion of 26 amino acid residues of the mature peptide at the N-terminal partially decreased the toxicity. Deleting PFD or MAD or SRA-interacting domain abolished toxicity, while, overexpressing each domain alone could not cause toxicity to the host cells. Deletion of the G2 sites while retaining G1 sites in the risk state resulted in persistent toxicity. Either deletion or exchanging the BH3 domain in the PFD led to complete loss of the toxicity in this experimental platform. Adding G0 to either G1 or G2 did not attenuate the toxicity of the either moiety. These results indicate that the integrity of the mature APOL1 protein is indispensable for its toxicity. Our study not only reveals the contribution of each domain of the APOL1 protein to cell injury, but also highlights some potential suggested targets for drug design to prevent or treat APOL1-associated nephropathy.