Evolution of Renal-Disease Factor APOL1 Results in Cis and Trans Orientations at the Endoplasmic Reticulum That Both Show Cytotoxic Effects

Abstract

The recent and exclusively in humans and a few other higher primates expressed APOL1 (Apolipoprotein L1) gene is linked to African human trypanosomiasis (also known as African sleeping sickness) as well as to different forms of kidney diseases. Whereas APOL1’s role as a trypanolytic factor is well established, pathobiological mechanisms explaining its cytotoxicity in renal cells remain unclear. In this study, we compared the APOL family members using a combination of evolutionary studies and cell biological experiments to detect unique features causal for APOL1 nephrotoxic effects. We investigated available primate and mouse genome and transcriptome data to apply comparative phylogenetic and maximum likelihood selection analyses. We suggest that the APOL gene family evolved early in vertebrates and initial splitting occurred in ancestral mammals. Diversification and differentiation of functional domains continued in primates, including developing the two members APOL1 and APOL2. Their close relationship could be diagnosed by sequence similarity and a shared ancestral insertion of an AluY transposable element. Live cell imaging analyzes showed that both expressed proteins show a strong preference to localize at the endoplasmic reticulum (ER). However, glycosylation and secretion assays revealed that—unlike APOL2—APOL1 membrane insertion or association occurs in different orientations at the ER, with the disease-associated mutants facing either the luminal (cis) or cytoplasmic (trans) side of the ER. The various pools of APOL1 at the ER offer a novel perspective in explaining the broad spectrum of its observed toxic effects.

Endoplasmic reticulum-translocation is essential for APOL1 cellular toxicity

“…and Wakashin et al. ( Muller et al., 2021 ; Wakashin et al., 2020 ), or possibly the GFP-APOL1 construct used compared to our artificial ΔMSALFL APOL1, that might be more toxic to cells. Notably, we used the African background (E150, I228, K255) in all constructs, therefore the attenuated ΔMSALFL APOL1 toxicity cannot be explained by a different APOL1 haplotype ( Lannon et al., 2019 ).…”

“…These conflicting finding can be reconciled by considering that some studies have used APOL1 constructs that lack signal peptide and showed that the signal peptide was dispensable for APOL1 cellular toxicity ( Granado et al., 2017 ; Lan et al., 2015a ; Nichols et al., 2015 ; Thomson et al., 2014 ), while other studies used APOL1 constructs that harbored non-cleavable signal peptide ( Khatua et al., 2015 ). Granado et al., and recently Muller et al., used GFP- APOL1 constructs that lack the signal peptide and were still expressed in the ER while retaining the toxicity potential ( Granado et al., 2017 ; Muller et al., 2021 ). Muller et al.…”

“…Muller et al. have fused N glycosylation tag (GT, Glyco-tag) that only becomes glycosylated in the ER lumen and elegantly demonstrated that APOL1 protein in which the SP (aa1– 28) was replaced by an N-terminal GFP-tag was facing the cytoplasmic side of the ER but still retained APOL1 toxicity ( Muller et al., 2021 ). Their findings do not reconcile with our conclusion of reduced toxicity of APOL1 that faces the cytoplasmic face of the ER compared to isoforms which insert into the ER lumen.…”

The Roles of Fatty Acids and Apolipoproteins in the Kidneys

“…Recently, Zee et al showed that glomerular APOL1 expression or APOL1 risk alleles are associated with cellular/tissue changes in patients with FSGS [ 195 , 196 ]. ApoL2 is found mainly in the brain [ 197 ], but its function remains unknown in the kidneys. ApoL3 and ApoL4 are associated with cholesterol and sphingolipid transport/recycling to the plasma membrane in the lungs and other tissues [ 190 ], but their functions are also unknown in the kidneys.…”

paPAML: An Improved Computational Tool to Explore Selection Pressure on Protein-Coding Sequences

“…We also analyzed the classical abalone sperm lysin sequence dataset that contains verifiable amino acid sites under positive and purifying selection [ 17 ]. The last example refers to an apolipoprotein family (APOL) that recently was investigated for adaptive evolution [ 18 ].…”