Domain-Specific Antibodies Reveal Differences in the Membrane Topologies of Apolipoprotein L1 in Serum and Podocytes

Abstract

Significance Statement: Two variants of circulating APOL1, G1 and G2, protect against subspecies of Trypanosoma brucei infection, but concurrently confer a greater risk of CKD. Little is known about the topology of APOL1 in HDL particles or on the podocyte cells affected in kidney disease. Antibodies generated along the length of APOL1 revealed differences of the exposed domains between serum and podocyte surface APOL1 topologies. These are the first direct insights into APOL1 conformations in physiological settings and have implications for development of APOL1-targeted drugs for APOL1 nephropathies. Background: Circulating APOL1 lyses trypanosomes, protecting against human sleeping sickness. Two common African gene variants of APOL1, G1 and G2, protect against infection by species of trypanosomes that resist wild-type APOL1. At the same time, the protection predisposes humans to CKD, an elegant example of balanced polymorphism. However, the exact mechanism of APOL1-mediated podocyte damage is not clear, including APOL1's subcellular localization, topology, and whether the damage is related to trypanolysis. Methods: APOL1 topology in serum (HDL particles) and in kidney podocytes was mapped with flow cytometry, immunoprecipitation, and trypanolysis assays that tracked 170 APOL1 domain-specific monoclonal antibodies. APOL1 knockout podocytes confirmed antibody specificity. Results: APOL1 localizes to the surface of podocytes, with most of the pore-forming domain (PFD) and C terminus of the Serum Resistance Associated-interacting domain (SRA-ID), but not the membrane-addressing domain (MAD), being exposed. In contrast, differential trypanolytic blocking activity reveals that the MAD is exposed in serum APOL1, with less of the PFD accessible. Low pH did not detectably alter the gross topology of APOL1, as determined by antibody accessibility, in serum or on podocytes. Conclusions: Our antibodies highlighted different conformations of native APOL1 topology in serum (HDL particles) and at the podocyte surface. Our findings support the surface ion channel model for APOL1 risk variant–mediated podocyte injury, as well as providing domain accessibility information for designing APOL1-targeted therapeutics.