Abstract: SA-PO378

Domain-Specific Antibodies Reveal the Membrane Topology of ApoL1

Session Information

Category: Chronic Kidney Disease (Non-Dialysis)

  • 301 CKD: Risk Factors for Incidence and Progression

Authors

  • Gupta, Nidhi, Genentech, Inc., South San Francisco, California, United States
  • Peterson, Andrew S., Genentech, Inc., South San Francisco, California, United States
  • Scales, Suzie J., Genentech, Inc., South San Francisco, California, United States
  • Wang, Xinhua, Genentech, Inc., South San Francisco, California, United States
  • De Maziere, Ann, UMC Utrecht, Utrecht, Netherlands
  • Posthuma, George, UMC Utrecht, Utrecht, Netherlands
  • Moran, Paul, Genentech, Inc., South San Francisco, California, United States
  • Lipari, Michael Terry, Genentech, Inc., South San Francisco, California, United States
  • Kirchhofer, Daniel, Genentech, Inc., South San Francisco, California, United States
  • Klumperman, Judith, UMC Utrecht, Utrecht, Netherlands
  • Brezski, Randall J., Genentech, Inc., South San Francisco, California, United States
Background

ApoL1 (Apolipoprotein L1) is a component of the trypanolytic factor that circulates on HDL3b particles and protects against Trypanosoma brucei brucei infection. Two common African variants in ApoL1 (G1 and G2) additionally protect against Trypanosoma brucei rhodesiense, but also confer a greater risk of chronic kidney disease in homozygotes. ApoL1 contains three domains named for their roles in trypanolysis – a pore forming domain that forms ion channels leading to lysis; a membrane addressing domain responsible for HDL binding and lysosomal membrane insertion; and an SRA-interacting domain that binds to the serum resistance factor of T.b. rhodesiense. However, little is known about ApoL1 topology i.e. which domains are exposed on HDL particles and kidney podocytes, the susceptible cell type in chronic kidney disease.

Methods

We generated >100 monoclonal antibodies to ApoL1 to better understand its topology, localization and roles in kidney disease and trypanolysis. We probed ApoL1 membrane topology in cell lines using immunofluorescence on differentially permeabilized cells, electron microscopy and flow cytometry. ApoL1 domain accessibility in human serum was mapped by the ability of the antibodies to block trypanolysis.

Results

We obtained specific (non ApoL2 cross-reactive) antibodies to all three domains of ApoL1. ApoL1 localized to the endoplasmic reticulum in overexpressing kidney cells, with all three domains sequestered within the lumen, topologically equivalent to the cell surface. By contrast, ApoL2 (which lacks a signal sequence) localized to the cytoplasmic face of the endoplasmic reticulum. We found portions of all three domains of ApoL1 are exposed in serum, as judged by pull-down and inhibition of trypanolysis assays. However, on the podocyte cell surface, the membrane addressing domain of ApoL1 is not exposed.

Conclusion

ApoL1 topology differs between serum and kidney cells.

Funding

  • Commercial Support