Nidhi Gupta1,2, Xinhua Wang3, Xiaohui Wen1, Paul Moran4, Maciej Paluch5, Philip E Hass5, Amy Heidersbach1, Benjamin Haley1, Daniel Kirchhofer4, Randall J Brezski3, Andrew S Peterson1, Suzie J Scales6,2. 1. Department of Molecular Biology, Genentech, South San Francisco, California. 2. Department of Immunology, Genentech, South San Francisco, California. 3. Department of Antibody Engineering, Genentech, South San Francisco, California. 4. Department of Early Discovery Biochemistry, Genentech, South San Francisco, California. 5. Department of Protein Chemistry, Genentech, South San Francisco, California. 6. Department of Molecular Biology, Genentech, South San Francisco, California sscales@gene.com.
Abstract
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.
BACKGROUND: Circulating APOL1 lyses trypanosomes, protecting against humansleeping 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.
Authors: Moin A Saleem; Michael J O'Hare; Jochen Reiser; Richard J Coward; Carol D Inward; Timothy Farren; Chang Ying Xing; Lan Ni; Peter W Mathieson; Peter Mundel Journal: J Am Soc Nephrol Date: 2002-03 Impact factor: 10.121
Authors: John F O'Toole; William Schilling; Diana Kunze; Sethu M Madhavan; Martha Konieczkowski; Yaping Gu; Liping Luo; Zhenzhen Wu; Leslie A Bruggeman; John R Sedor Journal: J Am Soc Nephrol Date: 2017-11-27 Impact factor: 10.121
Authors: Luc Vanhamme; Françoise Paturiaux-Hanocq; Philippe Poelvoorde; Derek P Nolan; Laurence Lins; Jan Van Den Abbeele; Annette Pays; Patricia Tebabi; Huang Van Xong; Alain Jacquet; Nicole Moguilevsky; Marc Dieu; John P Kane; Patrick De Baetselier; Robert Brasseur; Etienne Pays Journal: Nature Date: 2003-03-06 Impact factor: 49.962
Authors: S L Hajduk; D R Moore; J Vasudevacharya; H Siqueira; A F Torri; E M Tytler; J D Esko Journal: J Biol Chem Date: 1989-03-25 Impact factor: 5.157
Authors: Anneli Cooper; Hamidou Ilboudo; V Pius Alibu; Sophie Ravel; John Enyaru; William Weir; Harry Noyes; Paul Capewell; Mamadou Camara; Jacqueline Milet; Vincent Jamonneau; Oumou Camara; Enock Matovu; Bruno Bucheton; Annette MacLeod Journal: Elife Date: 2017-05-24 Impact factor: 8.140
Authors: Justin Chun; Cristian V Riella; Hyunjae Chung; Shrijal S Shah; Minxian Wang; Jose M Magraner; Guilherme T Ribas; Hennrique T Ribas; Jia-Yue Zhang; Seth L Alper; David J Friedman; Martin R Pollak Journal: J Am Soc Nephrol Date: 2022-03-01 Impact factor: 14.978
Authors: Suzie J Scales; Nidhi Gupta; Ann M De Mazière; George Posthuma; Cecilia P Chiu; Andrew A Pierce; Kathy Hötzel; Jianhua Tao; Oded Foreman; Georgios Koukos; Francesca Oltrabella; Judith Klumperman; WeiYu Lin; Andrew S Peterson Journal: J Am Soc Nephrol Date: 2020-08-06 Impact factor: 10.121
Authors: Mark Ultsch; Michael J Holliday; Stefan Gerhardy; Paul Moran; Suzie J Scales; Nidhi Gupta; Francesca Oltrabella; Cecilia Chiu; Wayne Fairbrother; Charles Eigenbrot; Daniel Kirchhofer Journal: Commun Biol Date: 2021-07-27