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STIM1 knock-down decreases the affinity of obinutuzumab for CD20 by altering CD20 localization to Triton-soluble
1Department of Pharmacology and Brain, Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.
Abstract:
Obinutuzumab is thought to exert its effects through its high antibody-dependent cellular cytotoxicity (ADCC) via glyco-engineering of the Fc region. In addition, obinutuzumab causes direct binding-induced cell death (DCD) only by specifically binding to its target CD20, a Ca2+ channel. However, the specific features of CD20 related to obinutuzumab binding-induction of cell death are not clearly understood. In this study, we evaluated the relationship between the Ca2+ channel features of CD20 as a store-operated Ca2+ channel (SOC) and obinutuzumab binding-induced cell death. Ca2+ channel function and biochemical analysis revealed that CD20 is an Orai1- and stromal interaction molecule (STIM1)-dependent Ca2+ pore. However, binding of obinutuzumab on CD20 did not have any effect on Ca2+ influx activity of CD20; the direct cell death rate mediated by obinutuzumab binding was almost equivalent with or without the extracellular Ca2+ condition. Given the apparent interaction between STIM1 and CD20, we observed Triton-X solubilized obinutuzumab-bound CD20 accompanied by STIM1. Subsequently, obinutuzumab binding and cell death were decreased by STIM1 knock-down in Ramos B cells. Thus, STIM1 directly contributes to cell death by increasing the affinity of cells for obinutuzumab by transferring CD20 to the Triton-soluble membrane region.
Insights
Obinutuzumab induces cell death independently of CD20 calcium channel activity. Stromal interaction molecule 1 (STIM1) enhances obinutuzumab binding and cell death by interacting with CD20.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Obinutuzumab, a glyco-engineered antibody, targets CD20 and induces cell death through antibody-dependent cellular cytotoxicity (ADCC) and direct binding-induced cell death (DCD).
- The precise mechanisms by which CD20 binding by obinutuzumab triggers cell death remain incompletely understood, particularly concerning CD20's role as a calcium channel.
Purpose of the Study:
- To investigate the relationship between the calcium channel properties of CD20 and obinutuzumab-induced cell death.
- To elucidate the role of calcium influx and STIM1 in obinutuzumab's direct cell death mechanism.
Main Methods:
- Functional and biochemical analysis of CD20 as a store-operated calcium (SOC) channel.
- Assessment of obinutuzumab-induced cell death with and without extracellular calcium.
- Investigation of STIM1 interaction with CD20 and the effect of STIM1 knockdown on obinutuzumab binding and cell death.
Main Results:
- CD20 functions as an Orai1- and STIM1-dependent calcium pore.
- Obinutuzumab binding to CD20 did not alter CD20's calcium influx activity.
- Extracellular calcium was not required for obinutuzumab-mediated direct cell death.
- STIM1 knockdown reduced obinutuzumab binding and cell death, indicating STIM1's contribution.
Conclusions:
- Obinutuzumab-induced direct cell death is independent of CD20's calcium channel function.
- STIM1 plays a crucial role in obinutuzumab-mediated cell death by enhancing the affinity of CD20 for obinutuzumab, potentially by facilitating its transfer to a Triton-soluble membrane fraction.
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