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. 2021 Aug 6;8(4):ENEURO.0069-21.2021. doi: 10.1523/ENEURO.0069-21.2021

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Copyright © 2021 Huang and Park

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Figure 3. — Characterizing the release mechanism of GPC4 using Nluc-GPC4. A, After biotinylating the surface proteins of astrocytes expressing Nluc-GPC4, cells were further incubated in the fresh medium for 5 h. The released Nluc-GPC4 is collected from the medium, and the ratio of biotinylation of the released Nluc-GPC4 was measured (luciferase activity of avidin pulldown over medium input). As a control for the release from the cell surface, PI-PLC were treated during the incubation time. Both basal and PI-PLC induced GPC4 release occur from the cell surface. GPC4^sec, which does not contain the GPI-anchor signal peptide and is thus not GPI-anchored, is directly secreted without membrane attachment and lacks biotinylation (basal vs PI-PLC ANOVA p = 0.0079, Cohen’s d = 1.012). B, Ultracentrifugation of ACMs containing Nluc-GPC4 was used to test for GPC4 association with extracellular vesicles as a potential release mechanism. Supernatant (soluble) fraction and pellet (vesicular) fraction Nluc were normalized to input Nluc luminescence. Nluc-GPC4 did not associate with the pellet (vesicular) fraction. An exosome marker, CD9, was enriched in the pellet (vesicular) fraction (t test p < 0.0001, Cohen’s d = 7.829). C, alpha toxin (AT), which binds to the glycan core of GPI-anchors, was used to pull down GPI-anchored proteins released through lipase activity. PI-PLC, lipase released Nluc-GPC4 is used as a positive control, while GPC4^sec, lacking a GPI anchor, is used as a negative control. Nluc-GPC4 pulled down by AT is normalized to input Nluc-GPC4 measurements. Basal release shows partial binding to AT, indicating that ∼30% of Nluc-GPC4 is released through a lipase mechanism (PI-PLC vs basal release ANOVA p < 0.0001, Cohen’s d = 7.659); **p < 0.01, ***p < 0.001.