Fig 1.
The FBA substrate is efficiently cleaved by SPP/SPPLs.
A. Design of SPP assay substrate based on BRI2 transmembrane domain. A FLAG tag fused to the NH2-terminus of BRI21-81. Aβ1-25/K16A fused to the COOH-terminus of BRI21-81. Potential cleavage in the transmembrane domain releases the ectodomain fragment xAβ1-25/K16A. B. Western blotting of FBA ±SPPL transfected cell lysates with and without 10 μM (Z-LL)2 ketone. Blot detected with anti-FLAG M2 antibody. The intact FBA and ICDs are marked with arrows. C. SPP/SPPLs significantly increase xAβ1-25/K16A secretion from FBA transfected cells. xAβ1-25/K16A levels were determined with Aβ ELISA. D. The FBA cleavages conducted by SPPLs co-transfection are largely inhibited by 10 μM (Z-LL)2 ketone. E. Western blotting of SPPLs transiently transfected HEK cell lysates. Blots detected with antisera against SPPL2a, SPPL2b, hSPP, and pSPP respectively. Monomer bands are marked with arrows. All experiments repeated 3 times. Statistical analysis performed by 1-way ANOVA ((*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
Fig 2.
Each SPP/SPPL has preferential cleavage site on FBA.
A. Cell lysates of FBA-SPP/SPPL co-transfected cells are used for IP/MS with anti-FLAG M2 magnetic beads. Top and bottom panels are the spectra for cells with DMSO or 20 μM LY-411,575. Main peaks observed are labeled with molecular weight (Da). The peaks below 7000 Da and at ~8080 Da are non-specific. B. 10–20 ml of FBA/SPP co-transfected cell culture media is used for IP/MS with the anti-Aβ1–16 antibody AB5 bound to magnetic beads. Main peaks observed in the xAβ1-25/K16A (ECDs) are labeled with molecular weights. The peaks at ~4190 Da are non-specific. C. Schematic representation of the cleavages of FBA. Putative TMD is underlined. Open and solid arrows indicate observed ICDs and xAβ1-25/K16A respectively. “Minor” peaks labeled with * are not shown in these spectra, but have been observed in others (See S1 Fig).
Table 1.
Molecular weight of FLAG-BRI2 peptide detected in FBA/SPP co-transfected cell lysates.
Table 2.
Molecular weight of x-Aβ1-25/K16A peptide detected in FBA/SPP co-transfected cell media.
Fig 3.
GSIs selectively inhibit SPP/SPPLs cleavage.
A. Chemical structure of select GSIs. B. xAβ1-25/K16A secretion from SPPLs co-transfected cell are largely inhibited by (Z-LL)2 ketone, LY-411,575, L685,458 and GSI II. The activity of hSPP was also inhibited by Compound E and DBZ. The xAβ1-25/K16A concentrations in DMSO treated cultures are set as 100%. Statistical analysis performed by 1-way ANOVA ((*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). C. Western blot of FBA/SPPL2b (top) and FBA/hSPP (bottom) co-transfected cell with select GSIs.
Fig 4.
GSIs inhibit SPP/SPPLs in a dose-dependent manner.
FBA/SPPLs co-transfected HEK cells were treated with DMSO or GSIs as appropriate. Twenty-four hours later the medium was collected for assay by Aβ ELISA. xAβ1-25/K16A levels from DMSO-treated cells served as the control. All tests were repeated 3 times. Data were analyzed using GraphPad.
Table 3.
IC50s (nM) of select GSI on FBA/SPPL co-transfected cells.
Fig 5.
The selectivity of GSIs on SPPL is verified using non-overexpressing A20 cells.
A. Western blot of A20 cells treated with 25 μM (Z-LL)2 ketone or GSIs. Blot was developed using anti-CD74 antibody In-1. P37 is the intact CD74. P10 and P8 are the 10 kD and 8 kD CD74 NTFs. Accumulation of P8 indicates SPPL2a inhibition. B. Dose response of A20 cell to (Z-LL)2 ketone, LY-411,575, GSI II, DBZ and Compound E.