Article Figures & Data
Figures
- Figure 1 Sequence and structure of CD59 WT and mutants
(A) The sequences of WT CD59, point mutations Cys64Tyr and Asp24Val, and frameshift mutations Asp24Valfs* and Ala16Alafs*: The mature membrane surface CD59 primary sequence consists of 77 residues after removal of a 25-residue N-terminal signal sequence and a C-terminal GPI-anchoring signal (not shown). Colored arrows and boxes represent β-strands and α-helices, respectively, as observed in WT CD59 (see B). Point mutations are marked with an asterisk. The sequence of the 2 frameshift mutants reveals significantly shortened proteins that lack most of the CD59 sequence, in particular the GPI-anchoring signal. (B) The structure of CD59 highlights the vicinity of the point mutations to known sites of activity and interaction. The mutated Cys64 and Asp24 positions are highlighted as spheres and labeled. The 3 characterized interfaces of CD59 are marked by numbered crescents: (1) The classic site characterized originally described by Bodian et al.21 with the central Trp40 residue shown in sticks, (2) a loop spanning residues 20–24 that modulates CD59 activity20 (colored in white), and (3) the edge β-strand that interacts with ILY18 (to the left of the crescent). Disulfide bridges that stabilize CD59 are shown in sticks. Two solved structures (2j8b and 2uwr) are shown, highlighting the relative flexibility of the C-terminal helix (colored in magenta). (C) The frameshift mutants contain only a small part of the original CD59 sequence: The structure of CD59 is shown, with the region with a sequence in common with the Ala16Alafs* and Asp24Valfs* mutants colored in yellow and yellow-white, respectively. The I-TASSER model for the Asp24Valfs* sequence aligns 2 additional β-strands (colored in green, ending with the sphere at CD59 position Trp40), before it diverges, and the remaining part (in blue) is unique to CD59. GPI = glycosyl phosphatidylinositol; WT = wild type.
- Figure 2 Western blot profile
See Methods for details. Anti-myc was used in all assays for detection. (A) Western blot expression pattern of WT and mutants Cys64Tyr, Asp24Val, Asp24Valfs*, and Ala16Alafs*, as well as GFP. Anti-myc was used for detection (upper panel) and anti-tubulin as a loading control (lower panel). (B) Western blot expression pattern of WT and Cys64Tyr mutant and GFP after using the Endo H enzyme. (C) Western blot expression pattern of conditioned medium. (D) Proteosomal degradation profile. Transfected cells treated with bortezomib (Velcade) and lysate sample were separated on 15% polyacrylamide gels. PVDF membranes were used for protein transfer. Anti-tubulin was used as a loading control (lower panel). PVDF = polyvinylidene difluoride; WT = wild type.
- Figure 3 Surface and intracellular localization of hCD59 mutants
(A) Characterization of WT and mutant Cys64Tyr, Asp24Val, Asp24Valfs*, and Ala16Alafs* constructs by myc antibody (red) and DAPI (blue). Staining with myc tag and secondary antibodies was added after fixation without Triton. (B) Characterization of WT and Asp24Valfs* and Ala16Alafs* CD59 mutants by anti-myc antibodies with or without Triton permeabilization. Differential recognition of WT and the Asp24Valfs* and Ala16Alafs* CD59 mutants by anti-myc. In both procedures, anti-myc detected the WT constructs with or without Triton, but in the mutant construct, ER staining was seen only with Triton. (C) Characterization of WT and Asp24Valfs* and Ala16Alafs* CD59 mutants by anti-myc and PDI antibodies. Upper panel: myc antibody (red); middle panel: PDI antibody (green) and DAPI (blue); and lower panel: merge. Cells were treated with methanol for permeabilization. Scale bars = 50 μm. PDI = protein disulfide isomerase; WT = wild type.
- Figure 4 Mutant detection by anti-CD59 antibodies using flow cytometry and fluorescent microscopy
(A) A panel of anti-CD59 antibodies in Cys64Tyr and healthy control lymphoblasts: MEM43, HC1, BRIC229, YTH53.1, A35, 1.39, and rabbit polyclonal antibodies, assayed by flow cytometry. (B) Staining of WT and mutant constructs Cys64Tyr, Asp24Val, Asp24Valfs*, and Ala16Alafs* by BRIC229 or MEM43f (red), DAPI (blue). Procedures were performed by live staining without fixation and Triton treatment and assayed by fluorescent microscopy. Scale bars = 50 μm.
- Figure 5 Lysis functional assay in hCD59 mutants
WT and mutant constructs Cys64Tyr, Asp24Val, Asp24Valfs*, and Ala16Alafs* were transfected to CHO cells. Forty-eight hours after transfection, cells were marked by calcein AM, and calcein fluorescence of supernatants was read using the Cytation 3 Cell Imaging Multi-Mode Reader with the excitation filter set at 485 nm and emission filter at 530 nm. Percent lysis for each well was calculated as calcein release/total calcein loading (*p < 0.05, **p < 0.002, ANOVA and Student t test). WT = wild type.
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