Supplemental Experimental Procedures

Cell Assay for Hybridoma Screening- Human neuroblastoma SH-SY5Y cells stably expressing APP were grown on 24-well plates till 90% confluent. After wash, cell were treated with 100 μL hybridoma supernatants mixed with 100 μL fresh growth medium DMEM supplemented with 4.5 g/L glucose, 0.11 g/L sodium pyruvate and 15% fetal bovine serum. After 24 hours treatment, conditioned media were collected and centrifuged at 13,000 rpm for 10 min at 4 °C, the supernatants were analyzed by western blotting for sAPPβ and sAPPα using antibodies anti-sAPPβ polyclonal antibody (Covance) and 6E10 (Signet).

Isotyping, Subcloning and Antibody Purification- The isotypes of hybridoma clones 1A11, 5G7 and 14F10 were determined by Mouse Monoclonal Antibody Isotyping kit (Roche) according to the manufacturer’s instructions as IgG1 (1A11, 5G7) and IgG2b (14F10). Each hybridoma clone was subcloned four rounds by limiting dilution. Antibody production was performed by culturing hybridoma clone in Celline CL-1000 bioreactors (VWR) using DMEM medium supplemented with 4 mM glutamine, 4.5 g/L D-glucose and 15 % fetal bovine serum at 37°C with 5 % CO2 and 70 % relative humidity. The antibody were purified by affinity chromatography using protein G Sepharose 4 (Sigma) and dialyzed in PBS using dialysis membrane MWCO 6000~8000 Daltons (Spectrum). Aliquots of antibody were snap frozen by liquid nitrogen and stored at -80°C.

Cell Surface Biotinylation- HEK293 cells stably expressing human BACE1 construct were washed and incubated with ice-cold PBS supplemented with 0.5 mg/mL EZ-LinkTM Sulfo-NHS-SS-Biotin (Pierce) at 4 °C for 45 min. Cells were collected and homogenized using a cell-cracker in STE-buffer containing 5 mM Tris-HCl pH 7.6, 250 mM sucrose and 1 mM EGTA supplemented with complete protease inhibitor cocktail (Roche). The 1.000 x g pellets were solublized in 1% CHAPSO on ice for 1 hour. Insoluble material was removed by 130.000 x g ultracentrifugation for 30 min at 4 °C. The supernatants were subjected to immunoprecipitation using the 1A11 or 5G7 mAb. Immunoprecipitates were further analyzed by western blotting using mAb 10B8 (generated in this work, epitope is within 46-240 aa of BACE1) for total BACE1, or Streptavidin-HRP Conjugate for cell-surface BACE1. Metabolic labeling and Pulse-Chase Experiments- HEK293 cells in 6- well plate (~1 million cells per well) were transfected with the cDNA of wild type or mutant BACE1. Twenty-four hours after transfection, cells were preincubated in methionine/cysteine-free (starve) medium for 30 min after which they were incubated in starve medium supplemented with 700 µCi of [35S] methionine/cysteine for 10 min. After metabolic labeling, cells were quenched in starve medium supplemented with a 100 molar excess of cold methionine/cysteine. Cells were then chased in normal growth medium for a variable amount of time after which they were washed and lysed in radioimmunoprecipitation assay (RIPA) buffer (1% sodium deoxycholate, 0.1 % SDS, 1% Triton X-100, 5 mM EDTA, 50 mM Tris, pH 8, 150 mM NaCl), and immunoprecipitated with mAb 10B8 (epitope located within 46-240 aa of BACE1). Immunoprecipitates were eluted by heating in 1.4 x Laemmli sample buffer plus β-mercaptoethanol at 85 °C for 10 min. Samples were then electrophoresed on 4~12 % Bis-Tris SDS PAGE. After fixation, the gels were dried and exposed to a PhosphorImager screen for 24-48 hours. PhosphorImager screens were read on a Typhoon scanner and bands were quantified by Aida Image Analyser version 4.22. Dot blot and Western blotting analysis of antibody specificity- Brain extractions of adult wild type or Bace1(-/-) mice (1) were prepared by homogenization in lysate buffer containing PBS supplemented with 1 % Triton and complete protease inhibitor cocktail (Roche). Cell lysates from BACE1 cDNA transfected or nontransfected HEK293 cells were prepared by solubling total cell pellets in the same lysate buffer. Insoluble material was removed by 14.000 rpm for 15 min at 4 °C. Brain extracts or cell lysates were either dotted directly on nitrocellulose membranes (10 µg brain extracts for dot blot), or heated in 1 x Laemmli sample buffer plus β-mercaptoethanol for 10 min at 70 °C, and electrophoresed on 4~12 % Bis-Tris SDS PAGE followed by transfer to nitrocellulose membranes. The membranes were blotted with test mAbs 1A11, 5G7 or a commercial rabbit mAb D10E5 (Cell signaling) at a dilution of 1: 1000 (µg/mL) for 1 hour at RT. Enhanced chemiuminescence substrate was applied to develop the blots and the signals were detected by a LAS-3000 luminescent image.

Antibody Uptake Assays- Primary hippocampal neurons derived from wild type or Bace1(-/-) mouse embryos were cultured for 7 days and incubated with 200 µg/ mL test mAb 1A11 labeled with Alexa Fluor 488 Monoclonal Antibody Labeling kit (Invitrogen). The incubation was maintained at growth condition for 6 hours. During the last 30 min of the incubation, a LysoTracker Red DND-99 (Invitrogen) was added into the growth medium for labeling of acidic cellular compartments. After the incubation, neurons were washed and analyzed by a confocal fluorescence microscopy (Biorad Radiance) for cellular distributions of the test mAb and LysoTracker.

Confocal Fluorescence Microscopy- HEK293 cells stably expressing human BACE1 were grown on coverslips coated with 0.2 mg/mL poly-L-lysine. Cells were fixed in 4 % paraformaldehyde and permeabilized in 0.1 % Triton X-100. After blocking the cells with 5 % goat serum diluted in blocking buffer (PBS supplemented with 2 % FBS, 2 % BSA, and 0.2 % Gelatin) overnight at 4 °C, cells were incubated with test mAb 1A11 or 5G7 together with either a commercial rabbit anti-BACE1 pAb ab2077 (Abcam) or EEA1 (Sigma) diluted in blocking buffer for 2 hours at RT. Cells were then washed and incubated with Alexa Fluor 488 goat-anti-mouse IgG (Invitrogen) and Alexa Fluor 594 goat-anti-rabbit IgG (Invitrogen) diluted in blocking buffer for 45 min at RT. After wash, the coverslips were mounted on slides and sealed with nail polish. Immunofluorescent images were acquired by a confocal fluorescence microscopy (Biorad Radiance).                             

Supplemental Figures 

Supplemental Figure 1. Mutagenesis of residues on loops D and F abolished immunoreactivity of mAb 1A11 to BACE1 in immunoprecipitation. Cell extracts of HEK293 cells overexpressing WT or mutant BACE1were subjected to immunoprecipitation using mAb 1A11 or 5G7 (control). Immunoprecipitants or total cell extracts (Input) were analyzed by western blotting (on 4-12 % SDS-PAGE) using mAb 10B8 (the epitope located within 46-240 aa of BACE1). Mutagenesis of residues 376SQD378 to WAA on loop F (A) or residues 332QAG334 to AGA on loop D (B) abolished the immunoreactivity of 1A11.Notice that control mAb 5G7 did not recognize immature BACE1, which was reported to be acetylated at Lys299 (2) thus destroyed the binding epitope for mAb 5G7 (see Supplemental Fig. S5). The lower bands indicated by an arrow were fragments of mouse IgG used in immunoprecipitation.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Supplemental Figure 2. Structure and sequence alignment of BACE1 with related aspartyl proteases. (A) 3-D structures of BACE1, Pepsin, Cathepsin D and BACE2 were created using PDB files: 2G94, 2EWY, 1LYW and 1PSO, respectively. Loops D and F on BACE1, and the corresponding structures on Pepsin, Cathepsin D and BACE2, are indicated in pink color. (B) Loops D and F are conserved among BACE1 sequences, and differ from the sequence of BACE2. Alignment of BACE1 and BACE2 protein sequences was performed using CLUSTALW.  

 

 

 

 

 

 

 

 

 

 

Supplemental Figure 3. Quantification of purified BACE1: Fc enzyme by western blotting. (A) Serial dilution of purified BACE1: Fc was analyzed by western blotting using a BACE1-specific mAb 10B8 (epitope located within BACE1 46-240 aa). Enhanced chemiluminescence substrate was used to develop the blot, and the signal was detected by a LAS-3000 luminescent image analyzer and further quantified using Aida Image Analyzer v.4.22. (B) The western blotting signal was linear within protein range 1.7 to 55.5 ng (in red rectangle). Purified wt or mutant BACE1: Fc was quantified within the linear range of a standard curve generated on the same blot.  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Supplemental Figure 4. Colocalization of mAbs 1A11 and 5G7 with an anti-BACE1 rabbit pAb ab2077 (Abcam) and EEA1 (Sigma E4156). HEK293 cells stably expressing human BACE1 were stained with mAb 1A11 (A and B: a-d) or 5G7 (A and B: e-h) in green, ab2077 (A: a-h) in red, EEA1 (B: a-h) in red. Open square in (A and B: a) and (A and B: e) represent the zoom area shown in (A and B: b-d) and (A and B: e-h), respectively. Inserts in (A: b-d) and (A: f-h) highlight the overlapping staining between mAb 1A11 or 5G7 and ab2077. Arrowheads in inserts (B: b-d) and (B: f-h) indicate colocalization between MAb 1A11 or 5G7 and EEA1. Bar= 10 μm.

 

Supplemental Figure 5. Epitope mapping of mAb 5G7. (A) A schema for immunogen BACE1 46-460 (1#) and the fragments (2# - 13#). The numbers on the fragments represent the amino acids corresponding to the full length human BACE1 protein. (B) Western blotting analysis (10% SDS-PAGE) of mAb 1A11 against purified GST-BACE1 mutants. Immunogen BACE1 46-460 (1#) and mutants BACE1 240-460 (4#), 274-460 (10#), 46-390 (11#) and 296-460 (12#) are immunoreactive, whereas all the rest mutants are not (upper panel). As expected, the anti-GST antibody recognizes all recombinant proteins (lower panel). As mAb 5G7 recognizes BACE1 296-460 (12#) and 46-390 (11#), but not 314-460 (8#) and 46-380 (13#), residues between 296-314 and 380-390 are important for mAb binding. Similar to mAb 1A11, mAb 5G7 is predicted to recognize a “conformational epitope” on BACE1. (C) 3D structures of BACE1 296-314 and 380-390 are presented using PDB file 1FKN. Three residues K299, E303, and Q386 are spatially adjacent and form a “conformational epitope”. (D) Mutations K299Q/ E303D or Q386P were introduced into full length BACE1. Mutants were expressed in HEK293 cells, and the cell extracts were subjected to immunoprecipitation using mAb 5G7 or 1A11. The immunoprecipitants or total extracts (Input) were analyzed by western blotting using a BACE1-specific mAb 10B8. Mutation K299Q/ E303D or Q386P abolishes or strongly impairs the immunoreactivity of mAb 5G7 to BACE1, but have no effect on the immunoreactivity of mAb 1A11 to BACE1. 

 

 

 

 

 

 

 

 

 

Supplemental Figure 6. Uptake of mAb 1A11 in primary cultured hippocampal neurons. 7-days cultured hippocampal neurons were incubated with Alex488 conjugated mAb 1A11 and maintained at growth conditions for 6 hours (A), or co-incubated with LysoTracker Red DND-99 (staining acidic compartments) during the last 30 min of the 6 hours incubation (B). A significant higher uptake of Alex488 conjugated mAb 1A11 was observed in neurons derived from wild type mouse embryos (a) than in neurons derived from BACE1 knockout mouse embryos (b and c), indicating a specific uptake of mAb 1A11 against BACE1. Alex488 conjugated MAb 1A11 (d) and LysoTracker (e) show partial colocalization (f). Bar= 10 μm.

 

 

 

 

 

 

 

 

 

 

 

 

 

Supplemental Figure 7. Analysis of antibody specificity by dot blot and western blotting. (A) Dot blot using 10 µg of brain extract from wild type or BACE1 knockout mouse. mAb 1A11 strongly reacted to brain extract from wild type mouse but not to brain extract from BACE1 knockout mouse, indicating that mAb 1A11 is highly specific to BACE1. Notice that the weak signal detected from BACE1 knockout mouse brain extract (blotted with mAb 1A11) is background signal as derived from the negative control (no primary antibody), in which no primary antibody but only HRP conjugated goat-anti-mouse secondary antibody was used. mAb 5G7 and a commercial anti-BACE1 rabbit mAb D10E5 (Cell Signaling) were included as controls. Notice that D10E5 reacts with the BACE1 knock out extract. (B) Western blotting (4~12% Bis-Tris SDS PAGE) using 40 µg of brain extracts from wild type or BACE1 knockout mouse, and 30 µg of cell extracts from human BACE1 transfected or non transfected HEK293 cells. mAb 1A11 detected BACE1 (around 64 kDa) in the material from the transfected cells. The weaker bands around 51 kDa detected in the mouse brains are background signals which are also presented in the lanes stained with only the secondary antibodies. Blot for β-actin has a very short exposure time thus did not detect the background signals. As mAb 1A11 recognizes a conformational epitope (Fig. 4), it thus reacts strongly to BACE1 in dot blot (non denaturing condition), but only weakly in western blotting (denaturing condition).

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