recombinant human bace2  (R&D Systems)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Soluble VCAM-1 May Serve as a Pharmacodynamic CSF Marker to Monitor BACE2 Activity in Non-Human Primates

doi: 10.1016/j.mcpro.2025.101012

Figure Lengend Snippet: Western blot validation of VCAM-1 as BACE2 substrate. A , scheme of VCAM-1 cleavage by BACE2. Mapping of detected peptides ( B ) suggests that VCAM-1 is shed closely above the plasma membrane. Western blotting was conducted with a polyclonal antibody that specifically targets the ectodomain. The figure was created with BioRender.com . B , Western blot detection of sVCAM-1 in CSF from cynomolgus monkeys treated with IgG RSV, anti-BACE1, ATV:BACE1 or verubecestat (N = 4 in each group). Albumin served as loading control and was visualized by total protein staining. C , quantification of Western blot results in B . First, sVCAM-1 signal intensities were normalized to albumin. For each individual, the two pre-dose values were averaged, followed by calculation of the post-dose/pre-dose ratio. Data depict mean and SD. One-way ANOVA in combination with Tukey’s multiple comparisons test. Only significant differences are indicated. ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The peptide was dissolved in 50 mM sodium acetate pH 4.4, and 0.5 μg of the peptide were incubated with 0.5 μg recombinant human BACE2 (R&D Systems) overnight at 37 °C.

Techniques: Western Blot, Biomarker Discovery, Clinical Proteomics, Membrane, Control, Staining

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Soluble VCAM-1 May Serve as a Pharmacodynamic CSF Marker to Monitor BACE2 Activity in Non-Human Primates

doi: 10.1016/j.mcpro.2025.101012

Figure Lengend Snippet: Validation of VCAM-1 as BACE2 substrate in human CSF . A , Western blot detection of sVCAM-1 in CSF from participants of a clinical phase 1 study . Participants were either treated with placebo (N = 6) or with 550 mg verubecestat (N = 5), and CSF was sampled 2 h before (pre-dose, pre) and 24 h after treatment (post-dose, post). Albumin served as loading control and was visualized by total protein staining. B , quantification of Western blot results in A . sVCAM-1 signal intensities were first normalized to albumin. Then, the post-dose value for each individual was normalized to the corresponding pre-dose value. Data show mean and SD. Unpaired Student’s t test. ∗∗∗ p < 0.001.

Article Snippet: The peptide was dissolved in 50 mM sodium acetate pH 4.4, and 0.5 μg of the peptide were incubated with 0.5 μg recombinant human BACE2 (R&D Systems) overnight at 37 °C.

Techniques: Biomarker Discovery, Western Blot, Control, Staining

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Soluble VCAM-1 May Serve as a Pharmacodynamic CSF Marker to Monitor BACE2 Activity in Non-Human Primates

doi: 10.1016/j.mcpro.2025.101012

Figure Lengend Snippet: Identification of the BACE2 cleavage site in VCAM-1 . A , localization and length of sVCAM-1 peptides detected in NHP CSF (N = 4) after GluC digest in a semi-specific TermineR-based search using the Fragpipe software. Peptides were mapped to the UniProt full-length Macaca fascicularis VCAM-1 sequence. Protein domain annotations were annotated based on prediction by Phobius. Pre-dose and post-dose CSF from the verubecestat treatment group was analyzed, and peptide LFQ values in the post-dose samples were normalized to the corresponding pre-dose sample. Peptide coloring represents the log 2 post-dose/pre-dose ratio. Coloring in dark blue or dark red indicates peptides only detected in pre-dose or post-dose CSF, respectively. Peptides either detected in pre-dose or post-dose CSF, but not in both samples of the same animal (so that calculation of the individual post-dose/pre-dose ratio was not possible), are colored in dark blue and dark red . Unspecific peptide termini, not resulting from GluC cleavage, are highlighted by yellow triangles . B , sVCAM-1 peptide mapping for the same samples as in A after analysis in Spectronaut. C , the semi-specific peptide identified in A and B , which potentially marks the BACE2 cleavage site, aligns with both Macaca fascicularis and human VCAM-1. Ectodomain ( blue ) and transmembrane domain ( yellow ) annotation is based on Phobius prediction (NHP) and UniProt (human), respectively. Amino acids in the human sequence that differ from the NHP VCAM-1 sequence are colored in red . In an in vitro cleavage assay, a synthetic VCAM-1 peptide was cleaved by BACE2 at the same position as found for CSF sVCAM-1. D , total ion chromatograms of the BACE2 cleavage assay of the synthetic VCAM-1 peptide depicted in C . Total ion chromatograms are shown for one representative out of two experiments. E , MS2 spectrum for the BACE2-cleaved synthetic VCAM-1 peptide. F , MS2 spectrum (hyperscore = 24.7) for the semi-specific, potentially BACE2-cleaved VCAM-1 peptide identified in NHP CSF. G , sequence logo graphic generated with WebLogo of the amino acid alignment of published BACE1 and 2 substrates around the published cleavage sites . Positively charged amino acids are displayed in blue , negatively charged amino acids in red , polar amino acids and glycine in green or purple (glutamine, asparagine), and non-polar amino acids are shown in black . The height of the amino acid symbols indicates their frequency at this position.

Article Snippet: The peptide was dissolved in 50 mM sodium acetate pH 4.4, and 0.5 μg of the peptide were incubated with 0.5 μg recombinant human BACE2 (R&D Systems) overnight at 37 °C.

Techniques: Software, Sequencing, In Vitro, Cleavage Assay, Generated

Journal: Molecular neurodegeneration

Article Title: Identification of BACE2 as an avid ß-amyloid-degrading protease.

doi: 10.1186/1750-1326-7-46

Figure Lengend Snippet: Figure 1 Overview and outcome of functional screening for novel Aß-lowering proteases. A, Cartoon illustrating the overall design of the screen. Briefly, an arrayed collection of 352 protease-encoding cDNAs was cotransfected into CHO cells together with an APP-AP fusion construct. Following a medium change and overnight incubation, Aß40 levels, AP activity, and cytotoxicity (via MTT assay) were analyized in the resulting conditioned media. B, Results of the screen, expressed as [Aß40]/AP ratios normalized to intraplate controls. Data are mean of 4 replicates. Note that the largest decrease in Aß levels by far was acheived by BACE2. C, Confirmation of the results of the screen. Following scale-up and sequence verification, cDNAs encoding BACE2 and its homolog, BACE1, were cotransfected together with APP-AP into CHO cells. Consistent with the outcome of the medium-throughput screen, BACE2, but not BACE1, expression resulted in significant decreases in the levels of both Aß40 and Aß42. Data are mean ± SEM of 4 replicates, and are normalized to vector-only controls.

Article Snippet: Recombinant BACE2 (R&D Systems) and plasmin (EMD Biosciences) were purchased from commercial sources, while recombinant IDE and secreted NEP (i.e., lacking the transmembrane domain) were generated and purified as described [23].

Techniques: Functional Assay, Construct, Incubation, Activity Assay, MTT Assay, Sequencing, Expressing, Plasmid Preparation

Journal: Molecular neurodegeneration

Article Title: Identification of BACE2 as an avid ß-amyloid-degrading protease.

doi: 10.1186/1750-1326-7-46

Figure Lengend Snippet: Figure 2 BACE2 degrades Aß in vitro. A, Percent Aß remaining following incubation with different concentrations of recombinant BACE2 for various lengths of time. Data are mean ± SEM of 4 replicates, normalized to protease-free controls. B, Comparison the relative Aß-degrading ability of recombinant BACE2 vs. BACE1. Note that 24 h incubation with BACE1 was required to achieve approximately the same extent of degradation as effected by BACE2 in 10 min. Data are mean ± SEM of 3 replicates, normalized to protease-free controls. C, BACE2 activity is pH dependent. Percent Aß degradation catalyzed by equivalent amounts of BACE2 at different pH values. Data are mean ± SEM of 4 replicates. D, BACE2 does not degrade fibrillar Aß. Lack of effect of BACE2 (10 nM) on preformed Aß42 fibrils following incubation at 37°C for 5 d, as determined by thioflavin T fluorescence. Data are mean ± SEM of 3 replicates.

Article Snippet: Recombinant BACE2 (R&D Systems) and plasmin (EMD Biosciences) were purchased from commercial sources, while recombinant IDE and secreted NEP (i.e., lacking the transmembrane domain) were generated and purified as described [23].

Techniques: In Vitro, Incubation, Recombinant, Comparison, Activity Assay, Fluorescence

Journal: Molecular neurodegeneration

Article Title: Identification of BACE2 as an avid ß-amyloid-degrading protease.

doi: 10.1186/1750-1326-7-46

Figure Lengend Snippet: Figure 3 Determination of peptide bonds within Aß hydrolyzed by BACE2. Top, Summary of cleavage sites determined from data in A-F, showing the major site (block arrow) and two minor sites (arrow heads). At t = 0 (A, C), intact Aß42 (A) and Aß40 (C) represent the only species present. Following incubation of Aß42 and Aß40 with 5nM BACE2 for 1 h (B,D, respectively), the full-length Aß species are essentially completely absent and replaced by Aß34. E,F, Additional Aß cleavage products are produced following incubation with larger amounts of BACE2 (25 nM) for longer lengths of time. By 1 h (E), a new peak corresponding to Aß20 is produced. By 24 h (F), Aß20 becomes the major species present, and Aß19 is also produced. Double-charged fragments are denoted by “+”, and “*” represents the modification of a fragment by AEBSF, which leads to a 183-Da increase in MW, as previously reported [46].

Article Snippet: Recombinant BACE2 (R&D Systems) and plasmin (EMD Biosciences) were purchased from commercial sources, while recombinant IDE and secreted NEP (i.e., lacking the transmembrane domain) were generated and purified as described [23].

Techniques: Blocking Assay, Incubation, Produced, Modification

Journal: Molecular neurodegeneration

Article Title: Identification of BACE2 as an avid ß-amyloid-degrading protease.

doi: 10.1186/1750-1326-7-46

Figure Lengend Snippet: Figure 4 Overexpression of BACE2 in cells yields Aß fragments identical to those produced in vitro. A,B, Spectra of Aß fragments determined by IP/MS analysis of the conditioned media of CHO cells transfected with APP and empty vector (A) or APP and BACE2 (B) (see Methods). A, APP expression alone produces peaks corresponding to Aß42, Aß40, Aß39, Aß38 and Aß37. B, Co-expression of APP and BACE2 results in decreases in the relative abundance of the aforementioned Aß species and the appearance of three new fragments: Aß34, Aß20 and Aß19. Double-charged fragments are denoted by “+”, and “*” represents the modification of a fragment by AEBSF, which leads to a 183-Da increase in MW, as previously reported [46].

Article Snippet: Recombinant BACE2 (R&D Systems) and plasmin (EMD Biosciences) were purchased from commercial sources, while recombinant IDE and secreted NEP (i.e., lacking the transmembrane domain) were generated and purified as described [23].

Techniques: Over Expression, Produced, In Vitro, Protein-Protein interactions, Transfection, Plasmid Preparation, Expressing, Modification

Journal: Molecular neurodegeneration

Article Title: Identification of BACE2 as an avid ß-amyloid-degrading protease.

doi: 10.1186/1750-1326-7-46

Figure Lengend Snippet: Figure 5 Comparison of the efficacy of BACE2 relative to other well-established AßDPs in vitro and in cultured cells. A, Degradation of Aß in vitro by equivalent nominal concentrations (5 nM) of recombinant BACE2, IDE, NEP and plasmin. Note that BACE2 degrades Aß at a faster rate than NEP and plasmin, but not IDE. B,C, Effects on Aß40 (A) and Aß42 (C) levels following cotransfection of CHO cells with APP together with equivalent quantities of cDNAs encoding BACE2, ECE1b and IDE. In good agreement with the results in vitro (A), BACE2 lowers the levels of both Aß species to an extent exceeding NEP and ECE1b, but comparable to IDE. Data are mean ± SEM of 4 replications, normalized to controls cotransfected with empty vector (Vo).

Article Snippet: Recombinant BACE2 (R&D Systems) and plasmin (EMD Biosciences) were purchased from commercial sources, while recombinant IDE and secreted NEP (i.e., lacking the transmembrane domain) were generated and purified as described [23].

Techniques: Comparison, In Vitro, Cell Culture, Recombinant, Cotransfection, Plasmid Preparation

Journal: Molecular neurodegeneration

Article Title: Identification of BACE2 as an avid ß-amyloid-degrading protease.

doi: 10.1186/1750-1326-7-46

Figure Lengend Snippet: Figure 6 BACE2 is localized to intracellular compartments relevant to Aß degradation. A, Exogenous administration of fluorescently labeled Aß40 (green) to CHO cells results in accumulation at intracellular sites overlapping with lysosomes, as labelled by Lysotracker Red (red) and visualized by confocal microscopy. B, BACE2 is expressed in multiple intracellular compartments, including lysosomes. Distribution of GFP- tagged BACE2 (green) in cells labeled with Lysotracker Red (red) shows significant localization within lysosomes (yellow). C, BACE2 colocalizes with exogenously administered Aß. Confocal images showing significant overlap (yellow) between BACE2 (green) and fluorescently labeled Aß (red). For these experiments, cells were imaged within 5 minutes of washing in cold PBS to remove medium containing excess fluorescently labeled Aß. Note that the the majority of BACE2-GFP-expressing cells contianed very low levels of fluorescent Aß (see Figure 7), and the particular cell shown exhibited relatively high levels of internalized Aß, allowing us to highlight the overlap with BACE2.

Article Snippet: Recombinant BACE2 (R&D Systems) and plasmin (EMD Biosciences) were purchased from commercial sources, while recombinant IDE and secreted NEP (i.e., lacking the transmembrane domain) were generated and purified as described [23].

Techniques: Labeling, Confocal Microscopy, Expressing

Journal: Molecular neurodegeneration

Article Title: Identification of BACE2 as an avid ß-amyloid-degrading protease.

doi: 10.1186/1750-1326-7-46

Figure Lengend Snippet: Figure 7 BACE2 degrades Aß at intracellular sites. A, CHO cells expressing GFP-BACE2 (green, left), but not those expressing GFP alone (green, right), exhibit marked reductions in intracellular Aß (red). For these experiments, cells were loaded for 6 h with 400 nM fluorescently labeled Aß40, washed, then incubated at 37°C for 1 h prior to imaging by conventional fluorescence microscopy. B,C, BACE2 overexpression significantly lowers intracellular Aß. B, Quantification of intracellular pools of fluorescently labeled Aß40 in CHO cells 0 and 2 hours after loading. C, Relative levels of intracellular (unmodified) Aß40 in CHO cells 0 and 2 hours after loading, as quantified by ELISA. Data are mean ± SEM of 3 replicates, normalized to vector-only controls. *P <0.05 by Tukey’s multiple comparisons test.

Article Snippet: Recombinant BACE2 (R&D Systems) and plasmin (EMD Biosciences) were purchased from commercial sources, while recombinant IDE and secreted NEP (i.e., lacking the transmembrane domain) were generated and purified as described [23].

Techniques: Expressing, Labeling, Incubation, Imaging, Fluorescence, Microscopy, Over Expression, Enzyme-linked Immunosorbent Assay, Plasmid Preparation